The alkaloid conessine and analogues as potent histamine H3 receptor antagonists

Article abstract of DOI:10.1021/jm8003625

The naturally occurring alkaloid, conessine (6), was discovered to bind to histamine H₃ receptors in a radioligand-based high-throughput screen. Conessine displayed high affinity at both rat and human H₃ receptors (pK_i = 7.61 and 8.27) and generally high selectivity against other sites, including histamine receptors H₁, H₂, and H ₄. Conessine was found to efficiently penetrate the CNS and reach very high brain concentrations. Although the very slow CNS clearance and strong binding to adrenergic receptors discouraged focus on conessine itself for further development, its potency and novel steroid-based skeleton motivated further chemical investigation. Modification based on introducing diversity at the 3-nitrogen position generated a new series of H₃ antagonists with higher in vitro potency, improved target selectivity, and more favorable drug-like properties. One optimized analogue (13c) was examined in detail and was found to be efficacious in animal behavioral model of cognition.

Full text of DOI:10.1021/jm8003625

J. Med. Chem. 2008, 51, 5423–5430
5423
The Alkaloid Conessine and Analogues as Potent Histamine H3 Receptor Antagonists
Chen Zhao,* Minghua Sun, Youssef L. Bennani,† Sujatha M. Gopalakrishnan, David G. Witte, Thomas R. Miller,
Kathleen M. Krueger, Kaitlin E. Browman, Christine Thiffault, Jill Wetter, Kennan C. Marsh, Arthur A. Hancock,
Timothy A. Esbenshade, and Marlon D. Cowart
Department of Neuroscience Research, Global Pharmaceutical Research DiVision, Abbott Laboratories,
100 Abbott Park Road, Abbott Park, Illinois 60064-6123
ReceiVed March 31, 2008
The naturally occurring alkaloid, conessine (6), was discovered to bind to histamine H3 receptors in a
radioligand-based high-throughput screen. Conessine displayed high affinity at both rat and human H3 receptors
(pKi ) 7.61 and 8.27) and generally high selectivity against other sites, including histamine receptors H1,
H2, and H4. Conessine was found to efficiently penetrate the CNS and reach very high brain concentrations.
Although the very slow CNS clearance and strong binding to adrenergic receptors discouraged focus on
conessine itself for further development, its potency and novel steroid-based skeleton motivated further
chemical investigation. Modification based on introducing diversity at the 3-nitrogen position generated a
new series of H3 antagonists with higher in vitro potency, improved target selectivity, and more favorable
drug-like properties. One optimized analogue (13c) was examined in detail and was found to be efficacious
in animal behavioral model of cognition.
Introduction
Histamine plays an important role in different physiological
processes by acting through the four discovered histamine
receptors H1, H2, H3, and H4.1 The histamine H3 receptor is a
G-protein coupled receptor (GPCR), predominantly expressed
in the central nervous system (CNS), where it modulates the
release of multiple neurotransmitters including histamine,
dopamine, noradrenaline, acetylcholine, glutamate, and seroto-
nin.2 Histamine H3 receptor antagonists can stimulate the release
of other neurotransmitters and have been posited to offer a
promising approach to the treatment of several CNS disorders,3,4
including attention deficit hyperactivity (ADHD),5 sleep disor-
ders,6 epilepsy,7 schizophrenia,8 and obesity.9
The histamine H3 receptor was first described in 198310 and
later cloned in 1999.11 Antagonists of this receptor have been
divided into two broad structural groupssimidazole and non-
imidazole analogues.12 Most of the earliest potent compounds
Figure 1
were imidazole derivatives.13 Representative imidazole-contain-
ing compounds include 1 (thioperamide),14 2 (ciproxifan),15 and
3 (GT-2331)16 (Figure 1). While these antagonists have been
found to be effective in a number of cognition models in rodents,
they had several drawbacks, including reports of affinity for
other receptors, e.g. H4 receptors,17 relatively poor CNS pen-
etration, and most importantly, the known potential to perpetrate
drug-drug interactions, through binding of the imidazole moiety
to the heme-Fe engaged in hepatic cytochrome P450 enzymes.18
More recently, attention in the field has turned to the non-
imidazole class of H3-antagonists in the search for more drug-
like compounds. SAR studies across a number of series have
demonstrated that tertiary amines can effectively replace the
imidazole moiety, reducing the potential for CYP inhibition,
as well as promoting other drug-like properties, and better CNS
penetration.12,19 Most of the reported nonimidazole H3 antago-
nists possess an “aromatic ring-spacer-basic amine” motif:
Figure 2
(Ar-X(CH2)n-NR1R2). Notable examples of this class include
4 (ABT-239)20,21 and 5 (GSK-189254)22 (Figure 2). Compared
to the earlier imidazole-based ligands, these compounds offer
improvements in binding affinity (especially human H3), receptor
selectivity, CNS penetration, pharmacokinetic and metabolic
* To whom correspondence should be addressed. Phone: (847) 938-5292.
Fax: (847) 937-9195. E-mail: chen.zhao@abbott.com.
†
Present address: Vertex Pharmaceuticals, 130 Waverly Street, Cam-
bridge, Massachusetts 01239.
10.1021/jm8003625 CCC: $40.75
2008 American Chemical Society
Published on Web 08/07/2008

5424 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Zhao et al.
Table 1. Comparison of Conessine, Ciproxifan, and Thioperamide
Binding at Histamine Receptors and Other receptors
mean pKi ( SEM
receptor
human H3
rat H3
human cortex H3
rat cortex H3
human H1
human H2
human H4
human R2C4
human R2C10
rat 5HT1
conessine
ciproxifan
thioperamide
8.27 ( 0.10
7.91 ( 0.10
8.46 ( 0.34
7.61 ( 0.08
<5.00
<5.00
<5.00
7.98 ( 0.18
6.18 ( 0.19
5.72 ( 0.06
<5.00
<5.00
7.20 ( 0.05
9.29 ( 0.09
7.05 ( 0.06
9.20 ( 0.04
<5.00
<5.00
5.73 ( 0.09
7.20 ( 0.13
7.37 ( 0.07
4.78 ( 0.18
4.73 ( 0.10
6.52 ( 0.19
7.14 ( 0.06
8.44 ( 0.07
7.18 ( 0.08
8.15 ( 0.06
<5.00
<5.00
7.32 ( 0.25
6.46 ( 0.11
6.90 ( 0.08
4.95 ( 0.28
5.25 ( 0.39
5.64 ( 0.13
Figure 3
rat 5HT2
rat 5HT3
assays.32 Conessine had higher affinity for the human H3
receptor than rat H3 receptor, in contrast to the widely used
reference H3 antagonist standards, thioperamide and ciproxifan,
which are 10- and 100-fold less potent respectively at the human
H3 receptor than at the rat receptor. Conessine had very good
selectivity for H3 receptors compared to the other histamine
receptors. It displayed negligible binding affinity for human
histamine H1 and H2 receptors, similar to ciproxifan and
thioperamide. However, conessine is much more selective for
human H3 versus the H4 receptor (>1860-fold) than the
imidazole-based ciproxifan (30-fold selective) and thioperamide
(only 1.5-fold selective). Conessine was also found to exhibit
low binding affinity for most other rodent and human
G-protein coupled receptors and ligand activated ion channels,
with the notable exception of potent affinity for the human
alpha2C-adrenergic receptor subtype (pKi ) 7.98, Ki ) 10.6
nM; Table 1).
