Ergoline-derived inverse agonists of the human H3 receptor for the treatment of narcolepsy

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DOI: 10.1002/cmdc.201402055

Ergoline-Derived Inverse Agonists of the Human H3

Receptor for the Treatment of Narcolepsy

Yves P. Auberson,*^[a]Thomas Troxler,*^[a]Xuechun Zhang,*^[b]Charles R. Yang,^[b]

Markus Fendt,^[c]Dominik Feuerbach,^[a]Yu-Chih Liu,^[b]Bharat Lagu,^[d]Andreas Lerchner,^[a]

Mark Perrone,^[d]Lijun Lei,^[b]Chao Zhang,^[b]Chunxiu Wang,^[b]Tie-Lin Wang,^[b]and

Mark G. Bock^[d]

Ergoline

derivative

(6aR,9R)-4-(2-(dimethylamino)ethyl)-N-

with previously reported clinical candidates during the night

following administration. Modifications to the ergoline core at

positions 1, 6 and 8 were systematically investigated, and

derivative 23 (1-((4aR,8R,9aR)-8-(hydroxymethyl)-1-(2-((R)-2-

methylpyrrolidin-1-yl)ethyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-

fg]quinolin-6(1H)-yl)ethanone) was identified as a promising

lead compound. Derivative 23 has a desirable pharmacokinetic

profile and demonstrated efficacy by enhancing brain concen-

trations of tele-methylhistamine, a major histamine metabolite.

This validates the potential of the ergoline scaffold to serve as

a template for the development of H3R inverse agonists.

phenyl-9-(pyrrolidine-1-carbonyl)-6,6a,8,9-tetrahydroindolo[4,3-

fg]quinoline-7(4H)-carboxamide (1), a CXCR3 antagonist, also

inhibits human histamine H3 receptors (H3R) and represents

a structurally novel H3R inverse agonist chemotype. It displays

favorable pharmacokinetic and in vitro safety profiles, and

served as a lead compound in a program to explore ergoline

derivatives as potential drug candidates for the treatment of

narcolepsy. A key objective of this work was to enhance the

safety and efficacy profiles of 1, while minimizing its duration

of action to mitigate the episodes of insomnia documented

Introduction

The human H3 receptor (H3R) has been the subject of exten-

sive research as a potential drug target since its discovery in

1983^[1]and subsequent cloning in 1999.^[2]H3R inverse agonists

have been proposed as agents for the treatment of numerous

maladies,^[3]ranging from cardiovascular, inflammatory, respira-

tory and pain disorders to migraine, attention deficit hyperac-

tivity disorder (ADHD), as well as multiple sclerosis^[4]and Tour-

ette’s syndrome.^[5]A particular focus of research has been on

the development of H3R inverse agonists for the management

of sleep disorders such as narcolepsy.

and histamine levels in the cerebrospinal fluid (CSF). Narcolep-

tic patients suffer from sleep disturbances manifested as exces-

sive daytime sleepiness, frequent sleep-onset rapid eye move-

ment (SOREM) periods and sleep paralysis.^[7]They also experi-

ence cataplectic events, expressed as sudden episodes of

muscle weakness, which may be precipitated by strong emo-

tional responses or rapidly initiated physical activity.

Orexinergic neurons promote arousal via activation of the

histaminergic system in the tubero-mamillary nucleus, where

the presynaptic H3R limit histamine release and regulate wake-

fulness.^[8,9]A deficient orexin system results in narcolepsy,^[10]

and inverse agonists capable of dampening the histamine neg-

ative feedback at the H3R have the potential to be of thera-

peutic benefit by elevating histamine to promote wakefulness

and thus compensate for the loss of orexinergic tone.

The underlying cause of human narcolepsy is the degenera-

tion of orexinergic neurons,^[6]which leads to decreased orexin

[a] Dr. Y. P. Auberson, Dr. T. Troxler, Dr. D. Feuerbach, Dr. A. Lerchner

Novartis Institutes for BioMedical Research

Forum 1, Novartis Campus, 4056 Basel (Switzerland)

E-mail: yves.auberson@novartis.com

Orexin-deficient (orexinꢀ/ꢀ) mice exhibit behavioral arrests

similar to cataplectic episodes in humans, and therefore, these

mice are considered to be a suitable animal model for this con-

dition.^[11]H3R inverse agonists that decrease narcoleptic epi-

sodes in orexinꢀ/ꢀ mice^[12]can reasonably be expected to re-

verse narcolepsy and possibly even cataplexy in humans.

Pitolisant (Figure 1), the most advanced H3R inverse agonist

clinical candidate, was shown to be efficacious in the treat-

ment of narcolepsy.^[13]After one week treatment with pitoli-

sant, narcoleptic patients showed a significant decrease in the

sleepiness score, as well as sleep suppression. This suggests

that by elevating brain histamine transmission, an H3R inverse

agonist can be used for treating narcolepsy. The prolonged du-

ration of action of pitolisant (T_1/2=11 h), however, leads to the

thomas.troxler@novartis.com

[b] Dr. X. Zhang, Dr. Ch. R. Yang, Dr. Y.-Ch. Liu, Dr. L. Lei, Dr. C. Zhang,

C. Wang, Dr. T.-L. Wang

Shanghai ChemPartner Co., Ltd

No. 5 Building, 998 Halei Rd, Zhangjiang Hi-Tech Park

Pudong New Area, Shanghai, 201203 (China)

E-mail: xchzhang@chempartner.cn

[c] Prof. M. Fendt

Institute for Pharmacology & Toxicology

Center of Behavioral Brain Sciences

Otto-von-Guericke-University Magdeburg

Leipziger Str. 44, 39120 Magdeburg (Germany)

[d] Dr. B. Lagu, Dr. M. Perrone, Dr. M. G. Bock

Novartis Institutes for BioMedical Research

Novartis Pharma AG, 250 Massachusetts Ave, Cambridge, MA 02139 (USA)

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rapid clearance, would lead to the selection of a candidate ful-

filling the desired profile.

Results and Discussion

Chemistry

The modifications of the ergoline skeleton in lead compound

1 were focused on positions 1, 6 and 8. As depicted in

Scheme 1, to access position-6 analogues, the penultimate

compound (8) was prepared by treating ergoline-8-carboxylic

acid methyl ester 2^[21]with isocyanatobenzene to afford urea 3.

Hydrolysis of the methyl ester, followed by coupling with pyr-

rolidine in the presence of 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tet-

ramethyluronium hexa- fluorophosphate (HATU) gave carboxa-

mide 4. Alkylating 4 with isopropyl 2-bromoacetate yielded

the isopropyl acetate (5), which was then reduced and mesy-

lated to afford 7. Displacement of the mesylate with dimethyl-

amine also simultaneously unmasked the 7-amino group to

provide amine 8. The latter compound was reacted with iso-

cyanatobenzene and acetyl chloride to afford 9a and 9b, re-

spectively. Compound 11 was obtained from 8 via the activat-

ed 4-nitrophenyl carbamate intermediate 10.

The syntheses of 15a–g with variations in position 1 are de-

picted in Scheme 2. Ergoline-8-carboxylic acid 12 was sequen-

tially coupled with pyrrolidine and dimethylcarbamic chloride

to afford 14. Target molecules 15a–g were readily obtained

from compound 14, introducing the side chain in a manner

similar to the procedure described for compounds 9a and 9b.

The low isolated yields for some analogues were primarily at-

tributable to their poor solubility in purification solvents.

Compounds 18a–d, with variations on position 8, were ac-

cessed as illustrated in Scheme 3. Starting from ergoline-8-car-

Figure 1. Clinical candidate H3R inverse agonists.

undesirable effect of insomnia.^[14,15]Other drug candidates,

such as PF-03654746 (T_1/2=9–18 h),^[16]MK-0249 (T_1/2=14 h),^[17]

JNJ-31001074 (bavisant; T_1/2=13 to 20 h),^[18]and ABT-288

(T_1/2=40–61 h)^[19]also have prolonged duration of action. This

severely limits their administration at doses that are efficacious

for treating narcolepsy while avoiding insomnia. The recently

described clinical candidate AZD5213 displays a half-life of ap-

proximately 5 h.^[20]Clinical positron emission tomography (PET)

imaging confirmed its fast receptor binding and rapid disen-

gagement kinetics in the human brain, resulting in a pharmaco-

kinetic profile predicted to be compatible with daytime effica-

cy and a limited risk of insomnia during the following night.

The objective of the present work was to identify a com-

pound with the potential to demonstrate efficacy in narcolepsy

patients, without concomitant insomnia during the following

night. Our working hypothesis was predicated on the notion

that achieving a rapid, high H3R occupancy, combined with

Scheme 1. Variation of the substituent at position 6. Reagents and conditions: a) isocyanatobenzene, CH₂Cl₂, RT, 12 h, 90%; b) 1m NaOH, THF/H₂O (4:1), RT,

2 h, 89%; c) pyrrolidine, HATU, Et₃N, CH₂Cl₂, RT, o/n, 73%; d) isopropyl 2-bromoacetate, TBAI, NaOH, CH₂Cl₂, 08C!RT, o/n, 90%; e) LiBH₄, THF, 08C!RT, o/n,

63.5%; f) MsCl, Et₃N, CH₂Cl₂, 08C!RT, 4 h; g) Me₂NH, MeCN, 508C, sealed tube, o/n, 68% (two steps); h) For 9a: isocyanatobenzene, CH₂Cl₂, RT, 12 h, 16%; For

9b: AcCl, DIPEA, CH₂Cl₂, 08C!RT, 4 h, 37%; i) 4-nitrophenyl chloroformate, DIPEA, CH₂Cl₂, 08C!RT, o/n, 74%; j) MeNH₂, DIPEA, DMAP, CH₂Cl₂, RT, o/n, 22%.

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Figure 2. Comparison of the core structures of ergoline (1) and lysergic acid

diethylamide (LSD).

hibitor,^[22]also has potent affinity for human H3 receptors (K_i=

8.3 nm). Its selectivity against a panel of more than 100 recep-

tors, proteases, and kinases (data not shown), its favorable

pharmacokinetic properties in rats,^[23a]and its unique structure

relative to previously reported H3R inverse agonists provided

the impetus to further explore the optimization of its proper-

ties. Inverse agonism of 1 was proven in GTPgS assays (data

not shown), based on the well-described activation of hista-

mine release by H3R inverse agonists.^[1]Additional incentive to

pursue the investigation of 1 as an H3R inverse agonist was

provided by preliminary in vivo experiments, which confirmed

that it shows histaminergic activities in rats after oral adminis-

tration^[23b]and strongly decreased the narcoleptic episodes in

orexin-deficient mice (Figure 3).

Scheme 2. Variation of the side chain at position 1. Reagents and conditions:

a) HATU, pyrrolidine, DMF, RT, o/n, 76%; b) dimethylcarbamic chloride,

DIPEA, DMAP, MeCN, 658C, 4 h, 71%; c) isopropyl 2-bromoacetate, TBAI,

NaOH, CH₂Cl₂, 08C!RT, o/n, 67%; d) LiBH₄, THF, 08C!RT, o/n, 81%; e) MsCl,

Et₃N, CH₂Cl₂, 08C!RT, o/n, 55%; f) amines, MeCN, 508C, o/n, 10–68%.

At the outset of the derivatization effort, we identified and

sought to address several potential deficiencies associated

with the ergoline derivative 1. As is evident in Figure 2, essen-

tially the entire structure of lysergic acid diethylamide (LSD) is

embedded within the lead compound 1. Therefore, one con-

cern was that the demonstrated selectivity of 1 versus seroto-

nin, adrenergic and dopamine receptors (Table 1) could be

eroded subsequent to derivatization, and that these analogues

or their corresponding metabolites might display unwanted

LSD-like mind-altering effects.^[24]Another concern was the

boxylic acid 12, acylation of position 6 with acetic anhydride

afforded 16. Esterification of position 8 with benzyl chloride,

and alkylation of position 1 with isopropyl 2-bromoacetate, fol-

lowed by debenzylation under hydrogenation conditions, gave

acid 17. The latter was coupled with various amines, and the

isopropyl ester group was reduced, mesylated and

reacted with (R)-2-methylpyrrolidine to yield products

18a–d.

