Novel spirotetracyclic zwitterionic dual H1/5-HT2A receptor antagonists for the treatment of sleep disorders

7778 J. Med. Chem. 2010, 53, 7778–7795

DOI: 10.1021/jm100856p

Novel Spirotetracyclic Zwitterionic Dual H₁/5-HT_2AReceptor Antagonists for the

Treatment of Sleep Disorders

Massimo Gianotti,*^,†Maurizio Botta, Stephen Brough,^‡Renzo Carletti,^†Emiliano Castiglioni,^†Corrado Corti,^†

Michele Dal-Cin,^†Sonia Delle Fratte,^†Denana Korajac,^§Marija Lovric,^§Giancarlo Merlo,^†Milan Mesic,^§Francesca Pavone,^†

Laura Piccoli,^†Slavko Rast,^§Maja Roscic,^§Anna Sava,^†Mario Smehil,^§Luigi Stasi,^{†,^}Andrea Togninelli, and

Mark J. Wigglesworth^‡

^†Neurosciences CEDD, GlaxoSmithKline, Medicines Research Centre, Via A. Fleming 4, 37135 Verona, Italy, ^‡GlaxoSmithKline, New Frontiers

Science Park, Third Avenue, Harlow, Essex, CM19 5AW, U.K., ^§Integrated Research Unit, IRU Chemistry, Prilaz Baruna Filipovica 29,

10000 Zagreb, Croatia, and Dipartimento Farmaco Chimico Tecnologico, Universitaꢀdegli Studi di Siena, Via Aldo Moro 2, I-53100 Siena, Italy.

^{^}Present address: Rottapharm Madaus, R&D Division, Via Valosa di Sopra 9, 20052 Monza, Italy.

Received July 9, 2010

Histamine H₁and serotonin 5-HT_2Areceptors mediate two different mechanisms involved in sleep

regulation: H₁antagonists are sleep inducers, while 5-HT_2Aantagonists are sleep maintainers. Starting

from 9⁰a, a novel spirotetracyclic compound endowed with good H₁/5-HT_2Apotency but poor

selectivity, very high Cli, and a poor P450 profile, a specific optimization strategy was set up. In partic-

ular, we investigated the possibility of introducing appropriate amino acid moieties to optimize the

developability profile of the series. Following this zwitterionic approach, we were able to identify several

advanced leads (51, 65, and 73) with potent dual H₁/5-HT_2Aactivity and appropriate develop-

ability profiles. These compounds exhibited efficacy as hypnotic agents in a rat telemetric sleep model

with minimal effective doses in the range 3-10 mg/kg po.

Introduction

treatments for sleep disorders include the use of prescription

hypnotics, such as benzodiazepines. However, these drugs

may be habit-forming, lose their effectiveness after extended

use, and metabolize more slowly for certain designated groups,

resulting in persisting medicative effects.

Other treatments include over-the-counter antihistamines,

e.g., diphenhydramine and dimenhydrinate. These medicines

are not designed to be strictly sedative in their activity, and as

such, this method of treatment has been associated with a

number of adverse side effects, e.g., persistence of the sedating

medication after the prescribed time of treatment or the so-

called “hangover effect”. Many of these side effects result

fromnonspecific activityinthe periphery as well asinthe CNS

during this period of extended medication.

Insomnia, defined as difficulty in initiating and/or main-

taining sleep, is one of the most common central nervous

system (CNS^a) disorders. According to the National Sleep

Foundation’s 2010 Sleep in America Poll, the majority of

those surveyed report that they only get a good night’s sleep a

few nights per week or less, with some ethnic groups doing

more poorly than others.¹The incidence of insomnia in the

general population is between 10-30%, and approximately

50% of cases complain of serious daytime consequences, such

as inability to concentrate, reduced energy, and memory

problems. When this persists for more than 1 month, without

an associated mental disorder or physical problem, it is

classified as primary insomnia.²

It has been suggested that brain histamine is involved in the

regulation of the sleep-wake cycle, arousal, cognition, and

memory primarily through action at the H₁receptors, produ-

cing a reduction of sleep latency in both preclinical⁴and

clinical studies.⁵On the other hand, selective blockade of

the 5-HT_2Areceptor has been proven in both preclinical⁶and

clinical⁷studies to be efficacious in reducing wake after sleep

onset (WASO) and increasing slow wave sleep (SWS) and

total sleep time (TST), therefore providing consolidation of

sleep. Itcanbe hypothesized thatthe combinationof thesetwo

mechanisms may provide an improved hypnotic profile versus

the marketed gold standard benzodiazepine-like drugs.⁸

As part of a broad drug discovery strategy aimed at the

identification of novel hypnotic agents,⁹we reasoned on

the possibility of optimizing (1S,3R)-5⁰,11⁰-dihydrospiro-

[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-yl(dimethyl)-

amine (9⁰a, Figure 1), a novel spirotetracyclic compound

Common symptoms of those suffering with a sleep disorder

include abnormal sleep behavior and difficulties in one or

more of falling asleep, remaining asleep, sleeping for adequate

lengths of time, and achieving restorative sleep.³Available

*To whom correspondence should be addressed. Phone: þ39-045-

8219524. Fax: þ39-045-8218196. E-mail: masgian@libero.it.

^aAbbreviations: CNS, central nervous system; hERG, human ether-

a-go-go-related gene K^þchannel; PK, pharmacokinetic; P450, cyto-

chrome P450; Cli, intrinsic clearance; fu, fraction unbound; MW,

molecular weight; clogD, calculated log D; PSA, polar surface area;

CHO, Chinese hamster ovary; HEK, human embryonic kidney; Fpo,

oral bioavailability; Br:Bl, brain-blood; Clb, blood clearance; Vss,

distribution volume; FLIPR, fluorescent imaging plate reader; SWS,

slow wave sleep; TST, total sleep time; ECG, electrocardiogram; EMG,

electromiogram; CT18, circadian time 18; REM, rapid eye movement;

NREM, nonrapid eye movement; RO, receptor occupancy; EWG,

electron withdrawing group; RCM, ring closing metathesis; TFA,

trifluoroacetic acid; DMP, Dess-Martin periodinane; HPLC, high

performance liquid chromatography; NT, not tested.

r

pubs.acs.org/jmc

Published on Web 10/13/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7779

Figure 1. In vitro and physicochemical data of compound 9⁰a. Superscript letters indicate the following: (a) functional pK_imeasured in

FLIPR (fluorescent imaging plate reader) assay;²⁶(b) pharmacokinetics; (c) intrinsic clearance in human (h) and rat (r) liver microsomes

(mL/min/g liver); (d) CYPEX (see experimental for details); (e) fraction unbound in rat brain; (f) ACD logD 7.4, version 11; (g) A²; (h)

dofetilide binding. See Experimental Section for details.

endowed with promising dual H₁/5-HT_2Ain vitro activity.¹⁰

Compound 9⁰a represents a low molecular weight lead,

although it is characterized by poor selectivity toward human

adrenergic R₁receptors and the human ether-a-go-go-related

gene (hERG) potassium channel as well as by suboptimal

physiochemical parameters. When preliminary pharmacoki-

netic data were gathered, a very high rat in vitro intrinsic

clearance (Cli), a low free fraction measured in vitro in homo-

genized brain tissue (Br-fu), and a strong inhibition of the 2D6

isoform of the human CYP450 were also observed. An

additional trait of 9⁰a is its antagonism of the 5-HT_2Band

5-HT_2Creceptors. While there is some evidence of slight

increases in wakefulness and motor activity after blockade of

the 5-HT_2Breceptor,¹¹no significant alteration in the percen-

tage distribution of any sleep stage, arousal and light SWS

(SWS1), was observed with the inhibition of the 5-HT_2C

receptor.¹²Therefore, it appears that the presence of 5-HT_2B

and 5-HT_2Cactivity in 9⁰a and in general in our compounds

may not represent a potential issue for their hypnotic profile.

It is well-known that the structure of H₁receptor antago-

nists can tolerate zwitterion moieties,¹³as they have been

intentionally introduced to reduce the sedating properties of

centrally acting first generation antihistamines by reducing

their brain penetrating capacities. Furthermore, in the second

generation H₁antagonists (e.g., cetirizine and fexofenadine),

the introduction of the zwitterion group was responsible for

a reduction of the drug-drug interactions and enhanced

receptor selectivity profiles with respect to first generation

drugs.¹⁴Despite the fact that zwitterions were initially intro-

duced to limit brain penetration of the first generation anti-

histamines, scientists at Hypnion, now Lilly, recently demon-

strated that zwitterionic structures can penetrate the brain and

induce hypnotic activity. In fact, they shaped their sleep pro-

gram strategy by incorporating zwitterion moieties into known

H₁/5-HT_2Aantagonist scaffolds.¹⁵This approach led Hypnion

to quickly progress several compounds into the clinical phase,

one of which, HY-10275 (LY-2624803) whose structure has

not yet been disclosed, successfully achieved its proof of

concept as a sleep drug.¹⁶

identify a novel synthetic route to successfully prepare com-

pounds 43-73 and 74-77 (Scheme 1). The synthesis of com-

pound 9⁰a has already been reported in a previous communi-

cation,¹⁰while herein we illustrate an optimized synthesis,

which has been adapted for the purpose of introducing sub-

stituents on the scaffold (such as R=Cl or F) and different

heteroatoms (such asX=O andS) inplace ofthe carbonin the

benzhydrylic position in the lower part of the compound.

Thus, starting from the appropriately substituted ketone 1,¹⁷

the first step of the synthesis was a potassium tert-butoxide

promoted bis-alkylation with allyl bromide.¹⁸In the case of 1c

and 1d the reaction led directly to the C-diallyl compounds 3c

and 3d, respectively, whereas a mixture of the corresponding

compounds 3 and C-allyl/O-allyl compound 2 was observed,

whenstartingfrom 1a, 1b, and 1e. Nevertheless, the mixtureof

2 and 3 was readily converted into the single compound 3 by

Claisen rearrangement.¹⁹The diallylation reaction is a crucial

step because it permits the construction of the quaternary

center. As reported, the intermediates 3 were then converted

via ring-closing metathesis (RCM), in good yields, into inter-

mediates 4, which underwent hydroboration/oxidation to

afford a mixture of compounds 5 and 6, which in turn was

oxidize using Dess-Martin periodinane (DMP) to provide 7

in excellent yields. The bis-ketone 7 could be either trans-

formed into the key intermediate 8 via a regioselective hydro-

genation reaction or processed through a reductive amination

reaction with an amino acid ester and hydrolysis of the

ensuing ester to give the final compounds 74-77.

The reduction of the benzylic ketone by hydrogenation is

probably the step of the synthesis that would most warrant

improvement, as the reaction usually necessitates harsh con-

ditions to proceed, such as a large excess of palladium on

carbon, high pressure and lengthy reaction times; nonetheless,

the recovery of desired product is good. Many alternative

reaction conditions were assessed to reduce this ketone, for

instance, a Wolf-Kishner reaction or triethylsilane in tri-

fluoroacetic acid (TFA), but a lack of reactivity or degrada-

tion was typically observed. In general, the removal of the

benzylic keto group has proven to be difficult from oxygen

and carbon-bridged scaffolds (X=O, C) and impossible from

sulfur-bridged compounds (X=S) and in some cases when the

aromatic ring is substituted (R ¼ H).

On the basis of this information, we considered introducing

a series of constrained/hindered amino acid derivatives to the

top region of this novel template with the aim of optimizing

compound 9⁰aby eradicating the observed developability issues.

In this article the optimization process of the initial lead 9⁰a,

which resulted in the identification of compounds 51, 65, and

73, will be described in detail. These compounds demon-

strated suitable overall profiles for a hypnotic with in vivo

activity in an animal model of sleep disorders.

Compounds 8 are racemic mixtures that can be resolved by

preparative chiral HPLC. For example, intermediates 8a, 8b,

and 8c have been resolved and the two enantiomers have been

named 8a (ent 1), 8a (ent 2), 8b (ent 1), 8b (ent 2), and 8c

(ent 1), 8c (ent 2) depending on their respective retention times

in the corresponding HPLC separation. For compounds 8a

(ent 1) and 8a (ent 2) the absolute stereochemistry was

determined by means of ab initio vibrational circular dichro-

ism (VCD) analysis;¹⁰hence, 8a (ent 1) was assigned as the

(1R)-enantiomer and 8a (ent 2) as the (1S)-enantiomer.

Chemistry

The spirocyclic skeleton of our lead is an unexplored

scaffold in organic chemistry; therefore, it was necessary to

7780 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Gianotti et al.

Scheme 1. General Synthetic Procedure for the Preparation of Compounds 9 and 43-77^a

^a(i) tert-Butanol, potassium tert-butoxide, allyl bromide, 35 ꢀC (starting from 1c and 1d, the reaction led directly to 3); (ii) Claisen rearrangement; (iii)

degassed CH₂Cl₂, Grubbs second generation catalyst, room temp; (iv) BH₃in THF, room temp, H₂O, then NaOH 3 M, H₂O₂, room temp; (v) DMP, dry

CH₂Cl₂, room temp; (vi) THF/acetic acid, Pd/C, H₂, room temp; (vii) dimethylamine or appropriate amino esters (R¹; see Tables 1-3), DCE,

NaBH(OAc)₃, room temp; (viii) KOH, MeOH/H₂O. (ix) To obtain 8b: (a) H-Cube hydrogenation (30 atm at 60 ꢀC); (b) DMP, dry CH₂Cl₂, room temp.