As can be seen in the data compiled in Table 2, conessine
was found to act as an antagonist of H3 receptor in cell- and
tissue-based functional assays.32 These functional assays assess
the ability of compounds to block H3 agonist-stimulated GTPγS
binding, adenylate cyclase activity or Ca2+ intracellular increase
(FLIPR). Consistent with the binding potency trends, conessine
had high functional potency at the human H3 receptor, several-
fold higher than ciproxifan and thioperamide, in human H3
GTPγS and human H3 FLIPR assays, but several-fold weaker
potency at rodent receptor (adenylate cyclase and electric field
stimulated guinea pig ileum tissue assay).
Figure 4
profiles, and especially lower potential for CYP inhibition than
imidazole derivatives.
Although early nonimidazole H3 antagonists displayed high
binding affinity and drug-like properties, there was a desire for
greater structural diversity. This was one of our motivations
for carrying out a high-throughput screen (HTS) to search for
novel structural classes of H3 antagonists. One particularly
intriguing hit that emerged from this early HTS screen was
compound 6, conessine (Figure 3), a natural product based on
a steroidal skeleton.23 Conessine is an alkaloid with a history
of use in folk medicine24 and has even been reported to have
some activity as an antibiotic agent.25
Natural products have over the years served as a rich source
of pharmacologically active agents. Some of these have oc-
casionally been developed into drugs, but more commonly they
have some shortcomings that must be corrected during an
optimization process requiring substantial structural modifica-
tion. All in all, the interesting structures and properties of natural
products make them an important source of inspiration and leads
for drug discovery efforts that resulted in clinically useful
entities, e.g., paclitaxel26 and morphine.27
Three natural products have been reported as H3 receptor
antagonists. In fact, one of the earliest nonimidazole H3
antagonists was aplysamine (7)28 (Figure 4), an alkaloid present
in a marine sponge. This compound is a weak functional H3
receptor antagonist, with an IC50 of 0.34 µg/mL (0.83 µM) in
a guinea pig ileum assay. Another natural product from the
marine sponge Verongula gigantean, verongamine (8)29 (Figure
4), also displayed weak H3 receptor activity (IC50 of 0.19 µg/
mL (0.50 µM) in a guinea pig ileum assay). More recently,
carcinine, an imidazole-containing dipeptide (ꢀ-alanyl histamine)
from the crustacean Carcinus maenas, has been found to be
moderately active as a histamine H3 ligand (rat H3 Ki ) 294
nM).30 Compared to these natural products, conessine is a much
more potent and selective H3 antagonist. Its novel structure may
serve as a starting point for the design of new classes of H3
antagonists.31
In a pharmacokinetic study in rat, conessine efficiently crossed
the brain-blood barrier, achieving high brain/plasma ratios (46-
to 114-fold) at all time points (Table 3). The absolute brain
concentrations reached a very high level of 6570 ng/gm one
hour after 1 mg/kg ip dosing. Such efficient brain penetration
is often deemed an advantage of a CNS-targeted drug, however,
the extremely slow rate of CNS clearance is atypical and was
deemed undesirable. Even after 24 h, 4140 ng/gm (>60%)
remained in brain, not much changed from the one-hour time
point. The prospect for CNS accumulation and consequent
elevated risk for toxicity upon repeated dosing presented a
potential liability. We believe it is likely that the high CNS
concentrations and low CNS clearance arose from the lipophilic
dibasic nature of the molecule. An additional drawback of this
dibasic character of conessine is the potential to induce toxicity
due to phospholipidosis, as it has been reported that dibasic
compounds are more likely to induce phospholipidosis than
monobasic congeners.33 Overall, therefore, despite favorable
high potency, conessine (6) itself lacks good drug-likeness.
However, structural characteristics of this alkaloidal core, a
novel highly rigid skeleton with no aromatic or heteroaromatic
Results and Discussion
Conessine exhibited high affinity for both human and rat
histamine H3 receptors, with pKi values of 8.27 and 7.61 (Ki )
5.37 and 24.5 nM), respectively (Table 1), in radioligand binding

Conessine and Analogues as Histamine H3 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5425
Table 2. Comparison of Antagonist Potencies of Conessine, Thioperamide, and Ciproxifan in Histamine H3 Receptor Functional Assays
mean ((SEM) [n]
functional assay
conessine
ciproxifan
thioperamide
human H3 GTPγS (pKb)
rat H3 GTPγS (pKb)
human H3 adenylate cyclase (pKb)
rat H3 adenylate cyclase (pKb)
human H3 FLIPR (pKb)
rat H3 FLIPR (pKb)
7.96 ( 0.03 [3]
ND
6.14 ( 0.00 [1]
6.99 ( 0.07 [3]
8.08 ( 0.11 [4]
ND
7.07 ( 0.13 [4]
8.78 ( 0.12 [5]
6.59 ( 0.04 [9]
9.20 ( 0.10 [4]
6.84 ( 0.08 [12]
9.42 ( 0.11 [8]
8.12 ( 0.56 [21]
7.39 ( 0.04 [4]
8.13 ( 0.14 [5]
6.10 ( 0.12 [6]
7.61 ( 0.14 [3]
6.82 ( 0.06 [11]
9.11 ( 0.14 [4]
8.44 ( 0.49 [20]
EFS guinea pig ileum (pA2)
6.55 ( 0.31 [17]
Table 3. Mean Blood and Brain Concentrations of Conessine 6 after a
1 mg/kg Concomitant ip Dosing in Rat
binding affinity (hH3 Ki ) 19.7 nM) of the parent compound
conessine 6 (hH3 Ki ) 5.31 nM). In contrast, lactam 10, with
the basic dimethylamine moiety, displayed more than 100-fold
weaker affinity (hH3 Ki ) 543 nM). This comparison of 9 versus
10 strongly suggested that the basic nitrogen in the pyrrolidine
ring is critical for H3 binding in conessine and conessine-like
steroid structures (Scheme 1), whereas the basic nitrogen at the
3-position of the steroid skeleton was not important for H3
binding. On the basis of these findings, the principle strategy
to probe the SAR relied on retaining the conessine skeleton
constant and introducing diversity at the 3-nitrogen position.
The key intermediate des-methyl-conessine (isoconessimine,
11)36 was easily prepared from the Troc-conessine 9, following
a reported literature method.37 Modification of the 3-position
of conessine targeted five categories of substituents to be
appended to the nitrogen: amides, N-aryls, carbamates, ureas,
and sulfonamides (Scheme 2). Analogues with equal or better
potency than conessine were found in several series. Of these,
the most interesting examples are shown in Table 4 as
compounds 12a-12m.
1 h
5 h
24 h
plasma (µg/mL)
blood (µg/mL)
brain (µg/g)
Cb/Cp (ratio)
FRBC
0.148
0.444
6.57
46.0
0.819
0.089
0.306
6.80
95.4
0.849
0.037
0.102
4.14
114.4
0.805
Scheme 1. Preparation and Binding Affinity of Conessine
a
Analogues
Among the five series of analogues, amides were found
particularly interesting due to high potency and the easy access
to reagents for diversification. The 2-(thiophen-2-yl)acetamide
12f displayed a 2-fold increase in both rat and human binding
affinity and good CNS penetration and clearance compared to
conessine, but unfortunately this compound had only 4.5% oral
bioavailability in rat.
