Structure-Affinity Relationships of 2,3,4,5-Tetrahydro-1 H-3-benzazepine and 6,7,8,9-Tetrahydro-5 H-benzo[7]annulen-7-amine Analogues and the Discovery of a Radiofluorinated 2,3,4,5-Tetrahydro-1 H-3-benzazepine Congener for Imaging GluN2B Subunit-Containi

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Structure−Aﬃnity Relationships of 2,3,4,5-Tetrahydro‑1H‑3-

benzazepine and 6,7,8,9-Tetrahydro‑5H‑benzo[7]annulen-7-amine

Analogues and the Discovery of a Radioﬂuorinated 2,3,4,5-

Tetrahydro‑1H‑3-benzazepine Congener for Imaging GluN2B

Subunit-Containing N‑Methyl‑^D‑aspartate Receptors

†

Hazem Ahmed,^†,∥Ahme_†d Haider,^†,∥Jasmine Varisco, Maja Stankovic, Rahel Wallimann,

́

†

†,‡

̈

Stefan Gruber, Irina Iten, Surya Hane, Adrienne Muller Herde, Claudia Keller, Roger Schibli,

Dirk Schepmann,^§Linjing Mu,^†,‡Bernhard Wunsch,^§and Simon M. Ametamey*^,†

̈

^†Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland

^‡Department of Nuclear Medicine, University Hospital Zurich, 8091 Zurich, Switzerland

^§Institute of Pharmaceutical and Medicinal Chemistry, University of Mu

̈ ̈

nster, Corrensstr. 48, 48149 Munster, Germany

S

* Supporting Information

ABSTRACT: Aspiring to develop a positron emission tomography

(PET) imaging agent for the GluN2B subunits of the N-methyl-^D-

aspartate receptor (NMDAR), a key therapeutic target for drug

development toward several neurological disorders, we synthesized a

series of 2,3,4,5-tetrahydro-1H-3-benzazepine and 6,7,8,9-tetrahydro-

5H-benzo[7]annulen-7-amine analogues. After in vitro testing via

competition binding assay and autoradiography, [¹⁸F]PF-NB1

emerged as the best performing tracer with respect to speciﬁcity

and selectivity over σ1 and σ2 receptors and was thus selected for

further in vivo evaluation. Copper-mediated radioﬂuorination was

accomplished in good radiochemical yields and high molar activities.

Extensive in vivo characterization was performed in Wistar rats

comprising PET imaging, biodistribution, receptor occupancy, and

metabolites studies. [¹⁸F]PF-NB1 binding was selective to GluN2B-rich forebrain regions and was speciﬁcally blocked by the

GluN2B antagonist, CP-101,606, in a dose-dependent manner with no brain radiometabolites. [¹⁸F]PF-NB1 is a promising

ﬂuorine-18 PET tracer for imaging the GluN2B subunits of the NMDAR and has utility for receptor occupancy studies.

especially neural plasticity as well as learning and memory.³⁻⁵

INTRODUCTION

■

In several neuropathological conditions, whether acute or

chronic, an elevation of the glutamate synaptic concentration

levels can transform glutamate from a vital neurotransmitter for

survival to a dangerous neurotoxin, triggering an apoptotic

cascade.^6,7This neurotoxic process has been found to be

associated with several CNS disorders such as Alzheimer’s

disease, vascular dementia, Parkinson’s disease, depression,

stroke, and schizophrenia.⁸⁻¹⁰Numerous drugs have been

developed as ion channel blockers; however, they exhibited

severe side eﬀects due to hampering of physiological neuronal

functions and oﬀ-target binding.¹¹Consequently, subunit-

targeting antagonistsmore speciﬁcally, GluN2B antago-

nistshave recently emerged as potential therapeutic agents

for Parkinson’s disease, cerebral ischemia, neuropathic pain,

and depression in preclinical assessments.¹²⁻¹⁸These selective

L-glutamate is the main excitatory neurotransmitter in the

central nervous system (CNS). Glutamatergic receptors can be

generally grouped into two classes: ionotropic (iGluRs)

receptors (further subdivided into three subtypes: N-methyl-

D-aspartate receptors (NMDARs), kainate receptors, and 2-

amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid re-

ceptors) and metabotropic (mGluRs) receptors (further

subdivided into 3 groups: groups I, II, and III).¹NMDARs

are heterotetrameric transmembrane proteins that can bear

three diﬀerent types of subunits: GluN1 subunit (eight splice

variants, GluN1a−h), GluN2 subunit (four diﬀerent genes,

GluN2A−D), and GluN3 subunit (two diﬀerent genes,

GluN3A−B). Functional NMDARs typically require the

binding of coagonist glycine and agonist glutamate to GluN1

and GluN2 subunits, respectively. Upon activation, the

membrane is depolarized, prompting the inﬂux of calcium

ions and the propagation of the signals across the synapses.²

NMDARs play a fundamental role in neuronal development,

Received: May 26, 2019

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Figure 1. Structures of ifenprodil (a) and (b) of our previously reported [¹¹C]Me-NB1 and (R)-[¹¹C] Me-NB1 radiotracers as conformationally

restricted analogues of ifenprodil.³¹⁻³³

GluN2B antagonists possess a better safety proﬁle when

compared with the ion channel blockers. This is mainly due to

less cerebellum-related side eﬀects such as cognitive and motor

eﬀects due to the poor expression of the GluN2B subunit in

the cerebellum.^19,20The binding site of these antagonists is

situated at the interface of the N-terminal domains of GluN1

and GluN2B subunits. This unique binding site was termed the

“ifenprodil binding site” after ifenprodil (Figure 1a)a sigma-

1 receptor (σ1R) antagonistwas found to inhibit NMDARs

bearing the GluN2B subunit in the 90’s.²¹While the GluN1

subunit shows homogenous expression across the brain, the

GluN2B pattern of expression is heterogeneous depending on

both the brain’s maturity and age.⁵Although ifenprodil is

nonselective and metabolically unstable, it served as a lead

compound for the next generation of GluN2B antagonists and

as a model for deducing the binding mode and pocket for such

ligands from the crystal structure of the GluN1 and GluN2B

dimers complexed with ifenprodil (PDB code 3QEL).^22,23The

forefront challenge facing GluN2B ligands is the oﬀ-target

binding, especially the σ1R, a distinctive ligand-regulated

molecular chaperone in the endoplasmic reticulum that

regulates the activity of NMDARs.^24,25Nevertheless, several

promising and more selective GluN2B antagonists such as CP-

101,606 and CERC-303 (MK-0657) have been developed and

evaluated in clinical trials.^12,26Contrary to the high expect-

ations of these drugs, several clinical trials showed no

signiﬁcant beneﬁt as of yet in patients.^27,28

Positron emission tomography (PET) is a noninvasive

imaging modality that can functionally assess biological targets

using probes with appropriate aﬃnity and selectivity, labeled

with positron-emitting radionuclides. For the development of

GluN2B therapeutic agents, it can be a pivotal tool to address

two essential questions: (1) Does the drug engage GluN2B

subunit-containing NMDARs in vivo? (2) If yes, what is the

required dose that can show a satisfactory pharmacological

response and what is the receptor occupancy for such a

response?.²⁹Additionally, side eﬀects can be minimized when

PET imaging receptor occupancy studies are employed.³⁰

So far, reported Glun2B subunit imaging probes have

suﬀered from four major drawbacks: brain radiometabolites,

substantial oﬀ-target binding, low brain uptake, and brain

binding patterns inconsistent with reported GluN2B expres-

sion patterns.³⁴While no clinically validated GluN2B PET

radiotracer exists, our group recently succeeded in developing

the (R)-enantiomer of [¹¹C]Me-NB1 (K_i(GluN2B) = 5.4 nM,

K_i(σ1) = 182 nM, K_i(σ2) = 554 nM), a 2,3,4,5-tetrahydro-

1H-3-benzazepin-1-ol GluN2B PET radiotracer (Figure 1b)

that lacks the aforementioned limitations.³¹Although we

demonstrated a successful receptor occupancy (RO) study

using [¹¹C]Me-NB1 with therapeutic GluN2B antagonists, the

major inherent limitation of [¹¹C]Me-NB1 is the short physical

half-life of carbon-11 (physical half-life = 20.3 min), which

precludes its transport to imaging centers without an onsite

cyclotron. As such, our eﬀorts are currently focused on the

development of a ﬂuorine-18 (physical half-life = 109.8 min)

labeled GluN2B subunit tracer, where ﬂuorine-18 oﬀers better

spatial resolution than carbon-11 and allows distribution of the

tracer to nearby PET centers/hospitals (satellite distribu-

tion).³⁵Our preliminary attempts led to (R)-[¹⁸F]OF-Me-

NB1, a ﬂuorine-18 analogue of Me-NB1.³⁶In the course of

that work, it came to light that the (R)-enantiomers of 2,3,4,5-

tetrahydro-1H-3-benzazepin-1-ols possess a prominent selec-

tivity toward the GluN2B subunit, which was conﬁrmed in a

study with the enantiomers of Me-NB1³¹and other 2,3,4,5-

tetrahydro-1H-3-benzazepin-1-ol-based analogues.³⁷Despite

encouraging preclinical ﬁndings observed with (R)-[¹⁸F]OF-

Me-NB1, the radioﬂuorinated probe exhibited a relatively fast

washout from the brain, thus limiting its utility for GluN2B-

speciﬁc brain imaging.

In the current study, we report the structure−aﬃnity

relationship of a series of derivatives of 2,3,4,5-tetrahydro-

1H-3-benzazepines and its closely related structural class of

compounds, namely, 6,7,8,9-tetrahydro-5H-benzo[7]annulen-

7-amines, that emerged as potential GluN2B antagonists.³⁸We

sought to explore the 6,7,8,9-tetrahydro-5H-benzo[7]annulen-

7-amines as potential PET tracers and anticipated that it could

be an escape route to the enantiomerically pure restrictions

posed by the 2,3,4,5-tetrahydro-1H-3-benzazepine derivatives

that we have investigated so far. Rigorous investigation of

ifenprodil-like allosteric ligands resulted in a robust pharma-

cophore model comprising three essential cornerstones: (1) a

secondary or tertiary basic amine that is protonated under

physiological conditions, (2) a substituted aromatic ring with

an electron-donating functionality, and (3) a second aromatic

ring, whether substituted or not.³⁹From these investigations,

racemic 14 (PF-NB1) and compound 27 emerged as the best

performing compounds considering their GluN2B binding

aﬃnity and selectivity over σ1Rs. Surprisingly, even though

compound 27 exhibited high nanomolar aﬃnity of 2.1 nM and

a reasonable 50-fold selectivity over σ1R, autoradiographic

evaluation of [¹⁸F]27 revealed a homogenous distribution

pattern in the rat brain. For compound [¹⁸F]14 ([¹⁸F]PF-

NB1) and its enantiomers, (R)-[¹⁸F]14 ((R)-[¹⁸F]PF-NB1)

and (S)-[¹⁸F]14 ((S)-[¹⁸F]PF-NB1), we observed a heteroge-

neous binding distribution that was in accordance with the

known expression pattern of GluN2B.³Although (R)-[¹⁸F]PF-

NB1 exhibited a slightly higher in vitro binding aﬃnity, the

remarkable autoradiograms with [¹⁸F]PF-NB1 prompted us to

select this radioligand for further in vivo evaluation using PET

imaging, biodistribution, receptor occupancy, and metabolite

studies in rodents.

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Figure 2. Modiﬁcation sites pursued for 7- and 9-alkoxy-2,3,4,5-tetrahydro-1H-3-benzazepine derivatives are indicated with substituents R¹−R².

Scheme 1. Synthesis of Reference Compound 3

Scheme 2. Synthesis of Linkers 11a and 11b

synthesis of phenol 2.⁴¹O-alkylation of 2 aﬀorded ﬂuoropropyl

ether 3 in 76% yield.

The ﬂuorinated mesylates, 11a and 11b, were synthesized as

RESULTS AND DISCUSSION

■

Chemistry. 2,3,4,5-Tetrahydro-1H-3-benzazepines. For

the 2,3,4,5-tetrahydro-1H-3-benzazepin-1-ol scaﬀold, it has

shown in Scheme 2 applying a nine-step synthetic approach

been shown that the benzylic alcohol is crucial for binding to

under the same conditions and in similar yields. With the aim

the GluN2B subunit as it exhibits a direct polar interaction

to introduce steric hindrance, alcohols 4a and 4b were

protected with a bulky methyl succinate ester moiety, thereby

favoring subsequent para-directed Friedel−Crafts acylation.

This strategy aﬀorded keto acids 5a and 5b in 74 and 52%

yields, respectively. Cleavage of the ester functionalities in 5a

and 5b was achieved by treatment with sodium hydroxide

with serine 132 of the GluN1 subunit; consequently, we did

not pursue any modiﬁcations at this structural moiety.^23,40

Similarly, the length of the alkyl chain was not modiﬁed as it

was previously shown that a 4-carbon chain length provides an

optimal distance between the central amino moiety and the

terminal phenyl ring;³²hence, further modiﬁcations were

under reﬂux to aﬀord alcohols 6a and 6b in quantitative yields.

Reduction of ketones 6a and 6b under modiﬁed Clemmensen

conditions⁴²with zinc and concentrated HCl yielded an

inseparable mixture of phenylbutanoic and phenylbutenoic

acid products in a ratio of 1:4. The mixture was subjected to

palladium-catalyzed hydrogenation to yield intermediates 7a

and 7b exclusively in overall yields of 49 and 79%, respectively.

The free alcohols were mesylated using mesyl chloride;

interestingly, we observed an unexpected simultaneous

methylation of the carboxylic acid, yielding methyl esters 8a

envisioned either at the 7-methoxy functionality or on the

phenyl ring (Figure 2). All modiﬁcations of the phenyl ring

were exclusively carried out at the para-position. Furthermore,

we investigated shifting the methoxy group from the 7-position

to the 9-position.

Demethylation of previously synthesized Me-NB1 by our

group³³was accomplished using BBr₃to aﬀord phenol 2 in

61% yield (Scheme 1). Late-stage demethylation circumvented

the need to undertake additional protection and deprotection

steps of the phenolic hydroxyl group described in the original

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Scheme 3. Synthesis of Reference Compounds 14, 15, and 16 with a Methoxy Moiety in 7-Position

Scheme 4. Synthesis of Reference Compound 21 with a 9-Methoxy Moiety

Figure 3. Alkylamino and piperazinyl substituted 6,7,8,9-tetrahydro-5H-benzo[7]annulene derivatives with possible sites of modiﬁcations indicated

with R³−R⁵.

and 8b.⁴³Fluorination of 8a by using the KF/kryptoﬁx 2.2.2

system did not yield the desired ﬂuorinated product; however,

using CsF as a ﬂuorinating agent in tert-amyl alcohol aﬀorded

intermediate 9a in 25% yield. Compound 9b, on the other

hand, was obtained using KF/kryptoﬁx 2.2.2 in a higher yield

of 33%. Subsequent reduction of the ester aﬀorded ﬂuorinated

alcohols 10a and 10b in quantitative and 78% yields,

respectively. In the ﬁnal step, alcohols 10a and 10b were

mesylated to aﬀord 11a and 11b in 82 and 90% yields,

respectively.

(PF-NB1) in 61% yield. Separation of the enantiomers by

chiral high-performance liquid chromatography (HPLC)

yielded the two enantiomers, (S)-14 and (R)-14. The circular

dichroism (CD) spectra for both enantiomers were recorded

and compared to the enantiomers of Me-NB1, to which we

previously determined the absolute conﬁguration by means of

X-ray crystallography (Supporting Information, Figure 1).³¹

Key intermediate 12 was reacted with the mesylates, 11a and

11b, to aﬀord reference compounds 15 and 16 in comparable

yields of 51 and 54%, respectively (Scheme 3).

N-alkylation of the commercially available key intermediate

12 with custom synthesized [4-(4-ﬂuorophenyl)butyl] meth-

anesulfonate⁴⁴aﬀorded intermediate 13 in 49% yield. Further

treatment with BBr₃yielded phenolic reference compound 14

For the synthesis of 9-methoxy derivative 21 (Scheme 4),

ketone 18³²was reduced with sodium borohydride to aﬀord

alcohol 19 in quantitative yield. The tosyl protecting group was

cleaved using activated magnesium to give secondary amine 20

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Scheme 5. Synthesis of Reference Compounds 24−28

Scheme 6. Synthesis of Reference Compounds 30−32

Scheme 7. Syntheseis of Reference Compounds 35, 37, and 38

in 70% yield. In the ﬁnal step, N-alkylation of the secondary

amine 20 with [4-(4-ﬂuorophenyl)butyl] methanesulfonate

gave the reference compound 21 in 25% yield.

6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-amines.

