Design and synthesis of novel tricyclic benzoxazines as potent 5-HT 1A/B/D receptor antagonists leading to the discovery of 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4 h-imidazo[5,1-c][1,4] benzoxazine-3-carboxamide (GSK588045)

J. Med. Chem. 2010, 53, 5827–5843 5827

DOI: 10.1021/jm100482n

Design and Synthesis of Novel Tricyclic Benzoxazines as Potent 5-HT_1A/B/DReceptor Antagonists

Leading to the Discovery of 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-imidazo-

[5,1-c][1,4]benzoxazine-3-carboxamide (GSK588045)

Steven M. Bromidge,*^,†Roberto Arban,^‡Barbara Bertani,^‡Silvia Bison,^‡Manuela Borriello,^‡Paolo Cavanni,^‡

Giovanna Dal Forno,^‡Romano Di-Fabio,^‡Daniele Donati,^‡,§Stefano Fontana,^‡Massimo Gianotti,^‡Laurie J. Gordon,^†

Enrica Granci,^‡Colin P. Leslie,^‡Luca Moccia,^‡Alessandra Pasquarello,^‡Ilaria Sartori,^‡Anna Sava,^‡

Jeannette M. Watson,^†Angela Worby,^†Laura Zonzini,^‡and Valeria Zucchelli^‡

^†Neurosciences CEDD, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, U.K., and

^‡Medicines Research Centre, Via A. Fleming 4, Verona 37135, Italy. ^§Present address: Nerviano Medical Sciences Srl, Via Pasteur 10,

20014 Nerviano, Milan, Italy.

Received April 20, 2010

Bioisoteric replacement of the metabolically labile N-methyl amide group of a series of benzoxazinones

with small heterocyclic rings has led to novel series of fused tricyclic benzoxazines which are potent

5-HT_1A/B/Dreceptor antagonists with and without concomitant human serotonin transporter (hSerT)

activity. Optimizing against multiple parameters in parallel identified 6-{2-[4-(2-methyl-5-quinolinyl)-1-

piperazinyl]ethyl}-4H-imidazo[5,1-c][1,4]benzoxazine-3-carboxamide (GSK588045) as a potent 5-HT_1A/B/D

ꢀ

receptor antagonist with a high degree of selectivity over human ether-a-go-go related gene (hERG)

potassium channels, favorable pharmacokinetics, and excellent activity in vivo in rodent pharmaco-

dynamic (PD) models. On the basis of its outstanding overall profile, this compound was progressed as a

clinical candidate with the ultimate aim to assess its potential as a faster acting antidepressant/anxiolytic

with reduced side-effect burden.

Introduction

neurons.⁵They are widely distributed in the brain and in

addition to serotonin transporters (SerT) are known to have a

major role in the control of synaptic 5-HT levels.⁵Blockade of

5-HT_1A/B/Dautoreceptors, with or without concomitant SerT

inhibition, rapidly increases brain 5-HT levels and conse-

quently should provide a fast onset of antidepressant/anxio-

lytic action relative to current therapies.⁶

We have previously reported several series of potent

5-HT_1A/B/Dreceptor antagonists both with and without addi-

tional hSerT reuptake inhibitory activity including 1 (SB-

649915) (Figure 1).⁷More recently we disclosed a series of

6- and 8-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-

3(4H)-ones with interesting in vitro and in vivo profiles

(such as 2-5).^8,9This article reports the further optimization

and evolution of these compounds which has resulted in the

identification of novel series of tricyclic benzoxazines culmi-

nating in the identification of 20 (GSK588045). This com-

pound demonstrated an outstanding overall profile with in

vivo activity in different animal models of anxiety and depres-

sion and has been progressed as a clinical candidate.

Chemistry. The synthesis of the 6-substituted (2 and 3)

and 8-substituted (4 and 5) compounds has been previously

described.^8,9The unsubstituted 4H-imidazo[2,1-c][1,4]ben-

zoxazine 6 was prepared as shown in Scheme 1. Thus, 8-(2-

propen-1-yl)-2H-1,4-benzoxazin-3(4H)-one⁹30 was con-

verted into the 3-(methylthio)-2H-1,4-benzoxazine 32 via

the corresponding thione 31. Reaction of 32 with amino

acetaldehyde dimethyl acetal afforded the aminoacetal 33,

which readily cyclized to the desired 6-(2-propen-1-yl)-4H-

imidazo[2,1-c][1,4]benzoxazine 34 on heating with HCl.

Over the past decades, a wealth of preclinical and clinical

evidence has confirmed a link between extracellular levels of

serotonin (5-HT) and a plethora of psychiatric indications, in

particular anxiety and depression.¹In fact, enhanced seroto-

nergicneurotransmissionhas becomethe unifying mechanism

of action of modern day antidepressants and the selective

serotonin reuptake inhibitors (SSRIs^a) have become estab-

lished as the most effective antidepressant agents in current

clinical use.²Despite the success of SSRIs, one undesirable

characteristic is a long latency to therapeutic onset which is

hypothesized to be due to the requirement for desensitization

of 5-HT₁autoreceptors to maintain increased 5-HT levels.³

Thisis consistentwithpreclinicalneurochemical studies which

have demonstrated that SSRIs such as paroxetine require

chronic dosing (14 to 21 days) in order to enhance extracel-

lular 5-HT levels in guinea pig dentate gyrus and rat frontal

cortex but have no effect after acute administration.⁴

5-HT₁autoreceptors are located on both the cell bodies

(5-HT_1A, 5-HT_1B, and 5-HT_1Dreceptor subtypes) and nerve

terminals (5-HT_1Band 5-HT_1Dreceptor subtypes) of 5-HT

*To whom correspondence should be addressed. Phone: þ44 (0)1279

627031. Fax: þ44 (0)1279 622371. E-mail: stevebromidge@gmail.com.

^aAbbreviations: hSerT, human serotonin transporter; hERG, human

ꢀ

ether-a-go-go related gene; SSRIs, selective serotonin reuptake inhibi-

tors; SAR, structure-activity relationships; PD, pharmacodynamic;

PK, pharmacokinetic; TI, therapeutic index; LHS, left-hand side; IA,

intrinsic activity; CYP450, cytochrome P450; DDI, drug-drug interac-

tions; Vss, volumes of distribution; HEK, human embryo kidney; GSH,

glutathione; CHO, Chinese hamster ovary.

r

2010 American Chemical Society

Published on Web 06/30/2010

pubs.acs.org/jmc

5828 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

Figure 1. Chemical structures of different series of 5-HT _1A/B/Dantagonists.

Scheme 1. Synthetic Route for Compounds 6 and 7^a

^a(a) Lawesson’s reagent, toluene, reflux, 1 h; (b) MeI, KOH, acetone, reflux, 2 h; (c) NH₂CH₂CH(OMe)₂, EtOH, reflux, 9 h; (d) conc HCl, MeOH,

reflux, 3 h; (e) (i) OsO₄, 4-methylmorpholine N-oxide, acetone/H₂O, room temp, 36 h; (ii) aq Na₂SO₃, room temp, 30 min; (f) 2-methyl-5-piperazin-1-

ylquinoline, 1,2-dichloroethane, NaB(OAc)₃H, room temp, 3 h; (g) NaH, 1-chloro-2-propanone, DMF, 0 °C to room temp, 7 h; (h) NH₄OAc, AcOH,

microwave 150 °C, 10 min; (i) OsO₄, NaIO₄, THF/water, room temp, 2 h.

Oxidative cleavage of the pendant vinyl of 34 with osmium

tetraoxide/sodium periodate to the aldehyde 35 followed by

reductive amination with 2-methyl-5-(1-piperazinyl)quino-

line completed the synthesis. The 2-methyl analogue 7 was

similarly prepared from 30 but by an alternative cyclization

strategy via the ketone 36 which was condensed with ammo-

nium acetate to 37, followed by oxidative cleavage of the

vinyland reductiveamination asabove toafford7 (Scheme1).

The 1,2-dimethyl 8 and 2-trifluoromethyl 9 4H-imidazo-

[2,1-c][1,4]benzoxazine analogues were also prepared indir-

ectly from 30 via the corresponding ketones 39 and 46,

respectively, as described above for 7 except that cyclization

of the imidazole ring was carried out as the final steps

(Scheme 2).

The unsubstituted 4H-imidazo[5,1-c][1,4]benzoxazine 10

was also prepared from the key intermediate 30 as shown in

Scheme 3. Thus, treatment of 30 with diethyl chlorophos-

phate and potassium t-butoxide followed by ethyl isocya-

noacetate smoothly afforded the 4H-imidazo[5,1-c][1,4]-ben-

zoxazine-3-ethylcarboxylate 47.¹⁰Saponification gave the acid

48, which was decarboxylated to 49 and converted to the

desired product 10 as described above. The acid 48 also served

as a key intermediate for the preparation of the amides 17 and

24. The 4H-imidazo[5,1-c][1,4]benzoxazine-3-ethylcarboxylate

47 was itself converted to the final compound 14 (Scheme 3).

To rapidly investigate the amidic portion of this class of

compounds, a slightly modified route was set up starting

from the advanced ester intermediate 14 (Scheme 4): amides 17,

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5829

Scheme 2. Synthetic Route for Compounds 8 and 9^a

^a(a) 3-Chloro-2-butanone, K₂CO₃, acetone, reflux, 4 h; (b) OsO₄, NaIO₄, THF/water, room temp, 2 h; (c) 2-methyl-5-piperazin-1-ylquinoline, 1,2-

dichloroethane, NaB(OAc)₃H, room temp, 3 h; (d) NH₄OAc, AcOH, microwave 150 °C, 10 min; (e) NaH, methyl bromoacetate, DMF, 0 °C to room

temp, 6 h; (f) NaOH, MeOH, room temp, 4 h; (g) (i) oxalyl chloride, DMF, toluene, 60 °C, 3 h; (ii) trifluoroacetic anhydride, pyridine, room temp, 2 h.

Scheme 3. Synthetic Route for Compounds 10, 14, 17, and 24^a

^a(a) KO^tBu, diethyl chlorophosphate, DMF, 0 °C, 10 min then ethyl isocyanoacetate, 60 °C, 6 h; (b) OsO₄, NaIO₄, THF/water, room temp, 2 h;

(c) 2-methyl-5-piperazin-1-ylquinoline, 1,2-dichloroethane, NaB(OAc)₃H, room temp, 3 h; (d) NaOH, MeOH, 60 °C, 30 min; (e) 1,2-dichlorobenzene,

microwave 250 °C, 10 min; (f) HOBT, EDC, NHR₁R₂, DMF/CH₂Cl₂, room temp, 4 h; (g) (i) OsO₄, 4-methylmorpholine N-oxide, acetone/H₂O, room

temp, 36 h; (ii) NaIO₄, THF/H₂O, room temp, 1 h.

19-23, 25-29 were obtained via the acid 65 by standard

coupling methods. In addition, the methyl ketone 15 and oxime

16 were prepared via the Weinreb amide 66, while the nitrile 18

was obtained via the corresponding primary amide 20.

The next step of the exploration was directed to the in-

vestigation of different fused heterocyclic moieties. Thus, the

1-methyl-4H-[1,2,4]triazolo[3,4-c][1,4]benzoxazine 11 and the

4H-tetrazolo[5,1-c][1,4]benzoxazine 13 were prepared starting

from the benzoxazine-3(4H)-thione 31 prepared as described

above. Thus, 31 was converted tothe hydrazone54, whichwas

treated with trimethyl orthoacetate or sodium nitrite respec-

tively to afford the triazole 55 or the tetrazole 57, which were

then converted by the methods described above to the final

compounds 11 and 13, respectively (Scheme 5).

5830 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

Scheme 4. Synthetic Route for Compound 15-16, 18-19, 21-23, and 25-29^a

^a(a) NaOH, MeOH/H₂O, microwave 120 °C, 5 min; (b) DIPEA, TBTU, hexamethyldisilazane, DMF, room temp, 1 h; (c) (CF₃)₂CO, pyridine/THF,

0 °C, 1 h; (d) DIPEA, TBTU, NHR₁R₂, DMF, room temp, 1 h; (e) DIPEA, TBTU, NHMeOMe HCl, DMF, room temp, 1 h; (f) MeMgBr, THF, 0 °C,

3

1 h; (g) NH₂OMe HCl, pyridine/EtOH, reflux, 2 h.

3

Scheme 5. Synthetic Route for Compounds 11 and 13^a

^a(a) NH₂NH₂H₂O, EtOH, 80 °C, 2 h; (b) MeC(OMe)₃, microwave 150 °C, 10 min; (c) (i) OsO₄, 4-methylmorpholine N-oxide, acetone/H₂O, room

temp, 36 h; (ii) NaIO₄, THF/H₂O, room temp, 1 h; (d) 2-methyl-5-piperazin-1-ylquinoline, 1,2-dichloroethane, NaB(OAc)₃H, room temp, 3 h;

3

(e) NaNO₂, aq HCl, 5 °C, 4 h; (f) OsO₄, NaIO₄, THF/water, room temp, 2 h.