Additional SAR studies of amides pointed us to conessine
analogues with appended amino acid side chains, which showed
significant improvements (>10 fold) in binding potencies
compared with conessine (6) (Table 5). A good representative,
exemplifying this series, was the N-methyl-D-valine analogue
13c. It showed high affinity at both human and rat H3 receptors
(Ki ) 0.21 and 2.57 nM), and was selective against other
histamine receptor subtypes (H1, H2, and H4, Ki > 10000 nM).
In a more general screen, compound 13c exhibited minimal or
no binding affinity (Ki > 10000 nM) to most receptors (e.g.,
dopamine, serotonin, and cholinergic subtypes), except for potent
binding to one off-target receptor (muscarinic-M1, Ki ) 11 nM).
In particular, 13c had reduced affinity for the human alpha2C
adrenergic receptor (R2C4 Ki ) 77.6 nM, hH3/R2C4 ) 370) and
greatly improved selectivity compared to conessine 6 (R2C4 Ki
) 10.6 nM, hH3/R2C4 ) 2.0). The compound was further
characterized in functional assays and showed potent (Kb ) 4
nM) H3 antagonism in adenylate cyclase and Ca2+ flux assays
and inverse agonism (EC50 3.3 nM) in a GTPγS assay.38,39 The
compound was highly orally bioavailable in rats (F ) 65%),
and demonstrated efficient CNS penetration (brain/blood ranges
12-49×), with a very long half-life (77 h). The compound was
found to have no notable CNS side effects at 1.3-13 mg/kg
(ip). It was also free of genotoxicity40 and did not inhibit CYP
enzymes (IC50 > 10 µM). Although the core structure of
compound 13c is a steroid, no binding (Ki > 30000 nM) to
a Reagents and conditions: (i) Troc-Cl, benzene, reflux, 4h, 48%; (ii)
NBS, acetone/H2O, 48h, rt, 85%.
moiety, may confer advantages in selectivity, toxicity, pharma-
cokinetics, or metabolic profile and lead to a novel class of H3
antagonists.
Based on the fact that all reported structural families of potent
nonimidazole H3 antagonists have at a minimum one basic
amine, we hypothesized that it might be possible to alter or
eliminate one of the two basic sites present in conessine without
compromising the H3 potency. To determine which of the two
basic nitrogen atoms might be necessary, compounds 9 and 10
were synthesized as shown in Scheme 1. Conessine was
demethylated by treatment with Troc-Cl in refluxing benzene
to give 47% yield of carbamate 9.34 The lactam 10 was prepared
in 85% yield by treating conessine 6 with an oxidizing agent
(NBS in aqueous acetone), using the procedure reported by
Bhutani et al.35
Testing of the resulting analogues in H3 receptor radioligand
binding assays revealed that carbamate 9, which retains only
the basic nitrogen of the pyrrolidine ring, maintained the high

5426 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Zhao et al.
a
Scheme 2. Preparation of Conessine analogues 12a-12m
a Reagents and conditions: (i) acid chloride, Et3N, CH2Cl2; (ii) aryl bromide, Pd2(dba)3, BINAP, NaOtBu; (iii) chloroformate, Et3N, CH2Cl2, or 4-nitrophenyl-
ester, Et3N, THF; (iv) carbamoyl chloride, Et3N, CH2Cl2; (v) sulfonyl chloride, Et3N, CH2Cl2.
Table 4. Binding Affinities of Conessine 6 and Its Derivatives 11 and 12a-12m at Human and Rat H3Ra
compound
R group
human H3 pKi ( SEM
human H3 Ki (nM)
rat H3 pKi ( SEM
rat H3 Ki (nM)
6
11
Me
H
8.27 ( 0.10
7.42 ( 0.01
6.96 ( 0.29
7.22 ( 0.10
7.91 ( 0.18
7.40 ( 0.22
7.79 ( 0.05
8.48 ( 0.21
8.78 ( 0.04
7.22 ( 0.13
7.30 ( 0.15
7.51 ( 0.28
7.63 ( 0.17
8.11 ( 0.12
7.72 ( 0.10
5.37
38.0
110
7.61 ( 0.08
6.52 ( 0.00
6.59 ( 0.09
6.42 ( 0.16
7.42 ( 0.11
6.71 ( 0.13
6.81 ( 0.09
7.95 ( 0.18
7.62 ( 0.09
6.59 ( 0.39
6.53 ( 0.25
6.62 ( 0.25
6.85 ( 0.01
7.21 ( 0.16
6.86 ( 0.11
24.5
302
257
380
38.0
195
155
11.2
24.0
257
295
240
141
61.7
138
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
COMe
CO(cyclopropyl)
COPh
CO(Ph-4-CN)
CO(4-Py)
COCH2(thiophen-2-yl)
Ph-4-CN
CO2Me
CONMe2
SO2NMe2
SO2-Et
60.3
12.3
39.8
16.2
3.31
1.66
60.3
50.1
30.9
23.4
7.76
19.0
SO2-Ph
SO2-(Ph-4-CN)
a Binding potencies were assessed by displacement of 3H-N-R-methyl histamine. The human H3 values were from cloned human H3 expressed in C6
cells, while rat H3 values were from rat cortical membranes. The pKi (-log Ki) ( the standard error of the mean (SEM) are reported.
glucocorticoid, androgen, estrogen, or progesterone receptors
was detected. Since it was reported that dibasic compounds have
sometimes been associated with induction of phospholipidosis,33
compound 13c was scrutinized in an in vitro phospholipidosis
assay. It only weakly induced phospholipidosis (P.I. ) 0.42 at
6.25 µM), where the P.I. is the phospholipidosis index,33 a ratio
of the test compound’s propensity to induce phospholipidosis
compared to the reference standard toxin amiodarone (P.I. )
1.00 at 10 µM). In further in vivo studies, compound 13c was
found to be effective in a 24 h inhibitory avoidance behavioral
model tested in mice at 0.01 mg/kg (ip), consistent with the
fact that many histamine H3 receptor antagonists with in vitro
potency in the similar range have been shown to improve
performance in a number of rodent behavioral models.3,20,41
In summary, the conessine analogues described in this paper
represent a novel structural class of histamine H3 receptor
antagonistssaliphatic azasteroids. They are highly potent, se-
lective, and some members of the class were found to have good
drug likeness by most measures.
Experimental Section
Chemistry Methods. Unless otherwise noted, all commercially
available solvents, chemicals, and reagents were used without
purification. 1H NMR spectra were obtained on a Varian Mercury
plus 300 or Varian UNITY plus 300 MHz instrument, with chemical
shifts (δ, ppm) determined using TMS as internal standard.
Abbreviations used in description of NMR spectra: s ) singlet, d
) doublet, t ) triplet, dd ) double doublet, dt ) double triplet, m
) multiplet, br ) broad singlet. Mass spectra were obtained on a
Kratos MS-50 instrument, and unless otherwise indicated, all MS
instruments were operated in the +APCI or +DCI mode to detect
positively charged ions. Elemental analysis was performed by
Quantitative Technologies, Inc. Flash column chromatography was

Conessine and Analogues as Histamine H3 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5427
Table 5. Binding Affinities of Conessine 6 and Its Derivatives 13a-13e at Human and Rat H3Ra
a Binding potencies were assessed by displacement of 3H-N-R-methyl histamine. The human H3 values were from cloned human H3 expressed in C6
cells, while rat H3 values were from rat cortical membranes. The pKi (-log Ki) ( the standard error of the mean (SEM) are reported.
performed with prepacked ISCO cartridges. Thin-layer chroma-
tography was performed on 250 mm silica gel 60 glass-backed
plates from Merck with F254 as indicator.
clumps that were gently broken up with a glass rod. The dark,
nonpyrophoric Pb/Cd couple was filtered, washed with water and
acetone, then vaccuum-dried, crushed, and stored in a closed vessel.