6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-amines are struc-

tural analogues of 2,3,4,5-tetrahydro-1H-3-benzazepines. The

key diﬀerence between the two leads is the endo or exo

position of the N-atom. It was shown for 6,7,8,9-tetrahydro-

5H-benzo[7]annulen-7-amines that removal of the benzylic

alcohol enhanced aﬃnity toward the GluN2B subunit, while

discarding the phenolic ether slightly increased selectivity

toward GluN2B subunits.³⁸The modiﬁcation sites that we

pursued are depicted in Figure 3. Conformational restriction of

the linker into a piperazine ring was shown to enhance

selectivity while retaining aﬃnity.⁴⁵

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Scheme 8. Synthesis of Reference Compound 41

Scheme 9. Synthesis of Pinacol Boronic Ester 43

Scheme 10. Synthesis of Pinacol Boronic Esters 48, (S)-48, and (R)-48

The reductive amination of the commercially available

ketone 22 followed a reported procedure³⁸that led to 6,7,8,9-

tetrahydro-5H-benzo[7]annulen-7-amine derivatives 23, 26,

and 27 in very good to excellent yields of 94, 89, and 81%,

respectively (Scheme 5). Methylation of secondary amines 23

and 27 aﬀorded the respective tertiary methylamine reference

compounds 24 and 28. To avoid overmethylation and

formation of quaternary ammonium salts, Eschweiler−Clarke

methylation was performed using formaldehyde and formic

acid.⁴⁶Fluoroalkylation of compound 23 using (2-ﬂuoroethyl)

tosylate gave reference compound 25 in 50% yield.

For reference compound 41 (Scheme 8), the previous

reductive amination approach of ketone 22 was not pursued

because of the extremely poor yields. The previously reported

explanation proposed that the unfavorable steric hindrance

caused by the four methylene moieties adjacent to the

intermediate iminium ion would hinder the reductive

amination. However, this explanation seems to be unfounded

given that the reductive amination of nitro 29 with a piperazine

derivative was accomplished as depicted above in Scheme 6. A

possible explanation could be that the resonance structure of

the nitro group helps stabilize the ﬂow of electrons involved in

the formation of the iminium intermediate. Instead, a

roundabout nucleophilic substitution strategy was followed.⁴⁵

The nucleophilic substitution of nosylate 40⁴⁵with the

piperazine derivative aﬀorded compound 41 in 32% yield.

Precursors for Radiosynthesis. The synthesis of the 6,7,8,9-

tetrahydro-5H-benzo[7]annulen-7-amine precursor 43 for

ﬂuorine-18 labeling is depicted in Scheme 9. Reductive

amination of ketone 22 aﬀorded secondary amine 42 in 30%

yield. Miyaura borylation conditions were applied to aryl

bromide 42, followed by Boc protection of the secondary

amine to aﬀord boronic ester precursor 43 in 34% yield.⁴⁹

The 2-nitro compound 29⁴⁷(Scheme 6) was treated under

reductive amination reaction conditions to generate piperazine

30 in a moderate yield of 41%. Secondary amine 31⁴⁷was

methylated under Eschweiler−Clarke methylation conditions

to aﬀord the tertiary amine 32 in 91% yield.

The reaction pathways leading to target compounds 35, 37,

and 38 are depicted in Scheme 7. Acylation of primary amine

33⁴⁸under Schotten−Baumann conditions with the respective

acid chlorides provided amides 34 and 36 in 32 and 35%

yields, respectively. Further reductive amination with the

corresponding primary amines led to reference compounds 35,

37, and 38 in 85, 71, and 72% yields, respectively.

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Table 1. Binding Aﬃnities and clog P Values of 2,3,4,5-Tetrahydro-1H-3-benzazepin-1-ol Derivatives

compound

R¹

R²

R³

K_i(hGluN1/GluN2B) SEM (nM)

K_i(σ1) SEM (nM)

clog P

Me-NB1

3

14

OCH₃

OCH₂CH₂CH₂F

OH

H

F

5.4 0.4

524

182

174

4.42

4.90

3.98

20

4

1200

a

10.4 3.9

544

a

(R)-14

(S)-14

15

16

21

OH

OCH₃

H

OCH₃

F

3.0 1.4

1130

218

79

75

420

3.98

4.72

5.25

4.56

a

F

CH₂CH₂F

CH₂CH₂CH₂F

F

42.0 23.3

929

8

2

308

104

4

a

K_ivalues determined using rat brain homogenates.

Table 2. Binding Aﬃnities and clog P Values for 6,7,8,9-Tetrahydro-5H-benzo[7]annulene Derivatives

The synthetic pathway leading to pinacol boronic ester 48

precursor for radioﬂuorination is shown in Scheme 10. Alcohol

45⁵⁰was mesylated and subsequently coupled with secondary

amine 12 via N-alkylation to aﬀord tertiary amine 46 in 56%

yield. The secondary alcohol of compound 46 was then

protected with acetic anhydride to provide acetic acid ester 47

in 69% yield. Palladium-catalyzed cross-coupling of iodo 47

and bis(pinacolato)diboron in the presence of KOAc (Miyaura

borylation conditions) gave pinacol boronic ester 48 in 79%

yield.⁴⁹The enantiomers of the racemic precursor 48 were

separated by chiral HPLC to yield the enantiomerically pure

precursors, (S)- and (R)-48, as previously described for other

tracers.³⁶

Structure−Aﬃnity Relationships. 2,3,4,5-Tetrahydro-

1H-3-benzazepines. The IC₅₀values were determined

experimentally and were transformed into their respective K_i

values (inhibition constants reﬂecting the binding aﬃnity)

using the Cheng−Prusoﬀ equation. Table 1 summarizes the K_i

values for binding at GluN2B subunits and σ1Rs as well as the

clog P values for the 2,3,4,5-tetrahydro-1H-3-benzazepine

derivatives. clog P is a lipophilicity indicator and is one of

the descriptors used to predict blood−brain barrier pene-

tration.⁵¹2,3,4,5-Tetrahydro-1H-3-benzazepine derivatives

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Scheme 11. Radiosynthesis of [¹⁸F]27

Scheme 12. Radiosynthesis of [¹⁸F]PF-NB1

have been shown to generally exhibit high selectivity over

several CNS receptors including σ2Rs, while oﬀ-target binding

to σ1Rs has been reported, in which case a slight structural

modiﬁcation can alter the binding proﬁle of a ligand from a

GluN2B subunit-binder to a σ1R selective ligand.³²

Elongation of the ﬂuoroalkoxy chain at the 7-position

(compound 3) diminished the aﬃnity by 3-fold compared with

values toward both receptors. The nonﬂuorinated analogue of

compound 27 was reported to exhibit a K_ivalue of 16 4 nM

to the GluN2B subunit as well as σ1 and σ2R K_ivalues of 150

and 27 12 nM, respectively.⁴⁵Adding a ﬂuorine atom at the

para-position of the phenyl ring in 27 had a dramatic impact

on the GluN2B subunit aﬃnity and selectivity over σ2Rs;

GluN2B subunit aﬃnity was enhanced nearly 8-fold, while

selectivity over σ2R soared from almost 2-fold to 120-fold. For

compounds 24 and 28, selectivity over σ1R was lost upon

methylation of the secondary amine compared with com-

pounds 27 and 23.⁴⁵Alkylating the secondary amine with a

ﬂuoroethyl chain in compound 25 restored the selectivity while

maintaining a high binding aﬃnity toward the GluN2B

subunit, indicating that larger substituents on the secondary

amine enhanced the aﬃnity. However, we did not attempt to

label this compound as it was highly lipophilic. An analogue of

24 with R¹as OBn was recently reported to have a K_iof 3.6

2.2 nM toward GluN2B as well as K_ivalues of 206 and 110

32 nM toward σ1 and σ2Rs, respectively.⁴⁸Even though this

analogue possessed attractive aﬃnity and selectivity attributes,

its clog P was exceedingly high (6.60); therefore, as an

alternative, we substituted the OBn with a benzamide

functionality. Unfortunately, the GluN2B subunit aﬃnity was

lost for compound 37. Addition of ﬂuorine on either of the

phenyl rings (compounds 35 and 38) could restore neither the

aﬃnity nor the selectivity. It is known from previous reports

that the conformational restriction of the alkyl chain linker by

cyclization especially using the cyclic diamine, piperazine, has a

positive impact on the GluN2B subunit aﬃnity.⁴⁵With this in

mind, we synthesized alkyl diamine derivative 26 to investigate

the role of a secondary amine functionality with no

conformational restriction; however, aﬃnity toward the

GluN2B subunit diminished, and selectivity over σ2Rs was

lost. We next added a ﬂuorine atom to the phenyl moiety of

the piperazine derivative that led to compound 41; however,

the ﬂuorine eﬀect we observed previously with regard to

selectivity was greatly reduced. Furthermore, we synthesized

ﬂuorinated nitro derivatives 30 and 32 since 2-nitro derivatives

were reported to exhibit high GluN2B subunit aﬃnity.⁴⁷

Although they were highly aﬃne, they displayed poor

selectivity. From this series of compounds, we selected the

best performing compound 27, which showed high GluN2B

subunit binding aﬃnity and good selectivity over σ1 and σ2Rs,

for radioﬂuorination.

the reported K_ivalue of 162

22 nM for the ﬂuoroethoxy

analogue.⁵²The decrease in aﬃnity follows a trend and shows

that the longer the chain, the less tolerable the binding pocket

seems to be.

Compound 14 (PF-NB1), which was obtained by addition

of a ﬂuorine to the phenyl ring at the para- position and

cleavage of the methyl group in the 7-methoxy functionality,

revealed a K_ivalue that approaches the reported K_ivalue of 14

1.5 nM for NB1⁴¹and is more than 2-fold higher than the

previously reported methylated analogue compound 13

(Scheme 3).³⁶On the other hand, selectivity over σ1Rs

remarkably increased from the reported σ1R K_ivalue of 194

nM for NB1⁴¹to 1200 nM for compound 14. This result was

surprising considering that the methylated derivative, PF-Me-

NB1, exhibited a high K_iof 12 nM toward σ1Rs, as previously

reported.³⁶Keeping the 7-methyl ether functionality and

introducing a ﬂuoroethyl substituent at the para-position of the

phenyl ring (compound 15) led to a dramatic reduction in

GluN2B subunit binding aﬃnity and loss of selectivity over

σ1Rs. Surprisingly, the ﬂuoropropyl derivative 16 showed a 3-

fold improved K_iof 308 nM relative to 15 and comparable

selectivity, suggesting that the decrease in aﬃnity could be

nonsterically related. The 9-alkoxy derivative 21 showed the

second-best aﬃnity and selectivity out of this class of

compounds. Since PF-NB1 outperformed all of the other

2,3,4,5-tetrahydro-1H-3-benzazepine derivatives, we separated

the enantiomers and determined the K_ivalues of the

corresponding enantiomers (S)-14 and (R)-14 as well as the

racemic mixture using rat brain homogenates. The selectivity

of 14 in rats was similar to that of the human GluN2B subunit

with 54- and 60-fold selectivity factors, respectively. Although

(R)-14 was better, all three forms still possessed suitable

aﬃnity and selectivity. Further evaluation of the 3 compounds

was carried out using in vitro autoradiography after 14, (S)-14,

and (R)-14 were radiolabeled with ﬂuorine-18.

6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-amines. Table

2 summarizes the respective K_ivalues and clog P data of the

6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine derivatives.

This class of compounds was shown to exhibit oﬀ-target

binding to both σ1 and σ2Rs; thus, we measured the IC₅₀

Radiochemistry. Radioﬂuorination of the boronic ester

precursors followed a Cu(II)-mediated aromatic nucleophilic

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Figure 4. Representative in vitro autoradiograms of [¹⁸F]PF-NB and [¹⁸F]27 obtained from rat coronal brain sections. Cx, cortex; Th, thalamus;

Cb, cerebellum; CPu, caudate putamen (striatum); Hp, hippocampus.

Figure 5. In vitro autoradiograms of (A) [¹⁸F]PF-NB1, (B) (R)-[¹⁸F]PF-NB1, and (C) (S)-[¹⁸F]PF-NB1 in rat coronal brain sections. Positive and

negative control blockers were used. Solutions of 1 μM antagonists, CP-101,606 and CERC-301, were employed to establish the speciﬁcity of the

tracers toward the GluN2B subunit. Solutions of 1 μM SA4503, ﬂuspidine, and PB28 were used as negative controls to conﬁrm selectivity to the

ifenprodil binding site.

substitution reaction in analogy to a previously published

procedure.⁵³

thesis time from EOB until the end of synthesis was 139 min

on average. The identities of [¹⁸F]27, [¹⁸F]PF-NB1, (R)-

[¹⁸F]PF-NB1, and (S)-[¹⁸F]PF-NB1 were conﬁrmed by

coinjection with their corresponding nonradioactive reference

compounds.

The radiosynthesis of the 6,7,8,9-tetrahydro-5H-benzo[7]-

annulene tracer [¹⁸F]27 depicted in Scheme 11 involved a one-

pot, two-step reaction sequence encompassing radioﬂuorina-

tion of 45, followed by Boc removal and semipreparative

HPLC puriﬁcation. The radiochemical yield (RCY) of [¹⁸F]27

was 54% (decay-corrected), and the molar activity amounted

to 88 GBq/μmol (n = 1). Radiochemical purity was greater

than 95%, which was appropriate for further biological

experiments. The total radiosynthesis time from the end of

bombardment (EOB) until the end of synthesis was 135 min.

The radiosynthesis of [¹⁸F]PF-NB1 was accomplished in

two steps (Scheme 12). The ﬁrst step yielded intermediate

[¹⁸F]50 in 14−17% RCYs (decay-corrected). [¹⁸F]50 was

trapped on a Sep-Pak C18 Plus cartridge and eluted with

absolute ethanol. The solvent was evaporated by azeotropic

drying using extra-dry MeCN. This step was necessary as water

quenches the Lewis acid used in the succeeding reaction.

Simultaneous cleavage of the methyl and acetyl protecting

groups was achieved by using BBr₃, and the ﬁnal product was

puriﬁed by semipreparative HPLC. The radiosyntheses of (R)-

[¹⁸F]PF-NB1 and (S)-[¹⁸F]PF-NB1 were accomplished in a

similar fashion using the corresponding pinacolboronate

precursors, (R)-48 and (S)-48. Radiochemical purity was

greater than 95%, and molar activity amounted to 210 39

GBq/μmol (n = 5) for [¹⁸F]PF-NB1. The ﬁnal product

In Vitro Autoradiography. Coronal Wistar rat whole

brain sections were used to establish the in vitro binding

characteristics of [¹⁸F]PF-NB1, (R)-[¹⁸F]PF-NB1, (S)-

[¹⁸F]PF-NB1, and [¹⁸F]27. While (R)-[¹⁸F]PF-NB1, (S)-

[¹⁸F]PF-NB1, and [¹⁸F]PF-NB1 were found to accumulate

heterogeneously in GluN2B-rich regions such as the cortex,

thalamus, hippocampus, and striatum,⁵[¹⁸F]27 was found to

bind homogenously across the whole rat brain (Figure 4). This

homogenous binding pattern can be explained by the high

lipophilicity of [¹⁸F]27 (clog P = 5.05). The high nonspeciﬁc

binding exhibited by [¹⁸F]27 precluded its further evaluation.

In Figure 5 are depicted the autoradiograms of (R)-[¹⁸F]PF-

NB1, (S)-[¹⁸F]PF-NB1, and [¹⁸F]PF-NB1. The distribution of

the radiotracers followed the GluN2B subunit expression

proﬁle in rats with the highest accumulation in the cortex,

striatum, thalamus, and hippocampus as described in the

literature.^3,5The lowest accumulation was detected in the

cerebellum, a region with low GluN2B expression.³Blocking

experiments with GluN2B antagonists, CP-101,606 and

CERC-301, conﬁrmed the speciﬁcity of the tracers toward

GluN2B. σ1R blockers, SA4503 and ﬂuspidine, as well as σ2R

blocker PB28, did not compete with either [¹⁸F]PF-NB1, (R)-

[¹⁸F]PF-NB1, or (S)-[¹⁸F]PF-NB1 binding, suggesting that

none of the three radiotracers binds to σ1R or σ2R. Contrary

activities ranged from 0.56 to 1.2 GBq and had 4

1.1%

isolated RCYs (decay-corrected, n = 5). The total radiosyn-

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to previous results from our group, we did not observe a

notable diﬀerence between the two enantiomers with regard to

selectivity and speciﬁcity. We selected racemic [¹⁸F]PF-NB1

for further in vivo testing given the remarkable autoradiograms,

which obviates the need for tedious chiral separation.

In Vivo PET Imaging. PET Imaging in Wistar Rats.

Dynamic PET imaging was performed on anesthetized Wistar

rats for 90 min under baseline conditions, and for blocking

experiments, CP-101,606 (1, 3, 5, and 10 mg/kg, GluN2B IC₅₀

= 11 nM⁵⁴) was injected 1 min before the intravenous

injection of [¹⁸F]PF-NB1. Figure 6 shows coronal, sagittal, and

axial images of the rat brain under baseline and blockade

conditions. Under baseline conditions, a heterogeneous

accumulation of [¹⁸F]PF-NB1 in the rat brain, which is

consistent with the known expression pattern of GluN2B

subunits in the brain, was observed. This accumulation was

inhibited by the GluN2B antagonist, CP-101,606, suggesting

speciﬁc binding of [¹⁸F]PF-NB1 to GluN2B subunit-

containing NMDARs.

The corresponding time−activity curves (TACs) comparing

tracer uptake under baseline and blockade conditions are

shown in Figure 7A. TACs for speciﬁc brain regions were

generally higher in the cortex, hippocampus, striatum, and

thalamus relative to the cerebellum (Supporting Information,

Figure 2). There was a remarkable diﬀerence in the washout

rate between [¹⁸F]PF-NB1 and the previously reported

[¹¹C]Me-NB1, (R)-[¹¹C]Me-NB1, and (R)-[¹⁸F]OF-Me-

NB1,^31,33,36in which case the washout rate of [¹⁸F]PF-NB1

was slower than the other radiotracers so far reported in our

group. Using CP-101,606, an experimental GluN2B targeting

drug that showed eﬃcacy in clinical trials,^12,55,56we assessed

concentration-dependent receptor occupancy of CP-101,606 at

doses spanning between 1 and 10 mg/kg (Figure 7B).

