ω-Phthalimidoalkyl Aryl Ureas as Potent and Selective Inhibitors of Cholesterol Esterase

Accepted Article

Title: ω-Phthalimidoalkyl aryl ureas as potent and selective inhibitors of

cholesterol esterase

Authors: Florian M. Dato, Miriam Sheikh, Rocky Z. Uhl, Alexandra

W. Schüller, Michaela Steinkrüger, Peter Koch, Jörg-Martin

Neudörfl, Michael Gütschow, Bernd Goldfuss, and Markus

Pietsch

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content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201800388

Link to VoR: http://dx.doi.org/10.1002/cmdc.201800388

A Journal of

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10.1002/cmdc.201800388

ChemMedChem

FULL PAPER

ω-Phthalimidoalkyl aryl ureas as potent and selective inhibitors

of cholesterol esterase

Florian M. Dato,^[a,b]Dr. Miriam Sheikh,^[a]Rocky Z. Uhl,^[a]Alexandra W. Schüller,^[a,b]Dr. Michaela

Steinkrüger,^[a]Dr. Peter Koch,^[a]Dr. Jörg-Martin Neudörfl,^[b]Prof. Michael Gütschow,^[c]Prof. Bernd

Goldfuss,*^[b]and Dr. Markus Pietsch*^[a]

Abstract: Cholesterol esterase (CEase), a serine hydrolase thought

to be involved in atherogenesis and thus coronary heart disease, is

considered as a target for inhibitor development. We investigated

recombinant human and murine CEases with a new fluorometric

assay in a structure-activity-relationship study applying a small

library of ω-phthalimidoalkyl aryl ureas. The urea motif with an

attached 3,5-bis(trifluoromethyl)phenyl group and the aromatic

character of the ω-phthalimide residue were most important for

inhibitory activity. In addition, an alkyl chain composed of three or

four methylene groups, connecting the urea and phthalimide

moieties, was found to be an optimal spacer for inhibitors. The so

optimized compounds 2 (1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-(1,3-

dioxoisoindolin-2-yl)propyl)urea) and 21 (1-(3,5-bis(trifluoromethyl)-

phenyl)-3-(4-(1,3-dioxoisoindolin-2-yl)butyl)urea) exhibit dissociation

constants (K_i) of 1-19 µM on the two CEases and show either a

competitive (2 at the human, 21 at the murine enzyme) or otherwise

a non-competitive mode of inhibition. Two related serine hydrolases,

monoacylglycerol lipase and fatty acid amide hydrolase, were

inhibited by ω-phthalimidoalkyl aryl ureas to a lesser extent.

possible implications in the development of atherosclerosis and,

thus, coronary heart disease (CHD) render this hydrolytic

enzyme an interesting drug target.^[7]Intestinal CEase facilitates

the absorption of cholesteryl esters and cholesterol, while a high

plasma activity of this enzyme was found to be correlated with

increased total plasma cholesterol and low-densitiy lipoprotein

(LDL) cholesterol levels.^[7-8]Furthermore, plasmatic CEase may

also contribute to CHD by converting “larger and less

atherogenic LDL to smaller and more atherogenic LDL

subspecies”^[8]and by being involved in the generation of

cholesterol crystals in LDL aggregates, which are thought of as

a hallmark of atherosclerotic plaques and represent a possible

source of inflammation.^[9-11]These results and further findings

from in vitro and in vivo studies led to the hypothesis that CEase

inhibitors might lower plasmatic LDL levels and thus may act as

anti-atherosclerotic agents.^[12-16]The majority of the initially

developed CEase inhibitor classes contain electrophilic sites

which are attacked by the active-site serine residue (e.g. boronic

acids,^[17]carbamates,^[17-21]phosphoisocoumarines,^[22]and

thieno[1,3]oxazin-4-ones 29 and 30,^[23]Scheme 1), while

recently reported rhodanine^[24]and thiazolidinedione^[24]inhibitors

as well as 2-(1H-indol-3yl)-4-phenylquinolines^[25]do not appear

to modify the catalytic triad (Ser-His-Asp).^[26-27]

Introduction

In recent years, there has been much effort in designing small

molecule inhibitors for enzymes of the serine hydrolase

family.^[1-3]In this context, we are particularly interested in the

bile-salt activated cholesterol esterase (CEase, EC 3.1.1.13), a

non-specific serine hydrolase with a vast variety of substrates

including cholesteryl esters, tri-, di- and monoacylglycerols,

phospholipids, vitamin A esters,^[4]and lipid amides.^[5-6]Its

Scheme 1. Structures of CEase inhibitors enzymatically characterized in this

study: ω-phthalimidoalkyl aryl ureas 2 and 21 and thieno[1,3]oxazin-4-ones

29 and 30.

[a]

Dr. M. Pietsch, F. M. Dato,^#Dr. M. Sheikh,^#R. Z. Uhl, A. W.

Schüller, Dr. M. Steinkrüger, Dr. P. Koch

Institute II for Pharmacology, Center of Pharmacology,

Medical Faculty

University of Cologne

Gleueler Strasse 24, 50931 Cologne (Germany)

Email: markus.pietsch@uk-koeln.de

In this study, we present the synthesis and characterization of

ω-phthalimidoalkyl aryl ureas 2 and 21 (Scheme 1) as reversible

inhibitors of recombinantly expressed murine and human

CEases. Structure-activity relationship studies on a small library

[b]

F. M. Dato, A. W. Schüller, Dr. J.-M. Neudörfl, Prof. Dr. B. Goldfuss

Institute of Organic Chemistry, Department of Chemistry

University of Cologne

of

derivatives

revealed

the

presence

of

a

3,5-

bis(trifluoromethyl)phenyl group attached to the urea to be

particularly advantageous for inhibition. Such introduction of

fluorine into bioactive molecules is a well-established strategy in

drug development.^[28]Fluorine and fluorine-containing groups

have severe polarizing effects on chemical bonds leading to

strong dipole moments and enhanced intra- and intermolecular

interactions. The fluorine introduction can be utilized to modulate

the acidity of adjacent moieties closed to fluorine and to adjust

the compounds’ lipophilicity. Moreover, spectroscopic,

Greinstrasse 4, 50939 Cologne (Germany)

Email: goldfuss@uni-koeln.de

Prof. Dr. M. Gütschow

Pharmaceutical Institute, Pharmaceutical Chemistry I

University of Bonn

An der Immenburg 4, 53121 Bonn (Germany)

These authors contributed equally to this work.

[c]

[#]

Supporting information for this article is given via a link at the end of

the document. ((Please delete this text if not appropriate))

10.1002/cmdc.201800388

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crystallographic and computational studies indicate that

covalently bound fluorine in organic molecules can act as a

hydrogen-bond acceptor.^[29-31]A blocking of potential oxidation

sites in order to prevent undesired metabolic pathways has been

realized, e.g. by replacing methyl-arene substituents by trifluoro-

methyl.^[29-31]Thus, pharmaceutical effectiveness, metabolic

stability and improved bioabsorption have been demonstrated

for several fluorinated drugs compared to their non-fluorinated

counterparts, and fluorophilic and fluorophobic environments

within target proteins have been determined, as exemplified in

the field of protease inhibitors.^[32-34]

In addition to potency, selectivity of enzyme inhibitors is crucial

for their usage as tools and therapeutics. To assess the

selectivity of CEase inhibitors, we chose to investigate two

serine hydrolases, i.e. human monoacylglycerol lipase (MAGL,

EC 3.1.1.23)^[35]and human fatty acid amide hydrolase (FAAH,

EC 3.5.1.99),^[36-38]which accept similar physiological substrates

to CEase, that is esters and amides of long chain fatty acids,

respectively,^[39-43]and represent the major degrading enzymes in

endocannabinoid metabolism.^[44-45]Furthermore, both MAGL and

FAAH are involved in several pathological conditions, amongst

them cancer, pain and inflammatory diseases, neurological and

cardiovascular disorders,^[46-55]and are, therefore, also

considered as targets for the development of therapeutic drugs.

Thorough pharmacological analysis of the three target

hydrolases CEase, MAGL and FAAH investigated in this study

requires the availability of robust assay systems allowing for the

kinetic assessment of the enzyme-inhibitor interaction. In this

context, spectrophotometric and fluorometric methods have

proven to be effective for the quantification of the enzymes’

activities. Here, we characterize 4-methylumbelliferyl butyrate

(4-MUB)^[56]and 4-nitrophenyl butyrate (4-NPB)^[57]as substrates

of CEase and MAGL, respectively, while N-(6-methoxypyridine-

3-yl)decanamide (D-MAP) has been introduced as FAAH

substrate in a previous report^[58](for structures of the three

substrates, see Figure S4, Supporting information).

Results and Discussion

Synthesis and structural characterization of phthalimide

derivatives

A series of 26 aryl urea derivatives containing either aromatic or

aliphatic phthalimide motifs were synthesized starting from the

corresponding phthalic anhydride and ω-amino acids with

various chain lengths to yield ω-phthalimido carboxylic acids 31-

38 (61-94%, Scheme 2). Formation of the urea moiety was

initiated by preparation of the respective acid chloride, followed

by its reaction with sodium azide and a Curtius rearrangement of

the acid azide. The formed isocyanate was then trapped with a

variety of anilines and piperazines to obtain compounds 1-26 in

low to moderate overall yields (3-62%) calculated from the

respective ω-phthalimido carboxylic acids. All but four of these

amines were commercially obtained, with 3’,5’-bis(trifluoro-

methyl)-substituted biphenylamines 39-41 (64-94%) being

prepared by Suzuki coupling of o-, m- and p-bromoaniline with

3’,5’-bis(trifluoromethyl)phenylboronic

acid,

respectively.

Furthermore, 1-(3,5-bis(trifluoromethyl)phenyl)piperazine 42

(51%) was synthesized from bis(2-chloroethyl)amine hydro-

chloride and 3,5’-bis(trifluoromethyl)aniline. To investigate the

influence of the urea motif on the inhibitory potency of the

compounds, the amides 27 (64%) and 28 (74%) corresponding

to the active CEase inhibitors 2 and 21 were obtained from the

phthalimido acids 33 and 34, respectively, under standard amide

coupling conditions using 1-ethyl-3-(3-dimethylaminopropyl)-

Scheme 2. Synthesis of compounds 1-28 and 39-42. Reagents and conditions: a) phthalic anhydride, neat, 3h, 170 °C; b) cis-hexahydrophthalic anhydride, cis-

1,2,3,6-tetrahydrophthalic anhydride, 3,4,5,6-tetrahydrophthalic anhydride or endo-3,6-methylene-1,2,3,6-tetrahydrophthalic anhydride, TEA, toluene, 14 h

reflux; c) 1. SOCl₂, toluene, 14 h, RT, 2. NaN₃, acetone/H₂O (4:1), 3 h, RT, 3. toluene, 20 min, reflux, 4. amine, 14 h, reflux; d) EDCHCl, HOBT, CH₂Cl₂, 14 h,

0 °CRT; e) 1. 3,5-bis(trifluoromethyl)phenylboronic acid, tri(o-tolyl)phosphine, K₂CO₃, Pd(OAc)₂, toluene/EtOH/H₂O (7:3:2), 1.5 h, 80 °C; f) bis(2-

chloroethyl)amine hydrochloride, diethyleneglycol monomethyl ether, 150 °C, 24 h.

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carbodiimide hydrochloride (EDCHCl) and 1-hydroxybenzo-

triazole hydrate (HOBT) (Scheme 2). The ¹H and ¹³C NMR

spectra of all intermediates and final products are shown in

Figures S32-S71 (Supporting information).

The structures of the aromatic and aliphatic phthalimido urea

derivatives were unambiguously proven by crystal structure

analysis of compounds 1, 2, 5, 9, 17, 20 and 24 (Tables S1-S7

and Figures S25-S31, Supporting information).