Henceforth, compound 8a (ent 1) is named 8a(R) and 8a

(ent 2) is named 8a(S).

data from primary assays. In order to better identify the

stereochemistry of the reported compounds, Table 1-3 also

contain details of the parent amino esters from which they

were derived and the parent spiroketone used in the reductive

amination step (Scheme 1, step vii).

The reductive amination reaction that leads to the forma-

tion of the second stereogenic center gave gooddiastereomeric

excess; in fact, the amine 9⁰a and the amino esters 10-38 and

39-42 were obtained with a diastereomeric ratio between 70/

30 and 90/10. The major diastereoisomer is identified with the

prime symbol (⁰) while the minor with the double prime (⁰⁰).

The separation of the two diastereoisomers was carried out at

this stageby using, also in this case, a preparativechiralHPLC

because of the difficulty of the separation by conventional

silica gel chromatography. The last step of the synthesis was

the hydrolysis of the esters 10-38 and 39-42 to give the final

amino acids 43-73 and 74-77 that were habitually purified

by chromatography on a C18 column.

Considering that in the final compounds there are two

stereogenic centers and consequentially four stereoisomers,

it was imperative to identify the most active isomer in order to

simplify the exploration. This analysis was carried out using

guvacine as amino ester and both the single known enantio-

mers of the spiroketone 8a, 8a(R) and 8a(S) (see Table 1). The

amino acid products obtained, 45 and 46, were tested as a

mixture of diastereoisomers, and it was clear that the mixture

46, obtained from 8a(S) via amino ester 13, was the most

active. The two diastereoisomers of 13 were then separated to

give the major 13⁰and the minor 13⁰⁰isomers. In this case we

observed that the major diastereoisomer (13⁰) gave the best

compound in terms of a mixed H₁/5-HT_2Aprofile (47), while

the minor diastereoisomer (13⁰⁰) gave 48, which is potent at H₁

but inactive at 5-HT_2A. Tables 1-3 illustrate the structures of

compounds (43-73 and 74-77) prepared according to the

procedures described in Scheme 1, along with corresponding

Results and Discussion

All the newly prepared compounds were assayed for their

agonist and antagonist activities using two kinds of functional

assays: a FLIPR (fluorescent imaging plate reader) assay,

using stably transfected Chinese hamster ovary (CHO) cells

expressing the human H₁receptor and stably transfected

SHSY5Y cells expressing the human 5-HT_2A, 5-HT_2B, and

5-HT_2Creceptors; a luminescence (Aequorin) assay using

frozen human embryonic kidney (HEK) cells stably express-

ing the human 5-HT_2Areceptor. Most of the 5-HT_2Adata

reported in Tables 1-3 were generated using the aequorin

assay, and those generated with the FLIPR assay are indicated

with a footnote. The well-known ketotifen²⁰and MDL-

100,907²¹were used as positive controls for H₁and 5-HT_2A

receptor antagonists; data obtained were consistent with the

corresponding affinities reported in the literature.

Before entering into a detailed discussion of the results

obtained, a few general observations can be made. In order to

combine the effect on sleep latency of a H₁antagonist with the

effect on sleep maintenance of a 5-HT_2Aantagonist, we

considered, in the first instance, balanced dual H₁/5-HT_2A

antagonist compounds to achieve comparable receptor occu-

pancy forthe two targets. Noagonistactivity wasobservedfor

any of the amino acids considered. High selectivity vs 5-HT_2B

and 5-HT_2Creceptors proved elusive; however, this was not

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7781

Table 1. Functional Activity (f-pK_i^a) at the Human H₁^b, 5-HT_2A^c, 5-HT_2B,^band 5-HT_2C^bReceptors, PSA^dand clogD^efor Compounds 43-67 ^j,k

7782 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Table 1. Continued

Gianotti et al.

^af-pK_i= functional pK_iobtained from the FLIPR and aequorin assays. SEM for H₁, 5-HT_2A, 5-HT_2B, and 5-HT_2Cdata sets is <0.1 with a minimum

of two replicates. ^bFLIPR = fluorescent imaging plate reader. See Experimental Section for assay details. ^cAequorin assay (intracellular Ca

luminescence). See Experimental Section for assay details. ^dA². ^eACD logD 7.4, version 11. ^fNot isolated. ^gWhen the reductive amination is performed with

a racemic amine (R¹in Scheme 1) and the chiral ketone 8a(S), the term “isomer 1” is used for the single stereoisomer with the shorter retention time in the

conditions of the chiral separation. Conversely the term “isomer 2” is used for the single stereoisomer with the longer retention time in the conditions of the chiral

separation. See Experimental Section for details. ^hWhen the reductive amination is performed with a chiral or achiral amine (R¹in the Scheme 1) and the racemic

ketone 8a, the term “isomer 1” is used for the single stereoisomer with the shorter retention time in the conditions of the chiral separation. Conversely the term

“isomer 2” is used for the single stereoisomer with the longer retention time in the conditions of the chiral separation. See Experimental Section for details. ⁱH₁

j

antagonist positive control. Symbol /represents the following: single unknown enantiomer. ^kSymbols ⁰and ⁰⁰represent the following: The major diastereoisomer

after reductive amination reaction was identified with the prime symbol (⁰) while the minor with the double prime (⁰⁰). ^l5-HT_2Aantagonist positive control.

considered a criterion to preclude compound progression

because of the minor role of these two receptors in sleep

regulation, as stated previously. With regards to selectivity

versus adrenergic R₁receptors and the hERG potassium

channel, which was a concern for our initial hit 9⁰a (Figure 1),

we never observed significant activity for any of the zwitter-

ionic compounds synthesized and tested in these assays (f-pK_i-

(hR_1A/B) < 6 in the FLIPR assay, hERG is discussed in detail

below). In terms of activity versus the primary targets we noticed

that the zwitterionic modification almost always retained the H₁

activity but reduced the 5-HT_2Apotency; thus, in light of these

preliminary results, it was clear that the challenge for the

exploration was to find balanced dual antagonist zwitterionic

compounds.

As highlighted above, the objective of this optimization

strategy was to identify suitable zwitterionic moieties able to

maximize the in vitro affinity versus both the H₁and 5-HT_2A

receptors and the selectivity toward undesired biological

targets enhancing the druglike characteristics of compound

9⁰a. Thus, the β-alanine derivative 43²²and the N-methyl

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7783

c

b

Table 2. Functional Activity (f-pK_i^a) at the Human H₁,^b5-HT_2A

(See Table 1 for Footnotes)

,

5-HT_2B

,

and 5-HT_2CReceptors, PSA,^dand clogD^efor Compounds 68-73

analogue 44, characterized by a reduced lipophilicity and

increased polar surface area (PSA) with respect to 9⁰a, were

readily prepared and tested as a mixture of diastereoisomers.

Both of them showed good potency at the H₁receptor

(f-pK_i=7.6 and 8.0 for 43 and 44, respectively), whereas the

tertiary amine 44 exhibited higher in vitro activity at the

5-HT_2Areceptor (f-pK_i=5.7 and 6.8 for 43 and 44, respec-

tively), although it proved to be less potent than the reference

compound 9⁰a (f-pK_i=7.9). Compound 43 was found to be

more selective than parent compound 9⁰a, whereas 44 retained

some in vitro activity at the 5-HT_2Creceptor (f-pK_i= 7.0).

Notably both compounds were found to be clean in terms of

CYP450 inhibition (IC₅₀>10 μM against all isoforms tested)

and affinity for the hERG ion channel (pIC₅₀< 4.2 and

pIC₅₀= 4.2 for 43 and 44, respectively). Further to these

promising initial results it was decided to concentrate the

exploration on the amino acid moiety with the aim of maxi-

mizing the in vitro affinity for the 5-HT_2Areceptor while

maintaining the improved developability properties observed.

To this end, a series of modified/constrained β, γ and δ-amino

acid derivatives shown in Table 1 was synthesized.

compound 47 emerged with good H₁activity (f-pK_i=7.6),

a satisfactory level of 5-HT_2Apotency (f-pK_i= 6.9), good

selectivity versus 5-HT_2B, and no selectivity over 5-HT_2C

( f-pK_i=6.8). Compound 47 was also found to be clean in

terms of CYP450 inhibition (IC₅₀> 10 μM against all iso-

forms tested) and affinity for the hERG ion channel (pIC₅₀<

4.4). The substitution of the tetracyclic scaffold was also

investigated, but the scope of the exploration was limited to

a specific position, following some indications from the

literature,²³due to synthetic feasibility. Therefore, compound

49 was synthesized and it was found to show high potency on

H₁(f-pK_i= 7.4), but unlike the unsubstituted analogue 47, it

was inactive as a 5-HT_2Areceptor antagonist. The exploration

continued on the tetrahydropyridine substructure and the

isoguvacine derivative 50 came out with suitable H₁/5-HT_2A

potencies (f-pK_i=7.4 and f-pK_i=7.1, respectively) and 5-HT_2B

selectivity. Some isonipecotic acid derivatives were also taken

into consideration, and the reference compound for this sub-

class, compound 51, maintained a good overall target profile

(f-pK_i= 7.3/f-pK_i= 7.1, on H₁/5-HT_2A, respectively) without

any selectivity versus 5-HT_2Band 5-HT_2Cas well as resulting to

be clean in terms of CYP450 (IC₅₀>10 μM against all isoforms

tested) and hERG ion channel inhibition (pIC₅₀<4.2). Sub-

stitution in the R-position of the acidic moiety of 51 led to

The results obtained for 43 and 44 suggested that the

exploration should be biased toward tertiary amines and, in

particular, attention was focused on cyclic amines. Thus,

7784 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Gianotti et al.

c

b

Table 3. Functional Activity (f-pK_i^a) at the Human H₁,^b5-HT_2Aand 5-HT_2CReceptors, PSA,^dand clogD^efor Compounds 74-77

(See Table 1 for Footnotes)

, 5-HT_2B,

compounds 52, 53, and 54 characterized by good H₁/5-HT_2A

antagonist potencies and increasing selectivity versus 5-HT_2B

and 5-HT_2Cpassing from methyl to fluorine substitution,

suggesting that electron withdrawing groups (EWG) in this

position successfully improve selectivity. Compound 54 also

shows a clean CYP450 profile (IC₅₀> 10 μM against all

isoforms tested) and selectivity over the hERG ion channel

(pIC₅₀<4.4). Homologation of compound 51 was not toler-

ated for 5-HT_2Areceptor antagonist activity (compound 55).

Moving to the nipecotic acid derivatives, compound 56

showed potent primary assay activity and 5-HT_2Bselectivity,

while its fluorine substituted analogue, compound 57, is selec-

tive versus both 5-HT_2Band 5-HT_2Creceptors, confirming

that an EWG in the R-position enhances selectivity but is

poorly active on H₁. The morpholine and piperazine deriva-

tives were also considered and synthesized; both of them are

selective over 5-HT_2B, but while compound 58 has well

balanced target potencies, a slight drop in the 5-HT_2Aactivity

was observed for 59.

A group of pyrrolidines was also studied, and in general, all

the compounds synthesized showed increased potency com-

pared to the piperidine derivatives previously described.

Compounds 60 and 61 have similar pharmacological profiles,

characterized by very high potency on the primary targets and

no selectivityatall versus 5-HT_2Band 5-HT_2Creceptors, while

the homologated compound 62 retains potency on H₁/

5-HT_2Awith a significantly reduced 5-HT_2Bactivity, display-

ing a different behavior with respect to the homologated

compound 55. Two [3.1.0] amino acidic fragments were also

investigated and the corresponding final compounds 63 and

64 synthesized; they show very interesting profiles with high

H₁antagonist potency. In particular 64 is characterized by a

hERG ion channel (pIC₅₀< 4.4), and a clean CYP450 profile

(IC₅₀> 10 μM against all isoforms tested).

The analysis of different cyclic secondary amines, which

started with piperidine, passing through pyrrolidines and

arriving at azetidine derivatives, is now close to completion.

Three compounds were prepared belonging to the latter class.

The first, compound 65, is the most active and balanced

derivative found (f-pK_i=8.2/f-pK_i=7.8, H₁/5-HT_2A, respec-

tively), even if it is not selective versus 5-HT_2C( f-pK_i=8.2).

Compound 65 also shows a clean CYP450 profile (IC₅₀> 10

μM against all isoforms tested) and selectivity over the hERG

ion channel (pIC₅₀< 4.3). The other two azetidine deriva-

tives, 66 and 67, are characterized by an unbalanced profile

versus 5-HT_2A, and the former is also characterized by

inactivity versus 5-HT_2C

.