An elegant route was established to the 1,2,3-triazole 12

starting with alkylation of 2-nitro-6-(2-propen-1-yl)phe-

nol 59 with 1-bromo-2-butyne followed by functional

group transformation of the nitro to the azide 62. Heating

the azide 62 gave smooth intramolecular 1,3-dipolar cyclo-

addition, generating the tricyclic system 63 in a single

step.¹¹Oxidative cleavage and reductive amination with

2-methyl-5-(1-piperazinyl)quinoline according to the gene-

ral procedures described above completed the synthesis

(Scheme 6).

Results and Discussion

We recently disclosed a series of 6-[2-(4-aryl-1-piperazinyl)-

ethyl]-2H-1,4-benzoxazin-3(4H)-ones as potent 5-HT_1A/B/D

receptor antagonists, a number of which had additional hSerT

reuptake inhibitory activity (Table 1).⁸Modulation of the

balance between the different target affinities allowed us to

identify compounds with interesting in vitro and in vivo

profiles. Unfortunately, the progression of these molecules

was precluded by the finding that they had significant inhibi-

tory activity at hERG potassium channels which has been

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5831

Scheme 6. Synthetic Route for Compound 12^a

^a(a) K₂CO₃, 1-bromo-2-butyne, acetone, reflux, 4 h; (b) Fe powder, AcOH, room temp, 16 h; (c) (i) NaNO₂, aq HCl, 0 °C, 30 min; (ii) NaN₃, H₂O,

5 °C, 30 min; (d) toluene, reflux, 2.5 h; (e) OsO₄, NaIO₄, THF/water, room temp, 2 h; (f) 2-methyl-5-piperazin-1-ylquinoline, 1,2-dichloroethane,

NaB(OAc)₃H, room temp, 3 h.

Table 1. Functional Activity (f-pK_ior pEC₅₀)

^a,bfor Human 5-HT_1A/B/DReceptors with Intrinsic Activity (IA), hSerT and hERG Binding Affinities

(pK_i)^band Rat PK Parameters for Benzoxazinones (2-5)

pK_i,^b

rat PK parameters^d

a,b

f-pK_ior *pEC₅₀

compd sub

R

5-HT_1A(IA) 5-HT_1B(IA) 5-HT_1D(IA) hSerT hERG Cli rat; hum^cmL/min/g liver CLb mL/min/kg Vss L/kg t_1/2

h Fpo % Br:Bl

2

3

4

5

6

H

10.2 (0.0)

Me 10.1 (0.3)

9.6 (0.0)

Me 9.8 (0.0)

6.6 (0.0)

8.7 (0.3)

9.0 (0.0)

8.4 (0.0)

*9.3 (0.6)

10.0 (0.0)

10.3 (0.0)

9.5 (0.0)

8.9

7.7

6.5

8.0

7.2

6.1

5.6

5.4

1.3; <0.5

0.8; 1.1

0.7; 1.0

1.0; 2.4

5

8

2.0

2.7

3.4

1.7

5.1

68

59

63

26

1.6

2.2

0.7

1.5

4.4

5.2

1.0

8

H

8

29

^aData from the agonist mode (pEC₅₀)* are reported for those compounds with intrinsic activity (IA) g 0.4 in this mode (i.e., partial or full agonists),

whereas, for those compounds with IA < 0.4 in the agonist mode (i.e., antagonists), data from the antagonist mode is reported ( f-pK_i). See Experimental

Section for assay details. ^bEach determination lies within 0.3 log units of the mean with a minimum of 3 replicates. See Experimental Section for assay

details. ^cIntrinsic clearance in liver microsomes. See Experimental Section for assay details. ^dIn vivo data determined by 0.5 mg/kg iv and 1 mg/kg po

administration in rat. Br:Bl at 1 h following iv dosing.

associated with increases in the QT interval prolongation and

the induction of potentially life-threatening ventricular arrhy-

thmias in the clinic (Torsades de Pointes).¹²For example, 2

had pK_iof 7.2 in a hERG binding assay which translated to

potent functional inhibition of hERG tail current in whole cell

electrophysiology studies.¹³Interestingly, the corresponding

NMe analogue 3 showed 10-fold reduced hERG affinity rela-

tive to 2 despite increased lipophilicity, which was somewhat

surprising since lipohilicity has been positively correlated with

hERG.¹⁴This finding led us to speculate a specific binding

interaction between the lactam N-H and the hERG channel,

and we subsequently discovered that transposing the substitu-

tion point of the linker from the 6- to the 8-position of the

benzoxazinone ring selectively disrupted binding to the hERG

channel while maintaining the key target interactions.⁹Thus,

the 8-substituted analogues 4 and 5 were found to have similar

orimproved pan5-HT_1A/B/Dantagonistpotencies relativeto2

and 3 with significantly reduced hERG affinities (40- and

5-fold, respectively). Ofparticular note, the structure-activity

relationships (SAR) for hSerT was reversed with respect tothe

6-substituted series, and whereas the 6-substituted NH analo-

gue 2 demonstrated 15-fold higher hSerT affinity than the

corresponding NMe analogue 3, the 8-substituted NH analo-

gue 4 showed 30-fold lower hSerT affinity compared to the

corresponding NMe analogue 5.

Although 4 and 5 possessed interesting and distinct phar-

macological profiles (additional hSerT component in the case

of 5) of potential therapeutic utility with reduced hERG/QT

risk, pharmacokinetic (PK) studies revealed nonideal in vivo

profiles. Thus, in rat PK studies (Table 1), 4 demonstrated

high oral bioavailability combined with low blood clear-

ance, a good half-life, but a relatively modest brain to blood

ratio (0.7). In contrast, the corresponding N-methyl analo-

gue 5 achieved a higher brain to blood ratio (1.5) and good

brain exposures but was more rapidly cleared than 4, result-

ing in modest half-life and bioavailability. Further investi-

gation determined that N-demethylation was a principle

route of metabolism in rat as evidenced by the detection of

high circulating levels of the des-methyl analogue 4 follow-

ing oral dosing of 5. In addition, the observation that 5 was

also efficiently converted to 4 in vitro in the presence of both

rat and human liver microsomes with a higher intrinsic

clearance, with the latter suggested a potentially higher rate

of metabolism in man.

5832 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

Table 2. Functional Activity (f-pK_ior pEC₅₀)

^a,bfor Human 5-HT_1A/B/DReceptors with Intrinsic Activity (IA), hSerT and hERG Binding Affinities

(pK_i),^bMicrosomal Intrinsic Clearance and Human CYP450 Activity for Tricyclobenzoxazines (6-13)

^aData from the agonist mode (pEC₅₀)* are reported for those compounds with intrinsic activity (IA) g 0.4 in this mode (i.e., partial or full agonists),

whereas for those compounds with IA <0.4 in the agonist mode (i.e., antagonists), data from the antagonist mode is reported (f-pK_i). See Experimental

Section for assay details. ^bEach determination lies within 0.3 log units of the mean with a minimum of 3 replicates. See Experimental Section for assay

details. ^cCYPEX assay: IC₅₀g 5 μM against different isoforms unless stated. See Experimental Section for assay details. ^dIntrinsic clearance in liver

microsomes. See Experimental Section for assay details.

As a result of the above findings, we designed and imple-

mented medicinal chemistry strategies aimed at circumventing

metabolic amide dealkylation of 5 with the ultimate objective

of identifying potent metabolically stable and brain penetrant

5-HT_1A/B/Dreceptor antagonists with or without additional

hSerT reuptake inhibitory activity together with further in-

creased therapeutic index (TI) over hERG. Several approaches

were investigated to this end including modification of the

nature of the N-alkyl group and modulation of the electronic

properties of the benzoxazinone system, but the most success-

ful tactic proved to be incorporation of the amide N-methyl

into a fused heterocyclic ring.

We initially targeted a range of tricyclic fused heterocycles

based on electrostatic potential similarity to 5 in combination

with chemical feasibility. This exploration was undertaken

with the left-hand side (LHS) of the molecule fixed as the 5-(4-

ethyl-1-piperazinyl)-2-methylquinoline since we had previously

found that this conferred a favorable overall balance of proper-

ties and in particular afforded low intrinsic activity (IA) at

5-HT_1A/B/Dreceptors.⁸Encouragingly, a number of new hetero-

tricyclic ring systems rapidly emerged with interesting profiles

such as the fused imidazo, triazolo, and tetrazolo-benzoxa-

zines (6-12). The isomeric 1,2- and 1,5-fused imidazobenzox-

azines (6 and 10) demonstrated similar target profiles to 5, dis-

playing excellent 5-HT_1A/B/Dantagonist potencies and mod-

erate hSerT affinity (Table 2). However, the hERG affinities

and the microsomal human intrinsic clearances were similar or

increased relative to 5. In addition, significant human cyto-

chrome P450 (CYP450) inhibitory activities emerged, particu-

larly in the case of 10, indicating a high risk of potential drug-

drug interactions (DDI) in a clinical setting.¹⁵

The 1,3,4- and 1,2,3-triazoles (11 and 12) showed increased

5-HT_1A/B/Dantagonist potencies together with clean CYP450

profiles and reduced intrinsic clearance in rat and human

microsomes compared to 5 (Table 2). Whereas 12 had similar

hSerT and hERG affinities to 5, compound 11 had reduced

affinities at both. On the basis of the increased separation

between 5-HT_1A/B/Dpotencies and hERG, 11 was progressed

to rat PK studies (Table 5). Encouragingly, the lower micro-

somal clearance translated to reduced in vivo clearance and

increased oral bioavailability and half-life relative to 5,

although the brain to blood ratio was significantly reduced.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5833

a,b

Table 3. Functional Activity (f-pK_ior pEC₅₀

)

for human 5-HT_1A/B/DReceptors with Intrinsic Activity (IA), hSerT and hERG binding affinities

(pK_i),^bMicrosomal Intrinsic Clearance and Human CYP450 Activity for 3-Substituted-4H-imidazo[5,1-c][1,4]benzoxazines (14-18)

^aData from the agonist mode (pEC₅₀)* are reported for those compounds with intrinsic activity (IA) g 0.4 in this mode (i.e., partial or full agonists),

whereas for those compounds with IA <0.4 in the agonist mode (i.e., antagonists), data from the antagonist mode is reported (f-pK_i). See Experimental

Section for assay details. ^bEach determination lies within 0.3 log units of the mean with a minimum of 3 replicates. See Experimental Section for assay

details. ^cCYPEX assay: IC₅₀g 5 μM against different isoforms unless stated. See Experimental Section for assay details. ^dIntrinsic clearance in liver

microsomes. See Experimental Section for assay details.

The fused tetrazolo-benzoxazine 13 also maintained a good

overall target profile together with low intrinsic clearance

although 5-HT_1A, 5-HT_1B, and hSerT potencies were some-

what reduced relative to 5 and a significant increase in hERG

affinity was evident (Table 2). The increased hERG affinity of

13 (and the 1,2,3-triazole 12) compared to the 2-methyltria-

zole 11 is suggestive of a specific H-bond interaction between

the additional nitrogen in the tetrazole ring system and the

hERG channel.

On the basis of the above promising results, the fused

imidazobenzoxazines (6 and 10) were selected for further

optimization and the introduction of different substituents

into key positions of the imidazole rings was undertaken with

the aim of increasing metabolic stability by blocking putative

sites of metabolism and reducing CYP450 interactions. Sub-

stitution of the 4 and 5 position of the fused 1,2-imidazo-

benzoxazine system with methyl (8 and 7) maintained target

potencies but increased hERG with no reduction in intrinsic

clearance. The 4-trifluoromethyl analogue 9 did show signifi-

cantly reduced intrinsic clearance combined with a promising

target profile but at the cost of dramatically increased hERG

affinity.

The 3 position of the fused 1,5-imidazo-benzoxazine 10 was

hypothesized to be a site of potential metabolism, and there-

fore the introduction of small electron withdrawing groups

suchasnitrile, ketones, and amideswas undertakeninorder to

reduce lipohilicity with the aim of addressing both the clear-

anceand CYP450 issuesand furtherincreasingselectivityover

hERG. Encouragingly, the ethyl ester 14, methyl ketone 15,

ketoxime ether 16, and the dimethyl amide 17 all maintained

very similar target profiles across 5-HT_1A/B/Dand hSerT com-

pared to 10 but demonstrated significantly reduced hERG

affinity together with reduced intrinsic clearance and favorable

CYP450 inhibitory profiles (Table 3). The cyano derivative 18

also maintained a good overall profile, although hSerT affinity

and selectivity between 5-HT_1A/B/Dand hERG were reduced.