To quantify the amount of product formed by substrate

hydrolysis, the fluorescence of 4-methylumbelliferone and the

absorbance of 4-nitrophenol were determined in dependence on

the compounds’ concentration under assay conditions (Figures

S5 and S6, Supporting information). Next, we identified the

range of enzyme concentration which results in a proportional

increase in enzymatic activity (Figure 1; Figure S7, Supporting

information). Such linear interdependence of activity and

concentration is crucial for the identification and characterization

of enzyme inhibitors. As a result, 40 ng mL^–1mCEase, 15 ng

mL^–1hCEase, 500 ng mL^–1bCEase and 20 ng mL^–1hMAGL

were chosen for the characterization of both enzyme-substrate

and enzyme-inhibitor interactions. For hFAAH, an enzyme

Eukaryotic expression of pancreatic murine and human

CEase

CEases from murine and human pancreas (mCEase and

hCEase) carrying a C-terminal double Strep II tag were over-

expressed in HEK 293-EBNA cells and secreted into the

supernatant. Purification of the target enzymes was done by

one-step affinity chromatography of the supernatant on

StrepTactin sepharose yielding 15 mg mCEase and 3.5 mg

hCEase per liter supernatant, respectively. While the present

study describes the recombinant expression of mCEase for the

first time, the obtained yield of hCEase could be compared to

literature data and was found to be superior to that of Loomes et

al.^[59](1 mg enzyme per liter supernatant) who used baby

hamster kidney cells. The purity and identity of the expressed

proteins were analyzed by SDS-PAGE on a 10% polyacrylamide

gel and Western blot analysis, respectively (Figure S3,

Supporting information). Both mCEase and hCEase showed

high purity but exhibited protein double bands with estimated

molecular masses of 74 and 130-140 kDa, respectively. Spilburg

et al.^[60]found molecular masses of 68 and 118 kDa for isolated

pancreatic mCEase and hCEase, respectively. The difference in

the mass to the recombinant CEases can be attributed to the

double Strep II tag with its thrombin cleavage site having a

weight of 4 kDa and a possible variation in glycosylation due to

expression in HEK 293 EBNA cells.^[60-62]The detected double

band has been observed both by Spilburg et al.^[60]and Hansson

et al.^[63]for isolated and recombinant pancreatic hCEase which

was explained as being caused by variations at the N-

glycosylation site of the protein since a mutant protein lacking

this particular site (N187Q) did not exhibit such double band.^[63]

Figure 1. Range of mCEase (black) and hCEase (red) where the enzymatic

rate is proportional to the enzyme concentration. Experiments were done with

200 µM 4-MUB. Mean values ± SEM from 2-3 experiments, each performed in

triplicate are shown.

A linear dependency was found in the range

3.6-89.1 ng mL^–1mCEase (slope: 15.7 ± 0.9 nmol min^–1µg^–1, n = 3) and

3.2-31.7 ng mL^–1hCEase (slope: 35.7 ± 0.2 nmol min^–1µg^–1, n = 3). For

bCEase, a slope of 0.531 ± 0.018 nmol min^–1µg^–1(n = 3) was determined in

the range 50-500 ng mL^–1under the same conditions (data not shown).

concentration of 1 µg mL^–1had previously been identified by

Dato et al.^[58]as suitable for such experiments.

The hydrolysis of 4-MUB and 4-NPB by the three pancreatic

CEases and hMAGL, respectively, was further characterized by

determination of the Michaelis constant, K_m, and the maximum

velocity, V_max(Table 1). Consumption of both substrates

followed Michaelis-Menten kinetics and showed small but not

negligible non-enzymatic hydrolysis (Figure 2 and Figure S8,

Supporting information), which had to be considered in the

following inhibition experiments. For the three CEases, we

determined values of K_min the range 65-223 µM, which are quite

in agreement with reported Michaelis constants obtained for the

hydrolysis of the related substrate 4-NPB by CEases from

different species.^{[18, 64-67]}However, with respect to V_max(and the

estimated turnover number k_cat), 4-NPB appears to be superior

in comparison to 4-MUB.^{[65, 67]}For the hydrolysis of 4-NPB by

hMAGL, K_m(184 µM) and V_max(269 µmol min^–1mg^–1; 20 ng mL^–1

MAGL corresponds to a maximum concentration of ~0.6 nM

under the assumption of a molecular weight of 34 kDa, leading

to a minimum k_catof 149 s^–1) were compared to the respective

parameters of the hMAGL-catalyzed hydrolysis of the related 4-

MUB^[68-69]and 4-nitrophenyl acetate (4-NPA).^[57]While similar

values of K_mhave been reported for these two MAGL substrates

Kinetic assays to determine the inhibition of CEases,

hMAGL and hFAAH

The enzymatic activity of the three pancreatic CEases, i.e.

mCEase, hCEase, and bovine CEase (bCEase), and the

endocannabinoid-degrading enzyme hMAGL was determined by

means of fluorometric and chromogenic assays using the

substrates 4-MUB^[56]and 4-NPB,^[57]respectively (Figure S4,

Supporting information), with the enzyme-substrate interaction

being kinetically characterized in the present study for the first

time. hFAAH was analyzed with the newly developed fluorogenic

substrate D-MAP (Figure S4, Supporting information), whose

properties towards hFAAH and suitability for inhibition

experiments have been recently described in detail by Dato et

al.^[58]

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(134-200 µM), the value of V_max(as well as the estimated value

of k_cat) obtained for 4-NPB exceeds those reported for 4-NPA

(52.2 µmol min^–1mg^–1)^[57]and 4-MUB (1.13 s^–1and 31.4 s^–1)^[68-69]

by at least a factor of ~5.

pre-incubation were supported by results of Björklund et al.^[72]

Validation of the hFAAH assay was done with the active site-

directed compounds URB597 (irreversible inhibitor)^[73]and TC-

F2 (reversible inhibitor)^[74]as described by Dato et al.^[58]

Table 1. Characterization of the enzyme-substrate interaction. Mean

Screening of phthalimide derivatives for inhibition of

mCEase, hMAGL and hFAAH

values ± SEM with the number of experiments are shown.

enzyme

substrate

K_m/ µM

V_max/ µMol min^–1mg^–1

n

Investigation of the phthalimide derivatives’ 1-28 ability to act as

serine hydrolase inhibitors was initially performed on mCEase,

hMAGL and hFAAH at a single inhibitor concentration of 25 µM

and substrate concentrations of 200 µM 4-MUB (2.7  K_m),

200 µM 4-NPB (1.1  K_m) and 10 µM D-MAP (1.2  K_m),

respectively. The results are shown in Tables 2-4.

mCEase

hCEase

4-MUB

74.4 ± 10.5

65.3 ± 3.4

7.73 ± 1.08

37.7 ± 1.5

6

3

bCEase

hMAGL

hFAAH

4-MUB

4-NPB

223 ± 22

184 ± 14

8.43 ± 0.90

1.30 ± 0.11

7

4

269 ± 16

a

D-MAP^

(9.08 ± 0.83)10^-3



First, the substitution pattern of the phenyl ring at the urea motif

was varied (compounds 1-17, Table 2), with 3,5-bis-

(trifluoromethyl) (2) being most favorable for inhibition of

mCEase. The two trifluoromethyl groups in 2 cause, among

other effects,^[75-77]distinct acidification of both the phenyl-

substituted NH group and the adjacent aromatic ortho-protons

increasing their ability to participate in hydrogen bonds. This

acidification is reflected by a downfield shift of the respective

[a] Values taken from Dato et al.^[58]

The higher values of V_max(k_cat) found for both CEase and MAGL

for the hydrolysis of 4-NPB in comparison to 4-MUB can be

attributed to the greater reactivity of the former substrate, which

is caused by the activating effect of the 4-nitrophenolate leaving

group on the carbonyl carbon atom of the ester bond.^[70]

1

signals in the H NMR spectrum (2: NH, δ = 9.37 ppm; o-arH, δ

= 8.05 ppm vs. 1: NH, δ = 8.54 ppm; o-arH, δ = 7.63 ppm).^[78-80]

Although the 4-nitro and 3,5-bis(nitro) group(s) mediate even

more pronounced effects (14: NH, δ = 9.38 ppm; o-arH, δ = 8.12

ppm; 7: NH, δ = 9.67 ppm; o-arH, δ = 8.67 ppm), compounds 14

and 7 show much decreased inhibitory activity towards mCEase.

This might be attributed to the strong intermolecular coordination

of the nitro group(s) to the urea motif (also occurring in an

aqueous environment),^[80-81]which would lead to dimer or

multimer formation and thus impair binding to the enzyme.

Acidification of both the phenyl-substituted NH and the aromatic

ortho-protons is less pronounced (compared to 2) in compounds

1 and 3-6 which do not inhibit mCEase. The same applies to

some extent to the para-substituted derivatives 8-13.

Introduction of a 1,4-phenylene linker between the urea NH

group and the 3,5-bis(trifluoromethyl)phenyl moiety leads to a

dramatic decrease in inhibitory potency on mCEase (2 vs. 17)

which is even more pronounced in case of 1,2- (15) and 1,3-

phenylene linkers (16). A detrimental effect on inhibition of the

serine hydrolase is also observed when exchanging the

aforementioned NH group in 2 for a piperazine moiety in 19

(Table 2). Since a 3,5-bis(trifluoromethyl)phenyl-substitued urea

was found to be most advantageous for mCEase inhibition, this

motif was kept constant and the aromatic phthalimide in 2 was

replaced by hydrogenated (bicyclic and tricyclic) moieties (23-26,

Table 3). The loss of both aromaticity and planar geometry of

the phthalimide backbone results in a decreased binding

capacity towards mCEase. Finally, the active mCEase inhibitor 2

was modified by varying the spacer length between the

phthalimide and urea motifs (20-22, Table 4). While shortening

the spacer from three to two methylene groups leads to a

complete loss of inhibitory activity towards mCEase (2 vs. 20),

increasing the spacer length to four or five methylene groups

only slightly decreases this activity (2 vs. 21, 22).

Figure 2. Determination of the values of K_mand V_maxfor the CEase-catalyzed

hydrolysis of 4-MUB (black symbols, mCEase, 40 ng mL^–1; red symbols,

hCEase, 15 ng mL^–1). Plot of the total rates (circles) and the rates of the non-

enzymatic hydrolysis (squares) versus the concentration of 4-MUB. Mean

values ± SEM from 2-6 experiments, each performed in duplicate or triplicate,

are shown. A Michaelis-Menten analysis using both rates gave K_m= 74.4 ±

10.5 µM, V_max= 0.309 ± 0.043 µM min^–1(n = 6) for mCEase and K_m= 65.3 ±

3.4 µM, V_max= 0.565 ± 0.022 µM min^–1(n = 3) for hCEase. The respective

analysis of the bCEase (500 ng mL^–1)-catalyzed hydrolysis of 4-MUB resulted

in K_m= 223 ± 23 µM and V_max= 0.650 ± 0.054 µM min^–1(n = 7, data not

shown).

To validate the three assays for the search of new compounds

capable of impairing the enzymes’ activity, reported inhibitors

were investigated. For the pancreatic CEases, the alternate

substrate inhibitors 29 and 30 were kinetically characterized

(Table 5),^[23]with the dissociation constants, K_i, obtained for the

inhibition of the bovine enzyme being in agreement with the

respective values obtained with the substrate 4-NPB.^[23]

Inhibition of hMAGL by the irreversible acting compound

JZL 184^[71]was evaluated in dependence on the pre-incubation

time of enzyme and inhibitor (Figure S9, Supporting information).

Here, the values of IC₅₀, i.e. the inhibitor concentration causing a

50% decrease in enzymatic activity, determined without and with

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Table 2. Structure-activity relationships in dependence on the aniline/phenylpiperazinyl moiety.

