Bioorganic & Medicinal Chemistry Letters 21 (2011) 5310–5314

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Strategies to lower the Pgp efflux liability in a series of potent indole azetidineMCHR1 antagonists

Kai Lu a, Yu Jiang a, Bin Chen a, Eman M. Eldemenky a, Gil Ma a, Mathivanan Packiarajan a,Gamini Chandrasena a, Andrew D. White a, Kenneth A. Jones b, Boshan Li b, Sang-Phyo Hong a,⇑a Department of Chemical and Pharmacokinetic Sciences, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USAb Synaptic Transmission Disease Biology Unit, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 May 2011Revised 6 July 2011Accepted 6 July 2011Available online 14 July 2011

Keywords:MCHPgpIndolyl azetidineDihydroindolyl azetidineCNS penetration

0960-894X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.bmcl.2011.07.020

⇑ Corresponding author. Tel.: +1 201 350 0152; faxE-mail address: sph@lundbeck.com (S.-P. Hong).

A series of potent indolyl azetidine rMCHR1 antagonists were found to show poor CNS penetration due toPgp efflux. We envisioned a strategy which included: lowering basicity; changing the conformationalflexibility motif; and removal of a hydrogen bond donor, in an attempt to optimize this property whilemaintaining target receptor efficacy. This work resulted in mitigation of Pgp efflux, and led us to identify1-dihydroindolyl azetidine derivatives with CNS penetration and excellent rMCHR1 binding affinity.

� 2011 Elsevier Ltd. All rights reserved.

NHN

N

O

O

rMCHr1, Ki = 11 nMhERG IC50 = 12 µM

Melanin-concentrating hormone (MCH) is a cyclic neuropeptideconsisting of 19 amino acids originally isolated from the pituitarygland of the salmon.1 MCH is produced predominantly by neuronsin the lateral hypothalamus and zona incerta which project broadlythroughout the brain of mammals.2 The biological function of MCHis mediated by its interaction with two G protein-coupled recep-tors known as MCHR1 and MCHR2.3 Over the last 20 years, studieshave suggested that MCH plays a key role in the regulation of foodintake and stress in rodents.4 For example, ICV injection of MCHreportedly stimulates food intake5 and chronic administration re-sults in an increase in body weight.6 The link between MCHR1and the function of MCH on feeding is illustrated with several stud-ies on MCHR1 KO mice. These mice lacking the gene encoding MCHare lean, hypophagic and maintain an elevated metabolic rate.7

Conversely, mice over-expressing the MCH gene are susceptibleto obesity and insulin resistance. In addition, MCH seems to playa role in the regulation of mood and stress. A number of groupshave disclosed high affinity MCHR1 antagonists with efficacy inbehavioral models of depression and anxiety.8 The weight of evi-dence suggests that small molecule antagonists of the MCH1receptor could be useful in the treatment of obesity or mood disor-ders. The promising in vitro and in vivo pharmacology of publishedMCHR1 antagonists has solidified the MCH1 receptor as an attrac-tive target for a number of potential disease indications.9

All rights reserved.

: +1 201 261 0623.

In an effort to discover potent MCHR1 antagonists, we identifiedindolyl azetidines, exemplified by compound 1 (Fig. 1), which havegood in vitro properties and are effectively devoid of hERG channelaffinity issues.10 Compound 1, however, exhibits poor CNS penetra-tion apparently due to facile Pgp efflux. Herein, we describe ourlead optimization efforts, focused on mitigating the Pgp efflux,with indolyl azetidine and dihydroindolyl azetidine rMCHR1antagonists.

In order to monitor the efflux properties of the compounds, weconducted an MDR1-MDCK11 permeability experiment with com-pound 1 to investigate its Pgp liability (Fig. 2). The apparent per-meability measured bi-directionally, after 2 h incubation, was0.19 � 10�6 cm/s (PappA-to-B) and 26.3 � 10�6 cm/s (PappB-to-A).This resultant high efflux ratio (144) with good recovery, 72%

1

Figure 1. Profile of rMCHR1 antagonist 1.

Figure 2. MDR1-MDCK profile and B/P ratio in +/�Pgp mice after iv administration of compound 1.

K. Lu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5310–5314 5311

(A-to-B) and 71% (B-to-A), respectively, portrays the apparentaffinity for the Pgp transporter system. Furthermore, in the PgpKO mice, a 6–8 h window is required to reach high brain levels sug-gesting the compound also has relatively poor intrinsic permeabil-ity which may confound the Pgp efflux data.12 Supporting thishypothesis, the brain to plasma ratio, after a 15 mg/kg iv dose,was substantially improved (B/P = 2.7) over 8 h in Pgp knock-outmice relative to a low brain to plasma ratio (B/P = 0.3) in wild types(see Supplementary data). The almost 10-fold improvement in B/Pratio in Pgp KO mice (B/P = 2.7 vs wild type) underscores the strongPgp substrate affinity of compound 1.