Cyclization of 16 with (E)-N’-hydroxyacetimidamide

gave the 1,2,4-oxadiazole derivative 19. The latter

compound was subjected to a similar sequence of re-

actions as described above for 18a–d to yield com-

pound 20. Beginning with compound 17, selective

reduction of the isopropyl ester group with lithium

borohydride, followed by esterification with metha-

nol and thionyl chloride gave methyl ester 21. Mesy-

lation and displacement of the mesylate with (R)-2-

methylpyrrolidine afforded 22. Straightforward reduc-

tion of the methyl ester led to alcohol 23, and there-

after to the methyl ether 24.

Pharmacological evaluation

Figure 3. Effect of a single 30 mgkg^ꢀ1dose of orally administered 1 on behavioral arrests

in orexinꢀ/ꢀ mice. a) The mean total number and b) the time course of behavior arrests

The present work had its genesis with the observa-

*

in orexin-deficient mice after oral administration of vehicle ( ) or compound 1 at

30 mgkg^ꢀ1

(

tion that ergoline derivative 1 (Figure 2), previously

*

). Compound 1 significantly decreased the number of behavioral arrests

reported as a CXC chemokine receptor 3 (CXCR3) in- (Wilcoxon matched-pairs signed rank test: W=ꢀ21.00, p=0.03).

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coming associated with ergoline

derivative 1 was the potential to

induce phospholipidosis, a prob-

lem common to many cationic

amphiphilic compounds acting

at H3R.^[26]Both 1 and 9a in-

duced strong signals in an in

vitro phospholipidosis assay,^[27]

(204% and 218%, respectively,

versus the positive control amio-

darone at 50 mm; Table 2). This

activity was eliminated by trans-

forming the phenyl urea in

1 and 9a to the less lipophilic

acetamide 9b and dimethyl urea

15a.

The aim of the ensuing opti-

mization effort was to identify

derivatives that would maintain

an H3R affinity profile similar to

9b and 15a, while displaying

pharmacokinetic properties pre-

dictive of a short duration of

action in man. Along these lines,

exploration of the side chain

substituents at position 1 (15a–

g) indicated that the dimethyl

(15a) to diethyl (15b) transfor-

mation increased affinity to both

H3R and cardiac hERG channels

(Table 2). Affinity for the latter

was decreased by cyclization to

Scheme 3. Variation of the substituent at position 8. Reagents and conditions: a) Et₃N, Ac₂O, THF, 08C!RT, o/n,

86%; b) CbzCl, Et₃N, DMAP, 08C!RT, 1 h, 71%; c) isopropyl 2-bromoacetate, TBAI, NaOH, CH₂Cl₂, 08C!RT, o/n,

79%; d) H₂, Pd/C THF, RT, o/n, 74%; e) R₁R₂NH, HATU, DIPEA, DMF, RT, 4 h, 78–88%; f) LiBH₄, THF, 08C!RT,

o/n; g) MsCl, Et₃N, CH₂Cl₂, 08C!RT, o/n, 82–88%; h) (R)-2-methylpyrrolidine, MeCN, Et₃N, 508C, o/n, 31–35%; i) (E)-

N’-hydroxyacetimidamide, HATU, DIPEA, RT!1008C, 4 h, 60%; j) isopropyl 2-bromoacetate, NaOH, TBAI, 08C!RT,

o/n, 80%; k) LiAlH₄, THF, ꢀ788C, 10 min, 70%; l) Et₃N, CH₂Cl₂, MsCl, 08C!RT, 4 h, 75%; m) (R)-2-methylpyrrolidine,

MeCN, 608C, o/n, 85%; n) 1. LiBH₄, THF, 08C!RT, o/n; 2. AcOH, 08C, 30 min, 44%; o) SOCl₂, CH₃OH, 08C!RT, 4 h,

83%; p) MsCl, Et₃N, CH₂Cl₂, 08C!RT, 4 h, 88%; q) (R)-2-methylpyrrolidine, MeCN, Et₃N, 508C, o/n, 33%; r) LiBH₄,

THF, 08C!RT, o/n, 40%; s) CH₃I, NaH, THF, RT, o/n, 23%.

a

piperidine ring (15c); this

change had minimal impact on

the interaction with H3R, and it

decreased hERG liability. De-

creasing the pK_aof the piperi-

chemical reactivity of the d-ring double bond common to

both 1 and LSD, which renders them photosensitive and sus-

ceptible to Michael addition.^[25]To circumvent these issues, we

probed the hydrogenation of this double bond, which led to

chemically more stable derivatives in line with published data

for dihydro-LSD (lumi-LSD).^[25]Thus the transformation of 1 to

9a yielded a compound that retains full H3R activity but dis-

plays dramatically diminished affinity for serotonin, adrenergic

and dopamine receptors, as well as for CXCR3. A final short-

dine amine by changing the ring to morpholine (15d) was det-

rimental to affinity, whereas contracting the cycle to the pyrro-

lidine ring (15e) enhanced potency. The best side chain sub-

stituent proved to be 2-methylpyrrolidine, with the R isomer

(15 f) being slightly more potent than the S isomer (15g), and

parallels observations made with other H3R inverse agonists,

such as ABT-239.^[28]

Profiling of 15 f confirmed that it had no significant affinity

for hERG channels and gave no measureable signal in the

Table 1. Affinity profile of lead compound 1, lysergic acid diethylamide (LSD) and 9a as inverse agonist for H3R and agonist at serotonin, adrenergic and

dopamine receptors.

Compd

K_i[nm]

H3R

IC₅₀[nm]

a1A

5HT1A

5HT2A

5HT2B

5HT2C

b1

b2

D1

8700

D2

D3

1

8.3

23

n.t.

18200

>30000

7700

>30000

4900

>30000

30

2800

>30000

>10000

>30000

220

>20000

>30000

140

>20000

>30000

740

10000

>30000

120

>20000

>30000

27

9a

LSD^[a]

>30000

1.1

2.7

5.5

180

[a] Data for LSD from the literature.^[24]Note that the hallucinogenic effects of LSD are attributed mostly to its strong partial agonist effects at 5-HT2A recep-

tors. Specific 5-HT2A agonist drugs are hallucinogenic, and 5-HT2A-specific antagonists largely block the stimulatory activity of LSD.^[32]

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Table 2. In vitro and in vivo properties of H3R inverse agonists.

Compd

hH3R K_i[nm]^[a]

cAMP binding

CXCR3

IC₅₀[mm]

pK_a

hERG

[%]^[b]

PLD

[%]^[c]

logP

MDR1-MDCK^[d]

B–A

Brain

level^[e]

tMeHA^[f]

% induction

A–B

ratio

1

9a

9b

11

0.2

0.7

24

8.3

23

275

97

0.062

11

>100

7.8

7.9

8.0

-

17

13

4

204

218

0

3.7

4.6

1.7

–

23

8

–

15a

15b

15c

15d

15e

15 f

15g

18a

18b

18c

18d

20

178

252

73

129

4307

214

>100

8.0

8.6

8.4

5.9

8.6

8.3

8.7

–

14

76

10

16

14

18

15

2

11

19

20

18

20

16

ꢀ7

8

0

–

0

–

0

–

0

–

1

–

1

0

–

0

2.6

3.5

3.0

2.6

3.3

3.9

2.3

–

3.2

–

2.6

0.7

0.9

1.5

18.0

23.8

7.8

9.9

–

72.2

–

43.1

19.4

24.2

49.9

28.5

32.8

48.7

37.7

–

22.9

–

16.7

28.9

26.1

32.7

1.58

1.38

6.3

3.81

–

5.1

7.6

535

-

5.3

0.2

1.7

0.9

2.0

2.5

0.7

1.0

1.4

0.5

1.2

6.8

–

6.8

>100

95^[f]

–

86^[f]

–

80

13

51

39

9.1

13

21

19

22

102

–

>100

–

30

–

56

59

18

4087

–

69

–

103

64

129

82

–

8.3

–

2.15

8.3

8.2

8.5

8.6

6.3

2.3

2.8

2.6

2.8

3.0

1.1

22

23

24

Bavisant

[a] cAMP and K_ivalues were determined from 2–3 samples; [b] Patch-clamp assay, % inhibition at 10 mm; [c] PLD: phospholipidosis, % of control (amiodar-

one) at 50 mm; [d] Permeability values expressed as P_appꢁ10^ꢀ6cms^ꢀ1in a Madin Darby Canine Kidney (MDCK) cell line transfected with the human multi-

drug resistance (MDR1) gene; [d] Brain concentration in ngg^ꢀ1, 1 h after oral administration of 10 mgkg^ꢀ1; [e] % increase of tele-methylhistamine brain

concentration 1 h after oral administration of 10 mgkg^ꢀ1, compared to vehicle group (n=5 rats per dose group); [f] Measured 0.5 h after oral administra-

tion of 10 mgkg^ꢀ1

.

phospholipidosis assay. Testing 15 f in Madin Darby Canine

Kidney (MDCK) cells transfected with the human multidrug re-

sistance (MDR1) gene showed that it had limited membrane

permeability due to strong Pgp transporter-mediated efflux.

Accordingly, after oral administration of 15 f to rats (10 mgkg^ꢀ1),

brain levels were low, despite rapid absorption and high plasma

levels (2895 ngmL^ꢀ1after 30 min). Nonetheless, 15 f elicited

a very pronounced pharmacodynamic effect, manifested in an

increased brain concentration of the major histamine metabo-

lite tele-methylhistamine (tMeHA), by more than two-fold, two

hours post-dosing. This encouraging result hinted at the possi-

bility that an even more robust interaction on histaminergic

neurons might be achievable with better brain penetrable er-

goline derivatives.

nition element for Pgp transporter-mediated efflux, their re-

placement is a frequently used strategy to decrease efflux and

enhance brain exposure.^[29]As a first step, the amide was re-

placed with a 1,2,4-oxadiazole ring (20), a classical amide bioi-

sostere.^[30]This modification dramatically improved the perme-

ation ratio in the MDCK assay, indicating decreased affinity for

the Pgp transporter. The simple replacement of the pyrrolidin-

ylamide with a methyl ester (22) confirmed that the amide

plays a limited role in terms of binding as well as cAMP inhibi-

tion to H3R and can be removed without loss of affinity. Sur-

prisingly, 22 did not display markedly enhanced brain penetra-

tion relative to 18d (56 vs 30 ngg^ꢀ1, 1 h after 10 mgkg^ꢀ1p.o.).

However, 22 was equally effective in increasing tMeHA levels

in vivo (103% increase vs vehicle group, 1 h after 10 mgkg^ꢀ1

p.o.) as the H3R antagonist clinical candidate, bavisant (82%

increase). This is a striking result, since 22 reaches a brain con-

centration in rats that is 73-fold lower than bavisant (56 vs

4087 ngg^ꢀ1). This difference can’t be explained by a fivefold

ratio in affinity for human H3R, and might indicate that bavi-

sant is functionally weaker at rat as compared with human re-

ceptors. Although ester 22 displays a desirable pharmacokinet-

ic profile (F=33%; T_1/2=0.6 h), it comes at the cost of de-

creased chemical stability—it was completely degraded in so-

lution at pH 7.4 at 808C within two days, thereby precluding

its further development.

Toward this end, the optimized side chain in 15 f was com-

bined with acetamide 9b to give 18a. The latter retained a pro-

file similar to 15 f, but displayed poor transcellular transport

properties. To address this issue, we turned our focus toward

exploring variations of the pyrrolidinyl amide. Replacement of

the pyrrolidine with primary, mono- or di-methylated amides

yielded analogues 18b–d. Although they maintained high af-

finity for H3R, these molecules have low permeability and are

rapidly effluxed. Measurement of the rat brain concentration of

the most potent compound, 18d, one hour after oral adminis-

tration, confirmed its low brain exposure. These amides never-

theless showed a clear pharmacological effect by increasing

tMeHA concentrations in the frontal cortex.

Compared with 22, methyl ether 24 displays very low brain

levels after one hour, contrasting with its high tMeHA induc-

tion. This suggests a strong initial efficacy, followed by a very

rapid clearance of 24, likely resulting from its demethylation to

the active metabolite 23. Interestingly, 23 has a profile similar

We reasoned that modulation of Pgp efflux, leading to even

modest improvements in brain exposure, could lead to corre-

spondingly higher efficacies. Since carbonyl amides are a recog-

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to 22 and was proven to be stable in solution. It has no signifi-

cant cross-reactivity against a panel of 80 ion channels, GPCRs

(including 5HT2A and 5HT2B up to 30 mm, and H4R, 25% in-

hibition at 10 mm), or kinases and proteases (data not shown).