[a]

Residual enzymatic activity (%) at [I] = 25 µM

Compd

R¹

R²

R³

R⁴

X

mCEase

hMAGL

hFAAH

1

2

H

NH

107 ± 1

108 ± 3

83.2 ± 3.0

78.2 ± 3.8

CF₃

5.25 ± 1.63

64.1 ± 10.4

3

H

Y

H

Me

OMe

F

H

Me

OMe

F

NH

Pip

94.7 ± 6.3

103 ± 3

105 ± 0

110 ± 3

103 ± 3

104 ± 6

80.5 ± 5.4

116 ± 2

105 ± 4

107 ± 4

107 ± 1

112 ± 2

104 ± 7

85.3 ± 4.6

72.5 ± 2.0

100 ± 5

90.2 ± 5.6

106 ± 3

65.2 ± 3.2

77.3 ± 1.7

78.0 ± 2.6

79.4 ± 3.0

84.7 ± 2.0

82.7 ± 1.2

86.0 ± 4.1

81.6 ± 3.7

84.7 ± 2.7

82.4 ± 2.9

85.1 ± 3.3

82.6 ± 2.7

75.6 ± 2.8

81.5 ± 2.9

84.7 ± 1.8

171 ± 5

4

H

5

H

88.4 ± 5.9

100 ± 8

6

Cl

NO₂

H

Cl

NO₂

H

7

H

85.3 ± 1.5

92.5 ± 2.7

100 ± 3

8

CF₃

Me

OMe

F

9

H

10

11

12

13

14

15

16

17

18

19

H

90.4 ± 3.1

101 ± 3

H

Cl

Br

NO₂

H

64.0 ± 7.2

94.5 ± 9.4

76.5 ± 1.0

87.8 ± 2.0

73.5 ± 7.8

58.2 ± 4.6

101 ± 1

H

Y

H

Y

H

80.9 ± 0.8

84.6 ± 2.5

CF₃

H

CF₃

95.3 ± 1.9

[a] Mean ± SEM (n = 3-4)

Furthermore, the importance of the urea motif in compounds 2

and 21 for mCEase inhibition was investigated by replacing the

NH at the aliphatic site for a methylene group, leading to the

almost inactive amides 27, 28 (Table 4).

Inhibition of hMAGL and hFAAH by phthalimide derivatives 1-28

(25 µM) was found to be weak to moderate, with compounds 2

and 21 (both active on mCEase) reducing the activity of the two

hydrolases to 64.1% at the most (Table 3). Although the related

compound 22 was slightly more potent on hMAGL (residual

activity of 49%), it was not further investigated on this enzyme

due to its promiscuous behavior towards mCEase (see below).

and v₀represent the enzymatic rate of substrate hydrolysis in

the presence and absence of inhibitor, I, while the Hill slope, n_H,

defines the steepness of the curve. In accordance with the

results shown in Table 4, compounds 2, 21 and 22 inhibited

mCEase with values of IC₅₀of 3.10 µM, 5.30 µM and 9.88 µM,

respectively. To exclude promiscuous inhibition, the experiments

were repeated in the presence of 0.01% (v/v) Triton X-100 to

dissolve aggregates formed by inhibitor molecules in aqueous

solutions.^[82]While the IC₅₀values of compounds 2 and 21 were

only slightly affected by the detergent (7.47 µM and 3.27 µM,

respectively, Figure 3), the respective value of phthalimide 22

was considerably increased by a factor of 6.2 to 61.6 µM (Figure

S10, Supporting information), characterizing the latter compound

as promiscuous inhibitor.^[82]Due to its detergent-sensitive

behavior, compound 22 was excluded from further experiments.

Both 2 and 21 also showed activity towards hCEase (Table 4

and Figure S11, Supporting information), inhibiting the enzyme

with values of IC₅₀of 23.9 µM and 2.29 µM, respectively.

Characterization of ω-phthalimidoalkyl aryl ureas as

inhibitors of murine and human CEases

On the basis of the results from the initial screening, the ω-

phthalimidoalkyl aryl ureas 2, 21 and 22 were selected for

further characterization on mCEase by determination of IC₅₀,

which was calculated by the equation v_E= v₀/(1+([I]/IC₅₀)^nH)

(Figures 3 and S10, Supporting information). In this equation, v_E

To gain deeper insights into the interaction of inhibitors 2, 21, 29

and 30 with murine and human CEases, we determined their

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dissociation constants K_iand modes of inhibition (MOI) by

varying both the substrate and inhibitor concentrations (Table 5).

The reported inhibitors 29 and 30 showed distinct species-

specific differences in activity. This was most obvious for

compound 29, which binds ~100-fold tighter to mCEase (K_i=

34.1 nM) than to hCEase. The two inhibitors show non-

competitive behavior towards the bovine and murine

isoenzymes in the presence of 4-MUB, with the enzyme-

substrate-inhibitor (ESI) complex formed between bCEase and

30 being inactive.

Table 3. Structure-activity relationships in dependence on the

(modified) phthalimide moiety.

[a]

Residual enzymatic activity (%) at [I] = 25 µM

Compd

mCEase

hMAGL

hFAAH

2

5.25 ± 1.63

89.1 ± 9.2

82.9 ± 10.2

64.1 ± 10.4

80.6 ± 4.5

72.4 ± 2.3

78.2 ± 3.8

87.0 ± 0.7

88.8 ± 1.0

23

24

25

26

71.0 ± 9.5

64.8 ± 2.9

84.8 ± 7.2

86.7 ± 1.6

91.0 ± 1.6

96.6 ± 10.2

[a] Mean ± SEM (n = 3-4).

Table 4. Structure-activity relationships in dependence on spacer length

and the urea/amide motif.

[a]

Residual enzymatic activity (%) at [I] = 25 µM

Compd

n

X

mCEase

hMAGL

hFAAH

Figure 3. Inhibition of mCEase (40 ng mL^–1)-catalyzed hydrolysis of 4-MUB

(200 µM, 2.7  K_m) by compounds 2 (A) and 21 (B) in the absence (black) and

presence (red) of 0.01% (v/v) Triton X-100. Data shown are mean values ±

SEM of percentage enzymatic rates from three separate experiments, each

performed in triplicate. A) Non-linear regression gave values of IC₅₀of 3.10 ±

0.82 µM and 7.47 ± 1.09 µM and Hill slopes, n_H, of 1.61 ± 0.03 and 1.37 ± 0.05

in the absence and presence of Triton X-100, respectively (mean values ±

SEM n = 3). Both IC₅₀and n_Hare significantly changed (P values of 0.0328

and 0.0169, respectively) in the presence of detergent (unpaired Student’s t-

test). B) Non-linear regression gave values of IC₅₀of 5.30 ± 0.15 µM and 3.27

± 0.67 µM and Hill slopes, n_H, of 1.31 ± 0.01 and 1.19 ± 0.04 in the absence

and presence of Triton X-100, respectively (mean values ± SEM n = 3). While

the IC₅₀value of 21 is significantly reduced (P = 0.0416) in the presence of

detergent, the two Hill slopes are non-significantly different (P > 0.05) from

each other (unpaired Student’s t-test).

20

1

2

NH

108 ± 5

97.4 ± 2.9

86.3 ± 2.8

78.2 ± 3.8

2^[b]

5.25 ± 1.63

64.1 ± 10.4

21^[b]

3

4

2

3

NH

15.6 ± 3.1

22.5 ± 0.3

71.8 ± 8.9

90.8 ± 1.3

68.5 ± 5.3

49.3 ± 6.7

91.1 ± 5.0

81.3 ± 3.0

85.0 ± 2.0

85.1 ± 3.1

87.5 ± 3.2

93.0 ± 2.6

22

NH

27

CH₂

28

[a] Mean ± SEM (n = 3-4). b Residual enzymatic activity of hCEase: 2,

43.9 ± 3.6%; 21, 5.46 ± 0.33%.

Analysis was done as shown in Figure 4 by a global fit according

to Equation 1 (including the constraints mentioned in the

footnotes of Table 5 and the Experimental Section) of the data in

the respective Michaelis Menten plot. Further information with

regard to the MOI of the compounds was obtained by means of

the plots shown in Figures S12-S24 (Supporting information).

In contrast, the ESI complex of bCEase with 29 and the ESI

complexes of mCEase with 29 and 30 are still able to form

product with the values of k_catbeing reduced by more than 95%.

Such activity of an ESI complex is revealed by hyperbolic

inhibition, which is even more

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Table 5. Kinetic characterization of CEase inhibitors including the dissociation constant of the enzyme-inhibitor complex (K_i) and the mode of inhibition (MOI).^[a]

mCEase

hCEase

bCEase

Compd

K_i(µM)

MOI

K_i(µM)

MOI

K_i(µM)

MOI

2

1.19 ± 0.07

nc

c

10.0 ± 0.5

11.8 ± 1.9

4.34 ± 0.84^[f]

c

n.d.

hnc

21

18.7 ± 3.3

nc

hmt

n.d.

29^[b,c]

0.0341 ± 0.0018^[d]

hnc

0.664 ± 0.030^[h]

30^[b,c]

0.262 ± 0.027^[e]

hnc

4.31 ± 0.48^[g]

hnc

1.68 ± 0.14^[i]

nc

[a] Mean ± SEM (n = 3-4). [b] K_ivalues for 29 (0.63 µM) and 30 (2.15 µM) were previously determined by Pietsch & Gütschow^[23]on bCEase by a

spectrophotometric assay using the chromogenic substrate 4-NPB. [c] Data were analyzed according to an equation of hyperbolic mixed-type inhibition (Equation

1). [d] α was set to 1,  = 0.00644 ± 0.00357. [e] α was set to 1,  = 0.0463 ± 0.0177. [f] α = 5.46 ± 0.96,  = 0.450 ± 0.164. [g] α was set to 1,  = 0.116 ± 0.016.

[h] α was set to 1,  = 0.0445 ± 0.0184. [i] α was set to 1,  was set to 0. Abbreviations: c, competitive; hmt, hyperbolic mixed-type; hnc, hyperbolic non-

competitive; nc, linear non-competitive; n. d., not determined.

Figure 4. Determination of K_iand MOI for the inhibition of mCEase (40 ng mL^–1; A, C) and hCEase (15 ng mL^–1; B, D) by compounds 2 (A, B) and 21 (C, D).

Shown are Michaelis-Menten plots with mean values ± SEM of enzymatic rates from three to four separate experiments, each performed in duplicate or

triplicate. Concentrations of compound 2: 0 µM (), 1 µM (), 2 µM (), 3 µM (), 4 µM (), 5 µM (), 6 µM (), and 7 µM (). Concentrations of compound 21:

0 µM (), 1.25 µM (), 2.5 µM (), 5 µM (), 10 µM (), 20 µM (), 30 µM (), and 40 µM (). Analysis was done according to equations of either competitive

or linear non-competitive inhibition (see Experimental Section) and results are listed in Table 5.

pronounced for the interaction of the two compounds with

hCEase. Here the respective ESI complexes hydrolyse 4-MUB

with values of k_catof 45% (29) and 12% (30) of that of the

uninhibited enzyme. While 30 shows non-competitive behavior,

binding of 29 to the free hCEase is ~5 times more favored than

to the hCEase-4-MUB complex, resulting in mixed-type inhibition

with a pronounced competitive component. The new ω-

phthalimidoalkyl aryl ureas 2 and 21 act with slightly larger

values of K_i(10.0 µM and 11.8 µM, respectively) on hCEase

compared to the two reference compounds 29 and 30, but lead

to complete inhibition of the enzyme. Furthermore, the difference

in the spacer length between the phthalimide and the urea

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moieties does not influence binding to the human isoenzyme.

This is different for mCEase, where shortening of the linker by

one methylene group (21 vs. 2) leads to a more than 15-fold

decrease in K_i(18.7 µM vs. 1.19 µM). With regards to the MOI of

the new compounds on the two CEase isoenzymes, no explicit

conclusion can be drawn, since both inhibitors show inconsistent

behavior in the presence of the substrate 4-MUB (competitive

and non-competitive inhibition).

by means of a new spectrophotometric method and a recently

published fluorescence assay, respectively.

Taken together, the results presented herein suggest that the ω-

phthalimidoalkyl aryl ureas 2 and 21 may be promising starting

points for further structural optimization to obtain potent and

selective tools that can be used to shed light on the role of

CEase in pathological processes.

