Development of a Novel Nonpeptidic 18F-Labeled Radiotracer for in Vivo Imaging of Oxytocin Receptors with Positron Emission Tomography

Article

pubs.acs.org/jmc

18

Development of a Novel Nonpeptidic F‑Labeled Radiotracer for in

Vivo Imaging of Oxytocin Receptors with Positron Emission

Tomography

Barbara Wenzel,*^,†,#Jan Mollitor,^‡,#Winnie Deuther-Conrad,^†Sladjana Dukic-Stefanovic,^†

Mathias Kranz,^†Chrysoula Vraka,^§Rodrigo Teodoro,^†Robert Gunther,^†Cornelius K. Donat,^†

̈

Friedrich-Alexander Ludwig,^†Steﬀen Fischer,^†Rene Smits,^‡Wolfgang Wadsak,^§Markus Mitterhauser,^§

Jorg Steinbach,^†Alexander Hoepping,^‡and Peter Brust^†

̈

^†Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Research Site Leipzig, Department of

Neuroradiopharmaceuticals,, Permoserstr. 15, 04318 Leipzig, Germany

^‡ABX Advanced Biochemical Compounds GmbH, 01454 Radeberg, Germany

^§Radiopharmacy and Experimental Nuclear Medicine, Division of Nuclear Medicine, Medical University of Vienna, A-1090 Vienna,

Austria

ABSTRACT: With the aim of imaging and quantiﬁcation of

oxytocin receptors (OTRs) in living brain using positron

emission tomography (PET), we developed a ¹⁸F-labeled small

molecule radiotracer and investigated its in vivo pharmacoki-

netics in mice and pig. [¹⁸F]6b (K_D= 12.3 nM) was

radiolabeled by a two-step procedure using a microwave

system with radiochemical yields of 26.9 4.7%. Both organ

distribution and small animal PET studies revealed limited

brain uptake of [¹⁸F]6b in mouse (mean SUV of 0.04 at 30

min pi). Besides, signiﬁcant radioactivity uptake in the pituitary

gland was observed (SUV of 0.7 at 30 min pi). In a dynamic

PET study in one piglet, we detected a higher uptake of

[¹⁸F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro

autoradiographic studies on porcine brain sections indicated interaction of [¹⁸F]6b with several oﬀ-target receptors.

quantitative data on OTR expression in human brain are

available yet. A semiquantitative study on monkey brain

revealed comparatively high expression in hippocampus and

moderate expression in nucleus basalis of Meynert and some

cortical and hindbrain regions.⁵In rats, however, beside the

basal ganglia and the hypothalamus, OTRs are also abundantly

expressed (∼200−400 fmol [³H]OT/mg protein) in the

olfactory system (anterior olf nucl), the bed nucleus of stria

terminalis, and the peduncular cortex.^2,6,7In addition, Freund-

Mercier et al.⁸have shown that neurophysins rather than OTR

are involved in binding of [³H]oxytocin in the neural lobe of

the pituitary.

Central behavioral eﬀects of the OT system are extensively

investigated in animals⁹⁻¹¹and in humans.¹²⁻¹⁵The

involvement in social and maternal behavior, trust and

empathy, partnership bonding, sexual behavior, stress-related

behavior, and learning and memory has been demonstra-

ted.^12,16Moreover, the impact of oxytocin on psychiatric

disorders such as schizophrenia,¹⁷depression,¹⁸and autism^19,20

is the object of recent studies. Research in this ﬁeld is mainly

1. INTRODUCTION

The small peptide oxytocin consists of nine amino acids

arranged in a disulﬁde-bonded cyclic structure with a short

“tail”. It is mainly synthesized in the paraventricular nuclei of

the hypothalamus as a large prohormone, cleaved into the

biologically active oxytocin and neurophysin I during passage in

the neurohypophysis, and secreted upon activation of the

neurosecretory cells by multiple physiological stimuli.¹It binds

to the oxytocin receptor (OTR), a 389 amino acid polypeptide

(human OTR) belonging to the G-protein coupled receptor

(GPCR) family. OTRs are peripherally expressed mainly in the

uterus, the mammary gland, the ovary, the testis, the prostate,

the kidney, the heart, and bone.²In the uterus, they mediate the

contracting eﬀect of oxytocin, the ﬁrst discovered action of this

hormone. Beside peripheral physiological functions,^2,3oxytocin

also plays an important role in the central nervous system,

where it acts as putative neurotransmitter and neurohormone in

diﬀerent brain regions. The OTR expression in brain is strongly

species-dependent,²which hampers the comparability between

data obtained in animals and human. Main OTR expressing

brain regions in human are basal ganglia, hypothalamic nuclei,

brain stem, basal nucleus of Meynert, and the lateral septal

nucleus.²Although investigated by autoradiography,⁴no

Received: August 18, 2015

A

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Figure 1. Reported PET radiotracers for OTR (I-a, I-b, and II); 6 as lead structure.

Scheme 1. Synthetic Route of Series A Derivatives 6a−6f and Precursor 7

a

Reagents and conditions: (a) B(OMe)₃, n-BuLi, THF, −78 °C to rt; (b) methyl 4-bromo-3-methylbenzoate, Pd(PPh₃)₄, Na₂CO₃, H₂O, EtOH,

toluene, 14 h, 90 °C; (c) 1 M NaOH, THF, 12 h, reﬂux; (d) (i) SOCl₂, 2 h, reﬂux, (ii) 10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine (8),

Hunig’s base, CH Cl , 0 °C to rt, overnight; (e) trichloroacetyl chloride, Hunig’s base, CH Cl , 2 h, 0 °C to rt achieving intermediates 15, 18, and

̈

2

19; (f) 2.5 M NaOH, acetone, 12 h, rt; (g) amidation with the corresponding amine (for 6a,b,g, diethanolamine; for 6c, diethanolamine and DAST,

CH₂Cl₂, 12 h, rt; for 6d,6e, 2-ﬂuoroethylamine; for 6f, morpholine) HOBt/EDCI, COMU, or TBTU, 12−20 h, rt; (h) NaH, CH₃I, DMF, 1 h, rt; (i)

MOMBr, TBAC, Hunig’s base, CH Cl , 24 h, 0 °C to rt achieving intermediate 16; (j) H , Pd(OH) /C, MeOH, 20 h, rt achieving intermediate 17;

(k) TsCl, triethylamine, 4-DMAP, CH₂Cl₂, 12 h, 0 °C to rt.

̈

2

conducted by behavioral monitoring after drug administration

or functional magnetic resonance imaging (fMRI), the latter

based on changes in the blood ﬂow in certain brain regions.¹⁴

An investigation of disease-speciﬁc changes in the expression of

OTR in the brain has been limited so far.

Positron emission tomography (PET) is a noninvasive

imaging method that can provide selective information on a

single biological target or biochemical process, when a target

speciﬁc radiotracer is used. Therefore, a radiotracer, binding

speciﬁcally to the OTR, would allow direct quantiﬁcation of

these receptors in the living brain and open new options for

diagnosis and therapy monitoring of the diseases mentioned

above.

In 2012 and 2013, Smith et al. reported for the ﬁrst time

structurally diﬀerent PET tracers intended for OTR imaging

(see I-a, I-b, and II in Figure 1), labeled with ﬂuorine-18 and

carbon-11, the most commonly used short-lived positron

emitting radionuclides.²¹⁻²³These compounds are based on

two nonpeptidic antagonists of the OTR developed by Merck

Research Laboratories in the early 1990s as drug candidates for

premature labor.^24,25Although these small molecule radio-

tracers showed appropriate physicochemical properties regard-

ing blood−brain barrier (BBB) permeability, the authors

observed a very poor brain uptake in rats and cynomolgus

monkeys insuﬃcient for OTR imaging.

In parallel to this work, we started to develop a ¹⁸F-labeled

PET radiotracer for imaging of OTR in brain. As lead structure,

N,N-bis(2-hydroxyethyl)-10-(2-methyl-2′-(triﬂuoromethyl)-

[1,1′-biphenyl]-4-carbonyl)-10,11-dihydro-5H-benzo[e]-

pyrrolo[1,2-a][1,4]diazepine-3-carboxamide, 6 (WAY-

162720;²⁶Figure 1), was selected, an OTR antagonist

described to be able to penetrate the BBB in mice.²⁷We

synthesized two series of derivatives and determined the

binding aﬃnity toward human OTR (hOTR) in vitro as well as

their lipophilicity (log D) and permeability values (P_m) by

diﬀerent HPLC methods. The most promising candidate was

labeled with [¹⁸F]ﬂuoride and investigated by in vitro

autoradiography on pig brain slices. Furthermore, the

pharmacokinetics of the novel ¹⁸F-labeled PET radiotracer

B

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a

Scheme 2. Synthetic Route of Series B Derivatives 12a−12d, 13a, 13b, and 14

a

Reagents and conditions: (a) (i) SOCl , 1 h, reﬂux; (ii) Hunig’s base, CH Cl , and 1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine for 12a−d, 4-

̈

2

piperidone for 13a,b, and intermediate 24, N-Boc-piperazine for 14 and intermediate 25, 4 h, 0 °C to rt; (b) triﬂuoroacetic acid, CH₂Cl₂, 2.5 h, rt;

(c) 2-bromoethanol, K₂CO₃, CH₃CN, 12 h, reﬂux; (d) diethanolamine, Na(OAc)₃BH, AcOH, 1,2-dichloroethane, 12 h, rt; (e) trichloroacetyl

chloride, Hunig’s base, CH Cl , 2 h, 0 °C to rt achieving intermediate 20; (f) 2.5 M NaOH, acetone, 12 h, rt achieving intermediate 21; (g)

̈

2

amidation with the corresponding amine (12a, diethanolamine; 12b, N-methylethanolamine; 12c and intermediate 22, N-Boc-piperazine; 12d and

intermediate 23, 4-amino-1-Boc-piperidine) HOBt/EDCI, COMU, or TBTU, 12−20 h, rt; (h) 12c,d, triﬂuoroacetic acid, CH₂Cl₂, 2,5 h, rt.

was investigated in mice and pig by ex vivo analyses and

dynamic PET studies.

carboxylic acids (5a−5c). The ﬁnal amidation could be

eﬀectively achieved by treating 5a−5c with 1-ethyl-3-(3-

dimethylamino-propyl) carbodiimide hydrochloride (EDCI)

and 1-hydroxybenzotriazole (HOBt) as coupling agents

followed by the addition of the corresponding amine. For

some substrates, the use of (1-cyano-2-ethoxy-2-

oxoethylidenaminooxy)dimethylamino-morpholino-carbenium

hexaﬂuorophosphate (COMU) or (benzotriazol-1-yl)-

N,N,N′,N′-tetramethyluronium tetraﬂuoroborate (TBTU)

proved to be superior to the combination of EDCI/HOBt.

For some of the derivatives, the synthetic route was slightly

changed. Thus, the synthesis of 6c was accomplished by

ﬂuorination of 6 using one equivalent of diethylaminosulfur

triﬂuoride (DAST). Compound 6e was synthesized by N-

methylation of 6d. Derivative 7 with the 4-tosyloxyethyl side

chain was synthesized as a precursor compound for the

intended radiosynthesis of [¹⁸F]6b. The synthetic route started

from the benzyl protected 2-(2-bromophenyl)-ethanol 1g,

which was converted to the corresponding boronic acid 2g for

subsequent Suzuki coupling. After haloform type reaction and

amidation with diethanolamine, the two primary hydroxyl

groups of the resulting derivative 6g were protected as

methyloxymethyl ether to avoid side reactions during radio-

synthesis. To introduce an appropriate leaving group for

nucleophilic radioﬂuorination, the benzyl protecting group of

the ethoxy side chain was removed by hydrogenolysis and

replaced by a tosylate function (7).

2. RESULTS AND DISCUSSION

2.1. Organic Chemistry. The synthesis of the new

derivatives is based on the synthesis of 6 described previously.²⁶

Key steps of the synthetic routes are a Suzuki coupling between

appropriately substituted phenylboronic acids and methyl 4-

bromo-3-methylbenzoate to form the desired biaryl framework,

followed by an amidation using a tricyclic benzodiazepine

(series A, Scheme 1) or other N-heterocycles like piperazine,

piperidine, and pyrrolopyrazine (series B, Scheme 2).

All derivatives of series A (6a−6f) contain a primary,

secondary, or cyclic alkyl side chain at the N-heterocyclic unit

with varying substituents and side chain length. To additionally

investigate the inﬂuence of the substituents at the biphenyl ring

system, diﬀerent ﬂuorine and alkyl ﬂuorine substituted systems

were synthesized with respect to the intended ¹⁸F-ﬂuorination.

In general, the synthesis of the compounds 6a−6f of series A

(Scheme 1) started with diﬀerently substituted 2-bromophenyl

substrates (1a−1b), which were converted to the correspond-

ing boronic acids (2a−2b; 2c was commercially available) using

n-BuLi and trimethyl borate. Following Suzuki cross coupling

with commercially available methyl 4-bromo-3-methylbenzoate

aﬀorded the corresponding biaryl units (3a−3c). To couple the

biaryl units with the benzopyrrolodiazepine moiety, the methyl

ester functionality was hydrolyzed and converted to its acid

chloride. Subsequent reaction with 10,11-dihydro-5H-pyrrolo-

[2,1-c][1,4]benzodiazepine, 8, synthesized according to re-

ported procedures,²⁸⁻³⁰provided the desired biaryl function-

alized benzopyrrolodiazepines (4a−4c). To introduce a

carboxyl group solely at the 3-position of the tricycle, aromatic

acylation with trichloroacetyl chloride in the presence of

The synthetic route for the series B derivatives 12a−12d,

13a, 13b, and 14 is shown in Scheme 2. The biaryl carboxylic

acid 9, obtained after ﬂuorination of 2-(2-bromo-phenyl)-

ethanol followed by Suzuki coupling with the corresponding

boronic acids 2b and subsequent methyl ester hydrolyses,

served as starting material. For the synthesis of 12a−12d, the

reaction sequence of carboxylation and amide formation was

applied. The required 1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine

Hunig’s base was performed followed by a haloform type

̈

reaction with aqueous NaOH to give the corresponding

C

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was synthesized as reported³¹via in situ formation of

formaldehyde imine of 2-(1H-pyrrol-1-yl)ethanamine, followed

by an intramolecular aza-Friedel−Crafts reaction. The synthesis

of 12a and 12b was accomplished by amidation of the

carboxylic acid with the appropriate secondary amine. To gain

access to 12d, the 4-amino-1-Boc-piperidine was used for

amidation followed by Boc deprotection. In the same way,

mono-Boc-protected piperazine was introduced to obtain 12c.

Boc-protected piperazine was also utilized for the synthesis of

14. After amidation and deprotection, the secondary amine 11

was ﬁnally alkylated with 2-bromoethanol to obtain the product

14. Compound 13a was synthesized by amide formation with

4-piperidone followed by reductive amination using diethanol-

amine. A side product of this reaction obtained by reduction of

the ketone functionality to the corresponding secondary

alcohol was derivative 13b.

diﬀusion of molecules through the BBB is the lipophilicity with

a log P or log D value in the range of 1 to 4.³⁵⁻³⁷The log D

values of the derivatives of series A (Table 1) were determined

according to the HPLC method of Donovan and Pescatore.³³

The log D values of 6, 6a, and 6b are in the range of 3.1−3.3,

which indicates at least a moderate passage across the BBB.^35,36

Compounds 6c−6f are considerably more lipophilic with values

of >4.1. On one hand, a higher lipophilicity is assumed to

enhance the passive diﬀusion across biological membranes but

also increases plasma protein binding which might decrease

drug availability. In addition, P. Wils and co-workers³⁸have

shown that the transport of a drug across intestinal epithelial

cells is decreased with increasing lipophilicity (log D values

ranging from 3.5 to 5.2), a phenomenon which was explained

by successive aqueous−lipid interphases that a higher lipophilic

drug has to cross. Correspondingly, a parabolic relationship

exists between lipophilicity and brain uptake,^39,40making

derivatives with higher log D values unsuitable as ligands for

PET imaging in brain.

Lipophilicity is only one of several physicochemical

parameters when discussing BBB permeability, and recent

studies have shown that log D values are not generally

appropriate predictors for BBB penetration.⁴¹Therefore, we

performed in addition permeability (P_m) measurements

according to Taillardat-Bertschinger et al. using immobilized

artiﬁcial membrane (IAM) chromatography.^34,42Those IAMs

mimic the lipid environment of cell membranes and are formed

by synthetic phospholipid analogues covalently bound on the

silica support. This modiﬁed stationary phase is packed as a

solid matrix in a liquid chromatography column. The calculated

P_mvalue correlates with the retention time of the tested

compound in the chromatogram. For the IAM measurements

of the derivatives of series A, an IAM.PC.DD2 column was used

in isocratic mode with phosphate buﬀer and acetonitrile in

diﬀerent ratios. Data analysis was performed as described in

detail in the Experimental Section. As basis, a series of 20

reference compounds was investigated which comprise

molecules known to penetrate the BBB by passive diﬀusion⁴³

2.2. Binding Aﬃnity and Estimation of BBB Perme-

ability in Vitro. For determination of the binding aﬃnities of

the new derivatives toward hOTR, competitive binding studies

were performed using a cell line stably transfected with hOTR

gene and [³H]oxytocin as radioligand. The calculated K_ivalues

are summarized in Table 1.