With the aim of increasing PSA and further reducing

lipophilicity, we decided to introduce heteroatoms in the scaf-

fold and a number of oxepine derivatives were synthesized

(Table 2). Learnings from the previous exploration were trans-

ferred to the new oxepine scaffold, and the first two com-

pounds prepared, guvacine derivative 68 and isoguvacine

derivative 69, displayed some very interesting features. In

particular, a trend of selectivity versus 5-HT_2Band 5-HT_2C

,

driven by the oxepine scaffold, is evident, even if there is also a

trend toward reduced potency on the primary targets. In fact

69, besides compound 54 mentioned above, was the only

compound synthesized that combines selectivity (f-pK_ie 5.7/

f-pK_i=6.0, on 5-HT_2B/5-HT_2C, respectively) with appropriate

potencies on the desired targets (f-pK_i=7.3/f-pK_i=7.6, on H₁/

5-HT_2A, respectively). Some piperidine derivatives were also

considered in this case, and three compounds, 70, 71, and 72,

were prepared. Unfortunately, they did not withstand the

reduction of potency derived from the oxepine scaffold, show-

ing moderate activities on the target receptors. On the other

well balanced profile (f-pK_i= 7.5/f-pK_i= 7.5, onH₁/5-HT_2A,

respectively), with selectivity versus 5-HT_2B(f-pK_i= 6.4), the

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7785

Table 4. Summary of the Affinity (pK_i) of Compounds at the Rat

Native H₁/5-HT_2AReceptors Determined Using Radioligand Binding

po administration through measurement of the brain to

blood AUC_0-8hratio. For 47 brain penetration was mea-

sured at 1 h following iv dosing. After iv administration, all

the compounds showed low clearance, in agreement with the

in vitro results except for 54. The volume of distribution

(Vss) was low for 47 and moderate for the other compounds,

and the half-life (t_1/2) was between 1 and 3 h except for

compound 64 where it was longer (t_1/2=5.3 h). After oral

administration, all the compounds displayed moderate to

high bioavailability (Fpo) with the exception of 54 (Fpo =

22%), and the brain penetration generally ranged from 0.1 to

0.5. Only compounds 64 and 69 showed higher brain pene-

tration of 2.2 and 1.8, respectively. Progression of some

compounds was determined on the basis of these results:

Compound 54 was stopped because of its inferior in vivo

profile, characterized by low bioavailability combined with

high blood clearance and also low brain penetration. Com-

pound 64 was stopped as well, although it did not present any

major PK issues, as the half-life of 5.3 h did not reflect the

hypnotic profile required.

pK_i^a

b

c

compd

r-H₁

r-5-HT_2A

47

51

54

64

65

69

73

8.06

8.33

7.91

8.27

8.25

7.70

7.85

6.93

7.47

6.45

7.16

7.48

6.98

7.47

^apK_ivalues are the mean of two independent experiments performed

in duplicate. The difference between the averaged pK_ivalues was lower

than 0.35. ^b[³H]Pyrilamine binding to rat cortical membranes. ^c[³H]-

Ketanserin binding to rat cortical membranes.

hand, the azepine derivative 73 was very well tolerated (f-pK_i=

7.3/f-pK_i= 7.5, on H₁/5-HT_2A, respectively) and displayed low

activity for the 5-HT_2Breceptor (f-pK_i= 6.1) and no selectivity

versus the 5-HT_2Creceptor (f-pK_i=7.5). Compound 73 showed

a clean CYP450 profile (IC₅₀> 10 μM against all isoforms

tested) and selectivity over the hERG ion channel (pIC₅₀<4.3).

Table 3 reports some efforts of structural diversification.

Fixing the carbonyl group next to the spiro system, substitu-

tion on the phenyl ring on the right-hand side (RHS), and

differentheteroatoms inthe benzhydrylicposition inthe lower

part of the compound (X = C, O, and S) were considered. A

comparison between compounds 47, 68, and 74, representa-

tive examples from the three scaffolds considered in this

exploration, showed how, starting from the carbocyclotetras-

piro, then considering the oxepine, and finally moving to the

last scaffold described with a carbonyl group, the H₁activity

was basically maintained but the 5-HT_2Aaffinity dropped in

the last case. The only compound, in this last subseries, able to

maintain a balanced overall target profile, albeit with moder-

ate potency, was 76, characterized by a fluorine on the RHS

phenyl ring and a sulfur on the lower part.

Despite their promising profile, compounds 47 and 69

were put on hold because of a developability concern related

to the potential toxicity associated with their tetrahydropyr-

idine substructures.²⁴

In Vivo Pharmacodynamic Profile. The hypnotic effects of

the remaining compounds (51, 65, and 73) were assessed

using telemetric recording of electroencephalogram (EEG)

and electromiogram (EMG) in the rat. Simultaneous record-

ing of EEG and EMG allows the accurate derivation of the

following sleep parameters: awake, nonrapid eye movement

(NREM) sleep, and REM sleep. The test compound’s effects

were assessed for 5 h immediately after treatment in the

active phase, starting at circadian time [CT] 18 (lights off at

circadian time 12) of the rat light-dark cycle. CT18 was

chosen to allow a maximal window to assess the hypnotic

effects of the compound.

H₁and 5-HT_2AReceptor Binding in Rat Cortex. Competi-

tion studies were performed to determine test compound

affinity at both H₁and 5-HT_2Areceptors in membranes from

rat cortex by using [³H]pyrilamine and [³H]ketanserin as

radioligands, respectively. Data obtained for the most

advanced compounds, 47, 51, 54, 64, 65, 69, and 73, are

reported in Table 4. In general, the pK_ivalues obtained in

binding studies for 5-HT_2Awere in good agreement with the

f-pK_ivalues found in the functional assay in human recom-

binant cells (Tables 1 and 2), whereas the affinity values for

the H₁receptor were higher than functional potencies. This

difference could depend on the species (rat versus human)

and/or different conditions of the assays, such as the ionic

composition of the incubation media or the incubation time.

Pharmacokinetic Studies. Compounds with favorable in

vitro profiles, able to combine H₁and 5-HT_2Apotencies and

exhibit selectivity versus 5-HT_2Band/or 5-HT_2C, were pro-

gressed through the screening cascade and evaluated in rat

pharmacokinetic studies, both in vitro and in vivo (Table 5).

All the amino acids considered for progression displayed

very low values of intrinsic clearance in rat and human

microsomes, low CYP450 inhibitory potencies, thus mini-

mizing the possibility of drug-drug interactions, and increased

fraction unbound in brain with respect to the starting lead

amine 9⁰a. The in vivo pharmacokinetics were investigated

in male rat following intravenous (iv) and oral (po) adminis-

tration, in a cassette dosing experimental design. Brain pene-

tration was determined, in almost all the experiments, after

The three compounds were tested orally in this paradigm

at 1, 3, and 10 mg/kg. All of them showed dose related

increases in total sleep time (TST) during the 5 h of observa-

tion, reaching a statistically significant effect at 3 mg/kg for

compound 65 (p < 0.01) and at 10 mg/kg for compounds 51

(p<0.01) and 73 (p<0.01). No effect was observed on the

sleep latency (Table 6). An effect of H₁antagonists on sleep

latency has been reported in the literature^4,25where com-

pounds were tested in a disturbed sleep model; taken together

withour results, thesedatasuggestthat H₁/5HT_2Aantagonists

may act on sleep onset in disturbed sleep conditions.

Ex Vivo Receptor Occupancy Study. Two compounds, 51

and 65, were characterized for their receptor occupancy

(RO) in rats in order to understand the level of occupancy

achieved at both target receptors at doses where 51 and 65

have been shown to produce a hypnotic effect in rats (CT18

model). Three doses were tested (1, 3, and 10 mg/kg, po, 2 h

pretreatment). A time point of 2 h after treatment was chosen

on the basis of preliminary PK data indicating T_maxoccurred

at 2 h for both compounds with plasma exposures C_max=216

ng/mL for compound 51 (at 1 mg/kg po) and C_max= 626 ng/

mL for compound 65 (at 3 mg/kg po). At this time point the

maximal RO values are expected. In the RO study, both

compounds showed a linear plasma exposure at T_maxacross

the three doses tested (compound 51 showing 119, 303, and

755 ng/mL at 1, 3, and 10 mg/kg, respectively; compound 65

showing 127, 417, and 1217 ng/mL at 1, 3, and 10 mg/kg,

respectively).

7786 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Table 5. Rat Pharmacokinetic Profile for Compounds

Gianotti et al.

^aIntrinsic clearance in liver microsomes, rat and human. ^bCYPEX assay. ^cfu = fraction unbound in brain-blood rat. ^dIn vivo data determined by

0.5 mg/kg iv and 1 mg/kg po administration in rat for compounds 51, 64, 73 and by 1 mg/kg iv and 3 mg/kg po administration in rat for compounds 47,

54, 65 and 69. Br:Bl measured by brain to blood AUC_0-8hratio following po dosing for 47 at 1 h following iv dosing.

Table 6. Sleep Promoting Effects of Compounds 51, 65, and 73 Administered Orally at 1, 3, and 10 mg/kg in Rat during Active Phase (Starting CT18)^a

compd

dose

veh

TST (min)

NREM sleep latency (min) NREM sleep over 5 h (min) REM sleep latency (min) REM sleep over 5 h (min)

51

90.2 ( 10.7

54.2 ( 13.9

69.8 ( 20.3

51.4 ( 11.5

34.6 ( 6.8

39.8 ( 7.1

58.1 ( 11.7

51.6 ( 6.0

33.5 ( 7.2

40.4 ( 6.3

37.8 ( 7.1

37.9 ( 7.5

31.5 ( 5.7

80.9 ( 8.5

84.7 ( 6.4

94.2 ( 8.1

103.7 ( 9.8*

79.4 ( 5.6

70.3 ( 12.8

83.6 ( 21.0

77.9 ( 13.6

83.8 ( 18.0

75.3 ( 13.9

77.8 ( 23.9

81.6 ( 9.8

64.5 ( 8.9

57.0 ( 18

9.3 ( 3.3

9.7 ( 1.7

1 mg/kg 94.4 ( 7.4

3 mg/kg 105.2 ( 9.1

10 mg/kg 105.2 ( 9.1*

11.0 ( 2.1

12.6 ( 2.0

12.7 ( 3.2

10.7 ( 2.1

16.1 ( 1.9

14.6 ( 2.2

10.5 ( 1.3

13.3 ( 2.3

15.1 ( 2.7*

16.2 ( 1.7*

65

73

veh

92.1 ( 5.8

1 mg/kg 98.1 ( 10.9

3 mg/kg 134.7 ( 9.8**

10 mg/kg 145.2 ( 11.5**

87.4 ( 9.3

118.6 ( 9.1**

130.5 ( 10.1**

105.6 ( 21

107.3 ( 6.6

123.2 ( 12

139.5 ( 10.5**

veh

116.1 ( 7.6

1 mg/kg 120.7 ( 7.8

3 mg/kg 138.3 ( 12.9

10 mg/kg 155.7 ( 11.6**

67.0 ( 12.3

77.7 ( 12.1

63.4 ( 8.2

^a(/) p < 0.05 and (//) p < 0.01 for each dose versus corresponding vehicle as determined by one-way ANOVA followed by Dunnett’s test. Values are

expressed as the mean ( SEM; n = 8. See Experimental Section for assay details.

[³H]Ketanserin was used as ligand for the 5-HT_2Arecep-

tor, whereas [³H]pyrilamine was used for the H₁receptor.

Compound 51 showed receptor occupancy levels of 0%, 5.6%,

and 18% for the 5-HT_2Areceptor and 30%, 35%, and 55% for

the H₁receptor. Similarly, compound 65 showed levels of

0.9%, 24%, and 47% for the 5-HT_2Areceptor and 16%, 45%,

and 66% for the H₁receptor. The two compounds 51 and 65

showed efficacy in the rat sleep model (CT18) at 10 mg/kg po

and at 3 mg/kg po, respectively (Table 6), thus suggesting that

in order to reach a hypnotic profile, a combination of a low RO

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7787

of both H₁and 5-HT_2Areceptors is needed; in particular a

5-HT_2Areceptor occupancy of ∼20% is sufficient to observe

the in vivo readout.

The receptor-ligand binding assays were performed using

[ethylene-3H]ketanserin hydrochloride (NET-791, 2.22-3.33 TBq/

mmol, Perkin-Elmer) for 5-HT_2Areceptor binding and [pyridinyl-5-

3H]pyrilamine (TRK608, 1.1 TBq/moml, Amersham) for H₁recep-

tor binding. Binding experiments were carried out in deep-well 96-

well plates in a total volume of 0.5 mL/well containing the radi-

oligand (final concentration of 1 nM [³H]ketanserin or 0.6 nM

[³H]pyrilamine), tissue suspension (50 μg/well for 5-HT_2Areceptor

or 150 μg/well for H₁receptor), various concentrations of test com-

pound, and the appropriate buffer (50 mM Tris-HCl, 10 mM MgCl₂,

pH7.4, for 5-HT_2Areceptor or 50 mM HEPES, pH 7.4, for H₁

receptor). The mixture was incubated with shaking at 37 ꢀC for 1 h

for 5-HT_2Areceptor binding or at room temperature for 2 h for H₁

receptor binding.

Samples were rapidly filtered through Whatman GF/B glass-

fiber filters presoaked with 0.3% PEI using a cell harvester and

then washed 4 times with 4 mL of ice-cold 0.9% NaCl buffer.

The filters were dried and counted in a liquid scintillation

counter (Tri-Carb 2900TR, Perkin-Elmer). Nonspecific binding

was determined by the presence of 10 μM mianserin or cetirizine

for 5-HT_2Aor H₁receptor, respectively.

Conclusions

A newspirotetracyclic classofdual H₁/5-HT_2Aantagonists,

together with the lead optimization process, has been pre-

sented. Starting from an unselective and poorly developable

lead (9⁰a), a zwitterionic approach successfully delivered high

quality leads characterized by low risk of CYP450 inhibition

and low affinity vs the hERG potassium channel and adre-

nergicreceptors. Onthe basisoftheirfavorableoverall profile,

several leads (51, 65, and 73), with equivalent in vitro poten-

cies and in vivo profiles, were identified and progressed in

the screening cascade toward a pharmacodynamic model of

sleep disorders (rat CT18 sleep model), where they exhibited a

robust effect, thus confirming a possible application as an

alternative therapy to currently available hypnotic drugs with

the potential for a reduced side effect burden.