Compounds 2 and 21 are, to our knowledge, the first CEase

inhibitors that contain phthalimide and urea motifs, which are

both essential for inhibitory potency, and additionally exhibit

selectivity over other serine hydrolases. So far, the urea SMP-

028^[83]has been described as selective inhibitor of hormone-

sensitive CEase from different species (IC₅₀: rat, 1.01 µM;

monkey, 31.1 µM; human, 33.8 µM),^[83]whereas, no phthalimide-

containing compound is known to reduce CEase’s activity. The

same applies for MAGL and FAAH, where (several) ureas but no

phthalimide derivative have been characterized as inhibitors

yet.^[84-88]In this context, it should be noted that a large variety of

MAGL and FAAH inhibitors bearing piperidinyl, piperazinyl,

azetidinyl, triazole and triazolopyridine urea scaffolds,

respectively, have been developed by several pharmaceutical

companies, causing (selective or “dual”) irreversible inactivation

of these two enzymes.^{[44, 89-91]}

Experimental Section

Chemistry

General: All reactions were carried out in oven-dried glassware under an

argon atmosphere using Schlenk techniques. Solvents were dried by

standard methods and distilled prior to use. Nuclear magnetic resonance

spectra were recorded on a Bruker Avance 300 MHz. ¹H and ¹³C NMR

spectra were processed with MestReNova v.9.0.1 (Mestrelab Research,

Santiago de Compostela, Spain). NMR chemical shifts () in parts per

million (ppm) were referenced to the residual solvent resonance relative

to tetramethylsilane (TMS). Signal multiplicities are given as singlet (s),

doublet (d), doublet of doublet (dd), triplet (t) and multiplet (m). GC-MS

spectra were recorded on a Varian GC-MS 4000 (Varian Inc., Palo Alto,

CA, USA) equipped with a J&W scientific DB-5ht column (Agilent

Technologies, Santa Clara, CA, USA). Infrared spectra were measured

on a Thermo Nicolet 380 FT-IR (Thermo Electron Cooperation, Waltham,

MA, USA). IR spectra were processed using OMNIC Specta (Thermo

Fisher Scientific, Waltham, MA, USA). Melting Points were determined

on a melting point apparatus MP 50 (Mettler Toledo, Gießen, Germany).

High and low resolution mass spectra were conducted on a Thermo

Fisher Scientific LTQ Orbitrap XL mass spectrometer equipped with a

FTMS Analyzer (Waltham, MA, USA). The crystal data were recorded on

a Bruker D8 Venture diffractometer system equipped with a micro focus

source (Cu). Crystal structures were prepared for publication with

CYLview v1.0.561 BETA (Legault, C. Y., Université de Sherbrooke, 2009,

http://www.cylview.org). All chemical reagents were purchased from TCI

(TCI Europe N.V., Zwijndrecht, Belgium), Acros Organics (Thermo Fisher

Scientific, Geel, Belgium), Sigma-Aldrich (Taufkirchen bei München,

Germany) or Carbolution Chemicals (St. Ingbert, Germany). Silica gel for

chromatography, 0.035-0.070 mm, 60 A was purchased from Acros

Organics (product number: 240360300, Thermo Fisher Scientific, Geel,

Belgium). Human recombinant N-terminally His-tagged fatty acid amide

hydrolase (hFAAH) expressed in Sf21 cells (product number: 10010183,

lot number: 0418822-4), human recombinant C-terminally hexahistidine-

tagged monoacylglycerol lipase (hMAGL) expressed in E. coli (product

number: 10007812, lot numbers: 0415588-1 and 0472146-1) and the

MAGL inhibitor JZL 184 (product number: 13158) were purchased from

Cayman Chemical (Ann Arbor, MI, USA). Purification of both pancreatic

murine and human CEases carrying a C-terminal double Strep II tag was

done on StrepTactin sepharose (product number: 2-1208-010, Iba,

Göttingen, Germany), with the two proteins being eluted with d-

desthiobiotin (product number: D1411, Sigma-Aldrich, Taufkirchen bei

München, Germany). Bovine pancreatic CEase (product number: C3766,

lot number: 051M7016V), 4-nitrophenyl butyrate (4-NPB, product

number: N9876), 4-methylumbelliferyl butyrate (4-MUB, product number:

19362), 4-methylumbelliferone (4-MU, product number: M1381), sodium

taurocholate hydrate (NaTC, product number: 86339) and anhydrous

dimethyl sulfoxide (DMSO, product numbers: 41647, 276855) were from

Sigma-Aldrich (Taufkirchen bei München, Germany). 4-Nitrophenol

(4-NP, product number: N/3200/48) was purchased from Fisher Scientic

UK (Loughborough, UK). cDNA of mCEase (BioCat, Heidelberg,

Germany) and hCEase (GeneCopoeiaTM, Rockville, USA) were

commercially obtained. CEase was detected on Western blots by means

Conclusions

The known thieno[1,3]oxazines-4-ones 29 and 30 previously

described as alternate substrate inhibitors of bovine CEase were

demonstrated in this study to exhibit high species-specific

differences in their inhibitory potency, reducing the activity of the

human CEase to a much lesser extent than those of the murine

and bovines isoenzymes due to increased dissociation

constants K_iand the formation of catalytically active enzyme-

inhibitor complexes. In the search for potent and selective

inhibitors of CEase, we applied a new assay using the

fluorogenic substrate 4-MUB on the recombinantly expressed

murine and human isoenzymes leading to the identification of ω-

phthalimidoalkyl aryl ureas 2, 21 and 22. Structure-activity

relationship studies revealed that the urea moiety itself as well

as an attached 3,5-bis(trifluoromethyl)phenyl group and the

aromatic character of the terminal phthalimide residue on the

alkyl chain are crucial for the inhibitory activity of these

compounds. The length of the alkyl spacer connecting the urea

and phthalimide motifs appeared to be less influencing on

CEase inhibition; however, an analysis in the presence of 0.01%

(v/v) Triton X-100 revealed that the C₅chain of compound 22

mediated promiscuous inhibition. Inhibitors 2 and 21 were

kinetically characterized on human and murine CEases yielding

values of K_iin the low to mid micromolar range with low to

moderate species-specific differences. In addition, our analyses

revealed complete but reversible inhibition of the two enzymes

and both competitive and non-competitive behavior. Both 2 and

21 showed, moreover, favorable selectivity for CEase over the

related human serine hydrolases MAGL and FAAH as examined

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of polyclonal rabbit anti-bile salt-activated lipase antibody (product

number: ab87431, Abcam, Cambridge, UK, 100 µg mL^–1) and polyclonal

swine anti-rabbit IgG antibody coupled to horseradish peroxidase (HRP)

(product number: P0399, Dako Denmark A/S, Glostrup, Denmark, 0.30 g

L^–1). WEST-ZOL^®plus Western Blot Detection System (product number:

16021, iNtRON Biotechnology, available via HISS Diagonistics GmbH,

Freiburg, Germany) was used for detection of HRP activity by

chemiluminescence visualized on Kodak Biomax X-ray films (Sigma,

Taufkirchen, Germany). The synthesis and structural characterization of

the CEase inhibitors 29 and 30 and the FAAH substrate D-MAP were

J = 11.2 Hz, 5.3 Hz, 2H), 1.79 (p, J = 6.6 Hz, 2H); ¹³C NMR (75 MHz,

DMSO-d₆) δ = 168.00, 154.76, 142.59, 134.29, 131.66, 130.55 (q, J =

32.3 Hz), 123.36 (q, J = 273.0 Hz), 122.94, 117.14, 113.34, 36.86, 35.25,

28.56; IR ṽ [cm^–1] = 3377 (w), 1700 (m), 1520 (m), 1387 (m), 1274 (s),

1175-1122 (m), 720 (m); HR-MS (ESI) m/z calcd for C₂₀H₁₅F₆N₃O₃[M +

H]⁺460.10904, found: 460.10900; Anal. calcd for C₂₀H₁₅F₆N₃O₃: C 52.30,

H 3.29, N 9.15, found: C 52.19, H 3.04, N 9.25.

1-(3',5'-Bis(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-3-(3-(1,3-

dioxoisoindolin-2-yl)propyl)urea (17): The reaction was performed

according to the general procedure with 0.70 g 4-(1,3-dioxoisoindolin-2-

yl)butanoic acid (3 mmol), 1.09 mL SOCl₂(1.78 g; 15 mmol), 0.20 g NaN₃

(3 mmol) and 0.916 g 3',5'-bis(trifluoromethyl)-[1,1'-biphenyl]-4-amine

(3 mmol). The crude product was purified via column chromatography

(65% MTBE/n-hexane) yielding a white solid (158 mg; 0.30 mmol; 10%):

R_f= 0.22 (65% MTBE/n-hexane); mp: 215 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ = 8.84 (s, 1H), 8.25 (s, 2H), 7.97 (s, 1H), 7.84 (m, 4H), 7.75

(d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 6.29 (t, J = 5.8 Hz, 1H), 3.63

previously reported by Pietsch & Gütschow^[23]and Dato et al.^[58]

,

respectively. All reagents were used without prior purification.

General procedure for the synthesis of ureas 1-26: To an ice-cold

solution of 1 eq. carboxylic acid in 20 mL toluene (abs.) 5 eq. SOCl₂was

slowly added via a syringe. The mixture was allowed to warm to room

temperature and stirred for 14 h. Then, the solvent was evaporated in

vacuo, the residue was dissolved in 20 mL acetone and 1 eq. NaN₃in

5 mL H₂O was added in one portion. The mixture was stirred at room

temperature for additional 3 h. The organic solvent was removed and the

aqueous solution was extracted with CH₂Cl₂. The organic phase was

separated and the solvent was evaporated in vacuo. The residue was

dissolved in 20 mL toluene and heated to reflux for 20 min. 1 eq. amine

was slowly added and the mixture was refluxed for additional 14 h. The

mixture was cooled to room temperature, the solvent was evaporated,

and the crude product was purified by flash chromatography and

recrystallized if mentioned.

(t, J = 6.9 Hz, 2H), 3.14 (q, J = 6.5 Hz, 2H), 1.78 (p, J = 6.9 Hz, 2H); ¹³

C

NMR (75 MHz, DMSO-d₆) δ = 168.00, 155.00, 142.48, 141.64, 134.31,

131.67, 130.85 (m, J = 32.9 Hz), 129.08, 127.64, 126.33, 123.42 (q, J =

275.1 Hz), 122.97, 119.75, 117.87, 36.78, 35.30, 28.86; IR ṽ [cm^–1] =

3319 (w), 1719 (s), 1635 (m), 1586 (m), 1401 (w), 1385 (m), 1284 (s),

1167 (m), 1119 (s), 1046 (m), 896 (m), 839 (m), 720 (s), 685 (m); HR-MS

(ESI) m/z calcd for C₂₆H₁₉F₆N₃O₃[M + H]⁺536.14034, found: 536.14026;

Anal. calcd for C₂₆H₁₉F₆N₃O₃: C 58.32, H 3.58, N 7.85, found: C 58.52,

H 3.89, N 7.86.

General procedure for the synthesis of phthalimide derivatives 31-

34: 1 eq. phthalic anhydride and 1 eq. amino acid were heated to 170 °C

under neat conditions. After stirring for 3 h, the mixture was allowed to

cool to room temperature. The residue was dissolved in CH₂Cl₂and

washed with 10% (w/w) HCl. The solvent was evaporated in vacuo and

the residue was recrystallized from ethanol.