Table 1. In Vitro Binding Aﬃnity, Lipophilicity, and

Permeability Data of New Derivatives

a

b

c

compd

K_i[nM]

lipophilicity (log D)

permeability (P_m)

Series A

6

8.7 1.1

21.5 8.8

13.3 7.1

17.7 5.1

96.6 7.5

27.3 6.4

66.3 14.9

3.21 0.02

3.31 0.04

3.14 0.01

4.89 0.30

4.18 0.18

5.20 0.36

4.08 0.16

Series B

3.21 1.23

4.49 0.80

6.28 5.64

24.70 8.81

34.20 9.32

25.71 3.49

7.09 3.06

6a

6b

6c

6d

6e

6f

d

12a

12b

12c

12d

13a

13b

14

>1000

nd

(e.g., raclopride 0.1

0.01, DASB 0.4

0.15, ﬂuoroethyl-

carfentanil 1.5 0.58, altanserin 2.2 0.8, and elacridar 4.0

2.7). Accordingly, P_mvalues between 0 and 4 obtained with this

test system allowed expectation of a good BBB penetration.⁴³

Table 1 shows the P_mvalues of the derivatives of series A.

Within this series, 6 is expected to be the compound with the

highest BBB permeability (P_mvalue of 3.21). As mentioned

earlier, this compound was described to be brain penetrable²⁷

and was therefore chosen as lead structure for the current

study. By contrast, the P_mvalues of compounds 6c, 6d, and 6e

are considerably higher (P_m= 24 to 34) and indicate poor

permeability. This could be caused by the high lipophilicity of

these derivatives hampering the transport of the compounds

across a membrane as mentioned.³⁸For the derivatives 6a, 6b,

and 6f, a moderate BBB permeability may be expected

according to P_mvalues between 4.5 and 7.

a

K_ivalues in nM (mean SD, n ≥ 2) were derived from IC₅₀values

according to the Cheng−Prusoﬀ equation;³²K_D([³H]oxytocin) =

b

2.56 0.95 nM. Log D values (mean SD, n ≥ 3, pH 7.4) were

determined by HPLC.³³P_mvalues (mean

S.D., n ≥ 3) were

c

determined by IAM-chromatography.³⁴nd: not determined.

d

Compounds 6a, 6b, and 6c bind with only slightly lower

aﬃnity to hOTR compared to the lead structure 6. This

indicates that minor structural modiﬁcations at the biphenyl

moiety and at the carbamide functionality are well accepted by

the receptor binding site. In contrast, a more pronounced

modiﬁcation of the substituents at the nitrogen of the

carbamide function results in a remarkable decrease in binding

aﬃnities (6d and 6f). Considering the compounds of series B,

we observed almost no relevant interaction with the hOTR (K_i

values of >1 μM), which is probably caused by the exchange of

the benzopyrrolodiazepine moiety.

On the basis of the highest in vitro binding aﬃnity in

combination with the moderate lipophilicity and permeability

data, 6b was selected as candidate for further ¹⁸F-labeling and in

vivo investigation.

2.3. Radiochemistry. The new radioligand [¹⁸F]6b was

prepared in a two-step synthesis by nucleophilic substitution of

a methoxymethyl (MOM) ether protected tosylate precursor

(7) using anhydrous K[¹⁸F]F-K_2.2.2-carbonate complex in

To estimate the BBB permeability of the new derivatives,

their lipophilicity and permeability was investigated using two

diﬀerent HPLC methods. An established criterion for a passive

D

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a

Scheme 3. ¹⁸F-Radiolabeling of [¹⁸F]6b

a

Reagents and conditions: (a) [¹⁸F]F⁻/K_2.2.2/K₂CO₃, ACN, with thermal heating (90 °C) or microwave heating (85−95 °C; 75 W); (b) 1 mL 2.0 M

HCl, with thermal heating (90 °C) or microwave heating (75−85 °C, 50 W).

Figure 2. (A) Semipreparative UV- and radio-HPLC chromatograms of [¹⁸F]6b (conditions: Reprosil-Pur C18-AQ, 250 mm × 10 mm, 44% ACN/

20 mM NH₄OAc_aq, 4 mL/min). (B) Analytical UV- and radio-HPLC chromatograms of the ﬁnal product of [¹⁸F]6b spiked with the reference 6b

(conditions: Reprosil-Pur C18-AQ, 250 mm × 4.6 mm, gradient with eluent A 10% ACN/20 mM NH₄OAc aq and eluent B 90% ACN/20 mM

NH₄OAc aq: 0−5′ 100% A, 5−10′ up to 55% B, 10−25′ 55% B, 25−30 up to 100% B, 30−40′ 100% B, 40−45′ up to 100% A, and 45−55′ 100% A).

acetonitrile followed by the deprotection of the two hydroxyl

groups using aqueous HCl (Scheme 3).

[¹⁸F]6b (B in Figure 2). Finally, the radiotracer was obtained

with a radiochemical purity of ≥97% in radiochemical yields of

20.9 4.1% (n = 4, decay corrected) with thermal heating and

26.9 4.7% (n = 6, decay corrected) with microwave heating

and speciﬁc activities between 35−160 GBq/μmol. Because of

the short reaction time and the slightly higher radiochemical

yield, the microwave heating system was preferred for the

radiosynthesis of [¹⁸F]6b.

The ¹⁸F-labeling process was investigated under thermal and

microwave heating. In general, higher labeling yields in shorter

reaction times were achieved under microwave conditions.

Using 75 W and a temperature interval of 85−95 °C, the

formation of the product was completed within 6−9 min and

labeling yields of 67.8

9.4% (n = 11) were achieved. By

contrast, under conventional heating at 90 °C, 15 min reaction

time was needed until no further increase of labeled product

was observed, resulting in labeling yields of 51.7 14.0% (n =

10). Beside [¹⁸F]ﬂuoride, no radioactive byproduct was

observed under both conditions tested. Moreover, the

precursor remained quite stable as proven by HPLC. The

deprotection step was also investigated with the two heating

methods and diﬀerent concentrations of hydrochloric acid (0.5,

1.0, and 2.0 M HCl each 1 mL). Using 1 mL of 2.0 M HCl and

microwave settings of 50 W/75−85 °C, the deprotection was

completed within 5 min. When lower concentrations of HCl

were used, longer reaction times were needed. Also for thermal

heating at 90 °C, 2.0 M HCl was most appropriate, however, 15

min reaction time was necessary to remove both protecting

groups quantitatively. With increasing reaction time the

formation of a byproduct (<5%) was observed, which appeared

to be slightly more lipophilic than the product according to

HPLC analysis.

The in vitro stability of the radiotracer was investigated by

incubation at 40 °C in the following solutions: (i) 0.9% aq

NaCl, (ii) phosphate-buﬀered saline (PBS), and (iii) pig plasma

samples. [¹⁸F]6b proved to be stable in all media, and no

deﬂuorination or degradation was observed within 30 min of

incubation time.

The n-octanol-buﬀer partition coeﬃcient (log D at pH 7.4)

of [¹⁸F]6b was determined for the n-octanol/PBS system by the

shake-ﬂask method. The obtained value of 2.87 0.15 (n = 3)

is slightly lower than the log D value of 3.14 determined by the

HPLC method for the nonradioactive reference compound 6b.

2.4. In Vitro Aﬃnity and Plasma Free Fraction of

[¹⁸F]6b. The equilibrium dissociation constant of [¹⁸F]6b was

determined in a homologous competitive binding experiment

with K_D= 12.3 nM, which is consistent with the above-reported

K_ivalue of 13.3 nM for the nonradioactive reference compound

6b.

The plasma free fraction f_pof [¹⁸F]6b, determined by

ultraﬁltration of a plasma sample prepared from pooled pig

blood and incubated with the radiotracer, was 0.02. This value

corresponds with the log D value and indicates suﬃcient

availability of the radiotracer for penetration of the BBB.

2.5. In Vitro Autoradiographic Studies. The distribution

of binding sites of [³H]oxytocin and [¹⁸F]6b in porcine brain is

shown in parts A and B of Figure 3, respectively. For [¹⁸F]6b,

we detected the following ranking order: chiasma opticus (CO)

For isolation of [¹⁸F]6b, the reaction mixture was neutralized

with appropriate amounts of 6.0 M NaOH and injected into the

semipreparative HPLC system. The product was collected at a

retention time of 27−30 min (A in Figure 2), puriﬁed using

solid phase extraction on an RP cartridge, and formulated in

sterile isotonic saline containing 10% of EtOH for better

solubility. Analytical HPLC of the ﬁnal product, spiked with the

unlabeled reference compound, conﬁrmed the identity of

E

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2.6. Metabolism of [¹⁸F]6b. The metabolism of [¹⁸F]6b

was investigated in plasma samples of female CD1 mice

obtained at 30 min pi of the radiotracer. Figure 4 shows a

typical analytical HPLC chromatogram of the metabolite proﬁle

of the radiotracer. For preparation of RP-HPLC samples, the

proteins were precipitated and extracted two times with ACN,

with a reproducible recovery of ∼82% of the starting

radioactivity in the supernatant. We assume that the radio-

activity remaining in the pellet mainly attributes to hydrophilic

radiometabolites. To verify this assumption, a plasma sample

was incubated in vitro with the parent radiotracer and

processed under identical conditions as the in vivo experiments.

As a result, in vitro more than 99% of the radiotracer could be

transferred into the supernatant, leading to the conclusion that

the radiotracer is recovered quantitatively also in the in vivo

samples. On the basis of this ﬁnding, we were able to correct

the percentage of intact radiotracer in dependence of the

recovery. Therefore, the intensities (mV/min; correlating to the

count rates) of each signal in the radio-HPLC chromatograms

were summed and related to 82%. This ratio was used to

calculate the percentage of intact radiotracer based on its peak

intensity in the chromatogram. Accordingly, 20% of intact

radiotracer is available in plasma at 30 min pi, which is slightly

less than the uncorrected value of 25%.

During the dynamic PET study in pig, we also determined

the fraction of nonmetabolized [¹⁸F]6b in plasma samples

taken at 2, 8, 30, and 60 min pi of the radiotracer and processed

them as described for the plasma samples of mouse. The

metabolite RP-HPLC proﬁle is comparable to the proﬁle

observed for the mouse samples at 30 min pi. However, for

samples collected at later time points, we detected a

considerable decrease of recovery of radioactivity ranging

from 97% at 2 min pi to 64% at 60 min pi. Therefore, the

percentage of intact radiotracer at each time point was

calculated as described and the corrected values are given in

Table 2. Accordingly, intact tracer accounts for 31% of total

activity in plasma at 30 min pi.

Figure 3. Distribution of the binding sites of (A) [³H]oxytocin (6 nM)

and (B) [¹⁸F]6b (∼10 nM) in porcine brain in vitro. Abbreviations:

Acb = nucleus accumbens, Cb = cerebellum, CC = corpus callosum,

Cd = nucleus caudatus, CO = chiasma opticus, Cx = cortex, CS =

colliculus superior, Fx = fornix, FR = formatio reticularis, Hip =

hippocampus, nP = nuclei pontis, Th = thalamus, PFC = prefrontal

cortex.

> fornix (Fx) > basal ganglia (Acb, Cd) ∼ brainstem (FR, nP)

> corpus callosum (CC) > cerebellum (Cb) ∼ hippocampus

(Hip) ∼ cortex (Cx) > prefrontal cortex (PFC). By contrast,

for [³H]oxytocin, a clearly diﬀerent pattern was detected: Hip >

Cx > Cb ∼ PFC > Cd > Th ∼ Acb > Fx ∼ CC > CO. This

quite sparse and very restricted distribution has also been

observed in the human and the brain of the titi monkey by

[

¹²⁵I]OVTA and [³H]oxytocin autoradiography.^4,44Therefore,

we supposed that these results suggest binding of [¹⁸F]6b to

both OTR as well as other targets. To test this assumption, we

performed appropriate competitive binding studies. We noticed

a considerable and overall reduction of binding sites of [¹⁸F]6b

by the GABA_Areceptor ligand THIP and a localized reduction

by oxytocin and the glutamate receptor ligand ^L-quisqualic acid.

In selected brain regions, the binding of [¹⁸F]6b was both

increased and decreased by the vasopressin receptor ligands

tolvaptan and SR49059 as well as the σ1/2 receptor ligand

siramesine (data not shown). We hypothesize that this

substantial oﬀ-target binding can be related to the presence

of the 1,4-benzodiazepine moiety in [¹⁸F]6b. In particular, 1,4-

benzodiazepine derivatives have been used among others as

anxiolytic, hypnotic, anticonvulsant, or antiarrhythmic drugs as

well as inhibitors or ligands of a variety of GPCRs such as

cholecystokinin, ﬁbrinogen, integrin, vasopressin, bradykinin, or

κ-opioid receptors.⁴⁵It might be expected that such features

hamper the applicability of benzodiazepine-related OTR ligands

such as [¹⁸F]6b for the PET imaging of OTR in the central

nervous system.

2.7. In Vivo Characterization of [¹⁸F]6b. To assess brain

uptake and pharmacokinetics of this type of tricyclic

benzodiazepine in more detail, biodistribution of [¹⁸F]6b was

investigated in female CD-1 mice 5 and 30 min after

intravenous injection of ∼220 kBq of the radioligand. With

0.41% ID/g (SUV = 0.11) at 5 min pi, a rather low brain uptake

was observed, followed by a fast washout (0.11% ID/g or SUV

= 0.03 at 30 min pi). In addition, we detected high uptake of

radioactivity in the pituitary gland (Figure 5; 8.36% ID/g, SUV

Figure 4. Analytical radio-HPLC chromatogram of a plasma sample of [¹⁸F]6b 30 min pi. Conditions: Reprosil-Pur C18-AQ; 250 mm × 4.6 mm;

ﬂow 1.0 mL/min; gradient mode see Experimental Section.

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Table 2. Percentage of [¹⁸F]6b in Plasma Samples of Pig

a

b

c

d

time of sampling [min pi] radioactivity recovery [%] peak area [¹⁸F]6b [mV/min] peak area total [mV/min] [¹⁸F]6b noncorr [%] [¹⁸F]6b corr [%]

2

97

77

69

64

461

55

467

77

99

71

44

16

96

55

31

10

8

30

60

76

172

70

11

a

b

c

Values are taken from radio-HPLC chromatograms. Sum of values of all signals taken from each radio-HPLC chromatogram. Percentage values

d

without correlation to the recovery of radioactivity. Percentage values with correlation to the recovery of radioactivity.

Figure 5. Organ distribution of [¹⁸F]6b in mice (data are presented as mean values sd; n = 2 per time point).

= 2.1 at 30 min pi), a region outside of the BBB with known

OTR expression.⁴⁶This uptake might indicate a speciﬁc

binding of [¹⁸F]6b to OTRs but could also be caused by the

binding to the oxytocin speciﬁc carrier protein neurophysin.⁸

The excretory organs small intestine and liver showed the

highest accumulation of radioactivity at 30 min pi (22.8% ID/g

and 12.1% ID/g, respectively). The increasing accumulation in

the femur may indicate some deﬂuorination of the radiotracer,

although OTR expression has also been found in bone

marrow.⁴⁷

To analyze the kinetics of the brain uptake of [¹⁸F]6b and to

assess an interaction of this particular tricyclic benzodiazepine

Figure 6. Representative image of a PET scan of [¹⁸F]6b at 60 min pi

of a brain of a female CD-1 mouse. The arrow is pointing to the

pituitary gland and the crosshairs designating the slice location of the

transverse and sagittal slice. (A) Transverse slice. (B) Sagittal slice.

with eﬄux transporters of the blood−brain barrier, pharmaco-

kinetics was further studied by dynamic PET scans under

baseline and blocking conditions in female CD-1 mice. The

results obtained by these PET studies revealed a very low

uptake of [¹⁸F]6b in the mouse brain during the 60 min

acquisition, with peak radioactivity in whole brain of SUV 0.07

at 5 and 0.04 at 60 min pi (Figure 6). A considerably higher

accumulation of radioactivity (SUV 0.7 at 60 min pi) was

detected in the pituitary gland. To investigate a possible

interaction of [¹⁸F]6b with the eﬄux transporter P-glycoprotein

(P-gp) as cause for the low brain uptake, additional PET studies

under pretreatment with the known P-gp inhibitor cyclo-

sporin⁴⁸were performed. However, neither in brain nor in the

pituitary gland of mice was an increase in the uptake of [¹⁸F]6b

observed.

radiotracers is species speciﬁcity as reﬂected by, e.g., higher

brain uptake of [¹¹C]GR205171 and [¹⁸F]altanserin in humans

and monkey than in rats⁴⁹and of the α7 nicotinic acetylcholine

receptor ligand [¹⁸F]NS14490 in pig than in mouse.^50,51To test

if such behavior is also relevant for the tricyclic benzodiazepines

under investigation in the current study, we decided to proceed

with the evaluation of [¹⁸F]6b in one pig.