A dose-dependent decrease of the SUVs with increasing CP-

101,606 doses was observed (Figure 7A). The SUVs were then

used to determine the in vivo receptor occupancy of CP-

101,606 (Figure 7B). The CP-101,606 dose required for 50%

receptor occupancy (D₅₀) was found to be 31 μmol/kg. This

study shows the utility of [¹⁸F]PF-NB1 in (1) visualizing target

engagement of GluN2B targeting drugs in vivo and (2)

estimating the required doses for GluN2B targeting drugs.

PET Imaging in Wild-Type and σ1R Knockout Mice. To

assess whether [¹⁸F]PF-NB1 exhibits oﬀ-target binding to

σ1Rs in vivo, pilot dynamic PET scans were performed using

σ1R knockout (σ1R-KO) and wild-type (WT) CD1 mice for

90 min. The resulting TACs are depicted in Figure 8. The

Figure 6. In vivo PET images of [¹⁸F]PF-NB1 in rat brain under

baseline and blockade (10 mg/kg CP-101,606 injected 1 min before

radiotracer injection) conditions superimposed on the MRI template

(PMOD). PET images were averaged from 30 to 90 min post tracer

injection. The color bar indicates standardized uptake value (SUV,

calculated as accumulated radioactivity (Bq) per g tissue divided by

injected dose per body weight).

Figure 7. (A) TACs representing whole brain accumulation of [¹⁸F]PF-NB1 (n = 3) presented as standardized uptake values (SUVs) from tracer

injection to 90 min postinjection. The speciﬁcity of binding and dose dependency was conﬁrmed by the injection of diﬀerent doses of the GluN2B

antagonist, C-P101,606 (1, 3, 5, and 10 mg/kg) (n = 1). (B) Receptor occupancy of the clinically experimental GluN2B antagonist CP-101,606

using [¹⁸F]PF-NB1 in Wistar rats calculated from experimental PET imaging of 90 min SUVs. A D₅₀of 31 μmol/kg was determined. The following

equations were used to calculate the receptor occupancy: (1) AUC = (b_max− b_min) × D₅₀/(D + D₅₀) + b_min. (2) RO = (b_max− AUC)/(b_max− b_min

)

× 100, where AUC is deﬁned as the area under the curve of the respective PET scan, D₅₀is the dose required to be injected to achieve 50%

receptor occupancy, D is the actual dose, RO is the receptor occupancy in %, and b_maxand b_minare the maximum and minimum binding,

respectively.

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TACs of [¹⁸F]PF-NB1 were found to be similar in both σ1R-

KO and WT mice, indicating that the brain uptake of [¹⁸F]PF-

NB1 is σ1R-independent.

5H-benzo[7]annulen-7-amines as GluN2B subunit ligands and

investigated their binding aﬃnities toward GluN2B subunits as

well as their selectivity over σRs. For the most promising

compounds, a robust radiosynthesis was established to aﬀord

[¹⁸F]PF-NB1, (R)-[¹⁸F]PF-NB1, (S)-[¹⁸F]PF-NB1, and [¹⁸F]

27 in good radiochemical yields, high molar activities, and

excellent radiochemical purities. Although 6,7,8,9-tetrahydro-

5H-benzo[7]annulen-7-amines showed high aﬃnity and

selectivity, they lacked the necessary structural polar features

that would label them as potential GluN2B imaging agents,

and thus this class of compounds would require further

optimization strategies. [¹⁸F]PF-NB1 is the ﬁrst radiolabeled

racemic 2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol deriva-

tive that shows high GluN2B subunit selectivity and speciﬁcity.

The use of [¹⁸F]PF-NB1 will obviate the need to perform

labor-intensive chiral HPLC separation. [¹⁸F]PF-NB1 ex-

hibited good in vivo performance in PET imaging studies

performed in rats and showed a remarkably slower washout

compared with our ﬁrst-generation 2,3,4,5-tetrahydro-1H-3-

benzazepin-1-ol tracers with no confounding brain radio-

metabolites. The results of the receptor occupancy with the

experimental GluN2B therapeutic drug CP-101,606 revealed a

dose dependency of blockade by [¹⁸F]PF-NB1. Taken

together, the results show that [¹⁸F]PF-NB1 is a promising

ﬂuorine-18 PET tracer with potential for clinical translation for

the imaging of the GluN2B subunits of the NMDARs and has

utility for studying GluN2B subunit receptor occupancy.

Figure 8. Typical TACs representing the whole brain uptake of

[¹⁸F]PF-NB1 in σ1R-KO and WT mice.

We conclude from these experiments that [¹⁸F]PF-NB1

exhibits high in vivo selectivity of binding to GluN2B subunits

over σ1R. Furthermore, these results conﬁrm the in vitro

selectivity observed from binding aﬃnity determinations and

the in vitro autoradiography experiments using ﬂuspidine, and

SA4503 as σ1R blocking agents.

Metabolite Studies. Previous tracers assessed by our

group from the 2,3,4,5-tetrahydro-1H-3-benzazepine class of

compounds showed no brain radiometabolites.^33,36To verify

that this holds true for the new radioligand, [¹⁸F]PF-NB1 was

injected into male Wistar rats, and blood samples at 5, 15, 30,

45, and 60 min postinjection, as well as brain extracts at 15, 30,

and 60 min, were taken and analyzed by radio-ultra-

performance liquid chromatography (UPLC). The analysis

showed only the presence of the intact parent radiotracer in

the brain up to 60 min postinjection. In Supporting

Information, Figure 3 are depicted the chromatograms of the

brain extracts. In blood, 3 radiometabolites, which were more

polar than the parent radioligand, were detected. At later time

points, the detection of radiometabolites was not possible due

to low radioactivity levels in the blood. This may be explained

by fast clearance of the radiotracer from the blood.

Biodistribution Studies. Ex vivo baseline (n = 4) and

blockade (n = 4) biodistribution experiments were carried out

at 45 min postinjection of [¹⁸F]PF-NB1 in male Wistar rats

using eliprodil as a GluN2B blocker. The 45 min postinjection

time point was selected based on the TACs of the baseline

PET scans. Similar to the PET results, [¹⁸F]PF-NB1 displayed

heterogeneous uptake in brain regions known to contain high

densities of GluN2B subunits with the cortex and thalamus

exhibiting the highest uptake. Compared with these brain

regions, the cerebellum showed a lower uptake of [¹⁸F]PF-

NB1: cortex/cerebellum (1.2, p = 0.0003), striatum/

cerebellum (1.1, p = 0.0045), and thalamus/cerebellum (1.1,

p = 0.014298). Both brain and body ex vivo biodistribution

results are summarized in the Supporting Information, Tables

1 and 2.

EXPERIMENTAL SECTION

■

Reagents and solvents were purchased from Sigma-Aldrich Chemie

GmbH (Germany), Acros Organics (Belgium), Fluka (Switzerland),

Merck (Germany), Chemieliva (China), Manchester Organics (U.K.),

or ABCR GmbH (Germany) and were used without further

puriﬁcation steps. The purchased solvents were of anhydrous grade

(puriss, dried over molecular sieves, H₂O < 0.005%) unless stated

otherwise. Solvents necessary for extractions, column chromatog-

raphy, and thin-layer chromatography (TLC) were acquired as

technical grade. Nonaqueous reactions were generally performed

under a nitrogen atmosphere using ﬂame-dried glassware and

standard syringe/septa methods. Reactions were magnetically stirred

and further monitored by TLC performed on Merck TLC glass sheets

(silica gel 60 F254). TLC Spots were visualized with UV light (λ =

254 nm). Chromatographic puriﬁcation of products was performed

using SiliaFlash P60 silica gel (Silicycle) for preparative column

chromatography (particle size 40−63 μm). Reactions at 0 °C were

carried out using an ice/water bath for cooling purposes.

The clog P values were calculated using CambridgeSoft software,

ChemDraw 15.0 (Perkin-Elmer). ¹H and ¹³C NMR spectra were

recorded on a Bruker Avance FT-NMR spectrometer (400 MHz),

and the chemical shifts (δ) are presented in ppm referenced to

tetramethylsilane (0 ppm). All coupling constants (J) are reported in

1

this work in Hz. Multiplicities in the H NMR spectra are generally

given as either s, singlet; d, doublet; dd, doublet of doublet; t, triplet;

dt, doublet of triplet; m, multiplet; or br, broad peak. All ¹³C NMR

spectra measured were completely proton decoupled.

High-resolution mass spectrometry (HRMS) was conducted on a

Bruker maXis (ESI-Qq-TOF-MS) spectrometer (Bruker Daltonik

GmbH, Germany), and the resulting data is reported in m/z.

Preparative HPLC of the ﬁnal compounds 3, 14, 15, 16, and 21 was

conducted with a Merck-Hitachi L7150 pump system and an Ultimate

XB-C18 column (150 mm × 21.2 mm, 5 mm) using a gradient system

of 10 mM aq. NH₄HCO₃(solvent A) and MeCN (solvent B); 0.0−

5.0 min, 20% B; 5.1−15.0 min, 20−50% B; 15.1−20.0 min, 50% B;

20.1−28.0 min, 50−95% B; 28.1−35.0 min, 95% B; 35.1−37.0 min,

95−20% B; 37.1−45.0 min, 20% B, with a ﬂow rate of 10 mL/min

and UV detection signal at 230 nm. For compounds 24, 25, 26, 27,

CONCLUSIONS

We have successfully designed and synthesized a series of

2,3,4,5-tetrahydro-1H-3-benzazepines and 6,7,8,9-tetrahydro-

■

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28, 30, 32, 35, 37, 38, and 41, the purity was determined using

analytical HPLC on a Merck Hitachi system comprising a D-7000

interface, an L-7100 pump, an L-7400 UV detector, and Ultimate XB-

C18 (4.6 mm × 150 mm, 3 μm) using generally a gradient system of

0.1% aq. triﬂuoroacetyl (TFA) (solvent A) and MeCN (solvent B);

0.0−7.0 min, 30−50% B; 7.1−15.0 min, 50% B; 15.1−20.0 min, 50−

60% B; 20.1−21.0 min, 60−95% B; 21.1−26.0 min, 95% B; 26.1−

27.0 min, 95−30% B; 27.1−32.0 min, 30% B with a ﬂow rate of 1

mL/min and UV detection signal at 254 nm. Enantiomeric

puriﬁcation of racemic precursor 48 was performed by semi-

preparative HPLC and a Reprosil Chiral-normal phase column (8

μm, 250 mm × 10 mm) using an isocratic system of hexane (solvent

A) and isopropanol (solvent B); 0.0−60.0 min, 2% B. Puriﬁcation of

the radiotracers was performed on a semipreparative Merck-Hitachi

L2130 HPLC system equipped with a radiation detector VRM 202

(Comecer, The Netherlands). For the puriﬁcation of (R)-, (S)- and

[¹⁸F]PF-NB1, an Agilent Eclipse XDB-C18 (9.4 mm × 250 mm, 5

μm) column was used with the following gradient solvent system:

0.1% aq. H₃PO₄(solvent A), MeCN (solvent B); 0.0−5.0 min, 20%

B; 5.1−25.0 min, 20−40% B; 25.1−28.0 min, 40% B; 28.1−30.0 min,

40−60% B; 30.1−33.0 min, 60−90% B; 33.1−36.0 min, 90% B;

36.1−38.0 min, 90−20% B; 38.1−42.0 min, 20% B. A ﬂow rate of 4

mL/min was used, and the UV signal was detected at 230 nm. For

quality control, an aliquot of the ﬁnal formulation was injected into an

analytical Agilent 1100 HPLC system mounted with a Raytest Gabi

Star radiodetector and an Atlantis T3 C18 reversed-phase column

(4.6 mm × 150 mm, 3 μm). The analysis gradient system was as

follows: 0.1% aq. TFA (solvent A), MeCN (solvent B); 0.0−10.0 min,

40−60% B; 10.1−11.0 min, 60−40% B; 11.1−15.0 min, 40% B, ﬂow

rate 1 mL/min and UV detection at 230 nm. For the semipreparative

HPLC puriﬁcation of [¹⁸F]27, an ACE-C18 column (250 mm × 10

mm, 5 μm) was used with the following conditions: 0.1% aq. H₃PO₄

(solvent A), MeCN (solvent B); 0.0−4.0 min, 20% B; 4.1−20.0 min,

20−60% B; 20.1−23.0 min, 60% B; 23.1−28.0 min, 60−80% B;

28.1−29.0 min, 80−5% B; 29.1−34.0 min, 5% B; 34.1−35.0 min, 5−

20% B; 35.1−38.0 min, 20% B with a ﬂow rate of 4 mL/min and UV

detection signal of 230 nm. For quality control, an aliquot from the

ﬁnal formulation was injected on a reversed-phase column

(LiChroCART 250-4, LiChrospher RP-18, 5 μm) with a ﬂow rate

of 1 mL/min and applying the following conditions: 0.1% aq. TFA

(solvent A), MeCN (solvent B); 0.0−2.0 min, 30% B; 2.1−15.0 min,

30−60% B; 15.1−17.1 min, 60% B; 17.1−20.1 min, 60−80% B;

20.1−21.1 min, 80−95% B; 21.1−25.1 min, 95% B; 25.1−26.0 min,

95−30% B; 26.1−28.1 min, 30% B with UV signal detection at 230

nm. Molar activity was calculated by comparing the UV intensity of

the formulated products against calibration curves of the correspond-

ing cold references of known concentrations. Purity of all biologically

tested compounds was ≥95%. Intermediates 23,⁴⁵29,⁴⁷31,⁴⁷33,⁴⁸

39,⁴⁵40,⁴⁵and 45⁵⁰were synthesized according to the published

literature.

155.2. HRMS (ESI) calculated for C₂₀H₂₆NO₂[M + H]⁺312.1958,

found 312.1958.

7-(3-Fluoropropoxy)-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-

benzo[d]azepin-1-ol (3). To a stirred solution of 2 (1.00 equiv, 33.0

mg, 0.106 mmol) in dimethylformamide (DMF, 0.4 mL), 3-

ﬂuoropropyl tosylate (1.22 equiv, 30.0 mg, 0.129 mmol) and

K₂CO₃(1.5 equiv, 24.0 mg, 0.159 mmol) were added. The reaction

mixture was stirred at 120 °C for 6 h and subsequently diluted with a

mixture of water and MeCN (1:1, 4.6 mL). The crude residue was

puriﬁed by preparative HPLC, and the title compound 3 was obtained

1

as a white solid (30.0 mg, 0.081 mmol, 76% yield). H NMR (400

MHz, CDCl₃): δ 7.33−7.23 (m, 2H), 7.21−7.15 (m, 3H), 7.11 (d, J =

7.8 Hz, 1H), 6.68−6.62 (m, 2H), 4.67 (t, J = 6.9 Hz, 1H), 4.60−4.53

(m, 2H), 4.29−4.11 (s, br, 1H), 4.07 (t, J = 6.9 Hz, 2H), 3.30−3.21

(m, 1H), 3.19−3.11 (m, 1H), 3.02−2.95 (m, 1H), 2.69−2.56 (m,

5H), 2.50 (d, J = 12.1 Hz, 1H), 2.40 (t, J = 11.9 Hz, 1H), 2.22−2.07

(m, 2H), 1.70−1.50 (m, 4H). ¹³C NMR (100 MHz, CDCl₃): δ 158.2,

149.1, 148.4, 142.4, 141.3, 129.7, 128.4, 128.0, 125.9, 120.4, 117.2,

111.0, 80.8 (d, J = 164.5), 72.4, 63.5, 60.9, 56.1, 36.9, 35.9, 31.0, 30.5,

29.2, 26.7. HRMS (ESI) calculated for C₂₃H₃₁FNO₂[M + H]⁺

372.2333, found 372.2337.

4-(4-Methoxy-4-oxobutanoyl)phenethyl methyl succinate (5a).

To an ice-cooled mixture of commercially available 4a (1.00 equiv,

6.90 mL, 57.3 mmol) in DCM (64 mL) was added portionwise

aluminum trichloride (1.20 equiv, 9.17 g, 68.8 mmol), followed by

methyl 4-chloro-4-oxobutyrate (1.10 equiv, 7.80 mL, 63.0 mmol).