4-(3,5-Bis(trifluoromethyl)phenyl)-N-(3-(1,3-dioxoisoindolin-2-

yl)propyl)piperazine-1-carboxamide (19): The reaction was performed

according to the general procedure with 0.70 g 4-(1,3-dioxoisoindolin-2-

yl)butanoic acid (3 mmol), 1.09 mL SOCl₂(1.78 g; 15 mmol), 0.20 g NaN₃

(3 mmol) and 0.895

g 1-(3,5-bis(trifluoromethyl)phenyl)piperazine

(3 mmol). The crude product was purified via column chromatography

(100% MTBE) yielding a white solid (556 mg; 1.05 mmol; 35%): R_f= 0.48

1

(100% MTBE); mp: 152 °C; H NMR (300 MHz, DMSO-d₆) δ = 7.84 (m,

General procedure for the synthesis of phthalimide derivatives 35-

38: 1 eq. phthalic anhydride, 1 eq. 4-aminobutyric acid and 0.1 eq. TEA

were dissolved in 20 mL toluene and heated to reflux for 14 h. Then, the

solvent was evaporated in vacuo, the residue was dissolved in CHCl₃

and washed with 10% (w/w) HCl. The solvent was removed and the raw

product was used in the next step without further purification.

4H), 7.47 (s, 2H), 7.29 (s, 1H), 6.65 (t, J = 5.2 Hz, 1H), 3.59 (t, J = 7.1 Hz,

2H), 3.39 (m, 8H), 3.08 (dd, J = 12.5 Hz, 6.5 Hz, 2H), 1.77 (m, 2H); ¹³C

NMR (75 MHz, DMSO-d₆) δ = 167.90, 157.28, 151.56, 134.31, 131.63,

131.05 (q, J = 33.0 Hz), 123.58 (q, J = 273.1 Hz), 122.94, 114.37, 110.12,

46.82, 42.89, 37.84, 35.55, 28.74; IR ṽ [cm^–1] = 3279 (w), 2937 (w), 2861

(w), 1711 (s), 1625 (m), 1551 (m), 1396 (s), 1274 (s), 1164-1123 (s), 993

(m), 964 (m), 865 (m), 718 (s), 683 (m); HR-MS (ESI) m/z calcd for

C₂₄H₂₂F₆N₄O₃[M + H]⁺529.16689, found: 529.16680; Anal. calcd for

C₂₄H₂₂F₆N₄O₃: C 54.55, H 4.20, N 10.60, found: C 54.65, H 3.89, N 10.74.

General procedure for the synthesis of biphenyl compounds 39-41:

To a mixture containing 35 mL toluene, 15 mL EtOH and 10 mL H₂O,

1 eq. bromoaniline, 1 eq. 3,5-bis(trifluoromethyl)phenylboronic acid,

0.05 eq. tri(o-tolyl)phosphine and 2 eq. K₂CO₃were added under an

argon atmosphere. The solution was degassed under reduced pressure

and purged with argon for 30 min. Then, 0.0125 eq. Pd(OAc)₂were

added and the mixture was heated to 80 °C for 1.5 h. After cooling to

room temperature, toluene and H₂O were added to the mixture, the

organic layer was separated, and the aqueous layer was extracted twice

with toluene. The organic layers were combined, washed with brine, and

the solvent was evaporated in vacuo. The crude product was purified by

flash chromatography.

1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-(1,3-dioxoisoindolin-2-

yl)butyl)urea (21): The reaction was performed according to the general

procedure with 0.74 g 5-(1,3-dioxoisoindolin-2-yl)pentanoic acid (3 mmol),

1.09 mL SOCl₂(1.78 g; 15 mmol), 0.20 g NaN₃(3 mmol) and 0.469 mL

3,5-bis(trifluoromethyl)aniline (0.687 g; 3 mmol). The crude product was

purified via column chromatography (40% EtOAc/n-hexane) yielding a

white solid (612 mg; 1.29 mmol; 43%): R_f= 0.32 (40% EtOAc/n-hexane);

mp: 169 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 9.18 (s, 1H), 8.06 (s, 2H),

7.84 (m, 4H), 7.52 (s, 1H), 6.48 (t, J = 5.5 Hz, 1H), 3.60 (t, J = 6.8 Hz,

2H), 3.12 (q, J = 6.4 Hz, 2H), 1.62 (m, 2H), 1.46 (m, 2H); ¹³C NMR

(75 MHz, DMSO-d₆) δ = 167.95, 154.77, 142.62, 134.33, 131.59, 130.54

(q, J = 32.5 Hz), 123.36 (q, J = 272.2 Hz) 122.96, 117.19, 113.29, 38.74,

37.18, 27.00, 25.43; IR ṽ [cm^–1] = 3332 (w), 2943 (w), 1712 (m), 1657

(m), 1561 (m), 1393 (m), 1275 (s), 1172 (m), 1129 (s), 1052 (m), 718 (s),

680 (m); HR-MS (ESI) m/z calcd for C₂₁H₁₇F₆N₃O₃[M + H]⁺474.12469,

found: 474.12531; Anal. calcd for C₂₁H₁₇F₆N₃O₃: C 53.28, H 3.62, N 8.88,

found: C 53.49, H 4.01, N 8.82.

1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(1,3-dioxoisoindolin-2-

yl)propyl)urea (2): The reaction was performed according to the general

procedure with 0.70 g 4-(1,3-dioxoisoindolin-2-yl)butanoic acid (3 mmol),

1.09 mL SOCl₂(1.78 g; 15 mmol), 0.20 g NaN₃(3 mmol) and 0.469 mL

3,5-bis(trifluoromethyl)aniline (0.687 g; 3 mmol). The crude product was

purified via column chromatography (45% EtOAc/n-hexane) yielding a

white solid (580 mg; 1.26 mmol; 42%): R_f= 0.37 (45% EtOAc/n-hexane);

mp: 180 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 9.37 (s, 1H), 8.05 (s, 2H),

7.83 (m, 4H), 7.51 (s, 1H), 6.51 (s, 1H), 3.62 (t, J = 6.6 Hz, 2H), 3.15 (dd,

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1-(3,5-Bis(trifluoromethyl)phenyl)-3-(5-(1,3-dioxoisoindolin-2-

yl)pentyl)urea (22): The reaction was performed according to the

general procedure with 0.78 g 6-(1,3-dioxoisoindolin-2-yl)hexanoic acid

(3 mmol), 1.09 mL SOCl₂(1.78 g; 15 mmol), 0.20 g NaN₃(3 mmol) and

0.469 mL 3,5-bis(trifluoromethyl)aniline (0.687 g; 3 mmol). The crude

product was purified via column chromatography (40% EtOAc/n-hexane)

yielding a white solid (779 mg; 1.60 mmol; 53%): R_f= 0.39 (40%

EtOAc/n-hexane); mp: 126 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 9.16

(s, 1H), 8.05 (s, 2H), 7.81 (m, 4H), 7.49 (s, 1H), 6.47 (t, J = 5.2 Hz, 1H),

3.57 (t, J = 6.9 Hz, 2H), 3.07 (m, 2H), 1.63 (m, 2H), 1.48 (m, 2H), 1.32 (m,

2H); ¹³C NMR (75 MHz, DMSO-d₆) δ = 167.91, 154.74, 142.65, 134.27,

131.58, 130.54 (q, J = 32.7 Hz), 123.41 (q, J = 172.3 Hz), 122.91, 117.13,

113.23, 38.96, 37.34, 29.11, 27.69, 23.60; IR ṽ [cm^–1] = 3341 (w), 2965

(w), 1705 (s), 1663 (m), 1580 (m), 1393 (m), 1272 (s), 1132 (s), 1059 (m),

875 (m), 718 (m); HR-MS (ESI) m/z calcd for C₂₂H₁₉F₆N₃O₃[M + H]⁺

488.14034, found: 488.14100; Anal. calcd for C₂₂H₁₉F₆N₃O₃: C 54.21,

H 3.93, N 8.62, found: C 54.23, H 4.38, N 8.83.

6.5 Hz, 2H), 2.23 (m, 4H), 1.68 (m, 6H); ¹³C NMR (75 MHz, DMSO-d₆) δ

= 170.77, 154.73, 142.61, 140.91, 130.58 (q, J = 32.6 Hz), 123.32 (q, J =

272.6 Hz), 117.16, 113.38, 36.83, 34.73, 28.73, 20.82, 19.48; IR ṽ [cm^–1

]

= 1704 (s), 1663 (s), 1571 (s), 1474 (s), 1386 (s), 1277 (s), 1269 (s),

1191 (s), 1161 (s), 1131 (s), 878 (s), 713 (s), 680 (s); HR-MS (ESI) m/z

calcd for C₂₀H₁₉F₆N₃O₃[M + Na]⁺486.1222, found: 486.1227; Anal. calcd

for C₂₀H₁₉F₆N₃O₃: C 51.84, H 4.13, N 9.07, found: C 51.90, H 4.48, N

9.09.

1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(1,3-dioxo-3a,4,7,7a-

tetrahydro-1H-4,7-methanoisoindol-2(3H)-yl)propyl)urea (26): The

reaction was performed according to the general procedure with 1.50 g

4-(1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindol-2(3H)-

yl)butanoic acid (6 mmol), 2.18 mL SOCl₂(3.57 g; 30 mmol), 0.39 g NaN₃

(6 mmol) and 0.938 mL 3,5-bis(trifluoromethyl)aniline (1.374 g; 6 mmol).

The crude product was purified via column chromatography (15%

acetone/toluene) yielding a white solid (85 mg; 0.18 mmol; 3%): R_f= 0.24

(15% acetone/toluene); mp: 86 °C; ¹H NMR (300 MHz, DMSO-d₆) δ =

9.36 (s, 1H), 8.07 (s, 2H), 7.54 (s, 1H), 6.47 (t, J = 5.7 Hz, 1H), 6.08 (s,

2H), 3.33 (m, 2H), 3.25 (t, J = 6.8 Hz, 4H), 3.02 (dd, J = 12.8 Hz, 6.5 Hz,

2H), 1.52 (m, 4H); ¹³C NMR (75 MHz, DMSO-d₆) δ = 177.61, 154.74,

142.62, 134.39, 130.61 (q, J = 32.2 Hz), 123.37 (q, J = 272.7 Hz), 117.18,

113.42, 51.79, 45.24, 44.27, 36.77, 35.30, 28.08; IR ṽ [cm^–1] = 1694 (s),

1387 (s), 1276 (s), 1172 (s), 1126 (s), 681 (s); HR-MS (ESI) m/z calcd for

C₂₀H₁₉F₆N₃O₃[M + Na]⁺498.1222, found: 498.1229; Anal. calcd for

C₂₀H₁₉F₆N₃O₃: C 53.06, H 4.03, N 8.84, found: C 53.84, H 4.33, N 8.71.

Compound still contains 0.67 eq. acetone from chromatographic

purification, which could not be removed. See ¹H NMR and ¹³C NMR

spectra in Figure S57, Supporting information.

1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(1,3-dioxohexahydro-1H-

isoindol-2(3H)-yl)propyl)urea (23): The reaction was performed

according to the general procedure with 0.72 g 4-(1,3-dioxohexahydro-

1H-isoindol-2(3H)-yl)butanoic acid (3 mmol), 1.09 mL SOCl₂(1.78 g; 15

mmol), 0.20 g NaN₃(3 mmol) and 0.469 mL 3,5-bis(trifluoromethyl)aniline

(0.687 g;

3 mmol). The crude product was purified via column

chromatography (15% acetone/toluene) yielding a white solid (220 mg;

0.47 mmol; 16%): R_f= 0.26 (15% acetone/toluene); mp: 120 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ = 9.38 (s, 1H), 8.08 (s, 2H), 7.54 (s, 1H), 6.50 (t,

J = 5.7 Hz, 1H), 3.40 (t, J = 6.9 Hz, 2H), 3.07 (q, J = 6.5 Hz, 2H), 2.93 (m,

2H), 1.66 (m, 6H), 1.33 (m, 4H); ¹³C NMR (75 MHz, DMSO-d₆) δ =

179.72, 154.77, 142.63, 130.59 (q, J = 32.2), 123.40 (q, J = 272.6 Hz),

117.18, 113.42, 38.90, 36.74, 35.44, 27.98, 23.21, 21.25; IR ṽ [cm^–1] =

1694 (s), 1388 (s), 1276 (s), 1172 (s), 1126 (s); HR-MS (ESI) m/z calcd

for C₂₀H₂₁F₆N₃O₃[M + Na]⁺488.1379, found: 488.1382; Anal. calcd for

C₂₀H₂₁F₆N₃O₃: C 51.62, H 4.55, N 9.03, found: C 51.75, H 4.58, N 9.29.