On the basis of their favorable in vitro profiles, 15, 17, and

18 were evaluated in rat PK studies (Table 5). Whereas 17 had

comparable in vivo clearance to 5, in the case of 15 and 18, the

lower microsomal clearance translated to reduced in vivo

clearance and, in combination with increased volumes of dis-

tribution (Vss), afforded significant increases in terminal half-

life. All three imidazobenzoxazines demonstrated good bioa-

vailability and favorable brain to blood ratios (consistent with

higher Vss). In an attempt to explain the disconnect between

the rat in vitro and in vivo clearance for 17, we hypothesized

that the dimethyl amide may be subject to metabolic demethyl-

ation in vivo. This putative route of metabolism was sup-

ported by the detection of high levels of a (M^þ-14) metabolite

in rat PK samples and led us to prepare a series of amide

analogues including cyclic tertiary amides (26-29), secondary

amides (19, 21-25) and the primary amide 20 (Table 4).

The secondary N-methyl amide 19 had an excellent overall

in vitro profile which was similar to that of 17 but with even

further reduced hERG affinity. Increasing the size and lipo-

philicity of the secondary N-alkyl substituent (21-25) main-

tained exquisitely high 5-HT_1A/B/Dantagonist potencies and

afforded modest gains in hSerT but with concomitant in-

creases in hERG affinities and increased intrinsic clearance

(particularly by human microsomes). The 4, 5, and 6-mem-

bered cyclic tertiary amides (26-28) also maintained impress-

ive 5-HT_1A/B/Dantagonist potencies together with increased

hSerT and hERG affinities, which, as above, was consistent with

increased lipohilicity. The morpholino analogue 29 was of par-

ticular interest, affording the lowest hERG affinity yet observed

in this series and low CYP450 inhibitory potencies together with

low intrinsic clearance. In addition, 29 displayed subnanomolar

antagonist potencies against 5-HT_1A/B/Dreceptors even though

5834 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

Table 4. Functional Activity (f-pK_ior pEC₅₀)

^a,bfor Human 5-HT_1A/B/DReceptors with Intrinsic Activity (IA), hSerT and hERG Binding Affinities

(pK_i),^bMicrosomal Intrinsic Clearance and Human CYP450 Activity for 3-Amido-4H-imidazo[5,1-c][1,4]benzoxazines (19-29)

^aData from the agonist mode (pEC₅₀)* are reported for those compounds with intrinsic activity (IA) g 0.4 in this mode (i.e., partial or full agonists),

whereas for those compounds with IA <0.4 in the agonist mode (i.e., antagonists), data from the antagonist mode is reported (f-pK_i). See Experimental

Section for assay details. ^bEach determination lies within 0.3 log units of the mean with a minimum of 3 replicates. See Experimental Section for assay

details. ^cCYPEX assay: IC₅₀g 5 μM against different isoforms unless stated. See Experimental Section for assay details. ^dIntrinsic clearance in liver

microsomes. See Experimental Section for assay details.

it had relatively modest hSerT affinity. The primary amide 20

also displayed a superb overall in vitro profile, comparable to

that of 17 but with 10-fold reduced hERG affinity together with

lower human intrinsic clearance.

understand their relative cardiovascular safety profiles in

terms of potential for QT interval prolongation, the effects of

the compounds on human cardiac potassium channels stably

expressed in HEK293 cells were investigated using a manual

patch clamp electrophysiology assay.¹³The compounds

inhibited hERG tail current with pIC₅₀values of 5.8, <5.0,

and 6.0, respectively, in broad agreement with dofetilide bind-

ing affinities. Compound 20 in particular demonstrated re-

markably low inhibitory potency affording insufficient inhibi-

tion of hERG tail current at the highest concentration tested

to allow IC₅₀calculation (pIC₂₅∼6.2). Furthermore, when

administered by iv infusion, 20 had no effect on QT interval

duration in an anaesthetised guinea pig model¹⁷up to the

maximum tested concentration of 7.7 μg/mL (solubility limit-

ed). Taken together, these data suggest that 20 possesses an

excellent therapeutic index between biologically active con-

centrations (see below) and those likely to evoke QT prolon-

gation via effects upon cardiac K^þion channels.

On the basis of their outstanding in vitro profiles, 19, 20,

and 29 were selected for further detailed characterization

initially in rat PK studies (Table 5). Encouragingly, all three

compounds showed good oral bioavailability and reduced

blood clearance, relative to 17, together with brain to blood

ratios >1 and high brain concentrations. The secondary amide

19 achieved the most impressive brain ratio (2.4), which may be

due to the formation of an intramolecular H-bond between the

amide NH and the imidazo nitrogen.¹⁶The availability of 19

allowed us to definitively confirm that this was a major meta-

bolite produced from the N-dimethyl tertiary amide 17 both in

vitro and in vivo (see above). In contrast, 19 was relatively

metabolically stable to demethylation as evidenced by the

detection of only minor traces of the corresponding primary

amide 20 both in vitro and in vivo.

DDI and Bioactivation Risk. Although both 19 and 20

exhibited comparable direct inhibition profiles against the major

human CYP450 enzymes during preliminary profiling (Table 4),

different profiles emerged with respect to bioactivation and DDI

Herg/QT risk. Compounds 19, 20, and 29 were originally

progressed on the basis of their low affinities for hERG

channels as assessed by a dofetilide binding assay. To better

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5835

risk during further detailed CYP3A4 inhibition profiling in

human liver microsomes using three different clinical probes

(Table 6). The secondary amide 19 and the primary amide 20

both displayed a low potential for interacting with human

CYP3A4, with 20 exhibiting the highest IC₅₀values for all

three probes (IC₅₀>88 μM). However, the results indicated a

decrease in IC₅₀values for the secondary amide 19 against the

midazolam probe (IC₅₀fold-change = 4) and the atorvastatin

probe (IC₅₀fold-change = 3), suggesting that 19 was a

metabolism-dependent inhibitor of CYP3A4 for midazolam

and atorvastatin in this assay. In contrast, the primary amide

20 gave no evidence of metabolism-dependent inhibition, up

to 100 μM concentration.

To qualitatively access the risk of reactive metabolite for-

mation, which is considered a risk factor for idiosyncratic

toxicity, the propensity of 19 and 20 to form glutathione

conjugates was also investigated.¹⁸Despite the closely related

chemical structures, in the case of 19 a glutathione adduct

(parent þ GSH) was observed following incubation with

GSH in the presence of human liver microsomes, with the

bioactivation site determined to be the methyl amidic group.

For the primary amide 20, the same GSH conjugate could not

be detected, highlighting a lower bioactivation risk relative to

19. On the basis of its superior overall profile, 20 was selected

for detailed in vitro and in vivo biological evaluation.

autoreceptors as assessed against Chinese hamster ovary

(CHO) cells expressing h5-HT_1Aor h5-HT_1Bor h5-HT_1D

receptors using [³H]-WAY100635 (5-HT_1A) and [³H]-5-HT

(5-HT_1Band 5-HT_1D) were high and in good agreement with

the functional potencies (Table 7). Comparable potencies

were also obtained for native tissue 5-HT_1Aand 5-HT_1B

receptors in rat, guinea pig, and marmoset cortex using [³H]-

WAY100635 and the 5-HT_1B/1Dreceptor antagonist radi-

oligand [³H]-GR125743, respectively. Under these condi-

tions, 5-HT_1Dreceptors are unlikely to contribute to the

binding of [³H]-GR125743 due to their relatively low expres-

sion in this brain region.

In vitro determination of function using [³⁵S]-GTPγS-SPA

binding to h5-HT_1A(HEK), h5-HT_1B(CHO), and h5-HT_1D

(CHO) cell membranes was also assessed. Increasing concen-

trations of 20 produced parallel rightward-shift of the agonist

(5-HT) concentration-response curves with the same max-

imal response in the presence and absence of the antagonist to

afford pA₂values in good agreement with the affinity con-

stants (pK_i) from receptor binding studies for all three recep-

tors (Table 7). 20 alone did not stimulate [³⁵S]-GTPγS binding

at any of the receptors (at concentrations up to 1 μM),

indicating that this compound does not have agonist proper-

ties at human recombinant 5-HT_1A, 5-HT_1B, and 5-HT_1D

receptors.

5-HT_1A/1B/1DRadioligand Binding and Functional Studies.

The affinities of 20 for human (h) recombinant 5-HT₁

Selectivity Profile. Compound 20 displayed good selec-

tivity (>100-fold) in a Cerep battery of more than 90

biological targets including GPCRs, ion channels, transpor-

ters, and enzymes with the exception of h-SERT and h-R_1A

adrenoceptors. Subsequently, 20 was determined to have a

pK_ivalues of 7.54 ( 0.07 (n = 3) and 8.56 ( 0.03 (n = 6) in

h-SERT (ligand [³H]-citalopram) and h-R_1Aadrenoceptor

(ligand [³H]-prazosin) filtration binding assays respectively.

Table 5. Rat Pharmacokinetic Profile for Compounds^a

compd

CLb^bmL/min/kg

Vss L/kg

t_1/2

h

Fpo %

Br:Bl

4

8

29

12

10

29

10

8

3.4

1.7

1.6

7.1

3.5

3.9

3.8

2.9

5.0

5.5

5.2

63

26

51

43

50

48

104

45

43

65

0.7

1.5

0.5

2.7

1.8

1.6

2.4

1.1

3.2

1.7

5

1.0

1.8

8.7

2.1

5.1

6.3

4.4

3.1

7.6

11

15

17

18

19

20

27

29

10

20

12

^aIn vivo data determined by 0.5 mg/kg iv and 1 mg/kg po adminis-

tration in rat. Br:Bl at 1 h following iv dosing. ^bIntrinsic clearance in

liver microsomes.

Table 6. In Vitro Metabolism-Dependent Inhibition of Three Clinical

Probes for Human CYP3A4 Using Human Liver Microsomes for

Compounds 19 and 20

19

20

clinical

probe

initial IC₅₀final IC₅₀fold- initial IC₅₀final IC₅₀fold-

(μM)^achange

(μM)

(μM)^achange

midazolam

atorvastatin

nifedipine

21

8

5

3

3.9

2.7

1.4

88

100

61

100

1.5

1.0

Figure 2. Effect of 20 on 8-OH-DPAT-induced hyperlocomotion

in rat. Data represent mean ( SEM of 8 animals per group; * p <

0.05, ** p < 0.01, ANOVA for completely randomized measures

versus 8-OH-DPAT group, followed by Dunnett’s test.

100

82

^aFollowing incubation.

Table 7. Summary of the Affinity (pK_i) of 20 for Human Recombinant and Native Tissue 5-HT_1A/1B/1DReceptors Determined Using Radioligand

Binding (Mean ( SEM) and Human Functional Potency (pA₂) Using [³⁵S]-GTPγS-SPA Binding

pK_ifor 5-HT_1A/1B/1Dreceptors

rat^a

guinea pig^a

marmoset^a

human^b

human functional potency^cpA₂

5-HT_1A

5-HT_1B

5-HT_1D

10.16 ( 0.14

8.49 ( 0.03

10.24 ( 0.09

9.80 ( 0.07

10.18 ( 0.12

9.81 ( 0.13

9.94 ( 0.07

9.10 ( 0.08

10.00 ( 0.08

9.69 ( 0.18

8.93 ( 0.05

9.77 ( 0.09

^aNative tissue binding to 5-HT_1Aand 5-HT_1Breceptors in rat, guinea pig, and marmoset cortex using [³H]-WAY100635 and the 5-HT_1B/1Dreceptor

antagonist radioligand [³H]-GR125743, respectively. ^bCHO cells expressing h5-HT_1Aor h5-HT_1Bor h5-HT_1Dreceptors using [³H]-WAY100635

(5-HT_1A) and [³H]-5-HT (5-HT_1Band 5-HT_1D). ^{c 35}S]-GTPγS-SPA binding to h5-HT_1A(HEK), h5-HT_1B(CHO) and h5-HT_1D(CHO).

[

5836 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

faster acting antidepressant/anxiolytic with reduced side-ef-

fect burden.

Experimental Section

Biological Test Methods. In Vitro Studies. a. Functional Potency:

Primary Screen at Human Recombinant 5-HT_1A, 5-HT_1B, and

5-HT_1DReceptors. The functional potency of the compounds was

determined by the following GTPγS binding assay. Cells used in

the study are Chinese hamster ovary (CHO) cells and human

embryo kidney (HEK293) cells transfected with DNA coding for

human receptors as follows: HEK293_5-HT_1A, CHO_5-HT_1B

,

and CHO_5-HT_1D. Test compounds were initially dissolved in

100% dimethyl sulfoxide (DMSO) to a concentration of 10 mM.