It inhibits the constitutive activity of the human H3R with a K_i

value of 1.2 nm in the GTPgS assay, demonstrating its inverse

agonism. A good permeability (Papp=26ꢁ10^ꢀ6cms^ꢀ1), with

a moderate efflux ratio of 1.8 was observed in Caco2 cells. In

MDCK-MDR1 cells, its passive permeability was similar (Papp=

28ꢁ10^ꢀ6cms^ꢀ1), but its efflux ratio of 6.3 is indicative of a Pgp

substrate. Compound 23 does not significantly bind to hERG

channels (16% at 10 mm) or inhibit common CYP450 enzymes

(<25% at 10 mm, 3A4, 2C9, 2D6, 1A2, 2C19). It displays rapid

absorption (brain T_max=0.5 h after 1 mgkg^ꢀ1p.o.) and a 54%

oral bioavailability in rats, and desirable pharmacodynamics

properties, including a fast elimination half-life (Table 3). The in

vitro clearance values are almost identical in rat and human

liver microsomes (CLint=59 and 58 mLmin^ꢀ1mg), suggesting

that 23 might also have a fast clearance in human.

whose overall profile confirms the possibility of combining de-

sirable selectivity, efficacy and pharmacokinetic properties in

a single molecule. Further optimization of ergoline derivatives

is anticipated to yield analogues with enhanced brain penetra-

tion and correspondingly higher pharmacological efficacy.

Experimental Section

Chemistry

General methods: All reagents and anhydrous solvents were ob-

tained from commercial sources and used as received. ¹H NMR

spectra were obtained on a Bruker 400 MHz instrument in the sol-

vent indicated, with tetramethylsilane (TMS) as an internal stan-

dard. Coupling constants (J) are reported in Hertz (Hz). Liquid chro-

matography/mass spectrometry (LC/MS) analyses were run on an

Agilent 1200 HPLC/6110 SQ system; mobile phase: 10 mm aq

NH₄HCO₃(A)/MeCN (B); gradient: 5% B for 0.2 min, increase to

95% B within 1.2 min, 95% B for 1.5 min, back to 5% B within

0.01 min; flow rate=1.8 mLmin^ꢀ1; XBridge C18 column (4.6ꢁ

50 mm, 3.5 mm), oven temperature: 508C. Compounds were puri-

fied by silica gel chromatography, and reactions were monitored

using thin-layer chromatography (TLC) at 254 nm. Preparative

HPLC conditions: XBridge Prep C18 column (10 mm OBD, 19ꢁ

250 mm); mobile phase: 0.1% aq NH₃(A); MeCN (B); gradient: 25

to 55% B in 10 min; flow rate=30 mLmin^-1.

Table 3. Pharmacokinetic properties of 23 after i.v. administration

(0.5 mgkg^ꢀ1).^[a]

Parameter

Mouse

Rat

Dog

(4aR,8R,9aR)-Methyl 6-(phenylcarbamoyl)-1,4,4a,6,7,8,9,9a-octa-

hydroindolo[1,14-fg]quinoline-8-carboxylate (3): Isocyanatoben-

zene (0.44 g, 3.7 mmol) was added to a solution of 2 (1 g,

3.7 mmol) in CH₂Cl₂(10 mL) at RT, then stirred for 2 h. The mixture

was washed with saturated aq NaHCO₃(10 mL) and brine (10 mL).

The organic layer was dried (anhyd MgSO₄), filtered, and concen-

trated in vacuo to give the title compound as a yellow solid (1.3 g,

90%): LC/MS: t_R=1.83 min, m/z=390.2 [M+H]⁺.

CL [Lh^ꢀ1kg^ꢀ1

]

10.2

2.27

0.21

49.2

2.57

2.78

1.20

196

2.29

3.67

1.44

219

Vd_SS[Lkg^ꢀ1

]

T_1/2,z[h]

AUC [hngmL^ꢀ1

]

1

[a] CL: total blood clearance; Vd_SS: volume of distribution at steady state;

T_1/2,z: apparent terminal half-life; AUC : area under the curve (t=0–1).

1

(4aR,8R,9aR)-N-Phenyl-8-(pyrrolidine-1-carbonyl)-4,4a,7,8,9,9a-

hexahydroindolo[1,14-fg]quinoline-6(1H)-carboxamide (4): To

a solution of 3 (1.3 g, 3.3 mmol) in THF/water (25 mL, v/v=4:1)

was added 1m aq NaOH (10 mL). The reaction mixture was stirred

at RT for 2 h. The mixture was adjusted to pH 5 with 1n aq HCl

and extracted with EtOAc (3ꢁ30 mL). The combined organic layers

were dried (anhyd MgSO₄), filtered and concentrated in vacuo to

give the crude intermediate as a yellow solid (1.1 g, 89%): LC/MS:

t_R=1.42 min, m/z=376.2 [M+1]⁺. This intermediate (750 mg,

2 mmol) was dissolved in CH₂Cl₂(20 mL), and the solution was

treated with Et₃N (606 mg, 6 mmol), 2-(7-azabenzotriazol-1-yl)-

1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (838 mg,

2.2 mmol) and pyrrolidine (296 mg, 4 mmol). The reaction mixture

was then stirred at RT overnight. After quenching with water

(20 mL), the resulting mixture was extracted with EtOAc (3ꢁ

50 mL). The organic layer was dried (anhyd MgSO₄), filtered, and

concentrated in vacuo. The resultant residue was purified by silica

gel chromatography (CH₂Cl₂/MeOH, 20:1) to yield 4 as a white

solid (0.71 g, 83%): LC/MS: t_R=1.85 min, m/z=429.3 [M+H]⁺.

Evaluation of the pharmacokinetic/pharmacodynamic prop-

erties in rats confirmed the efficacy of 23 in increasing brain

tMeHA levels (Figure 4a), with a brain-to-plasma concentration

ratio of 0.15 (Figure 4b). In the key experiment, an orally ad-

ministered single oral dose of 23 (10 mgkg^ꢀ1) to rats achieved

a maximal H3R occupancy in the brain of only 54%, indicative

of properties unlikely to provide robust wake-promoting activi-

ty.^[31]

The optimization of these novel ergoline structures to ach-

ieve higher receptor occupancy, while retaining a short dura-

tion of action, is ongoing in order to identify a candidate with

a suitable profile for clinical development.

Conclusions

The serendipitous discovery that ergoline derivative 1 displayed

H3R inverse agonist properties uncovered a novel chemotype

interacting with this target. However, the lead compound (1) is

encumbered with a number of limitations (e.g., chemical reac-

tivity, phospholipidosis, modest selectivity, and extended termi-

nal half-life), which preclude its development. Accordingly, its

physicochemical and pharmacological profiles were systemati-

cally improved by iterative structural modifications. The most

advanced molecule to emerge from this effort was ergoline 23,

Isopropyl 2-((4aR,8R,9aR)-6-(phenylcarbamoyl)-8-(pyrrolidine-1-

carbonyl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-

yl)acetate (5): A mixture of 4 (428 mg, 1 mmol), NaOH (400 mg,

10 mmol) and tetrabutylammonium iodide (TBAI, 37 mg, 0.1 mmol)

in CH₂Cl₂(20 mL) was cooled to 08C and treated slowly with iso-

propyl 2-bromoacetate (362 mg, 2 mmol). The reaction mixture

was then allowed to warm to RT while stirring overnight. The re-

sulting mixture was diluted with CH₂Cl₂(100 mL), washed with

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Figure 4. Effect of 23 compared with bavisant on tMeHA (panels a,c) and corresponding plasma and brain concentration (panels b,d), 1 h after oral adminis-

tration. a) Dose-dependent increase of brain (frontal cortex) tele-methyl histamine (tMeHA), a major metabolite of histamine, caused by 23; vehicle is 50 mm

citrate buffer. b) Brain (frontal cortex) (ngg^ꢀ1) and plasma (ngmL^ꢀ1) exposure of 23, 1 h after oral administration; a dose-dependent change can also be ob-

served, with plasma exposure many fold greater than brain exposure. c,d) Bavisant also elevated brain tMeHA, with about a fourfold higher exposure in the

brain than in plasma.

water (50 mL), saturated aq NaHCO₃(50 mL) and brine (80 mL).

The organic layer was dried (anhyd MgSO₄), filtered, and concen-

trated in vacuo. The crude product was purified by silica gel chro-

matography (petroleum ether/EtOAc, 2:1) to give the title com-

pound as a yellow oil (475 mg, 90%): LC/MS: t_R=1.95 min, m/z=

529.3 [M+H]⁺.

ic layer was dried (anhyd MgSO₄), filtered, and concentrated in va-

cuo to give crude 7 as a yellow solid (500 mg, 91%), which was

used directly in the next step without further purification: LC/MS:

t_R=1.91 min, m/z=551.2 [M+H]⁺.

((4aR,8R,9aR)-1-(2-(Dimethylamino)ethyl)-1,4,4a,6,7,8,9,9a-octa-

hydroindolo[1,14-fg]quinolin-8-yl)(pyrrolidin-1-yl)methanone (8):

To a solution of 7 (500 mg, 0.91 mmol) in MeCN (8 mL) was added

Me₂NH (648 mg, 8 mmol) under argon. The reaction mixture was

stirred at 508C overnight in a sealed tube. After cooling to RT, the

resulting mixture was diluted with EtOAc (20 mL) and washed with

water (15 mL). The organic layer was dried (anhyd MgSO₄), filtered,

and concentrated in vacuo to give the title compound as a white

solid (260 mg, 68%): LC/MS: t_R=1.54 min, m/z=381.3 [M+H]⁺.

(4aR,8R,9aR)-1-(3-Hydroxyethyl)-N-phenyl-8-(pyrrolidine-1-car-

bonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinoline-6(1H)-

carboxamide (6): LiBH₄(2m in THF, 3 mL, 6 mmol) was slowly

added to a mixture of 5 (800 mg, 1.5 mmol) in THF (15 mL) at 08C

under argon. The reaction mixture was stirred at 08C for 3 h, and

then at RT overnight. The resulting mixture was quenched by care-

ful addition of water (5 mL), stirred at RT for 30 min, and then ex-

tracted with EtOAc (3ꢁ30 mL). The combined organic layers were

dried (anhyd MgSO₄), filtered, and concentrated in vacuo to give

the title compound as a yellow solid (450 mg, 63.5%), which was

used without further purification: LC/MS: t_R=1.82 min, m/z=473.3

[M+H]⁺.

(4aR,8R,9aR)-1-(2-(Dimethylamino)ethyl)-N-phenyl-8-(pyrrolidine-

1-carbonyl)-

4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinoline-

6(1H)-carboxamide (9a): To a solution of 8 (380 mg, 1 mmol) in

CH₂Cl₂(10 mL) was added isocyanatobenzene (119 mg, 1 mmol) at

RT. The reaction mixture was stirred at RT for 12 h. The mixture

was diluted with CH₂Cl₂(100 mL) and washed with saturated aq

NaHCO₃(10 mL) and brine (10 mL). The organic layer was dried

(anhyd MgSO₄), filtered and concentrated in vacuo to give a crude

product, which was purified by preparative HPLC to afford the title

compound as a white solid (80 mg, 16%): ¹H NMR (400 MHz,

CDCl₃): d=9.87 (s, 1H), 7.45 (dd, J=8.6, 1.0 Hz, 2H), 7.27 (d, J=

2.0 Hz, 1H), 7.23 (s, 1H), 7.18–7.07 (m, 2H), 6.93 (t, J=7.3 Hz, 1H),

6.86–6.70 (m, 2H), 4.19 (dd, J=9.9, 5.8 Hz, 3H), 3.98 (td, J=11.1,

2-((4aR,8R,9aR)-6-(Phenylcarbamoyl)-8-(pyrrolidine-1-carbonyl)-

4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl

methanesulfonate (7): A mixture of 6 (472 mg, 1 mmol) and Et₃N

(303 mg, 3 mmol) in CH₂Cl₂(15 mL) was cooled to 08C. MsCl

(170 mg, 1.5 mmol) in CH₂Cl₂(5 mL) was added slowly, and the re-

sulting mixture was stirred at RT for 4 h. The organic solvent was

then removed in vacuo. The residue was diluted with CH₂Cl₂

(20 mL) and washed with saturated aq NaHCO₃(10 mL). The organ-

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3.6 Hz, 1H), 3.70–3.44 (m, 6H), 3.21 (t, J=11.8 Hz, 1H), 3.08–2.88

(m, 3H), 2.71 (dt, J=10.5, 5.4 Hz, 2H), 2.31 (d, J=2.2 Hz, 6H), 2.13–

1.92 (m, 4H), 1.58–1.48 ppm (m, 1H); LC/MS: t_R=1.88 min, m/z=

500.4 [M+H]⁺.