Recently, a rule of 4 has been formulated to predict relative pro-pensity for Pgp efflux liability.13 Compounds with a molecularweight <400, containing a total number of nitrogen and oxygenatoms <4, and a basic pKa

14 <8 are unlikely to be Pgp substrates.The presence of hydrogen bond donors is also deemed to be detri-mental. Compound 1 has a low molecular weight (385), but a highheteroatom count (N + O = 5), relatively high basicity (pKa = 8.7),one hydrogen bond donor, and five rotatable bonds. Not surpris-ingly, this indicates a reasonable chance of Pgp liability, as is borneout by the experimental data. We logically envisioned a designstrategy focused on lowering the pKa, reducing the number of het-ero atoms in the molecule, removing the hydrogen bond donor andchanging the overall conformational mobility to mitigate Pgpefflux.

The synthesis of the indolyl azetidine derivatives prepared toinvestigate optimization of these properties (1 and 5a–f) is out-lined in Scheme 1. Indolyl-hydroxyazetidine 3 was readily ob-tained via a coupling reaction between 6-bromo-indole (2) andN-Boc-azetidin-2-one in the presence of potassium hydroxide inrefluxing methanol. Subsequent LiAlH4 reduction of intermediate3 in refluxing THF resulted in selective dehydroxylation and con-comitant reduction of the Boc group to afford intermediate 4a in31% overall yield. Surprisingly the bromine functionality in themolecule remained intact under reductive conditions, presumablydue to the electron rich environment of the indole. Selective dehy-droxylation of intermediate 3 could be achieved without Boc re-moval with a 3:1 mixture of Et3SiH/TFA15 in DCM at 0 �C toafford 1-N-Boc-indolylazetidine 4b in good yield. Subsequent cop-per mediated coupling reactions between bromoindolyl-azetidines4a–c and the corresponding pyridones16 were carried out to affordcompounds 1 and 5a–f in good yields (78–92%). The couplingswere achieved by heating a mixture of CuI (0.5–1.0 equiv), trans-N,N0-dimethyl-cyclohexane-1,2-diamine (1.0 equiv) and K2CO3

(1.5–2.0 equiv) in DMF at 100 �C for 16 h. The substituents R3 forcompound 5d–f were installed before coupling using the corre-sponding alkyl halides (MeI, 2-fluoroethyl bromide or 2-methoxy-ethyl bromide) in the presence of base in DMF. The methylsubstituent at R2 of compounds 4c and 5c was installed with stan-dard alkylation conditions (KOtBu, MeI in THF) in quantitativeyield.

In general, it was found that the indolyl azetidine derivatives 1and 5a–f have good binding affinity for the rMCH1 receptor. (Table1) This series of analogs tolerates a variety of substituents at the R1,R2 and R3 positions. Interestingly, shorter and rigid ligands such asphenylpyridones (5b and f) still maintain good rMCHR1 bindingaffinity. The hydrogen bond donor is not essential for binding affin-ity as capping the indole NH with a methyl substituent (5c and f)resulted in similar binding affinities. The flexibility of binding po-tency in this template allowed us to investigate several differentapproaches to potentially mitigate the Pgp liability. First, we triedto modulate the hydrogen bond acceptor capacity of the benzyloxypyridone moiety by adding halogen substituents. The simple addi-tion of a fluorine substituent at the benzyloxy terminus of R1 (5a)slightly increased brain exposure, but the efflux ratio remainedhigh (150).17 Several other attempts to mitigate Pgp efflux by add-ing halogen atoms such as F or Cl at C3 of the pyridone ring or C5 ofthe indole core moiety were unsuccessful. Next, we turned ourattention to lowering the basicity of the template. Installation ofa methoxyethyl substituent at R3 (5d) slightly lowered basicity(pKa = 8.2) with a concomitant improvement in brain exposure.However, the efflux ratio (MDR1-MDCK = 88) remained high. Fur-ther lowering the basicity (pKa = 6.9) by placing a strong electronwithdrawing fluoroethyl substituent18 at R3 (5e) increased brainexposure further without measurably improving the efflux liability(ratio = 43). We also believed the flexible pyridone moiety played arole in poor CNS penetration by allowing the molecule to accom-modate a reasonable Pgp binding conformation. Under thishypothesis, the conformational flexibility of the molecule was re-duced by eliminating the hydroxymethyl linker (5b). This modifi-cation, to our surprise, retained MCH potency, but resulted inimproved total brain exposure. Furthermore, capping the hydrogenbond donor alone by methylation of indole NH (5c) did not appearto resolve the Pgp efflux based on a maintained poor B/P ratio(0.03). We then decided to apply a combined approach of confor-mational restraint; capping the hydrogen bond donor (indoleNH); and attenuation of the basicity (pKa = 7.4). This combined ap-proach (compound 5f) resulted in high total brain exposure with a