Although this single PET study in pig conﬁrmed a low BBB

permeability of [¹⁸F]6b with a maximum SUV of 0.34 in brain

at 30 min pi, this value reﬂects an about 10-fold higher brain

uptake in pig than in mouse (SUV 0.03−0.04 at 30 min pi).

Interestingly, the uptake in the olfactory bulb (SUV = 0.33 at

30 min pi) was considerably higher than in other brain regions,

which is consistent with a comparably high OTR expression

found in rats.⁷

Finally, [¹⁸F]6b failed to image brain OTR in vivo in mouse,

most probably due to lack of brain penetration. However, the

log D and P_mvalues at physiological pH are assumed to be

compatible with at least a moderate brain permeation of

[¹⁸F]6b. In addition, the absence of an eﬀect of cyclosporine on

the brain uptake of the radiotracer indicates no substantial

interaction of [¹⁸F]6b with the P-gp. However, an important

aspect of preclinical evaluation of brain targeting PET

Despite a slightly better penetration of [¹⁸F]6b across the

BBB in pig than in mouse, the total brain uptake is rather low

and hampers reliable imaging and quantiﬁcation of OTR in the

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living brain. To disclose the reason for the low brain

penetration is ambitious. As often reported, the magnitude of

brain uptake of a compound is mainly determined by its size,

lipophilicity, H-bonding capacity, polar surface area (PSA), and

molecular ﬂexibility.^36,37The lipophilicity of [¹⁸F]6b with an

experimentally determined log D value of 2.8 is in the range for

BBB penetrating compounds^35,36and gives therefore no

valuable information. However, with a molecular weight of

555 g/mol, the size of the compound is considerably higher

than the limit of 400 g/mol, reported by Waterbeemd et al. for

a set of 125 CNS-active and inactive drugs.³⁷In this study,

additional to the size dependency, also the PSA was speciﬁed as

signiﬁcant criterion for BBB penetration and estimated to be

<90 Å²for CNS-active drugs. Later on, Kelder et al. suggested

an even more stringent cutoﬀ of PSA < 60 Å²based on

calculations with a data set of more than 700 CNS-active

drugs.⁵²Therefore, with a calculated TPSA (topological PSA)

value⁵³of 86 Å²for [¹⁸F]6b, a passage across the BBB seems to

be not favored (TPSA values are comparable to PSA values⁵⁴).

Because the PSA is deﬁned as the surface area occupied by

nitrogen and oxygen atoms and the polar hydrogens attached to

them,⁵⁵it also reﬂects the hydrogen bonding capacity and

polarity of a compound. Thus, in particular, the two OH

groups, acting as H-bond donors, contribute to the high TPSA

value. Additionally, the free rotation and steric availability of

these aliphatic OH groups may hamper the diﬀusion across a

lipophilic membrane. We are aware that the requirements for

BBB penetration are more complex and not only simply related

to physicochemical properties. However, these considerations

may serve as a ﬁrst explanation for the observed poor brain

uptake of [¹⁸F]6b.

tracers for the oxytocin receptor because of the observed poor

brain penetration and limited target selectivity of its analogue

[¹⁸F]6b.

4. EXPERIMENTAL SECTION

4.1. Organic Chemistry. General. All reactions were carried out

under argon atmosphere, in dry glassware, with dried solvents and

anhydrous conditions, unless otherwise stated. Reagents were

purchased with a minimum commercial quality of 95% and used

without further puriﬁcation. Analytical thin layer chromatography

(TLC) was performed on 0.25 mm silica gel plates Alugram SIL G/

UV₂₅₄(Machery-Nagel, Germany). The spots were visualized by using

UV light or staining with ninhydrin, phosphomolybdic acid, or vanillin

followed by heating. Column chromatography for puriﬁcation of the

crude products was carried out on silica gel (60, particle size 0.040−

0.063 mm, Machery-Nagel). NMR spectra were recorded on Bruker

AV 500 Ultra instruments and calibrated using residual nondeuterated

solvents as an internal reference. Following abbreviations were used to

describe the multiplicities: s = singlet, d = doublet, t = triplet, q =

quartet, m = multiplet, dt = doublet of triplet, td = triplet of doublet,

dd = doublet of doublet.

Analysis of all ﬁnal compounds was performed by TLC, MS, and

¹H- and ¹³C NMR spectroscopy. The purity of the ﬁnal compounds

was ≥95% and was conﬁrmed by LC/MS analysis employing a

Thermo SCIENTIFIC Ultimate 3000 system consisting of a

quaternary pump, a diode array detector, and an autosampler.

Electrospray ionization mass spectra were obtained using a MSQ

mass detector (Thermo SCIENTIFIC). An Ascentic Express Peptide

ES-C18 column 150 mm × 2.1 mm (SUPELCO) was used in gradient

mode (eluent: acetonitrile/H₂O + 0.1% HCOOH (v); 5% to 100%

ACN over 12 min with a ﬂow rate of 0.4 mL/min), and the

chromatograms were monitored at 220 nm.

Chemical names of compounds were generated by ChemDraw

Ultra 10.0.

TPSA values were calculated using the free Molecular Property

Calculation service of Molinspiration (molinspiration.com).

3. SUMMARY AND CONCLUSION

With the 10,11-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]-

diazepine derivative 6b, a novel ligand with high aﬃnity for

the oxytocin receptor was synthesized and radiolabeled with

ﬂuorine-18 and the binding aﬃnity in vitro and pharmacoki-

netics in vivo were evaluated.

Synthesis of the Precursor 7. Benzyl 2-(2-Bromophenyl)ethyl

Ether (1g). A solution of 7.44 g (37 mmol) of 2-bromophenylethyl

alcohol in 60 mL of THF was added under argon atmosphere to an

ice-cooled suspension of 2.25 g (56 mmol, 60% in mineral oil) NaH in

60 mL of THF. After stirring for 30 min at rt, the suspension was

heated to 50 °C for 30 min and cooled to 0 °C for the addition of 5.4

mL (7.77 g, 45 mmol) benzyl bromide. The reaction mixture was

stirred at rt overnight. Then 50 mL of H₂O were slowly added and the

organic solvent was removed under reduced pressure. The aqueous

layer was extracted with diethyl ether, and the organic layer was dried

over Na₂SO₄. Column chromatography (petroleum ether (PE):

diethyl ether = 15:1) of the crude product aﬀorded 10 g (93%) of

1g as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ [ppm]: 3.09 (t, J

= 7.0 Hz, 2H), 3.73 (t, 7.0 Hz, 2H), 4.55 (s, 2H), 7.08 (td, J = 7.5 Hz,

J = 1.5 Hz, 1H), 7.24 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.27−7.36 (m,

6H), 7.54 (dd, J = 8.0 Hz, J = 1.0 Hz, 1H).

Benzyl 2-(2-Boronophenyl)ethyl Ether (2g). To a solution of 8.9 g

(31 mmol) of 1g in 150 mL of dry THF, n-BuLi (14.8 mL, 2.5 M in n-

hexane) was added at −78 °C and the mixture was stirred for 1 h at

this temperature. After addition of 4.1 mL (3.8 g, 37 mmol) of

trimethyl borate, the cooling bath was removed and the reaction

mixture was stirred at rt overnight, quenched with 100 mL of 1.0 M

aqueous HCl, and stirred for 1 h. The organic layer was separated, and

the aqueous layer was extracted with CH₂Cl₂(2 × 100 mL). The

combined organic layers were washed with brine and dried over

Na₂SO₄and the solvent was removed in vacuum. The obtained 7.9 g

(99%) of the crude product 2g was used in the next step without any

further puriﬁcation.

Because of the very low brain uptake of [¹⁸F]6b in mouse

and pig, this radiotracer is not suitable for examining the

oxytocin receptor in the living brain. The molecular size of

[¹⁸F]6b and the relatively high polar surface area with two

easily accessible OH groups might account for the low brain

penetration. This is also reﬂected by the in vitro permeability

(P_m) determined by IAM chromatography, which is not in the

ideal range of brain penetrating compounds.⁴³The lipophilicity,

characterized by the log D value, was not a good predictor for

BBB penetration of [¹⁸F]6b. Moreover, on the basis of

blockade PET studies in mice, we could show that [¹⁸F]6b

does not signiﬁcantly interact with the eﬄux transporter P-gp, a

further parameter that can strongly inﬂuence the brain uptake

of a compound.

Furthermore, in vitro autoradiographic blocking studies on

porcine brain indicated interaction of the radioligand at binding

sites of several oﬀ-target receptors, which hampers the

applicability of [¹⁸F]6b as speciﬁc OTR-PET tracer. This low

selectivity is assumed to be caused by the 1,4-benzodiazepine

moiety in [¹⁸F]6b, which is part of numerous drugs and ligands

for several GPCRs. However, as we have shown with the series

B compounds, this 1,4-benzodiazepine moiety is essential for

high aﬃnity to the OTR binding site.

2′-(2-Benzyloxyethyl)-2-methylbiphenyl-4-carboxylic Acid Methyl

Ester (3g). A stirred solution of 7.9 g (31 mmol) of 2g, 7.0 g (31

mmol) of methyl 4-bromo-3-methylbenzoate, and 14.2 g (134 mmol)

of Na₂CO₃in 360 mL of H₂O/ethyl alcohol/toluene (1:1:2) was

degassed for 30 min with argon. The catalyst Pd(PPh₃)₄(1.78 g, 1.5

Finally, we conclude that the selected OTR antagonist 6 is

not suitable as lead structure for the development of PET

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mmol) was added, and the obtained yellow solution was reﬂuxed at 90

°C for 14 h. After cooling to rt, 150 mL of ethyl acetate (EtOAc) was

added and the aqueous layer was separated. The organic layer was

washed with brine and dried over Na₂SO₄. After removal of the

solvents, the crude product was puriﬁed by column chromatography

(n-hexane:EtOAc = 10:1) to yield 9.5 g (85%) of 3g as colorless oil.

¹H NMR (500 MHz, CDCl₃) δ [ppm]: 2.06 (s, 3H), 2.60 (dt, J = 14.0

Hz, J = 7.0 Hz, 1H), 2.75 (dt, J = 14.0 Hz, J = 7.0 Hz, 1H), 3.45 (t, J =

7.5 Hz, 2H), 3.95 (s, 3H), 4.37 (s, 2H), 7.06 (dd, J = 7.5 Hz, J = 1.5

Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.16−7.18 (m, 2H), 7.24−7.36 (m,

6H), 7.85 (dd, J = 8.0 Hz, J = 1.5 Hz, 1H), 7.93 (s, 1H).

10,11-Dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine (8). Com-

pound 8 was synthesized according to the literature²⁸⁻³⁰by N-

alkylation of pyrrole-2-carboxaldehyde with 2-nitrobenzyl bromide

followed by reductive ring closure of 1-(2-nitrobenzyl)-2-pyrrolecar-

boxaldehyde. ¹H NMR (500 MHz, CDCl₃) δ [ppm]: 4.18 (br s, 1H),

4.46 (d, J = 4.0 Hz, 2H), 5.19 (s, 2H), 6.02−6.05 (m, 2H), 6.49 (dd, J

= 8.0 Hz, J = 1.0 Hz, 1H), 6.63 (td, J = 7.5 Hz, 1.0 Hz, 1H), 6.68−6.69

(m, 1H), 6.98−7.05 (m, 2H).

(br s, 2H), 5.98 (br s, 2H), 6.38 (d, J = 4.5 Hz, 1H), 6.86 (d, J = 8.0

Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.97 (dd, J = 7.5 Hz, J = 1.5 Hz,

1H), 7.01−7.07 (m, 2H), 7.12 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.15−

7.17 (m, 2H), 7.21−7.35 (m, 7H), 7.42 (dd, J = 7.5, J = 1.5 Hz, 1H),

7.47 (d, J = 4.0 Hz, 1H).

10-[2′-(2-Benzyloxyethyl)-2-methylbiphenyl-4-carbonyl]-10,11-

dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid

(5g). To a solution of 6.4 g (10 mmol) of 15 in 200 mL of acetone, 30

mL of 2.5 M aqueous NaOH was added and the mixture was stirred at

rt overnight. After removal of the solvent, 25 mL of 2.0 M aqueous

HCl was added and the mixture was extracted with EtOAc. The

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated to

remove the solvent. The crude product was puriﬁed by column

chromatography (PE:EtOAc = 1:1) to provide 4.5 g (83%) of 5g as a

white solid; mp 64−68 °C. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]:

1.83 (s, 3H), 2.39 (dt, J = 14.0 Hz, J = 7.0 Hz, 1H), 2.58 (dt, J = 14.0

Hz, J = 7.0 Hz, 1H), 3.30 (t, J = 7.0 Hz, 2H), 4.28 (s, 2H), 5.20 (br s,

2H), 5.95 (br s, 2H), 6.12 (d, J = 4.0 Hz, 1H), 6.77 (d, J = 4.0 Hz,

1H), 6.83−6.87 (m, 2H), 6.95−7.02 (m, 3H), 7.09 (t, J = 7.0 Hz, 1H),

7.16 (t, J = 7.0 Hz, 2H), 7.21−7.35 (m, 8H), 12.34 (br s, 1H).

10-[2′-(2-Benzyloxyethyl)-2-methylbiphenyl-4-carbonyl]-10,11-

dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid

Bis(2-hydroxyethyl)amide (6g). A mixture of 2.5 g (4.5 mmol) of 5g,

0.66 g (6.3 mmol) of diethanolamine, 0.88 g (6.5 mmol) of HOBt,

(5H,11H-Benzo[e]pyrrolo[1,2-a][1,4]diazepin-10-yl)-[2′-(2-benzy-

loxyethyl)-2-methylbiphenyl-4-yl]methanone (4g). To a stirred

solution of 9.5 g (26 mmol) of 3g in 190 mL of THF, a solution of

96 mL of 1.0 M aqueous NaOH was added and the mixture was stirred

under reﬂux overnight. The complete consumption of the starting

material was conﬁrmed by TLC (PE:n-hexane = 9:1). After removal of

THF, 1.0 M aqueous HCl was added to adjust a pH value of 3 and the

product was extracted with EtOAc. The combined organic layers were

dried over Na₂SO₄. Removal of the solvent aﬀorded 8.5 g (94%) of the

corresponding carboxylic acid as white solid, which was used directly

1.12 g (5.9 mmol) of EDCI, and 1.3 mL (0.99 g, 7.7 mmol) of Hunig’s

̈

base in 45 mL of DMF was stirred at rt overnight. The mixture was

concentrated, diluted with 75 mL of H₂O, and extracted three times

with 75 mL of EtOAc. The organic layer was washed twice with 50 mL

of a saturated aqueous NaHCO₃solution, dried over Na₂SO₄, ﬁltered,

and concentrated to remove the solvent. Puriﬁcation by column

chromatography (CH₂Cl₂:methanol = 95:5) aﬀorded 2.46 g (85%) of

1

in the next step; mp 61−64 °C. H NMR (500 MHz, DMSO-d₆) δ

[ppm]: 2.00 (s, 3H), 2.50 (dt, J = 14.0 Hz, J = 7.0 Hz, 1H), 2.67 (dt, J

= 14.0 Hz, J = 7.0 Hz, 1H), 3.40 (t, J = 7.0 Hz, 2H), 4.31 (s, 2H), 7.06

(dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.12−7.18 (m, 3H), 7.22−7.30 (m,

4H), 7.33 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.40 (m, 1H), 7.78 (dd, J =

8.0 Hz, J = 1.5 Hz, 1H), 7.87 (d, J = 1.5 Hz, 1H), 12.93 (br s, 1H).

A solution of 4.5 g (13 mmol) of 2′-(2-benzyloxy-ethyl)-2-methyl-

biphenyl-4-carboxylic acid in 25 mL of thionyl chloride was reﬂuxed

under argon atmosphere for 2 h. The excess of thionyl chloride was

removed under vacuum, and the residue was diluted three times with

10 mL of toluene followed by removal of the solvent. The resultant

acid chloride was dissolved in 25 mL of CH₂Cl₂and added dropwise

to an ice-cold solution of 2.8 g (15 mmol) of 8 and 5.1 mL (30 mmol)

1

6g as a white solid. H NMR (500 MHz, CDCl₃) δ [ppm]: 1.89 (s,

3H), 2.50 (td, J = 7.5 Hz, J = 13.5 Hz, 1H), 2.64 (dt, J = 7.0 Hz, J =

14.0 Hz, 1H), 3.36 (t, J = 7.5 Hz, 2H) 3.44 (br s, 2H), 3.77 (t, J = 5.0

Hz, 4H), 3.88 (br s, 4H), 4.33 (dd, J = 12.0 Hz, J = 14.5 Hz, 2H), 5.30

(br s, 2H), 5.47 (br s, 2H), 6.01 (d, J = 4.0 Hz, 1H), 6.51 (d, J = 4.0,

1H), 6.79−6.85 (m, 2H), 6.96 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 7.0 Hz,

1H), 7.10 (t, J = 7.0 Hz, 1H), 7.17−7.22 (m, 3H), 7.24−7.27 (m, 3H),

7.28−7.31 (m, 3H), 7.45 (dd, J = 1.5 Hz, J = 7.5 Hz, 1H).