This gives the 10% Pb/Cd couple (4.9 mmol Cd in 654 mg couple).
Then 10% Pb/Cd couple (1.2 g, 9 mmol Cd) was added to a
rapidly stirred mixture of compound 9 (800 mg, 1.54 mmol), THF
(6 mL), and aqueous 1N NH4OAc (6 mL). The mixture was stirred
for 5 h, then another portion of 10% Pb/Cd couple (1.0 g) was
added and then stirred overnight. The solid was filtered, the filtrate
was diluted with water, and adjusted the pH with saturated sodium
bicarbonate, then extracted with dichloromethane (3 × 50 mL).
The combined organic layers were dried over Na2SO4, filtered, and
evaporated to give crude product, which was purified by flash
chromatography (silica gel, 20:1:0.1 CH2Cl2/CH3OH/NH4OH) to
Methyl-((3S,3aS,5aS,5bR,9S,11aR,11bS,13aR)-2,3,11a-trimethyl-
2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-2-
aza-pentaleno[1,6a-a]phenanthren-9-yl)-carbamic Acid 2,2,2-
trichloro-ethyl Ester (9). 2,2,2-Trichloroethylchloroformate (5.94
g, 28 mmol) was added dropwise to a stirred solution of conessine
(6, 10 g, 28 mmol) in 200 mL of benzene. The reaction mixture
became a very thick gel. It was then heated at reflux for 4 h (oil
bath temperature 90 °C), then cooled down to room temperature,
quenched with water, adjusted the pH with 25 mL of saturated
sodium bicarbonate, and extracted with dichloromethane (3 × 100
mL). The combined organic layers were dried over Na2SO4, filtered,
and evaporated to give crude product, which was purified by flash
chromatography (silica gel, 20:1:0.1 CH2Cl2/CH3OH/NH4OH) to
1
provide 397 mg (75%) of the title compound. H NMR (CDCl3):
δ ppm 0.94 (s, 3H), 0.98 (m, 1H), 1.06 (d, J ) 6.1 Hz, 3H),
1.09-1.26 (m, 5H), 1.33-1.46 (m, 4H), 1.58-1.67 (m, 3H),
1.72-1.91 (m, 6H), 2.05-2.12 (m, 2H), 2.22 (s, 3H), 2.46 (s, 3H),
3.01 (d, J ) 10.4 Hz, 1H), 5.35 (m, 1H). MS (DCI/NH3): m/z 343
(M + H)+.
N-Methyl-N-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-trimeth-
yl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-
2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-acetamide (12a). Com-
pound 11 (12 mg, 0.035 mmol) and triethylamine (15 µL, 3.0 equiv)
were dissolved in dichloromethane (1 mL). Acetyl chloride (3.8
µL, 0.053 mmol, 1.5 equiv) was added to it dropwise. The mixture
was stirred at room temperature for 2 h, then quenched with water
and extracted with dichloromethane (3 × 5 mL). The combined
organic layers were dried over sodium sulfate and concentrated to
1
provide 8.8 g (61%) of the title compound. H NMR (CDCl3): δ
ppm 0.96 (s, 3H), 1.04-1.26 (m, 7H), 1.34-1.43 (m, 3H), 1.55 (s,
3H), 1.64-1.92 (m, 9H), 2.04-2.13 (m, 2H) 2.21 (s, 3H),
2.35-2.52 9 (m, 2H), 2.91 (s, 3H), 3.89 (m, 1H), 4.74 (s, 2H),
5.35 (m, 1H). MS (DCI/NH3): m/z 517 (M + H)+.
Methyl-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-trimethyl-
2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-2-
aza-pentaleno[1,6a-a]phenanthren-9-yl)-amine (11). Preparation
of 10% Pb/Cd couple:37 yellow lead oxide (PbO, 108 mg,
0.49mmol) was dissolved in 5 mL of 50% aqueous AcOH, and the
solution was slowly added to a vigorously stirred suspension of
Cd dust (100 mesh, 546 mg, 4.9 mmol) in deionized water (10
mL). The Cd darkened as Pb deposited on its surface and formed

5428 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Zhao et al.
give the crude product, which was then purified by flash chroma-
tography (silica gel, 20:1:0.1 CH2Cl2/CH3OH/NH4OH) to provide
10.5 mg (78.0%) of the title compound. 1H NMR (CDCl3): δ ppm
0.93 (m, 3H), 1.08 (m, 3H), 1.31 (m, 4H), 1.57 (m, 3H), 1.68 (m,
3H), 1.87 (m, 7H), 2.10 (m, 3H), 2.23 (m, 3H), 2.37 (m, 1H), 2.52
(m, 1H), 2.80 (m, 3H), 2.87 (m, 3H), 3.35 (m, 1H), 3.73 (m, 1H),
5.36 (m, 1H). MS (DCI/NH3): m/z 385 (M + H)+.
6H), 1.63-1.68 (m, 4H), 1.79-1.85 (m, 3H), 1.93-1.20 (m, 2H),
2.06-2.13 (m, 3H), 2.43-2.52 (m, 2H), 2.88 (s, 3H), 3.63 (m,
1H), 5.39 (m, 1H), 6.69 (d, J ) 8.80 Hz, 2H), 7.44 (d, J ) 8.80
Hz, 2H). MS (DCI/NH3): m/z 444 (M + H)+.
Methyl-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-trimethyl-
2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-2-
aza-pentaleno[1,6a-a]phenanthren-9-yl)-carbamic acid methyl
ester (12h). Compound 12h (11.0 mg, 79.0%) was prepared by
using the procedure for making compound 12a, except substituting
methyl chloroformate for acetyl chloride. 1H NMR (CDCl3): δ ppm
0.92 (s, 3H), 1.02-1.39 (m, 7H), 1.53 (s, 3H), 1.56 (s, 3H),
1.69-1.91 (m, 8H), 1.98-2.13 (m, 2H), 2.21-2.26 (m, 2H),
2.37-2.55 (m, 2H), 2.65 (m, 1H), 2.80 (d, 3H), 3.36 (m, 1H), 3.69
(s, 3H), 3.76 (m, 1H), 5.33 (m, 1H). MS (DCI/NH3): m/z 401 (M
+ H)+.
1,1,3-Trimethyl-3-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-
trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahy-
dro-1H-2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-urea (12i). Com-
pound 11 (15.0 mg, 0.044 mmol) and triethylamine (25 µL, 4.0
equiv) were dissolved in dichloromethane (1 mL). Dimethylcar-
bamyl chloride (6 µL, 0.065 mmol, 1.5 equiv) was added to it
dropwise. The mixture was stirred at room temperature overnight.
The resulting clear solution was directly loaded on silica gel column
and eluted with 20:1:0.1 CH2Cl2/CH3OH/NH4OH to provide 14.0
mg (76.9%) of the title compound. 1H NMR (CDCl3): δ ppm 0.95
(s, 3H), 1.04-1.39 (m, 10H), 1.55 (s, 6H), 1.60-1.92 (m, 10H),
2.02-2.10 (m, 2H), 2.20 (m, 2H), 2.35 (m, 1H), 2.45-2,55 (m,
1H) 2.72 (s, 3H), 2.78 (s, 3H), 3.47 (m, 1H), 5.35 (m, 1H). MS
(DCI/NH3): m/z 414 (M + H)+.