Isopropyl 2-((4aR,8R,9aR)-6-(dimethylcarbamoyl)-8-(pyrrolidine-

1-carbonyl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-

1(4H)-yl)acetate: To a solution of 14 (1.9 g, 4.9 mmol) in CH₂Cl₂

(160 mL) was slowly added NaOH (1.96 g, 49 mmol), TBAI (0.36 g,

0.98 mmol) and isopropyl 2-bromoacetate (3.55 g, 19.6 mmol) at

08C. The reaction mixture was then stirred at RT overnight. The

precipitate was filtered out, and the filtrate was sequentially

washed with water (50 mL), saturated aq NaHCO₃(50 mL) and

brine (80 mL). The organic layer was dried (anhyd MgSO₄), filtered

and concentrated in vacuo to give the title compound as a yellow

oil (1.6 g, 67%): LC/MS: t_R=1.91 min, m/z=481.2 [M+H]⁺.

1-((4aR,8R,9aR)-1-(2-(Dimethylamino)ethyl)-8-(pyrrolidine-1-car-

bonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-6(1H)-yl)e-

thanone (9b): A mixture of 8 (75 mg, 0.2 mmol) and N,N-diisopro-

pylethylamine (DIPEA, 52 mg, 0.4 mmol) in CH₂Cl₂(20 mL) was

cooled to 08C. The mixture was slowly treated with AcCl (19 mg,

0.24 mmol). The reaction mixture was stirred at 08C to RT for 4 h.

The solvent was removed in vacuo, and the residue was purified

by preparative HPLC to give the title compound as a yellow oil

(31 mg, 37%): ¹H NMR (400 MHz, CDCl₃): d=7.16–7.10 (m, 2H),

6.81–6.83 (m, 2H), 4.17 (t, J=7.2 Hz, 3H), 4.08–4.01 (m, 1H), 3.59–

3.55 (m, 2H), 3.51–3.48 (m, 3H), 3.39–3.35 (m, 1H), 3.23 (t, J=

11.2 Hz, 1H), 3.03–2.91 (m, 2H), 2.79–2.67 (m, 3H), 2.30 (s, 6H),

2.27 (s, 3H), 2.05–1.89 ppm (m, 5H); LC/MS: t_R=1.75 min, m/z=

423.3 [M+H]⁺.

(4aR,8R,9aR)-1-(2-Hydroxyethyl)-N,N-dimethyl-8-(pyrrolidine-1-

carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinoline-6(1H)-

carboxamide: To a solution of 2-((4aR,8R,9aR)-6-(dimethylcarbamo-

yl)-8-(pyrrolidine-1-carbonyl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]-

quinolin-1(4H)-yl)acetate (2.1 g, 4.36 mmol) in THF (150 mL) was

added LiBH₄(2m in THF, 6.5 mL, 13 mmol) at 0^oC under argon. The

reaction mixture was stirred at 08C for 3 h, followed by RT over-

night. After cooling to 08C, AcOH (6 mL) was added slowly. The re-

sulting mixture was stirred at 08C for 30 min, then adjusted to

pH 8 with saturated aq NaHCO₃(40 mL) and extracted with EtOAc

(2ꢁ100 mL). The combined organic layers were washed with brine

(30 mL), dried (anhyd MgSO₄), filtered and concentrated in vacuo

to give the title compound as a brown oil (1.5 g, 81%): LC/MS: t_R=

1.71 min, m/z=425.1 [M+H]⁺.

(4aR,8R,9aR)-4-Nitrophenyl 1-(2-(Dimethylamino)ethyl)-8-(pyrro-

lidine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quino-

line-6(1H)-carboxylate (10): To a mixture of 8 (150 mg, 0.39 mmol)

in CH₂Cl₂(20 mL) was slowly added DIPEA (100 mg, 0.78 mmol)

and 4-nitrophenyl chloroformate (96 mg, 0.48 mmol) at 08C. The

resulting reaction mixture was stirred at RT overnight. The solvent

was removed in vacuo, and the crude product was purified by

prep-TLC (EtOAc/MeOH, 10:3, R_f=0.4) to give the title compound

as a yellow oil (160 mg, 74%).

2-((4aR,8R,9aR)-6-(Dimethylcarbamoyl)-8-(pyrrolidine-1-carbon-

yl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl

methanesulfonate: A mixture of (4aR,8R,9aR)-1-(2-hydroxyethyl)-

N,N-dimethyl-8-(pyrrolidine-1-carbonyl)-4,4a,7,8,9,9a-hexahydro-

indolo[1,14-fg]quinoline-6(1H)-carboxamide (0.9 g, 2.1 mmol) and

Et₃N (636 mg, 6.3 mmol) in CH₂Cl₂(50 mL) was cooled to 08C. MsCl

(460 mg, 3.2 mmol) in CH₂Cl₂(5 mL) was slowly added to the

cooled solution. The reaction mixture was stirred at 08C for 3 h,

followed by RT overnight. The solvent was removed, and the resi-

due was purified by silica gel chromatography (EtOAc/petroleum

ether, 2:1) to give the title compound as a white solid (580 mg,

55%): LC/MS: t_R=1.79 min, m/z=503.2 [M+H]⁺.

(4aR,8R,9aR)-1-(2-(Dimethylamino)ethyl)-N-methyl-8-(pyrroli-

dine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quino-

line-6(1H)-carboxamide (11): To

a mixture of 10 (150 mg,

0.28 mmol) and DIPEA (72 mg, 0.56 mmol)) in CH₂Cl₂(20 mL) was

added MeNH₂(0.28 mL, 0.56 mmol) and 4-dimethylaminopyridine

(DMAP, 7 mg, 0.056 mmol). The reaction mixture was stirred at RT

overnight. The solvent was removed in vacuo, and the residue pu-

rified by preparative HPLC to give the title compound as yellow oil

(26 mg, 22%): ¹H NMR (400 MHz, CD₃OD): d=7.18 (d, J=8.4 Hz,

1H), 7.12–7.08 (m, 1H), 6.91 (s, 1H), 6.85 (d, J=6.8 Hz, 1H), 4.59 (s,

1H), 4.25 (t, J=7.2 Hz, 2H), 3.89–3.84 (m, 1H), 3.68–3.61 (m, 3H),

3.56–3.41 (m, 3H), 3.29–3.24 (m, 2H), 3.17–3.04 (m, 2H), 2.83–2.72

(m, 6H), 2.30 (s, 6H), 2.08–2.01 (m, 2H), 1.96–1.90 (m, 2H), 1.70–

1.61 ppm (m, 1H); LC/MS: t_R=1.53 min, m/z=438.2 [M+H]⁺.

(4aR,8R,9aR)-1-(2-(Dimethylamino)ethyl)-N,N-dimethyl-8-(pyrroli-

dine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quino-

line-6(1H)-carboxamide (15a): To a solution of 2-((4aR,8R,9aR)-6-

(dimethylcarbamoyl)-8-(pyrrolidine-1-carbonyl)-4a,6,7,8,9,9a-hexa-

hydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl

methanesulfonate

((4aR,8R,9aR)-1,4,4a,6,7,8,9,9a-Octahydroindolo[1,14-fg]quinolin-

8-yl)(pyrrolidin-1-yl)methanone (13): To a solution of 12 (2 g,

7.8 mmol) in DMF (10 mL) was added pyrrolidine (3.2 mL,

39 mmol), HATU (4.4 g, 11.7 mmol) and Et₃N (3.2 mL, 23.4 mmol).

The reaction mixture was stirred at RT overnight, then diluted with

water (15 mL) and extracted with EtOAc (3ꢁ50 mL). The combined

organic layers were washed with brine (3ꢁ50 mL), dried (anhyd

MgSO₄), filtered and concentrated to give the title compound as

a brown solid (1.8 g, 76%), which was used without further purifi-

cation: LC/MS: t_R=1.67 min, m/z=310.2 [M+H]⁺.

(75 mg, 0.15 mmol) in MeCN (10 mL) was added Me₂NH (1.36 g,

16 mmol) under argon. The reaction mixture was stirred at 508C

overnight. After cooling to RT, the organic solvent was removed in

vacuo to yield the crude product. The residue was purified by

preparative HPLC to give 15a as a white solid (16 mg, 24%):

¹H NMR (400 MHz, CDCl₃): d=7.17–7.11 (m, 2H), 6.88–6.87 (m, 1H),

6.75 (s, 1H), 4.19 (t, J=7.2 Hz, 2H), 3.64–3.40 (m, 6H), 3.18–3.12 (m,

4H), 2.98 (s, 6H), 2.88–2.69 (m, 4H), 2.31 (s, 6H), 2.05–1.86 ppm (m,

5H); LC/MS: t_R=1.50 min, m/z=452.3 [M+H]⁺.

(4aR,8R,9aR)-N,N-dimethyl-8-(pyrrolidine-1-carbonyl)-4,4a,7,8,9,

9a-hexahydroindolo[1,14-fg]quinoline-6(1H)-carboxamide (14):

To a solution of 13 (1.7 g, 5.5 mmol) in MeCN (120 mL) was added

dimethylcarbamic chloride (0.65 g, 6.1 mmol), DIPEA (2.1 g,

16.5 mmol) and DMAP (0.13 g, 1.1 mmol). The reaction mixture

was stirred at 658C for 4 h, then cooled to RT, diluted with CH₂Cl₂

(50 mL) and washed with water (50 mL) and brine (50 mL). The or-

ganic layer was dried (anhyd MgSO₄), filtered and concentrated to

yield the title compound as a yellow solid (1.5 g, 71%): LC/MS: t_R=

1.80 min, m/z=381.1 [M+H]⁺.

(4aR,8R,9aR)-1-(2-(Diethylamino)ethyl)-N,N-dimethyl-8-(pyrroli-

dine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quino-

line-6(1H)-carboxamide(15b): Reaction of 2-((4aR,8R,9aR)-6-(dime-

thylcarbamoyl)-8-(pyrrolidine-1-carbonyl)-4a,6,7,8,9,9a-hexahydro-

indolo[1,14-fg]quinolin-1(4H)-yl)ethyl methanesulfonate and Et₂NH

in a manner similar to 15a but using 200 mg starting material

(0.4 mmol) gave 15b as a white solid (95 mg, 50%): ¹H NMR

(400 MHz, CDCl₃): d=7.18–7.16 (d, 2H), 6.88–6.87 (m, 1H), 6.76 (s,

1H), 4.17 (t, J=7.2 Hz, 2H), 3.62–3.41 (m, 5H), 3.20–3.14 (m, 4H),

3.00 (s, 6H), 2.87–2.81 (m, 4H), 2.62 (q, J=7.2 Hz, 4H), 2.05–2.02

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(m, 2H), 1.93–1.92 (m, 2H), 1.85–1.82 (m, 2H), 1.07–1.04 ppm (t, J=

7.2 Hz, 6H); LC/MS: t_R=1.97 min, m/z=480.3 [M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]-

quinoline-8-carboxylic acid (16): To

a solution of 12 (5 g,

19.5 mmol) in THF (200 mL) at 08C was slowly added Et₃N (43 mL,

156 mmol) and Ac₂O (18.5 mL, 97.6 mmol). The reaction mixture

was stirred at RT overnight. The precipitate was collected by filtra-

tion to yield crude 16 as a white solid (5 g, 86%), which was used

without further purification: LC/MS: t_R=1.46 min, m/z=299.1

[M+H]⁺.