N-(3,5-Bis(trifluoromethyl)phenyl)-5-(1,3-dioxoisoindolin-2-

yl)pentanamide (27): To

bis(trifluoromethyl)aniline (0.458 g;

a

stirred solution of 0.31 mL 3,5-

mmol), 0.593 5-(1,3-

2

g

dioxoisoindolin-2-yl)pentanoic acid (2.4 mmol) and 0.31 g 1-hydroxy-

benzotriazole hydrate (2.3 mmol) in 15 mL CH₂Cl₂was added 0.48 g

EDCHCl (2.5 mmol) in one portion at 0 °C. The mixture was allowed to

warm to room temperature, stirred for additional 14 h and was then

washed three times with a saturated Na₂CO₃solution. The organic layer

was separated and the solvent was removed in vacuo. The crude product

was purified by flash chromatography (1% acetone/CHCl₃) yielding a

white solid (588 mg; 1.28 mmol; 64%): R_f= 0.26 (1% acetone/CHCl₃);

mp: 143 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 10.53 (s, 1H), 8.22

(s, 2H), 7.83 (m, 4H), 7.68 (s, 1H), 3.60 (t, J = 6.2 Hz, 2H), 2.40 (t, J =

6.6 Hz, 2H), 1.63 (m, 4H); ¹³C NMR (75 MHz, DMSO-d₆) δ = 172.00,

167.92, 141.01, 134.30, 131.58, 130.67 (q, J = 32.8 Hz), 123.14 (q, J =

271.7 Hz), 122.93, 118.53, 115.66, 37.11, 35.81, 27.40, 21.96; IR ṽ [cm^–

¹] = 3674 (m), 2987-2901 (s), 1706 (m), 1394 (m), 1275 (m), 1066 (s),

891 (m), 725 (m); HR-MS (ESI) m/z calcd for C₂₁H₁₆F₆N₂O₃[M + H]⁺

459.11379, found: 459.11486; Anal. calcd for C₂₁H₁₆F₆N₂O₃: C 55.03,

H 3.52, N 6.11, found: C 55.36, H 3.78, N 6.17.

1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(1,3-dioxo-3a,4,7,7a-

tetrahydro-1H-isoindol-2(3H)-yl)propyl)urea (24): The reaction was

performed according to the general procedure with 0.71 g 4-(1,3-dioxo-

3a,4,7,7a-tetrahydro-1H-isoindol-2(3H)-yl)butanoic acid (3 mmol), 1.09

mL SOCl₂(1.78 g; 15 mmol), 0.20 g NaN₃(3 mmol) and 0.469 mL 3,5-

bis(trifluoromethyl)aniline (0.687 g; 3 mmol). The crude product was

purified via column chromatography (10% acetone/toluene) yielding a

white solid (119 mg; 0.26 mmol; 9%): R_f= 0.26 (10% acetone/toluene);

mp: 94 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 9.38 (s, 1H), 8.08 (s, 2H),

7.54 (s, 1H), 6.47 (t, J = 5.7 Hz, 1H), 5.85 (m, 2H), 3.38 (t, J = 6.9 Hz,

2H), 3.15 (m, 2H), 3.02 (q, J = 6.5 Hz, 2H), 2.29 (dd, J = 67.8 Hz, 14.2

Hz, 4H), 1.59 (p, J = 6.8 Hz, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ =

180.23, 154.75, 142.62, 130.59 (q, J = 32.4 Hz), 127.77, 123.98 (q, J =

273.3 Hz), 117.19, 113.49, 38.58, 36.57, 35.80, 27.95, 23.19; IR ṽ [cm^–1

]

= 1696 (s), 1676 (s), 1389 (s), 1276 (s), 1130 (s), 680 (s); HR-MS (ESI)

m/z calcd for C₂₀H₁₉F₆N₃O₃[M + Na]⁺486.1222, found: 486.1226; Anal.

calcd for C₂₀H₁₉F₆N₃O₃: C 51.84, H 4.13, N 9.07, found: C 52.16, H 4.66,

N 9.19.

N-(3,5-Bis(trifluoromethyl)phenyl)-6-(1,3-dioxoisoindolin-2-

yl)hexanamide (28): To

bis(trifluoromethyl)aniline (0.458 g;

a

stirred solution of 0.31 mL 3,5-

mmol), 0.627 6-(1,3-

2

g

1-(3,5-Bis(trifluoromethyl)phenyl)-3-(3-(1,3-dioxo-4,5,6,7-tetrahydro-

1H-isoindol-2(3H)-yl)propyl)urea (25): The reaction was performed

according to the general procedure with 1.42 g 4-(1,3-dioxo-4,5,6,7-

tetrahydro-1H-isoindol-2(3H)-yl)butanoic acid (6 mmol), 2.18 mL SOCl₂

dioxoisoindolin-2-yl)hexanoic acid (2.4 mmol) and 0.31 g 1-hydroxy-

benzotriazole hydrate (2.3 mmol) in 15 mL CH₂Cl₂was added 0.48 g

EDCHCl (2.5 mmol) in one portion at 0 °C. The reaction was allowed to

warm to room temperature and stirred for additional 14 h and was then

washed three times with a saturated Na₂CO₃solution. The organic layer

was separated and the solvent was removed in vacuo. The crude product

was purified by flash chromatography (1% acetone/CHCl₃) yielding a

white solid (699 mg; 1.48 mmol; 74%): R_f= 0.26 (1% acetone/CHCl₃);

mp: 146 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 10.50 (s, 1H), 8.22

(s, 2H), 7.80 (m, 4H), 7.68 (s, 1H), 3.57 (t, J = 7.0 Hz, 2H), 2.35 (t, J =

(3.57 g; 30 mmol), 0.39

g NaN₃(6 mmol) and 0.938 mL 3,5-

bis(trifluoromethyl)aniline (1.374 g; 6 mmol). The crude product was

purified via column chromatography (10% acetone/toluene) yielding a

white solid (240 mg; 0.52 mmol; 9%): R_f= 0.36 (10% acetone/toluene);

mp: 145 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 9.34 (s, 1H), 8.07 (s, 2H),

7.53 (s, 1H), 6.47 (t, J = 5.8 Hz, 1H), 3.42 (t, J = 6.9 Hz, 2H), 3.08 (q, J =

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7.3 Hz, 2H), 1.61 (m, 4H), 1.31 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ

= 172.17, 167.87, 141.02, 134.26, 131.54, 130.65 (q, J = 32.8 Hz),

123.19 (q, J = 272.9 Hz), 122.87, 118.51, 115.62, 37.23, 36.16, 27.65,

25.74, 24.24; IR ṽ [cm^–1] = 3668 (w), 3354 (w), 2988-2901 (m), 1700 (s),

1569 (w), 1385 (s), 1278 (s), 1188-1050 (s), 887 (m), 721 (m), 605 (s);

HR-MS (ESI) m/z calcd for C₂₂H₁₈F₆N₂O₃[M + H]⁺473.12944, found:

473.12963; Anal. calcd for C₂₂H₁₈F₆N₂O₃: C 55.94, H 3.84, N 5.93, found:

C 55.90, H 3.81, N 5.42.

white solid (4.24 g; 17.8 mmol; 89%); mp: 84 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ = 12.07 (s, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.20 (m, 6H), 1.70

(m, 6H); ¹³C NMR (75 MHz, DMSO-d₆) δ = 173.78, 170.74, 140.85, 36.32,

30.85, 23.64, 20.86, 19.50; LR-MS (ESI) m/z calcd for C₁₂H₁₅NO₄[M - H]^-

236.1, found: 236.1.

4-(1,3-Dioxo-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindol-2(3H)-

yl)butanoic acid (38):^[95]The reaction was performed according to the

general procedure with 2.06 g 4-aminobutyric acid (20 mmol) and 3.28 g

endo-3,6-methylene-1,2,3,6-tetrahydrophthalic anhydride (20 mmol). The

product was isolated as a white solid (3.03 g; 12.2 mmol; 61%); mp:

94 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 12.06 (s, 1H), 6.05 (t, J =

1.8 Hz, 2H), 3.31 (m, 2H), 3.24 (m, 4H), 2.13 (t, J = 7.4 Hz, 2H), 1.56 (m,

4H); ¹³C NMR (75 MHz, DMSO-d₆) δ = 177.48, 173.71, 134.33, 51.81,

45.21, 44.24, 36.90, 30.83, 22.69; LR-MS (ESI) m/z calcd for C₁₃H₁₅NO₄

[M - H]^-248.1, found: 248.1.

4-(1,3-Dioxoisoindolin-2-yl)butanoic acid (32):^[92]The reaction was

performed according to the general procedure with 4.13 g 4-aminobutyric

acid (40 mmol) and 5.92 g phthalic anhydride (40 mmol). The product

was isolated as a white solid (8.74 g; 37.5 mmol; 94%); mp: 115 °C (lit.

1

117 °C); H NMR (300 MHz, DMSO-d₆) δ = 12.06 (s, 1H), 7.81 (m, 4H),

3.59 (t, J = 6.8 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 1.81 (p, J = 7.0 Hz, 2H);

¹³C NMR (75 MHz, DMSO-d₆) δ = 173.90, 168.00, 134.29, 131.69,

122.97, 36.95, 31.02, 23.37; LR-MS (ESI) m/z calcd for C₁₂H₁₁NO₄[M -

H]^-232.1, found: 232.0.

3',5'-Bis(trifluoromethyl)-[1,1'-biphenyl]-4-amine (41):^[96]The reaction

was performed according to the general procedure with 1.72

g

5-(1,3-Dioxoisoindolin-2-yl)pentanoic acid (33):^[93]The reaction was

performed according to the general procedure with 2.00 g 5-aminovaleric

acid (17 mmol) and 2.52 g phthalic anhydride (17 mmol). The product

was isolated as a white solid (3.48 g; 14.1 mmol; 83%); mp: 120 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ = 7.83 (m, 4H), 3.56 (t, J = 6.7 Hz, 2H),

2.23 (t, J = 7.1 Hz, 2H), 1.54 (m, 4H); ¹³C NMR (75 MHz, DMSO-d₆) δ =

174.21, 167.94, 134.36, 131.59, 122.99, 37.11, 33.07, 27.46, 21.82;

LR-MS (ESI) m/z calcd for C₁₃H₁₃NO₄[M - H]^-246.1, found: 246.1.

4-bromoaniline (10 mmol), 2.58 g 3,5-bis(trifluoromethyl)phenylboronic

acid (10 mmol), 0.15 g tri(o-tolyl)phosphine (0.5 mmol), 2.76 g K₂CO₃(20

mmol) and 0.028 g Pd(OAc)₂(0.13 mmol). The crude product was

purified via column chromatography (10% EtOAc/n-hexane) yielding a

light brown solid (2.80 g; 9.19 mmol; 92%): R_f= 0.32 (10% EtOAc/n-

hexane); mp: 85 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 8.14 (s, 2H), 7.84

(s, 1H), 7.56 (d, J = 8.6 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 5.51 (s, 2H);

¹³C NMR (75 MHz, DMSO-d₆) δ = 149.96, 143.19, 130.75 (q, J = 32.4

Hz), 127.89, 125.26, 123.54 (q, J = 264.1 Hz), 123.52, 118.44, 114.15;

GC-MS (EI, 70 eV): t_r(min) = 12.1; m/z (%): 286 (28), 305 (100) [M]⁺.