Cyclopropanecarboxylicacidmethyl-((3S,3aS,5aS,5bR,9S,11aR,
13aR)-2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,
13-hexadecahydro-1H-2-aza-pentaleno[1,6a-a]phenanthren-9-
yl)-amide (12b). Compound 12b (12.8 mg, 88.6%) was prepared
by using the procedure for making compound 12a, except substitut-
1
ing cyclopropane carbonyl chloride for acetyl chloride. H NMR
(CDCl3): δ ppm 0.75 (m, 4H), 0.96 (s, 3H), 0.98 (s, 3H), 1.00-2.60
(m, 23H), 2.87 (s, 3H), 3.04 (s, 3H), 3.94 (m, 1H), 4.40 (m, 1H),
5.38 (m, 1H). MS (DCI/NH3): m/z 411 (M + H)+.
N-Methyl-N-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-trimeth-
yl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-
2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-benzamide (12c). Com-
pound 12c (13.0 mg, 83.1%) was prepared by using the procedure
for making compound 12a, except substituting benzoyl chloride
for acetyl chloride. 1H NMR (CDCl3): δ ppm 0.93 (s, 3H),
1.02-1.50 (m, 7H), 1.53 (s, 3H), 1.56 (s, 3H), 1.83-2.52 (m, 14H),
2.79 (d, J ) 4.4 Hz, 3H), 3.02 (m, 1H), 3.35 (m, 1H), 3.74 (m,
1H), 5.32 (m, 1H), 7.33-7.40 (m, 5H). MS (DCI/NH3): m/z 447
(M + H)+.
4-Cyano-N-methyl-N-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-
trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-
1H-2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-benzamide (12d). Com-
pound 12d (8.0 mg, 48.5%) was prepared by using the procedure
for making compound 12a, except substituting 4-cyanobenzoyl
chloride for acetyl chloride. 1H NMR (CDCl3): δ ppm 0.95 (d,
3H), 1.00-1.48 (m, 7H), 1.53 (s, 3H), 1.56 (s, 3H), 1.84-2.65
(m, 14H), 2.79 (d, J ) 4.4 Hz, 3H), 3.02 (m, 1H), 3.35 (m, 1H),
3.72 (m, 1H), 5.33 (m, 1H), 7.46 (m, 2H), 7.70 (d, J ) 8.14 Hz,
2H). MS (DCI/NH3): m/z 472 (M + H)+.
N-Methyl-N-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-trimeth-
yl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-
2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-isonicotinamide (12e).
Compound 12e (8.8 mg, 56.2%) was prepared by using the
procedure for making compound 12a, except substituting isonico-
tinoyl chloride hydrochloride for acetyl chloride. 1H NMR (CDCl3):
δ ppm 0.91 (s, 3H), 0.96 (s, 3H), 1.00-2.75 (m, 21H), 2.80 (m,
3H), 3.03 (s, 3H), 3.27 (m, 1H), 3.71 (m, 1H), 4.47 (m, 1H), 5.30
(m, 1H), 7.39 (d, J ) 4.07 Hz, 2H), 8.71 (d, J ) 4.07 Hz, 2H). MS
(DCI/NH3): m/z 448 (M + H)+.
1,1-Dimethyl-3-methyl-3-((3S,3aS,5aS,5bR,9S,11aR,13aR)-
2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexa-
decahydro-1H-2-aza-pentaleno[1,6a-a][phenanthren-9-yl)-sul-
famide (12j). Compound 12j (10.0 mg, 50.5%) was prepared by
using the procedure for making compound 12i, except substituting
dimethylsulfomoyl chloride for dimethylcarbamyl chloride. 1H
NMR (CDCl3): δ δ ppm 0.93 (s, 3H), 0.99-1.44 (m, 10H), 1.55
(s, 6H), 1.62-1.92 (m, 10H), 2.02-2.12 (m, 2H), 2.22 (m, 2H),
2.35 (m, 1H), 2.42-2,53 (m, 1H) 2.76 (s, 3H), 2.77 (s, 3H), 3.57
(m, 1H), 5.38 (m, 1H). MS (DCI/NH3): m/z 450 (M + H)+.
Ethanesulfonic acid methyl-((3S,3aS,5aS,5bR,9S,11aR,13aR)-
2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexa-
decahydro-1H-2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-
amide (12k). Compound 12k (12.5 mg, 82.0%) was prepared by
using the procedure for making compound 12i, except substituting
1
ethanesulfonyl chloride for dimethylcarbamyl chloride. H NMR
(CDCl3): δ δ ppm 0.91 (s, 3H), 1.06-1.26 (m, 10H), 1.34 (t, J )
7.5 Hz, 3H), 1.53 (s, 3H), 1.55 (s, 3H), 1.66-1.90 (m, 10H), 2.08
(m, 1H), 2.24 (m, 1H), 2.52 (m, 1H), 2.83 (s, 3H), 2..96 (q, J )
7.46 Hz, 2H), 3.72 (m, 1H), 5.36 (m, 1H). MS (DCI/NH3): m/z
435 (M + H)+ and 452 (M + NH4)+.
N-Methyl-N-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-trimeth-
yl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-
2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-benzenesulfona-
mide (12l). Compound 12l (12.5 mg, 82.0%) was prepared by using
the procedure for making compound 12i, except substituting
benzenesulfonyl chloride for dimethylcarbamyl chloride. 1H NMR
(CDCl3): δ ppm 0.85 (s, 3H), 0.95 (m, 1H), 1.03 (d, 3H), 1.10-1.45
(m, 9H), 1.50-1.90 (m, 9H), 2.03 (m, 1H), 2.18 (m, 3H), 2.34 (m,
2H), 2.78 (s, 3H), 2.95 (m, 1H), 3.76 (m, 1H), 5.22 (m, 1H), 7.48
(m, 2H), 7.57 (m, 1H), 7.81 (m, 2H). MS (DCI/NH3): m/z 483 (M
+ H)+ and 500 (M + NH4)+.
4-Cyano-N-methyl-N-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-
trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadeca-hy-
dro-1H-2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-benzenesulfona-
mide (12m). Compound 12m (20.0 mg, 89.5%) was prepared by
using the procedure for making compound 12i, except substituting
4-cyanobenzenesulfonyl chloride for dimethylcarbamyl chloride.
1H NMR (CDCl3): δ ppm 0.88 (s, 3H), 1.04-2.39 (m, 10H), 1.55
(s, 6H), 1.60-1.84 (m, 7H), 2.02 (m, 1H), 2.19 (m, 2H), 2.33 (m,
2H), 2.81 (s, 3H), 2.96 (m, 1H), 3.75 (m, 1H), 5.25 (m, 1H), 7.80
(m, 2H), 7.93 (m, 2H). MS (DCI/NH3): m/z 508 (M + H)+.