(4aR,8R,9aR)-N,N-dimethyl-1-(2-(piperidin-1-yl)ethyl)-8-(pyrroli-

dine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quino-

line-6(1H)-carboxamide (15c): Reaction of 2-((4aR,8R,9aR)-6-(dime-

thylcarbamoyl)-8-(pyrrolidine-1-carbonyl)-4a,6,7,8,9,9a-hexahydro-

indolo[1,14-fg]quinolin-1(4H)-yl)ethyl methanesulfonate and piperi-

dine in a manner similar to 15a gave 15c as a white solid (31 mg,

42%): ¹H NMR (400 MHz, CDCl₃): d=7.18–7.16 (m, 2H), 6.88–6.87

(m, 1H), 6.77 (s, 1H), 4.21 (t, J=7.6 Hz, 2H), 3.60–3.41 (m, 5H),

3.20–3.14 (m, 4H), 3.00 (s, 6H), 2.87–2.70 (m, 4H), 2.48 (br s, 4H),

2.01–1.80 (m, 6H), 1.78–1.65 ppm (m, 6H); LC/MS: t_R=1.99 min, m/

z=492.3 [M+1]⁺.

(4aR,8R,9aR)-Benzyl-6-acetyl-1,4,4a,6,7,8,9,9a-octahydroindolo-

[1,14-fg]quinoline-8-carboxylate: To

a mixture of 16 (5 g,

16.8 mmol) and Et₃N (7.5 mL, 50.3 mmol) in CH₂Cl₂(150 mL) was

added CbzCl (3.8 mL, 26.8 mmol) slowly at 0^oC. After stirring for

5 min, DMAP (0.2 g, 1.68 mmol) was added. The reaction mixture

was stirred at RT for 1 h, and then the solvent was removed in va-

cuo. The residue was diluted with CH₂Cl₂(100 mL) and washed

with saturated aq NaHCO₃(30 mL) and brine (30 mL). The organic

layer was dried (anhyd MgSO₄), filtered and concentrated to give

the crude product, which was used directly in the next step with-

out further purification (4.6 g, 71%): LC/MS: t_R=1.92 min, m/z=

389.2 [M+1]⁺.

(4aR,8R,9aR)-N,N-dimethyl-1-(2-morpholinoethyl)-8-(pyrrolidine-

1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinoline-

6(1H)-carboxamide (15d): Reaction of 2-((4aR,8R,9aR)-6-(dimethyl-

carbamoyl)-8-(pyrrolidine-1-carbonyl)-4a,6,7,8,9,9a-hexahydro-

indolo[1,14-fg]quinolin-1(4H)-yl)ethyl methanesulfonate and mor-

pholine in a manner similar to 15a gave 15d as a white solid

(46 mg, 62%): ¹H NMR (400 MHz, CD₃OD): d=7.19–7.09 (m, 2H),

6.87–6.86 (m, 2H), 4.24 (t, J=6.8 Hz, 2H), 3.70–3.64 (m, 6H), 3.47–

3.42 (m, 3H), 3.22–3.18 (m, 2H), 3.04–2.96 (m, 10H), 2.74–2.71 (m,

3H), 2.50–2.48 (m, 4H), 2.06–2.03 (m, 2H), 1.95–1.92 (m, 2H), 1.67–

1.70 ppm (m, 1H); LC/MS: t_R=1.19 min, m/z=494.1 [M+H]⁺.

(4aR,8R,9aR)-Benzyl 6-acetyl-1-(2-isopropoxy-2-oxoethyl)-1,4,4a,

6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxylate: To

a

solution

of

(4aR,8R,9aR)-benzyl-6-acetyl-1,4,4a,6,7,8,9,9a-

octahydroindolo[1,14-fg]quinoline-8-carboxylate (1.9 g, 4.9 mmol)

in CH₂Cl₂(160 mL) was slowly added NaOH (1.96 g, 49 mmol) and

TBAI (0.36 g, 0.98 mmol), followed by isopropyl 2-bromoacetate

(3.55 g, 19.6 mmol) at 0^oC. The resulting reaction mixture was

stirred at RT overnight. After addition of ice water (50 mL), the or-

ganic layer was separated, dried (anhyd MgSO₄), filtered and con-

centrated in vacuo to yield the crude product. The residue was pu-

rified by silica gel chromatography (EtOAc/petroleum ether, 1:5) to

give the title compound as a yellow solid (1.9 g, 79%): LC/MS: t_R=

2.05 min, m/z=489.2 [M+H]⁺.

(4aR,8R,9aR)-N,N-Dimethyl-1-(2-(pyrrolidin-1-yl)ethyl)-8-(pyrroli-

dine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quino-

line-6(1H)-carboxamide (15e): Reaction of 2-((4aR,8R,9aR)-6-(dime-

thylcarbamoyl)-8-(pyrrolidine-1-carbonyl)-4a,6,7,8,9,9a-hexahydro-

indolo[1,14-fg]quinolin-1(4H)-yl)ethyl methanesulfonate and pyrroli-

dine in a manner similar to 15a gave 15e as a white solid (23 mg,

32%): ¹H NMR (400 MHz, CDCl₃): d=7.22–7.06 (m, 2H), 6.87(dd,

J=4.7, 1.9 Hz, 1H), 6.75 (s, 1H), 4.23 (t, J=7.2 Hz, 2H), 3.61–3.41

(m, 5H), 3.25–3.08 (m, 4H), 2.99 (s, 6H), 2.91–2.76 (m, 4H), 2.57 (m,

4H), 2.12–1.78 ppm (m, 10H); LC/MS: t_R=1.88 min, m/z=478.3

[M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-1-(2-isopropoxy-2-oxoethyl)-1,4,4a,6,7,8,

9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxylic acid (17): To

a solution of (4aR,8R,9aR)-benzyl-6-acetyl-1-(2-isopropoxy-2-oxoeth-

yl)-1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxylate

(0.93 g, 1.9 mmol) in THF (300 mL) was added 10% Pd/C (100 mg,

0.19 mmol). The reaction mixture was stirred under an H₂atmos-

phere at RT overnight. The resulting mixture was filtered through

Celite, and the filter cake was washed with THF (50 mL). The filtrate

was concentrated in vacuo to give the crude product, which was

washed with hexane/EtOAc (50 mL, v/v=30:1) to give the title

compound as a white solid (0.56 g, 74%): LC/MS: t_R=1.56 min,

m/z=399.1 [M+H]⁺.

(4aR,8R,9aR)-N,N-dimethyl-1-(2-((R)-2-methylpyrrolidin-1-yl)eth-

yl)-8-(pyrrolidine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo-

[1,14-fg]quinoline-6(1H)-carboxamide

(15 f):

Reaction

of

2-((4aR,8R,9aR)-6-(dimethylcarbamoyl)-8-(pyrrolidine-1-carbonyl)-

4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl metha-

nesulfonate and (R)-2-methylpyrrolidine in a manner similar to 15a

gave 15 f as a white solid (43 mg, 58%): ¹H NMR (400 MHz,

CD₃OD): d=7.10–7.18 (m, 2H), 6.88–6.86 (m, 2H), 4.25–4.22 (m,

2H), 3.70–3.60 (m, 3H), 3.45–3.32 (m, 3H), 3.32–3.29 (m, 5H), 3.22–

3.14 (m, 4H), 2.97–2.93 (m, 4H), 2.45–2.52 (m, 2H), 2.22–2.19 (m,

1H), 2.06–2.03 (m, 5H), 1.86–1.76 (m, 3H), 1.19–1.40 ppm (m, 5H);

LC/MS: t_R=1.98 min, m/z=492.3 [M+H]⁺.

Isopropyl

2-((4aR,8R,9aR)-6-acetyl-8-carbamoyl-4a,6,7,8,9,9a-

hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)acetate: To a solution

of 17 (239 mg, 0.6 mmol) in DMF (10 mL) was added NH₄Cl

(255 mg, 4.8 mmol), DIPEA (744 mg, 6 mmol) and HATU (456 mg,

1.2 mmol). The reaction mixture was stirred at RT for 4 h. After di-

luting with water (10 mL), the resulting mixture was extracted with

CH₂Cl₂(3ꢁ10 mL). The combined layers were dried (anhyd MgSO₄),

filtered and concentrated in vacuo to yield the crude product as

a yellow oil (200 mg, 84%), which was used directly in the next

step without further purification: LC/MS: t_R=1.73 min, m/z=398.2

[M+H]⁺.

(4aR,8R,9aR)-N,N-Dimethyl-1-(2-((S)-2-methylpyrrolidin-1-yl)eth-

yl)-8-(pyrrolidine-1-carbonyl)-4,4a,7,8,9,9a-hexahydroindolo-

[1,14-fg]quinoline-6(1H)-carboxamide

(15g):

Reaction

of

2-((4aR,8R,9aR)-6-(dimethylcarbamoyl)-8-(pyrrolidine-1-carbonyl)-

4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl metha-

nesulfonate and (S)-2-methylpyrrolidine in a manner similar to 15a

but using 150 mg starting material (0.3 mmol) gave 15g as a white

solid (85 mg, 58%): ¹H NMR(400 MHz, CD₃OD): d=7.20–7.10 (m,

2H), 6.88–6.86 (m, 2H), 4.26–4.22 (m, 2H), 3.75–3.62 (m, 3H), 3.51–

3.40 (m, 3H), 3.30–3.28 (m, 5H), 3.20–3.08 (m, 4H), 3.02–2.78 (m,

4H), 2.50–2.35 (m, 2H), 2.22–2.19 (m, 1H), 2.06–1.85 (m, 5H), 1.72–

1.60 (m, 3H), 1.38–1.22 (m, 1H), 1.08 (t, J=7.0 Hz, 1H), 1.05 ppm

(d, J=6.0 Hz, 3H); LC/MS: t_R=1.98 min, m/z=492.3 [M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-1-(2-hydroxyethyl)-1,4,4a,6,7,8,9,9a-octa-

hydroindolo[1,14-fg]quinoline-8-carboxamide: To a solution of

isopropyl

2-((4aR,8R,9aR)-6-acetyl-8-carbamoyl-4a,6,7,8,9,9a-hexa-

hydroindolo[1,14-fg]quinolin-1(4H)-yl)acetate (1.65 g, 4.15 mmol) in

THF (150 mL) was slowly added LiBH₄(2m in THF, 6.5 mL,

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13 mmol) at 08C. The reaction mixture was stirred at RT overnight.

The reaction was then re-cooled to 08C, AcOH (60 mL) was slowly

added, and the resulting mixture was stirred for an additional

30 min. After dilution with EtOAc (150 mL), the pH was adjusted to

8 with saturated aq NaHCO₃(40 mL), followed by washing with

brine (30 mL). The organic layer was dried (anhyd MgSO₄), filtered,

and concentrated in vacuo to give the title compound as a brown

oil (0.9 g, 64%), which was used without further purification:

LC/MS: t_R=1.63 min, m/z=342.3 [M+H]⁺.

After cooling to 08C, AcOH (6 mL) was slowly added to the mix-

ture, which was stirred at 08C for an additional 30 min. The pH of

the resulting mixture was adjusted to 8 with saturated aq NaHCO₃

(40 mL), and extracted with EtOAc (2ꢁ100 mL). The combined or-

ganic layers were washed with brine (30 mL), dried (anhyd MgSO₄),

filtered and concentrated in vacuo to give the title compound as

a brown oil (481 mg, 31%): LC/MS: t_R=1.22 min, m/z=356.2

[M+H]⁺.

2-((4aR,8R,9aR)-6-Acetyl-8-(methylcarbamoyl)-4a,6,7,8,9,9a-hexa-

hydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl methanesulfonate: A

2-((4aR,8R,9aR)-6-Acetyl-8-carbamoyl-4a,6,7,8,9,9a-hexahydro-

indolo[1,14-fg]quinolin-1(4H)-yl)ethyl

methanesulfonate:

solution

of

(4aR,8R,9aR)-6-acetyl-1-(2-hydroxyethyl)-N-methyl-

(4aR,8R,9aR)-6-acetyl-1-(2-hydroxyethyl)-1,4,4a,6,7,8,9,9a-octahydro-

indolo[1,14-fg]quinoline-8-carboxamide (716 mg, 2.1 mmol) and

Et₃N (636 mg, 6.3 mmol) were combined in CH₂Cl₂(50 mL) and

cooled to 0^oC. MsCl (460 mg, 3.2 mmol) in CH₂Cl₂(5 mL) was

slowly added to the mixture, which was stirred at 08C for 3 h, and

then overnight at RT. The organic solvent was removed in vacuo to

give the title compound as a yellow oil (0.7 g, 82%), which was

used directly in the next step without further purification. LC/MS:

t_R=1.03 min, m/z=420.2 [M+H]⁺.