Serial dilution of the test compounds in 100% DMSO was carried

out using a Biomek FX in 384 well assay plates so that the final

top concentration of test compound was 3 μM in the assay. Test

compounds were added at 1.0% total assay volume (TAV) to a

solid, white, 384-well assay plate (Costar). Fifty percent TAV of

precoupled (for 90 min at RT) membranes (5 ug/well), wheat-

germ agglutinin polystyrene scintillation proximity assay beads

(RPNQ0260 Amersham International) (0.25 mg/well) in 20 mM

HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl₂, and 10 μM GDP

were added. The third addition was a 20% TAVaddition of either

buffer (agonist format) or EC₈₀of the agonist 5HT, prepared in assay

buffer (antagonist format). The assay was started by the addition of

29% TAV of GTPγ[³⁵S] 0.38 nM final (37 MBq/ml, 1160 Ci/mmol,

Amersham). After all additions, assay plates were spun down for

1 min at 1000 rpm. Assay plates were counted on a Viewlux, 613/55

filter, for 5 min, between 2 and 6 h after the final addition.

Figure 3. Effect of 20 on SKF-99101H-induced hypothermia in

guinea pig. Data represent mean ( SEM of 8 animals per group;

** = p < 0.01, ANOVA for completely randomized measures

versus vehicle treated animals, followed by Dunnett’s test.

Table 8. ED₅₀and EC₅₀for 20 in ex Vivo 5-HT_1Aand 5-HT_1BReceptor

Occupancy Studies in Guinea Pig Cortex

ED₅₀mg/kg

EC₅₀ng/mL

5-HT_1A([³H]-WAY100635 binding)

5-HT_1B([³H]-GR125743 binding)

0.2

0.1

9.0

4.5

In Vivo PD Profile. Compound 20 was determined to have

potent activity in both 5-HT_1Aand 5-HT_1Bagonist driven

PD models in rat and guinea pig, respectively. In the 5-HT_1A

driven PD model,^7b20 significantly reversed 8-OH-DPAT-

induced hyperlocomotion in rats in a dose-dependent manner

(Figure 2), with an ED₅₀of 0.05 mg/kg po (plasma EC₅₀= 3.1

ng/mL). In the 5-HT_1Bdriven PD model¹⁹20 significantly

reversed SKF-99101-induced hypothermia in the guinea pig in a

dose dependent manner (Figure 3), with an ED₅₀of 0.11 mg/kg

po (extrapolated EC₅₀= 2.3 ng/mL in plasma). In 5-HT_1Aand

5-HT_1B, ex vivo receptor occupancy (RO) studies in guinea pig

using [³H]-WAY100635 (5-HT_1A) and[³H]-GR125743 (5-HT_1B)

as radioligands, compound 20 afforded ED₅₀values of 0.2 and

0.1 mg/kg, respectively, (EC₅₀9.0 and 4.5 ng/g in cortex) which

are in line with the above PD data (Table 8, Figure 4).

b. Receptor Affinities at Human Recombinant 5-HT_1A

,

5-HT_1B, and 5-HT_1DReceptors. The affinities of the compounds

for the human recombinant 5-HT_1A, 5-HT_1B, and 5-HT_1Drecep-

tors were determined by the following assays. Cell pellets deriving

from three clones of Chinese hamster ovary (CHO) cells stably

transfected with the human 5-HT_1A, 5-HT_1B, and 5-HT_1Drecep-

tors were resuspended in 10 volumes of 50 mM HEPES, 1 mM

EDTA pH 7.4 in the presence of 1 μM leupeptin, 25 μg/mL

bacitracin, 1 mM PMSF, and 2 μM pepstain A (homogenization

buffer). The cells were homogenized by a Glass Waring blender,

and the resulting suspension spun at 500g for 20 min. The super-

natant was then centrifuged at 48000g for 30 min and the final

pellet was resuspended in homogenization buffer and homoge-

nized again. The final suspension was distributed into aliquots and

store at -80 °C. Protein content in the membrane preparations

was evaluated with BioRad protein assay. The day of the experi-

ment, membrane aliquots were thawed and resuspended in Tris-

Mg buffer (50 mM Tris/HCl, pH 7.7, 10 mM MgCl₂, 1 g/L ascor-

bic acid, and 5 μM pargiline) and homogenized with Polytron

immediately before use. Assays were performed into deep-well 96-

well plates in a total volume of 0.5 mL/well. For 5-HT_1Abinding

assay, 10 μg of protein of membranes were incubated with 0.5-

1 nM [³H]-WAY100635 (TRK1034, 2.85 TBq/mmol, Amersham)

and test drug, at 37 °C for 45 min, in Tris-Mg buffer supplemented

with 30 μM GppNHp (guanosine 5⁰-[β,γ-imido]triphosphate).

For 5-HT_1Band 5-HT_1Dbinding assays, 10 μg protein of mem-

branes were incubated with 3 nM [³H]-5-HT (TRK1006, 4.00

TBq/mmol, Amersham) and test drug, at 37 °C for 45 min, in Tris-

Mg buffer. Nonspecific binding (NSB) was defined using 10 μMof

WAY100635 or 5-HT for 5-HT_1Aand 5-HT_1B/D, respectively.

Incubation was stopped by rapid filtration and washes with ice-

cold 50 mM Tris buffer, pH 7.7, onto GF/B Unifilter plate (Perkin-

Elmer) using a Perkin-Elmer Harvester. Radioactivity was mea-

sured by Topcount (Perkin-Elmer) scintillation counting. pK_i

values of test drugs were calculated according to the Cheng-

Prusoff equation from the IC₅₀, generated by an iterative least-

squares curve fitting program (GraphPad Prism) and the K_D

(equilibrium dissociation constant of the radioligand) determined

at each receptor type (5-HT_1A, 5-HT_1B, and 5-HT_1D) through

saturation experiments.

Evidence supporting fast-onset antidepressant/anxiolytic

activity was obtained from both neurochemical studies and

validated rodent and primate animal models of anxiety

which will be the subject of a separate publication.

Conclusions

In conclusion, the bioisosteric replacement of the metabo-

lically labile N-methyl amide group of a series of benzoxazi-

nones with small heterocyclic rings has led to novel series of

fused tricyclic benzoxazines, many of which are potent

5-HT_1A/B/Dreceptor antagonists both with and without con-

comitant hSerT activity. On the basis of their initial profiles

fused imidazobenzoxazines (6 and 10) were selected for

further exploration. Optimizing against multiple parameters

in parallel identified several promising molecules which were

progressed into more extensive characterization to enable the

selection of the highest quality development candidate possi-

ble. In particular, 20 emerged as a potent 5-HT_1A/B/Dreceptor

antagonist with a high degree of selectivity over hERG

potassium channels, favorable PK, and excellent activity in

vivo in rodent PD models. On the basis of its outstanding

overall profile, 20 was selected for progression as a clinical

candidate with the ultimate aim to assess its potential as a

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5837

Figure 4. Occupancy at 5-HT_1Aand 5-HT_1Breceptors by 20 in guinea pig cortex together with brain and plasma exposure.

c. Receptor Affinity at Native 5-HT_1Aand 5-HT_1B/DReceptors

Determined in Brain Cortex Tissue from Rat, Guinea Pig, and

Marmoset. Frozen brain cortex tissue samples from rat, guinea

pig, and marmoset were thawed and resuspended in 20 volumes

(v/w) of 50 mM Tris buffer, pH 7.7. Each tissue was homo-

genized with a Potter homogenizer and then spun at 48000g for

15 min. The pellet was resuspended in 20 volumes of 50 mM

Tris buffer and homogenized with a Polytron homogenizer. The

homogenate was incubated at 37 °C in a shaking water bath for

20 min. After a further homogenization, the tissue homogenate

was centrifuged again at 48000g for 15 min. The supernatant

was discarded, and the membrane pellet was finally resuspended

in 4 volumes of 50 mM Tris buffer, distributed into tubes, and

immediately frozen on dry ice and stored at -80 °C until use.

Protein content was evaluated with BioRad protein assay.

The affinity of test compounds for 5-HT_1Areceptor in rat,

guinea pig, and marmoset brain cortex was assessed through

competition binding experiments using the 5-HT_1A-selective

antagonist radioligand [³H]-WAY100635 in low agonist affinity

conditions, i.e. in thepresence of 100 μM GPP(NH)p, a GTP

analogue which promotes the transition of receptors to the

inactive (G-protein uncoupled) state. Competition binding ex-

periments were carried out in deep-well 96-plate format in a final

volume of 0.5 mL/well of assay buffer (Tris 50 mM, pH 7.7

containing 0.2 mM ascorbic acid and 100 μM Gpp(NH)p) in the

presence of 0.4-0.7 nM [³H]-WAY100635. NSB was deter-

mined in the presence of 10 μM WAY100635. The binding

reaction was started through the addition of the membranes

(∼30-40 μg brain cortex membrane protein/well) and allowed

to proceed for 45 min at 37 °C in a shaking water bath. At the

end of the incubation, the reaction was stopped by filtration

through glass fiber sheet (GF/C) presoaked in 0.3% PEI,

followed by washes with ice cold 50 mM Tris buffer, pH 7.7,

using a 96 way-harvester (Brandel M96/R). Radioactivity re-

tained on the filters was determined by conventional liquid

scintillation spectrometry.

assessed by using [³H]citalopram binding assay performed in

recombinant epithelial pig kidney cells stably transfected with

human SERT (hSERT/LLCPK). Cell pellet was homogenized

in 30-50 volumes of assay buffer (50 mM Tris, 120 mM NaCl,

5 mM KCl, 10 μM pargyline, 0.1% ascorbic acid, pH = 7.7) and

centrifuged at 48000g for 20 min at 4 °C. The pellet was then

resuspended in the same volume of assay buffer and after incu-

bation at 37 °C for 20 min centrifuged as before. The final pellet

was resuspended in assay buffer at ∼0.2 mg protein/mL (as

determined with BioRad protein assay).

The binding assay was performed in deep-well 96-well plate

and the following additions were used up to a total assay volume

of 400 μL: 100 μL of compound dilution or assay buffer (to define

total binding, TB) or 10 μM fluoxetine (NSB), 100 μL of [³H]-

citalopram (0.25 nM, TRK-1068, 296TBq/mmol, Amersham)

and 200 μL of membranes diluted in assay buffer (2 μg of protein/

well). Membranes were added last to initiate the reaction and

incubated at room temperature for 2 h. The reaction was then

stopped by rapid filtration through GF/B filter-plate (Unifilter,

Perkin-Elmer), presoaked in 0.5% polyethylenimmine (PEI)

using a Perkin-Elmer harvester. Wells were washed three times

with cold 0.9% NaCl solution. The dried plates were counted

using TopCount (Perkin-Elmer) liquid scintillation counter.

pK_ivalues were calculated according to the Cheng-Prusoff

equation from the IC₅₀and the K_Ddetermined at human SERT

through saturation experiments.

Receptor Selectivity. The receptogram screen was performed

by Cerep assessing the binding of 10 μM on 83 G-Protein

coupled receptors, channels, enzymes, and transporters.

hERG-[³H]-Dofetilide Binding Assay. hERG activity was

measured using [³H]-dofetilide binding in a scintillation proxi-

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

PerkinElmer Viewlux imager.

CYP450 Inhibition CYPEX Assay. Inhibition (IC₅₀) of hu-

man CYP1A2, 2C9, 2C19, 2D6, and 3A4 was determined using

Cypex Bactosomes expressing the major human P450s. A range

of concentrations (0.1, 0.2, 0.4, 1, 2, 4, and 10 μM) of test com-

pound were prepared in methanol and preincubated at 37 °C for

10 min in 50 mM potassium phosphate buffer (pH 7.4) contain-

ing recombinant human CYP450 microsomal protein (0.1 mg/

mL; Cypex Limited, Dundee, UK) and probe-fluorescent sub-

strate. The final concentration of solvent was between 3 and

4.5% of the final volume. Following preincubation, NADPH

regenerating system (7.8 mg glucose-6-phosphate, 1.7 mg NADP,

and 6 units 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

rateof metabolite production(AFU/min) was determinedat each

concentration of compound and converted to a percentage of the

mean control rate using Magellan (Tecan software). The inhibi-

tion (IC₅₀) of each compound was determined from the slope of

the plot using Grafit v5 (Erithacus software, UK). Miconazole

The affinity of test compound for native 5-HT_1B/Dreceptors in

brain cortex was assessed through competition binding experi-

ments essentially as described before but using the 5-HT_1B/D

antagonist radioligand [³H]-GR125743 (0.3 nM, TRK-1046,

2.96 TBq/mmol, Amersham) in an assay buffer containing: Tris

50 mM, pH 7.7, 10 mMMgCl₂, 0.2mM ascorbic acid, and 100 nM

of a cocktail of four blockers (8-OH-DPAT, SB-243213, SB-

258585, and SB-269970) of serotonergic receptors (5-HT_1A,

5-HT_2C, 5-HT₆, and 5-HT₇, respectively). NSB was determined

in presence of 10 μM serotonin.

pK_ivalue of the test drug was calculated according to the

Cheng-Prusoff equation from the IC₅₀and the K_Ddetermined

in the three animal species evaluated (rat, guinea pig, and

marmoset) through saturation experiments.

d. [³H]Citalopram Binding Assay for Human Recombinant

Serotonin Transporter (SERT). The affinity of the compounds

to bind the reuptake site of serotonin transporter (SERT) was

5838 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

was added as a positive control to each plate. CYP450 isoform

substrates used were ethoxyresorufin (ER; 1A2; 0.5 μM), 7-meth-

oxy-4-triflouromethylcoumarin-3-acetic acid (FCA; 2C9; 50 μM),

3-butyryl-7-methoxycoumarin (BMC; 2C19; 10 μM), 4-methyla-

minomethyl-7-methoxycoumarin(MMC; 2D6;10μM), diethoxy-

flourescein (DEF; 3A4; 1 μM), and 7-benzyloxyquinoline (7-BQ;

3A4; 25 μM). The test was performed in three replicates.