10-[2′-(2-Benzyloxyethyl)-2-methylbiphenyl-4-carbonyl]-10,11-

dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid

Bis(2-methoxymethoxyethyl)amide (16; Intermediate of Reaction

Step (i) in Scheme 1). A solution of 1.39 g (2.2 mmol) of 6g in 30 mL

of Hunig’s base in 100 mL of CH Cl . The cooling bath was removed,

̈

of CH Cl was cooled on ice, and 1.6 mL (9.5 mmol) of Hunig’s base,

̈

2

and the mixture was stirred at rt overnight. After hydrolysis with 50

mL of H₂O, the aqueous layer was extracted three times with CH₂Cl₂.

The combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuum. The crude product was puriﬁed by column

chromatography (PE:EtOAc = 4:1 to 1:1) to obtain 5.6 g (84%) of 4g

65 mg (0.22 mmol) of tetrabutylammonium chloride, and 0.77 mL

(9.5 mmol) of bromomethyl methyl ether were added. The resultant

solution was allowed to warm to rt and stirred overnight. The mixture

was diluted with H₂O, and the separated aqueous layer was extracted

with CH₂Cl₂. The combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated in vacuum. Flash chromatography

(PE:EtOAc = 1:1) of the crude product aﬀorded 1.4 g (87%) of 16

as an orange oil. ¹H NMR (500 MHz, CDCl₃) δ [ppm]: 1.83 (s, 3H),

2.38 (td, J = 7.5 Hz, J = 13.5 Hz, 1H), 2.57 (dt, J = 7.0 Hz, J = 14.0 Hz,

1H), 3.20−3.26 (m, 6H), 3.30 (t, J = 7.0 Hz, 2H), 3.58−3.73 (m, 8H),

4.27 (s, 2H), 4.57 (br s, 3H), 4.63 (br s, 1H), 5.20 (br s, 2H), 5.35 (br

s, 2H), 6.05 (d, J = 3.5 Hz, 1H), 6.29 (d, J = 3.5 Hz, 1H), 6.83−6.88

(m, 2H), 6.96 (d, J = 7.0 Hz, 1H), 6.98−7.02 (m, 2H), 7.09 (t, J = 7.0

Hz, 1H), 7.16 (d, J = 7.0 Hz, 2H), 7.21−7.35 (m, 8H).

1

as colorless oil. H NMR (500 MHz, DMSO-d₆) δ [ppm]: 1.83 (s,

3H), 2.38 (dt, J = 14.0 Hz, J = 7.0 Hz, 1H), 2.57 (dt, J = 14.0 Hz, J =

7.0 Hz, 1H), 3.30 (t, J = 7.0 Hz, 2H), 4.27 (s, 2H), 5.21 (br s, 2H),

5.31 (br s, 2H), 5.93 (dd, J = 3.0 Hz, J = 3.0 Hz, 1H), 5.97 (br s, 1H),

6.83−6.86 (m, 2H), 6.90−6.97 (m, 2H), 7.00−7.07 (m, 2H), 7.10−

7.17 (m, 3H), 7.21−7.36 (m, 7H), 7.45 (d, J = 7.5 Hz, 1H).

1-[10-[2′-(2-Benzyloxyethyl)-2-methylbiphenyl-4-carbonyl]-

10,11-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-3-yl]-2,2,2-

trichloroethanone (15; Intermediate of Reaction Step (e) in Scheme

1). To an ice-cold solution of 5.5 g (11 mmol) of 4g in 115 mL of

10-[2′-(2-Hydroxyethyl)-2-methylbiphenyl-4-carbonyl]-10,11-di-

hydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid

Bis(2-methoxymethoxyethyl)amide (17; Intermediate of Reaction

Step (j) in Scheme 1). To a degassed solution of 860 mg (1.18 mmol)

of compound 16 in 60 mL of methanol, Pd(OH)₂/C (200 mg, 20 wt

%, wet, 0.28 mmol) was added. The mixture was vigorously stirred at

rt under a hydrogen atmosphere (balloon pressure) for 20 h. TLC

analysis (100% EtOAc) indicated the complete consumption of the

starting material. The reaction mixture was ﬁltered through Celite and

washed with methanol. The ﬁltrate was concentrated and dried in

vacuum to give 750 mg (99%) of the titled compound as yellowish

viscous oil. The intermediate 17 was used in the next step without any

further puriﬁcation. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 1.86 (s,

CH Cl , Hunig’s base was added (3.6 mL, 2.7 g, 22 mmol) under

̈

2

argon atmosphere. After the addition of 3.8 mL (6.2 g, 34 mmol) of

trichloro acetyl chloride over 10 min, the cooling bath was removed

and the mixture was stirred at rt overnight. The reaction mixture was

diluted with 50 mL of H₂O, and the aqueous layer was extracted twice

with CH₂Cl₂. The combined organic layers were washed with 100 mL

of 1.0 M aqueous HCl and 50 mL of brine, dried over Na₂SO₄, ﬁltered,

and concentrated to remove the solvent. Column chromatography

(PE:EtOAc = 2:1) aﬀorded 6.4 g (92%) of the product 15 as a

1

colorless solid; mp 80−85 °C. H NMR (500 MHz, DMSO-d₆) δ

[ppm]: 1.84 (s, 3H), 2.38 (dt, J = 14.0 Hz, J = 7.0 Hz, 1H), 2.57 (dt, J

= 14.0 Hz, J = 7.0 Hz, 1H), 3.30 (t, J = 7.0 Hz, 2H), 4.28 (s, 2H), 5.30

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3H), 2.24−2.31 (m, 1H), 2.41−2.47 (m, 1H), 3.24−3.30 (m, 8H),

3.62−3.74 (m, 8H), 4.47 (t, J = 5.0 Hz, 1H), 4.55−4.78 (m, 4H), 5.15

(br s, 2H), 5.35 (br s, 2H), 6.05 (d, J = 3.5 Hz, 1H), 6.29 (d, J = 3.5

Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 7.5 Hz, 1H), 7.02 (d, J

= 7.5 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.21

(td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.25 (s, 1H), 7.27 (td, J = 7.5 Hz, 1.5

Hz, 1H), 7.31 (dd, J = 7.5 Hz, 1.5 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H).

Toluene-4-sulfonic Acid 2-(4′-[3-[Bis(2-methoxymethoxy-ethyl)-

carbamoyl]-5H,11H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10-car-

bonyl]-2′-methylbiphenyl-2-yl)ethyl Ester (7). A solution of 178 mg

(0.93 mmol) of p-toluenesulfonyl chloride in 1 mL of CH₂Cl₂was

added under argon atmosphere to an ice-cooled solution of 104 mg

(0.16 mmol) of compound 17, 0.13 mL (0.93 mmol) of triethylamine,

and 2 mg (0.02 mmol) of Steglich base (4-DMAP) in 3 mL of CH₂Cl₂.

The mixture was stirred at rt overnight. The progress of the reaction

was monitored by TLC (n-hexane:EtOAc = 1:1). After complete

consumption of 17, the reaction mixture was diluted with H₂O and

extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated in vacuum. The resulting residue was

puriﬁed by column chromatography (n-hexane:EtOAc = 2:3) to give

using 0.35 g (0.57 mmol) of 18, product 5a was isolated as a white

solid in 99% yield (0.29 g) and used in the next step without further

puriﬁcation. ¹H NMR (500 MHz, CDCl₃) δ [ppm]: 1.93 (s, 3H), 5.32

(br s, 2H), 5.98 (br s, 2H), 6.11 (d, J = 4.0 Hz, 1H), 6.79 (d, J = 7.0

Hz, 1H), 6.86 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H), 6.91 (d, J = 8.0 Hz,

1H), 6.97−7.00 (m, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.09 (d, J = 4.0 Hz,

1H), 7.12−7.19 (m, 2H), 7.26 (s, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.72

(dd, J = 9.0 Hz, J = 5.5 Hz, 1H).

10-(5′-Fluoro-2-methyl-2′-triﬂuoromethylbiphenyl-4-carbonyl)-

10,11-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carbox-

ylic Acid Bis(2-hydroxyethyl) Amide (6a). A mixture of 290 mg (0.57

mmol) of 5a, 75 mg (0.72 mmol) of diethanol amine, 86 mg (0.63

mmol) of HOBt, 127 mg (0.66 mmol) of EDCI, and 0.15 mL (113

mg, 0.87 mmol) of Hunig’s base in 4 mL of DMF was stirred at rt

̈

overnight. The mixture was concentrated, and the residue was diluted

with 7 mL of H₂O and extracted three times with 7 mL of EtOAc. The

organic layer was washed twice with 5 mL of a saturated aqueous

NaHCO₃solution, dried over Na₂SO₄, ﬁltered, and concentrated to

remove the solvent. Column chromatography (CH₂Cl₂:methanol =

95:5) followed by crystallization from EtOAc to remove traces of

impurities aﬀorded 190 mg (56%) of 5a as a white solid; mp 100−107

1

101 mg (78%) of 7 as a white solid; mp 75−80 °C. H NMR (500

1

MHz, CDCl₃) δ [ppm]: 1.76 (s, 3H), 2.38 (td, J = 7.5 Hz, J = 13.5 Hz,

1H), 2.41 (s, 3H), 2.59 (dt, J = 7.0 Hz, J = 14.0 Hz, 1H), 3.25 (br s,

6H), 3.58−3.73 (m, 8H), 3.78−3.90 (m, 2H), 4.57 (br s, 3H), 4.64 (br

s, 1H), 5.20 (br s, 2H), 5.36 (br s, 2H), 6.05 (d, J = 3.5 Hz, 1H), 6.30

(d, J = 3.5 Hz, 1H), 6.67 (d, J = 7.5 Hz, 1H), 6.82 (d, J = 6.0 Hz, 1H),

6.94−6.96 (m, 2H), 7.02 (t, J = 7.0 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H),

7.22 (s, 1H), 7.23−7.29 (m, 3H), 7.35 (d, J = 7.0 Hz, 1H), 7.41 (d, J =

8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H). ¹³C NMR (125 MHz, DMSO-

d₆) δ [ppm]: 19.18, 20.95, 31.53, 45.97, 47.03, 54.59, 55.06, 64.65,

65.22, 69.70, 91.44, 92.66, 95.64, 107.61, 110.51, 125.07, 126.67,

126.80, 126.98, 127.28, 127.57, 128.39, 128.41, 128.63, 128.91, 129.12,

129.21, 129.61, 129.97, 132.03, 133.52, 134.67, 134.69, 134.82, 140.13,

141.30, 141.40, 144.73, 163.73, 169.17. LC/MS retention time: 10.80

min. MS (ESI) m/z = 796.2 [M + H]⁺.

°C. H NMR (500 MHz, DMSO-d₆) δ [ppm]: 1.88 (s, 3H), 3.48−

3.65 (m, 8H), 4.82 (t, J = 5.0 Hz, 2H), 5.25 (br s, 2H), 5.34 (br s, 2H),

6.03 (d, J = 3.0 Hz, 1H), 6.30 (d, J = 3.5 Hz, 1H), 6.87 (d, J = 6.0 Hz,

1H), 6.93 (d, J = 7.5 Hz, 1H), 7.01−7.07 (m, 2H), 7.13−7.18 (m,

2H), 7.28 (s, 1H), 7.38 (d, J = 7.0 Hz, 1H), 7.44 (td, J = 8.0 Hz, J = 2.5

Hz, 1H), 7.88 (dd, J = 9.0 Hz, J = 5.5 Hz, 1H). ¹³C NMR (125 MHz,

DMSO-d₆) δ [ppm] 19.29, 45.89, 47.03, 58. 45, 58. 56, 107.50, 110.23,

115.10 (d, J = 21.6 Hz), 118.30 (d, J = 22.1 Hz), 122.27, 123.50 (q, J =

29.9 Hz), 124.44, 124.51, 127.01, 127.36, 128.21, 128.44, 128.71,

128.75, 128.98 (m), 129.07, 129.29, 134.70, 135.07, 135.64, 138.42,

141.32, 142.03 (d, J = 7.6 Hz), 162.32, 163.76, 164.33, 169.04. LC/MS

retention time: 8.94 min. MS (ESI) m/z = 596.4 [M + H]⁺.

Synthesis of 6b. 2-(2-Fluoroethyl)phenyl Bromide (1b). A mixture

of 5 g (25 mmol) of 2-(2-hydroxyethyl)phenyl bromide and 3.5 mL

(27 mmol) of DAST in 75 mL of dry CH₂Cl₂was stirred at rt for 20 h.

The reaction mixture was diluted with 150 mL of saturated aqueous

NaHCO₃solution and stirred for additional 30 min. The CH₂Cl₂layer

was separated, and the aqueous layer was extracted with CH₂Cl₂. The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated to remove the CH₂Cl₂. Puriﬁcation by column

chromatography (PE:EtOAc = 9:1) provided 3.2 g (63%) of 1b as a

Synthesis of 6a. 5′-Fluoro-2-methyl-2′-triﬂuoromethylbiphenyl-

4-carboxylic Acid Methyl Ester (3a). The compound was synthesized

using 1.0 g (4.8 mmol) of 5-ﬂuoro-2-(triﬂuoromethyl)phenylboronic

acid according to the procedure for the synthesis of 3g. The obtained

crude product was puriﬁed by column chromatography (n-

hexane:EtOAc = 9:1) to yield 1.4 g (91%) of 3a as a colorless oil.

¹H NMR (500 MHz, CDCl₃) δ [ppm]: 2.05 (s, 3H), 3.86 (s, 3H),

7.04 (d, J = 6.5 Hz, 1H), 7.10 (t, J = 6.5 Hz, 1H), 7.30 (s, 1H), 7.42−

7.48 (m, 2H), 7.89 (dd, J = 7.5 Hz, J = 5.5 Hz, 1H).

1

colorless liquid. H NMR (500 MHz, CDCl₃) δ [ppm]: 3.19 (dt, J =

22.5 Hz, J = 7.0 Hz, 2H), 4.66 (dt, J = 47.5 Hz, J = 6.5 Hz, 2H), 7.12

(td, J = 7.5 Hz, 2.0 Hz, 1H), 7.25−7.31 (m, 2H), 7.56 (dd, J = 7.5 Hz,

J = 1.0 Hz, 1H).

2-(2-Fluoroethyl)phenylboronic Acid (2b). According to the

procedure of 2g described above, 3.0 g (15 mmol) of 1b, 80 mL of

dry THF, 7.2 mL of n-BuLi (2.5 M in n-hexane, 18 mmol), and 2.0 mL

(18 mmol) of trimethyl borate reacted to give 2.0 g (79%) of the

boronic acid 2b as a clear, colorless oil. The crude product was used in

the next step without any further puriﬁcation.

(5H,11H-Benzo[e]pyrrolo[1,2-a][1,4]diazepin-10-yl)-(5′-ﬂuoro-2-

methyl-2′-triﬂuoromethylbiphenyl-4-yl)methanone (4a). According

to the procedure described for the synthesis of 4g, compound 3a was

hydrolyzed and 0.96 g (3.2 mmol) of the resulting carboxylic acid was

used to react with 0.89 g (4.8 mmol) of 8. The obtained crude product

was puriﬁed by column chromatography (n-hexane:EtOAc = 4:1) to

yield 1.4 g (94%) of 4a as a colorless oil. ¹H NMR (500 MHz, DMSO-

d₆) δ [ppm]: 1.89 (s, 3H), 5.10 (br s, 2H), 5.30 (br s, 2H), 5.93 (dd, J

= 3.0 Hz, J = 3.0 Hz, 1H), 5.97 (br s, 1H), 6.83 (t, J = 2.5 Hz, 1H),

6.91−6.95 (m, 2H), 7.04 (d, J = 6.5 Hz, 1H), 7.10 (t, J = 6.5 Hz, 1H),

7.16−7.21 (m, 2H), 7.30 (s, 1H), 7.42−7.48 (m, 2H), 7.89 (dd, J = 7.5

Hz, J = 5.5 Hz, 1H).