The mixture was stirred at 0 °C for 30 min, quenched with ice water

(400 mL), and extracted with DCM (3 × 300 mL). The combined

organic layers were washed with brine (2 × 150 mL) and dried over

anhydrous magnesium sulfate. Solvents were removed under reduced

pressure to give the methyl phenethyl succinate quantitatively. Methyl

phenethyl succinate (1.00 equiv, 12.8 g, 54.3 mmol) was stirred at 0

°C in DCM (60 mL) before adding aluminum trichloride (5.50 equiv,

39.8 g, 299 mmol) portionwise, followed by methyl 4-chloro-4-

oxobutyrate (1.30 equiv, 8.70 mL, 70.6 mmol). The mixture was

stirred at 0 °C for 30 min, quenched with ice water (300 mL) and

extracted with DCM (3 × 200 mL). The combined organic layers

were washed with brine (2 × 100 mL) and dried over anhydrous

magnesium sulfate, and solvents were removed under reduced

pressure. The resulting crude was puriﬁed by ﬂash column

chromatography (silica gel; Hex/EtOAc 6:4) to give 5a (14.0 g,

40.2 mmol, 74%). ¹H NMR (400 MHz, CDCl₃): δ 2.59 (s, 4H), 2.75

(t, J = 6.5 Hz, 2H), 2.99 (t, J = 6.5 Hz, 2H), 3.29 (t, J = 6.5 Hz, 2H),

3.66 (s, 3H), 3.69 (s, 3H), 4.32 (t, J = 7.0 Hz, 2H), 7.31 (d, J = 8.2

Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ

28.1, 28.9, 29.1, 33.4, 35.1, 51.9, 52.0, 64.6, 128.4, 129.2, 135.1, 143.7,

172.3, 172.8, 173.3, 197.7. HRMS (ESI) calculated for C₁₈H₂₃O₇[M

+ H]⁺351.1438, found 351.1442.

3-(4-(4-Methoxy-4-oxobutanoyl)phenyl)propyl Methyl Succinate

(5b). To an ice-cooled mixture of commercially available 4b (1.00

equiv, 3.00 mL, 22.0 mmol) in DCM (25 mL) was added portionwise

aluminum trichloride (1.20 equiv, 3.52 g, 26.4 mmol), followed by the

addition of methyl 4-chloro-4-oxobutyrate (1.10 equiv, 3.00 mL, 24.2

mmol). The mixture was stirred at 0 °C for 30 min, quenched with ice

water (300 mL), and extracted with DCM (3 × 200 mL). The

combined organic layers were washed with brine (2 × 100 mL) and

dried over anhydrous magnesium sulfate. Solvents were removed

under reduced pressure to yield methyl (3-phenylpropyl) succinate

quantitatively. Methyl (3-phenylpropyl) succinate (1.00 equiv, 5.64 g,

22.5 mmol) was then dissolved in DCM (25 mL) at 0 °C before the

portionwise addition of aluminum trichloride (3.20 equiv, 9.61 g, 72.1

mmol) and methyl 4-chloro-4-oxobutyrate (1.30 equiv, 3.60 mL, 29.3

mmol) was carried out. The mixture was stirred at 0 °C for 30 min,

quenched with ice water (400 mL), and extracted with DCM (3 ×

200 mL). The combined organic layers were washed with brine (2 ×

100 mL) and dried over anhydrous magnesium sulfate. Solvents were

removed under reduced pressure, and the residue was puriﬁed by ﬂash

column chromatography (silica gel; Hex/EtOAc 6:4) to give 5b (4.29

Chemistry. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-benzo[d]-

azepine-1,7-diol (2, NB1). To a solution of commercially available

1 (1.00 equiv, 170 mg, 0.522 mmol) in dichloromethane (DCM) (7.2

mL), BBr₃(7.30 equiv, 3.80 mL, 1 M in DCM, 3.80 mmol) was added

dropwise at −78 °C. The reaction mixture was warmed to rt and

subsequently stirred for 4 h. Afterward, the reaction mixture was

poured into ice water (36 mL), and the organic phase was completely

evaporated under reduced pressure. The resultant mixture was diluted

with distilled water (100 mL) and subsequently extracted with DCM

(3 × 100 mL). The combined organic layers were dried over

anhydrous magnesium sulfate and concentrated under reduced

pressure. The crude was puriﬁed by preparative HPLC to give

compound 2 (100 mg, 0.318 mmol, 61%). ¹H NMR (400 MHz,

CDCl₃): δ 1.50−1.71 (m, 4H), 2.40 (t, J = 12.1 Hz, 1H), 2.49 (d, J =

12.06 Hz, 1H), 2.56−2.67 (m, 5H), 2.98 (dd, J1 = 6.0 Hz, J2 = 12.3

Hz, 1H), 3.11−3.28 (m, 2H), 4.57 (d, J = 6.7 Hz, 1H), 6.53−6.58 (m,

2H), 7.01 (d, J = 7.7, 1H), 7.15−7.21 (m, 3H), 7.27−7.31 (m, 2H).

¹³C NMR (100 MHz, CDCl₃): δ 26.7, 29.2, 36.7, 56.2, 59.7, 60.8,

1

g, 11.7 mmol, 52%). H NMR (400 MHz, CDCl₃): δ 1.92−2.02 (m,

72.5, 112.5, 117.6, 125.9, 128.5, 128.6, 130.1, 135.4, 141.5, 142.4,

2H), 2.63 (s, 4H), 2.71−2.78 (m, 4H), 3.3 (t, J = 6.8 Hz, 2H), 3.68

L

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(s, 3H), 3.69 (s, 3H), 4.11 (t, J = 6.5 Hz, 2H), 7.27 (d, J = 8.3 Hz,

2H), 7.91 (d, J = 8.3 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ =

28.2, 29.0, 29.2, 29.9, 32.3, 33.4, 51.9, 52.0, 64.0, 128.5, 128.8, 134.8,

147.2, 172.3, 172.9, 173.5, 197.7. HRMS (ESI) calculated for

C₁₉H₂₅O₇[M + H]⁺365.1595, found 365.1595.

then ﬁltered through a pad of celite and washed with MeOH (5 × 20

mL). The ﬁltrate was concentrated in vacuo to aﬀord compound 7b

(1.57 g, 7.07 mmol, 79%). ¹H NMR (400 MHz, (CD₃)₂CO): δ 1.74−

1.92 (m, 4H), 2.27 (t, J = 7.4 Hz, 2H), 2.58−2.66 (m, 4H), 3.54 (t, J

= 6.2 Hz, 2H), 7.11 (br s, 4H). ¹³C NMR (100 MHz, (CD₃)₂CO): δ

27.0, 31.7, 33.1, 34.6, 34.8, 61.1, 128.5, 128.5, 139.3, 140.1, 174.5.

HRMS (ESI) calculated for C₁₃H₁₈NaO₃[M + Na]⁺245.1148, found

245.1148.

4-(4-(2-Hydroxyethyl)phenyl)-4-oxobutanoic Acid (6a). To a

stirred solution of aq. NaOH (30%, 35 mL) and MeOH (526 mL)

was added 5a (1.00 equiv, 14.0 g, 40.0 mmol). The reaction mixture

was heated to reﬂux and stirred for 20 min. The pH was adjusted to 7

using conc. HCl (37% w/w in water), and the volatiles were

evaporated under reduced pressure. The resulting mixture was diluted

with aq. HCl (0.03 M, 300 mL) and extracted with EtOAc (3 × 300

mL). The combined organic layers were dried over anhydrous

magnesium sulfate, and the solvents were evaporated under reduced

pressure to give the title compound 6a (11.9 g, 40.0 mmol, quant.).

¹H NMR (400 MHz, (CD₃)₂CO): δ 2.71 (t, J = 6.4 Hz, 2H), 2.88 (t,

J = 6.70 Hz, 2H), 3.30 (t, J = 6.3 Hz, 2H), 3.80 (t, J = 6.8 Hz, 2H),

7.39 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H). ¹³C NMR (100

MHz, (CD₃)₂CO): δ 28.2, 33.7, 40.0, 63.2, 128.6, 130.0, 135.8, 146.3,

173.7, 198.2. HRMS (ESI) calculated for C₁₂H₁₄NaO₄[M + Na]⁺

245.0784, found 245.0783.

Methyl 4-(4-(2-((Methylsulfonyl)oxy)ethyl)phenyl)butanoate

(8a). To a solution of 7a (1.00 equiv, 2.20 g, 10.6 mmol) in DCM

(211 mL) was added dropwise methanesulfonyl chloride (5.00 equiv,

4.10 mL, 52.8 mmol), followed by the addition of tetraethylammo-

nium (TEA, 2.00 equiv, 2.90 mL, 21.1 mmol). The mixture was

stirred at rt for 30 min, diluted with EtOAc (150 mL), washed with

aq. HCl (0.03 M, 3 × 300 mL), and dried over anhydrous magnesium

sulfate. Solvents were removed under reduced pressure and the

residue was puriﬁed by ﬂash column chromatography (silica gel; Hex/

EtOAc 7:3, 1% AcOH) to obtain the title compound 8a (672 mg,

1

2.32 mmol, 22%). H NMR (400 MHz, (CD₃)₂CO): δ 1.84−1.93

(m, 2H), 2.31 (t, J = 7.5 Hz, 2H), 2.63 (t, J = 7.9 Hz, 2H), 2.98 (s,

3H), 3.03 (t, J = 6.7 Hz, 2H), 3.61 (s, 3H), 4.42 (t, J = 6.8 Hz, 2H),

7.17 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H). ¹³C NMR (100

MHz, (CD₃)₂CO): δ 27.4, 33.6, 35.2, 35.7, 37.0, 51.5, 71.5, 129.4,

129.9, 135.4, 141.1, 173.9. HRMS (ESI) calculated for C₁₄H₂₁O₅S [M

+ H]⁺301.1104, found 301.1105.

4-(4-(3-Hydroxypropyl)phenyl)-4-oxobutanoic Acid (6b). To a

stirred solution of aq. NaOH (30%, 7.2 mL) and MeOH (108 mL)

was added 5b (3.00 g, 8.23 mmol). The reaction mixture was heated

to reﬂux and stirred for 20 min. The pH was adjusted to 7 using conc.

HCl, and the volatiles were evaporated under reduced pressure. The

resulting mixture was diluted with aq. HCl (0.03 M, 300 mL) and

extracted with EtOAc (3 × 300 mL). The combined organic layers

were dried over anhydrous magnesium sulfate, and the solvents were

evaporated under reduced pressure to give the title compound 6b

Methyl 4-(4-(3-((Methylsulfonyl)oxy)propyl)phenyl)butanoate

(8b). To a solution of 7b (1.00 equiv, 1.57 g, 7.07 mmol) in DCM

(141 mL) was added dropwise methanesulfonyl chloride (5.00 equiv,

2.70 mL, 35.3 mmol), followed by the addition of TEA (2.00 equiv,

2.00 mL, 14.1 mmol). The mixture was stirred at rt for 30 min, diluted

with EtOAc (150 mL), washed with aq. HCl (0.03 M, 3 × 150 mL),

and dried over anhydrous magnesium sulfate. Solvents were removed

under reduced pressure and the residue was puriﬁed by ﬂash column

chromatography (silica gel; Hex/EtOAc 7:3, 1% AcOH) to obtain the

1

(2.57 g, 8.23 mmol, quant.). H NMR (400 MHz, (CD₃)₂CO): δ

1.79−1.88 (m, 2H), 2.70 (t, J = 6.5 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H),

3.29 (t, J = 6.5 Hz, 2H), 3.57 (t, J = 6.3 Hz, 2H), 7.37 (d, J = 8.3 Hz,

2H), 7.94 (d, J = 8.3 Hz, 2H). ¹³C NMR (100 MHz, (CD₃)₂CO): δ

28.0, 32.5, 33.6, 34.7, 61.2, 128.6, 129.3, 135.4, 148.9, 174.0, 198.0.

HRMS (ESI) calculated for C₁₃H₁₆NaO₄[M + Na]⁺259.0941, found

259.0944.

1

title compound 8b (974 mg, 3.11 mmol, 44%). H NMR (400 MHz,

(CD₃)₂CO): δ 1.83−1.93 (m, 2H), 1.99−2.07 (m, 2H), 2.30 (t, J =

7.4 Hz, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.71 (t, J = 8.0 Hz, 2H), 3.07 (s,

3H), 3.60 (s, 3H), 4.23 (t, J = 6.4 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H),

7.17 (d, J = 8.0 Hz, 2H). ¹³C NMR (100 MHz, (CD₃)₂CO): δ 27.6,

31.7, 31.9, 33.8, 35.3, 37.2, 51.6, 70.5, 129.4, 129.5, 139.4, 140.4,

174.0. HRMS (ESI) calculated for C₁₅H₂₂NaO₅S [M + Na]⁺

337.1080, found 337.1080.

4-(4-(2-Hydroxyethyl)phenyl)butanoic Acid (7a). To a solution of

6a (1.00 equiv, 9.29 g, 41.8 mmol) in water (50 mL), zinc (7.00

equiv, 19.1 g, 293 mmol) was added portionwise. Conc. HCl (21 mL)

was added dropwise, and the reaction mixture was reﬂuxed for 1 h.

The mixture was diluted with aq. HCl (0.03 M, 350 mL) and

extracted with EtOAc (3 × 350 mL). The combined organic layers

were dried over anhydrous magnesium sulfate, and solvents were

removed under reduced pressure. The residue was puriﬁed by ﬂash

column chromatography (silica gel; Hex/EtOAc 6:4, 1% AcOH) to

give a mixture of the inseparable oleﬁn and 7a. Therefore, the mixture

(4.18 g) and Pd/C (10%, 1.01 g, 10.7 mmol) were suspended in

MeOH (53 mL) and stirred at rt for 1 h under a H₂atmosphere. The

mixture was then ﬁltered through a pad of celite and washed with

MeOH (5 × 20 mL). The ﬁltrate was concentrated in vacuo to aﬀord

compound 7a (4.04 g, 19.4 mmol, 49%). ¹H NMR (400 MHz,

(CD₃)₂CO): δ 1.84−1.93 (m, 2H), 2.30 (t, J = 7.4 Hz, 2H), 2.62 (t, J

= 7.4 Hz, 2H), 2.77 (t, J = 7.1 Hz, 2H), 3.73 (t, J = 7.1 Hz, 2H), 7.12

(d, J = 8.1 Hz, 2H), 7.16 (d, J = 8.1 Hz, 2H). ¹³C NMR (100 MHz,

(CD₃)₂CO): δ 27.6, 33.6, 35.3, 39.9, 64.0, 129.2, 129.9, 138.0, 140.3,

173.8. HRMS (ESI) calculated for C₁₂H₁₆NaO₃[M + Na]⁺231.0992,

found 231.0991.

Methyl 4-(4-(2-Fluoroethyl)phenyl)butanoate (9a). To a solution

of 8a (1.00 equiv, 1.00 g, 3.33 mmol) in tert-amyl alcohol (12 mL),

CsF (3.00 equiv, 1.52 g, 9.99 mmol) was added portionwise. The

mixture was stirred at 80 °C for 6 h, cooled to rt, diluted with water

(200 mL), and extracted with DCM (3 × 200 mL). The combined

organic layers were dried over anhydrous magnesium sulfate, followed

by the removal of solvents under reduced pressure. The residue was

puriﬁed by ﬂash column chromatography (silica gel; Hex/EtOAc

1

95:5) to give the title product 9a (190 mg, 0.83 mmol, 25%). H

NMR (400 MHz, (CD₃)₂CO): δ 1.84−1.93 (m, 2H), 2.31 (t, J = 7.4

Hz, 2H), 2.62 (t, J = 7.9 Hz, 2H), 2.94 (t, J = 6.5 Hz, 1H), 2.99 (t, J =

6.5 Hz, 1H), 3.61 (s, 3H), 4.55 (t, J = 6.5 Hz, 1H), 4.67 (t, J = 6.5 Hz,

1H), 7.15 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H). ¹³C NMR

(100 MHz, (CD₃)₂CO): δ 27.6, 33.8, 35.3, 37.2, 51.6, 84.9, 129.5,

129.9, 136.1, 140.9, 174.0. HRMS (EI) calculated for C₁₃H₁₇FO₂

[M^+•] 224.1207, found 224.1210.

Methyl 4-(4-(3-Fluoropropyl)phenyl)butanoate (9b). A mixture of

kryptoﬁx 2.2.2 (3.30 equiv, 3.75 g, 9.95 mmol), KF (3.30 equiv, 578

mg, 9.95 mmol), and 8b (1.00 equiv, 948 mg, 3.02 mmol) was

dissolved in MeCN (38 mL) and stirred at 90 °C for 1 h. Afterward,

the mixture was poured into water (200 mL) and extracted with

DCM (3 × 200 mL). The combined organic layers were dried over

anhydrous magnesium sulfate and concentrated in vacuo. The crude

product was puriﬁed by ﬂash column chromatography (silica gel;

Hex/EtOAc 95:5) to aﬀord 9b (238 mg, 3.28 mmol, 33%). ¹H NMR

(400 MHz, (CD₃)₂CO): δ 1.84−1.92 (m, 2H), 1.93−2.03 (m, 2H),

2.31 (t, J = 7.5 Hz, 2H), 2.61 (t, J = 7.8 Hz, 2H), 2.69 (t, J = 8.0 Hz,

2H), 3.61 (s, 3H), 4.38 (t, J = 6.0 Hz, 1H), 4.50 (t, J = 6.0 Hz, 1H),

4-(4-(3-Hydroxypropyl)phenyl)butanoic Acid (7b). To a solution

of 6b (1.00 equiv, 1.76 g, 7.46 mmol) in water (8.6 mL), zinc (7

equiv, 3.410 g, 52.2 mmol) was added portionwise. Conc. HCl (3.7

mL) was added dropwise, and the reaction mixture was reﬂuxed for 1

h. The mixture was diluted with aq. HCl (0.03 M, 250 mL) and

extracted with EtOAc (3 × 250 mL). The combined organic layers

were dried over anhydrous magnesium sulfate, and solvents were

removed under reduced pressure. The residue was puriﬁed by ﬂash

column chromatography (silica gel; Hex/EtOAc 6:4, 1% AcOH) to

give an inseparable mixture of 7b and the oleﬁn. The mixture (1.52 g)

and Pd/C (10%, 0.345 g, 3.24 mmol) were suspended in MeOH (18

mL) and stirred at rt for 1 h under a H₂atmosphere. The mixture was

M

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7.14 (br s, 4H). ¹³C NMR (100 MHz, (CD₃)₂CO): δ 27.7, 31.6, 33.0,

33.8, 35.4, 51.6, 84.0, 129.4, 129.5, 139.9, 140.3, 174.1. HRMS (EI)

calculated for C₁₄H₁₉FO₂[M^+•] 238.1364, found 238.1366.

amine 12 (1.00 equiv, 30.0 mg, 0.155 mmol) in DMF (0.5 mL) was

added N,N-diisopropylethylamine (0.90 equiv, 24.0 μL, 0.140 mmol).