Radioligand binding data were analyzed by nonlinear regres-

sion analysis using GraphPad Prism 4.0 (GraphPad Software,

CA). Curve fitting from competition binding experiments was

determined by using a one site competition equation after

checking that the Hill slope in the four-parameter logistic

equation was not different from 1.0. In this condition, pK_i

values of test drugs were calculated according to the Cheng-

Prusoff equation from the IC₅₀, and the K_D(equilibrium dis-

sociation constant of the radioligand) was determined at each

receptor type (5-HT_2A, H₁) through saturation experiments.

Results are expressed as mean values of two separate experi-

ments performed in duplicate.

P450 CYPEX Assay. Inhibition (IC₅₀) of human CYP1A2,

2C9, 2C19, 2D6, and 3A4 was determined using Cypex Bacto-

somes expressing the major human P450s. A range of concen-

trations (0.1, 0.2, 0.4, 1, 2, 4, and 10 μM) of test compound were

prepared in methanol and preincubated at 37 ꢀC for 10 min in

50 mM potassium phosphate buffer (pH 7.4) containing recom-

binant human CYP450 microsomal protein (0.1 mg/mL; Cypex

Limited, Dundee, U.K.) and probe-fluorescent substrate. The

final concentration of solvent was between 3% and 4.5% of the

final volume. Following preincubation, NADPH regenerating

system (7.8 mg of glucose 6-phosphate, 1.7 mg of NADP, and

6 units of glucose 6-phosphate dehydrogenase/mL of 2% (w/v)

NaHCO₃; 25 μL) was added to each well to start the reaction.

Production of fluorescent metabolite was then measured over a

10 min time course using a Spectrafluor Plus plate reader. The

rate of metabolite production (AFU/min) was determined at

each concentration of compound and converted to a percentage

of the mean control rate using Magellan (Tecan software).

The inhibition (IC₅₀) of each compound was determined from

the slope of the plot using Grafit, version 5 (Erithacus software,

U.K.). Miconazole was added as a positive control to each plate.

CYP450 isoform substrates used were ethoxyresorufin (ER;

1A2; 0.5 μM), 7-methoxy-4-triflouromethylcoumarin-3-acetic

acid (FCA, 2C9, 50 μM), 3-butyryl-7-methoxycoumarin (BMC,

2C19, 10 μM), 4-methylaminomethyl-7-methoxycoumarin (MMC,

2D6, 10 μM), diethoxyfluorescein (DEF, 3A4, 1 μM), and 7-ben-

zyloxyquinoline (7-BQ, 3A4, 25 μM). The test was performed in

three replicates.

Experimental Section

Biological Test Methods. FLIPR screening assays for H₁,

5-HT_2A, 5-HT_2B, and 5-HT_2Cwere all run essentially as

described in Wigglesworth et al.²⁶

5-HT_2AAequorin Assay.²⁷Frozen human embryonic kidney

(HEK) cells stably expressing the human 5-HT_2Aserotonin

receptor and aequorin apoprotein were thawed and added

dropwise to an appropriate volume of warm DMEM media

(Gibco Invitrogen 41965-039) containing 10% dialyzed fetal

bovine serum (FBS) (Invitrogen, 05-4011DK). Cells were then

spun down at 1000 rpm for 5 min at room temperature. The

supernatant was poured off and the pellet resuspended in HBSS

buffer (Sigma kit H1387) supplemented with HEPES (Sigma

H0887), NaHCO₃(Sigma S8761), 0.1% pluronic acid F68

solution (Gibco Invitrogen 24040-032), and 0.1% bovine serum

albumin (CalBiochem 126609). A sample was taken and a cell

count performed. Cells were diluted down to 2.5 ꢀ 10⁶cells/mL

in loading buffer. Coelenterazine [5 μM] (Invitrogen C6780) was

added to the cell suspension and the cell vessel wrapped in foil.

The cell vessel was put on a windmill rotator (Bibby Stuart) and

left overnight at room temperature. Before assay, a sample was

taken and a cell count performed. Cells were diluted to an

appropriate final density immediately prior to assay. Plates

containing compounds (0.5 μL) were placed in a Lumilux, where

they were diluted in buffer (20 μL), before additions of cells

(20 μL) and a predetermined submaximal concentration of

5-HT (20 μL), while luminescence was monitored. Data were

analyzed using area under curve for the entire time course,

normalized to in-plate nominal high and low controls, and

fitted to a four-parameter logistic equation.

hERG ³H-Dofetilide Binding Assay. hERG activity was mea-

3

sured using H-dofetilide binding in a scintillation proximity

assay (SPA) format. The activity was measured with a Perkin-

Elmer Viewlux imager.

Receptor Binding Assays. The in vitro binding assays of H₁

and 5-HT_2Areceptors were performed using the rat cerebral

cortex. Male Spagrue-Dawley rats (200-250 g) were decapi-

tated, and the cerebral cortex was rapidly removed and homo-

genized in 20 volumes of ice-cold 50 mM Tris-hydrochloride

buffer, pH 7.4. The homogenate was centrifuged at 41000g for

15 min at 4 ꢀC. The pellet was resuspended in buffer, incubated

at 37 ꢀC for 15 min, and washed twice by the same procedure.

The final pellet was resuspended in 4 volumes of ice-cold buffer.

The final suspension was distributed into aliquots and stored at

-80 ꢀC. Protein content in the membrane preparations was

evaluated using the Bradford method (BCA protein assay,

PIERCE).

Intrinsic Clearance (Cli) Assay. Intrinsic clearance (Cli) values

were determined in rat and human liver microsomes. Test com-

pounds (0.5 μM) were incubated at 37 ꢀC for 30 min in 50 mM

potassium phosphate buffer (pH 7.4) containing 0.5 mg of

microsomal protein/mL. The reaction was started by addition

of cofactor (NADPH, 8 mg/mL). The final concentration of

solvent was 1% of the final volume. At 0, 3, 6, 9, 15, and 30 min,

an aliquot (50 μL) was taken, quenched with acetonitrile con-

taining an appropriate internal standard, and analyzed by

7788 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Gianotti et al.

HPLC-MS/MS. The intrinsic clearance (Cli) was determined

from the first-order elimination constant by nonlinear regres-

sion using Grafit, version 5 (Erithacus software, U.K.), cor-

rected for the volume of the incubation and assuming 52.5 mg of

microsomal protein/g liver for all species. Values for Cli were

expressed as (mL/min)/g liver. The lower limit of quantification

of clearance was determined to be when <15% of the com-

pound had been metabolized by 30 min, and this corresponded

to a Cli value of 0.5 (mL/min)/g liver. The upper limit was

50 (mL/min)/g liver.

(circadian time (CT) 18). Recordings were made for the sub-

sequent 5 h test period.

Results were expressed as mean value ( SEM. Statistical

analysis was performed by a one-way analysis of variance

followed by Dunnett’s test.

Ex Vivo Receptor Occupancy by Autoradiography. Sprague-

Dawley rats were acutely administered with vehicle (0.5%

HPMC po) or test compounds (1, 3, or 10 mg/kg po), and after

2 h they were sacrificed and brains were quickly removed and

frozen in precooled isopentane at -20 ꢀC.

In Vivo Studies. All experiments were prereviewed and

approved by a local animal care committee in accordance with

the guidelines of the “Principles of Laboratory Animal Care”

(NIH Publication No. 86-23, revised 1985) and with a project

license that was obtained according to Italian law (Art. 7,

Legislative Decree No. 116, January 27, 1992), which acknowl-

edges European Directive 86/609/EEC on the care and welfare of

laboratory animals.

In Vivo Pharmacokinetics. The pharmacokinetics and oral

bioavailability of test compounds were determined following iv

(bolus) and po (suspension) administration to male Sprague-

Dawley rats at 0.5-1 and 1-3 mg free base/kg, respectively.

Blood and brain samples were collected at intervals up to 24 h

after dosing. Blood and brain samples were subsequently assayed

for test compound concentration using a method based on protein

precipitation followed by HPLC-MS/MS analysis.

Rat Sleep Study. Male Sprague-Dawley rats (275-300 g,

Charles River Italy) were housed singly on a light-dark cycle

(15:00-03:00 h light) 1 week prior to surgery. Access to food and

water was allowed ad libitum.

In order to collect the biopotential signals, a miniature multi-

channel telemetric transmitter (TL10M3-F40-EET, Data Sciences

Int.) was implanted intraperitoneally into the animals.

H₁ex Vivo Receptor Occupancy. Coronal brain sections were

cut at 14 μm, mounted onto microscope slides, and incubated

with 1nM [³H]pyrilamine (TRK608, Amersham) in 50 mM Tris-

HCl, pH 7.5, buffer for 10 min at room temperature. Nonspe-

cific binding was determined in the presence of 10 μM pyrila-

mine. Sections were subsequently washed 4 ꢀ 2 min in ice-cold

incubation buffer and exposed to imaging plates for 7 days

and analyzed with a phosphoimager scanner (Fuji BAS-5000).

Receptor occupancy was measured in cerebral cortex.

5-HT_2Aex Vivo Receptor Occupancy. Coronal brain sections

were cut at 14 μm, mounted onto microscope slides and incu-

bated with 1 nM [³H]ketanserine (NET791, Perkin-Elmer) in

50 mM Tris-HCl þ 10 mM MgCl₂, pH 7.5, buffer for 10 min at

room temperature. Nonspecific binding was determined in the

presence of 10 μM mianserin. Sections were subsequently

washed 4 ꢀ 2 min in ice-cold incubation buffer and exposed to

imaging plates for 7 days and analyzed with a phosphoimager

scanner (Fuji BAS-5000). Receptor occupancy was measured in

cerebral cortex.

Chemistry. General Methods. Proton nuclear magnetic reso-

nance (¹H NMR) spectra were recorded either on Varian

instruments at 300, 400, or 500 MHz or on Bruker instruments

at 300, 400, or 500 MHz. Chemical shifts are reported in ppm (δ)

using the residual solvent line as internal standard. Splitting

patterns are designated as follows: s, singlet; d, doublet; t, triplet;

q, quartet; m, multiplet; b, broad. The NMR spectra were

recorded at a temperature ranging from 25 to 90 ꢀC. When

more than one conformer was detected, the chemical shifts for

the most abundant one is reported.

To allow recording of cortical electroencephalogram (EEG),

two electrodes were fixed permanently, with dental cement, to

the skull. They were directly in contact with the dura mater

through two drilled holes on the fronto-parietal region. Two

electrodes were fixed to the skeletal muscles of the neck for

recording electromyogram (EMG).

Mass spectra (MS) were run on a 4 II triple quadrupole

Agilent MSD 1100 mass spectrometer, operating in ES(þ) and

ES(-) ionization mode. The usage of this methodology is indi-

cated by “MS”.

Optical rotations were measured by using a Jasco DIP-360

digital polarimeter with a path length of 10 cm recorded at the

sodium D line.

After recovery from surgery, animals were maintained in their

home cage in a temperature controlled environment (21 ( 1 ꢀC)

with access to food and water ad libitum. Implanted animals

demonstrated a normal behavioral repertoire immediately after

recovery from surgery, but to allow normal sleep patterns to be

re-established, animals were utilized after 3 weeks. The environ-

mental conditions described above were maintained throughout

the sleep studies.

For the duration of the test period freely moving animals

remained in their home cages on individual receivers. EEG and

EMG signals were recorded continuously using DSI Dataquest

A.R.T.

The EEG trace, divided into 10 s epochs, was digitally trans-

formed (FFT transformation) to provide the power spectra of δ,

θ, R, and β bands in order to distinguish three different activity

patterns in the rat (awake, NREM sleep, and REM sleep). The

markers assigned by the automated scoring system (sleep stage,

DSI) were transferred to the EEG digital signal and subse-

quently confirmed by visual examination of the EEG and EMG

traces by trained operators, blind to the drug treatment.

Analysis of sleep parameters included latency to NREM sleep

(time interval to the first six consecutive NREM sleep epochs

after injection), latency to REM sleep (time interval to the first

REM sleep epoch after injection), and time spent wake, NREM

sleep, REM sleep, and total sleep time (TST).

Drug studies were carried out according to a randomized

paired crossover design where, in separate experimental ses-

sions, each animal received control and drug treatments. Com-

pounds were administered orally in a volume of 2 mL/kg in

doses of 1, 3, and 10 mg/kg. Animals were treated with com-

pound or respective vehicle 6 h after switch off of the light

HPLC-mass spectra were run on an Agilent LC/MSD 1100

mass spectrometer, operating in ES(þ) and ES(-) ionization

mode coupled with HPLC instrument Agilent 1100 series. LC/

MS ES(þ) was performed on a Supelcosil ABZ þ Plus (33 mm ꢀ

4.6 mm, 3 m) (mobile phase, 100% [water þ 0.1% formic acid]

for 1 min, then from 100% [water þ 0.1% formic acid] to 5%

[water þ 0.1% formic acid] and 95% acetonitrile in 5 min, finally

under these conditions for 2 min; T = 40 ꢀC; flow = 1 mL/min.

LC/MS ES(-) was performed on a Supelcosil ABZ þ Plus

(33 mm ꢀ 4.6 mm, 3 m) (mobile phase, 100% [water þ 0.05%

ammonia] for 1 min, then from 100% [water þ 0.05% ammonia]

to 5% [water þ 0.05% ammonia] and 95% acetonitrile in 5 min,

finally under these conditions for 2 min; T=40 ꢀC; flow = 1 mL/

min. In the mass spectra only one peak in the molecular ion

cluster is reported. The usage of this methodology is indicated by

“HPLC-MS” in the analytical characterization of the described

compounds.