NADP, 6 U glucose-6-phosphate dehydrogenase per mLof 2%

sodium bicarbonate), one positive substrate (acetaminophen).

Test compound and acetaminophen as positive control were

incubated at 100 μM at 37 °C for 1.5 h. Incubation mixtures

were collected at 0and 90in and quenched with 6% acetic acid in

acetonitrile. Assay mixtures were centrifuged (900g, 10 mn, 4 °C),

and 10 μL of the resulting supernatants were injected into the

HPLC-MS system.

CYP450 Inhibition Gentest Assay. Inhibition potential (IC₅₀)

of test compound for inhibition of human CYP1A2, 2C9, 2C19,

2D6, and 3A4 was determined. A range of concentrations (0.1,

0.33, 1, 3.3, 10, 33, and 100 μM) of test compound prepared in

methanol were preincubated at 37 °C for 10 min in 50 mM

potassium phosphate buffer (pH 7.4) containing 0.1, 0.16, 0.2,

or 0.4 mg recombinant human CYP450 microsomal protein

mL^-1(Gentest Corporation, USA) and substrate. Following

preincubation, 25 μL mL^-1of a NADPH regenerating system

(7.8 mg glucose 6-phosphate, 1.7 mg NADP, and 6 U glucose-6-

phosphate dehydrogenase mL^-12% w/v NaHCO₃) 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 (Tecan). The rate of metabolite

production (AFU/min) was determined for each concentration

of compound and converted to a percentage of the mean control

rate using Magellan v3.0 (Tecan software). The inhibitory

potential (IC₅₀) was determined from the slope of the plot using

Grafit v4.12 (Erithacus software, UK). Miconazole was added

as a positive control to each plate.

Intrinsic Clearance (Cli) Assay. Intrinsicclearance (CLi)values

were determined in rat and human liver microsomes. Test

compounds (0.5 μM) were incubated at 37 °C for 30 min in

50 mM potassium phosphate buffer (pH 7.4) containing 0.5 mg

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 contain-

ing an appropriate internal standard, and analyzed by HPLC-

MS/MS. The intrinsic clearance (CLi) was determined from the

first-order elimination constant by nonlinear regression using

Grafit v5 (Erithacus software, UK), corrected for the volume of

the incubation, and assuming 52.5 mg 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 deter-

mined to be when <15% of the compound had been metabo-

lized 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.

Patch Clamp hERG Electrophysiology Assay. On the days of

experimentation, a weighed amount of the test substance was

formulated in dimethyl sulfoxide (DMSO; lot no. U10781;

Sigma-Aldrich, UK), by shaking, to give a stock concentration

of 10 mM of the test compound. The 10 mM stock solution was

serially diluted in DMSO to give further stock solutions of 1 and

0.1 mM. Aliquots of these stock solutions were added to bath

solution to achieve final perfusion concentrations of 0.1, 1, and

10 μM test compound. The corresponding vehicle concentration

in all test substance perfusion solutions was 0.1% DMSO. Test

substance stock formulations were freshly prepared on each day

of experimentation, stored at room temperature, and protected

from light.

CYP450 Inhibition Using Human Liver Microsomes. A range

of concentrations (0.1, 0.33, 1, 3.3, 10, 33, and 100 μM) of test

compound were prepared in methanol. These were preincubated

at 37 °C for 20 min in 50 mM potassium phosphate buffer

(pH 7.4) containing pooled human liver microsomal protein

(0.1 mg/mL; Xenotech) and CYP3A4 substrate (midazolam,

atorvastatin or nifedipine; 2.5, 10, or 50 μM, respectively). To

start the reaction, NADPH regenerating system (7.8 mg glucose-

6-phosphate, 1.7 mg NADP and 6 units glucose-6-phosphate de-

hydrogenase/mL of 2% (w/v) NaHCO₃; 50 μL/mL) was added.

Following a 5 min incubation, reactions were terminated with the

addition of 250 μL acetonitrile.

Quantification of 1⁰-hydroxy midazolam, o-hydroxy atorvas-

tatin, and oxidized nifedipine metabolites was carried out using

validated HPLC-MS analytical methods. Inhibition (IC₅₀) was

determined using Grafit (Erithacus software, UK).

Reference Substance. E-4031 (batch no. MLE9446; Wako Pure

Chemical Industries Ltd.). Stock solutions of E-4031 (100 μM)

were prepared in reverse osmosis water, aliquoted, and stored at

approximately -20 °C until use. On the day of use, the 100 μM

stock solution was added to bath solution to give a final perfusion

concentration of 100 nM.

Metabolism-Dependent Inhibition of CYP3A4 in Human Liver

Microsomes. A range of concentrations (0.1, 0.33, 1, 3.3, 10, 33,

and 100 μM) of the test compound and troleandomycin (positive

control) were prepared in methanol. These were pre-incubated

at 37 °C for 20 min in 50 mM potassium phosphate buffer (pH 7.4)

containing pooled human liver microsomal protein (0.1 mg/mL;

Xenotech) and NADPH regenerating system (7.8 mg glucose-6-

phosphate, 1.7 mg NADP, and 6 units glucose-6-phosphate

dehydrogenase/mL of 2% (w/v) NaHCO₃; 50 μL/mL) [Cofactor

preincubation samples] or CYP3A4 substrate (midazolam, ator-

vastatin, or nifedipine; 2.5, 10, or 50 μM, respectively) [substrate

preincubation samples]. To start the reaction, CYP3A4 substrate

(midazolam, atorvastatin, or nifedipine; 2.5, 10, or 50 μM, re-

spectively) was added to the cofactor preincubation samples and

NADPH regenerating system (50 μL/mL) to the substrate pre-

incubation samples. Following 5 min incubation for midazolam

and 10 min incubation for atorvastatin or nifedipine, reactions

were terminated with the addition of 250 μL of acetonitrile.

Quantification of 1⁰-hydroxy midazolam, o-hydroxy atorvas-

tatin and oxidized nifedipine metabolites was carried out using

validated HPLC-MS analytical methods. Inhibition (IC₅₀) was

determined using Grafit (Erithacus software, UK) and TDI

assessed comparing the difference in IC₅₀between the cofactor

and substrate preincubation samples for each CYP3A4 substrate.

GSH Trapping. Incubations were carried out with human liver

microsomes (1 mg/mL), glutathione (10 mM), MgCl₂(3 mM),

NADPH generating system (7.8 mg glucose-6-phosphate, 1.7 mg

Bath and Pipette Solutions. The composition of bath solution

was (mM): NaCl 137, KCl 4, CaCl₂1.8, MgCl₂1.0, D-glucose 10,

N-2-hydroxyethylpiperazine-N⁰-2-ethanesulfonic acid (HEPES)

10, pH 7.4 with 1 M NaOH. Pipette solution was prepared in

batches, aliquoted, and stored frozen until the day of use. The

composition of pipet solution was (mM): KCl 130, MgCl₂1.0,

ethylene glycol-bis(ss-aminoethyl ether)-N,N,N⁰,N⁰-tetraacetic

acid (EGTA) 5, MgATP 5, HEPES 10, pH 7.2 with 1 M KOH.

Study Design. Cells (passage number: 48) were transferred to

the recording chamber and continuously perfused (at approxi-

mately 1-2 mL/min) with bath solution at room temperature.

High resistance seals (seal resistances >1.5 GΩ) were formed

between the patch electrodes (resistance range: 1.4-5.5 MΩ)

and individual cells. The membrane across the electrode tip was

then ruptured and the whole-cell patch-clamp configuration was

established. Once a stable patch had been achieved, recording

commencedinvoltage-clampmode, withthe cellinitially clamped

at -80 mV. Currents were evoked by stepping the membrane

potential to þ20 mV and then to -50 mV (tail current). Test

compound at 10, 1, and 0.1 μM was used to produce (if any)

inhibition of hERG tail current and therefore to investigate the

concentration-responserelationship(n = 3 cells/concentration).

The effect of the vehicle (0.1% DMSO) was investigated in three

cells. The effect of 100 nM E-4031 was investigated in two of the

vehicle treated cells to confirm the sensitivity of the test system to

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5839

an agent known to block hERG current. All perfusion solutions

were applied for approximately 10 min.

homogenizer. The homogenate was diluted to a final volume

of 100-fold the original wet weight of tissue and further homo-

genized by using a Polytron homogenizer. The resulting homo-

genate was directly used for the 5-HT_1B/Dbinding, while for

5-HT_1Abinding it was further diluted 1:1. For each cortex sample,

the protein concentration was determined by using the Bio-Rad

Protein Assay. Occupancy of 5-HT_1Aand 5-HT_1B/Dreceptors was

assessed through binding experiments using [³H]-WAY100635

and [³H]-GR125743, as described previously.

In Vivo Studies. All experimental procedures were carried out

in accordance with Italian law (Legislative Decree no. 116, 27

January 1992), which acknowledges the European Directive

86/609/EEC and were fully compliant with GlaxoSmithKline

policy and codes of practice on the care and use of laboratory

animals. Animals were housed under standard laboratory con-

ditions (23 ( 1 °C; lights on 0600-1800 h) with food and water

ad libitum.

Each single animal of the different groups (vehicle and com-

pound-treated) generated from each binding assay four values:

two total bound (TB) and two nonspecific bound (NSB). The

average of the NSB was subtracted from the TB of each brain

sample to obtain the specific binding (SB). Maximal SB values

werefromthe vehicle-treated animalsand SBgradually decreased

in the drug-treated animals with the increase of the dose. Percen-

tage of specific binding in drug-treated animals (% SB) was then

calculated and the % of receptor occupancy (RO) inversely

correlates with the % SB (i.e., 0% RO is in the vehicle group

and 100% ROis when the drug completely blocksthe radioligand

binding). The ED₅₀values were estimated bynonlinear regression

between % RO and the dose of compound administered. EC₅₀s

were estimated using % RO vs compound concentration in the

brain or in the plasma, as analytically determined by DMPK.

Anesthetized Guinea Pig Model for the Assessment of QT

Prolongation. Prior to the experiment, six male Hartley guinea

pigs (Charles River, France), weighing between 564 and 605 g on

the day of the test, were anaesthetised with urethane (1-1.5 g/kg

ip), tracheotomized, artificially ventilated with a tidal volume of

approximately 1 mL/100 g at a rate of 54 cycles/min, and surgi-

cally prepared for the experiment (i.e., insertion of catheters in

appropriate blood vessels, see below, and placement of surface

electrodes for lead II electrocardiogram recording). The ani-

mal’s temperature was kept constant between 36.8 and 38.0 °C.

Once physiological parameters were stabilized (approximately

30 min following surgery), each animal was given either the

vehicle (5% w/v dextrose in aqueous 0.9 w/v sodium chloride) or

test compound by the intravenous route (via the jugular vein)

over 15 min/treatment. The following parameters were recorded:

arterial pressure (via the carotid artery), heart rate, electrocardio-

graphy (i.e., RR, PQ, QRS, and QT interval duration; the QT

interval was corrected for heart rate changes according to

Fridericia, Bazett and Van de Water’s formula). Parameters

were recorded prior to dosing to establish baseline measure-

ments and continuously during the infusion period but data

were reported every 5 min during each infusion period. In

addition, blood samples (0.3 mL) were collected via the femoral

artery at the end of each infusion period for toxicokinetic

evaluations.

In Vivo Pharmacokinetics in Rat. The pharmacokinetics and

oral bioavailability of test compounds were determined follow-

ing iv (bolus) and po administration to male Sprague-Dawley

rats of the at 0.5 and 1 mg free base/kg, respectively. Blood

samples were assayed for test compound concentration using a

method based on protein precipitation followed by HPLC-MS/

MS analysis.