N-Methyl-2-thiophen-2-yl-N-((3S,3aS,5aS,5bR,9S,11aR,13aR)-
2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexa-
decahydro-1H-2-aza-pentaleno[1,6a-a]phenanthren-9-yl)-ace-
tamide (12f). Compound 12f (4.0 mg, 24.5%) was prepared by
using the procedure for making compound 12a, except substituting
1
2-thiopheneacetyl chloride for acetyl chloride. H NMR (CDCl3):
δ ppm 0.93 (s, 3H), 1.04 (d, 3H), 1.05-2.15 (m, 18H), 2.20 (s,
3H), 2.38 (m, 2H), 2.50 (m, 1H), 2.90 (d, 3H), 3.01 (m, 1H), 3.66
(m, 1H), 3.90 (d, 2H), 4.42 (m, 1H), 5.34 (m, 1H), 6.87 (m, 1H),
6.95 (m, 1H), 7.18 (m, 1H). MS (DCI/NH3): m/z 467 (M + H)+.
4-[Methyl-((3S,3aS,5aS,5bR,9S,11aR,13aR)-2,3,11a-trimethyl-
2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-2-aza-
pentaleno[1,6a-a]phenanthren-9-yl)-amino]-benzonitrile (12g). A
mixture of compound 11 (20 mg, 0.058 mmol), 4-bromobenzonitrile
(16 mg, 0.088 mmol), tris(dibenzylideneacetone)dipallidium (2.1
mg, 0.0023 mmol), racemic-2,2′-bis(diphenylphosphino)-1,1′-bi-
naphthyl (BINAP) (2.2 mg, 0.0035 mmol), and cesium carbonate
(29 mg, 0.088 mmol) in toluene (1 mL) were mixed and heated at
100 °C overnight. The reaction mixture was cooled, quenched with
water, and extracted with dichloromethane (3 × 5 mL). The
combined organic layer was dried over sodium sulfate, filtered, and
concentrated to give the crude product, which was purified by flash
chromatography (silica gel, 20:1:0.1 CH2Cl2/CH3OH/NH4OH) to
provide 12 mg (46%) of the title compound. 1H NMR (CDCl3): δ
ppm 0.99 (s, 3H), 1.09-1.27 (m, 7H), 1.38-1.42 (m, 2H), 1.55 (s,

Conessine and Analogues as Histamine H3 Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5429
2-Amino-3-(S)-methyl-pentanoicacidmethyl-((3S,3aS,5aS,5bR,
9S,11aR,11bS,13aR)-2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,
11,11a,11b,12,13-hexadecahydro-1H-2-aza-pentaleno[1,6a-a]ph-
enanthren-9-yl)-amide L-Tartrate (13a). A mixture of compound
11 (1.0 g, 2.92 mmol), N-Boc-L-isoleucine (811 mg, 3.51 mmol,
1.2 equiv), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
drochloride (840 mg, 4.38 mmol, 1.5 equiv), and 1-hydroxyben-
zotriazole (590 mg, 4.37 mmol, 1.5 equiv) in dichloromethane (15
mL) was stirred at room temperature overnight. The solvent was
evaporated under reduced pressure, and the residue was dissolved
in minimum amount of dichloromethane and purified by flash
chromatography (silica gel, 30:1:0.1 CH2Cl2/CH3OH/NH4OH) to
provide 1.57 g (96.9%) of the Boc protected title compound. It
was dissolved in a mixture dichloromethane (20 mL) and TFA (20
mL) and stirred at room temperature for 3 h. The solvent was
evaporated under reduced pressure and the residue was triturated
with dichloromethane (3×). The residue was then dissolved in ethyl
acetate, stirred with sodium bicarbonate powder for 30 min, and
filtered. The filtrate was concentrated and purified by flash
chromatography (silica gel, 25:1:0.1 CH2Cl2/CH3OH/NH4OH) to
provide 0.87 g (65.4%) of the free base. It was dissolved in
methanol (100 mL) and stirred with L-tartaric acid (573 mg, 3.82
mmol, 2 equiv) at room temperature overnight, concentrated, and
dried on high vacuum to provide 1.44 g (100%) of the title
compound. 1H NMR (300 MHz, DMSO-d6) δ ppm 0.80-0.96 (m,
9 H), 0.99-1.07 (m, 1H), 1.12 (d, J ) 6.44 Hz, 3H), 1.16-1.22
(m, 4H), 1.32-1.53 (m, 8H), 1.53-1.71 (m, 5H), 1.72-1.96 (m,
5H), 1.97-2.10 (m, 2H), 2.30 (d, J ) 11.53 Hz, 1H), 2.43 (s, 3H),
2.57 (m, 1H), 2.93 (s, 3H), 3.22 (d, J ) 11.19 Hz, 1H), 3.94 (s,
4H), 4.08 (t, J ) 6.27 Hz, 1H), 4.12 - 4.25 (m, 1H), 5.07 (br,
8H), 5.34 (m, 1H). MS (DCI/NH3): m/z 456 (M + H)+.
NH4OH) to provide 16.0 g (73.1%) of the title compound. 1H NMR
(CDCl3): δ ppm 0.87-1.02 (m, 10H), 1.15-1.26 (m, 4H),
1.34-1.51 (m, 6H), 1.55 (s, 6H), 1.65-1.91 (m, 4H), 1.80-1.93
(m, 4H) 1.98 (d, J ) 4.4 Hz, 3H), 2.13-2.64 (m, 4H), 2.85 (s,
2H), 2.95 (s, 3H), 4.30 (m, 1H), 5.00 (m, 1H), 5.36 (m, 1H), 6.30
(m, 1H). MS (DCI/NH3): m/z 498 (M + H)+.
2-((S)-Acetylamino)-N-methyl-3-phenyl-N-((3S,3aS,5aS,5bR,9S,
11aR,13aR)-2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,
12,13-hexadecahydro-1H-2-aza-pentaleno[1,6a-a]phenanthren-
9-yl)-propionamide (13e). Compound 13e (18.2 mg, 78.0%) was
prepared by using the procedure for making compound 13d, except
substituting N-acetyl-L-phenylalanine for N-acetyl-L-leucine. 1H
NMR (CDCl3): δ ppm 0.88 (m, 3H), 0.96 (m, 1H), 1.03 (s, 3H),
1.14-1.23 (m, 5H), 1.34-1.41 (m, 3H), 1.54 (m, 1H), 1.60-1.87
(m, 10H), 1.99 (s. 3H), 2.08 (m, 1H), 2.19 (s, 3H), 2.31-2.35 (m,
2H), 2.78 (s, 3H), 2.97 (m, 2H), 4.27 (m, 1H), 5.15 (m, 1H), 5.35
(m, 1H), 6.39 (m, 1H), 7.14-7.30 (m, 5H). MS (DCI/NH3): m/z
532 (M + H)+.
Method for 24 h Inhibitory Avoidance Test. Mice were in-
jected ip with test compound (or in the case of control animals,
with identical carrier vehicle) in a volume of 10 mL/kg and placed
back into their home cage. Twenty minutes afterward, a training
was begun by placing mice in the light side of a two-chambered
compartment. The latency to enter the adjoining dark compartment
was recorded; upon entering the darkened compartment, an inescap-
able footshock (0.2 mA, 1 s duration) was presented to the mouse.
In the memory retention test 24 h later, the mouse was retested
using methods identical to those on the training day, with the latency
to enter the darkened compartment recorded, with learning evi-
denced by a statistically significant (p < 0.05) increase in the latency
to enter the darkened chamber.