The resulting mixture was stirred for 5 min at rt, and 4-(4-

ﬂuorophenyl)butyl methanesulfonate (0.90 equiv, 34.0 mg, 0.140

mmol) in DMF (0.5 mL) was subsequently added dropwise. The

resulting solution was stirred at 90 °C for 3 h and diluted with a

mixture of water and MeCN (1:1, 4.5 mL). Puriﬁcation was

performed by preparative HPLC to give the title compound 13 as a

white solid (26.0 mg, 0.076 mmol, 49%). ¹H NMR (400 MHz,

CDCl₃): δ 7.16−7.07 (m, 3H), 7.00−6.93 (m, 2H), 6.68−6.62 (m,

2H), 4.57 (d, J = 6.9 Hz, 1H), 3.78 (s, 3H), 3.31−3.26 (m, 1H),

3.19−3.11 (m, 1H), 3.02−2.95 (m, 1H), 2.69−2.56 (m, 5H), 2.50 (d,

J = 12.1 Hz, 1H), 2.40 (t, J = 11.9 Hz, 1H), 1.70−1.49 (m, 4H), signal

for the OH is not visible. ¹³C NMR (100 MHz, CDCl₃): δ 161.4 (d, J

= 243.1 Hz), 159.1, 141.4, 138.0, 135.7, 129.8 (d, J = 9.2 Hz, 3C),

116.8, 115.17 (d, J = 21.5 Hz, 2C), 110.4, 72.6, 61.0, 59.7, 56.3, 55.3,

37.1, 35.1, 29.4, 26.8. HRMS (ESI) calculated for C₂₁H₂₇FNO₂[M +

H]⁺344.2020, found 344.2023.

4-(4-(2-Fluoroethyl)phenyl)butan-1-ol (10a). To a solution of 9a

(1.00 equiv, 182 mg, 0.812 mmol) in tetrahydrofuran (THF, 1.4 mL)

was added lithium aluminum hydride (2.00 equiv, 62.0 mg, 1.62

mmol) at 0 °C. The mixture was then allowed to stir at rt for 30 min.

The resulting mixture was diluted with water (100 mL) and extracted

with DCM (3 × 100 mL). The combined organic layers were dried

over anhydrous magnesium sulfate and the solvents were evaporated

under reduced pressure to give 10a (165 mg, 0.812 mmol, quant.). ¹H

NMR (400 MHz, (CD₃)₂CO): δ 1.49−1.57 (m, 2H), 1.62−1.71 (m,

2H), 2.60 (t, J = 7.7 Hz, 2H), 2.93 (t, J = 6.5 Hz, 1H), 2.98 (t, J = 6.5

Hz, 1H), 3.41 (t, J = 5.3 Hz, 1H), 3.52−3.58 (m, 2H), 4.54 (t, J = 6.1

Hz, 1H), 4.66 (t, J = 6.1 Hz, 1H), 7.14 (d, J = 8.1 Hz, 2H), 7.18 (d, J

= 8.1 Hz, 2H). ¹³C NMR (100 MHz, (CD₃)₂CO): δ 28.8, 33.4, 36.0,

37.2, 62.4, 84.9, 129.4, 129.8, 135.6, 141.8. HRMS (EI) calculated for

C₁₂H₁₇FO [M^+•] 196.1258, found 196.1256.

4-(4-(3-Fluoropropyl)phenyl)butan-1-ol (10b). To a solution of

9b (1.00 equiv, 232 mg, 0.974 mmol) in THF (1.5 mL), lithium

aluminum hydride (2.00 equiv, 73.9 mg, 1.95 mmol) was added

portionwise at 0 °C. The mixture was then allowed to stir at rt for 30

min. The resulting mixture was diluted with water (250 mL) and

extracted with DCM (3 × 250 mL). The combined organic layers

were dried over anhydrous magnesium sulfate and the solvents were

evaporated under reduced pressure to give 10b (160 mg, 0.760 mmol,

78%). ¹H NMR (400 MHz, (CD₃)₂CO): δ 1.49−1.58 (m, 2H),

1.58−1.62 (m, 2H), 1.89−2.03 (m, 2H), 2.59 (t, J = 7.9 Hz, 2H),

2.68 (t, J = 7.9 Hz, 2H), 3.41 (t, J = 5.4 Hz, 1H), 3.52−3.58 (m, 2H),

4.38 (t, J = 6.0 Hz, 1H), 4.50 (t, J = 6.0 Hz, 1H), 7.13 (s, 4H). ¹³C

NMR (100 MHz, (CD₃)₂CO): δ 28.8, 31.6, 33.1, 33.5, 36.0, 62.4,

83.9, 129.2, 129.4, 139.5, 141.2. HRMS (EI) calculated for C₁₃H₁₉FO

[M^+•] 210.1414, found 210.1412.

3-(4-(4-Fluorophenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]-

azepine-1,7-diol (14, PF-NB1). Compound 13 (1.00 equiv, 118 mg,

0.343 mmol) was dissolved in DCM (5.0 mL), followed by dropwise

addition of BBr₃in DCM (7.30 equiv, 1 M, 2.5 mL, 2.50 mmol) while

cooling the reaction mixture to −78 °C. The resulting mixture was

stirred for 4 h at rt and subsequently quenched by the addition of ice

water (5.0 mL). DCM was evaporated under reduced pressure, and an

extraction was performed with aq. NaOH (1 M, 25 mL) and ethyl

acetate (3 × 25 mL). The combined organic layers were concentrated

under reduced pressure, and the resulting crude was puriﬁed by

preparative HPLC to give the title compound 14 (30.0 mg, 0.085

1

mmol, 61% yield). H NMR (400 MHz, CDCl₃): δ 7.15−7.07 (m,

2H), 7.00−6.92 (m, 3H), 6.53−6.46 (m, 2H), 5.70−5.15 (s, br, 2H),

4.57 (d, J = 6.9 Hz, 1H), 3.22−3.08 (m, 2H), 2.99−2.88 (m, 1H),

2.69−2.50 (m, 6H), 2.40 (t, J = 11.9 Hz, 1H), 1.70−1.49 (m, 4H).

¹³C NMR (100 MHz, CDCl₃): δ 161.4 (d, J = 243.1), 155.8, 141.3,

4-(4-(2-Fluoroethyl)phenyl)butyl Methanesulfonate (11a). To a

solution of 10a (1.00 equiv, 160 mg, 0.815 mmol) in DCM (16 mL),

methanesulfonyl chloride (5.00 equiv, 0.32 mL, 4.08 mmol) was

added dropwise. After 5 min of stirring at rt, TEA (2.00 equiv, 0.23

mL, 1.63 mmol) was added, and the reaction mixture was allowed to

stir for an additional 30 min at rt. The mixture was diluted with

EtOAc (100 mL), washed with aq. HCl (0.03 M, 3 × 100 mL), and

dried over anhydrous magnesium sulfate. Solvents were removed

under reduced pressure and the crude was puriﬁed by ﬂash column

chromatography (silica gel; Hex/EtOAc 8:2) to obtain the title

138.0, 134.6, 130.0, 129.8 (d, J = 8.4 Hz, 2C), 117.8, 115.2 (d, J =

21.6 Hz, 2C), 112.7, 72.6, 61.0, 59.7, 56.3, 36.5, 35.1, 29.4, 26.7.

HRMS (ESI) calculated for C₂₀H₂₅FNO₂[M + H]⁺330.1864, found

330.1864.

3-(4-(4-(2-Fluoroethyl)phenyl)butyl)-7-methoxy-2,3,4,5-tetrahy-

dro-1H-benzo[d]azepin-1-ol (15). To a solution of commercially

available amine 12 (1.00 equiv, 30.0 mg, 0.155 mmol) in DMF (0.5

mL) was added dropwise N,N-diisopropylethylamine (0.90 equiv, 24

μL, 0.140 mmol), and the resulting mixture was stirred for 5 min at rt.

A solution of 4-(4-(2-ﬂuoroethyl)phenyl)butyl methanesulfonate

(0.90 equiv, 38.0 mg, 0.140 mmol) in DMF (0.5 mL) was added

dropwise. The resulting mixture was stirred at 90 °C for 3 h and

subsequently diluted with water and MeCN (1:1, 4.5 mL).

Puriﬁcation was carried out by preparative HPLC to give the title

1

compound 11a (184 mg, 0.668 mmol, 82%). H NMR (400 MHz,

(CD₃)₂CO): δ 1.69−1.79 (m, 4H), 2.65 (t, J = 7.2 Hz, 2H), 2.93 (t, J

= 6.4 Hz, 1H), 2.99 (t, J = 6.4 Hz, 1H), 3.06 (s, 3H), 4.26 (t, J = 6.2

Hz, 2H), 4.54 (t, J = 6.5 Hz, 1H), 4.67 (t, J = 6.5 Hz, 1H), 7.17 (d, J =

8.3 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H). ¹³C NMR (100 MHz,

(CD₃)₂CO): δ 28.2, 29.5, 35.5, 37.1, 37.3, 71.1, 84.9, 129.4, 129.9,

136.0, 141.2. HRMS (ESI) calculated for C₁₃H₂₃FNO₃S [M+NH₄]⁺

292.1377, found 292.1380.

1

compound 15 (29.0 mg, 0.078 mmol, 51% yield). H NMR (400

MHz, CDCl₃): δ 7.18−7.05 (m, 5H), 6.67−6.62 (m, 2H), 4.68 (t, J =

6.7 Hz, 1H), 4.60−4.53 (m, 2H), 3.78 (s, 3H), 3.31−3.21 (m, 1H),

3.19−3.11 (m, 1H), 3.06−2.95 (m, 3H), 2.70−2.56 (m, 5H), 2.50 (d,

J = 12.1 Hz, 1H), 2.40 (t, J = 11.9 Hz, 1H), 1.70−1.50 (m, 4H), signal

for the OH is not visible. ¹³C NMR (100 MHz, CDCl₃): δ 159.1,

141.4, 140.8, 135.7, 134.5, 129.8, 129.1 (2C), 128.7 (2C), 116.8,

110.4, 84.3 (d, J = 168.8), 72.6, 61.0, 59.7, 56.3, 55.3, 37.1, 35.1, 31.0,

29.4, 26.8. HRMS (ESI) calculated for C₂₃H₃₁FNO₂[M + H]⁺

372.2333, found 372.2338.

3-(4-(4-(3-Fluoropropyl)phenyl)butyl)-7-methoxy-2,3,4,5-tetra-

hydro-1H-benzo[d]azepin-1-ol (16). Commercially available amine

12 (1.00 equiv, 30.0 mg, 0.155 mmol) was dissolved in DMF (0.5

mL), followed by the dropwise addition of N,N-diisopropylethylamine

(0.90 equiv, 24 μL, 0.140 mmol), and the resulting mixture was

stirred for 5 min at rt. A solution of 4-(4-(2-ﬂuoropropyl)phenyl)-

butyl methanesulfonate (0.90 equiv, 40.0 mg, 0.140 mmol) in DMF

(0.5 mL) was added dropwise. The resulting mixture was stirred at 90

°C for 3 h and subsequently diluted with water and MeCN (1:1, 4.5

mL). Puriﬁcation was carried out by preparative HPLC to give the

title compound 16 (32.0 mg, 0.083 mmol, 54% yield). ¹H NMR (400

MHz, CDCl₃): δ 7.18−7.09 (m, 5H), 6.68−6.62 (m, 2H), 4.56 (d, J =

4-(4-(3-Fluoropropyl)phenyl)butyl Methanesulfonate (11b). To a

solution of 10b (1.00 equiv, 157 mg, 0.747 mmol) in DCM (15 mL),

methanesulfonyl chloride (5 equiv, 0.29 mL, 3.73 mmol) was added

dropwise. After 5 min of stirring at rt, TEA (2.00 equiv, 0.21 mL, 1.49

mmol) was added, and the reaction mixture was allowed to stir for an

additional 30 min at rt. The mixture was diluted with EtOAc (100

mL), washed with aq. HCl (0.03 M, 3 × 100 mL), and dried over

anhydrous magnesium sulfate. The solvents were removed under

reduced pressure and the crude was puriﬁed by ﬂash column

chromatography (silica gel; Hex/EtOAc 8:2) to obtain the title

1

compound 11b (193 mg, 0.672 mmol, 90%). H NMR (400 MHz,

(CD₃)₂CO): δ 1.69−1.81 (m, 4H), 1.81−1.90 (m, 2H), 2.62−2.71

(m, 4H), 3.06 (s, 3H), 4.25 (t, J = 6.0 Hz, 2H), 4.38 (t, J = 5.9 Hz,

1H), 4.50 (t, J = 5.9 Hz, 1H), 7.15 (s, 4H). ¹³C NMR (100 MHz,

(CD₃)₂CO): δ 28.3, 29.5, 31.6, 33.0, 35.5, 37.1, 71.1, 83.8, 129.3,

129.4, 139.7, 140.6. HRMS: calculated for C₁₄H₂₅FNO₃S [M+NH₄]⁺

306.1534, found 306.1534.

3-(4-(4-Fluorophenyl)butyl)-7-methoxy-2,3,4,5-tetrahydro-1H-

benzo[d]azepin-1-ol (13). To a solution of commercially available

N

DOI: 10.1021/acs.jmedchem.9b00812

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Article

6.9 Hz, 1H), 4.46 (dt, J₁= 47.1 Hz, J₂= 5.8 Hz, 2H), 3.78 (s, 3H),

3.31−3.21 (m, 1H), 3.19−3.11 (m, 1H), 3.06−3.00 (m, 1H), 2.72 (t,

J = 7.6 Hz, 2H), 2.68−2.56 (m, 5H), 2.50 (d, J = 12.1 Hz, 1H), 2.40

(t, J = 11.9 Hz, 1H), 2.08−1.92 (m, 2H), 1.70−1.50 (m, 4H), signal

for the OH is not visible. ¹³C NMR (100 MHz, CDCl₃): δ 159.1,

141.4, 140.7, 138.6, 135.8, 129.8, 128.6 (2C), 128.6 (2C), 116.8,

110.4, 83.3 (d, J = 164.6), 72.6, 61.0, 59.7, 56.3, 55.3, 37.1, 36.6, 35.1,

32.3, 29.8, 26.8. HRMS (ESI) calculated for C₂₄H₃₃FNO₂[M + H]⁺

386.2490, found 386.2492.

mixture of water and MeCN (1:1, 4.5 mL). Puriﬁcation was

performed by preparative HPLC to give the title compound 21 (9.0

mg, 0.026 mmol, 25% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.15−

7.07 (m, 3H), 6.99−6.92 (m, 2H), 6.75 (d, J = 7.8, 1H), 6.67 (d, J =

7.8, 1H), 5.51 (d, J = 6.9 Hz, 1H), 3.80 (s, 3H), 3.36−3.27 (m, 1H),

3.26−3.19 (m, 1H), 3.07−3.00 (m, 1H), 2.71−2.55 (m, 5H), 2.42 (d,

J = 12.1 Hz, 1H), 2.35 (t, J = 11.9 Hz, 1H), 1.70−1.50 (m, 4H), signal

for the OH is not visible. ¹³C NMR (100 MHz, CDCl₃): δ 161.3 (d, J

= 243.1), 157.6, 142.1, 138.0, 131.4, 129.8, 129.8, 128.4, 122.9, 115.3,

115.1, 109.7, 62.3, 60.1, 59.7, 56.1, 56.1, 37.1, 35.1, 29.4, 26.8. HRMS

(ESI) calculated for C₂₁H₂₇FNO₂[M + H]⁺344.2020, found

344.2022.

9-Methoxy-3-tosyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-one

(18). The synthesis and puriﬁcation of ketone 18 were performed

according to the published procedure but with minor modiﬁcations.³²

Acid 17 (1.00 equiv, 50.0 g, 138 mmol) was dissolved in DCM (500

mL), and the solution was cooled to 0 °C. Subsequent addition of

P₂O₅(5.00 equiv, 98.0 g, 690 mmol) and stirring at 0 °C for 24 h was

followed by addition of NaOH (3%) to give a pH of 13 and extraction

with DCM (3 × 500 mL). Puriﬁcation with ﬂash column

chromatography (silica gel; DCM:EtOAc 20:1) and iterative

recrystallization from EtOH gave the title compound 18 (920 mg,

N-Methyl-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]-

annulen-7-amine (24). Formic acid (10.0 equiv, 118 μL, 3.08 mmol)

was added to a solution of 23 (1.00 equiv, 86.0 mg, 0.308 mmol) in

formaldehyde (10.0 equiv, 229 μL, 3.08 mmol), which was reﬂuxed at

100 °C for 16 h. The solvent was subsequently evaporated under

reduced pressure and diluted with 2 M aq. NaOH (100 mL), which

was extracted with chloroform (4 × 100 mL). The combined organic

layers were washed with brine (200 mL), dried over anhydrous

sodium sulfate, ﬁltered, and concentrated under reduced pressure.