Alternatively mass directed analytical HPLC (Agilent tech-

nology HP1100) was carried out using a 19 mm ꢀ100 mm or

30 mm ꢀ100 mm, 5 μm, reversed phase Waters Atlantis column

as the stationary phase and a gradient from water þ 0.1%

formic acid to acetonitrile þ 0.1% formic acid as the eluent. The

HPLC system was monitored by DAD array detector and an

Agilent 110MSD mass spectrometer. The LC elution method

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7789

(using Zorbax Eclipse XDB, 4.6 mm ꢀ 150 mm, 5 μm C8 column)

was the following: 15 min method at 25 ꢀC, mobile phase composed

of different CH₃CN/H₂O-HCOOH 0.1% mixtures at a flow rate

of 1 mL/min (all solvents were HPLC grade, Fluka).

Alternatively HPLC spectra were performed using a reversed-

phase liquid chromatography (ProStar 210/215 PrepStar218)

and UV-vis detector (ProStar 325). The LC elution method

(using Varian Polaris 5 C-18, 150 mmꢀ4.6 mm) was the fol-

lowing: 15 min method at 25 ꢀC, mobile phase composed of

different CH₃CN/H₂O-HCOOH 0.1% mixtures at a flow rate

of 1 mL/min (all solvent were HPLC grade, Fluka).

ZQTM mass spectrometer (Waters) operating in positive and

negative electrospray ionization modes ES(þ) and ES(-) (mass

range 100-1000).

A set of acidic and basic semipreparative gradients have been

used.

Method A consisted of the following chromatographic acidic

conditions for up to 30 mg of crude: column, 100 mm ꢀ 21.2 mm

SupelcosilTM ABZ þ Plus (5 μm particle size); mobile phase, A

[water þ 0.1% formic acid]/B [acetonitrile þ 0.1% formic acid];

flow rate, 20 mL/min; gradient, 5% B for 1 min, 95% B in 9 min,

100% B in 3.5 min.

Alternatively HPLC spectra were performed using a Waters

2690 apparatus at 25 ꢀC using a 3 mm ꢀ100 mm, 3.5 μm,

reversed phase X-Terra C-18 column as the stationary phase

and a gradient from 5% [water þ 0.1% formic acid] to 90%

[acetonitrile þ 0.1% formic acid] during 19.5 min or 20%

[water þ 0.1% formic acid] to 95% [acetonitrile þ 0.1% formic]

during 19 min as the eluent. Flow rate was 0.5 mL/min (all

solvents were HPLC grade, Merck). The HPLC system was

monitored by DAD array detector at 254 nm and a Micromass

Quattromicro mass spectrometer.

Total ion current (TIC) and DAD UV chromatographic

traces together with MS and UV spectra associated with the

peaks were taken on a UPLC/MS Acquity system equipped with

2996 PDA detector and coupled to a Waters Micromass ZQTM

mass spectrometer operating in positive or negative electro-

spray ionization mode. LC/MS ES(() was performed using an

Acquity UPLC BEH C18 column (50 mm ꢀ 21 mm, 1.7 μm

particle size) and column temperature of 40 ꢀC (mobile phase A,

(water þ 0.1% formic acid); mobile phase B, (acetonitrile þ

0.075% formic acid); flow rate of 1.0 mL/min; gradient, t =

0 min 3% B, t=0.05 min 6% B, t=0.57 min 70% B, t=1.4 min

99% B, t=1.45 min 3% B). The usage of this methodology is

indicated by “UPLC/MS” in the analytic characterization of the

described compounds.

GC-MS (Varian Saturn 2000) was carried out using a Varian

Chrompack CP-Sil low bleed/MS 30 m ꢀ 0.25 mm, 0.5 μm

column as the stationary phase and helium (2 mL/min) as the

carrier gas. Injector temperature was 270 ꢀC, and column

temperature was increased from 200 to 300 ꢀC at a rate of

10 ꢀC/min and then held at 300 ꢀC for 5 min. Mass detection was

performed using chemical ionization (CH₃CN) in the range

from 200 m/z to 450 m/z.

For reactions involving microwave irradiation, a Personal

Chemistry Emrys optimizer was used.

For hydrogenation reactions, H-Cube continuous-flow

hydrogenation reactor occasionally was used.

Flash silica gel chromatography was carried out on silica gel

230-400 mesh (supplied by Merck AG Darmstadt, Germany)

or over Varian Mega Be-Si prepacked cartridges or over

prepacked Biotage or Isolute Flas silica cartridges. Alternatively

chromatographic purifications were performed on columns

packed with Merck 60 silica gel, 23-400 mesh, for flash tech-

nique. Thin-layer chromatography was carried out using

Merck TLC plates Kieselgel 60F-254 and visualized with UV

light, 5% phosphomolybdic acid, and aqueous potassium per-

manganate. SPE-SCX cartridges are ion exchange solid phase

extraction columns by supplied by Varian. The eluent used

with SPE-SCX cartridges is methanol followed by 2 N ammo-

nia solution in methanol. Oasis HLB extraction cartridges

are ion exchange solid phase extraction columns supplied by

Waters. The eluent used with HLB cartridges is water followed

by methanol.

In a number of preparations, purification was performed

using either Biotage manual flash chromatography (Flashþ)

or automatic flash chromatography (Horizon) systems. All

these instruments work with standard Biotage Silica cartridges.

In a number of preparations, purification was performed on a

mass-directed autopurification (MDAP) system Fraction Lynx

equipped with Waters 2996 PDA detector and coupled with a

Method B consisted of the following chromatographic acidic

conditions for up to 100 mg of crude: column, 150 mm ꢀ 30 mm

XTerra Prep MS C18 (10 μm particle size); mobile phase, A

[water þ 0.1% formic acid]/B [acetonitrile þ 0.1% formic acid];

flow rate, 40 mL/min; gradient, 1% B to 100% B in 7 min lasting

for 7.5 min.

Method C consisted of the following chromatographic basic

conditions for up to 100 mg of crude: column, 150 mm ꢀ 30 mm

XTerra Prep MS C18 (10 μm particle size); mobile phase, A

[water þ 10 mM ammonium carbonate (adjusted to pH 10 with

ammonia)]/B [acetonitrile]; flow rate, 40 mL/min; gradient,

10% B for 0.5 min, 95% B in 12.5 min.

General procedures for the preparation of compounds 9 and

43-77 are shown in Scheme 1. The purity of the compounds

reported in the manuscript was established through HPLC and/

or ¹H NMR methodologies. All final compounds reported in the

manuscript have a purity of >95%.

11,11-Di-2-propen-1-yl-5,11-dihydro-10H-dibenzo[a,d ]cyclohep-

ten-10-one (3a). The starting ketone 5,11-dihydro-10H-dibenzo-

[a,d ]cyclohepten-10-one (1a, 10 g, 48 mmol, whose preparation

has been described in J. Med. Chem. 2004, 47, 4202-4212) was

suspended in tert-butanol (100 mL) and heated to 35 ꢀC. Potas-

sium tert-butoxide (13.5 g, 120 mmol) and allyl bromide (16.6 mL,

192 mmol) were added at this temperature, and the pink suspen-

sion was stirred for 1 h. The suspension was cooled to room

temperature and quenched with NH₄Cl (saturated solution). The

mixture was partitioned between diethyl ether and water. The

organic phase was separated, dried over N₂SO₄, and evaporated in

vacuum to give the crude material (13 g) as an orange oil as a

mixture of C-diallyl (3a) and C-allyl/O-allyl (2a), which was

progressed to the Claisen rearrangement without any further

purification/characterization. The neat mixture (13 g) was heated

at 185 ꢀC for 15 min to give the desired product (3a, 13 g) that was

used in the next step without any further purification. MS (ESI)

m/z: 311 [M þ Na]^þ. ¹H NMR (400 MHz, chloroform-d) δ:

2.75-2.96 (m, 4H), 3.94 (s, 2H), 4.92-5.04 (m, 4H), 5.42-5.55(m,

2H), 7.10-7.35 (m, 8H).

8-Chloro-11,11-di-2-propen-1-yldibenzo[b,f ]oxepin-10(11H)-

one (3d). The solution of KO^tBu (2.47 g,22 mmol) in 100 mL of

the BuOH was stirred for 10 min under a stream of argon at

t

room temperature. Then 8-chlorodibenzo[b,f ]oxepin-10(11H)-

one (1 g, 4.1 mmol; for preparation see J. Med. Chem. 1980,

23 (5), 494-501) and allyl bromide (9.31 mL,21,8 mmol) were

added. The mixture was then heated at 60 ꢀC for 3 h. After the

mixture was cooled to room temperature, a saturated solution of

NaHCO₃(150 mL) was added in one portion. The mixture was

left under magnetic stirring for an additional 15 min. The

precipitate was filtered off, and the aqueous phase was then

extracted using ethyl acetate (3 ꢀ 50 mL). Collected organic

layers were washed with saturated NaHCO₃solution, dried with

anhydrous Na₂SO₄, and evaporated, giving a crude product (3d,

1

1.8 g) as an oil. GC-MS m/z: 325 [M þ 1] ^þ. H NMR (400

MHz, chloroform-d) δ: 2.85-3.01 (m, 4H), 5.04-5.12 (m, 4H),

5.62-5.76 (m, 2H), 7.14-7.30 (m, 7H).

Spiro[cyclopent-3-ene-1,10⁰-dibenzo[a,d]cyclohepten]-11⁰(5⁰H)-

one (4a). To a solution of 11,11-di-2-propen-1-yl-5,11-dihydro-

10H-dibenzo[a,d ]cyclohepten-10-one (3a, 1.00 g, 3.47 mmol) in

degassed DCM (1 L) was added Grubbs second generation

catalyst (15 mol %, 0.44 g) under argon atmosphere at room

7790 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Gianotti et al.

temperature. The reaction mixture was stirred at room tempera-

ture overnight. The dark solution was then adsorbed on silica gel

(10equivwtrelative tocatalyst) andpassedthrougha pad ofsilica

gel (petroleum ether/diethyl ether 1/1). The filtered solution was

stirred with activated charcoal (50 equiv wt relative to product)

for 12 h. After the carbon was filtered, the filtrate was concen-

trated in vacuum and purified by silica gel column chromatogra-

phy (petroleum ether/diethyl ether = 9/1) to give the title

compound (4a, 748 mg) as a white solid. MS (ESI) m/z: 283

[M þ Na]^þ. ¹H NMR (400 MHz, chloroform-d) δ: 2.92-2.99 (m,

2H), 3.56-3.63 (m, 2H), 4.36 (m, 2H), 5.71-5.74 (m, 2H),

7.09-7.51 (m, 7H), 7.78-7.88 (m, 1H).

2⁰-Chloro-3H,11⁰H-spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-

3,11⁰-dione (7d). 2⁰-Chloro-11⁰H-spiro[cyclopent-3-ene-1,10⁰-di-

benzo[b,f ]oxepin]-11⁰-one (4d, 2.1 g, 7.08 mmol) was melted in

anhydrous THF (100 mL) under argon atmosphere, and 1 M

solution of BH₃-THF (8 mL, 8 mmol) was added dropwise. The

mixture was left for 4 h under stirring at room temperature.

After 4 h, H₂O (20 mL) and 10% NaOH (10 mL) were added

followed by 30% H₂O₂(5 mL). The mixture was stirred over-

night and then diluted with H₂O (40 mL) and extracted with

diethyl ether. Organic layer was washed with brine, dried, and

evaporated, giving a crude product as a mixture of 5d and 6d

(2 g). The mixture was dissolved in dry DCM (100 mL), and

Dess-Martin (triacetoxyperiodinane) (9 g, 21.31 mmol) was

added. The mixture was stirred overnight under argon atmo-

sphere. The reaction was worked up by washing three times with

NaOH (10% aqueous solution). Water layers were washed with

DCM and combined organic layers were dried (Na₂SO₄/

MgSO₄) and evaporated to give the crude title compound (7d,

2.13 g), which was used in the next step without any further

purification. HPLC-MS m/z: 312.89 [M þ 1]^þ; t_R=8.44 min.

GC-MS m/z: 313 [M þ 1]^þ. ¹H NMR (400 MHz, chloroform-d)

δ: 2.18-2.39 (m, 2H), 2.41-2.47 (m, 1H), 2.76-2.79 (d, 1H),

3.07-3.12 (m, 1H), 7.25-7.38 (m, 5H), 7.50-7.52 (m, 1H),

8.01-8.02 (d, 1H).

2⁰-Chloro-11⁰H-spiro[cyclopent-3-ene-1,10⁰-dibenzo[b,f ]oxepin]-

11⁰-one (4d). An amount of 3 L of dry DCM was degassed with

argon, and 8-chloro-11,11-di-2-propen-1-yldibenzo[b,f ]oxepin-

10(11H)-one (3d, 6.78 g, 20.8 mmol) and Grubbs second genera-

tion catalyst (15.5 mol %, 1.275 g) were added. The solution was

stirred under argon atmosphere for 8 h, and then it was passed

through a prepacked silica gel column and evaporated. The crude

oil obtained (6.5 g) was purified by silica gel column chromatog-

raphy (n-hexane/ethyl acetate = 9:0.2), and the fraction of the

product that was obtained gave a precipitate after suspension in

hexane to give the title compound (4d, 3.5 g). GC-MS m/z: 297

[M þ 1] ^þ. ¹H NMR (400 MHz, chloroform-d) δ: 2.92-3.95 (d,

2H), 3.42-3.46 (d, 2H), 5.72 (s, 2H), 7.20-7.35 (m, 6H), 7.45-

7.47 (m, 1H).