Chemistry. General Methods. Proton magnetic resonance

(NMR) spectra were recorded either on Varian instruments at

300, 400, or 500 MHz, or on a Bruker instrument at 300 MHz.

Chemical shifts are reported in ppm (δ) using the residual

solvent line as internal standard. Splitting patterns are designed

as 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. Mass

spectra (MS) are typically taken on a 4 II triple quadrupole mass

spectrometer (Micromass UK) or on a Agilent MSD 1100 mass

spectrometer, operating in ES (þ) and ES (-) ionization mode or

on an Agilent LC/MSD 1100 mass spectrometer, operating in ES

(þ) and ES (-) ionization mode coupled with HPLC instrument

8-OH-DPAT-Induced Hyperlocomotion.²⁰Male Sprague-

Dawley rats (250-350 g, Charles River, Italy) were used. Rat

locomotory activity was assessed as the total distance (cm)

traveled by each rat in the test arena over a 30 min period

using Digiscan analyzer system (Omnitech, model RXYZCM-8,

Columbus, OH). Rats were placed individually into clear plexiglas

boxes, measuring 40 cm ꢀ 40 cm ꢀ 30.5 cm and covered with a

perforated plexiglas lid. Infrared monitoring sensors were located

around the perimeter walls. Data were collected and analyzed by a

Digiscan analyzer (Omnitech, model DCM-4, Columbus, OH),

which in turn transferred information to a computer. Total

distance was recorded during 30 min test period.

The animals were treated orally with vehicle or compound 20

at 0.03, 0.1, or 0.3 mg/kg 120 min before a subcutaneous (sc)

injection of 0.2 mg/kg of 8-OH-DPAT. The standard 5-HT_1A

receptor antagonist WAY-100,635 (Fletcher, 1996) was used as

positive control (0.03 mg/kg sc 30 min before test).

SKF99101H-Induced Hypothermia. Male Dunkin Hartley

guinea pigs (300-400 g, Harlan, Germany) were used. Core body

temperature was monitored on freely moving animals by using a

telemetric transponders (IPTT-200 BioMedic Data System, Sea-

ford, DE) implanted sc in the dorso-cervical area four days before

test. Body temperature was recordedbyplacingthe scanner(DAS

5007 BioMedic Data System, Seaford, DE) above the transpon-

der until a stable reading was achieved.

The animals were treated orally with vehicle or compound

20 at 0.1, 0.3, or 1 mg/kg 60 min before sc administration of

10 mg/kg of SKF-99101H a standard 5-HT_1/7agonist. Tem-

perature measurements were taken immediately prior to SKF-

99101H dosing (time 0) and every 30 min for a total of 180 min.

Data are expressed as a fall in the peak temperature (°C) com-

pared to body temperature at time 0 (just before SKF-99101H

administration).

Drugs. Compound 20 was suspended in Methocel 0.5% (w/v,

HPCM Colorcon, Dow Chemical Company, Midland, MI).

8-OH-DPAT was purchased by Sigma (Italy) and dissolved in

saline before use. WAY-100,635 (N-{2-[4-(2-methoxyphenyl)-1-

piperaziny]ethyl}-N-(2-pyridinyl) cyclohexane-carboxamide

trihydrochloride) was synthesized in house and dissolved in sterile

water before use. SKF-99101H (3-2-dimethylaminoethyl-4-chloro-

5-propoxyindole hemifumarate) was synthesized in house and dis-

solved in saline before use. All compounds were prepared with

correction for salt form such that doses cited refer to mg/kg of active

principle.

Measurement of the Occupancy at 5-HT_1Aand 5-HT_1B/D

Receptors Using ex Vivo Binding on Guinea Pig Brain Cortex

Homogenate. The test compound was dissolved in 0.5% meth-

ocel and administered per os (po) to male guinea pigs (about

250 g, strain DH, Harlan), dosed at 0.03, 0.1, 1, and 3 mg/kg po;

a further group was run with vehicle. For each dose condition,

four animals were used. The receptor occupancy determination

was performed 1 h after the administration. Animals were

sacrificed by decapitation, and the brain was quickly removed

and the cerebral cortex was dissected. One-fourth of the tissue

was used by the DMPK Department for the analytical quanti-

fication of the amount of the test compound in the cortex.

Remaining tissue was processed for binding experiments.

The cortex sample from each single animal of the different

experimental groups was weighed and homogenized in 30 mL

of 50 mM Tris Buffer pH 7.7 using a glass-glass Potter

5840 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

Agilent 1100 series. LC/MS-ES (þ): analysis performed on a

Supelcosil ABZ þPlus (33 mm ꢀ 4.6 mm, 3 μm) (mobile phase:

100% [water þ 0.1% HCO₂H] for 1 min, then from 100% [water þ

0.1% HCO₂H] to 5% [water þ 0.1% HCO₂H] and 95% [CH₃CN]

in 5 min, finally under these conditions for 2 min; T = 40 °C;

flux = 1 mL/min; LC/MS-ES (-): analysis performed on a

Supelcosil ABZ þPlus (33 mm ꢀ 4.6 mm, 3 μm) (mobile phase:

100% [water þ 0.05% NH₃] for 1 min, then from 100%

[water þ0.05% NH₃to 5% [water þ 0.05% NH₃] and 95%

[CH₃CN] in 5 min, finally under these conditions for 2 min; T =

40 °C; flux = 1 mL/min]; in the mass spectra, only one peak in

the molecular ion cluster is reported.

AcOEt in cyclohexane to afford the title compound (93 mg,

41%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ: 7.2 (m,

1H), 6.9 (bs, 2H), 5.9 (m, 1H), 5 (m, 2H), 4.5 (s, 2H), 3.3 (d, 2H),

2.5 (s, 3H). MS: m/z 277.3 [M þ H]^þ.

6-(2-Propen-1-yl)-4H-imidazo[2,1-c][1,4]benzoxazine (34). A

mixture of intermediate 33 (90 mg, 0.33 mmol) and conc HCl

(0.8 mL) in MeOH (1 mL) was heated at reflux for 3 h. After

concentration in vacuo the residue was dissolved in CH₂Cl₂

(20 mL), washed with an aqueous saturated solution of NaH-

CO₃(15 mL), dried over Na₂SO₄, and evaporated in vacuo. The

crude material was purified by chromatography on silica gel

eluting with 35% AcOEt in cyclohexane to afford the title com-

1

DAD chromatographic traces, mass chromatograms, and

mass spectra were taken on a UPLC/MS AcquityTM system

coupled with a Micromass ZQTM mass spectrometer operating

in ESI positive or negative. The phases used are: (A) H₂O/ACN

95/5 þ 0.1% TFA; (B) H₂O/ACN 5/95 þ 0.1% TFA. The

gradient was: (t = 0 min) 95%A 5%B, (t = 0.25) 95%A 5%B,

(t = 3.30) 100%B, (t = 4.0) 100%B, followed by 1 min of re-

conditioning. 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 silica cartridges. SPE-SCX cartridges

are ion exchange solid phase extraction columns supplied by

Varian. The eluent used with SPE-SCX cartridges is methanol

followed by 2N ammonia solution in methanol. In a number of

preparations, purification was performed using either Biotage

manual flash chromatography (Flashþ) or automatic flash

chromatography (Horizon, SP1) systems. All these instruments

work with Biotage Silica cartridges. SPE-Si cartridges are silica

solid phase extraction columns supplied by Varian. Microwave

reactions were carried out in a Personal Chemistry Emrys

Optimiser (300 W). All reactions were carried out under anhy-

drous nitrogen or argon atmosphere using standard Schlenk

techniques. Most chemicals and solvents were analytical grade

and used without further purification.

pound (65 mg, 92%) as a yellow solid. H NMR (300 MHz,

CDCl₃) δ: 7.4 (bs, 1H), 7.2 (bs, 2H), 7.05 (bs, 2H), 5.95 (m, 1H),

5.3 (s, 2H), 5.1 (m, 2H), 3.45 (d, 2H). MS: m/z 213.3 [M þ H]^þ.

4H-Imidazo[2,1-c][1,4]benzoxazin-6-ylacetaldehyde (35).

4-Methylmorpholine N-oxide (218 mg, 1.87 mmol) and osmium

tetroxide (225 μL of a 4% by wt solution in water, 0.035 equiv)

were added to a solution of intermediate 34 (200 mg, 0.94 mmol)

in a 8:1 mixture of acetone/water (17.5 mL). The reaction was left

under stirring overnight before additional osmium tetroxide

(200 μL, 0.03 equiv) and 4-methylmorpholine N-oxide (250 mg,

2.1 mmol) were added to drive the reaction to completion. After a

further 24 h, the reaction was quenched with a saturated aqueous

solution of sodium sulfite (75 mL). After 30 min stirring, the

mixture was extracted with AcOEt (3 ꢀ 75 mL) and the combined

organic layers were dried over Na₂SO₄and concentrated in vacuo

to give a 7:3 mixture (by NMR) of the desired product 35 and 4H-

imidazo[2,1-c][1,4]benzoxazine-6-carbaldehyde, which was used

without purification in the next step.

4H-Imidazo[2,1-c][1,4]benzoxazin-6-ylacetaldehyde 35. ¹H

NMR (300 MHz, CDCl₃) δ: 9.8 (s, 1H), 7-7.4 (m, 5H), 5.25

(s, 2H), 3.8 (s, 2H). MS m/z 215.3 [M þ H]^þ.

4H-Imidazo[2,1-c][1,4]benzoxazine-6-carbaldehyde. ¹H NMR

(300 MHz, CDCl₃) δ: 10.5 (s, 1H), 7.7 (m, 1H), 7.5 (m, 1H), 7-7.4

(m, 3H), 5.4 (s, 2H). MS m/z 201.3 3 [M þ H]^þ.

6-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-imidazo-

[2,1-c][1,4]benzoxazine Dihydrochloride (6). 2-Methyl-5-piperazin-

1-yl-quinoline (68 mg, 0.25 mmol) was added to a 7:3 mixture of

intermediate 35 and 4H-imidazo-[2,1-c][1,4]benzoxazine-6-carbal-

dehyde (50 mg, 0.2 mmol) in dry 1,2-dichloroethane (5 mL) at room

temperature. After 15 min, sodium triacetoxyborohydride (52 mg,

0.25 mmol) was added and the resulting reaction mixture was

stirred for 3 h, then quenched with a saturated aqueous solution of

NH₄Cl (20 mL) and extracted with CH₂Cl₂(3 ꢀ 20 mL). The

combined organics were dried over Na₂SO₄and concentrated in

vacuo. The crude products were separated by chromatography on

silica gel eluting with 2% MeOH in CH₂Cl₂to afford the free base

of the title compound (14 mg; 20% yield) and the byproduct

6-{[4-(2-methyl-5-quinolinyl)-1-piperazinyl]methyl}-4H-imidazo-

[2,1-c][1,4]benzoxazine (14 mg, 50% yield). The free base of 6 was

dissolved in dry MeOH (0.5 mL), and HCl (2.2 equiv of a 1.25 M

solution in methanol) was slowly added at 0 °C. The resulting

suspension was stirred at 0 °C for 4 h, followed by evaporation of

the volatiles and trituration with Et₂O to give the title compound

The purity of the compounds reported in the manuscript was

established through HPLC methodology. All the compounds

reported in the manuscript have a purity >95%.

General Synthetic Procedures. The preparation of com-

pounds 6-29 are shown in Schemes 1-6. Experimental and

analytical data for compounds 6, 10, 11-13, and 19-20 are

described below.

8-(2-Propen-1-yl)-2H-1,4-benzoxazine-3(4H)-thione (31). A

mixture of 8-(2-propen-1-yl)-2H-1,4-benzoxazin-3(4H)-one 30

(2 g, 10.5 mmol) and Lawesson’s reagent (2.2 g, 5.3 mmol) in dry

toluene (35 mL) was heated at reflux for 1 h. The mixture was

cooled to room temperature and the solvent evaporated in vacuo.

The crude material was purified by chromatography on silica gel

eluting with 10% cyclohexane in AcOEt to afford the title

1

compound (1.9 g, 88%) as a white solid. H NMR (300 MHz,

CDCl₃) δ: 9.6 (s, 1H), 6.85 (m, 2H), 6.75 (m, 1H), 5.8 (m, 1H),

5.05 (m, 2H), 4.75 (s, 2H), 3.35 (d, 2H).