1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxamide

(0.20 g, 0.56 mmol) and Et₃N (0.22 mL, 1.56 mmol) in CH₂Cl₂

(25 mL) was cooled to 08C. MsCl (89 mg, 0.78 mmol) in CH₂Cl₂

(5 mL) was slowly added, and the resulting reaction mixture was

warmed to RT and stirred for 4 h. The solvent was removed in va-

cuo to yield the crude product, which was diluted with CH₂Cl₂

(50 mL). The organic layer was washed with saturated aq NaHCO₃

(30 mL), dried (anhyd MgSO₄), filtered and concentrated in vacuo

to give the title compound as a yellow solid (0.21 g, 88%): LC/MS:

t_R=1.64 min, m/z=433.9 [M+H]⁺.

1-((4aR,8R,9aR)-1-(2-((R)-2-Methylpyrrolidin-1-yl)ethyl)-8-(pyrroli-

dine-1-carbonyl)-,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-

6(1H)-yl)ethanone (18a): The title compound was prepared as

(4aR,8R,9aR)-6-Acetyl-N-methyl-1-(2-((R)-2-methylpyrrolidin-1-

yl)ethyl)-1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-

carboxamide (18c): To a mixture of 2-((4aR,8R,9aR)-6-acetyl-8-

(methylcarbamoyl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-

1(4H)-yl)ethyl methanesulfonate (147 mg, 0.34 mmol) and Et₃N

(0.22 mL, 1.6 mmol) in MeCN (15 mL) was added (R)-2-methylpyrro-

lidine (275 mg, 3.2 mmol) under argon. The reaction mixture was

stirred at 508C overnight. The solvent was removed in vacuo to

yield the crude product, which was purified by preparative HPLC

to give 18c as a white solid (47 mg, 33%): ¹H NMR (400 MHz,

CDCl₃): d=7.20–7.14 (m, 2H), 6.88–6.86 (m, 2H), 4.62–4.59 (m,1H),

4.25 (t, J=7.6 Hz, 2H), 3.88 (m, 1H), 3.29–3.17 (m, 4H), 3.08–2.92

(m, 3H), 2.88–2.84 (d,3H), 2.55–2.37 (m, 2H), 2.26–2.19 (m, 4H),

1.78–1.70 (m, 5H), 1.50–1.40 (m, 2H), 1.11–1.10 ppm (d, J=6.0 Hz,

3H); LC/MS t_R=1.77 min, m/z=423.3 [M+H]⁺.

1

a white solid (100 mg, 35%) in a manner similar to 18d: H NMR

(400 MHz, CDCl₃): d=7.20–7.04 (m, 2H), 6.83 (s, 2H), 4.32–3.92 (m,

4H), 3.69–3.44 (m, 5H), 3.38 (d, J=11.9 Hz, 1H), 3.30–3.11 (m, 3H),

3.08–2.89 (m, 2H), 2.81–2.70 (m, 1H), 2.58–2.45 (m, 2H), 2.33–2.26

(m, 4H), 2.08–1.74 (m, 8H), 1.52–1.40 (m, 1H), 1.12 ppm (d, J=

5.9 Hz, 3H); LC/MS: t_R=1.74 min, m/z=463.1 [M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-1-(2-((R)-2-methylpyrrolidin-1-yl)ethyl)-

1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxa-

mide (18b): To a solution of 2-((4aR,8R,9aR)-6-acetyl-8-carbamoyl-

4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl metha-

nesulfonate (461 mg, 1.1 mmol) in MeCN (10 mL) was added (R)-2-

methylpyrrolidine (468 mg, 5.5 mmol). The reaction mixture was

stirred at 508C overnight. The reaction mixture was cooled, and

the solvent was removed in vacuo to yield the crude product. Pu-

rification by preparative HPLC yielded 18b as a white solid

(150 mg, 33%): ¹H NMR (400 MHz, CD₃OD): d=7.21 (d, J=8.4 Hz,

1H), 7.12 (t, J=7.2 Hz, 1H), 6.94 (s, 1H), 6.86 (d, J=7.8 Hz, 1H),

4.59 (m, 1H), 4.32–4.26 (m, 2H), 3.99–3.98 (m, 1H), 3.60 (m, 1H),

3.19–3.15 (m, 2H), 2.97–2.84 (m, 3H), 2.57–2.46 (m, 2H), 2.30–2.22

(m, 4H), 1.97–1.94 (m, 1H), 1.81–1.77 (m, 2H), 1.64–1.61 (m, 1H),

1.60–1.46 (m, 2H), 1.14–1.12 (d, J=6.4 Hz, 3H), 0.96–0.92 ppm (m,

1H); LC/MS: t_R=1.71 min, m/z=409.3 [M+H]⁺.

Isopropyl 2-((4aR,8R,9aR)-6-acetyl-8-(dimethylcarbamoyl)-4a,6,7,

8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)acetate: To a so-

lution of 17 (239 mg, 0.6 mmol) in DMF (10 mL) was added

Me₂NH·HCl (154 mg, 1.9 mmol), DIPEA (300 mg, 2.4 mmol) and

HATU (456 mg, 1.2 mmol). The reaction mixture was stirred at RT

for 4 h, then diluted with water (10 mL) and extracted with CH₂Cl₂

(3ꢁ10 mL). The combined organic layers were dried (anhyd

MgSO₄), filtered, and concentrated in vacuo to yield the crude

product as a yellow oil (200 mg, 78%), which was used directly in

the next step without further purification: LC/MS: t_R=1.87 min,

m/z=426.2 [M+H]⁺.

Isopropyl 2-((4aR,8R,9aR)-6-acetyl-8-(methylcarbamoyl)-4a,6,7,8,

9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)acetate: To a solu-

tion of 17 (0.24 g, 0.6 mmol) in DMF (10 mL) was added MeNH₂

(74 mg, 2.4 mmol), DIPEA (0.3 g, 2.4 mmol) and HATU (0.46 g,

1.2 mmol). The reaction mixture was stirred at RT for 4 h. After di-

luting with water (10 mL), the resulting mixture was extracted with

CH₂Cl₂(3ꢁ20 mL). The combined organic layers were dried (anhyd

MgSO₄), filtered and concentrated in vacuo to give the crude prod-

uct (217 mg, 88%), which was used directly in the next step with-

out further purification: LC/MS: t_R=1.78 min, m/z=412.3 [M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-1-(2-hydroxyethyl)-N,N-dimethyl-1,4,4a,6,7,

8,9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxamide:

To

a mixture of isopropyl 2-((4aR,8R,9aR)-6-acetyl-8-(dimethylcarbamo-

yl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl) acetate

(0.89 g, 2.1 mmol) in THF (80 mL) was slowly added LiBH₄(2 m in

THF, 3.1 mL, 6.2 mmol) at 08C under argon. The reaction mixture

was stirred at RT overnight, then cooled to 0^oC before slowly

adding AcOH (30 mL). The mixture was then stirred for an addi-

tional 30 min, and then adjusted to pH 8 with saturated aq

NaHCO₃(40 mL). The mixture was extracted with EtOAc (2ꢁ80 mL),

and the combined organic layers were dried (anhyd MgSO₄), fil-

tered and concentrated in vacuo to give the title product as

a brown oil (319 mg, 41 %), which was used in the next step with-

out further purification: LC/MS: t_R=1.62 min, m/z=370.1 [M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-1-(2-hydroxyethyl)-N-methyl-1,4,4a,6,7,8,

9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxamide: To a solu-

tion of isopropyl 2-((4aR,8R,9aR)-6-acetyl-8-(methylcarbamoyl)-

4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)acetate

(1.65 g, 4.36 mmol) in THF (150 mL) was added LiBH₄(2m in THF,

6.5 mL, 13 mmol). The reaction mixture was stirred at RT overnight.

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2-((4aR,8R,9aR)-6-Acetyl-8-(dimethylcarbamoyl)-4a,6,7,8,9,9a-

hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl methanesulfo-

nate: A solution of MsCl (460 mg, 3.2 mmol) in CH₂Cl₂(5 mL) was

slowly added to a mixture of (4aR,8R,9aR)-6-acetyl-1-(2-hydroxyeth-

yl)-N,N-dimethyl-1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-

8-carboxamide (775 mg, 2.1 mmol) and Et₃N (636 mg, 6.3 mmol) in

CH₂Cl₂(50 mL) at 08C. The reaction mixture was stirred at 08C for

3 h, then warmed to RT and stirred overnight. The organic solvent

was removed in vacuo to give the crude product, which was puri-

fied by silica gel chromatography (EtOAc/petroleum ether, 2:1) to

afford the title compound as a white solid (790 mg, 84%): LC/MS:

t_R=1.69 min, m/z=448.1 [M+H]⁺.

organic extracts were dried (anhyd MgSO₄), filtered, and concen-

trated in vacuo to give the title compound as a yellow solid

(560 mg, 70%): LC/MS: t_R=1.59 min, m/z=381.1 [M+H]⁺.

2-((4aR,8R,9aR)-6-Acetyl-8-(3-methyl-1,2,1-oxadiazol-2-yl)-4a,6,7,

8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl methane-

sulfonate:

A mixture of 1-((4aR,8R,9aR)-1-(2-hydroxyethyl)-8-(3-

methyl-1,2,1-oxadiazol-2-yl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]-

quinolin-6(1H)-yl)ethanone (0.18 g, 0.52 mmol) and Et₃N (0.22 mL,

1.56 mmol) in CH₂Cl₂(25 mL) was cooled to 08C, and MsCl (89 mg,

0.78 mmol) in CH₂Cl₂(5 mL) was slowly added to the mixture. After

stirring at RT for 4 h, the reaction was quenched with saturated aq

NaHCO₃(30 mL). The aqueous and organic phases were separated,

and the organic layer was dried (anhyd MgSO₄), filtered, and con-

centrated in vacuo to give the title compound as a yellow solid

(0.24 g, 75%): LC/MS: t_R=1.68 min, m/z=459.1 [M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-N,N-dimethyl-1-(2-((R)-2-methylpyrrolidin-

1-yl)ethyl)-1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-

carboxamide (18d): (R)-2-Methylpyrrolidine (468 mg, 5.5 mmol)

was added to a solution of 2-((4aR,8R,9aR)-6-acetyl-8-(dimethylcar-

1-((4aR,8R,9aR)-8-(3-Methyl-1,2,1-oxadiazol-2-yl)-1-(2-((R)-2-

methylpyrrolidin-1-yl)ethyl)-4,4a,7,8,9,9a-hexahydroindolo [1,14-

bamoyl)-4a,6,7,8,9,9a-hexahydro

indolo[1,14-fg]quinolin-1(4H)-

yl)ethyl methanesulfonate (492 mg, 1.1 mmol) in MeCN (10 mL).

The reaction mixture was stirred at 508C overnight. The solvent

was removed in vacuo to give the crude product, which was puri-

fied by preparative HPLC to yield 18d as a white solid (150 mg,

31%): ¹H NMR (400 MHz, CDCl₃): d=7.17–7.14 (m, 2H), 6.84–6.83

(m, 2H), 4.23 (t, J=7.6 Hz, 2H), 4.15–4.04 (m, 2H), 3.48–3.36 (m,

2H), 3.26–3.15 (m, 7H), 2.99–2.97 (m, 4H), 2.78–2.72 (m, 1H), 2.52–

2.48 (m, 1H), 2.42–2.23 (m, 5H), 2.02–1.73 (m, 4H), 1.43–1.40 (m,

1H), 1.12–1.10 ppm (d, J=6.0 Hz, 3H); LC/MS: t_R=1.85 min, m/z=

437.2 [M+H]⁺.

fg]quinolin-6(1H)-yl)ethanone

2-((4aR,8R,9aR)-6-acetyl-8-(3-methyl-1,2,1-oxadiazol-2-yl)-4a,6,7,8,

(20):

To

a

mixture

of

9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ethyl methanesulfo-

nate (230 mg, 0.5 mmol) in MeCN (10 mL) was added (R)-2-methyl-

pyrrolidine (212 mg, 2.5 mmol) under argon. The reaction mixture

was stirred at 608C overnight. The solvent was removed in vacuo,

and the crude product was purified by preparative HPLC to give

the title compound as a yellow solid (190 mg, 85%): ¹H NMR

(400 MHz, CDCl₃): d=7.20–7.17 (m, 2H), 6.88–6.87 (m, 2H), 4.27–

4.13 (m, 4H), 3.90–3.68 (m, 1H), 3.62–3.58 (m, 1H), 3.40–2.90 (m,

6H), 2.60–2.50 (m, 1H), 2.43–2.30 (m, 4H), 2.22–2.20 (m, 1H), 2.12

(s, 3H), 2.02–1.73 (m, 4H), 1.52–1.37 (m, 1H), 1.13–1.09 ppm (m,

3H); LC/MS: t_R=1.86 min, m/z=448.2 [M+H]⁺.