The resulting crude was puriﬁed via ﬂash column chromatography

(silica gel; Hex/EtOAc 6:4) to yield the title compound 24 (72.0 mg,

1

2.67 mmol, 11% yield). H NMR (400 MHz, CDCl₃): δ 7.47−7.42

(m, 2H), 7.33−7.27 (m, 1H), 7.18−7.12 (m, 2H), 6.78 (d, J = 7.8,

1H), 6.70 (d, J = 7.8, 1H), 4.09 (s, 2H), 3.76 (s, 3H), 3.53 (t, J = 6.9,

2H), 2.82 (t, J = 6.9, 2H), 2.39 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 202.5, 157.6, 143.2, 137.8, 135.9, 133.1, 129.8 (2C), 126.9

(2C), 126.2, 120.7, 111.1, 55.9, 55.4, 46.6, 32.1, 21.7. HRMS (ESI)

calculated for C₁₈H₂₀NO₄S [M + H]⁺346.1108, found 346.1105.

9-Methoxy-3-tosyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol

(19). Compound 18 (1.00 equiv, 450 mg, 1.30 mmol) was suspended

in MeOH (6.8 mL), and NaBH₄(2.00 equiv, 99.0 mg, 2.61 mmol)

was added portionwise to the suspension. The reaction mixture was

stirred for 2 h at rt. The solvent was evaporated under reduced

pressure. Subsequently, water (250 mL) was added, and an extraction

was carried out with DCM (3 × 250 mL). The combined organic

layers were dried over anhydrous magnesium sulfate and then

concentrated under reduced pressure to give the title compound 19

1

0.245 mmol, 80% yield). H NMR (400 MHz, CDCl₃): δ 7.31−7.26

(m, 2H), 7.23−7.15 (m, 3H), 7.15−7.09 (m, 4H), 2.84−2.61 (m,

7H), 2.50−2.41 (m, 2H), 2.23 (s, 3H), 2.11−2.02 (m, 2H), 1.80 (p, J

= 7.6 Hz, 2H), 1.43−1.30 (m, 2H). ¹³C NMR (101 MHz, CDCl₃): δ

142.8 (2C), 142.6, 129.0 (2C), 128.5 (2C), 128.4 (2C), 126.4 (2C),

125.8, 67.8, 53.3, 37.8, 33.8, 33.6 (2C), 30.3, 30.0 (2C). HRMS (ESI)

calculated for C₂₁H₂₈N [M + H]⁺294.2216, found 294.2218.

N-(2-Fluoroethyl)-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-

benzo[7]annulen-7-amine (25). 2-Fluoroethyl 4-methylbenzenesul-

fonate (1.50 equiv, 176 mg, 0.805 mmol) and Cs₂CO₃(1.50 equiv,

262 mg, 0.805 mmol) were added to a solution of 23 (1.00 equiv, 150

mg, 0.537 mmol) in DMF (0.4 mL). The solution was left to stir

overnight at 90 °C. Thereafter, the reaction mixture was diluted with

ice-cold water (110 mL) and extracted with DCM (3 × 35 mL).

Afterward, the combined organic layers were washed with brine (50

mL), dried over anhydrous sodium sulfate, ﬁltered, and concentrated

at 65 °C under reduced pressure. The crude was puriﬁed via ﬂash

column chromatography (silica gel; DCM) to yield the title

1

(440 mg, 1.27 mmol, 97% yield). H NMR (400 MHz, CDCl₃): δ

7.70−7.62 (m, 2H), 7.33−7.27 (m, 2H), 7.17−7.12 (m, 1H), 6.78 (d,

J = 7.8, 1H), 6.67 (d, J = 7.8, 1H), 5.61 (d, J = 6.9, 1H), 4.09−4.00

(m, 1H), 3.94−3.87 (m, 1H), 3.79 (s, 3H), 3.48−3.39 (m, 1H), 3.53,

3.12−3.05 (m, 1H), 2.95−2.74 (m, 3H), 2.38 (s, 3H). ¹³C NMR (100

MHz, CDCl₃): δ 157.6, 143.2, 140.3, 135.9, 129.8 (2C), 129.1, 128.6,

126.9 (2C), 122.9, 109.7, 64.2, 55.9, 51.0, 48.1, 35.2, 21.7. HRMS

(ESI) calculated for C₁₈H₂₁NNaO₄S [M + Na]⁺370.1083, found

370.1083.

1

compound 25 (88.0 mg, 0.270 mmol, 50% yield). H NMR (400

MHz, CDCl₃) δ 7.19−7.13 (m, 2H), 7.10−7.01 (m, 7H), 4.49 (d, J =

47.6 Hz, 2H), 4.34−3.92 (m, 3H), 3.16−2.58 (m, 6H), 2.53−2.39

(m, 2H), 2.03−1.88 (m, 2H), 1.85−1.72 (m, 2H), 1.53−1.39 (m,

2H). ¹³C NMR (101 MHz, CDCl₃): δ 142.1 (2C), 141.5, 129.1 (2C),

128.4 (2C), 128.3 (2C), 126.6 (2C), 126.0, 82.0 (d, J = 170.7 Hz),

64.2 (d, J = 19.6 Hz), 60.4, 43.1, 33.5, 33.2 (2C), 32.9 (2C), 32.2.

HRMS (ESI) calculated for C₂₂H₂₉FN [M + H]⁺326.2279, found

326.2277.

9-Methoxy-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol (20). To a

stirred solution of 19 (1.00 equiv, 190 mg, 0.547 mmol) in MeOH

(12 mL) was added activated Mg (50.0 equiv, 664 mg, 27.3 mmol)

portionwise, and the reaction mixture was reﬂuxed for 24 h. The

reaction mixture was ﬁltered, followed by the addition of aq. HCl (0.1

M, 2.0 mL). The solvents were evaporated under reduced pressure,

and the crude was redissolved in DCM (200 mL). Aq. NaOH (3%)

was added, and the aqueous phase was extracted with DCM (3 × 200

mL). The combined organic layers were dried over magnesium sulfate

and evaporated under reduced pressure. The crude product was

puriﬁed by ﬂash column chromatography (silica gel; DCM/MeOH

8:2, 4% triethylamine) to yield the title compound 20 (74.0 mg, 0.383

N¹-Phenyl-N²-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-yl)-

ethane-1,2-diamine (26). A solution of ketone 22 (1.00 equiv, 120

mg, 0.749 mmol) and N¹-phenylethane-1,2-diamine (1.50 equiv, 150

μL, 1.12 mmol) in DCM (29 mL) was stirred for 30 min, followed by

the addition of sodium triacetoxyborohydride (2.10 equiv, 333 mg,

1.57 mmol) and subsequent vigorous stirring overnight at rt. The

reaction mixture was diluted with sat. aq. NaHCO₃(30 mL) and was

extracted with DCM (3 × 30 mL). The organic phase was washed

with brine (30 mL), dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. The crude was puriﬁed by

gravity column chromatography (silica gel; Hex/EtOAc 7:3, 1% TEA)

1

mmol, 70% yield). H NMR (400 MHz, CDCl₃): δ 7.13−7.06 (m,

1H), 6.75 (d, J = 7.8, 1H), 6.67 (d, J = 7.8, 1H), 5.51 (d, J = 6.9, 1H),

3.79 (s, 3H), 3.40−3.32 (m, 2H), 3.29−3.22 (m, 1H), 2.95−2.73 (m,

3H), signals for the OH and NH are not visible. ¹³C NMR (100 MHz,

CDCl₃): δ 157.6, 143.2, 131.4, 129.1, 122.9, 109.7, 64.2, 55.9, 52.8,

49.4, 38.9. HRMS (ESI) calculated for C₁₁H₁₆NO₂[M + H]⁺

194.1176, found 194.1177.

1

to aﬀord compound 26 (187 mg, 0.667 mmol, 89% yield). H NMR

(400 MHz, CDCl₃) δ 7.12−7.04 (m, 2H), 7.00 (s, 4H), 6.61 (t, J =

7.3 Hz, 1H), 6.56−6.51 (m, 2H), 3.14−3.08 (m, 2H), 2.87−2.80 (m,

2H), 2.75−2.54 (m, 5H), 2.05−1.95 (m, 2H), 1.28−1.19 (m, 2H),

signals for the NH are not visible. ¹³C NMR (101 MHz, CD₃OD): δ

148.6, 142.1 (2C), 130.3 (2C), 130.0 (2C), 127.9 (2C), 119.3, 114.4

(2C), 62.7, 44.7, 41.5, 32.3 (2C), 31.7 (2C). HRMS (ESI) calculated

for C₁₉H₂₅N₂[M + H]⁺281.2012, found 281.2014.

3-(4-(4-Fluorophenyl)butyl)-9-methoxy-2,3,4,5-tetrahydro-1H-

benzo[d]azepin-1-ol (21). To a stirred solution of 20 (1.00 equiv,

20.0 mg, 0.103 mmol) in DMF (0.5 mL) was added dropwise DIPEA

(0.90 equiv, 16 μL, 0.093 mmol). The resulting mixture was stirred

for 5 min at rt before 4-(4-ﬂuorophenyl)butyl methanesulfonate (0.90

equiv, 23.0 mg, 0.093 mmol) in DMF (0.5 mL) was added dropwise.

The resulting solution was stirred at 90 °C for 3 h and diluted with a

O

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Journal of Medicinal Chemistry

Article

N-(3-(4-Fluorophenyl)propyl)-6,7,8,9-tetrahydro-5H-benzo[7]-

annulen-7-amine (27). A solution of ketone 22 (1.00 equiv, 100 mg,

0.624 mmol) and 3-(4-ﬂuorophenyl)propan-1-amine (2.00 equiv, 191

mg, 1.25 mmol) in DCM (6.1 mL) was stirred for 30 min before

sodium triacetoxyborohydride (2.00 equiv, 265 mg, 1.25 mmol) was

added. The reaction mixture was vigorously stirred for 18 h, followed

by dilution of the mixture with sat. aq. NaHCO₃(30 mL) and

subsequent extraction with DCM (3 × 30 mL). The combined

organic phases were washed with brine (30 mL), dried over

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure. The crude was puriﬁed with ﬂash chromatography (silica

gel; DCM/MeOH 98.5:1.5, 1% TEA) to aﬀord the title compound 27

¹H NMR (400 MHz, CDCl₃) δ 7.99−7.95 (m, 2H), 7.31−7.15 (m,

6H), 2.93 (dd, J = 14.4, 7.6 Hz, 2H), 2.78−2.69 (m, 3H), 2.66−2.60

(m, 2H), 2.47−2.41 (m, 2H), 2.22 (s, 3H), 2.14−2.02 (m, 2H), 1.78

(p, J = 7.6 Hz, 2H), 1.42−1.30 (m, 2H). ¹³C NMR (101 MHz,

CDCl₃): δ 150.7, 146.5, 144.3, 142.5, 129.8, 128.5 (2C), 128.4 (2C),

125.8, 123.8, 121.6, 67.2, 53.2, 37.6, 33.7, 33.5, 33.4, 30.2, 29.6, 29.5.

HRMS (ESI) calculated for C₂₁H₂₇N₂O₂[M + H]⁺339.2067, found

339.2070.

4-Fluoro-N-(7-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)-

benzamide (34). Amine 33 (1.00 equiv, 160 mg, 0.913 mmol) was

dissolved in DCM (1.8 mL) and cooled to 0 °C. TEA (1.00 equiv,

140 μL, 0.913 mmol) was added to the solution, followed by 4-

ﬂuorobenzoyl chloride (0.99 equiv, 110 μL, 0.911 mmol) slowly

dropwise and was subsequently stirred for 11 h at rt. The mixture was

diluted with DCM (25 mL), washed with sat. aq. NaHCO₃(3 × 10

mL) and then with brine (15 mL), dried over anhydrous sodium

sulfate, ﬁltered, and concentrated under reduced pressure. The crude

was puriﬁed by ﬂash column chromatography (silica gel; Hex/EtOAc

6:4, 1% aq. NH₃) to aﬀord compound 34 (87.0 mg, 0.291 mmol, 32%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.92−7.85 (m, 2H), 7.60 (d, J

= 2.3 Hz, 1H), 7.40 (dd, J = 8.1, 2.3 Hz, 1H), 7.22 (d, J = 8.1 Hz,

1H), 7.20−7.15 (m, 2H), 2.95−2.84 (m, 4H), 2.65−2.56 (m, 4H),

signal for the NH is not visible. ¹³C NMR (101 MHz, CDCl₃) δ

211.2, 165.1 (d, J = 252.4 Hz), 164.8, 141.7, 137.2, 136.8, 130.0 (2C),

129.5 (d, J = 9.6 Hz, 2C), 121.2, 118.9, 116.1 (d, J = 22.4 Hz, 2C),

44.8, 44.6, 30.9, 30.2. HRMS (ESI) calculated for C₁₈H₁₇FNO₂[M +

H]⁺298.1238, found 298.1241.

4-Fluoro-N-(7-((3-phenylpropyl)amino)-6,7,8,9-tetrahydro-5H-

benzo[7]annulen- 2-yl)benzamide (35). A solution of 34 (1.00

equiv, 75.0 mg, 0.252 mmol) and 3-phenylpropan-1-amine (2.00

equiv, 72 μL, 0.504 mmol) in DCM (9.0 mL) was stirred for 30 min

before sodium triacetoxyborohydride (2.00 equiv, 107 mg, 0.504

mmol) was added and vigorously stirred for 18 h at rt. Sat. aq.

NaHCO₃(100 mL) was added, and the reaction mixture was

extracted with DCM (3 × 60 mL). The organic phase was washed

with brine (200 mL), dried over anhydrous sodium sulfate, ﬁltered,

and concentrated under reduced pressure. The crude was puriﬁed by

ﬂash column chromatography (silica gel; DCM/MeOH 9:1, 1% aq.

NH₃) to aﬀord compound 35 (90.0 mg, 0.215 mmol, 85% yield). ¹H

NMR (400 MHz, CDCl₃) δ 7.89−7.80 (m, 2H), 7.43 (d, J = 2.1 Hz,

1H), 7.37−7.23 (m, 3H), 7.21−7.15 (m, 3H), 7.14−7.04 (m, 3H),

2.91 (t, J = 10.4 Hz, 1H), 2.84−2.70 (m, 4H), 2.70−2.59 (m, 4H),

2.30−2.09 (m, 2H), 1.99 (p, J = 7.6 Hz, 2H), 1.49−1.35 (m, 2H),

signals for the NH are not visible. ¹³C NMR (101 MHz, CDCl₃) δ

164.9 (d, J = 252.7 Hz), 164.7, 143.4, 142.0, 139.2, 135.9 (2C), 131.4,

129.7, 129.5 (d, J = 9.1 Hz, 2C), 128.5 (2C), 126.0 (2C), 121.1,

118.1, 115.9 (d, J = 22.7 Hz, 2C), 61.4, 46.5, 34.3, 34.2, 33.8 (2C),

32.4, 31.8. HRMS (ESI) calculated for C₂₇H₃₀FN₂O [M + H]⁺

417.2337, found 417.2344.

1

(150 mg, 0.504 mmol, 81% yield). H NMR (400 MHz, CDCl₃) δ

7.17−7.08 (m, 6H), 7.00−6.93 (m, 2H), 2.88−2.61 (m, 9H), 2.12−

2.01 (m, 2H), 1.84−1.73 (m, 2H), 1.35−1.23 (m, 2H), signal for the

NH is not visible. ¹³C NMR (101 MHz, CDCl₃): δ 161.4 (d, J =

243.8 Hz), 142.7 (2C), 137.9, 129.8 (d, J = 8.6 Hz, 2C), 129.0 (2C),

126.3 (2C), 115.2 (d, J = 20.4 Hz, 2C), 61.4, 46.6, 34.7 (2C), 33.0,

32.4 (2C), 29.8. HRMS (ESI) calculated for C₂₀H₂₅FN [M + H]⁺

298.1966, found 298.1969.

N-(3-(4-Fluorophenyl)propyl)-N-methyl-6,7,8,9-tetrahydro-5H-

benzo[7]annulen-7-amine (28). Formic acid (17.0 equiv, 0.5 mL,

13.03 mmol) was added to a solution of 27 (1.00 equiv, 228 mg,

0.767 mmol) in formaldehyde (17.0 equiv, 1.0 mL, 13.03 mmol),

which was reﬂuxed at 100 °C for 16 h. The solvent was subsequently

evaporated under reduced pressure, diluted with 2 M aq. NaOH (300

mL), and extracted with chloroform (4 × 300 mL). The combined

organic layers were washed with brine (500 mL), dried over

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure. The resulting crude was puriﬁed via ﬂash column

chromatography (silica gel; Hex/EtOAc 5:5) to yield compound 28

1

(118 mg, 0.379 mmol, 49% yield). H NMR (400 MHz, CDCl₃) δ

7.20−7.09 (m, 6H), 7.00−6.91 (m, 2H), 2.84−2.57 (m, 7H), 2.43 (t,

J = 7.3 Hz, 2H), 2.23 (s, 3H), 2.11−2.01 (m, 2H), 1.76 (p, J = 7.3 Hz,

2H), 1.41−1.30 (m, 2H). ¹³C NMR (101 MHz, CDCl₃): δ 161.3 (d, J

= 242.4 Hz), 142.8 (2C), 138.1, 129.8 (d, J = 8.2 Hz, 2C), 129.0

(2C), 126.4 (2C), 115.1 (d, J = 21.3 Hz, 2C), 67.9, 53.1, 37.8, 33.6

(2C), 32.9, 30.4, 29.9 (2C). HRMS (ESI) calculated for C₂₁H₂₇FN

[M + H]⁺312.2122, found 312.2117.