2,2,2-Trichloro-1-[10-(5′-ﬂuoro-2-methyl-2′-triﬂuoromethylbi-

phenyl-4-carbonyl)-10,11-dihydro-5-benzo[e]pyrrolo[1,2-a][1,4]-

diazepin-3-yl]ethanone (18; Intermediate of Reaction Step (e) in

Scheme 1). The compound was synthesized using 0.30 g (0.60 mmol)

of 4a according to the procedure for the synthesis of 15. The obtained

crude product was puriﬁed by column chromatography (n-

hexane:EtOAc = 4:1) to yield 0.35 g (95%) of the titled product as

a colorless oil. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 1.89 (s, 3H),

5.10 (br s, 2H), 5.30 (br s, 2H), 5.93 (dd, J = 3.0 Hz, J = 3.0 Hz, 1H),

5.97 (br s, 1H), 6.39 (d, J = 4.0 Hz, 1H), 6.79 (d, J = 7.0 Hz, 1H), 7.04

(d, J = 6.5 Hz, 1H), 7.10 (t, J = 6.5 Hz, 1H), 7.16−7.21 (m, 2H), 7.30

(s, 1H), 7.42−7.48 (m, 2H), 7.89 (dd, J = 7.5 Hz, J = 5.5 Hz, 1H).

10-(5′-Fluoro-2-methyl-2′-triﬂuoromethylbiphenyl-4-carbonyl)-

10,11-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carbox-

ylic Acid (5a). According to the procedure for the synthesis of 5g and

2′-(2-Fluoroethyl)-2-methylbiphenyl-4-carboxylic Acid Methyl

Ester (3b). The compound was synthesized using 136 mg (0.8

mmol) of 2b according to the procedure for the synthesis of 3g. The

obtained crude product was puriﬁed by column chromatography

(EtOAc:n-hexane = 1:19) to give 170 mg (78%) of compound 3b as a

1

colorless oil. H NMR (500 MHz, CDCl₃) δ [ppm] 2.05 (s, 3H),

2.55−2.65 (m, 1H), 2.73−2.83 (m, 1H), 3.87 (s, 3H), 4.38 (t, J = 6.5

Hz, 1H), 4.47 (t, J = 6.5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 1H), 7.25 (d, J =

8.0 Hz, 1H), 7.33 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.38 (td, J = 7.5 Hz,

J = 1.5 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.83 (dd, J = 7.5 Hz, J = 1.5

Hz, 1H), 7.92 (s, 1H).

(5H,11H-Benzo[e]pyrrolo[1,2-a][1,4]diazepin-10-yl)-[2′-(2-ﬂuo-

roethyl)-2-methylbiphenyl-4-yl]methanone (4b). According to the

procedure described for the synthesis of 4g, compound 3b was

hydrolyzed and 1.03 g (4.0 mmol) of the resulting carboxylic acid was

used to react with 8. The obtained crude product was puriﬁed by

crystallization from EtOAc to give 1.60 g (99%) of 4b as a colorless

crystalline solid. ¹H NMR (500 MHz, CDCl₃) δ [ppm]: 1.86 (s, 3H),

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2.41−2.51 (m, 1H), 2.62−2.72 (m, 1H), 4.23 (t, J = 6.5 Hz, 1H), 4.32

(t, J = 6.5 Hz, 1H), 5.10 (br s, 2H), 5.31 (br s, 2H), 5.93 (dd, J = 3.5

Hz, J = 2.0 Hz, 1H), 5.97 (br s, 1H), 6.84 (t, J = 2.5 Hz, 1H), 6.89−

6.95 (m, 2H), 7.01 (d, J = 6.5 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 7.09

(t, J = 7.5 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 7.25−7.34 (m, 3H), 7.37

(d, J = 7.5 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H).

(500 MHz, DMSO-d₆) δ [ppm]: 1.85 (s, 3H), 2.83 (t, J = 5.0 Hz, 1H),

2.87−2.91 (m, 3H), 4.26 (t, J = 5.5 Hz, 2H), 4.46 (dt, J = 47.5 Hz, J =

5.0 Hz, 2H), 5.20 (br s, 2H), 5.93 (br s, 2H), 6.16 (d, J = 3.5 Hz, 1H),

6.85 (d, J = 3.5 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz,

1H), 7.00−7.08 (m, 2H), 7.15 (td, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.22 (d,

J = 7.5 Hz, 1H), 7.27 (s, 1H), 7.37 (dd, J = 7.5 Hz, J = 1.0 Hz, 1H),

7.60 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.80 (d, J = 8.0 Hz,

1H). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm]: 19.39, 45.99 (br s),

46.21, 47.39, 48.63 (d, J = 20 Hz), 63.12, 83.35 (d, J = 162 Hz),

109.25, 117.80, 121.46, 121.53, 123.73 (q, J = 272 Hz), 124.56, 125.90

(q, J = 5 Hz), 126.87 (q, J = 29 Hz), 127.15, 128.13, 128.35, 128.47,

128.59, 129.29, 130.37, 131.14, 132.19, 134.08, 134.87, 135.10, 138.86

(q, J = 1.5 Hz), 139.82, 141.15, 160.37, 169.17. LC/MS retention

time: 9.39 min. MS (ESI) m/z = 580.1 [M + H]⁺.

Synthesis of 6d−6f. 10-(2-Methyl-2′-triﬂuoromethylbiphenyl-4-

carbonyl)-10,11-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-

3-carboxylic Acid (5c).²⁶Compound 5c was synthesized in ﬁve steps

starting from 5.2 g (27 mmol) of commercially available 2c and 6 g

(26 mmol) of methyl 4-bromo-3-methylbenzoate according to the

published procedure for the synthesis of 6.²⁶The obtained crude

product was puriﬁed by crystallization from EtOAc to give 6.5 g (50%)

of 5c as a white solid; mp 230−234 °C. ¹H NMR (500 MHz, DMSO-

d₆) δ [ppm]: 1.85 (3H, s), 5.19 (br s, 2H), 5.95 (br s, 2H), 6.12 (d, J =

4.0 Hz, 1H), 6.77 (d, J = 4.0 Hz, 1H), 6.87 (d, J = 7.5 Hz, 1H), 6.91

(d, J = 7.5 Hz, 1H), 7.01−7.07 (m, 2H), 7.15 (t, J = 7.5 Hz, 1H), 7.22

(d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.34 (d, J = 7.0 Hz, 1H), 7.60 (t, J =

7.5 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 12.33

(br s, 1H).

2,2,2-Trichloro-1-[10-[2′-(2-ﬂuoroethyl)-2-methylbiphenyl-4-car-

bonyl]-10,11-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-3-yl]-

ethanone (19; Intermediate of Reaction Step (e) in Scheme 1). The

compound was synthesized from 1.6 g (3.9 mmol) of 4b according to

the procedure for the synthesis of 15. The obtained crude product was

puriﬁed by column chromatography (EtOAc:n-hexane = 1:4) to give

1

2.1 g (95%) of the titled compound as a white solid. H NMR (500

MHz, CDCl₃) δ [ppm]: 1.87 (s, 3H), 2.42−2.51 (m, 1H), 2.63−2.73

(m, 1H), 4.23 (t, J = 7.0 Hz, 1H), 4.33 (t, J = 7.0 Hz, 1H), 5.31 (br s,

2H), 5.99 (br s, 2H), 6.39 (d, J = 4.0 Hz, 1H), 6.89−6.93 (m, 2H),

7.02 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.04−7.12 (m, 2H), 7.18 (td; J =

7.5 Hz, J = 1.5 Hz, 1H), 7.28 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.30−

7.35 (m, 2H), 7.37 (d, 7.5 Hz, 1H), 7.44 (dd, 7.5 Hz, J = 1.5 Hz, 1H),

7.47 (d, J = 4.5 Hz, 1H).

10-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-carbonyl]-10,11-dihy-

dro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid (5b).

The compound was synthesized using 2.1 g (3.7 mmol) of 19

according to the procedure for the synthesis of 5g. The obtained crude

product was puriﬁed by column chromatography (EtOAc:n-hexane =

1

1:4) to give 1.6 g (92%) of 5b as a white solid; mp 184−190 °C. H

NMR (500 MHz, CDCl₃) δ [ppm]: 1.86 (s, 3H), 2.42−2.51 (m, 1H),

2.62−2.73 (m, 1H), 4.23 (t, J = 7.0 Hz, 1H), 4.33 (t, J = 7.0 Hz, 1H),

5.19 (br s, 2H), 5.94 (br s, 2H), 6.12 (d, J = 4.0 Hz, 1H), 6.77 (d, J =

4.0 Hz, 1H), 6.86 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 7.00−

7.06 (m, 3H), 7.14 (t, J = 7.0 Hz, 1H), 7.25−7.38 (m, 5H), 12.34 (br

s, 1H).

10-(2-Methyl-2′-triﬂuoromethylbiphenyl-4-carbonyl)-10,11-dihy-

dro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid (2-

Fluoroethyl)amide (6d). To a solution of 200 mg (0.41 mmol) of 5c

in 3.5 mL of THF, a mixture of 0.2 mL (0.98 mmol) Hunig’s base, 66

̈

mg (0.66 mmol) of 2-ﬂuoroethylamine hydrochloride, and 170 mg

(0.53 mmol) of TBTU was added. The mixture was stirred at rt

overnight and diluted with 5 mL of a 0.5 M aqueous NaHCO₃solution

followed by extraction with EtOAc. The combined organic layers were

dried over Na₂SO₄, ﬁltered, and concentrated in vacuum. Puriﬁcation

by column chromatography (n-hexane:EtOAc = 2:1) aﬀorded 176 mg

10-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-carbonyl]-10,11-dihy-

dro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid Bis-

(2-hydroxyethyl)amide (6b). To an ice-cooled solution of 100 mg

(0.21 mmol) of 5b and 0.05 mL (0.42 mmol) of N-methylmorpholine

in 2 mL of DMF, 91.4 mg of COMU (0.21 mmol) was added. After

stirring for 10 min, a solution of 0.02 mL (0.21 mmol) of

diethanolamine in 1 mL of DMF was added dropwise. The resultant

bright-yellow solution was stirred for additional 30 min before the

cooling bath was removed. The mixture was allowed to warm to rt

overnight. The complete consumption of the starting material was

conﬁrmed by TLC (CH₂Cl₂:methanol = 95:5), and the reaction

mixture was diluted with 5 mL of EtOAc. The organic layer was

subsequently washed with aqueous KHSO₄(5 wt %), aqueous

NaHCO₃(5 wt %), and brine and dried over Na₂SO₄. After ﬁltration

and removal of the solvent under reduced pressure the crude product

was puriﬁed by column chromatography (CH₂Cl₂:methanol = 97:3) to

give 73 mg (61%) of 6b as a white solid; mp 90−94 °C. ¹H NMR (500

MHz, DMSO-d₆) δ [ppm]: 1.86 (s, 3H), 2.43−2.52 (m, 1H), 2.62−

2.72 (m, 1H), 3.56 (br s, 8H), 4.23 (t, J = 6.5 Hz, 1H), 4.32 (t, J = 6.5

Hz, 1H), 4.83 (t, J = 5.0 Hz, 2H), 5.20 (br s, 2H), 5.34 (br s, 2H), 6.03

(d, J = 3.5 Hz, 1H), 6.30 (d, J = 3.5 Hz, 1H), 6.85−6.91 (m, 2H),

7.00−7.07 (m, 3H), 7.14 (t, J = 7.5 Hz, 1H), 7.25−7.29 (m, 2H), 7.32

(td, J = 1.5 Hz, J = 8.0 Hz, 1H), 7.37 (d, J = 7.5 Hz, 2H). ¹³C NMR

(125 MHz, DMSO-d₆) δ [ppm]: 19.32, 33.10 (d, J = 20 Hz), 45.83,

47.03, 58.55, 82.76 (d, J = 166 Hz), 107.50, 110.22, 125.11, 126.48,

126.97, 127.35, 127.55, 128.38, 128.51, 128.73, 128.91, 129.11, 129.60,

129.66, 134.05, 134.10, 134.77, 134.92, 140.26, 141.46, 141.62, 163.75,

169.23. LC/MS retention time: 8.61 min. MS (ESI) m/z = 556.2 [M +

H]⁺.

1

(81%) of 6d as a white solid; mp 197−200 °C. H NMR (500 MHz,

DMSO-d₆) δ [ppm]: 1.84 (s, 3H), 3.52 (dq, J = 30.0 Hz, J = 5.5 Hz,

2H), 4.52 (dt, J = 47.5 Hz, J = 5.0 Hz, 2H), 5.16 (br s, 2H), 5.94 (br s,

2H), 6.07 (d, J = 4.0 Hz, 1H), 6.72 (d, J = 4.0 Hz, 1H), 6.83 (d, J = 7.5

Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 6.98−7.03 (m, 2H), 7.12 (td, J = 7.5

Hz, J = 1.0 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.26 (s, 1H), 7.32 (dd, J

= 7.5 Hz, J = 1.0 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.69 (t, J = 7.5 Hz,

1H), 7.80 (d, J = 7.5 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ

[ppm]: 19.38, 45.89, 46.14 (br s), 64.79, 82.21 (d, J = 164.8 Hz),

108.45, 112.35, 123.74 (q, J = 272.4 Hz), 124.56, 125.75, 125.89 (q, J

= 4.9 Hz), 126.89 (q, J = 29.3 Hz), 126.92, 128.11, 128.23, 128.35,

129.29, 131.09, 131.15, 132.17, 135.07, 135.31, 135.37, 138.90 (q, J =

1.6 Hz), 139.73, 141.00, 161.49, 169.16. LC/MS retention time: 10.16

min. MS (ESI) m/z = 536.1 [M + H]⁺.

10-(2-Methyl-2′-triﬂuoromethylbiphenyl-4-carbonyl)-10,11-dihy-

dro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-carboxylic Acid (2-

Fluoroethyl)methyl Amide (6e). To a stirred solution of 164 mg (0.3

mmol) of 6d in 0.3 mL of DMF, 44 mg (1.8 mmol, 60 wt % in mineral

oil) of NaH was added followed by the addition of 0.11 mL (1.8

mmol) of methyl iodide. After stirring for 1 h at rt, the complete

consumption of the reaction was conﬁrmed by TLC analysis (n-

hexane:EtOAc = 3:2). The mixture was quenched with 1 mL of H₂O

followed by extraction with EtOAc. The combined organic layers were

dried over Na₂SO₄, ﬁltered, and concentrated in vacuum. The crude

product was puriﬁed by column chromatography (n-hexane:EtOAc =

3:1) to obtain 80 mg (47%) of 6e as a white solid; mp 165−168 °C.

¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 1.84 (s, 3H), 3.11 (br s,

Synthesis of 6c. 10-(2-Methyl-2′-triﬂuoromethylbiphenyl-4-car-

bonyl)-10,11-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-3-

carboxylic Acid (2-Fluoroethyl)-(2-hydroxyethyl)amide (6c). To a

solution of 50 mg (0.09 mmol) of 6 (synthesized according to

literature²⁶) in 0.4 mL of dry CH₂Cl₂, 0.01 mL (0.1 mmol) of DAST

was added. The mixture was stirred at rt overnight. TLC analysis

conﬁrmed the complete consumption of the starting material.

Puriﬁcation via column chromatography (CH₂Cl₂:methanol = 95:5)

provided 32 mg (64%) of 6c as a white solid; mp 82−85 °C. ¹H NMR

3H), 3.80 (dt, J = 26.5 Hz, J = 5.0 Hz, 2H), 4.65 (dt, J = 47.5 Hz, J =

5.0 Hz, 2H), 5.17 (br s, 2H), 5.41 (br s, 2H), 6.07 (d, J = 3.5 Hz, 1H),

6.32 (d, J = 3.5 Hz, 1H), 6.86−6.92 (m, 2H), 7.00−7.08 (m, 2H), 7.15

(t, J = 7.5 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.37 (d, J =

7.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 7.80 (d,

K

DOI: 10.1021/acs.jmedchem.5b01080

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

J = 8.0 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm]: 19.48,

45.88, 46.96, 80.87, 82.18, 107.72, 111.59, 120.46, 122.64, 124.51,

124.82, 125.87 (q, J = 5.0 Hz, 1C), 126.19, 126.54, 126.77, 126.99,

127.23, 128.11, 128.35, 128.45, 128.68, 129.10, 129.24, 129.66, 131.14,

132.17, 134.70, 135.08, 135.22, 138.87 (q), 139.78, 141.32, 163.28,

169.14. LC/MS retention time: 10.33 min. MS (ESI) m/z = 550.1 [M

+ H]⁺.

1.5 g (76%) of the titled product as a yellowish foam; mp 87−92 °C.

¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 2.04 (s, 3H), 2.61−2.72

(m, 1H), 2.77−2.88 (m, 1H), 4.88 (br s, 2H), 4.45 (dt, J = 47.5 Hz, J =

6.5 Hz, 2H), 4.46 (t, J = 5.5 Hz, 2H), 4.91 (br s, 2H), 6.34 (br s, 1H),

7.13 (dd, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 7.33

(td, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.37−7.41 (m, 2H), 7.44−7.51 (m,

3H).