2-Amino-3-(R)-methyl-pentanoicacidmethyl-((3S,3aS,5aS,5bR,
9S,11aR,11bS,13aR)-2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,
11,11a,11b,12,13-hexadecahydro-1H-2-aza-pentaleno[1,6a-a]ph-
enanthren-9-yl)-amide L-Tartrate (13b). Compound 13b (104.3
mg, 59.2%) was prepared by using the procedure for making
compound 13a, except substituting N-Boc-D-isoleucine for N-Boc-
Supporting Information Available: Combustion analysis of
compounds 12a-12m and 13a-13e and graphical result of
compound 13c in the rodent inhibitory avoidance behavioral model.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
L-isoleucine. H NMR (300 MHz, DMSO-d6) δ ppm 0.82-0.96
(m, 9 H), 1.03-1.07 (m, 1H), 1.15 (d, J ) 6.78 Hz, 3H), 1.21-1.45
(m, 12H), 1.57-1.68 (m, 5H), 1.77-1.95 (m, 5H), 1.98-2.09 (m,
2H), 2.40 (d, J ) 8.48 Hz, 1H), 2.45 (s, 3H), 2.57 (m, 1H), 2.93
(s, 3H), 3.27 (d, J ) 11.19 Hz, 1H), 3.99 (s, 4H), 4.13 (m, 1H),
4.15-4.31 (m, 1H), 5.20 (br, 8H), 5.30 (m, 1H). MS (DCI/NH3):
m/z 456 (M + H)+.
References
(1) (a) Hill, S. J.; Ganellin, C. R.; Timmerman, H.; Schwartz, J. C.;
Shankley, N. P.; Young, J. M.; Schunack, W.; Levi, R.; Haas, H. L.
International Union of Pharmacology. XIII. Classification of Histamine
Receptors. Pharmacol. ReV. 1997, 49, 253–278. (b) Nakamura, T.;
Itadani, H.; Hidaka, Y.; Ohta, M.; Tanaka, K. Molecular Cloning and
Characterization of a new Human Histamine Receptor, HH4R.
Biochem. Biophys. Res. Commun. 2000, 279, 615–620.
(2) (a) Schlicker, E.; Malinowska, B.; Kathmann, M.; Gothert, M.
Modulation of Neurotransmitter Release via Histamine H3 Heterore-
ceptors. Fundam. Clin. Pharmacol. 1994, 8, 128–137. (b) Morisset,
S.; Rouleau, A.; Ligneau, X.; Gbahou, F.; Tardivel-Lacombe, J.; Stark,
H.; Schunack, W.; Ganellin, C. R.; Schwartz, J.-C.; Arrang, J.-M. High
constitutive activity of native H3 receptors regulates histamine neurons
in brain. Nature 2000, 408, 860–864. (c) Brown, R. E.; Stevens, D. R.;
Haas, H. L. The physiology of brain histamine. Prog. Neurobiol. 2001,
63, 637–672.
(3) Esbenshade, T. A.; Fox, G. B.; Cowart, M. D. Histamine H3 receptor
antagonists: preclinical promise for treating obesity and cognitive
disorders. Mol. InterVentions 2006, 6, 77–88.
(4) (a) Leurs, R.; Blandina, P.; Tedford, C.; Timmerman, H. Therapeutic
potential of histamine H3 receptor agonists and antagonists. Trends
Pharmacol. Sci. 1998, 19, 177–183. (b) Stark, H.; Schlicker, E.;
Schunack, W. Developments of histamine H3-receptor antagonists.
Drugs Future 1996, 21, 507–520.
(5) Oades, R. D. Attention deficit disorder with hyperactivity (ADDH):
The contribution of catecholaminergic activity. Prog. Neurobiol. 1987,
29, 365–391.
(6) Monti, J. M. Involvement of histamine in the control of the waking
state. Life Sci. 1993, 53, 1331–1338.
(7) Vohora, D.; Pal, S. N.; Pillai, K. K. Histamine and selective H3-receptor
ligands: a possible role in the mechanism and management of epilepsy.
Pharmacol., Biochem. BehaV. 2001, 68, 735–741.
(8) Velligan, D. I.; Miller, A. L. Cognitive dysfunction in schizophrenia
and its importance to outcome: the place of atypical antipsychotics in
treatment. J. Clin. Psychiatry 1999, 60, 25–28.
3-(R)-Methyl-N-methyl-2-methylamino-N-((3S,3aS,5aS,5bR,9S,
11aR,13aR)-2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,
12,13-hexadecahydro-1H-2-aza-pentaleno[1,6a-a]phenanthren-
9-yl)-butyramide L-Tartrate (13c). Compound 13c (3.2, 80.0%)
was prepared by using the procedure for making compound 13a,
except substituting N-Boc-D-valine for N-Boc-L-isoleucine. 1H NMR
(CDCl3): δ 0.95 ppm (m, 12H), 1.00-2.20 (m, 19H), 2.25 (s, 3H),
2.29 (s, 3H), 2.40-2.80 (m, 3H), 2.91 (d, 3H), 3.05 (m, 3H), 3.65
(m, 1H), 4.48 (m, 1H), 5.38 (m, 1H). 1H NMR (300 MHz, DMSO-
d6) δ ppm 0.91-0.96 (m, 9 H), 1.03-1.07 (m, 1H), 1.15 (d, J )
6.78 Hz, 3H), 1.22-1.40 (m, 8H), 1.46-1.54 (m, 1H), 1.60-1.70
(m, 5H), 1.77-1.97 (m, 5H), 2.01-2.12 (m, 2H), 2.35 (s, 3H),
2.57 (m, 1H), 2.94 (s, 3H), 3.11 (m, 1H), 3.17 (s, 3H), 3.32 (d, J
) 11.53 Hz, 1H), 3.88 (m, 1H), 4.00 (s, 4H), 4.19 - 4.26 (m, 1H),
4.76 (br, 8H), 5.34 (m, 1H). MS (DCI/NH3): m/z 456 (M + H)+.
2-Acetylamino-4-(S)-methyl-pentanoic acid methyl-((3S,3aS,
5aS,5bR,9S,11aR,11bS,13aR)-2,3,11a-trimethyl-2,3,3a,4,5,5a,5b,
6,8,9,10,11,11a,11b,12,13-hexadecahydro-1H-2-aza-pentaleno[1,6a-a]-
phenanthren-9-yl)-amide (13d). A mixture of compound 11 (15
m g, 0.044 mmol), N-acetyl-L-leucine (9.0 mg mg, 0.053 mmol,
1.2 equiv), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
drochloride (13 mg, 0.068 mmol, 1.5 equiv), and 1-hydroxyben-
zotriazole (9 mg, 0.068 mmol, 1.5 equiv) in dichloromethane (1
mL) and THF (0.5 mL) was stirred at room temperature overnight.
The solvent was evaporated under reduced pressure, and the residue
was dissolved in minimum amount of dichloromethane and purified
by flash chromatography (silica gel, 30:1:0.1 CH2Cl2/CH3OH/
(9) (a) Hancock, A. A. H3 receptor antagonists/inverse agonists as anti-
obesity agents. Curr. Opin. InVest. Drugs 2003, 4, 1190–1197. (b)

5430 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Zhao et al.
Hancock, A. A.; Bush, E. N.; Jacobson, P. B.; Faghih, R.; Esbenshade,
T. A. Histamine H3 antagonists in models of obesity. Inflammation
Res. 2004, 53, S47-S48.
(28) Pompni, S. A.; Gullo, V. P.; Horan, A. C.; Patel, M. G.; Coval, S.
U.S. Patent 5,352,7071994.
(29) (a) Pompni, S. A.; Gullo, V. P.; Horan, A. C.; Patel, M. G.; Coval, S.