Mixture of 3-Hydroxyspiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-11⁰(5⁰H)-one (5a) and 5⁰,11⁰-Dihydrospiro[cyclo-

pentane-1,10⁰-dibenzo[a,d ]cycloheptene]-3,11⁰-diol (6a). 1 M borane

solution in THF (0.77 mL, 0.77 mmol) was added dropwise to

a stirred solution of spiro[cyclopent-3-ene-1,10⁰-dibenzo-

[a,d ]cyclohepten]-11⁰(5⁰H)-one (4a, 0.200 g, 0.77 mmol) in anhy-

drous THF (1.6 mL) at room temperature under an N₂atmo-

sphere, and the mixture was stirred at room temperature for

2.5 h. Water (0.08 mL) was then added dropwise, followed by

3 M sodium hydroxide (0.10 mL). Hydrogen peroxide (0.12 mL,

35%) was then added at a rate to maintain the temperature

between 30 and 50 ꢀC, and the reaction mixture was stirred for

16 h at room temperature. Diethyl ether (1.6 mL) was added to

the reaction mixture, and the organic phase was washed with

brine and water. The organic solvent was evaporated to give a

residue which, for analytical purposes, was purified by silica gel

column chromatography (5/1 petroleum ether/diethyl ether),

affording 3-hydroxyspiro[cyclopentane-1,10⁰-dibenzo[a,d ]cyclo-

hepten]-11⁰(5⁰H)-one (5a, 0.091 g) and 5⁰,11⁰-dihydrospiro[cyclo-

pentane-1,10⁰-dibenzo[a,d ]cycloheptene]-3,11⁰-diol (6a, 0.079 g).

3-Hydroxyspiro[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-

11⁰(5⁰H)-one (5a). MS (ESI) m/z: 279 [M þ 1]^þ, 301 [M þ Na]^þ,

261 [M - H₂O]^þ, 579 [2M þ Na]^þ. ¹H NMR (400 MHz, chloro-

form-d) δ: 1.66-1.92 (m, 2H), 2.12-2.21 (m, 1H), 2.32-2.48

(m, 1H), 2.85-2.95 (m, 1H), 3.21-3.30 (m, 1H), 4.35-4.44 (m,

3H), 7.10-7.46 (m, 7H), 7.91-7.95 (m, 1H).

5⁰,11⁰-Dihydro-3H-spiro[cyclopentane-1,10⁰-dibenzo[a,d ]cyclo-

hepten]-3-one (8a). In a Parr apparatus, 3H-spiro[cyclopentane-

1,10⁰-dibenzo[a,d ]cycloheptene]-3,11⁰(5⁰H)-dione (7a, 2.200 g,

7.96 mmol) was dissolved in THF (40 mL). AcOH (10.00 mL)

and wet Pd/C (10% (w/w), 50% w/w of water content) (4.24 g,

3.98 mmol) were added, and the mixture was hydrogenated

(0.016 g, 7.96 mmol) under 5 atm pressure for 5 days. During

this time three portions of Pd/C (2.5 g, 10% (w/w), 50% w/w of

water content) were added. The palladium was then filtered over

Celite and the solvent evaporated to afford the title compound

(8a, 2.2 g) asa racemic mixture. MS(ESI) m/z: 285 [Mþ Na]^þ. ¹H

NMR (400 MHz, chloroform-d) δ: 2.24-2.32 (m, 2H), 2.51-

2.62 (m, 4H), 3.09-3.16 (m, 2H), 4.11-4.21 (m, 2H), 7.04-7.35

(m, 8H).

The racemic mixture of 5⁰,11⁰-dihydro-3H-spiro[cyclopen-

tane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-one (8a) was submitted

for preparative chiral HPLC (column, Chiralpak IA (25 cm ꢀ

2.0 cm), 5 μm; bobile phase, n-hexane/(ethanol/methanol

50/50) 96/4% v/v; flow rate, 14 mL/min; UV, 225 nm; 19 mg/

inj in CH₂Cl₂/ethanol/methanol/hexane) and gave two enantio-

mers:

(-)-(1R)-5⁰,11⁰-Dihydro-3H-spiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-one(8a(R) (ent 1)). [R]²_D⁰-83ꢀ (c 0.88, CHCl₃)

(optical rotation was measured on a different batch); retention

time = 12.5 min (683 mg).

(þ)-(1S)-5⁰,11⁰-Dihydro-3H-spiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-one (8a(S) (ent 2)). [R]²_D⁰þ83ꢀ (c 0.93,

CHCl₃) (optical rotation was measured on a different batch);

retention time = 14.0 min (655 mg).

5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]cycloheptene]-

3,11⁰-diol (6a). MS (ESI) m/z: 303 [M þ Na]^þ. ¹H NMR (400 MHz,

chloroform-d) δ: 1.83-2.43 (m, 5H), 2.61-2.71 (m, 1H), 3.81-3.89

(m, 1H), 4.47-4.61 (m, 2H), 5.03 (s, 1H), 7.03-7.61 (m, 8H).

3H-Spiro[cyclopentane-1,10⁰-dibenzo[a,d ]cycloheptene]-3,11⁰-

(5⁰H)-dione (7a). To a solution of Dess-Martin triacetoxyper-

iodinane (0.38 g, 0.9 mmol) a mixture of 3-hydroxyspiro-

[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-11⁰(5⁰H)-one and

5⁰,11⁰-dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohep-

tene]-3,11⁰-diol (5a and 6a, 0.100 g, 0.36 mmol) was added in dry

DCM (8 mL). The reaction mixture was left at 25 ꢀC for 3.5 h.

The mixture was diluted with DCM (10 mL) and washed with

NaOH (1 N) and then brine. The organic layer was dried over

Na₂SO₄and after solvent evaporation gave the title compound

(7a, 92 mg). MS (ESI) m/z: 299 [M þ Na]^þ. ¹H NMR (400 MHz,

chloroform-d) δ: 2.26-2.49 (m, 3H), 2.76-2.84 (m, 1H), 3.26-

3.46 (m, 2H), 4.32-4.51 (m, 2H), 7.16-7.48 (m, 7H), 7.92-7.96

(m, 1H).

3H,11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-3-one (8c).

THF (10 mL) and glacial acetic acid (5 mL) were mixed in a Paar

bottle, and 2⁰-chloro-3H,11⁰H-spiro[cyclopentane-1,10⁰-dibenzo[b,

f]oxepin]-3,11⁰-one (7d, 100 mg, 0.319 mmol) and Pd/C (100 mg)

were added. The mixture was shaken in the Paar apparatus under H₂

atmosphere (6 bar) at room temperature for 2 days. Then the

mixture was filtered through a membrane filter and evaporated.

The residue was dissolved in ethyl acetate (10 mL) and washed with

NaHCO₃(saturated solution 3 ꢀ 15 mL). Organic layer was dried

(Na₂SO₄/MgSO₄) and evaporated to give an oil that crystallized

from MeOH to give the title compound (8c, 91.2 mg) as a racemic

mixture. GC-MS m/z: 265 [M þ 1]^þ. ¹H NMR (400 MHz, chloro-

form-d) δ: 2.33-2.49 (m, 4H), 2.62-2.66 (d, 1H), 2.74-2.78

(d, 1H), 3.07-3.18 (m, 2H), 7.02-7.27 (m, 8H).

The racemic mixture of 3H,11⁰H-spiro[cyclopentane-1,10⁰-

dibenzo[b,f ]oxepin]-3-one (8c, 550 mg) was submitted for

preparative chiral HPLC (column=Chiralcel OJ-H; mobile

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7791

phase = n-hexane/isopropanol 88/12; flow rate =14 mL/min;

UV, 225 nm; 25 mg/inj) and gave two enantiomers:

and the mixture was stirred for an additional 4 h. The mixture

was washed with saturated aqueous NaHCO₃solution, then

brine, and the organic phase was separated and concentrated

under vacuum. The crude product was purified using a SCX

cartridge, eluting first with DCM, second with MeOH, and third

with NH₃in MeOH (2 M). After solvent evaporation the desired

compound (13, 94 mg) was obtained as mixture of diastereoi-

somers (∼85/15). UPLC/MS R_f=0.66; m/z (ES) 388.2 [M þ H]^þ.

The diastereomeric mixture of methyl 1-(5⁰,11⁰-dihydrospiro-

[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-yl)-1,2,5,6-

tetrahydro-3-pyridinecarboxylate (13, 94 mg) was submitted for

chiral HPLC purification (preparative chromatographic condi-

tions: column = Chiralcel OD-H; mobile phase = n-hexane/

ethanol 75/25% v/v; flow rate = 0.8 mL/min; DAD=210-340

nm; CD=225 nm) to give 13⁰and 13⁰⁰.

3H,11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-3-one (8c

(ent 1)). Retention time=17.1 min (171 mg).

3H,11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-3-one (8c

(ent 2)). Retention time=19.7 min (180 mg).

(1S)-N,N-Dimethyl-5⁰,11⁰-dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-amine (9a). Dimethylamine (2.0 M in THF, 0.09

mL, 0.172 mmol, 1.5 equiv) and (þ)-(1S)-5⁰,11⁰-dihydro-3H-spiro-

[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-one (8a(S), 30 mg,

0.115) were mixed in DCE and glacial acetic acid (2 drops). After

20 min at room temperature NaBH(OAc)₃(29 mg, 0.137 mmol, 1.2

equiv) was added and the resulting reaction mixture was stirred at

room temperature from 6 h. The workup was done by adding

NaHCO₃(saturated solution) and then by extracting with DCM.

Products were purified by silica gel column chromatography.

The reaction mixture was quenched with NaHCO₃(saturated

aqueous solution) and extracted with dichloromethane. The

organic layers were combined, dried (Na₂SO₄), and concen-

trated in vacuo. The crude product was purified by SCX, afford-

ing the title compound (9, 21 mg) as a mixture of diastereoi-

somers (∼85/15).

Methyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylate

1

(13⁰). Retention time = 8 min (70 mg). H NMR (400 MHz,

chloroform-d) δ: 6.98-7.37 (m, 9H), 4.27 (d, 2H), 4.02 (d, 2H),

3.72-3.79 (m, 3H), 3.36-3.45 (m, 1H), 3.21-3.34 (m, 2H),

3.05-3.19 (m, 2H), 2.55-2.73 (m, 2H), 2.34-2.50 (m, 2H),

2.20-2.33 (m, 2H), 2.08-2.19 (m, 1H), 1.80-2.05 (m, 3H).

Methyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylate

This diastereomeric mixture was submitted for chiral HPLC

purification (preparative SFC chromatographic conditions:

column, Chiralcel OD-H, 25 cm ꢀ 2.0 cm; pressure, 172 bar;

flow rate, 22 mL/min; UV detection, CD 220 nm; modifier,

ethanol þ 0.1% isopropylamine 10%) to give two diastereoi-

somers:

(1S,3R)-N,N-Dimethyl-5⁰,11⁰-dihydrospiro[cyclopentane-1,10⁰-

dibenzo[a,d ]cyclohepten]-3-amine (9⁰a). Retention time = 14.05

min (5.1 mg). ¹H NMR (400 MHz, chloroform-d) δ: 1.87-2.22

(m, 6H), 2.33 (s, 6H), 2.84 (m, 1H), 3.15 (d, 1H), 3.27 (d, 1H), 4.04

(d, 1H), 4.25 (d, 1H), 7.01-7.32 (m, 8H).

1

(13⁰⁰). Retention time=10 min (11 mg). H NMR (400 MHz,

chloroform-d) δ: 7.39-7.52 (m, 1H), 7.09-7.27 (m, 6H), 6.99-

7.09 (m, 2H), 4.24 (d, 1H), 4.06 (d, 1H), 3.75 (s, 3H), 3.01-3.40

(m, 5H), 2.54-2.74 (m, 2H), 2.25-2.46 (m, 2H), 1.87-2.19

(m, 4H).

Ethyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-4-piperidinecarboxylate (16). To a solu-

tion of (þ)-(1S)-5⁰,11⁰-dihydro-3H-spiro[cyclopentane-1,10⁰-

dibenzo[a,d ]cyclohepten]-3-one (8a(S), 74 mg, 0.27 mmol) and

ethyl isonipecotate (0.082 mL, 0.534 mmol) in 1,2-dichlo-

roethane (4 mL) was added AcOH (2 drops). The reaction mix-

ture was stirred at room temperature for 1 h, and then NaBH-

(OAC)₃(85 mg, 0.400 mmol) was added. The resulting mixture

was stirred overnight. The mixture was diluted with DCM and

washed with a saturated solution of NaHCO₃, brine. The phases

were separated, and the organic layer was concentrated under

vacuum. The crude mixture was purified by passing through a

12 g Si-redisep cartridge (eluting with cHex/EtOAc, from 100:0

to 80:20) to afford the title compound (16, 82 mg) as mixture

of two diastereoisomers. UPLC/MS R_f= 0.63; m/z (ES) 404.2

[M þ H]^þ.