3-(Methylthio)-8-(2-propen-1-yl)-2H-1,4-benzoxazine (32). To

a mixture of intermediate 31 (100 mg, 0.49 mmol) and KOH

(69 mg, 1.23 mmol) in acetone (2 mL) was added methyl iodide

(46 μL, 0.74 mmol) in two portions 15 min apart. The reaction

mixture was heatedunder reflux for 2 h, cooled, filtered toremove

KI, and evaporated in vacuo. The crude material was purified by

chromatography on silica gel eluting with 10% cyclohexane in

AcOEt to afford the title compound (70 mg, 65%) as a colorless

oil. ¹H NMR (300 MHz, CDCl3) δ: 7.2 (m, 1H), 6.9 (bs, 2H), 5.9

(m, 1H), 5 (m, 2H), 4.5 (s, 2H), 3.3 (d, 2H), 2.5 (s, 3H). MS: m/z

220.2 [M þ H]^þ.

1

as a yellow solid. H NMR (500 MHz, DMSO-d₆) δ: 11.07 (bs,

1H), 8.78 (m, 1H), 8.05 (s, 1H), 7.88 (m, 2H), 7.7 (m, 2H), 7.38 (m,

1H), 7.25 (bs, 1H), 7.24 (d, 1H), 7.18 (t, 1H), 5.43 (s, 2H), 3.8-3.2

(m, 12H), 2.84 (s, 3H). MS m/z 426.3 [M þ H]^þ.

6-{[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]methyl}-4H-

imidazo[2,1-c][1,4]benzoxazine dihydrochloride. ¹H NMR

(500 MHz, DMSO-d₆) δ: 10.64 (bs, 1H), 8.8 (bs, 1H), 8.0 (m, 1H),

7.9 (bs, 3H), 7.8 (bs, 1H), 7.7 (d, 1H), 7.1-7.4 (m, 3H), 5.5 (s, 2H),

4.5 (s, 2H), 3.8-3.1 (m, 8H), 2.7 (s, 3H). MS m/z: 412.4 [M þ H]^þ.

Ethyl 6-(2-Propen-1-yl)-4H-imidazo[5,1-c][1,4]benzoxazine-3-

carboxylate (47). Diethyl chlorophosphate (0.15 mL, 1.8 mmol)

was added to a solution of intermediate 30 (170 mg, 0.9 mmol)

and potassium t-butoxide (110 mg, 0.9 mmol) in dry DMF (5 mL)

at 0 °C.After 10min, a solutionofethylisocyanoacetate (0.15 mL,

1.35 mmol) and potassium t-butoxide (152 mg, 1.35 mmol) in dry

N-[2,2-Bis(methyloxy)ethyl]-8-(2-propen-1-yl)-2H-1,4-benzox-

azin-3-amine (33). A mixture of intermediate 32 (180 mg, 0.82

mmol) and aminoacetaldehyde dimethylacetal (134 μL, 1.23

mmol) in dry EtOH (5 mL) was heated at reflux for 9 h. The

volatiles were evaporated in vacuo and the crude product was

purified by chromatography on silica gel eluting with 40%

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5841

DMF(2mL) wasadded. The reactionmixturewasstirredat 60°C

for 6 h and then cooled and quenched with water (5 mL). The

DMF was evaporated in vacuo and the crude product purified by

SPE-SI cartridge eluting with 30% AcOEt in cyclohexane to

NaOH (3 mL of a 10% aq solution), and the resulting white

suspension was heated for 5 min under microwave irradiation at

120 °C. The resulting pale-yellow solid was filtered off, suspended in

water (15 mL), and the solution was neutralized with an aqueous

solution of AcOH. The resulting off-white solid precipitate was

filtered and washed with Et₂O (3 ꢀ 20 mL) affording the title

compound (0.168 g, 81%). ¹H NMR (300 MHz, DMSO-d₆) δ: 8.49

(bs, 1H), 8.3(d, 1H), 7.7(d, 1H), 7.55 (bs, 1H), 7.35(d, 1H), 7.2(d,

1H), 7.07 (m, 2H), 5.5 (s, 2H), 3.05-2.75 (m, 15H). MS m/z 470.3

[M þ H]^þ.

1

afford the title compound (132 mg, 52%) as a white solid. H

NMR (300 MHz, CDCl₃) δ: 8.2 (s, 1H), 7.55 (d, 1H), 7.3 (d, 1H),

7.2 (t, 1H), 6.25 (m, 1H), 5.7(s, 2H), 5.2-5.3(m,2H), 4.65 (q, 2H),

3.65 (d, 2H), 1.6 (t, 3H). MS m/z 285.2 [M þ H]^þ.

Ethyl 6-(2-Oxoethyl)-4H-imidazo[5,1-c][1,4]benzoxazine-3-

carboxylate (51). Intermediate 47 (77 mg, 0.27 mmol) was

treated with osmium tetroxide according to the general proce-

dure described for 38 (D28) to afford the title compound (52 mg,

6-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-imidazo-

[5,1-c][1,4]benzoxazine-3-carboxamide Dihydrochloride (20). To a

stirred solution of compound 65 (30 mg, 0.06 mmol) and DIPEA

(0.012 mL) in DMF (1 mL) was added TBTU (22.6 mg, 0.07

mmol), and the resulting solution was stirred for 1 h at room

temperature. Hexamethyldisilazane (0.013 mL, 0.07 mmol) was

added and the solution stirred for 1 h. The excess of amine was

scavenged by using a PS-isocyanate resin, and after filtration of the

resin and evaporation of the solvent, the crude product was purified

by SPE cartridge (silica gel) eluting with 3% MeOH in CH₂Cl₂to

afford the free base of the title compound as a white solid. The

free base was dissolved in dry MeOH and treated with HCl (2.1

equiv of 1N solution in Et₂O) at 0 °C. Evaporation of solvent and

trituration with Et₂O gave the final compound (22 mg, 65%). ¹H

NMR (300 MHz, DMSO-d₆) δ: 11.4 (bs, 1H), 9.05 (d, 1H), 8.59 (s,

1H), 8.08 (d, 1H), 8.01 (t, 1H), 7.91 (d, 1H), 7.84 (dd, 1H), 7.51-

7.49 (bd, 2H), 7.33 (bs, 1H), 7.26 (d, 1H), 7.15 (t, 1H), 5.59 (s, 2H),

3.81-3.72 (bd, 2H), 3.53-3.49 (bd, 4H), 3.43-3.39 (bd, 4H),

3.22-3.17 (m, 2H), 2.97 (s, 3H). MS m/z 469.4 [M þ H]^þ.

General Procedure for Amide Formation (19, 21-23 and

25-29). To a stirred solution of intermediate 65 (1 equiv) and

DIPEA (1.1 equiv) in DMF was added TBTU (1.1 equiv) and

the resulting solution was stirred for 1 h at room temperature.

The desired amine (1.1 equiv) was added and the solution stirred

for 1 h. The crude solution was applied to a SPE-SCX cartridge

(eluting with MeOH followed by 2N ammonia solution in

MeOH). After evaporation of solvent from the ammonia frac-

tions and trituration with Et₂O, the free base of the desired

compound was isolated in pure form. The free base was dis-

solved in dry MeOH and treated with HCl (2.1 equiv of 1N

solution in Et₂O) at 0 °C. Evaporation of solvent and trituration

with Et₂O gave the final compounds in pure form.

1

67%) as a white solid. H NMR (300 MHz, CDCl₃) δ: 9.8 (s,

1H), 8 (s, 1H), 7.4 (t, 1H), 7-7.1 (m, 2H), 5.5 (s, 2H), 4.35 (q,

2H), 3.8 (s, 2H), 1.4 (t, 3H).

Ethyl 6-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-

imidazo[5,1-c][1,4]benzoxazine-3-carboxylate (14). A mixture of

intermediate 51 (D6) (50 mg, 0.17 mmol) was reacted with 2-methyl-

5-piperazin-1-yl-quinoline (58 mg, 0.21 mmol) as in the general

procedure described for 6 of to afford the free base of the title

compound (61 mg, 70%) as a white solid. ¹H NMR (300 MHz,

CDCl₃) δ: 8.4 (d, 1H), 8 (s, 1H), 7.7 (d, 1H), 7.55 (t, 1H), 7.3 (d,

1H), 7.2 (m, 1H), 7.1 (d, 1H), 6.95-7.05 (m, 2H), 5.55 (s, 2H), 4.4

(q, 2H), 2.7-3.15 (m, 12H), 3.7 (s, 3H), 1.4 (t, 3H). MS m/z 498.5

[M þ H]^þ.

The free base (20 mg, 0.04 mmol) was dissolved in dry MeOH

(1 mL) and treated with HCl (0.068 mL of a 1.25N solution in

MeOH, 0.084 mmol) at 0 °C. The resulting suspension was stirred

at 0 °C for 4 h. Evaporation of the volatiles and trituration with

Et₂O gave the dihydrochloride of the title compound (20 mg,

87%) as a yellow solid. ¹H NMR (500 MHz, DMSO-d₆) δ: 10.67

(bs, 1H), 8.8-8.6 (bs, 1H), 7.89 (dd, 1H), 7.82 (s, 2H), 7.64 (bs,

1H), 7.35 (s, 1H), 7.29 (dd, 1H), 7.2 (d, 1H), 4.3 (q, 2H), 3.76 (dd,

2H), 3.6-3.3 (m, 8H), 2.8 (s, 3H), 1.33 (t, 3H).

6-(2-Propen-1-yl)-4H-imidazo[5,1-c][1,4]benzoxazine-3-carboxylic

Acid (48). A solution of intermediate 47 (900 mg, 3.17 mmol) in a 1:1

mixture of MeOH and 1 M solution of NaOH (60 mL) was stirred at

60 °C for 30 min. The cooled reaction mixture was neutralized with

AcOH and then cooled to 0 °C. The crude product was collected by

filtration, washed with MeOH, and dried to afford the title com-

pound (570 mg, 70%) as a white solid. ¹H NMR (300 MHz, CDCl₃)

δ: 8.55 (s, 1H), 7.75 (d, 1H), 7.05-7.15 (m, 2H), 5.8 (m, 1H), 5.5 (s,

2H), 5.0-5.1 (m, 2H), 3.45 (d, 2H).

N-Methyl-6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-

4H-imidazo[5,1-c][1,4]benzoxazine-3-carboxamide Dihydrochloride

(19). The title compound was prepared in 44% yield from inter-

mediate 65 following the general procedure for amide formation

6-(2-Propen-1-yl)-4H-imidazo[5,1-c][1,4]benzoxazine (49). A

mixture of intermediate 48 (120 mg, 0.47 mmol) in 1,2-dichloro-

benzene (1.5 mL) was irradiated in a microwave reactor (300 W,

250 °C, 10 min). The solvent was removed by SPE-SCX car-

tridge (eluting with MeOH followed by 2N ammonia solution in

MeOH) to afford the title compound (61 mg, 100%) as a white

1

using methylamine (2N solution in THF). H NMR (300 MHz,

DMSO-d₆) δ: 11.29 (bs, 1H), 9.05 (d, 1H), 8.60 (s, 1H), 8.07-7.97

(m, 2H), 7.89(d, 1H), 7.83(dd, 1H), 7.49(d, 1H), 7.37(bs, 1H), 7.24

(t, 1H), 7.15 (t, 1H), 5.59 (s, 2H), 3.70-3.38 (m, 10H), 3.22-3.15

(m, 2H), 2.96 (s, 3H), 2.74(d, 3H). MS m/z 483.4 [M þ H]^þ.

1-Methyl-6-(2-propen-1-yl)-4H-[1,2,4]triazolo[3,4-c][1,4]ben-

zoxazine (55). A solution of intermediate 31 (1.38 g, 6.71 mmol)

in absolute EtOH (50 mL) was added dropwise over 1 h to a

solution of hydrazine monohydrate (8 mL) in absolute EtOH

(50 mL) at 80 °C. The resulting reaction mixture was stirred at

reflux for a further 40 min and concentrated in vacuo to afford

(3E)-8-(2-propen-1-yl)-2H-1,4-benzoxazin-3(4H)-one hydra-

zone 54, which was immediately used in the following step

without purification. The hydrazone 54 was mixed with tri-

methyl orthoacetate (15 mL) and heated with stirring at 150 °C

for 10 min under microwave irradiation. The resulting reaction

mixture was evaporated in vacuo and purified by flash chro-

matography on silica gel, eluting with 5% MeOH in CH₂Cl₂to

give the title compound (0.78 g, 51%) as a pale-yellow solid. ¹H

NMR (300 MHz, CDCl₃) δ: 7.36 (d, 1H), 7.11-7.03 (m, 2H),

6.01-5.84 (m, 1H), 5.25 (s, 2H), 5.08-5.01 (m, 2H), 3.43 (d,

2H), 2.76 (s, 3H). MS m/z 228.20 [M þ H]^þ.

1

solid. H NMR (300 MHz, CDCl₃) δ: 8.6 (s, 1H), 7.6 (d, 1H),

7.2-7.35 (m, 3H), 6.25 (m, 1H), 5.5 (s, 2H), 5.35 (m, 2H), 3.65 (d,

2H). MS m/z 213.2 [M þ H]^þ.