1-(4-Fluorophenyl)-4-(2-nitro-6,7,8,9-tetrahydro-5H-benzo[7]-

annulen-7-yl)piperazine (30). A solution of 29 (1.80 equiv, 150 mg,

0.731 mmol) and 1-(4-ﬂuorophenyl)piperazine (1.00 equiv, 73.0 mg,

0.406 mmol) in DCM (0.4 mL) was stirred for 30 min before sodium

triacetoxyborohydride (1.80 equiv, 155 mg, 0.731 mmol) was added

to the mixture and subsequently stirred vigorously overnight. The

reaction mixture was diluted with sat. aq. NaHCO₃(30 mL) and

extracted with DCM (3 × 30 mL). The combined organic layers were

washed using brine (30 mL), dried over anhydrous sodium sulfate,

and evaporated under reduced pressure. The resulting crude was

puriﬁed via ﬂash chromatography (silica gel; DCM/MeOH 100:0,

99:1). The titled compound 30 was then obtained by decanting using

N-(7-Oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)-

benzamide (36). Amine 33 (1.00 equiv, 160 mg, 0.913 mmol) was

dissolved in DCM (1.8 mL) and cooled to 0 °C. TEA (1.00 equiv,

140 μL, 0.913 mmol) was added to the solution, followed by benzoyl

chloride (0.99 equiv, 106 μL, 0.911 mmol) slowly dropwise, and the

reaction was stirred for 11 h at rt. The mixture was diluted with DCM

(25 mL), washed with sat. aq. NaHCO₃(3 × 10 mL) and then with

brine (15 mL), dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. The crude was puriﬁed by ﬂash

column chromatography (silica gel; Hex/EtOAc 6:4, 1% aq. NH₃) to

1

MeCN and water (61.0 mg, 0.165 mmol, 41% yield). H NMR (400

MHz, CDCl₃) δ 8.02−7.94 (m, 2H), 7.26 (d, J = 8.1 Hz, 1H), 7.00−

6.83 (m, 4H), 3.19−2.92 (m, 6H), 2.85−2.59 (m, 7H), 2.24−2.08

(m, 2H), 1.54−1.42 (m, 2H). ¹³C NMR (101 MHz, CDCl₃): δ 157.3

(d, J = 238.8 Hz), 150.6, 148.2, 146.6, 144.2, 129.9, 123.9, 121.7,

118.0 (d, J = 7.3 Hz, 2C), 115.6 (d, J = 21.9 Hz, 2C), 67.6, 51.0 (2C),

48.8 (2C), 33.1, 33.0, 29.8, 29.7. HRMS (ESI) calculated for

C₂₁H₂₅FN₃O₂[M + H]⁺370.1925, found 370.1927.

N-Methyl-2-nitro-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-

benzo[7]annulen-7-amine (32). Formic acid (17.0 equiv, 0.3 mL,

8.91 mmol) was added to a solution of 31 (1.00 equiv, 170 mg, 0.524

mmol) in formaldehyde (17.0 equiv, 0.7 mL, 8.91 mmol) and was

reﬂuxed at 100 °C for 16 h. The solvent was evaporated under

reduced pressure, and the residue was resuspended in 2 M aq. NaOH

(300 mL) and extracted with chloroform (4 × 100 mL). Combined

organic layers were washed using brine (100 mL), dried over

anhydrous sodium sulfate, and concentrated in vacuo. The residue

was puriﬁed via ﬂash chromatography (silica gel; DCM/MeOH 95:5,

1% NH₃) to aﬀord compound 32 (161 mg, 0.476 mmol, 91% yield).

1

aﬀord compound 36 (90.0 mg, 0.323 mmol, 35% yield). H NMR

(400 MHz, CDCl₃) δ 7.89−7.84 (m, 2H), 7.62 (d, J = 2.2 Hz, 1H),

7.58−7.53 (m, 1H), 7.52−7.46 (m, 2H), 7.42 (dd, J = 8.1, 2.2 Hz,

1H), 7.24−7.18 (m, 1H), 2.93−2.80 (m, 4H), 2.64−2.52 (m, 4H),

signal for the NH is not visible. ¹³C NMR (101 MHz, CDCl₃) δ

211.3, 165.9, 141.6, 137.0, 135.0, 132.6, 132.0, 130.0, 129.0 (2C),

127.1 (2C), 121.2, 118.9, 44.8, 44.6, 30.8, 30.2. HRMS (ESI)

calculated for C₁₈H₁₈NO₂[M + H]⁺280.1332, found 280.1333.

N-(7-((3-Phenylpropyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]-

annulen-2-yl)benzamide (37). A solution of 36 (1.00 equiv, 79.0 mg,

0.281 mmol) and 3-phenylpropan-1-amine (2.00 equiv, 80 μL, 0.562

P

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mmol) in DCM (10 mL) was stirred for 30 min before sodium

triacetoxyborohydride (2.00 equiv, 119 mg, 0.562 mmol) was added

and vigorously stirred for 16 h at rt. Sat. aq. NaHCO₃(100 mL) was

added, and the reaction mixture was extracted with DCM (3 × 60

mL). The combined organic phases were washed with brine (200

mL), dried over anhydrous sodium sulfate, ﬁltered, and concentrated

under reduced pressure. The crude was puriﬁed by ﬂash column

chromatography (silica gel; DCM/MeOH 9:1, 1% aq. NH₃) to aﬀord

2H), signal for the NH is not visible. ¹³C NMR (101 MHz, CDCl₃) δ

142.2, 131.6 (2C), 130.3 (2C), 129.1 (4C), 126.5 (2C), 119.8, 61.5,

45.8, 33.5, 33.0 (2C), 32.3 (2C), 31.0. HRMS (ESI) calculated for

C₂₀H₂₅BrN [M + H]⁺358.1165, found 358.1167.

Tert-butyl(6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-yl)(3-(4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propyl)-

carbamate (43). Compound 42 (1.00 equiv, 236 mg, 0.658 mmol),

4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.20

equiv, 201 mg, 0.790 mmol), PdCl₂(dppf)-DCM Adduct (0.05

equiv, 27.2 mg, 0.033 mmol), and potassium acetate (4.00 equiv, 258

mg, 2.63 mmol) were suspended in DMF (5.8 mL) and stirred at 100

°C for 5 h. The reaction mixture was then cooled and diluted with

water (200 mL) and subsequently extracted with methyl-tert-butyl

ether (MTBE, 3 × 60 mL). The combined organic phases were

washed with brine (200 mL), dried over anhydrous sodium sulfate,

ﬁltered, and concentrated under reduced pressure. The brown crude

was dried overnight under high vacuum. The crude (249 mg) was

dissolved in MeCN (2.1 mL) before adding di-tert-butyl dicarbonate

(0.40 mL, 1.84 mmol), N,N-dimethylpyridin-4-amine (15.0 mg, 0.123

mmol), and TEA (0.30 mL, 1.84 mmol). The mixture was stirred for

17 h at rt. Afterward, the mixture was concentrated under reduced

pressure, and the residue was dissolved in EtOAc (5 mL), washed

once with sat. aq. NH₄Cl (5 mL), once with water (5 mL), and then

once with brine (5 mL). The organic phase was then dried over

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure. The crude was puriﬁed by ﬂash column chromatography

(silica gel; Hex/EtOAc 9:1) to aﬀord compound 43 (293 mg, 0.579

mmol, 34% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J = 7.9 Hz,

2H), 7.16−7.08 (m, 6H), 4.34−4.18 (m, 1H), 3.04 (d, J = 52.9 Hz,

2H), 2.87−2.66 (m, 4H), 2.52 (t, J = 7.6 Hz, 2H), 2.03−1.92 (m,

2H), 1.80 (p, J = 7.6 Hz, 2H), 1.45 (s, 9H), 1.33 (s, 12H), 1.29−1.23

(m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 155.3, 145.6, 142.3, 135.1

(2C), 129.1 (4C), 127.8 (2C), 126.6 (2C), 83.8, 79.5 (2C), 64.2,

43.2, 34.0 (2C), 33.4 (2C), 33.2, 32.4, 28.7 (3C), 25.0 (4C). HRMS

(ESI) calculated for C₃₁H₄₅BNO₄[M + H]⁺506.3442, found

506.3454.

3-(4-(4-Iodophenyl)butyl)-7-methoxy-2,3,4,5- tetrahydro-1H-

benzo[d]azepin-1-ol (46). TEA (1.80 equiv, 4.00 mL, 28.6 mmol)

was added to a solution of 45 (1.00 equiv, 4.39 g, 15.9 mmol)

dissolved in DCM (160 mL). Under cooling with ice, MsCl (1.03

equiv, 1.30 mL, 16.5 mmol) was added dropwise. After 5 min of

stirring, the reaction mixture was allowed to come to rt, and stirring

was continued for 30 min. The reaction mixture was quenched with

water (200 mL) and extracted with DCM (3 × 200 mL). The

combined organic layers were washed with brine, dried over

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure. The resulting residue and 12 (350 mg, 1.81 mmol) were

dissolved in DMF (18 mL) before the dropwise addition of N,N-

diisopropylethylamine (276 μL, 1.58 mmol). The reaction mixture

was reﬂuxed at 90 °C for 2.5 h before the mixture was diluted with 1

mM aq. NaOH (400 mL) and extracted with DCM (3 × 300 mL).

The combined organic layers were washed with brine (500 mL), dried

over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. The crude was puriﬁed by ﬂash column

chromatography (silica gel; Hex/EtOAc 8:2 to 2:8, 0.1% aq. NH₃),

resulting in the title compound 46 (459 mg, 1.08 mmol, 56% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.63−7.55 (m, 2H), 7.10 (d, J = 8.0

1

compound 37 (80.0 mg, 0.200 mmol, 71% yield). H NMR (400

MHz, CD₃OD) δ 7.94−7.89 (m, 2H), 7.60−7.54 (m, 1H), 7.53−7.47

(m, 2H), 7.46−7.39 (m, 2H), 7.30−7.25 (m, 2H), 7.23−7.14 (m,

3H), 7.10 (d, J = 8.0 Hz, 1H), 2.93−2.64 (m, 9H), 2.24−2.09 (m,

2H), 1.87 (p, J = 7.6 Hz, 2H), 1.34−1.21 (m, 2H), signals for the NH

are not visible. ¹³C NMR (101 MHz, CD₃OD) δ 165.6, 143.9, 143.0,

139.9, 138.0, 136.4, 132.8, 130.2, 129.6 (2C), 129.5 (2C), 129.4

(2C), 128.6 (2C), 127.0, 122.9, 120.2, 62.8, 47.0, 34.5 (2C), 34.3,

33.3, 32.6, 31.9. HRMS (ESI) calculated for C₂₇H₃₁N₂O [M + H]⁺

399.2431, found 399.2432.

N-(7-((3-(4-Fluorophenyl)propyl)amino)-6,7,8,9-tetrahydro-5H-

benzo[7]annulen-2-yl)benzamide (38). A solution of 36 (1.00 equiv,

9.00 mg, 0.032 mmol) and 3-(4-ﬂuorophenyl)propan-1-amine (2.00

equiv, 10 μL, 0.064 mmol) in DCM (1.2 mL) was stirred for 30 min

before sodium triacetoxyborohydride (2.00 equiv, 14.0 mg, 0.064

mmol) was added and vigorously stirred for 18 h at rt. Sat. aq.

NaHCO₃(15 mL) was added, and the reaction mixture was extracted

with DCM (3 × 10 mL). The combined organic phases were washed

with brine (30 mL), dried over anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. The crude was puriﬁed by

gravity column chromatography (silica gel; Hex/EtOAc 9:1, 1% aq.

NH₃) to aﬀord compound 38 (10.0 mg, 0.023 mmol, 72% yield). ¹H

NMR (400 MHz, CD₃OD) δ 7.39−7.34 (m, 2H), 7.32−7.27 (m,

2H), 7.25−7.18 (m, 3H), 7.04−6.98 (m, 2H), 6.83 (d, J = 8.0 Hz,

1H), 6.47 (d, J = 2.4 Hz, 1H), 6.38 (dd, J = 8.0, 2.4 Hz, 1H), 2.89 (tt,

J = 10.8, 3.3 Hz, 1H), 2.81−2.75 (m, 2H), 2.71−2.62 (m, 6H), 2.20−

2.07 (m, 2H), 1.86 (p, J = 7.6 Hz, 2H), 1.27−1.15 (m, 2H), signals

for the NH are not visible. ¹³C NMR (101 MHz, CDCl₃) δ 166.0,

161.6 (d, J = 244.1 Hz), 142.0, 137.3, 136.8, 136.1, 135.1, 131.9,

129.9 (d, J = 8.3 Hz, 3C), 128.8 (2C), 127.2 (2C), 121.0, 118.4, 115.5

(d, J = 21.9 Hz, 2C), 61.8, 44.6, 32.4 (2C), 32.1 (2C), 31.4, 29.9.

HRMS (ESI) calculated for C₂₇H₃₀FN₂O [M + H]⁺417.2337, found

417.2331.

1-(4-Fluorophenyl)-4-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-

7-yl)piperazine (41). A solution of 40 (1.00 equiv, 235 mg, 0.676

mmol) and 1-(4-ﬂuorophenyl)piperazine (4.90 equiv, 0.6 mL, 3.31

mmol) in MeCN (15 mL) was stirred vigorously for 16 h at 80 °C.

The reaction mixture was concentrated in vacuo and puriﬁed by

gravity column chromatography (silica gel; DCM/MeOH 99:1, 1%

NH₃) to aﬀord the title compound 41 (70.0 mg, 0.216 mmol, 32%

yield). ¹H NMR (400 MHz, CDCl₃) δ 7.15−7.10 (m, 4H), 7.01−6.82

(m, 4H), 3.17−3.05 (m, 4H), 2.93−2.70 (m, 9H), 2.22−2.08 (m,

2H), 1.53−1.41 (m, 2H). ¹³C NMR (101 MHz, CDCl₃): δ 157.3 (d, J

= 238.6 Hz), 148.3, 142.7 (2C), 129.1, 128.5 (2C), 126.4, 117.9 (d, J

= 8.5 Hz, 2C), 115.6 (d, J = 22.1 Hz, 2C), 68.4, 51.0 (2C), 48.7 (2C),

33.4 (2C), 30.2 (2C). HRMS (ESI) calculated for C₂₁H₂₆FN₂[M +

H]⁺325.2075, found 325.2079.

N-(3-(4-Bromophenyl)propyl)-6,7,8,9-tetrahydro-5H-benzo[7]-

annulen-7-amine (42). A solution of commercially available ketone

22 (1.00 equiv, 371 mg, 2.31 mmol) and 3-(4-bromophenyl)propan-

1-amine (2.00 equiv, 710 μL, 4.63 mmol) in DCM (8.2 mL) was

stirred for 30 min before sodium triacetoxyborohydride (1.50 equiv,

738 mg, 3.48 mmol) was added and vigorously stirred for 16 h at rt.

Sat. aq. NaHCO₃(90 mL) was added, and the reaction mixture was

extracted with DCM (3 × 75 mL). The combined organic phases

were washed with brine (180 mL), dried over anhydrous sodium

sulfate, ﬁltered, and concentrated under reduced pressure. The crude

was puriﬁed by ﬂash column chromatography (silica gel; DCM/

MeOH 9:1, 1% aq. NH₃) to aﬀord compound 42 (248 mg, 0.691

Hz, 1H), 6.98−6.90 (m, 2H), 6.72−6.59 (m, 2H), 4.57 (d, J = 6.7 Hz,

1H), 3.78 (s, 3H), 3.32−3.21 (m, 1H), 3.19−3.09 (m, 1H), 3.03−

2.94 (m, 1H), 2.65 (dd, J = 15.2, 5.9 Hz, 1H), 2.59 (t, J = 7.2 Hz,

4H), 2.50 (d, J = 11.7 Hz, 1H), 2.40 (t, J = 11.7 Hz, 1H), 1.66−1.51

(m, 4H), signal for OH is not visible. ¹³C NMR (101 MHz, CDCl₃) δ

159.1, 142.0, 141.3, 137.5 (2C), 135.7, 130.7 (2C), 129.9, 116.7,

110.3, 90.9, 72.6, 60.9, 59.7, 56.3, 55.3, 37.0, 35.4, 29.0, 26.7. HRMS

(ESI) calculated for C₂₁H₂₇INO₂[M + H]⁺452.1081, found

452.1083.