(1S,3S)-N,N-Dimethyl-5⁰,11⁰-dihydrospiro[cyclopentane-1,10⁰-

dibenzo[a,d ]cyclohepten]-3-amine (9⁰⁰a). Retention time = 17.39

min (1.9 mg). ¹H NMR (400 MHz, chloroform-d) δ: 1.85-2.27

(m, 6H), 2.36 (s, 6H), 2.89 (m, 1H), 3.07 (d, 1H), 3.19 (d, 1H), 4.07

(d, 1H), 4.23 (d, 1H), 7.03-7.25 (m, 7H), 7.43-7.49 (m, 1H).

Methyl 1-((1R)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylate (12).

To a solution of (-)-(1R)-5⁰,11⁰-dihydro-3H-spiro[cyclopentane-

1,10⁰-dibenzo[a,d ]cyclohepten]-3-one (8a(R), 60 mg, 0.229 mmol)

in 1,2-DCE (2 mL) were added methyl 1,2,5,6-tetrahydro-3-pyridi-

necarboxylate (40.6 mg, 0.229 mmol) and DIPEA (0.044 mL, 0.252

mmol). After the mixture was stirred for 10 min at room tempera-

ture, AcOH (2 drops) and NaBH(OAc)₃(72.7 mg, 0.343 mmol)

were added. The reaction mixture was stirred at room temperature

overnight. The mixture was washed with saturated aqueous NaH-

CO₃solution and then brine. The layers were separated and the

organic layer was evaporated under vacuum to afford a yellow oil,

which was dissolved in methanol and purified using a 1 g SCX

cartridge (eluting first with MeOH and then 2 M NH₃in MeOH).

Evaporation of the solvent gave the title product (12, 55 mg) as a

mixture of diastereoisomers as yellow foam. UPLC/MS R_f=0.63;

m/z (ES) 388.1 [M þ H]^þ. ¹H NMR (400 MHz, chloroform-d) δ:

6.95-7.37 (m, 9H), 4.15-4.31 (m, 1H), 3.92-4.13 (m, 1H), 3.65-

3.85 (m, 3H), 2.99-3.47 (m, 5H), 2.52-2.75 (m, 2H), 1.79-2.51

(m, 8H).

This diastereomeric mixture of ethyl 1-(5⁰,11⁰-dihydrospiro-

[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-yl)-4-piper-

idinecarboxylate (16) was submitted for chiral chromatography

purification (preparative chromatographic conditions: column =

Chiralcel OD-H; mobile phase = n-hexane/isopropanol 85/15%

v/v; flow rate=1 mL/min; DAD=210-340 nm; CD=225 nm) to

give 16⁰.

Ethyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-4-piperidinecarboxylate (16⁰). Only the

major diastereoisomer was isolated: retention time=7 min (60

mg). ¹H NMR (400 MHz, chloroform-d) δ: 6.84-7.48 (m, 8H),

3.86-4.40 (m, 3H), 2.74-3.44 (m, 4H), 1.62-2.48 (m, 15H),

1.16-1.39 (m, 3H).

Methyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylate

(13). To a solution of (þ)-(1S)-5⁰,11⁰-dihydro-3H-spiro[cyclo-

pentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-one, (8a(S),100 mg,

0.38 mmol) in 1,2-dichloroethane (5 mL) were added methyl

1,2,5,6-tetrahydro-3-pyridinecarboxylate hydrochloride (67.7

mg, 0.381 mmol) and DIPEA (0.073 mL, 0.419 mmol). After

the mixture was stirred for 10 min at room temperature, AcOH

(0.044 mL, 0.762 mmol) and NaBH(OAC)₃(121 mg, 0.572

mmol) were added. The reaction mixture was stirred at room

temperature overnight. Further guvacine methyl esther hydro-

cloride (0.3 equiv) and NaBH(OAC)₃(0.5 equiv) were added,

Ethyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-4-fluoro-4-piperidinecarboxylate (19). To

a solution of (þ)-(1S)-5⁰,11⁰-dihydro-3H-spiro[cyclopentane-

1,10⁰-dibenzo[a,d ]cyclohepten]-3-one (8a(S), 58 mg, 0.221 mmol)

and ethyl 4-fluoro-4-piperidinecarboxylate (prepared as described

in WO/2002/032893²⁸) (58.1 mg, 0.332 mmol) in 1,2-dichlor-

oethane (3 mL) was added a drop of AcOH. The reaction mixture

was stirred at room temperature for 0.5 h, and then NaBH(OAC)₃

(70.3 mg, 0.332 mmol) was added. The resulting reaction mixture

was stirred for 3 h, quenched with NaHCO₃(saturated aqueous

solution), and extracted with dichloromethane. The organic layers

were combined, dried (Na₂SO₄), and concentrated in vacuo.

7792 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Gianotti et al.

The crude product was purified by flash chromatography on silica

gel (20 g), eluting with a gradient of dichloromethane/methanol

99.5/0.5 to 99/1 to afford the title compound (19, 30 mg) as a

mixture of diastereoisomers (∼10/90).

The diastereomeric mixture (30, 4.5 g) was submitted for

chiral HPLC purification (semipreparative SFC conditions:

column = Chiralcel OD-H; modifier = ethanol 15%; flow

rate=2.5 mL/min; pressure=120 bar; temperature=38 ꢀC;

detector = 220 nm) to give two single diastereoisomers, but only

the major distereoisomer was characterized:

This diastereomeric mixture was submitted for chiral

HPLC purification (preparative chromatographic conditions:

column = Chiralcel OJ-H; mobile phase = n-hexane/EtOH 85/

15% v/v; flow rate = 0.8 mL/min; DAD = 210-340 nm; CD =

225 nm) to give 19⁰.

Methyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-3-azetidinecarboxylate (30⁰). Retention

time = 4.9 min (2.62 g). H NMR (400 MHz, chloroform-d)

1

Ethyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-4-fluoro-4-piperidinecarboxylate (19⁰). Only

the major diastereoisomer was isolated: retention time = 21.9 min

(21mg). UPLC/MSR_f= 0.70; m/z(ES) 422.2 [M þ H]^þ. ¹HNMR

(400 MHz, chloroform-d) δ: 7.31-7.32 (m, 1H), 7.14-7.21 (m,

6H), 7.00-7.08 (m, 1H), 4.20-4.35 (m, 3H), 4.02 (d, 1H), 3.31 (d,

1H), 3.12 (d, 1H), 2.97-3.08 (m, 2H), 2.74-2.90 (m, 1H),

1.78-2.49 (m, 12H), 1.34 (t, 3H).

δ: 7.30-7.02 (m, 8 H), 4.13 (q, 2 H), 3.75 (s, 3 H), 3.63-3.57 (m,

2 H), 3.39-3.08 (m, 6 H), 2.15-1.65 (m, 6 H).

Methyl 1-(11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-

3-yl)-1,2,3,6-tetrahydro-4-pyridinecarboxylate (34). To 3H,11⁰H-

spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-3-one (8c,100 mg,

0.378 mmol) in DCE (5 mL) were added methyl 1,2,3,6-tetra-

hydro-4-pyridinecarboxylate (81 mg, 0.574 mmol) HCl salt and

DIPEA (0.066mL, 0.378 mmol). After the mixture was stirred for

10 min, sodium triacetoxyborohydride (120 mg, 0.567 mmol) and

acetic acid (2 drops) were added. The mixture was left stirring

overnight. The reaction was quenched with NaHCO₃. The

organic layer was separated and the water phase extracted with

DCM. The combined organic layers were dried over Na₂SO₄and

the solvent was evaporated to give the title compound (34, 150

mg) as a mixture of two diastereoisomeric racemates. m/z (ES):

390.2 [M þ H]^þ.

Ethyl 3-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-3-azabicyclo[3.1.0]hexane-1-carboxylate

(29). To a solution of (þ)-(1S)-5⁰,11⁰-dihydro-3H-spiro[cyclo-

pentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-one (8a(S), 70 mg,

0.267 mmol) and ethyl 3-azabicyclo[3.1.0]hexane-1-carboxylate

(83 mg, 0.534 mmol, for preparation see WO/2007/055093²⁹) in

1,2-dichloroethane (5 mL) under nitrogen was added AcOH

(2 drops). The reaction mixture was stirred at room temperature

for 1 h 30 min, and then NaBH(OAC)₃(85 mg, 0.400 mmol) was

added. The resulting mixture was stirred overnight. The mixture

was diluted with DCM. The organic phase was then washed with

saturated solution of NaHCO₃, then brine and concentrated

under vacuum. The crude product was purified through Si-

redisep cartridge (12 g), eluting with cHex/EtOAc (from 100:0

to 80:20) to afford the title compound (29, 82 mg) as a mixture

of two diastereoisomeric racemates. UPLC/MS R_f= 0.67; m/z

(ES) 402.2 [M þ H]^þ.

This mixture was submitted for chiral HPLC separation (pre-

parative chromatographic conditions: column = Chiralcel OJ-H;

mobile phase = n-hexane/(ethanol þ 0.1% isopropylamine) 93/7%

v/v; flow rate = 14 mL/min; DAD = 225 nm) to give 34⁰isomer 1

and 34⁰isomer 2 (the minor isomers were not characterized).

Methyl 1-(11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-

3-yl)-1,2,3,6-tetrahydro-4-pyridinecarboxylate (34⁰, Isomer 1).

Retention time = 17.23 min (28 mg). ¹H NMR (400 MHz,

chloroform-d) δ: 7.26-7.32 (m, 1 H), 7.00-7.25 (m, 7 H),

6.87-6.94 (m, 1 H), 3.76 (s, 3 H), 3.05-3.32 (m, 4 H),

2.85-2.99 (m, 1 H), 2.59-2.71 (m, 2 H), 2.38-2.52 (m, 3 H),

2.14 (d, 5 H).

Methyl 1-(11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-

3-yl)-1,2,3,6-tetrahydro-4-pyridinecarboxylate (34⁰, Isomer 2).

Retention time = 26.42 min (27 mg). ¹H NMR (400 MHz,

chloroform-d) δ: 7.27-7.33 (m, 1 H), 7.01-7.24 (m, 7 H),

6.87-6.95 (m, 1 H), 3.76 (s, 3 H), 3.06-3.31 (m, 4 H),

2.83-2.99 (m, 1 H), 2.60-2.70 (m, 2 H), 2.38-2.51 (m, 3 H),

2.14 (s, 5 H).

This mixture was submitted for chiral HPLC separation

(preparative chromatographic conditions: column = Chiralcel

OJ-H; mobile phase = n-hexane/ethanol/isopropanol 96/2/2%

v/v⁰; flow rate = 1 mL/min; DAD=210-340 nm; CD=225 nm)

to obtain 29⁰⁰isomer 1 and 29⁰⁰isomer 2; minor isomers were not

characterized.

Ethyl 3-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-3-azabicyclo[3.1.0]hexane-1-carboxylate

1

(29⁰Isomer 1). Retention time=13.62 min (28 mg). H NMR

(400 MHz, chloroform-d) δ: 7.29-7.34 (m, 1 H), 7.08-7.24 (m,

6 H), 7.00-7.07 (m, 1 H), 4.22 (d, 1 H), 4.14 (q, 2 H), 4.04 (d,

1 H), 3.21-3.29 (m, 1 H), 3.08-3.16 (m, 3 H), 2.96-3.06 (m,

1 H), 2.68 (d, 1 H), 2.41-2.51 (m, 1 H), 1.77-2.15 (m, 7 H),

1.44-1.51 (m, 1 H), 1.29-1.37 (m, 1 H), 1.25 (t, 3 H).

Methyl 1-(11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-

3-yl)-3-azetidinecarboxylate (38). 3H,11⁰H-Spiro[cyclopentane-

1,10⁰-dibenzo[b,f]oxepin]-3-one (8c (ent 2), 70 mg, 0.265 mmol)

and methyl 3-azetidinecarboxylate hydrochloride (48.2 mg,

0.318 mmol) in acetonitrile (4 mL) were stirred under nitrogen

to give a colorless solution. After the mixture was stirred

for 30 min at room temperature, NaBH(OAC)₃(84 mg, 0.397

mmol) was added. The mixture was stirred overnight. Water

was added. The solution was concentrated under reduced

pressure and the aqueous layer extracted with DCM. The

phases were separated on a hydrophobic frit, and the combined

organic solvent was evaporated. The product was purified

using a NH₂5 g column, eluting with EtOAc/cyclohexane

1:9 to give the title compound (38, 85 mg) as a mixture of

two diastereoisomers. UPLC/MS R_f=0.65; m/z (ES) 364.06

[M þ H]^þ.

Ethyl 3-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-3-azabicyclo[3.1.0]hexane-1-carboxylate

(29⁰Isomer 2). Retention time = 17.76 min (26 mg). ¹H NMR

(400 MHz, chloroform-d) δ: 7.26-7.32 (m, 1 H), 7.08-7.25 (m,

6 H), 6.98-7.07 (m, 1 H), 4.12-4.26 (m, 3 H), 4.05 (d, 1 H),

3.21-3.29 (m, 2 H), 3.14 (d, 1 H), 2.97-3.07 (m, 2 H), 2.78 (d,

1 H), 2.33-2.42 (m, 1 H), 1.80-2.13 (m, 7 H), 1.43-1.54 (m,

1 H), 1.33-1.39 (m, 1 H), 1.28 (t, 3 H).

Methyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-3-azetidinecarboxylate (30). A mixture of

(þ)-(1S)-5⁰,11⁰-dihydro-3H-spiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-one (8a(S), 5.45 g, 20.77 mmol) and methyl 3-aze-

tidinecarboxylate hydrochloride (3.78 g, 24.9 mmol) in dry DCM

(25 mL) was stirred for 15 min. NaBH(OAC)₃(8.81 g, 41.5 mmol)

was then added and the reaction mixture stirred overnight,

quenched with NaHCO₃(saturated aqueous solution), and

extracted with dichloromethane. The organic layers were combined,

dried (Na₂SO₄), and concentrated in vacuo. The crude product was

purified by flash chromatography on silica gel, eluting with a

gradient of MeOH in dichloromethane (from 0% to 3%) to afford

the title compound (30, 6.7 g) as a mixture of two diastereoisomers.