NHBr

NHBr

N

HO

Boc

2 31, R1 = PhCH2O, R2 = H5a, R1 = 2-F-PhCH2O, R2 = H5b, R1 = 4-Cl-Ph, R2 = H5c, R1 = PhCH2O, R2 = Me

a bf

NHBr

N

4a

NN

N

O

R1R2

NBr

NBoc

4b, R = H4c, R = Me

5d, R1 = PhCH2O, R2 = H, R3= MeO(CH 2)25e, R1 = PhCH2O, R2 = H, R3 = F-(CH2)25f, R1 = 4-F-Ph, R2 = Me, R3 = F-(CH2)2

NN

NR3

O

R1R2

c

d, e, f

g

R

g

Scheme 1. Synthesis of indolyl azetidine derivatives. Reagents and conditions: (a) N-Boc-azetidin-2-one, KOH, MeOH, reflux, 41%; (b) LiAlH4, THF, reflux, 76%; (c) Et3SiH/TFA(3:1), DCM, 0 �C, quant; (d) TFA/DCM, quant (crude); (e) K2CO3, R3-halides, DMF, 20–40%; (f) pyridones,16 CuI, trans-N,N0-dimethyl-cyclohexane-1,2-diamine, K2CO3, DMF,100 �C, 78–92%; and (g) KOtBu, 18-C-6, MeI, THF, quant.

Table 1rMCHR1 binding affinity of indolyl azetidine derivatives, their B/P exposure, and MDR1-MDCK efflux ratios

NN

NR3

O

R1R2

No. R1 R2 R3 pKaa rMCH1, Ki (nM) Brain (ng/g)b Plasma (ng/mL)b PappA-to-B (�10�6 cm/s) PappB-to-A (�10�6 cm/s) Effluxd

1 PhCH2O H Me 8.7 11 11 48 0.2 26.3 1445a 2-F–PhCH2O H Me 8.7 6.7 22 140 0.2 25.3 1505b 4-Cl–Ph H Me 8.7 7.8 100 350 0.9 53.7 605c PhCH2O Me Me 8.8 11 110c 4000c — — —5d PhCH2O H MeO(CH2)2 8.2 5.2 49 310 0.4 34.1 885e PhCH2O H F-(CH2)2 6.9 4.3 130 620 1.4 58.9 435f 4-F–Ph Me F–(CH2)2 7.4 9.0 810 1000 7.1 45.4 6.4

a Calculated base pKa.b Rat PK at 10 mg/kg, PO, 4 h.c Rat PK at 10 mg/kg, SC, 4 h.d MDR1-MDCK efflux ratio (PappB-to-A/PappA-to-B).

5312 K. Lu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5310–5314

significant improvement in B/P ratio (0.8) and markedly lower ef-flux (PappB-to-A/PappA-to-B = 6.4). However, the efflux ratio,although improved, still needed to be optimized to further mitigatethe Pgp liability. The persistent Pgp efflux with indolyl azetidinederivatives, in light of the progress, inspired us to modify the in-dole core itself to achieve our goals. We envisioned adihydroindole, which has similar overall topology, but no hydrogenbond donor, thus potentially reducing the Pgp affinity further.

The synthesis of dihydroindolyl azetidine derivatives (9a–e) isoutlined in Scheme 2. Reductive amination of commercially avail-able 6-bromo-dihydroindole 6 and N-Boc-azetidine-2-one readilyafforded 7 in good yield. The copper catalyzed coupling of 6-bro-modihydroindole azetidine 7 with the corresponding pyridonesgave 8. Subsequent Boc deprotection and reductive amination(R2 = Me) or alkylation (R2 = F-ethyl) resulted in dihydroindolylazetidine derivatives 9b–e. Alternatively, 9b–e were synthesized