3-(4-(2-Bromophenyl)butyl)-7-methoxy-2,3,4,5-tetrahydro-1H-

benzo[d]azepin-1-yl Acetate (47). Hydroxyl 46 (1.00 equiv, 455 mg,

1.01 mmol) and DMAP (0.22 equiv, 27.0 mg, 0.222 mmol) were

dissolved in DCM (25 mL), before the dropwise addition of TEA

1

mmol, 30% yield). H NMR (400 MHz, CDCl₃) δ 7.41−7.36 (m,

2H), 7.13−7.03 (m, 6H), 2.88−2.68 (m, 6H), 2.67−2.58 (m, 3H),

2.21−2.06 (m, 2H), 1.90 (p, J = 7.6 Hz, 2H), 1.37 (q, J = 11.4 Hz,

Q

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Journal of Medicinal Chemistry

Article

(4.10 equiv, 0.60 mL, 4.15 mmol) and acetic anhydride (2.00 equiv,

0.20 mL, 2.02 mmol). After stirring at rt overnight, the reaction

mixture was diluted with sat. aq. NaHCO₃(50 mL) and extracted

with DCM (3 × 75 mL). The combined organic layers were washed

with brine (180 mL), dried over anhydrous sodium sulfate, ﬁltered,

and concentrated under reduced pressure. The crude was puriﬁed by

ﬂash column chromatography (silica gel; Hex/EtOAc 7:3, 0.1% aq.

NH₃) aﬀording 47 (341 mg, 0.692 mmol, 69% yield). ¹H NMR (400

MHz, CDCl₃) δ 7.65−7.48 (m, 2H), 7.18 (d, J = 8.2 Hz, 1H), 6.97−

6.86 (m, 2H), 6.76−6.60 (m, 2H), 5.90−5.81 (m, 1H), 3.79 (s, 3H),

3.10 (dd, J = 14.1, 9.4 Hz, 1H), 2.96 (dd, J = 12.6, 7.5 Hz, 1H), 2.88−

2.77 (m, 1H), 2.76−2.60 (m, 3H), 2.55 (q, J = 8.5, 7.9 Hz, 4H), 2.12

(s, 3H), 1.66−1.56 (m, 2H), 1.55−1.43 (m, 2H). ¹³C NMR (101

MHz, CDCl₃) δ 170.3, 159.3, 142.3, 137.4 (3C), 131.5, 130.7 (2C),

128.6, 116.0, 110.5, 90.8, 75.0, 58.4 (2C), 55.3, 55.2, 36.0, 35.4, 29.1,

26.5, 21.5. HRMS (ESI) calculated for C₂₃H₂₉INO₃[M + H]⁺

494.1187, found 494.1179.

7-Methoxy-3-(4-(4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-

yl)phenyl)butyl)-2,3,4,5 tetrahydro-1H-benzo[d]azepin-1-yl Ace-

tate (48). Compound 47 (1.00 equiv, 310 mg, 0.628 mmol),

4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.20

equiv, 197 mg, 0.777 mmol), potassium acetate (4.00 equiv, 246

mg, 2.51 mmol), and PdCl₂(dppf)-DCM adduct (0.05 equiv, 27.2 mg,

0.033 mmol) were dissolved in DMF (7.2 mL) and stirred under

reﬂux at 100 °C for 5 h before the reaction mixture was diluted with

water (100 mL) and extracted with MTBE (3 × 225 mL). The

combined organic layers were washed with brine (600 mL), dried

over anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. The crude was puriﬁed by ﬂash column

chromatography (silica gel; Hex/EtOAc 7:3, 0.1% aq. NH₃) aﬀording

48 (246 mg, 0.499 mmol, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ

7.72 (d, J = 8.0 Hz, 2H), 7.18 (dd, J = 8.0, 6.2 Hz, 3H), 6.74−6.59

(m, 2H), 5.86 (d, J = 6.4 Hz, 1H), 3.78 (s, 3H), 3.10 (dd, J = 14.1, 8.9

Hz, 1H), 2.97 (dd, J = 12.6, 7.4 Hz, 1H), 2.81 (dd, J = 14.5, 7.9 Hz,

1H), 2.72−2.59 (m, 5H), 2.58−2.52 (m, 2H), 2.11 (s, 3H), 1.63 (p, J

= 7.2 Hz, 2H), 1.56−1.45 (m, 2H), 1.34 (s, 12H). ¹³C NMR (101

MHz, CDCl₃) δ 170.3, 159.3, 146.1, 142.4, 135.0 (2C), 131.5, 128.7

(2C), 128.0, 115.9, 110.5, 83.7 (2C), 75.1, 60.5, 58.6, 58.5, 55.3, 55.2,

36.1, 36.0, 29.2, 26.6, 25.0 (4C), 21.5. HRMS (ESI) calculated for

C₂₉H₄₁BNO₅[M + H]⁺494.3077, found 494.3082.

In Vitro Binding Assay. The binding assay for GluN2B

containing NMDARs followed previously published procedures.^38,57

In brief, mouse L(tk−) cells carrying the stably transfected

dexamethasone-inducible eukaryotic expression vectors pMSG

NR1a and pMSG NR2B in a 1:5 ratio were employed. The

competitive binding assay was performed with the radioligand

[³H]ifenprodil (60 Ci/mmol, PerkinElmer) using standard 96-well

multiplates (Diagonal, Muenster, Germany). For the aﬃnity

determination toward σ1 and σ2 receptors, a previously published

and well-established binding assay was used.⁵⁸⁻⁶⁰The radioligands

used were [³H](+)-pentazocine and [³H]di-o-tolylguanidine for σ1

and σ2 receptor binding assays, respectively. Triplicate measurements

are reported with standard error of measurement (SEM). Alter-

natively, whole rat brain membranes were used for IC₅₀testing of the

compounds for the determination of the aﬃnity toward σ1Rs as

previously reported.⁶¹The whole brain rat membranes were prepared

as previously described.⁶²For determination of IC₅₀toward GluN2B

subunit-containing NMDARs, a dilution series of 8 diﬀerent

concentrations of the compounds in question was prepared, ranging

from 30 pM to 30 μM. [³H]ifenprodil was added to the diﬀerent

dilutions of the test ligand together with brain membranes (1 mg/mL

total protein concentration) in HEPES buﬀer (30 mM 4-(2-

hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt

(HEPES), 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM

MgCl₂, pH 7.4) in a total volume of 200 μL. Nonspeciﬁc binding was

determined in the presence of CP-101,606, while total binding was

attained in the exclusive presence of buﬀer and brain membranes. The

test samples were incubated at 25 °C for 60 min under mechanical

shaking (110 rpm). Termination of the incubation and removal of the

free radioligand were achieved by the further addition of 3 mL of

HEPES buﬀer, followed by vacuum ﬁltration through glass microﬁber

ﬁlter paper (Whatman, GF/C 25 mm, preincubated in 0.05%

polyethylenimine for 30 min). Afterward, scintillation ﬂuid (10 mL/

scintillation vial, Ultima Gold, Perkin-Elmer) was added to the ﬁlters,

and the activity was measured by a Packard 2200CA TRI-CARB

liquid scintillation analyzer.

Radiochemistry. [¹⁸F]ﬂuoride ions were produced by bombard-

ment of 98% enriched ¹⁸O-water via the e ¹⁸O(p,n)¹⁸F nuclear

reaction using a Cyclone 18/9 cyclotron (18-MeV; IBA Belgium).

Aqueous [¹⁸F]ﬂuoride was trapped on a preconditioned anion-

exchange cartridge (Waters SepPak Accell QMA cartridge carbonate)

and then eluted with a solution of kryptoﬁx 2.2.2 (6.3 mg/mL),

K₂C₂O₄(1 mg/mL), and K₂CO₃(0.1 mg/mL) in MeCN/H₂O (4:1,

0.9 mL), followed by azeotropic drying with MeCN (3 × 0.8 mL).⁵³

The reacti-vial was purged with air (20 mL), and the residue was

redissolved in a solution of 6−8 mg of boronic ester precursor, either

45 or 48, and 14 mg of Cu(OTf)₂(py)₄in 0.3 mL of dry

dimethylacetamide (DMA). The resulting solution was stirred at

120 °C for 20 min.

The Boc-protected [¹⁸F]49 crude was diluted with 2 mL of water/

MeCN (1:1) and then treated with 0.7 mL of 7 M HCl for 10 min at

95 °C. The formed [¹⁸F]27 was puriﬁed via semipreparative HPLC.

The collected fractions were diluted with 35 mL of water and trapped

on a C18 Light cartridge (Waters, preconditioned with 5 mL of EtOH

and 5 mL of water). The cartridge was washed with water (5 mL),

followed by product elution with 0.5 mL of EtOH into the ﬁnal

formulation vial. Finally, the product was formulated with 5% EtOH

in water for injection.

For the acetyl-protected [¹⁸F]50, the reaction mixture was diluted

with 35 mL of water, before trapping the intermediate on a C18 Plus

cartridge (Waters, preconditioned with 5 mL of MeCN and 5 mL of

water). After washing the cartridge with water (5 mL), the product

was eluted with 2 mL of EtOH. The solvent was evaporated at 95 °C

for 5 min, followed by azeotropic drying with MeCN (3 mL × 0.8

mL). The residue was cooled down in an ice bath before redissolving

it in 0.4 mL of DCM. The vial was kept in an ice bath, and 1 mL of

BBr₃(1 M in DCM) was added simultaneously, followed by stirring at

rt for 15 min. The DCM was evaporated, and the crude was then

redissolved in aq. 0.1% H₃PO₄/MeCN (5:1, 3 mL). Subsequently, the

product was puriﬁed by semipreparative HPLC. Collected fractions

were diluted with 35 mL of water, and the product was trapped on a

C18 Light cartridge (Waters, preconditioned with 5 mL of EtOH and

5 mL of water). The cartridge was washed with 5 mL of water,

followed by product elution with 0.5 mL of absolute EtOH. The

product was formulated with 5% EtOH in WFI to aﬀord [¹⁸F]PF-

NB1. Enantiomeric purity was not tested postradiosynthesis for (R)-

and (S)- [¹⁸F]PF-NB1.

In Vitro Autoradiography. Adult rat or mouse brain tissues

embedded in optimum cutting temperature medium were prepared as

10 μm-thick coronal sections on a cryostat (Cryo-Star HM 560 MV;

Microm, Thermo Scientiﬁc, Wilmington, DE). Slices were adsorbed

on SuperFrost Plus glass slides (Menzel, Braunschweig, Germany)

and subsequently stored at −20 °C. At the start of autoradiography,

slices were thawed for 15 min on ice and subsequently preconditioned

in an aqueous buﬀer comprising 30 mM HEPES, 0.56 mM MgCl₂,

110 mM NaCl, 5 mM KCl, 3.3 mM CaCl₂, and 0.1% fatty acid free

bovine serum albumin (pH 7.4, 0 °C) for 10 min. The slides were left

to dry for 2−3 min at rt and then incubated with 2.9−3.1 nM of (R)-

[¹⁸F]PF-NB1, (S)-[¹⁸F]PF-NB1, [¹⁸F]PF-NB1, or [¹⁸F]27 for 20 min

at rt in a humidiﬁed chamber. For blockade experiments, 1 μM of

GluN2B ligands, CERC-301 (IC₅₀for GluN2B = 3.6 nM⁶³) and CP-

101,606 (IC₅₀for GluN2B = 11 nM⁵⁴), was used. For the negative

control, 1 μM of σ1R ligands, SA4503 (IC₅₀for σ1R = 17.4 nM⁶⁴)

and ﬂuspidine (K_ifor σ1R = 0.59 nM⁶⁵), as well as the σ2R blocker

PB28 (K_iσ1R = 0.68 nM⁶⁶), was used. The slices were decanted and

washed for 5 min in an aqueous buﬀer containing 30 mM HEPES,

0.56 mM MgCl₂, 110 mM NaCl, 5 mM KCl, 3.3 mM CaCl₂, and 0.1%

fatty acid free bovine serum albumin (pH 7.4, 0 °C). Subsequently,

the slices were washed (2 × 3 min) in an aqueous buﬀer containing

30 mM HEPES, 0.56 mM MgCl₂, 110 mM NaCl, 5 mM KCl, 3.3 mM

R

DOI: 10.1021/acs.jmedchem.9b00812

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

CaCl₂(pH 7.4), and (2 × 5 s) in distilled water. After drying the

slices for 5 min at rt, they were exposed to a phosphor imager plate

(Fuji, Dielsdorf, Switzerland) for 35−40 min. The ﬁlms were scanned

by a BAS5000 reader (Fuji), and data analysis was performed using

Ahmed Haider: 0000-0002-5204-4473

nsch: 0000-0002-9030-8417

Simon M. Ametamey: 0000-0003-4285-6731

Bernhard Wu

̈

AIDA 4.50.010 software (Raytest Isotopenmessgerate GmbH,

Straubenhardt, Germany).

Author Contributions

^∥H.A. and A.H. contributed equally.

In Vivo PET Imaging. Animal care and experiments were

compliant with Swiss Animal Welfare legislation and were authorized

by the Veterinary Oﬃce of the Canton Zurich, Zurich, Switzerland.

Wistar male rats were purchased from Charles River (Sulzfeld,

Germany), while CD1 and σ1R-KO mice were purchased from

Envigo (Barcelona, Spain). Wistar rats (317−423 g) and mice (40.5−

44.1 g) were anesthetized using isoﬂurane and scanned inside a PET/

CT scanner (Super Argus, Sedecal, Madrid, Spain) upon tail-vein

injection of 30.13−34.68 MBq, 0.7−1.1 nmol/kg (rats, scan time 0−

90 min postinjection), or 12.11−14.87 MBq, 1.99−3.80 nmol/kg

(mice, scan time 1−91 min postinjection), of [¹⁸F]PF-NB1. For

anatomical alignment, PET scans were followed by computed

tomography (CT). For blockade studies, escalating doses of CP-

101,606 (1, 3, 5 and 10 mg/kg) were injected into Wistar rats 1 min

prior to tracer injection. The resulting data were reconstructed in

user-deﬁned time frames with a voxel size of 0.3875 × 0.3875 × 0.775

mm³³as previously detailed by our group.³³Time−activity curves for

speciﬁc brain regions of interest were deﬁned on an MRI T2 template

by PMOD v3.9 (PMOD Technologies, Zurich, Switzerland). The

results are given as standardized uptake values (SUVs), which is the

decay-corrected tissue radioactivity normalized to the injected

radioactivity and body weight.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This project was supported by the Swiss National Science

Foundation Grant Nos. 310030E-160403/1 and

310030E_182872/1. The Table of Contents (TOC) graphic

was constructed using biorender.com. Dr. Jose Miguel Vela is

acknowledged for providing our group with the σ1R-KO mice.

ABBREVIATIONS

■

NMDARs, N-methyl-D-aspartate receptors; σRs, sigma recep-

tors; BBr3, boron tribromide; HCl, hydrochloric acid; KF,

potassium ﬂuoride; CsF, cesium ﬂuoride; KOAc, potassium

acetate; RCY, radiochemical yield; EOB, end of bombardment;

MeCN, acetonitrile; SUV, standardized uptake value; Bq,

becquerel; TACs, time−activity curves; RO, receptor occu-

pancy; KO, knockout; EtOAc, ethyl acetate; EtOH, ethanol;

NH₄HCO₃, ammonium hydrogen carbonate; H₃PO₄, phos-

phoric acid; K₂CO₃, potassium carbonate; CDCl₃, deuterated

chloroform; Hex, hexane; MeOH, methanol; NaOH, sodium

hydroxide

Metabolite Study. Wistar rats were injected with 72.8−436 MBq

(0.95−14.75 nmol/kg) of [¹⁸F]PF-NB1. Brain homogenate extracts at

predeﬁned times (15, 30, and 60 min) and blood (5, 15, 30, 45, 60

min) were acquired and evaluated by radio-ultraperformance liquid

chromatography (UPLC).

Biodistribution Study. Eight male Wistar rats (4 baseline and 4

blockade animals) were injected with [¹⁸F]PF-NB1 via tail-vein

injection (20.0−26.9 MBq, 0.33−0.57 nmol/kg) and sacriﬁced by

decapitation under anesthesia using isoﬂurane at 45 min postinjection.

For blockade experiments, 2 mg/kg eliprodil in a vehicle of aqueous

glucose (5%), NaCl (0.45%), and citric acid (1 mM) was injected

momentarily prior to tracer injection. Organs were separated and

weighed and radioactivity was measured in a γ-counter (Perkin-Elmer,

Schwerzenbach, Switzerland). Biodistribution results are reported in

the Supporting Information section as % normalized injected dose/

gram tissue. For statistical analysis, an independent two-tailed paired

Student’s test assuming a normal distribution of the data set was

employed to determine statistical probability values.

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* Supporting Information

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General procedures; circular dichroism (CD) spectra of

(S)/(R)-PF-NB1 and -Me-NB1; time−activity curves

(TACs) of [¹⁸F]PF-NB1 in diﬀerent brain regions of

interest; ex vivo metabolite study of [¹⁸F]PF-NB1 in rat

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Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: simon.ametamey@pharma.ethz.ch.

■

ORCID

Hazem Ahmed: 0000-0001-7047-5202

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DOI: 10.1021/acs.jmedchem.9b00812

J. Med. Chem. XXXX, XXX, XXX−XXX

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Structure-Affinity Relationships of 2,3,4,5-Tetrahydro-1 H-3-benzazepine and 6,7,8,9-Tetrahydro-5 H-benzo[7]annulen-7-amine Analogues and the Discovery of a Radiofluorinated 2,3,4,5-Tetrahydro-1 H-3-benzazepine Congener for Imaging GluN2B Subunit-Containi

DOI: 10.1021/acs.jmedchem.9b00812

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