UPLC/MS R_f= 0.61; m/z (ES) 362.11 [M þ H]^þ.

The diastereomeric mixture (38) was submitted for chiral

HPLC purification (semipreparative chiral SFC conditions:

column = Chiralpak AD-H; modifier = ethanol þ 0.1% isopropy-

lamine, 10%; flow rate = 52 mL/min; pressure = 120 bar; tem-

perature = 38 ꢀC; DAD = 220 nm) to give 38⁰(only the major dias-

tereoisomer was characterized).

Methyl 1-(11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-

3-yl)-3-azetidinecarboxylate (38⁰). Retention time = 7.0 min (19

mg). ¹H NMR (400 MHz, chloroform-d) δ: 7.29-7.00 (m, 8 H),

3.73 (s, 3 H), 3.58-2.94 (m, 8 H), 2.21-1.58 (m, 6 H).

Article

1-((1R)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7793

3.60-3.96 (m, 3 H), 2.85-3.36 (m, 4 H), 1.86-2.74 (m, 7 H),

1.10-1.39 (m, 2 H).

cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic Acid

Hydrochloride Salt (45). To a solution of methyl 1-((1R)-5⁰,

11⁰-dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-

3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylate (12, 55 mg,

0.142 mmol) in methanol/water 3:1 (2.4 mL) was added LiOH

(16.99 mg, 0.710 mmol), and the reaction mixture was left to stir

for 4 h. Further LiOH (17 mg, 0.71 mmol) was added, and the

mixture was left to stir overnight. The organic solvent was

evaporated under vacuum and the aqueous phase washed with

DCM. The reaction mixture was slowly acidified with 3 N HCl,

checking the pH of the solution. A white precipitate formed at

pH 1. The solid was filtered and dried under vacuum. The solid

was triturated from EtOAc to give the title compound (45, 41

mg). UPLC/MS R_f= 0.62; m/z (ES) 374.3 [M þ H]^þ. ¹H NMR

(400 MHz, chloroform-d) δ: 6.95-7.37 (m, 9H), 4.15-4.31 (m,

1H), 3.92-4.13 (m, 1H), 3.65-3.85 (m, 3H), 2.99-3.47 (m, 5H),

2.52-2.75 (m, 2H), 1.79-2.51 (m, 8H).

(-)-1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-4-piperidinecarboxylic Acid (51). LiOH

(17.80 mg, 0.743 mmol) was added to a solution of ethyl

1-((1S)-5⁰,11⁰-dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-yl)-4-piperidinecarboxylate (16⁰, 60 mg, 0.149

mmol) in ethanol (5 mL) and water (1 mL). The reaction mixture

was left to stir at 70 ꢀC for 4 h, overnight at room temperature,

and then at 70 ꢀC for further for 3 h. The ethanol was evaporated

under vacuum, and the mixture was acidified with 3 N HCl (until

pH 1 was reached). The solution was purified using an HLB

cartridge (6 g) by eluting first with water and then MeOH to

afford the title compound (51, 55 mg). [R]²_D⁰-18.6ꢀ (c 0.63,

MeOH) (optical rotation was measured on a different batch and

for the HCl salt). UPLC/MS R_f= 0.62; m/z (ES) 376.2 [M þ

H]^þ. ¹H NMR (400 MHz, chloroform-d) δ: 7.30-7.44 (m, 1 H),

6.99-7.23 (m, 7 H), 3.95-4.25 (m, 2 H), 3.14-3.71 (m, 6 H),

1.80-2.70 (m, 12 H).

Methyl 1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo-

[a,d ]cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylate (46).

Methyl 1-((1S)-5⁰,11⁰-dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylate (13, 66

mg, 0.17 mmol) was dissolved in methanol (1.8 mL)/water (0.6

mL), and LiOH (20.39 mg, 0.852 mmol) was added. The reaction

mixture was left under stirring overnight. Methanol was evaporated

under vacuum and the aqueous phase washed with DCM. The

aqueous phase was acidified with 3 N HCl, checking the pH of the

solution (until pH 1), but only a few milligrams of the desired

compound precipitated. The compound was retained by the organic

phase (DCM). Solvent was removed to give the title compound

(46, 25 mg) as free base. UPLC/MS R_f= 0.63; m/z(ES) 374.17 [M þ

1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-yl)-4-fluoro-4-piperidinecarboxylic Acid (54). To

a solution of ethyl 1-((1S)-5⁰,11⁰-dihydrospiro[cyclopentane-

1,10⁰-dibenzo[a,d ]cyclohepten]-3-yl)-4-fluoro-4-piperidinecar-

boxylate (19⁰, 21 mg, 0.050 mmol) in THF (1.5 mL) and water

(0.5 mL) was added LiOH (4.77 mg, 0.199 mmol), and the

mixture was heated under reflux for 4 h. The solvent was concen-

trated at reduced pressure, and the residue was dissolved in water

and then neutralized with HCl (1 M). This mixture was purified by

C18 column (10 g) to give the title compound (54, 14 mg) as a white

solid. UPLC/MS R_f= 0.64; m/z (ES) 394.2 [M þ H]^þ. ¹H NMR

(500 MHz, chloroform-d) δ:7.00-7.40 (m, 8 H), 4.15 (d, 1 H), 4.06

(d, 1 H), 3.71-3.85 (m, 1 H), 3.51-3.70 (m, 1 H), 3.29-3.44 (m,

1 H), 3.31 (d, 1 H), 3.17 (d, 1 H), 2.84-3.03 (m, 2 H), 2.49-2.83 (m,

4 H), 2.11-2.36 (m, 5 H), 1.79-2.00 (m, 1 H).

1

H]^þ. Data reported for the major diastereoisomer: H NMR (400

MHz, chloroform-d) δ 7.30-7.40 (m, 1H), 7.09-7.22 (m, 5H),

6.96-7.07 (m, 2H), 3.97-4.24 (m, 2H), 3.59-3.85 (m, 2H),

3.44-3.56 (m, 1H), 3.14-3.37 (m, 2H), 2.92-3.09 (m, 2H),

1.84-2.75 (m, 5H).

3-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-yl)-3-azabicyclo[3.1.0]hexane-1-carboxylic Acid

(64). To a mixture of ethyl 3-((1S)-5⁰,11⁰-dihydrospiro[cyclo-

pentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-yl)-3-azabicyclo[3.1.0]-

hexane-1-carboxylate (29⁰isomer 2, 26 mg, 0.065 mmol) in

ethanol (5 mL) and water (1 mL) was added KOH (14.53 mg,

0.259 mmol). The resulting mixture was heated at 100 ꢀC in a

microwave reactor (Personal Chemistry Emrys optimizer) for

30 min. The solvent was then evaporated under vacuum and the

mixture acidified with 3 N HCl (until pH 1 was reached). The

solution was purified with a HLB cartridge (6 g), eluting first

with water and then MeOH to afford the title compound (64,

18.8 mg). UPLC/MS R_f= 0.63; m/z (ES) 374.2 [M þ H]^þ. ¹H

NMR (400 MHz, chloroform-d) δ: 7.40-7.57 (m, 1 H),

7.08-7.25 (m, 5 H), 6.97-7.08 (m, 2 H), 4.02-4.19 (m, 2 H),

3.88-4.02 (m, 1 H), 3.52-3.84 (m, 3 H), 3.21-3.37 (m, 2 H),

3.05-3.18 (m, 1 H), 2.53-2.72 (m, 1 H), 2.34-2.53 (m, 1 H),

2.03-2.28 (m, 4 H), 1.73-1.97 (m, 2 H), 1.58-1.71 (m, 1 H).

1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-yl)-3-azetidinecarboxylic Acid Formate Salt (65).

In a 50 mL round-bottomed flask was dissolved methyl 1-((1S)-

5⁰,11⁰-dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-

3-yl)-3-azetidinecarboxylate (30⁰, 66 mg, 0.183 mmol) in metha-

nol (5 mL) and water (2.5 mL) to give a colorless solution. KOH

(41.0 mg, 0.730 mmol) was added, and the mixture was stirred at

room temperature overnight. MS monitoring showed that the

reaction was complete. Solvent was removed and the residue taken

up with HCl 1 M and passed through an HLB 6 g column (water

and MeOH to elute). Then a second purification was done by

Fraction Lynx acid method to give the title compound as a yellow

solid (65, 57.9 mg). UPLC/MS t_R= 0.58; m/z (ES) 348.08 [M þ

1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic Acid

(47). To a solution of methyl 1-((1S)-5⁰,11⁰-dihydrospiro-

[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-yl)-1,2,5,6-tet-

rahydro-3-pyridinecarboxylate (13⁰, 70 mg, 0.181 mmol) in

methanol (6 mL) and water (1 mL) was added LiOH (21.63

mg, 0.903 mmol). The reaction mixture was left to stir at

room temperature overnight. Further LiOH (1.2 equiv) and

water (0.5 mL) were added, and the resulting mixture was

stirred at 40 ꢀC for 6 h. The methanol was then evaporated

under vacuum, and the reaction mixture was acidified with

3 N HCl (until pH ∼ 1). This solution was purified using an

HLB cartridge (5 g) by eluting first with water and then

MeOH to give the title compound (47, 56 mg). UPLC/MS

R_f= 0.61; m/z (ES) 374.2 [M þ H]^þ. ¹H NMR (400 MHz,

chloroform-d) δ: 7.30-7.40 (m, 1H), 7.09-7.22 (m, 5H),

6.96-7.07 (m, 2H), 3.97-4.24 (m, 2H), 3.59-3.85 (m, 2H),

3.44-3.56 (m, 1H), 3.14-3.37 (m, 2H), 2.92-3.09 (m, 2H),

1.84-2.75 (m, 5H).

1-((1S)-5⁰,11⁰-Dihydrospiro[cyclopentane-1,10⁰-dibenzo[a,d ]-

cyclohepten]-3-yl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic Acid

(48). To a solution of methyl 1-((1S)-5⁰,11⁰-dihydrospiro-

[cyclopentane-1,10⁰-dibenzo[a,d ]cyclohepten]-3-yl)-1,2,5,6-tet-

rahydro-3-pyridinecarboxylate (13⁰⁰, 11 mg, 0.028 mmol) in

methanol (1.5 mL) and water (0.15 mL) was added LiOH

(3.40 mg, 0.142 mmol). The reaction mixture was left to stir

overnight at room temperature and then at 40 ꢀC for 4 h. The

methanol was evaporated under vacuum, and the reaction

mixture was acidified with 3 N HCl, checking the pH of the

solution (until pH 1). This solution was purified using an HLB

cartridge (2 g) by eluting first with water and then MeOH to give

the title compound (48, 5.8 mg). UPLC/MS R_f= 0.61; m/z (ES)

374.2 [M þ H]^þ. ¹H NMR (400 MHz, chloroform-d) δ:

7.47-7.73 (m, 1 H), 6.86-7.33 (m, 8 H), 3.98-4.28 (m, 2 H),

1

H]^þ. H NMR (400 MHz, DMSO-d₆) δ: 7.33-6.94 (m, 8 H),

4.17-3.99 (m, 2 H), 3.48-3.37 (m, 2 H), 3.26-3.15 (m, 5 H),

3.14-3.06 (m, 1 H), 2.01-1.90 (m, 2 H), 1.85-1.61 (m, 4 H).

1-(11⁰H-Spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-3-yl)-

1,2,3,6-tetrahydro-4-pyridinecarboxylic Acid (69). To a solution

7794 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Gianotti et al.

of methyl 1-(11⁰H-spiro[cyclopentane-1,10⁰-dibenzo[b,f ]oxepin]-

3-yl)-1,2,3,6-tetrahydro-4-pyridinecarboxylate (34⁰isomer 2, 27

mg, 0.069 mmol) in methanol (2 mL) were added water (1 mL)

and lithium hydroxide (1.660 mg, 0.069 mmol). The mixture

was heated at 45 ꢀC for 4 h. The MeOH was evaporated and the

water phase acidified with HCl (2 N in water) until pH < 1. The

suspension was purified by C18 cartridge (5 g) by using water and

then MeOH as eluent to obtain, after solvent evaporation, a cream

solid (17 mg). The product was further purified by Fraction Lynx

HPLC to give the title compound as a white solid (69, 8.2 mg).

m/z (ES): 376.1 [M þ H]^þ. ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.32-7.39 (m, 1 H), 7.04-7.30 (m, 7 H), 6.74-6.84 (m, 1 H),

3.40-2.20 (m, 15 H).

ChemMedChem. [Online early access]. DOI: 10.1002/cmdc.201000280.

Published Online: Oct 4, 2010.

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Supporting Information Available: Experimental procedures

and supporting data for compounds 43, 44, 49, 50, 52, 53,

55-63, 66-68, and 70-72. This material is available free of

charge via the Internet at http://pubs.acs.org.

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Article Doi

Novel spirotetracyclic zwitterionic dual H1/5-HT2A receptor antagonists for the treatment of sleep disorders

DOI: 10.1021/jm100856p

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Full text of DOI:10.1021/jm100856p

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