6-(1,3-Dioxoisoindolin-2-yl)hexanoic acid (34):^[92]The reaction was

performed according to the general procedure with 5.25

g

6-aminohexanoic acid (40 mmol) and 5.92 g phthalic anhydride (40

mmol). The product was isolated as a white solid (7.28 g; 27.9 mmol;

70%); mp: 109 °C (lit. 109 °C); H NMR (300 MHz, DMSO-d₆) δ = 11.97

(s, 1H), 7.83 (m, 4H), 3.55 (t, J = 7.1 Hz, 2H), 2.18 (t, J = 7.3 Hz, 2H),

1.54 (m, 4H), 1.27 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ = 174.36,

167.91, 134.33, 131.59, 122.97, 37.26, 33.44, 27.69, 25.78, 24.05; LR-

MS (ESI) m/z calcd for C₁₄H₁₅NO₄: [M - H]^-260.1, found: 260.1.

1-(3,5-Bis(trifluoromethyl)phenyl)piperazine (42): According to Liu et

al.,^[97]a solution of 13.21 g bis(2-chloroethyl)amine hydrochloride (74

mmol) and 11.56 mL 3,5-bis(trifluoromethyl)aniline (16.96 g, 74 mmol) in

20 mL diethyleneglycol monomethyl ether was heated to 150 °C for 24 h.

The mixture was allowed to cool to room temperature and dissolved in

150 mL hot MeOH. The solution was cooled to room temperature and

400 mL Et₂O was added. The white precipitate was filtered off, treated

with aqueous Na₂CO₃and extracted with EtOAc. The solvent was

evaporated in vacuo and the product was isolated as a white solid (11.25

g; 37.7 mmol; 51%): Mp: 105 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 7.40

(s, 2H), 7.23 (s, 1H), 3.22 (m, 4H), 2.83 (m, 4H), 2.43 (s, 1H); ¹³C NMR

(75 MHz, DMSO-d₆) δ = 152.17, 131.06 (q, J = 32.1 Hz), 123.62 (q, J =

272.8 Hz), 113.91, 109.69, 48.15, 45.29; GC-MS (EI, 70 eV): t_r(min) =

12.8; m/z (%): 256 (100), 298 (22) [M]⁺.

1

4-(1,3-Dioxohexahydro-1H-isoindol-2(3H)-yl)butanoic acid (35): The

reaction was performed according to the general procedure with 2.06 g

4-aminobutyric acid (20 mmol) and 3.08

g cis-hexahydrophthalic

anhydride (20 mmol). The product was isolated as a white solid (4.23 g;

17.6 mmol; 88%); mp: 86 °C; ¹H NMR (300 MHz, DMSO-d₆) δ = 12.08

(s, 1H), 3.39 (t, J = 6.9 Hz, 2H), 2.91 (m, 2H), 2.20 (t, J = 7.3 Hz, 2H),

1.64 (m, 6H), 1.33 (m, 4H); ¹³C NMR (75 MHz, DMSO-d₆) δ = 179.63,

173.80, 38.87, 37.15, 30.91, 23.17, 22.68, 21.23; LR-MS (ESI) m/z calcd

for C₁₂H₁₇NO₄[M - H]^-238.1, found: 238.1.

Biochemistry

Cloning of mCEase WT and full-length hCEase cDNA into the vector

pCEP-Pu/BM40SP: The cDNA of hCEase was a customized clone

containing 16 tandem repeating units at the C terminus, NheI and BamHI

restriction sites, and a dummy base at the NheI restriction site to avoid a

frameshift in the open reading frame (GeneCopoeia^TM, Rockville, MD,

USA). Furthermore, the signal peptide (5’-ATGCTCACCATGGGG-

CGCCTGCAACTGGTTGTGTTGGGCCTCACCTGCTGCTGGGCAGTG-

GCGAGTGCC-3’) and the stop codon were removed. For mCEase

(BioCat, Heidelberg, Germany) these modifications were performed by

PCR. The PCR followed a two-step protocol with a denaturation step at

98 °C for 30 s as well as a combined annealing and extension step (25

cycles 98 °C for 10 s and 72 °C for 35 s), ending with a final extension for

10 min at 72 °C. The PCR product, i.e. the mCEase DNA insert, was

subsequently applied to a 1% agarose gel in a Tris-acetate-EDTA buffer,

visualized with 0.5 µg mL^–1ethidium bromide solution, excised from the

gel and purified with a GeneJET gel extraction kit. The eukaryotic

episomal expression vector pCEP-Pu/BM40SP was used for eukaryotic

4-(1,3-Dioxo-3a,4,7,7a-tetrahydro-1H-isoindol-2(3H)-yl)butanoic acid

(36):^[94]The reaction was performed according to the general procedure

with 2.06 g 4-aminobutyric acid (20 mmol) and 3.04 g cis-1,2,3,6-

tetrahydrophthalic anhydride (20 mmol). The product was isolated as a

white solid (3.90 g; 16.4 mmol; 82%); mp: 105 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ = 12.08 (s, 1H), 5.85 (m, 2H), 3.37 (t, J = 6.8 Hz, 2H), 3.12

(m, 2H), 2.39 (m, 2H), 2.17 (m, 4H), 1.64 (p, J = 7.1 Hz, 2H); ¹³C NMR

(75 MHz, DMSO-d₆) δ = 180.07, 173.71, 127.75, 38.50, 37.43, 30.67,

23.14, 22.60; LR-MS (ESI) m/z calcd for C₁₂H₁₅NO₄[M - H]^-236.1, found:

236.1.

4-(1,3-Dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)butanoic

(37): The reaction was performed according to the general procedure

with 2.06 4-aminobutyric acid (20 mmol) and 3.04 3,4,5,6-

tetrahydrophthalic anhydride (20 mmol). The product was isolated as a

acid

g

10.1002/cmdc.201800388

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FULL PAPER

expression of CEases. This vector is a pCEP4 (ThermoFisher Scientific)

derivative modified by introduction of sequences encoding puromycin

resistance and the double Strep II tag. Furthermore, pCEP-Pu/BM40SP

contains a stop codon in the multiple cloning site (harboring NheI and

XhoI restriction sites) and the signal peptide BM40.^[98]pCEP-Pu/BM40SP

was kindly provided by Prof. Dr. Manuel Koch and PD Dr. Frank Zaucke

(Center for Biochemistry, Medical Faculty, University of Cologne).^[99]Both

cDNA clones, i.e. mCEase and hCEase, were cloned as sticky-end

fragments into the expression vector pCEP-Pu/BM40SP. Confirmation of

the right orientation of the mCEase and hCEase constructs was

conducted by restriction mapping with PstI. The plasmids were purified

with NucleoBond Xtra Midi kit (Macherey-Nagel, Düren, Germany). The

gene sequences of mCEase and hCEase in the eukaryotic expression

vector pCEP-Pu/BM40SP as well as the final, full sequences of the

recombinant proteins are provided in Figures S1 and S2 (Supporting

information), respectively.

two fluorophores 4-methylumbelliferone (4-MU) and 5-amino-2-

methoxypyridine (MAP) were excited at wavelengths of λ_ex= 360 nm and

310 nm, respectively, and emission was detected at wavelengths of λ_em

=

460 nm and 400 nm, respectively, applying a sensitivity of 35. Stock

solutions of compounds 1-28 (5 mM) were prepared in DMSO. Kinetic

experiments were performed over a period of 5-10 min and progress

curves were analyzed by linear regression with the software Gen 5

v.1.11.5. All values were determined in n experiments and given as mean

values ± standard error of the mean (SEM). Data analysis was done with

GraphPad Prism v5.04 for Windows (GraphPad Software, San Diego, CA,

USA).

CEase assay: Assay buffer was 100 mM sodium phosphate, 100 mM

NaCl, pH 7.0, which was preheated to 30 °C. Analysis of compounds 2,

21 and 22 for promiscuous inhibition was done in the presence of 0.01%

(v/v) Triton X-100. For this purpose, 1% (v/v) Triton X-100 in assay buffer

was freshly prepared and further diluted to 0.0286% (v/v) Triton X-100

with the same buffer. NaTC (12 mM) was dissolved in assay buffer and

kept at 30 °C. Stock solutions (stored at -80 °C) of mCEase (0.865 and

1.43 mg mL^–1), hCEase (1.01 and 1.21 mg mL^–1) and bCEase (5 mg

mL^–1) were prepared in enzyme buffer, i.e. 100 mM sodium phosphate,

pH 7.0, and diluted with the same buffer (ice-cold) immediately before

starting the measurement. Reference inhibitors 29 and 30 (10 mM) and

4-methylumbelliferone (4-MU, 20 mM) were dissolved and further diluted

in DMSO; stock solution of 4-MUB (20 mM) was freshly prepared in

DMSO for every measurement and diluted with DMSO. The second

substrate dilution prepared with assay buffer (1:4) was immediately done

before starting the measurement. The assays were performed in a total

volume of 200 µL in the presence of 6 mM NaTC and 5% (v/v) DMSO.

The CEase assay mixture contained 70 µL assay buffer (with or without

0.0286% (v/v) Triton X-100), 100 µL NaTC (12 mM) solution, 5 µL

inhibitor solution or DMSO, and 5 µL enzyme solution or enzyme buffer;

the enzymatic reaction was started by addition of 20 µl 4-MUB in assay

buffer with 25% (v/v) DMSO. All measurements were performed in

duplicate or triplicate in the presence and the absence of CEase.

Expression of mCEase and hCEase in HEK293-EBNA cells: Adherent

HEK293-EBNA cells were cultivated in Dulbecco's Modified Eagle

Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and a

combination of 100 IU mL^–1penicillin and 100 µg mL^–1streptomycin

(PenStrep), at 37 °C and 5% CO₂(DMEM, FCS and PenStrep were

obtained from Gibco, Life Technologies, Dreireich, Germany and Pan

Biotech, Aidenback, Germany.) Wild type cells were splitted 1:5 twice a

week into 75 cm²tissue culture flasks (BD Biosciences, Bedford, MA,

USA). To express mCEase and hCEase, cells (50-70% confluent in

75 cm²falcon flasks) were transfected with 12 µg plasmid DNA using

24 µl TurboFect (Thermo Fisher Scientific, St. Leon Rot, Germany) in

1200 µl serum-free DMEM. Before being exposed to the transfection

complex, the cells were washed with serum-free DMEM. The transfection

complex was added drop-wise to the cells (kept in 5 mL serum-free

DMEM) and incubated for 4 h at 37 °C. Then, 20 mL DMEM with 10%

FCS was added to the flask, which was exchanged for 13 mL selection

medium, i.e. DMEM containing 10% FCS and puromycin (3 µg mL^–1

,

Gold Biotechnology, St. Louis, MO, USA), after 24 h. Upon reaching

100% confluence, the transfected cells were first transferred from 75 cm²

to 175 cm²tissue flasks (BD Biosciences, Bedford, MA, USA). After

having reached 100% confluence in 175 cm²flasks, all cells were

passaged to 175 cm²Nunclon Delta Surface triple tissue culture flasks

(Thermo Fisher Scientific, St. Leon Rot, Germany). After the cells had

reached approximately 100% confluence in the 175 cm²triple flasks, the

content of FCS in DMEM was reduced from 10% to 2%. The cell

supernatant was collected every two days, pooled, and centrifuged at

4 °C for 7 min. Collection of the supernatant was performed over 2 weeks.

To purify mCEase and hCEase, the cell supernatant was applied to a

self-assembled 1 mL StrepTactin sepharose column. After washing the

StrepTactin sepharose with PBS buffer pH 7.8 (10 column volumes),

elution of the protein was conducted with PBS buffer pH 7.8 containing

2.5 mM d-desthiobiotin. All steps were done at a flow rate of 0.1-0.25 mL

min^–1. Protein-containing fractions were tested for hydrolytic activity

towards 4-MUB, with activity-showing fractions being pooled. The buffer

composition was adjusted to 100 mM sodium phosphate buffer, pH 7.0,

the solution was aliquoted, shock frozen with liquid nitrogen, lyophilized

on a freeze dryer and stored at -80 °C.

The absorbance of 4-MU (2-20 µM) depending on the compounds´

concentrations was investigated in the absence of CEase by adding 100

µL NaTC (12 mM), 5 µL DMSO, 5 µL enzyme buffer and 5 µL 4-MU (80-

800 µM) in DMSO to 85 µL assay buffer.