HN

Br

N

Br

NBoc

N

N

NBoc

O

R1

N

N

NR2

O

R16 7 8

9a, R1 = PhCH2O, R2 = H9b, R1 = PhCH2O, R2 = Me

, R1 = PhCH2O, R2 = F-(CH2)29d9c

, R1 = 4-Cl-Ph, R2 = Me9e, R1 = 4-Cl-Ph, R2 = F-(CH2)2

a b c

9b-e

c, b

Scheme 2. Synthesis of dihydroindolyl azetidine derivatives. Reagents and conditions: (a) N-Boc-azetidin-2-one, NaBH(OAc)3, DCM, 93%; (b) pyridones,16 CuI, trans-N,N0-dimethyl-cyclohexane-1,2-diamine, K2CO3, DMF, 100 �C, quant (crude); and (c) TFA, DCM; then formaldehyde, NaBH(OAc)3, DCM (or) 2-fluoroethyl bromide, K2CO3, DMF, 14–31% (for two steps).

Table 2rMCHR1 binding affinity of dihydroindolyl azetidine derivatives, their B/P exposure, and MDR1-MDCK efflux ratios

N

N

NR2

O

R1

No. R1 R2 pKaa rMCH 1, Ki (nM) Brain (ng/g)b Plasma (ng/mL)b PappA-to-B (�10�6 cm/s) PappB-to-A (�10�6 cm/s) Effluxc

9a PhCH2O H 10.4 2.4 27 130 — — —9b PhCH2O Me 8.6 2.5 780 500 2.6 36.5 149c PhCH2O F–(CH2)2 6.9 4.9 520 420 17.7 38.2 2.29d 4-Cl–Ph Me 8.6 4.9 600 190 — — —9e 4-Cl–Ph F–(CH2)2 6.9 6.5 1100 650 28.9 30.1 1

a Calculated base pKa.b Rat PK at 10 mg/kg, PO, 4 h.c MDR1-MDCK efflux ratio (PappB-to-A/PappA-to-B).

K. Lu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5310–5314 5313

by the reverse synthetic sequence (i.e., Boc deprotection, alkylationor reductive amination, and then copper catalyzed coupling).

To our satisfaction, the dihydroindolyl azetidine analogs 9a–e(Table 2) have similarly potent rMCHR1 antagonist binding affini-ties to the indolyl azetidine derivatives (1 and 5a–f). The benzyloxyanalog 9a, unsubstituted at the azetidine terminus shows a low to-tal brain exposure presumably due to high basicity (pKa = 10.4). Amethyl substituent at R2 of the azetidine (9b) attenuates basicity(pKa = 8.6) significantly versus compound 9a and thus the totalbrain exposure of compound 9b is substantially improved. How-ever, the efflux is still somewhat high (ratio = 14). We further low-ered the basicity by installing a 2-fluoroethyl group at R2 (9c,pKa = 6.9). The Pgp efflux (ratio = 2.2) of compound 9c was signifi-cantly improved and now deemed acceptable. However, to our dis-may, the total brain exposure of compound 9c is lower than that ofcompound 9b, presumably due to an increase in lipophilicity(c log D7.4 = 4.4 vs 3.1, respectively) and rotatable bonds (5 vs 7,respectively). Subsequent limiting of conformational flexibility byinstalling a 4-chlorophenyl at R1 resulted in good brain exposure(9d, B/P >3). Further lowering the basicity (pKa = 6.9) with a 2-flu-oroethyl substituent at R2, as before, improved brain exposure

additionally (9e). The MDR1-MDCK data (PappB-to-A/PappA-to-

B = 1) of compound 9e suggests it will be effectively devoid of aPgp efflux. Compound 9e was subsequently advanced in our inves-tigation of MCHR1 ligands, due to good rMCHR1 binding affinity,high brain exposure and apparent lack of a Pgp efflux, the resultsof which will be reported in due course.

In summary, we have described our lead optimization efforts toidentify the potent rMCHR1 antagonist 9e with apparent lack ofPgp efflux liability. The combined strategies to solve the Pgp effluxissue involved lowering the basicity, reducing conformational flex-ibility, reducing heteroatom count, and removing a hydrogen bonddonor. This optimally engineered derivative maintained good bind-ing affinity against rMCH1 receptor with excellent CNS exposure,allowing us to examine the utility of MCHR1 antagonists aspotential CNS therapeutics.

Acknowledgments

The authors would like to thank Arifa Husain, ChristineG. Mazza, Lingyun Wu, Mohammad R. Marzabadi and Marc Labellefor their helpful discussion within the team. We also thank Asanthi

5314 K. Lu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5310–5314

Pieris and Manuel Cajina for performing PK studies and bioanaly-sis, respectively. We also thank for Chi Zhang for the PAMPAanalysis.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bmcl.2011.07.020.

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17. Difluorination of the phenyl group also did not improve CNS exposure relativeto 5a.

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