1-((4aR,8R,9aR)-8-(3-Methyl-1,2,1-oxadiazol-2-yl)-4,4a,7,8,9,9a-

hexahydroindolo[1,14-fg]quinolin-6(1H)-yl)ethanone (19): DIPEA

(8.8 g, 68 mmol), HATU (12.9 g, 34 mmol) and (E)-N’-hydroxyaceti-

midamide (3.8 g, 51 mmol) were added to a solution of 16 (5 g,

17 mmol) in DMF (50 mL). The reaction mixture was stirred at RT

for 1 h, and then at 1008C for 3 h. After cooling to RT, the reaction

mixture was quenched with water (30 mL) and extracted with

EtOAc (3ꢁ60 mL). The combined organic layers were dried (anhyd

MgSO₄), filtered, and concentrated in vacuo to give the crude

product, which was purified by silica gel chromatography (EtOAc/

petroleum ether, 1:1) to afford 19 as a yellow solid (3.4 g, 60%):

LC/MS: t_R=1.63 min, m/z=337.2 [M+H]⁺.

(4aR,8R,9aR)-6-Acetyl-1-(2-hydroxyethyl)-1,4,4a,6,7,8,9,9a-octa-

hydroindolo[1,14-fg]quinoline-8-carboxylic acid: LiBH₄(2m in

THF, 6.5 mL, 13 mmol) was slowly added to a solution of 17

(1.65 g, 4.36 mmol) in THF (150 mL) at 08C under argon. The result-

ing reaction mixture was then stirred at RT overnight. After re-cool-

ing to 08C, AcOH (6 mL) was slowly added to the reaction mixture,

which was stirred for an additional 30 min before adjusting the pH

to 8 with saturated aq NaHCO₃(40 mL). The reaction mixture was

extracted with EtOAc (2ꢁ100 mL), and the combined organic

layers were washed with brine (30 mL), dried (anhyd MgSO₄), fil-

tered, and concentrated to give the crude product as a brown oil

(620 mg, 44%), which was used directly in the next step without

further purification: LC/MS: t_R=1.23 min, m/z=343.2 [M+H]⁺.

Isopropyl 2-((4aR,8R,9aR)-6-acetyl-8-(3-methyl-1,2,1-oxadiazol-2-

yl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-1(4H)-yl)ace-

tate: A mixture of 19 (2.5 g, 7.4 mmol), NaOH (2.7 g, 35.5 mmol)

and TBAI (0.54 g, 1.48 mmol) in CH₂Cl₂(50 mL) was cooled to 08C.

Isopropyl 2-bromoacetate (2.7 g, 14.8 mmol) was slowly added and

the reaction mixture, which was then stirred at RT overnight. The

precipitate was removed by filtration, and the filtrate was washed

with water (100 mL). The organic layer was dried (anhyd MgSO₄),

filtered, and concentrated in vacuo. Purification by silica gel chro-

matography (petroleum ether/EtOAc, 2:1) gave the title compound

as a yellow oil (2.6 g, 80%), which was used in the next step with-

out further purification: LC/MS: t_R=1.83 min, m/z=437.3 [M+H]⁺.

(4aR,8R,9aR)-Methyl

9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxylate (21): SOCl₂

(173 mg, 1.46 mmol) was slowly added to solution of

6-acetyl-1-(2-hydroxyethyl)-1,4,4a,6,7,8,

a

(4aR,8R,9aR)-6-acetyl-1-(2-hydroxyethyl)-1,4,4a,6,7,8,9,9a-octahydro-

indolo[1,14-fg]quinoline-8-carboxylic acid (100 mg, 0.29 mmol) in

CH₃OH (15 mL) at 08C. The reaction mixture was stirred at RT for

4 h. The solvent was removed in vacuo to yield the crude product,

which was diluted with EtOAc (20 mL). The organic layer was

washed with saturated aq NaHCO₃(15 mL), dried (anhyd MgSO₄),

filtered and concentrated in vacuo to give the title compound as

a yellow solid (86 mg, 83%), which was used in the next step with-

out further purification: LC/MS: t_R=1.69 min, m/z=357.2 [M+H]⁺.

1-((4aR,8R,9aR)-1-(2-Hydroxyethyl)-8-(3-methyl-1,2,1-oxadiazol-

2-yl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg] quinolin-6(1H)-yl)-

ethanone: A suspension of LiAH₄(0.24 g, 6.3 mmol) in THF (10 mL)

was cooled to ꢀ788C. Isopropyl 2-((4aR,8R,9aR)-6-acetyl-8-(3-

methyl-1,2,1-oxadiazol-2-yl)-4a,6,7,8,9,9a-hexahydroindolo[1,14-fg]-

quinolin-1(4H)-yl)acetate (1 g, 2.1 mmol) was slowly added at

ꢀ788C under argon. After stirring at ꢀ788C for 10 min, the mixture

was quenched with the careful addition of water (5 mL), then fil-

tered through Celite, and the filter cake washed with THF (20 mL).

The filtrate was extracted with EtOAc (3ꢁ100 mL). The combined

(4aR,8R,9aR)-Methyl

6-acetyl-1-(2-(methylsulfonyloxy)ethyl)-

1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-carboxyl-

ate: A mixture of 21 (0.19 g, 0.52 mmol) and Et₃N (0.22 mL,

1.56 mmol) in CH₂Cl₂(25 mL) was cooled to 08C. MsCl (170 mg,

1.5 mmol) in CH₂Cl₂(5 mL) was slowly added to the mixture, which

was then stirred at RT for 4 h. The organic solvent was removed in

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vacuo to yield the crude product. The residue was diluted with

CH₂Cl₂(50 mL), washed with saturated aq NaHCO₃(30 mL) and

concentrated in vacuo to give the title compound as a yellow solid

(0.22 g, 88%), which was used in the next step without further pu-

rification: LC/MS: t_R=1.68 min, m/z=435.2 [M+H]⁺.

sence of increasing concentrations (10-point dose titrations with

3-fold serial dilutions) of test compound to determine H3R compe-

tition binding. The binding incubations (120 min at 288C) were

performed in a final volume of 0.1 mL buffer (50 mm Tris pH 7.5,

5 mm MgCl₂). (R)-(ꢀ)-a-Methylhistamine thioperamide (10 mm) was

used to define nonspecific binding. All binding reactions were ter-

minated by transferring 70 mL binding reaction from reaction plate

into gel filtration plates (Zeba 96-well spin desalting plates,

Thermo Scientific), followed by centrifugation at 1000g for 2 min

to collect the protein with bound radioligand. Microscint-40

(200 mL) was added to determine the bound radiolabel by using

a Wallac Microbeta Trilux 2450 microplate liquid scintillation coun-

ter (PerkinElmer). IC₅₀values and Hill slopes were determined by

using GraphPad Prism: log(inhibitor) vs responseꢀvariable slope.

The corresponding K_ivalues were calculated using the Chang–

Prusoff equation: K_i=IC₅₀/{1+([radioligand]/K_d)}.

(4aR,8R,9aR)-Methyl

6-acetyl-1-(2-((R)-2-methylpyrrolidin-1-

yl)ethyl)-1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]quinoline-8-

carboxylate (22): To a mixture of (4aR,8R,9aR)-methyl-6-acetyl-1-(2-

(methylsulfonyloxy)ethyl)-1,4,4a,6,7,8,9,9a-octahydroindolo[1,14-fg]-

quinoline-8-carboxylate (139 mg, 0.32 mmol) and Et₃N (0.22 mL,

1.6 mmol) in MeCN (15 mL) was added (R)-2-methylpyrrolidine

(275 mg, 3.2 mmol) under argon. The reaction mixture was then

stirred at 508C overnight. The solvent was removed to yield the

crude product, which was purified by preparative HPLC to give 22

1

as a white solid (50 mg, 33%): H NMR (400 MHz, CD₃OD): d=7.20

(d, J=8.4 Hz, 1H), 7.12 (t, J=7.2 Hz, 1H), 6.92 (s, 1H), 6.83 (d, J=

6.8 Hz, 1H), 4.33–4.22 (m, 2H), 4.08–4.03 (m, 1H), 3.76 (s, 3H),

3.54–3.49 (m, 1H), 3.40–3.37 (s, 2H), 3.26–3.16 (m, 3H), 3.05–2.96

(m, 1H), 2.90–2.81 (m, 2H), 2.51–2.48 (m, 1H), 2.38–2.34 (m, 1H),

2.26–2.22 (m, 4H), 1.98–1.94 (m, 1H), 1.79–1.73 (m, 2H), 1.62–1.58

(m, 1H), 1.47–1.42 (m, 1H), 1.09–1.08 ppm (d, J=6.0 Hz, 3H);

LC/MS: t_R=1.76 min, m/z=424.1 [M+H]⁺.

Adenylate cyclase (cAMP) assay: In the presence of 10 mm 3-isobu-

tyl-1-methylxanthine, CHO-A8 cells stably expressing the full-length

human, mouse or rat H3R were incubated at 288C for 50 min with

increasing concentrations of test compound in the presence of

5 mm (human and mouse) or 1.5 mm (rat) (R)-a-methylhistamine

and 3 mm forskolin. cAMP formation was determined using the

Ultra LANCE cAMP assay kit (PerkinElmer). Plates were read using

an EnVision reader (PerkinElmer). Data were normalized to the

amount of cAMP produced in control wells and are expressed as

a percentage of inhibition. Experiments were run in duplicate, and

data were analyzed using GraphPad Prism to obtain IC₅₀values

and Hill slopes. pK_bvalues were determined by the generalized

Cheng–Prusoff equation.

1-((4aR,8R,9aR)-8-(Hydroxymethyl)-1-(2-((R)-2-methylpyrrolidin-

1-yl)ethyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-6(1H)-

yl)ethanone (23): To a solution of 22 (0.2 g, 0.48 mmol) in THF

(30 mL) was slowly added LiBH₄(2m in THF, 0.72 mL, 1.42 mmol) at

08C under argon. The reaction mixture was stirred at 08C for 3 h,

then at RT overnight. The reaction was quenched at 08C with the

careful addition of water (5 mL). After stirring for 30 min, the mix-

ture was extracted with EtOAc (3ꢁ50 mL). The combined organic

layers were dried (anhyd MgSO₄), filtered, and concentrated in

vacuo to give the crude product. Purification by preparative HPLC

In vivo PD/PK: Male Sprague–Dawley rats (Shanghai Laboratory

Animal Center, China) at the age of 8 weeks were used. They had

access to food and water ad libitum. All procedures were approved

by the Institutional Animal Care and Use Committee of Chempart-

ner Co., LTD (IACUC Protocol NO: A998HL0076). On the study day,

the body weights of each animal were recorded before dosing.

Compounds were dissolved in 50 mm citrate acid buffer and soni-

cated briefly until there was little or no suspension in the solution.

Different groups of animals (n=5 rats/group) were dosed orally

1 h or at other time points (1 mLkg^ꢀ1) before they were sacrificed

using CO₂. Oral bavisant was used as a reference in all studies.

1

yielded the title compound as a white solid (76 mg, 40%): H NMR

(400 MHz, CD₃OD): d=7.33 (d, J=8.4 Hz, 1H), 7.20 (t, J=7.6 Hz,

1H), 7.04 (s, 1H), 6.91 (d, J=7.6 Hz, 1H), 4.60 (t, J=6.4 Hz, 2H),

3.92–3.84 (m, 3H), 3.63–3.34 (m, 8H), 3.10–2.70 (m, 3H), 2.25–2.18

(m, 5H), 2.10–1.94 (m, 2H), 1.75–1.65 (m, 1H), 1.46 (d, J=6.8 Hz,

3H), 0.86–0.84 ppm (m, 1H); LC/MS: t_R=1.69 min, m/z=396.3

[M+1]⁺.