Investigation on the CEase-catalyzed hydrolysis of 4-MUB (200 µM)

depending on the enzyme’s concentration (mCEase: 0-89.1 ng mL^–1

,

hCEase: 0-31.7 ng mL^–1bCEase: 0-500 ng mL^–1) was done by adding

,

5 µL CEase in enzyme buffer or enzyme buffer to the assay mixture.

Rates in the presence of CEase were corrected by the mean value of the

rate without CEase and plotted versus the enzyme concentration. All

further experiments were done with 40 ng mL^–1mCEase, 15 ng mL^–1

hCEase and 0.5 µg mL^–1bCEase, respectively.

Investigations on the Michaelis constant, K_m, and the maximum rate, V_max

for the CEase-catalyzed substrate hydrolysis was performed for twelve

concentrations of 4-MUB in the range 20-200 µM by addition of 20 µL

substrate solution (200-2000 µM) in assay buffer with 25% (v/v) DMSO.

Both total rates and rates of non-enzymatic hydrolysis were plotted

versus the substrate concentration and included in parameter

determination.

,

K_iand the MOI for the inhibition of the three CEases by the reference

compounds 29 and 30 was determined in the presence of four to seven

inhibitor concentrations and four 4-MUB concentrations (25-100 µM).

Inhibitor concentrations of compound 29: mCEase, 0.075-2 µM; hCEase,

1-25 µM; bCEase: 0.6-2.4 µM. Inhibitor concentrations of compound 30:

mCEase, 0.1-2 µM; hCEase, 1-25 µM; bCEase: 1-5 µM. Rates obtained in

the presence of CEase were corrected by the mean values of the

respective rates without enzyme. An analysis according to an equation of

hyperbolic mixed-type inhibition (Equation 1):

Enzyme kinetics

General: All measurements were done at 30 °C on a BioTek Synergy™

2 multimode microplate reader with the software Gen 5 v.1.11.5 (Bad

Friedrichshall, Germany). The spectrophotometric and fluorometric

assays were performed in 96-well plates (BRANDplates®) with F-bottom

being either completely transparent (MAGL assay) or black with black

(FAAH assay) and transparent (CEase assay) bottom, respectively. The

chromophore 4-nitrophenole (4-NP) was investigated at λ = 405 nm; the

10.1002/cmdc.201800388

ChemMedChem

FULL PAPER

[ ]

푉

S

enzyme buffer or enzyme buffer. The reaction was started by addition of

20 µL 4-NPB (200-4500 µM) in assay buffer with 25% (v/v) DMSO.

Inhibition of hMAGL (20 ng mL^–1) by JZL 184 (0.75 nM-25 µM) in the

presence of 4-NPB (200 µM, 1.1  K_m) was studied in mixture of 170 µL

assay buffer, 5 µL enzyme (0.8 µg mL^–1) in enzyme buffer or enzyme

buffer, and 5 µL JZL 184 (30 nM-1000 µM) in DMSO. The reaction was

started after pre-incubation of hMAGL with JZL 184 for 0-45 min at 30 °C

in the BioTek Synergy™ 2 multimode microplate reader by addition of

20 µL 4-NPB (2000 µM) in assay buffer with 25% (v/v) DMSO. Rates

obtained in the presence of hMAGL were corrected by the mean values

of the respective rates without enzyme. The corrected rate in the

absence of JZL 184 was set to 100%. The percentage corrected rates

were analyzed according to the four parameter logistic equation: rate =

bottom + (top - bottom)/(1 + 10^((logIC₅₀- [I])^nH)), were IC₅₀, n_H, bottom

and top represent the inhibitor concentration, [I], causing 50% inhibition,

the Hill slope, and the lower and upper plateaus of the sigmoid dose-

response curve, respectively. Inhibition of hMAGL by compounds 1-28

(25 µM) was similarly investigated in the presence of 4-NPB (200 µM,

1.1  K_m) without pre-incubation starting the enzymatic reaction by

addition of substrate. Percentage residual activity was calculated in

relation to control experiments done in the absence of inhibitor.

푚푎푥

I +퐾

v_E=

,

[ ]

I +α퐾

i

[ ]

)+ S (

α퐾

(

)

[ ]

β I +α퐾

m

[ ]

β I +α퐾

i

hyperbolic non-competitive inhibition (α = 1 in Equation 1) and linear non-

competitive inhibition (α = 1,  = 0 in Equation 1), respectively, provided

the dissociation constant, K_i, and the parameters α and  both of which

describe the MOI.^[100]

Inhibitor concentration resulting in 50% inhibition, IC₅₀

, of 4-MUB

(200 µM) hydrolysis was determined on mCEase, hCEase, and bCEase

with seven to twelve concentrations of 29 and 30, respectively. Inhibitor

concentrations used on mCEase: 29, 0.1-2.5 µM; 30, 0.25-5 µM. Inhibitor

concentrations used on hCEase: 29, 5-200 µM; 30, 1-50 µM. Inhibitor

concentrations used on bCEase: 29, 0.5-10 µM; 30, 2.5-50 µM.

Percentage residual activities (corrected by the non-enzymatic

hydrolysis) were calculated in relation to the mean value of rate of the

respective control experiments done in the absence of inhibitor (set to

100%).

Screening for inhibition of CEase by compounds 1-28 (25 µM) was done

in the presence of 200 µM 4-MUB (mCEase: 2.7  K_m, hCEase: 3.1  K_m).

Values of IC₅₀for the inhibition of the mCEase- and hCEase-catalyzed 4-

MUB (200 µM) hydrolysis by 2, 21, and 22, respectively, were determined

with seven inhibitor concentrations. Inhibitor concentrations used on

mCEase (without Triton X-100): 2, 3-48 µM; 21, 1.25-80 µM; 22, 1.75-60

µM. Inhibitor concentrations used on hCEase: 2, 3-25 µM; 21, 1.25-80 µM.

To exclude promiscuous inhibition four to seven inhibitor concentrations

of 2 (3-48 µM), 21 (1.25-10 µM) and 22 (1.75-60 µM) were investigated on

mCEase in the presence of 0.01% (v/v) Triton X-100. Percentage

residual activities were calculated as described above.

Determination of K_iand the MOI for compounds 2 (1-7 µM) and 21 (1.25-

40 µM) on both mCEase and hCEase was done in the presence of four

concentrations of 4-MUB (25-100 µM) without inhibitor and in the

presence of 7 inhibitor concentrations, respectively. Rates obtained in the

presence of CEase were corrected by the mean value of the respective

rates without enzyme. An analysis according to an equation of linear non-

competitive inhibition (α = 1,  = 0 in Equation 1) and competitive

inhibition (α = ∞,  = 0 in Equation 1), respectively, provided the

dissociation constant, K_i.^[100]

FAAH assay: The assay was performed as described by Dato et al.^[58]

Briefly reactions were carried out in preheated (30 °C) 100 mM Na₂HPO₄/

NaH₂PO₄, pH 8.0 containing 1% (w/v) glycerol and 0.1% (w/v) Triton X-

100 (assay buffer) in the presence of 5% (v/v) DMSO. hFAAH stock

(0.5 mg mL^–1) was prepared in enzyme buffer consisting of 20 mM

HEPES containing 150 mM NaCl, 1 mM Na₂-EDTA, 1 mM DTT, 0.5%

(w/v) CHAPS and 20% (w/v) glycerol), stored at -80 °C, and freshly

diluted with the same buffer to 40 µg mL^–1before each measurement.

Stock solution (10 mM) of the substrate N-(6-methoxypyridin-3-

yl)decanamide (D-MAP) was prepared in DMSO and freshly pre-diluted

to 100 µM in assay buffer containing 25% (v/v) DMSO before each

measurement. Inhibition of hFAAH (1 µg mL^–1) was investigated in the

presence of 10 µM D-MAP (1.2  K_m) and 25 µM of compounds 1-28

without pre-incubation starting the enzymatic reaction by addition of

substrate. Percentage residual activity was calculated in relation to

control experiments done in the absence of inhibitor.

MAGL assay: Assay buffer was preheated (30 °C) 50 mM Tris-

HCl/NaOH, pH 7.4 containing 1 mM EDTA-Na₂. hMAGL stock (50 µg

mL^–1) was prepared in ice-cold enzyme buffer (50 mM HEPES/ NaOH,

pH 7.4 containing 100 mM NaCl, 5 mM MgCl₂, 0.1% (w/v) Triton X-100,

2 mM Na₂-EDTA and 25% (w/v) glycerol), stored at -80 °C, and freshly

diluted with the same buffer to 0.2-10 µg mL^–1before each measurement.

The substrate 4-NPB (20 mM) and the product 4-nitrophenol (4-NP,

10 mM) were dissolved in DMSO and freshly pre-diluted with assay

Acknowledgements

F. M. Dato and B. Goldfuss thank the Fonds der Chemischen

Industrie, the Deutsche Forschungsgemeinschaft (DFG GO-930-

13), Bayer AG, BASF AG, Wacker AG, Evonic AG, Raschig

GmbH, Symrise GmbH, Solvay GmbH, the OMG group, and

INEOS-Koeln for support. M. Sheikh, M. Steinkrüger and M.

Pietsch are grateful to the Graduate Program in Pharmacology

and Experimental Therapeutics of the University of Cologne and

the Bayer Health Care AG.

buffer to obtain

a solution with 25% (v/v) DMSO before each

measurement. Stock solution of the inhibitor JZL 184 (10 mM) was

freshly prepared in DMSO. The assays were performed in a total volume

of 200 µL in the presence of 5% (v/v) DMSO.

The absorbance of 4-NP (2-20 µM) depending on the compound´s

concentrations was investigated in the absence of hMAGL by adding

5 µL DMSO, 5 µL enzyme buffer and 20 µL 4-NP (20-200 µM) dissolved

in assay buffer with 25% (v/v) DMSO to 170 µL assay buffer.

Conflict of interest

To investigate the hMAGL-catalyzed hydrolysis of 4-NPB (200 µM)

depending on the enzyme’s concentration (5-40 ng mL^–1), 5 µL hMAGL

(0.2-1.6 µg mL^–1) in enzyme buffer or enzyme buffer was added to a

mixture of 5 µL DMSO and 170 µL assay buffer. The reaction was started

by addition of 20 µL 4-NPB (2000 µM) in assay buffer with 25% (v/v)

DMSO.

The authors declare no conflict of interest.

Keywords: Enzyme kinetics

Phthalimides • Rearrangement

• Hydrolases • Inhibitors •

Determination of the parameter K_mand V_maxfor the MAGL (20 ng mL^–1)-

catalyzed hydrolysis of 4-NPB (20-450 µM) was done with a mixture of

170 µL assay buffer, 5 µL DMSO and 5 µL enzyme (0.8 µg mL^–1) in

10.1002/cmdc.201800388

ChemMedChem

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10.1002/cmdc.201800388

ChemMedChem

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Entry for the Table of Contents

FULL PAPER

Phthalimide ureas against coronary

heart disease (CHD): A series of 26

phthalimide ureas was synthesized by

Curtius rearrangement. Investigations

of these compounds on the CHD-

relevant cholesterol esterase (CEase)

and related serine hydrolases resulted

in selective inhibition of CEase with K_i

values in the low micromolar range.

Florian M. Dato, Dr. Miriam Sheikh,

Rocky Z. Uhl, Alexandra W. Schüller, Dr.

Michaela Steinkrüger, Dr. Peter Koch,

Dr. Jörg-Martin Neudörfl, Prof. Michael

Gütschow, Prof. Bernd Goldfuss and Dr.

Markus Pietsch*

Page No. – Page No.

ω-Phthalimidoalkyl aryl ureas as

potent and selective inhibitors of

cholesterol esterase

Article Doi

ω-Phthalimidoalkyl Aryl Ureas as Potent and Selective Inhibitors of Cholesterol Esterase

DOI: 10.1002/cmdc.201800388

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Article abstract of DOI:10.1002/cmdc.201800388

Full text of DOI:10.1002/cmdc.201800388

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