[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-yl]-[3-(morpholino-4-car-

bonyl)-5H,11H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-10-yl]-

methanone (6f). A mixture of 150 mg (0.32 mmol) of 5b, 183 mg

(0.48 mmol) of 1-bis(dimethylamino)methylene-1H-1,2,3-triazolo-

[4,5-b]pyridinium-3-oxid hexaﬂuorophosphate, (HATU) 0.32 mL

2-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-carbonyl]-1,2,3,4-

tetrahydropyrrolo[1,2-a]pyrazine-6-carboxylic Acid (21; Intermedi-

ate of Reaction Step (f) in Scheme 2). According to the procedure for

the synthesis of 5g and using 1.2 g (2.4 mmol) of 20 in 50 mL of

acetone and 10 mL (2.5 M) of an aqueous NaOH solution, 900 mg

(91%) of the product was isolated as a white solid and used in the next

(1.9 mmol) of Hunig’s base, and 0.11 mL (1.28 mmol) of morpholine

̈

1

step without further puriﬁcation. H NMR (500 MHz, DMSO-d₆)) δ

dissolved in 15 mL of DMF was stirred at rt overnight. The mixture

was concentrated, and the residue was diluted with 7 mL of H₂O and

extracted three times with 7 mL of EtOAc. The organic layer was

washed twice with 5 mL of a saturated aqueous NaHCO₃solution,

dried over Na₂SO₄, ﬁltered, and concentrated to remove the solvent.

Column chromatography (n-hexane:EtOAc = 1:1) aﬀorded 158 mg

[ppm]: 2.03 (s, 3H), 2.60−2.71 (m, 1H), 2.76−2.87 (m, 1H), 3.83 (br

s, 2H), 4.41 (t, J = 7.0 Hz, 1H), 4.51 (t, J = 7.0 Hz, 1H), 4.53 (br s,

2H), 4.80 (br s, 2H), 6.05 (br s, 1H), 6.83 (br s, 1H), 7.13 (dd, J = 7.5

Hz, J = 1.5 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.33 (td, J = 7.5 Hz, 1.5

Hz, 1H), 7.35−7.40 (m, 2H), 7.44−7.46 (m, 2H), 12.07 (br s, 1H).

2-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-carbonyl]-1,2,3,4-

tetrahydropyrrolo[1,2-a]pyrazine-6-carboxylic Acid Bis(2-

hydroxyethyl)amide (12a). The compound was synthesized using

110 mg (0.28 mmol) of 21 and 52 mg (0.5 mmol) of diethanolamine

according to the procedure for the synthesis of 6b. The obtained crude

product was puriﬁed by column chromatography (CH₂Cl₂:methanol =

97:3) to yield 25 mg (18%) of 12a as a white foam; mp 72−75 °C. ¹H

NMR (500 MHz, DMSO-d₆)) δ [ppm]: 2.03 (s, 3H), 2.60−2.71 (m,

1H), 2.76−2.87 (m, 1H), 3.55 (br s, 8H), 3.70−4.00 (br s, 2H), 4.12

(t, J = 5.0 Hz, 2H), 4.46 (dt, J = 47.0 Hz, 6.5 Hz, 2H), 4.78 (br s, 4H),

5.96 (br s, 1H), 6.47 (br s, 1H), 7.13 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H),

7.19 (d, J = 7.5 Hz, 1H), 7.31−7.40 (m, 3H), 7.45 (br d, J = 7.5 Hz,

2H). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm]: 19.44, 30.57, 33.14

(d, J = 20.1 Hz), 37.85, 46.87, 54.79, 65.78, 66.11, 83.05 (d, J = 165.5

Hz), 103.34 (br s), 123.07, 123.66, 124.26, 124.30, 126.49, 127.63,

128.52, 128.63, 129.04, 129.35, 129.48, 134.36 (d, J = 6.0 Hz), 135.75,

140.42, 161.52, 162.38. LC/MS retention time: 7.87 min. MS (ESI)

m/z = 494.2 [M + H]⁺.

1

(92%) of 6f as a white solid; mp 190−193 °C. H NMR (500 MHz,

DMSO-d₆) δ [ppm]: 1.86 (s, 3H), 2.42−2.52 (m, 1H), 2.62−2.71 (m,

1H), 3.63 (br s, 8H), 4.28 (dt, J = 47.0 Hz, J = 6.5 Hz, 2H), 5.17 (br s,

2H), 5.43 (br s, 2H), 6.08 (d, J = 4.0 Hz, 1H), 6.28 (d, J = 4.0 Hz,

1H), 6.86 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 7.00−7.07 (m,

3H), 7.15 (td, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.25−7.29 (m, 2H), 7.32

(td, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.37 (d, J = 7.5 Hz, 1H), 7.42 (dd, J =

7.5 Hz, J = 1.0 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm]:

19.32, 33.1 (d, J = 20.4 Hz), 38.13, 45.84 (br s), 46.91, 66.14, 82.75 (d,

J = 165.7 Hz), 107.86, 111.65, 125.12, 125.64, 126.47, 127.00, 127.55,

128.42, 128.50, 128.71, 128.90, 129.16, 129.62, 129.65, 129.95, 134.06

(d, J = 6.5 Hz), 134.74, 134.80, 134.93, 140.25, 141.39, 141.64, 161.73,

169.23. LC/MS retention time: 9.81 min. MS (ESI) m/z = 538.2 [M +

H]⁺.

Synthesis of 12a and 12b. 2′-(2-Fluoroethyl)-2-methylbiphenyl-

4-carboxylic Acid (9). The compound was synthesized using 1.17 g

(4.3 mmol) of 3b according to the procedure for the synthesis of 4g.

After workup, 1.05 g (95%) of a white solid was obtained and used in

2-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-carbonyl]-1,2,3,4-

tetrahydropyrrolo[1,2-a]pyrazine-6-carboxylic Acid (2-

Hydroxyethyl)methyl Amide (12b). The compound was synthesized

using 110 mg (0.28 mmol) of 21 and 38 mg (0.5 mmol) of 2-

methylaminoethanol according to the procedure for the synthesis of

6b. The obtained crude product was puriﬁed by column chromatog-

raphy (CH₂Cl₂:methanol = 97:3) to yield 50 mg (39%) of 12b as a

1

the next step without any further puriﬁcation; mp 128−130 °C. H

NMR (500 MHz, DMSO-d₆) δ [ppm]: 2.04 (s, 3H), 2.56−2.66 (m,

1H), 2.74−2.84 (m, 1H), 4.37 (t, J = 6.5 Hz, 1H), 4.47 (t, J = 6.5 Hz,

1H), 7.10 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.32 (td, J =

7.5 Hz, J = 1.5 Hz, 1H), 7.38 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.44 (d, J

= 7.5 Hz, 1H), 7.82 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.90 (s, 1H).

(3,4-Dihydro-1H-pyrrolo[1,2-a]pyrazin-2-yl)-[2′-(2-ﬂuoroethyl)-2-

methylbiphenyl-4-yl]methanone (10). A solution of 1.6 g (6.2 mmol)

of 9 in 10 mL of thionyl chloride was reﬂuxed under argon atmosphere

for 1 h. The excess of thionyl chloride was removed under vacuum,

and the residue was treated three times with 5 mL of toluene followed

by removal of the solvent. The resultant acid chloride was dissolved in

10 mL of CH₂Cl₂and added dropwise to an ice-cold solution of 0.85 g

(7.0 mmol) of 1,2,3,4-tetrahydro-pyrrolo[1,2-a]pyrazine (synthesized

according to procedures described³¹) and 2.5 mL (13.6 mmol) of

1

white solid; mp 125−129 °C. H NMR (500 MHz, DMSO-d₆) δ

[ppm]: 2.03 (s, 3H), 2.60−2.71 (m, 1H), 2.76−2.87 (m, 1H), 3.06 (br

s, 3H), 3.53−3.58 (m, 4H), 3.78 (br s, 2H), 4.17 (t, J = 5.5 Hz, 2H),

4.46 (dt, J = 47.0 Hz, 6.5 Hz, 2H), 4.77 (br s, 3H), 5.97 (br s, 1H),

6.47 (br s, 1H), 7.13 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.19 (d, J = 7.5

Hz, 1H), 7.31−7.40 (m, 3H), 7.44 (d, J = 7.5 Hz, 2H). ¹³C NMR (125

MHz, DMSO-d₆) δ [ppm]: 19.45, 33.14 (d, J = 20.4 Hz), 44.05 (br s),

44.89 (br s), 54.79, 58.32, 83.05 (d, J = 165.5 Hz), 103.11, 112.23,

124.12, 124.25, 126.48, 127.62, 127.99, 128.50, 129.14, 129.34, 129.47,

134.36 (d, J = 6.0 Hz), 135.74, 140.42, 141.94, 162.90 (br s), 169.33

(br s). LC/MS retention time: 8.37 min. MS (ESI) m/z = 464.2 [M +

H]⁺.

Hunig’s base in 30 mL of CH Cl . The cooling bath was removed and

̈

2

the mixture stirred for 4 h at rt. After hydrolysis of the reaction with 50

mL of H₂O, the aqueous layer was extracted three times with CH₂Cl₂.

The combined organic layers were washed with an aqueous solution of

NaHCO₃, dried over Na₂SO₄, ﬁltered, and concentrated. The crude

product was puriﬁed by column chromatography (EtOAc:n-hexane =

Synthesis of 12c. 4-[2-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-

carbonyl]-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-6-carbonyl]-pi-

perazine-1-carboxylic Acid tert-Butyl Ester (22; Intermediate of

Reaction Step (g) in Scheme 2). The compound was synthesized

using 200 mg (0.5 mmol) of 21 and 170 mg (0.9 mmol) of 1-Boc-

piperazine according to the procedure for the synthesis of 6b. The

obtained crude product was puriﬁed by column chromatography (n-

hexane:EtOAc = 2:3) to yield 170 mg (59%) of the titled product as a

white solid; mp 95−98 °C. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]:

1.42 (s, 9H), 2.02 (s, 3H), 2.60−2.68 (m, 1H), 2.76−2.87 (m, 1H),

3.37 (br s, 4H), 3.61 (br s, 4H), 3.78 (br s, 2H), 4.17 (dd, J = 5.0 Hz, J

= 5.0 Hz, 2H) 4.41 (t, J = 6.5 Hz, 1H), 4.50 (t, J = 6.5 Hz, 1H), 4.78

(br s, 2H), 6.02 (br s, 1H), 6.42 (br s, 1H), 7.12 (dd, J = 7.5 Hz, J =

1.5 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.30−7.38 (m, 3H), 7.45 (d, J =

7.5 Hz, 2H).

1

1:9) to obtain 1.5 g (67%) of 10 as a white foam; mp 50−54 °C. H

NMR (500 MHz, CDCl₃) δ [ppm]: 2.08 (s, 3H), 2.68−2.78 (m, 1H),

2.84−2.91 (m, 1H), 3.90 (br s, 2H), 4.10 (br s, 2H), 4.43 (dt, J = 47.5

Hz, J = 6.5 Hz, 2H), 4.80 (br s, 2H), 5.90 (br s, 1H), 6.19−6.25 (m,

1H), 6.63 (t, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.17 (d, J = 8 Hz, 1H),

7.29−7.32 (m, 2H), 7.36−7.39 (m, 3H).

2,2,2-Trichloro-1-[2-[2′-(2-ﬂuoroethyl)-2-methylbiphenyl-4-car-

bonyl]-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-6-yl]ethanone (20;

Intermediate of Reaction Step (e) in Scheme 2). The compound

was synthesized using 1.4 g (3.9 mmol) of 10 according to the

procedure for the synthesis of 15. The obtained crude product was

puriﬁed by column chromatography (n-hexane:EtOAc = 2:1) to yield

L

DOI: 10.1021/acs.jmedchem.5b01080

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-yl]-[6-(piperazine-1-car-

bonyl)-3,4-dihydro-1H-pyrrolo[1,2-a]pyrazin-2-yl]methanone (12c).

To an ice-cooled solution of 170 mg (0.3 mmol) of 22 in 5 mL of

CH₂Cl₂, triﬂuoroacetic acid was added (0.6 mL, 0.8 mmol) under

argon atmosphere. After stirring at rt for 2 h, the mixture was diluted

with a saturated aqueous NaHCO₃solution. The aqueous layer was

extracted several times with CH₂Cl₂, the resultant organic layer washed

with brine and dried over Na₂SO₄. After ﬁltration, the solvent was

removed under reduced pressure and the residue was dried in vacuum

to give 600 mg (88%) of the titled compound. The obtained crude

product was used for the next step without further puriﬁcation. H

1

NMR (500 MHz, CDCl₃) δ [ppm]: 2.08 (s, 3H), 2.54 (br s, 4H),

2.67−2.78 (m, 1H), 2.80−2.90 (m, 1H), 3.94 (br s, 4H), 4.42 (dt, J =

47.0 Hz, J = 6.5 Hz, 2H), 7.09 (dd, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.18

(d, J = 7.5 Hz, 1H), 7.27−7.40 (m, 5H). ¹³C NMR (125 MHz,

CDCl₃) δ [ppm] 19.96, 33.82 (d, J = 20.6 Hz), 41.23 (br s), 46.51 (br

s), 83.31 (d, J = 168.5 Hz), 124.07, 126.69, 127.86, 128.71, 129.50,

129.70, 129.75, 134.19, 134.41 (d, J = 6.6 Hz), 136.79, 140.67, 142.99,

170.88, 206.62.

1

to give 120 mg (84%) of 12c as a white solid; mp 125−130 °C. H

NMR (500 MHz, DMSO-d₆) δ [ppm]: 2.03 (s, 3H), 2.60−2.67 (m,

1H), 2.69 (dd, J = 5.0 Hz, J = 5.0 Hz, 4H), 2.76−2.87 (m, 1H), 3.55

(dd, J = 5.0 Hz, J = 5.0 Hz, 4H), 3.78 (br s, 2H), 4.17 (dd, J = 5.0 Hz, J

= 5.0 Hz, 2H) 4.41 (t, J = 6.5 Hz, 1H), 4.50 (t, J = 6.5 Hz, 1H), 4.78

(br s, 2H), 5.99 (br s, 1H), 6.35 (br s, 1H), 7.13 (dd, J = 7.5 Hz, J =

1.5 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.31−7.39 (m, 3H), 7.44 (d, J =

7.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm]: 19.44, 33.42

(d, J = 20.4 Hz), 43.94 (brs), 44.89 (brs), 45.31 (brs), 45.46, 83.05 (d,

J = 165.1 Hz), 103.25 (brs), 112.11, 123.49, 124.27, 126.48, 127.62,

128.29 (brs), 128.53, 129.13, 129.35, 129.48, 134.36 (d, J = 6 Hz),

135.75, 140.41, 141.96, 161,41, 169.34 (brs). LC/MS retention time:

7.44 min. MS (ESI) m/z = 389.3 [M − C₄H₉N₂]⁺, 475.2 [M + H]⁺.

Synthesis of 12d. 4-([2-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-

carbonyl]-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-6-carbonyl]-

amino)piperidine-1-carboxylic Acid tert-Butyl Ester (23; Intermedi-

ate of Reaction Step (g) in Scheme 2). The compound was

synthesized using 200 mg (0.5 mmol) of 21 and 213 mg (0.9 mmol)

of 4-amino-1-Boc-piperidine according to the procedure for the

synthesis of 6b. The obtained crude product was puriﬁed by column

chromatography (n-hexane:EtOAc = 1:2) to yield 112 mg (38%) of

the titled product as a yellow solid. ¹H NMR (500 MHz, DMSO-d₆) δ

[ppm] 1.44 (s, 9H), 1.55−1.65 (m, 2H), 1.85−1.89 (m, 2H), 1.96 (s,

3H), 2.44−2.54 (m, 1H), 2.61−2.70 (m, 1H), 3.56 (br s, 4H), 3.71−

3.85 (br s, 2H), 3.81−3.90 (m, 1H), 4.38−4.41 (m, 3H), 4.50 (t, J =

6.5 Hz, 1H), 4.75 (br s, 2H), 6.03 (d, J = 3.5 Hz, 1H), 6.30 (d, J = 3.5

Hz, 1H), 7.14 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.18 (d, J = 7.5 Hz,

1H), 7.30−7.40 (m, 3H), 7.40−7.45 (m, 2H), 7.84 (d, J = 7.5 Hz,

1H). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm] 19.32, 33.10 (d, J =

20 Hz), 45.83, 47.03, 58.55, 82.76 (d, J = 166 Hz), 107.50, 110.22,

125.11, 126.48, 126.97, 127.35, 127.55, 128.38, 128.51, 128.73, 128.91,

129.11, 129.60, 129.66, 134.05, 134.10, 134.77, 134.92, 140.26, 141.46,

141.62, 163.75, 169.23.