1-((4aR,8R,9aR)-8-(Methoxymethyl)-1-(2-((R)-2-methylpyrrolidin-

1-yl)ethyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-6(1H)-

yl)ethanone (24): To a solution of 23 (126 mg, 0.32 mmol) in

MeCN (15 mL) was added CH₃I (70 mg, 0.48 mmol) and K₂CO₃

(0.22 g, 1.6 mmol) under argon. The reaction mixture was stirred at

RT overnight. The solvent was removed to yield the crude product,

which was purified by preparative HPLC to give the title com-

Blood sample collection and bioanalyses: A cardiac puncture was

performed to collect blood sample from the cardiac cavity. The col-

lected blood was immediately mixed with EDTA-K2 (20 mLmL^ꢀ1) to

avoid clotting. The blood samples in tubes were then centrifuged

(15 min, 6000 rpm), and the plasma transferred to new tubes and

cooled in dry ice then stored in a ꢀ708C freezer until bioanalysis

(usually within 2–3 days). A 30 mL aliquot of sample was added

pound as

a

white solid (30 mg, 23%): ¹H NMR (400 MHz,

[D₆]DMSO): d=7.23 (d, J=8.4 Hz, 1H), 7.10–7.04 (m, 2H), 6.80 (d,

J=8.4 Hz, 1H), 4.85–4.75 (m, 1H), 4.62–4.50 (m, 2H), 3.75–3.68 (m,

2H), 3.42–3.39 (m, 2H), 3.26–3.10 (m, 5H), 2.92–2.90 (m, 1H), 2.78–

2.70 (m, 3H), 2.59–2.50 (m, 1H), 2.18–2.10 (m, 1H), 2.05 (s, 3H),

1.93–1.72 (m, 4H), 1.32–1.28 (m, 2H), 1.18–1.16 (d, J=6.0 Hz, 3H),

0.68–0.66 ppm (m, 1H); LC/MS: t_R=1.68 min, m/z=410.2 [M+H]⁺.

with 30 mL of the internal standard (dexamethasone, 300 ngmL^ꢀ1

)

and then followed by 150 mL MeCN for protein precipitation. The

mixture was vortexed for 2 min and centrifuged at 12000 rpm for

5 min. The 5 mL supernatant was analyzed by using LC/MS–MS.

Frontal cortex brain tissue collection and bioanalyses: The rat brains

were taken out of the skull and rinsed with ice-cold saline. Resting

on top of a petri dish of ice, the frontal cortices were then re-

moved from the rest of the brain with a sharp blade. The wet

weight of the frontal cortex was immediately weighed and record-

ed. The frontal cortex samples were then kept in dry ice until

being transfer to a ꢀ708C freezer for storage. The brain sample

was homogenized for 2 min with 3 volumes (v/w) of homogenizing

solution (EtOH/PBS, 85:15), and then centrifuged at 12000 rpm for

Biology

General: LC/MS–MS analyses were performed on an Agilent 6410,

triple quadrupole mass spectrometer.

Radioligand binding assay: Human H3R membrane preparations in

CHO-K1 cells (PerkinElmer) were incubated with 1 nm [³H]-N-a-

methylhistamine ([³H]-NAMH, PerkinElmer) in the presence or ab-

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5 min. Finally, 60 mL supernatant was added into a 96-well plate

and 3 mL supernatant was analyzed by using LC/MS–MS.

taining 1.0 mm EDTA, pH 7.4). The compound and microsomes mix-

tures were loaded (30 mL) into 96-well assay plates and preincubat-

ed at 378C for 10 min. Metabolism was initiated at designated

time points (0 and 30 min) by adding 15 mL of prewarmed NADPH

stock solution (6 mm prepared in 0.1m potassium phosphate

buffer containing 1.0 mm EDTA, pH 7.4). At the end of the incuba-

tion, 135 mL of MeCN containing 200 ngmL^ꢀ1osalmide was added

into each well to stop the reaction. For the 0 min time point, the

NADPH solution was added after the system was quenched with

MeCN. The reaction mixtures were centrifuged at 3220 g for

10 min. The supernatants were mixed with Milli-Q water (1:1, v/v)

and analyzed by LC/MS–MS.

Ex vivo receptor occupancy assay: At specific times after oral treat-

ment, the animals were sacrificed and the frontal cortices were dis-

sected out on ice. The crude homogenate samples of rat frontal

cortex were used to measure H3R binding as described above with

[³H]-N-a-methylhistamine as the radioligand. The protein concen-

tration of each sample was determined by Pierce BCA protein

assay kit (Thermo, #23227). The cortical homogenate was added to

a 96-well plate containing [³H]-N-a-methylhistamine (0.1 nm), in

a volume of 0.2 mL (final protein concentration 4 mgmL^ꢀ1), incubat-

ed at 288C for 15 min, then the reaction was stopped by rapid fil-

tration using a filtration plate (Millipore multiscreen GF/B plate,

#F6HN58185). The filters were washed three times with ice-cold

buffer (50 mm Tris, pH 7.5), then mixed with 250 mL of Microscint-

40 to determine the bound radiolabel by using a Wallac Microbeta

Trilux 2450 microplate liquid scintillation counter. Nonspecific bind-

ing was determined in the presence of 10 mm thioperamide. Each

data point was obtained from a total of at least four animals. The

inhibition of specific [³H]NAMH binding, calculated relative to vehi-

cle-treated samples, was determined to provide an indication of re-

ceptor occupancy by the compound. The dose-dependent % re-

ceptor occupancy and time course were analyzed using GraphPad

Prism: Spikes, Points & collecting line. The dose–response was fit

using GraphPad Prism: log(inhibitor) vs response<ꢀvariable slope.

MDCK-MDR1 assay: Seeded and washed MDCK-MDR1 cells were

put in a 24-well plate. The transepithelial electrical resistance

(TEER) across the monolayer was measured at room temperature

for each well using a Millicell ERSohm meter. The cell monolayers

with TEER higher than 250 ohmcm²were used for permeability

assay. Donor solution containing test compound (in duplicate)

(10 mm) were loaded either to the apical chamber (600 mL) for A-to-

B (apical to basolateral) or to the basolateral chamber (900 mL) for

B-to-A (basolateral to apical) permeation. The receiver chambers

were loaded with buffered Hank’s balanced salt solution (HBSS)

(800 mL in the basolateral chamber for A-to-B permeation and

500 mL in the apical chamber for B-to-A permeation). All solutions

loaded to apical chambers also contained 5 mm Lucifer Yellow as

a quality control for monolayer integrity. An aliquot of 100 mL was

taken from all the donor chambers as time zero (T₀) samples

before transport was initiated. A second aliquot of 100 mL was also

taken at T₀from all apical chambers containing Lucifer Yellow.

Transport was initiated by placing the apical plate seeded with

MDCK-MDR1 monolayers onto the basolateral plate. The assem-

bled plates were kept in a culture incubator (5% CO₂and 95% rel-

ative humidity) at 378C for 90 min.

Aqueous stability: Test compounds were prepared as solutions in

potassium phosphate buffer (pH 7.4) or aqueous H₂SO₄(pH 1) at

14 mm, and stirred at RT and 808C, respectively for 7 days, with LC/

MS monitoring every 24 h. The samples for LC/MS were prepared

through fivefold dilution with MeOH/H₂O (5:1; v/v).

CYP450 time-dependent inhibition assay: Time-dependent CYP450

inhibition was assessed in parallel with reference inhibitors by

a preincubation and secondary incubation protocol. During prein-

cubation, an aliquot of 20 mL of 5 mm NADPH (+NADPH) or buffer

(ꢀNADPH) was added into 80 mL of a mixture of positive control,

test compound, or solvent, and P450 enzymes (1.25 mgmL^ꢀ1of

human liver microsomes or 62.5 pmolmL^ꢀ1of recombinant

CYP2C19). After 30 min, an aliquot of 20 mL from the preincubation

mixture was transferred into 180 mL of a secondary incubation mix-

ture containing substrates and 1 mm NADPH. The substrate con-

centrations for CYP1A2 (phenacetin at 75 mm), CYP2C9 (diclofenac

at 10 mm), and CYP3A4 (midazolam at 10 mm) were at reported K_m

values, and those for CYP2D6 (bufurolol at 40 mm) and CYP2C19

(S-mephenytoin at 100 mm) were at threefold the reported K_m

values. The secondary incubation was allowed for 15 min. The reac-

tions were quenched by transferring 60 mL of the aliquot from the

secondary incubation system into 120 mL MeCN containing the

stable isotopes of the metabolites for the corresponding CYP iso-

forms as internal standards. After 5 min vortexing, proteins were

precipitated by 15 min centrifugation at 3220 g. The resulting su-

pernatants were transferred into 96-well sample plates for LC/MS–

MS analysis. Changes in P450 enzyme activities were evaluated by

comparing the production of the metabolites based on the peak

area ratios (metabolite/internal standard) between the preincuba-

tion systems with and without inhibitor after normalization for the

activity loss in the absence of NADPH.

Caco-2 permeability: Caco-2 cells were seeded in a 24-well plate at

a density of 4.8ꢁ10⁴/well 21–27 days prior to the assay and main-

tained in culture (378C, 5% CO₂with 95% relative humidity) with

change of growth medium (minimum essential medium+10%

fetal bovine serum+1% non-essential amino acids) every other

day. On the assay day, Caco-2 monolayers were washed once with

HBSS containing 25 mm HEPES (pH 7.4), and the TEER across the

monolayer was measured at room temperature for each well using

a Millicell ERSohm meter. The cell monolayers with TEER higher

than 250 ohmcm²were used for permeability assay. The proce-

dures of permeability assay conducted in Caco-2 cells were the

same as those described in MDCK-MDR1 assay.

Test in orexin-deficient mice: Homozygous orexin-deficient mice

(n=11) were used for this study. For testing compound effects on

narcoleptic episodes, mice were treated immediately before lights-

off and then put into large Plexiglas boxes equipped with saw

dust bedding, a nest box, and enrichments such as wooden chew

block, transparent running wheel, transparent mouse tunnel, plas-

tic disc, and five glass marbles. The behavior of the mice was vid-

eotaped from above using infrared cameras. Videotapes were

scored offline by two independent experienced observers. Narco-

leptic episodes were defined as periods of total inactivity outside

the nest box, lasting longer than 10 s and preceded by activity last-

ing longer than 40 s.^[11,33]After a one-week recovery period in the

home cage, the animals were retested. Each animal was treated

with either saline or compound 1 (30 mgkg^ꢀ1) in a pseudo-

randomized order. An experimental session started 2 h before

lights-off when the animals were put into the exposure boxes. Im-

mediately before lights-off, the animals were treated and the video

Liver microsomal stability: Stock solutions of test compounds at

10 mm in DMSO were prepared and stored at ꢀ808C. Before the

assay, spiking solutions for the test compounds were prepared in

MeCN at 500 mm and then further diluted to 1.5 mm in microsomes

(0.75 mgmL^ꢀ1prepared in 0.1m potassium phosphate buffer con-

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[15] C. Inocente, I. Arnulf, H. Bastuji, A. Thibault-Stoll, A. Raoux, R. Reimao,

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recording was started (recording duration: 4 h). The animals were

left in the exposure box until the next morning. Then, they were

put back into the home cage. The first experimental session was

without any treatment to determine a pre-test baseline (pre-test).

Then, every animal was repeatedly tested with the different treat-

ment in a pseudo-randomized order. At the end, a further session

without any treatment was added to determine the post-test base-

line (post-test).

Acknowledgements

It is a pleasure to acknowledge the support and encouragement

of Drs. Karin Briner and Scott Biller.

[21] A. M. Crider, R. Grubb, K. A. Bachmann, A. K. Rawat, J. Pharm. Sci. 1981,

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Keywords: histamine receptors

narcolepsy · neurotransmitters

·

medicinal chemistry

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Received: March 5, 2014

Published online on May 21, 2014

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ChemMedChem 2014, 9, 1683 – 1696 1696

Article Doi

Ergoline-derived inverse agonists of the human H3 receptor for the treatment of narcolepsy

DOI: 10.1002/cmdc.201402055

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