4H-Imidazo[5,1-c][1,4]benzoxazin-6-ylacetaldehyde (50). The

title compound was prepared in 30% yield according to the

general oxidation procedure described for 38 starting from

intermediate 49 (60 mg, 0.28 mmol). ¹H NMR (300 MHz, CDCl₃)

δ: 9.8 (s, 1H), 8.15 (s, 1H), 7.7 (d, 1H), 7.3-7.45(m,3H),5.5(s,2H),

3.75 (s, 2H).

6-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-imidazo-

[5,1-c][1,4]benzoxazine Dihydrochloride (10). The title compound

was prepared in 40% yield following the general reductive amina-

tion procedure described for 6starting from intermediate 50 (20mg,

0.08 mmol) and 2-methyl-5-piperazin-1-yl-quinoline (20 mg, 0.1

1

mmol). H NMR (500 MHz, DMSO-d₆) δ: 10.64 (bs, 1H), 8.64

(s, 1H), 8.47 (d, 1H), 7.81 (d, 1H), 7.7 (s, 2H), 7.48 (d, 1H), 7.3-7.1

(m, 3H), 7.07 (s, 1H), 5.35 (s, 2H), 3.8-3.1 (m, 12H), 2.68 (s, 3H).

MS m/z 426.3 [M þ H]^þ.

6-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-imidazo-

[5,1-c][1,4]benzoxazine-3-carboxylic Acid (65). To a solution of

compound 14 (220 mg, 0.44 mmol) in MeOH (3 mL) was added

(1-Methyl-4H-[1,2,4]triazolo[3,4-c][1,4]benzoxazin-6-yl)acet-

aldehyde (56). Intermediate 55 (D24) (142 mg, 0.62 mmol) was

5842 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15

Bromidge et al.

treated with 4-methylmorpholine N-oxide (145 mg, 1.24 mmol)

and osmium tetroxide (0.20 mL of 4% by wt solution in water)

according to the procedure described for 53a to give the title

compound (50 mg, 35%) as a white solid which was used

AcOH (10 mL) was added iron powder (619 mg, 11.1 mmol,

4 equiv) at room temperature and the mixture was stirred for

16 h. The solvent was evaporated in vacuo, and AcOEt (50 mL)

was added to the residue. The precipitate was removed by filtra-

tion, and the filtrate was extracted successively with 1N NaOH

(10 mL) and brine (10 mL) before drying over MgSO₄and

evaporation in vacuo to afford the title compound (540 mg,

97%). ¹H NMR (300 MHz, CDCl₃) δ: 6.86 (m, 1H), 6.61-6.53

(m, 2H), 5.94 (m, 1H), 5.10-5.01 (m, 2H), 4.44 (m, 2H), 3.82 (bs,

2H), 3.40 (m, 2H), 1.87 (m, 3H). MS m/z 202.10 [M þ H]^þ.

1-Azido-2-(2-butyn-1-yloxy)-3-(2-propen-1-yl)benzene (62). A

solution of NaNO₂(72 mg, 1.05 mmol, 1.05 equiv) in water

(1 mL) was added dropwise to a suspension of intermediate 61

(201 mg, 1 mmol) in 4N HCl (3 mL) under vigorous stirring and

ice/water cooling. The mixture was then neutralized by addition

of aqueous NaHCO₃, and a solution of sodium azide (65 mg,

1 mmol, 1 equiv) in water (1 mL) was slowly added at 5 °C. After

30 min, the mixture was extracted with Et₂O (3 ꢀ 20 mL) and the

combined organics dried over MgSO₄and evaporated under

reduced pressure to give the title azide (222 mg, 98%), which was

1

without further purification. H NMR (300 MHz, CDCl₃) δ:

9.78 (s, 1H), 7.49 (d, 1H), 7.15-7.12 (m, 2H), 5.28 (s, 2H), 3.82

(s, 2H), 2.79 (s, 3H). MS m/z 230.20 [M þ H]^þ.

1-Methyl-6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-

4H-[1,2,4]triazolo[3,4-c][1,4]benzoxazine dihydrochloride (11).

The title compound was prepared in 71% yield following the

general reductive amination procedure described for 6 starting

from intermediate 56 (45 mg, 0.2 mmol) and 2-methyl-5-piper-

1

azin-1-yl-quinoline (54 mg, 0.24 mmol). H NMR (500 MHz,

DMSO-d₆) δ: 11.21 (bs, 1H), 8.92 (bs, 1H), 7.97 (bs, 2H), 7.81

(bd, 1H), 7.72 (d, 1H), 7.45 (bd, 1H), 7.34 (d, 1H), 7.25 (t, 1H),

5.46 (s, 2H), 3.75 (d, 2H), 3.3-3.6 (m, 8H), 3.24 (m, 2H), 2.91 (s,

3H), 2.74 (s, 3H). MS m/z 441.3 [M þ H]^þ.

6-(2-Propen-1-yl)-4H-tetrazolo[5,1-c][1,4]benzoxazine (57). A

solution of hydrazine hydrate (380 μL, 12.2 mmol) in dry THF

(2 mL) was treated dropwise with a solution of intermediate 31

(500 mg, 2.44 mmol) in THF (25 mL) at 20 °C. After stirring for

1.5 h, the solvent was removed under reduced pressure to afford

the intermediate hydrazone 54, which was used in the following

step without any further purification. To a suspension of

hydrazone 54 (495 mg, 2.44 mmol) in 0.5N HCl (15 mL), a

solution of NaNO₂(252 mg, 3.66 mmol) in water (2 mL) was

added dropwise at 5 °C. After stirring for 4 h, the mixture was

neutralized with a saturated aqueous solution of NaHCO₃and

extracted with CH₂Cl₂(3 ꢀ 30 mL). The combined organic

layers were dried over Na₂SO₄and concentrated in vacuo. The

crude product was purified by flash chromatography on silica

gel eluting with 20% EtOAc in cyclohexane to afford the title

compound (235 mg, 45%). ¹H NMR (400 MHz, CDCl₃) δ: 7.85

(d, 1H), 7.28-7.16 (m, 2H), 6.01-5.94 (m, 1H), 5.65 (s, 2H),

5.14-5.08 (m, 2H), 3.47 (d, 2H). MS m/z 215.10 [M þ H]^þ.

4H-Tetrazolo[5,1-c][1,4]benzoxazin-6-ylacetaldehyde (58). The

title compound was prepared following the procedure of 38 from

intermediate 57 (235 mg, 1.09 mmol). The crude product was

purified by flash chromatography on silica gel eluting with

AcOEt/cyclohexane (4/6) to afford the title compound (144 mg,

61%). ¹H NMR (300 MHz, CDCl₃) δ: 3.77 (s, 2H) 5.56 (s, 2H)

7.14 (d, 2H) 7.90 (t, 1H) 9.72 (s, 1H).

1

used for the next step without further purification. H NMR

(300 MHz, CDCl₃) δ: 7.02 (m, 1H), 6.96-6.87 (m, 2H), 5.90 (m,

1H), 5.10-4.95 (m, 2H), 4.55 (s, 2H), 3.45 (m, 2H), 1.80 (s, 3H).

3-Methyl-6-(2-propen-1-yl)-4H-[1,2,3]triazolo[5,1-c][1,4]ben-

zoxazine (63). A solution of intermediate 62 (222 mg, 0.98 mmol)

in toluene (8 mL) was heated at reflux for 2.5 h. The solvent was

then evaporated under reduced pressure, and the residue was

purified by flash chromatography on silica gel eluting with 20%

AcOEt in cyclohexane to afford the title compound (183 mg,

82%). ¹H NMR (300 MHz, CDCl₃) δ: 7.86 (m, 1H), 7.10-7.00

(m, 2H), 5.90 (m, 1H), 5.25 (s, 2H), 5.05-4.98 (m, 2H), 3.39 (m,

2H), 2.33 (s, 3H). MS m/z 228.20 [M þ H]^þ.

(3-Methyl-4H-[1,2,3]triazolo[5,1-c][1,4]benzoxazin-6-yl)acet-

aldehyde (64). The title compound was prepared in 66% yield

according to the procedure of intermediate 38 starting from

intermediate 63 (183 mg, 0.806 mmol). The product was puri-

fied by flash chromatography on silica gel using AcOEt/cyclo-

hexane (2/3) as eluent. ¹H NMR (300 MHz, CDCl₃) δ: 9.72 (s,

1H), 7.97 (m, 1H), 7.1 (m, 2H), 5.26 (s, 2H), 3.74 (s, 2H), 2.33 (s,

3H). MS m/z 230.10 [M þ H]^þ.

3-Methyl-6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-

4H-[1,2,3]triazolo [5,1-c][1,4]benzoxazine dihydrochloride (12).

The title compound was prepared in 81% yield following the

general reductive amination procedure described for 6 starting

from intermediate 64 (60 mg, 0.262 mmol). The crude product

was purified by flash chromatography on silica gel eluting with a

gradient of 1-3% MeOH in CH₂Cl₂to afford the free base of the

title compound (93 mg, 81%). Treatment with HCl (2.2 equiv of

1.25N solution in MeOH) in 4:1 MeOH/CH₂Cl₂(5 mL) at 0 °C

gave the title compound as a solid. ¹H NMR (500 MHz, DMSO-

d₆) δ: 10.51 (bs, 1H), 8.62 (bs, 1H), 7.93 (d, 1H), 7.78 (bs, 2H),

7.60 (bs, 1H), 7.37 (d, 1H), 7.32 (bs, 1H), 7.24 (t, 1H), 5.58 (s,

2H), 3.75-3.10 (m, 12H), 2.76 (s, 3H), 2.34 (s, 3H). MS m/z

441.20 [M þ H]^þ.

6-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4H-tetra-

zolo[5,1-c][1,4]benzoxazine Dihydrochloride (13). The title com-

pound was prepared in 77% yield following the general reductive

amination procedure described for 6 starting from intermediate 58

(47 mg, 0.218 mmol) and 2-methyl-5-(1-piperazinyl)quinoline

(74 mg, 0.326 mmol). The crude product was purified by flash chro-

matography on silica gel eluting with 2% MeOH in CH₂Cl₂to

afford the free base of the title compound (72 mg, 77%). Treatment

with HCl (2.2 equiv of 1.25 M solution in MeOH) in 2:1 MeOH/

CH₂Cl₂(3mL) at0°C gave the title compound as a yellow solid. ¹H

NMR (500 MHz, DMSO-d₆) δ: 2.72 (s, 3H) 3.18-3.56 (m, 10H)

3.66-3.78 (m, 2H) 5.84-5.97 (m, 2H) 7.22-7.32 (m, 2H) 7.43 (d,

1H) 7.47-7.63 (m, 1H) 7.67-7.83 (m, 2H) 7.89 (dd, 1H) 8.36-8.75

(m, 1H) 11.24 (bs, 1H). MS m/z 428.00 [M þ H]^þ.

Acknowledgment. We thank Dr. Carla Marchioro and her

analytical group for the high quality support received and all

other colleagues who helped in generating the data reported in

this manuscript.

2-(2-Butyn-1-yloxy)-1-nitro-3-(2-propen-1-yl)benzene (60). A

mixture of 2-nitro-6-(2-propen-1-yl)phenol 59 (500 mg, 2.79

mmol), 1-bromo-2-butyne (269 μL, 3.07 mmol, 1.1 equiv), and

K₂CO₃(424 mg, 3.07 mmol, 1.1 equiv) in acetone (20 mL) was

heated at reflux for 4 h. The mixture was cooled to room

temperature before the solids were filtered off and washed with

acetone (30 mL). The combined organics were concentrated in

vacuo, and the crude product was purified by flash chromato-

graphy on silica gel eluting with 10% AcOEt in cyclohexane to

afford the title compound (640 mg, 99%). ¹H NMR (300 MHz,

CDCl₃) δ: 7.65 (m, 1H), 7.39 (m, 1H), 7.13 (m, 1H), 5.90 (m, 1H),

5.11-5.02 (m, 2H), 4.60 (m, 2H), 3.49 (m, 2H), 1.77 (s, 3H).

[2-(2-Butyn-1-yloxy)-3-(2-propen-1-yl)phenyl]amine (61). To

a solution of intermediate 60 (640 mg, 2.77 mmol) in glacial

Supporting Information Available: Experimental and sup-

porting data for compounds 7-9, 15-18, and 21-29. This

material is available free of charge via the Internet at http://

pubs.acs.org.

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6-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones:

Article Doi

Design and synthesis of novel tricyclic benzoxazines as potent 5-HT 1A/B/D receptor antagonists leading to the discovery of 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-4 h-imidazo[5,1-c][1,4] benzoxazine-3-carboxamide (GSK588045)

DOI: 10.1021/jm100482n

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Full text of DOI:10.1021/jm100482n

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