2-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-carbonyl]-1,2,3,4-

tetrahydropyrrolo[1,2-a]pyrazine-6-carboxylic Acid Piperidin-4-yl

Amide (12d). To a stirred solution of 112 mg (0.2 mmol) of 23 in

5 mL of CH₂Cl₂, 0.5 mL (0.7 mmol) of triﬂuoroacetic acid was added

under argon atmosphere. After stirring for 2.5 h at rt, the complete

consumption of the reaction was conﬁrmed by TLC analysis

(CH₂Cl₂:methanol = 9:1) and the mixture was diluted with a

saturated aqueous NaHCO₃solution. The aqueous layer was extracted

several times with EtOAc, and the resultant organic layer washed with

brine and dried over Na₂SO₄. After ﬁltration, the solvent was removed

under reduced pressure and the residue treated with diethyl ether. The

precipitate was ﬁltered and dried in vacuum to give 70 mg (71%) of

12d as a yellow solid; mp 119−123 °C. ¹H NMR (500 MHz, DMSO-

d₆) δ [ppm]: 1.53−1.61 (m, 2H), 1.82−1.86 (m, 2H), 2.03 (s, 3H),

2.60−2.71 (m, 1H), 2.76−2.87 (m, 3H), 3.18 (d, J = 13.0 Hz, 2H),

3.70−3.93 (m, 3H), 4.39−4.43 (m, 3H), 4.51 (t, J = 6.5 Hz, 1H), 4.77

(br s, 2H), 5.98 (br s, 1H), 6.88 (br s, 1H), 7.13 (dd, J = 7.5 Hz, J =

1.5 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.31−7.40 (m, 3H), 7.42−7.46

(m, 2H), 7.85 (d, J = 7.5 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ

[ppm]: 19.47, 28.46, 33.17 (d, J = 20.3 Hz), 42.25, 43.61, 44.69 (br s),

45.47, 83.08 (d, 165.1 Hz), 103.65 (br s), 112.53, 124.18, 124.28,

126.51, 127.66, 128.53, 129.16, 129.38, 129.50. 134.38 (d, J = 5.8 Hz),

135.77, 140.44, 142.00, 160.49, 169.33. LC/MS retention time: 7.57

min. MS (ESI) m/z = 489.2 [M + H]⁺.

{4-[Bis(2-hydroxyethyl)amino]-piperidin-1-yl]-[2′-(2-ﬂuoroethyl)-

2-methylbiphenyl-4-yl]methanone (13a) and (2′-(2-Fluoroethyl)-2-

methyl-[1,1′-biphenyl]-4-yl)(4-hydroxypiperidin-1-yl)methanone

(13b). To a stirred solution of 370 mg (1.1 mmol) of 24 and 130 mg

(1.2 mmol) of diethanolamine in 4 mL dichloroethane, Na(OAc)₃BH

(340 mg, 1.6 mmol) and acetic acid (72 mg, 1.2 mmol) were added.

The mixture was stirred at rt overnight and diluted with 10 mL of a

saturated aqueous NaHCO₃solution. The aqueous layer was extracted

with CH₂Cl₂and the organic layer dried over Na₂SO₄. After ﬁltration

and removal of the solvent in vacuum, the crude product was puriﬁed

by column chromatography (CH₂Cl₂:methanol:triethyl amine =

90:10:1) to give both compounds as white solids in a ratio of 1:3;

30 mg (6%) of 13a (R_f= 0.3; CH₂Cl₂:methanol = 95:5) and 90 mg

(24%) of 13b (R_f= 0.7; CH₂Cl₂:methanol = 95:5).

1

13a: mp 55−60 °C. H NMR (500 MHz, DMSO-d₆) δ [ppm]

1.35−1.45 (m, 2H), 1.65−1.85 (m, 2H), 2.01 (s, 3H), 2.57−2.69 (m,

5H), 2.75−2.86 (m, 3H), 3.05 (br s, 2H), 3.40 (br s, 4H), 3.72 (br s,

1H), 4.36 (br s, 1H), 4.45 (dt, 47.5 Hz, J = 6.5 Hz, 2H), 4.55 (br s,

1H), 7.12 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H),

7.25 (d, J = 7.5 Hz, 1H), 7.31 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.33 (s,

1H), 7.37 (td, J = 7.5, J = 1.5 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H). ¹³C

NMR (125 MHz, DMSO-d₆) δ [ppm]: 19.46, 33.13 (d, J = 20 Hz),

40.97 (br s), 46.55 (br s), 52.98, 54.79, 59.10, 59.50 (br s), 83.04 (d, J

= 165 Hz), 123.84, 126.47, 127.57, 128.13, 129.17 (d, J = 4.9 Hz),

129.44, 134.31, 134.36, 135.27, 135.55, 140.50, 141.34, 168.66. LC/

MS retention time: 6.94 min. MS (ESI) m/z = 429.2 [M + H]⁺.

1

13b: mp 132−134 °C. H NMR (500 MHz, DMSO-d₆) δ [ppm]

1.35−1.45 (m, 2H), 1.65−1.85 (m, 2H), 2.01 (s, 3H), 2.57−2.69 (m,

1H), 2.75−2.85 (m, 1H), 3.21 (br s, 2H), 3.57 (br s, 1H), 3.72−3.78

(m, 1H), 4.03 (br s, 1H), 4.45 (dt, J = 47.0 Hz, J = 6.5 Hz, 2H), 4.79

(d, J = 4.0 Hz, 1H), 7.11 (dd, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.14 (d, J =

7.5 Hz, 1H), 7.23 (dd, J = 7.5 Hz, J = 1.0 Hz, 1H), 7.29−7.33 (m, 2H),

7.36 (td, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H). ¹³C

NMR (125 MHz, DMSO-d₆) δ [ppm] 19.44, 33.13 (d, J = 20.3 Hz),

33.75 (br s), 34.41 (br s), 44.56 (br s), 65.41, 83.03 (d, J = 165.1 Hz),

123.77, 126.46, 127.56, 128.06, 129.19, 129.42, 134.34 (d, J = 6.4 Hz),

135.38, 135.54, 140.50, 141.29, 168.67. LC/MS retention time: 7.96

min. MS (ESI) m/z = 342.2 [M + H]⁺.

Synthesis of 14. 4-[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-car-

bonyl]-piperazine-1-carboxylic Acid tert-Butyl Ester (25; Intermedi-

ate of Reaction Step (a) in Scheme 2). The compound was

synthesized using 258 mg (1.0 mmol) of 9 and 230 mg (1.2 mmol) of

N-Boc-piperazine according to the procedure for the synthesis of 6b.

The obtained crude product was puriﬁed by column chromatography

(n-hexane:EtOAc = 3:2) to yield 414 mg (97%) of the titled product

1

as a white solid; mp 55−58 °C. H NMR (500 MHz, DMSO-d₆) δ

[ppm]: 1.42 (s, 9H), 2.01 (s, 3H), 2.59−2.69 (m, 1H), 2.75−2.86 (m,

1H), 3.40 (br s, 6H), 3.57 (br s, 2H), 4.45 (dt, J = 47.0 Hz, J = 6.5 Hz,

2H), 7.10 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H),

7.28 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.32 (td, J = 7.5 Hz, J = 1.5 Hz,

1H), 7.35−7.38 (m, 2H), 7.44 (d, J = 7.5 Hz, 1H).

[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-yl]-piperazin-1-yl-metha-

none (11). To a stirred solution of 900 mg (2.1 mmol) of 25 in 50 mL

of CH₂Cl₂under argon atmosphere, 5 mL (65 mmol) of triﬂuoroacetic

acid was added After stirring for 2.5 h at rt, the complete consumption

of 25 was conﬁrmed by TLC analysis (n-hexane:EtOAc = 1:1) and the

mixture was diluted with a saturated aqueous NaHCO₃solution. The

aqueous layer was extracted several times with EtOAc, and the

resultant organic layer washed with brine and dried over Na₂SO₄. After

ﬁltration, the solvent was removed under reduced pressure and the

Synthesis of 13a and 13b. 1-[2′-(2-Fluoroethyl)-2-methylbiphen-

yl-4-carbonyl]-piperidin-4-one (24; Intermediate of Reaction Step

(a) in Scheme 2). The compound was synthesized using 500 mg (2.0

mmol) of 9 and 338 mg (2.2 mmol) of 4-piperidone monohydrate

hydrogen chloride according to the procedure for the synthesis of 6b

M

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Journal of Medicinal Chemistry

Article

residue was treated with diethyl ether. The precipitate was ﬁltered and

dried in vacuum to give 700 mg (99%) of 11 as a white solid. ¹H NMR

(500 MHz, DMSO-d₆) δ [ppm]: 2.01 (s, 3H), 2.58−2.69 (m, 1H),

2.75−2.85 (m, 4H), 3.20−3.70 (m, 6H), 4.45 (dt, J = 47.0 Hz, J = 6.5

Hz, 2H), 7.10 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.15 (d, J = 7.5 Hz,

1H), 7.23−7.27 (m, 1H), 7.30−7.38 (m, 3H), 7.44 (d, J = 7.5 Hz,

1H).

μg/mL) together with a mixture of toluene (0.5 mL) and triphenylene

(5 mg) as internal standards in methanol (50 mL) to a short polymeric

ODP-50 column (20 mm × 4 mm, 5 μm, Shodex, Showa Denko

Europe GmbH, Munich, Germany) using a linear gradient from 10%

methanol and 90% 10 mM sodium phosphate buﬀer to 100%

methanol within 9.4 min at a ﬂow rate of 1.5 mL/min and a pH of 7.4.

Permeability (P_m) measurements were performed according to

Taillardat-Bertschinger et al. using immobilized artiﬁcial membrane

(IAM) chromatography.^34,42For IAM measurements, the same HPLC

system and an IAM.PC.DD2 (150 mm × 4.6 mm, Regis Technologies

Inc., Morton Grove, USA) column were used isocratically with 10 mM

phosphate buﬀer and acetonitrile in diﬀerent ratios (50/50, 55/45, 60/

40, and 65/35) adjusted to pH 7.0 and 1 mL/min ﬂow rate. P_mvalues

are obtained by dividing membrane partition coeﬃcient K_mby the

molecular weight of the tracer candidates. K_mwas calculated on the

basis of the capacity factors k_IAMand correction of the column

conditions (total volume of solvent within column (V_m) and volume of

interphase (V_s)), at which the k_IAMfactors are calculated and

extrapolated to 100% aqueous phase.

[2′-(2-Fluoroethyl)-2-methylbiphenyl-4-yl]-[4-(2-hydroxyethyl)-

piperazin-1-yl]methanone (14). To a stirred solution of 300 mg (0.9

mmol) of 11 in 7.5 mL of acetonitrile, 1.2 g (8.7 mmol) potassium

carbonate and 0.1 mL (1.5 mmol) of 2-bromoethanol were added.

After heating under reﬂux overnight, the consumption of the reactants

was observed by TLC analysis (CH₂Cl₂:methanol = 9:1) and

additional 0.05 mL (0.75 mmol) of 2-bromoethanol were added.

After further reﬂuxing for 4 h, the solvent was removed in vacuum and

the residue was diluted with EtOAc. The resultant organic layer was

washed with H₂O, dried over Na₂SO₄, ﬁltered, and concentrated in

vacuum. The crude product was puriﬁed by column chromatography

(CH₂Cl₂:methanol = 95:5) to yield 200 mg (59%) of 14 as a white

foam; mp 55−60 °C. ¹H NMR (500 MHz, DMSO-d₆) δ [ppm]: 2.01

(s, 3H), 2.43 (t, J = 6.0 Hz, 1H), 2.44 (br s, 5H), 2.58−2.69 (m, 1H),

2.75−2.85 (m, 1H), 3.40 (br s, 2H), 3.53 (q, J = 6.0 Hz, 2H), 3.62 (br

s, 2H), 4.38−4.43 (m, 2H), 4.50 (t, J = 6.5 Hz, 1H), 7.11 (dd, J = 7.5

Hz, J = 1.0 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H), 7.24 (dd, 7.5 Hz, J = 1.5

Hz, 1H), 7.30−7.34 (m, 2H), 7.36 (td, J = 7.5 Hz, J = 1.5 Hz, 1H),

7.44 (d, J = 7.5 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ [ppm]:

19.59, 33.17 (d, J = 20.3 Hz), 47.04, 52.99, 58.21, 59.81, 82.72 (d, J =

164.0 Hz), 123.42, 125.83, 126.94, 127.66, 128.55, 128.55, 128.78,

133.65 (d, J = 6.3 Hz), 134.26, 134.89, 139.73, 140.70, 167.71. LC/MS

retention time: 6.72 min. MS (ESI) m/z = 371.2 [M + H]⁺.

4.2. In Vitro Binding Studies. The studies were performed using

membrane homogenates prepared from CHO cells stably transfected

with human oxytocin receptor gene (hOTR-CHO; obtained from Bice

Chini, Instituto di Neuroscienze, Consiglio Nazionale delle Ricerche,

Milano, Italy). For the competitive binding studies, membrane

preparation was incubated with [³H]oxytocin (K_D= 2.56 nM

0.95 nM; [tyrosyl-2,6-³H],1676 GBq/mmol; PerkinElmer LAS GmbH,

Rodgau, Germany) at 1 nM and seven concentrations of test

compounds (10 pM to 10 μM) in buﬀer consisting of 50 mM

TRIS-HCl, pH 7.4, 5 mM MgCl₂, and 0.1% BSA for 60 min at 22 °C.

The binding was terminated by rapid ﬁltration (48-well semi-

automated cell harvester; Brandel, Gaithersburg, USA) using GF/B

glass ﬁber ﬁlter presoaked in 0.3% PEI for 60 min at rt. Radioactivity

trapped on the ﬁlters was counted by liquid scintillation counting

(Beckman LSC 6500; Beckman Coulter GmbH, Krefeld, Germany).

Nonspeciﬁc binding was deﬁned as binding remaining in the presence

of 10 μM oxytocin. The assays were performed two times, each in

duplicate. Experimental data were analyzed by nonlinear regression,

and IC₅₀curves generated by a one-site competition model. The K_i

values of the test compounds were calculated using the Cheng−Prusoﬀ

equation.

4.4. Determination of Plasma-Free Fraction. Binding of [¹⁸F]

6b to plasma proteins was estimated by ultraﬁltration. First, 1 mL of

plasma obtained from pig was spiked with 60 μL of saline containing

10 MBq [¹⁸F]6b and incubated for 30 min at 37 °C. The separation of

protein-bound and free [¹⁸F]6b was achieved by ﬁltration through

anisotropic hydrophilic Ultracel YM membrane (Amicon Centrifree,

30000 MW cutoﬀ; MerckMilliporeAmicon Inc., USA) as speciﬁed by

the manufacturer (2000g, 15 min, 21 °C). The radioactivity

concentrations in the plasma obtained before centrifugation (C) and

of the ultraﬁltrate (C_u) were estimated by gamma counting of

respective sample aliquots (Wallac 1470 Wizard 3″; PerkinElmer LAS

GmbH, Rodgau, Germany). The value of plasma free fraction f _Pwas

calculated according to the relation f _P(%) = C_u/C × 100.

4.5. Radiochemistry. No-carrier-added [¹⁸F]ﬂuoride was pro-

duced via the [¹⁸O(p,n)¹⁸F] nuclear reaction by irradiation of an

[¹⁸O]H₂O target (Hyox 18 enriched water, Rotem Industries Ltd.,

Israel) on a Cyclone 18/9 (iba RadioPharma Solutions, Belgium) with

ﬁxed energy proton beam using a Nirta [¹⁸F]ﬂuoride XL target.

Microwave assisted radioﬂuorination was performed in a standard 4

mL V vial using a Discover PETWave Microwave (CEM, NC, USA).

Radio thin-layer chromatography (radio-TLC) was performed on

silica gel (Polygram SIL G/UV₂₅₄) and aluminum oxide (Polygram

Alox N/UV₂₅₄) precoated plates with EtOAc (EE)/PE (PE) 5/1 (for

labeling product) and EE/EtOH 10/1 (for [¹⁸F]2), respectively. The

plates were exposed to storage phosphor screens (BAS-TR2025,

FUJIFILM Co., Tokyo, Japan) and recorded using a bioimaging

analyzer system (BAS-1800 II, FUJIFILM). Images were evaluated

with the BASReader and AIDA 2.31 software (raytest Isotopenmessg-

erate GmbH, Straubenhardt, Germany).

̈

Analytical chromatographic separations were performed on a

JASCO LC-2000 system, incorporating a PU-2080Plus pump, AS-

2055Plus auto injector (100 μL sample loop), and a UV-2070Plus

detector coupled with a gamma radioactivity HPLC detector (Gabi

The equilibrium dissociation constant K_Dof 6b was determined

performing a homologous binding experiment. [¹⁸F]6b (speciﬁc

activity: 160 GBq/μmol) at a radioactivity concentration of 57 kBq/

mL (identical to a chemical concentration of 0.35 nM) was

coincubated with various dilutions of 6b (ﬁnal concentration 0.1

nM−100 μM) with membranes obtained from hOTR-CHO cells in

buﬀer consisting of 50 mM TRIS-HCl, pH 7.4, 5 mM MgCl₂, and

0.1% BSA for 60 min at 22 °C. The binding was terminated by rapid

ﬁltration (48-well semiautomated cell harvester; Brandel, Gaithers-

burg, USA) using GF/B glass ﬁber ﬁlter presoaked in 0.3% PEI for 60

min at rt. Radioactivity trapped on the ﬁlters was counted by gamma-

counting (Wallac 1470 Wizard 3″; PerkinElmer LAS GmbH, Rodgau,

Germany). CPM data were analyzed by nonlinear regression, and an

IC₅₀curve generated by a one-site competition model. The K_Dvalue of

6b was calculated according to K_D= IC₅₀− [radioligand], valid for the

one-site homologous competition binding model.

Star, raytest Isotopenmessgerate GmbH). Data analysis was performed

̈

with the Galaxie chromatography software (Agilent Technologies)

using the chromatograms obtained at 254 and 272 nm, respectively.

Semipreparative HPLC separations were performed on a JASCO

LC-2000 system, incorporating a PU-2080-20 pump, a UV/vis-2075

detector coupled with a gamma radioactivity HPLC detector whose

measurement geometry was slightly modiﬁed (Gabi Star, raytest

Isotopenmessgerate GmbH), and a fraction collector (Advantec CHF-

̈

122SC). Data analysis was performed with the Galaxie chromatog-

raphy software (Agilent Technologies) using the chromatograms

obtained at 254 nm.

The ammonium acetate (NH₄OAc) concentration stated as 20 mM

NH₄OAc aq corresponds to the concentration in the aqueous

component of an eluent mixture.

Radiosyntheses. No-carrier-added [¹⁸F]ﬂuoride in 2 mL of H₂O

4.3. Determination of log D and P_mValues. The log D values

were determined by HPLC (Agilent 1100/1200 series) according to

Donovan and Pescatore.³³The compounds were injected (2 μL, 150

was trapped on a Chromaﬁx 30 PS-HCO₃⁻cartridge (Macherey-Nagel

GmbH & Co. KG, Duren, Germany). The activity was eluted with 300

̈

μL of an aqueous solution of potassium carbonate (K₂CO₃, 1.8 mg, 13

N

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Journal of Medicinal Chemistry

Article

μmol) into a 4 mL V vial and Kryptoﬁx 2.2.2 (K_2.2.2, 11 mg, 29 μmol)

in 1 mL of ACN was added. The aqueous [¹⁸F]ﬂuoride was

azeotropically dried under vacuum and nitrogen ﬂow within 7−10

min using a single mode microwave (75 W, at 50−60 °C, power

cycling mode). Two aliquots of ACN (2 × 1.0 mL) were added during

the drying procedure, and the ﬁnal complex was dissolved in 1000 μL

of ACN ready for labeling. The reactivity of the anhydrous K[¹⁸F]F-

K_2.2.2-carbonate complex as well as the reproducibility of the drying

procedure were checked via the standard reaction with 2 mg (5.4

μmol) of ethylene glycol ditosylate (Sigma-Aldrich, Germany) at 80

°C for 10 min in ACN. Thereafter, a solution of 2.0−2.5 mg of

precursor in 500 μL of ACN was added and the ¹⁸F-labeling was

performed under thermal heating (90 °C, 15 min) or microwave-

assisted irradiation (75W, 85−95 °C, 9 min, power cycling mode). To

analyze the reaction mixture and to determine labeling yields, samples

were taken for radio-HPLC and radio-TLC. Moreover, the stability of

the tosylate precursor was investigated under both heating conditions

used by HPLC analysis at diﬀerent time points of the reaction.

After cooling to <30 °C, hydrochloric acid was added and the

deprotection was performed by thermal heating (90 °C, 15 min, 1 mL

2.0 M HCl) or microwave irradiation (50W, 75−85 °C, 5 min, power

cycling mode, 1 mL 2.0 M HCl). Thereafter, the reaction mixture was

neutralized with aqueous 6.0 M NaOH and directly applied to an

isocratic semipreparative RP-HPLC for isolation of [¹⁸F]6b (44%

ACN/20 mM NH₄OAc_aq, 4 mL/min, Reprosil-Pur C18-AQ, 250 mm

× 10 mm; 5 μm; Dr. Maisch HPLC GmbH, Germany). The collected

radiotracer fraction was diluted with 40 mL of H₂O to perform ﬁnal

puriﬁcation by sorption on a Sep-Pak C18 light cartridge (Waters,

Milford, MA, USA) and successive elution with 0.75 mL of ethanol.

The solvent was reduced under a gentle argon stream and the desired

radiotracer formulated in sterile isotonic saline containing 10% EtOH

(v/v). The identity and radiochemical purity of [¹⁸F]6b was conﬁrmed

by radio-HPLC (gradient and isocratic mode) and radio-TLC (Alox

N/UV₂₅₄, EE/EtOH 10/1). For radio-HPLC, a Reprosil-Pur C18-AQ

column (250 mm × 4.6 mm; 5 μm; Dr. Maisch HPLC GmbH;

Germany) with ACN/20 mM NH₄OAc aq as eluent mixture and a

ﬂow of 1.0 mL/min was used (gradient: eluent A 10% ACN/20 mM

NH₄OAc aq; eluent B 90% ACN/20 mM NH₄OAc aq; 0−5 min 100%

A, 5−10 min up to 55% B, 10−25 min 55% B, 25−30 up to 100% B,

30−40 min 100% B, 40−45 min up to 100% A, and 45−55 min 100%

A; isocratic, 42% ACN/20 mM NH₄OAc_aq). Speciﬁc activity was

determined on the base of a calibration curve carried out under

isocratic HPLC conditions (42% ACN/20 mM NH₄OAc_aq) using

chromatograms obtained at 272 nm as an appropriate maximum of UV

absorbance.

10000 rpm. The precipitates were washed with 200 μL of solvent

mixture and subjected to the same procedure. The combined

supernatants (total volume between 1.0−1.5 mL) were concentrated

at 65 °C under argon ﬂow to a ﬁnal volume of approximately 100 μL

and analyzed by radio-TLC and analytical radio-HPLC. To determine

the percentage of radioactivity in the supernatants compared to total

activity, aliquots of each step as well as the precipitates were quantiﬁed

by gamma counting.

4.6. In Vitro Autoradiographic Studies. Brain sections (20 μm)

of ﬂash-frozen brains of female domestic pigs (Sus s. domestica, 6

weeks, 12−14 kg) were cut using a cryostat, thaw-mounted onto

microscope slides, and after air-drying stored at −80 °C until use.

Brieﬂy, the brain sections were allowed to thaw in air and rinsed twice

in 50 mM Tris buﬀer, pH 7.4, to remove endogenous ligand. The

sections were then incubated with the 6 nM [³H]oxytocin [tyrosyl-

2,6-³H], 1676 GBq/mmol; PerkinElmer LAS GmbH, Rodgau,

Germany) or ∼10 nM [¹⁸F]6b in TRIS buﬀer (50 mM TRIS-HCl,

5 mM NaCl, 0.1% BSA pH 7.4) for 60 min at room temperature.

Nonspeciﬁc binding was determined in the presence of 10 or 0.1 μM

oxytocin, respectively. Displacement of [¹⁸F]6b was also evaluated

with the following ligands: 1 μM ^L-quisqualic acid (glutamate receptor

ligand), 0.35 μM THIP (GABA_Areceptor ligand), 0.2 μM siramesin

(σ1/2 receptor ligand), 0.08 nM SR49059 (V_1Areceptor ligand), or 3

nM tolvaptan (V_1/2receptor ligand). Subsequently, the sections were

washed twice for 2 min in ice-cold TRIS buﬀer and rinsed for 5 s in

ice-cold distilled H₂O. The sections were rapidly dried in a stream of

cold air before being exposed to a tritium-sensitive imaging plate.

Developed autoradiographs were analyzed in a phosphor imager (Fuji

BAS 1800 II). The quantiﬁcation was performed by using 2D-

densitometric analysis (AIDA 2.31 software; raytest Isotopenmessg-

erate GmbH, Germany).

̈

4.7. In Vivo Studies in Mice. Animals for in vivo studies were

obtained from the Medizinisch-Experimentelles Zentrum, Universitat

̈

Leipzig. All procedures that include animals were approved by the

respective State Animal Care and Use committee and conducted in

accordance with the German Law for the Protection of Animals (TVV

08/13). For all in vivo studies in mice, female CD-1 mice, 10−12

weeks old, 20−25 g, were used.

Biodistribution of [¹⁸F]6b in Mice. Mice received an injection of

approximately 200 kBq [¹⁸F]6b with a speciﬁc activity of 70 GBq/

μmol in 200 μL of 0.9% saline into the tail vein. The animals were

anesthetized for blood and urine sampling and euthanized by luxation

of the cervical spine at 5 and 30 min pi (n = 2 per each time point).

The organs of interest were removed and weighed, and the

radioactivity was measured by gamma counting (Wallac 1470 Wizard

3″; PerkinElmer LAS GmbH, Rodgau, Germany). The percentage of

injected dose per gram of wet tissue (% ID/g wet weight) was

calculated.

In Vitro Stability and Partition Coeﬃcient. The in vitro stability of

[¹⁸F]6b was investigated by incubation of small tracer amounts (10−

15 MBq) at 40 °C in 1 mL of following solutions: (i) 0.9% aq NaCl,

(ii) PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄; pH = 7.4),

and (iii) pig plasma samples. At various time points, aliquots were

analyzed by radio-TLC and radio-HPLC.

PET/MR Studies of [¹⁸F]6b in Mice. Mouse small animal PET

acquisitions were obtained on a preclinical PET/MR system

(nanoScan PET/MR, Mediso Medical Imaging Systems, Budapest,

Hungary). Anesthesia was induced by exposing mice to 4% isoﬂurane

in air and then maintained by reducing the ratio to 1.5% for the

duration of the studies. The animal was placed in the PET scanner

followed by a 15 min MR scan. The subsequent 60 min PET scan was

started with the beginning of the intravenous injection of [¹⁸F]6b

(mean = 11.8 MBq; 10.4−13.1 MBq), and radioactivity concentration

was measured in sequential frames of 5 min duration. Activity volumes

were reconstructed with iterative reconstruction (OSEM, four

iterations, six subsets) including an MR-based attenuation and scatter

correction, achieving a reconstructed spatial resolution of 1.5 mm.⁴⁵

Additional experiments were similarly performed in mice preinjected

with cyclosporin (50 mg/kg iv, 60 min prior to radioligand

administration).

The partition coeﬃcient of [¹⁸F]6b was experimentally determined

for the n-octanol/PBS system by the shake-ﬂask method. Aliquots of

the formulated radiotracer product were diluted in the buﬀer (20−50

μL, 1:1000) and added to a mixture of 3.0 mL of pre-equilibrated n-

octanol and 3.0 mL of PBS. After shaking for 20 min at room

temperature, the samples were centrifuged (5000g, 5 min), and

duplicates of samples (0.5 mL each) of the organic as well as the

aqueous layers were measured in a gamma counter. Another duplicate

of samples (1 mL each) of the organic layer was subjected to the same

procedure until constant partition coeﬃcient values had been

obtained. All measurements were done in triplicate.

Radiotracer Metabolism of [¹⁸F]6b in Mice. Blood samples of

mouse were taken at 30 min after intravenous injection of 84 MBq of

[¹⁸F]6b (n = 2). Pig blood samples were taken at 2, 8, 30, and 60 min

after intravenous injection of 250 MBq of [¹⁸F]6b. Plasma was

obtained by centrifugation of blood at 12000 rpm at 4 °C for 10 min.

Protein precipitation was performed by addition of ice-cold ACN in a

ratio of 4:1 of organic solvent to plasma. The samples were vortexed

for 2 min, equilibrated on ice for 15 min, and centrifuged for 5 min at

4.8. PET Study in Pig. Animal procedure was approved by the

Animal Care and Use Committee of Saxony (TVV 08/13). One piglet

(6 weeks old) was used in this study. Anaesthesia and surgery of the

animal was performed as described previously.⁵⁶In brief, the animal

was premedicated with midazolam (1 mg·kg⁻¹im) followed by

induction of anesthesia with 3% isoﬂurane in 70% N₂O/30% O₂. All

O

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

incision sites were inﬁltrated with 1% lidocaine, and anesthesia was

maintained throughout the surgical procedure with 1.5% isoﬂurane. A

central venous catheter was introduced through the left external

jugular vein and used for the administration of the radiotracer and

drugs and for volume substitution with heparinized (50 IE·mL⁻¹)

lactated Ringer’s solution (2 mL·kg⁻¹·h⁻¹). An endotracheal tube was

inserted by tracheotomy for artiﬁcial ventilation (Servo Ventilator

900C, Siemens-Elema, Sweden) after immobilization with pancuro-

nium bromide (0.2 mg·kg⁻¹·h⁻¹). The artiﬁcial ventilation was

adjusted to maintain normoxia and normocapnia (Radiometer ABL

500, Copenhagen, Denmark). Polyurethane catheters (Ø 0.5 mm)

were advanced through the left and the right femoral arteries into the

abdominal aorta to withdraw arterial blood samples for regular

monitoring of blood gases and for radiotracer input function

measurements. Body temperature was monitored by a rectal

temperature probe and maintained at ∼38 °C by a heating pad.

After completion of surgery, anesthesia was maintained with 0.5%

isoﬂurane in 70% N₂O/30% O₂, and the animal was allowed to

stabilize for 1 h before PET imaging.

PET Scanning Protocol. PET imaging was performed according to

the protocol described recently.⁵⁷In brief, animals were scanned

position prone, with the head held in the aperture of a clinical

tomograph (ECAT EXACT HR+, CTI/Siemens) using a custom-

made head holder. For attenuation and scatter correction, transmission

scans were acquired using three rotating ⁶⁸Ge rod sources. [¹⁸F]6b

(225 MBq; A_s, 54 GBq/μmol) was applied in 10 mL saline as a 2 min

iv infusion using a syringe pump, followed by ﬂushing with 10 mL of

heparinized saline (50 IE·mL⁻¹). The emission recording started upon

initiation of the injection, and dynamic emission data were acquired

for a total of 120 min. Arterial blood was sampled continuously using a

peristaltic pump during the ﬁrst 20 min of the recording, followed by

manual sampling at 25, 30, 40, 50, 60, 90, and 120 min after injection.

After centrifugation, the plasma radioactivity concentration was

measured using a gamma counter (1470 Wizard, PerkinElmer,

Shelton, CT, USA) cross calibrated to the PET scanner. Additionally,

arterial blood was sampled manually at 4, 16, 30, and 60 min pi and

plasma obtained as described above to determine the fractions of

nonmetabolized [¹⁸F]6b (see below).

Neuroscienze, Milano, Italy, for providing the hOTR-CHO

cell line.

ABBREVIATIONS USED

ACN, acetonitrile; EtOAc, ethyl acetate; PE, petroleum ether;

Hunig’s base, N,N-diiosopropylamine; Steglich base, 4-

■

̈

dimethylaminopyridine; COMU, (1-cyano-2-ethoxy-2-

oxoethylidenaminooxy)dimethylamino-morpholino-carbenium

hexaﬂuorophosphate; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium tetraﬂuoroborate; HATU, 1-bis-

(dimethylamino)methylene-1H-1,2,3-triazolo[4,5-b]-

pyridinium-3-oxid hexaﬂuorophosphate; EDCI, 1-ethyl-3-(3-

dimethylamino-propyl) carbodiimide hydrochloride carbodii-

mide hydrochloride; HOBt, 1-hydroxybenzotriazole; DAST,

diethylaminosulfur triﬂuoride; TAC, time−activity curve; VOI,

volume of interest; SUV, standard uptake value; V_T,

distribution volume; BP, binding potential; PSA, polar surface

area; TPSA, topological polar surface area

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scatter, decay, and scanner-speciﬁc dead time, images were

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AUTHOR INFORMATION

■

Corresponding Author

*E-mail: b.wenzel@hzdr.de. Phone: +49 341 2341794637.

Author Contributions

^#B.W. and J.M. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The project was funded by Europaischer Fonds fur regionale

̈

Entwicklung (EFRE). We thank Dr. Karsten Franke for

providing [¹⁸F]ﬂuoride. We thank Dr. Achim Hiller, Juliane

Schaller, and Tina Spalholz for their assistance in the lab. We

are very grateful to Bice Chini from the Instituto di

P

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Development of a Novel Nonpeptidic 18F-Labeled Radiotracer for in Vivo Imaging of Oxytocin Receptors with Positron Emission Tomography

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