U.S. Patent US 5,217,9861993. (b) Mierzwa, R.; King, A.; Conover,
M. A.; Tozzi, S.; Puar, M. S.; Patel, M.; Coval, S. J. Verongamine, a
novel bromotyrosine-derived histamine H3-antagonist from the marine
sponge Verongula gigantean. J. Nat. Prod. 1994, 57, 175–177.
(30) Chen, Z.; Sakurai, E.; Hu, W.; Jin, C.; Kiso, Y.; Kato, M.; Watanabe,
T.; Wei, E.; Yanai, K. Pharmacological effects of carcinine on
histaminergic neurons in the brain. Br. J. Pharmacol. 2004, 143, 573–
580.
(31) (a) Cowart, M.; Sun, M.; Zhao, C.; Witte, D. G.; Miller, T. R.; Krueger,
K. M.; Browman, K.; Fox, G. B.; Bennani, Y. L.; Esbenshade, T. A.;
Hancock, A. A. A new family of histamine H3 receptor antagonists
based on a natural product: discovery, SAR, and properties of the
series. Inflamm. Res 2007, 56, S1-S2. (b) Santora, V. J.; Covel, J. A.;
Hayashi, R.; Hofilena, B. J.; Ibarra, J. B.; Pulley, M. D.; Weinhouse,
M. I.; Sengupta, D.; Duffield, J. J.; Semple, G.; Webb, R. R.; Sage,
C.; Ren, A.; Pereira, G.; Knudsen, J.; Edwards, J. E.; Suarez, M.;
Frazer, J.; Thomsen, W.; Hauser, E.; Whelanc, K.; Grottick, A. J. A
new family of H3 receptor antagonists based on the natural product
Conessine. Bio. Med. Chem. Lett. 2008, 18, 1490–1494.
(10) Arrang, J. M.; Garbarg, M.; Schwartz, j.C. Auto-inhibition of brain
histamine release mediated by a novel class (H3) of histamine receptor.
Nature 1983, 302, 832–837.
(11) Lovenberg, T. W.; Roland, B. L.; Wilson, S. J.; Jiang, X.; Pyati, J.;
Hurvar, A.; Jackson, M. R.; Erlander, M. G. Cloning and functional
expression of the human histamine H3 receptor. Mol. Pharmacol. 1999,
55, 1101–1107.
(12) Stark, H. Recent advances in histamine H3/H4 receptor. Expert Opin.
Ther. Pat. 2003, 13, 851–865.
(13) Stark, H.; Kathmann, M.; Schlicker, E.; Schunack, W.; Schlegel, B.;
Wolfgang, S. Medicinal chemical and pharmacological aspects of
imidazole-containing histamine H3 receptor antagonists. Mini ReV.
Med. Chem. 2004, 965–977.
(14) Arrang, J. M.; Garbarg, M.; Lancelot, J. C.; Lecomte, J. M.; Pollard,
H.; Robba, M.; Schunack, W.; Schwartz, J.-C. Highly potent and
selective ligands for histamine H3 receptors. Nature 1987, 327, 117–
123.
(15) Ligneau, X.; Lin, J.-S.; Vanni-Mercier, G.; Jouvet, M.; Muir, J. L.;
Ganellin, C. R.; Stark, H.; Elz, S.; Schunack, W.; Schwartz, J. C.
Neurochemical and Behavioural Effects of Ciproxifan, a Potent
Histamine H3 Receptor Antagonist. J. Pharmacol. Exp. Ther. 1998,
287, 658–666.
(16) Tedford, C. E.; Hoffmann, M.; Seyedi, N.; Maruyama, R.; Levi, R.;
Yates, S. L.; Ali, S. L.; Phillips, J. G. High antagonist potency of
GT-2227 and GT-2331, new histamine H3 receptor antagonists, in two
functional models. Eur. J. Pharmacol. 1998, 351, 307–311.
(17) (a) Esbenshade, T.; Krueger, K.; Denny, L.; Miller, T.; Kang, C.; Witte,
D.; Yao, B.; Black, L.; Bennani, Y.; Fox, G.; Hancock, A. A. Complex
pharmacological effects of GT-2331. Soc. Neurosci. Abstr. 2001, 27,
378–411. (b) Leurs, R.; Tulp, M. T.; Menge, W. M.; Adolfs, M. J.;
Zuiderveld, O. P.; Timmerman, H. Evaluation of the receptor
selectivity of the H3 receptor antagonists, iodophenpropit and thiop-
eramide: an interaction with the 5-HT3 receptor revealed. Br. J.
Pharmacol. 1995, 116, 2315–2321.
(18) (a) Lin, J.; Lu, A. Y. H. Inhibition and Induction of Cytochrome P450
and the Clinical Implications. Clin. Pharmacokinet. 1998, 35, 361–
390. (b) Yang, R.; Hey, T. A.; Aslanian, R.; Rizzo, C. A. Coordination
of Histamine H3 Receptor Antagonists with Human Adrenal Cyto-
chrome P450 Enzymes. Pharmacology 2002, 66, 128–135. (c) Zhang,
M.; Ballard, M. E.; Pan, P.; Roberts, S.; Faghih, R.; Cowart, M. D.;
Esbenshade, T. A.; Fox, G. B.; Decker, M. W.; Hancock, A. A.; Rueter,
L. E. Lack of cataleptogenic potentiation with non-imidazole H3
receptor antagonists reveals potential drug-drug interactions between
imidazole-based H3 receptor antagonists and antipsychotic drugs. Brain
Res. 2005, 1045, 142–149.
(19) Cowart, M. D.; Altenbach, R; Black, L.; Faghih, R.; Zhao, C.; Hancock,
A. A. Medicinal chemistry and biological properties of non-imidazole
histamine H3 antagonists. Mini ReV. Med. Chem. 2004, 979–992.
(20) Cowart, M. D.; Faghih, R.; Curtis, M. P.; Gfesser, G. A.; Bennani,
Y. L.; Black, L. A.; Pan, L.; Marsh, K. C.; Sullivan, J. P.; Esbenshade,
T. A.; Fox, G. B.; Hancock, A. A. (2-(2(R)-Methyl-pyrrolidine-1-
yl)ethyl)benzofuran-5-yl)-benzonitrile (ABT-239) and related 2-ami-
noethylbenzofuran H3 antagonists potently enhance cognition and
attention. J. Med. Chem. 2005, 48, 38–55.
(32) For assay conditions, see Esbenshade, T. A.; Krueger, K. M.; Miller,
T. R.; Kang, C. H.; Denny, L. I.; Witte, D. G.; Yao, B. B.; Fox, G. B.;
Faghih, R.; Bennani, Y.; Williams, M.; Hancock, A. A. Two Novel
and Selective Nonimidazole Histamine H3 Receptor Antagonists
A-304121 and A-317920: I. In Vitro Pharmacological Effects. J. Phar-
macol. Exp. Ther. 2003, 305, 887–896.
(33) (a) Ulrich, R. G.; Kilgore, K. S.; Sun, E. L.; Cramer, C. T.; Ginsburg,
L. C. An in vitro fluorescence assay for the detection of drug-induced
cytoplasmic lamellar bodies. Toxicol. Methods 1991, 1, 89–105. (b)
Gum, R. J.; Hickman, D.; Fagerland, J. A.; Heindel, M. A.; Gagne,
G. D.; Schmidt, J. M.; Michaelides, M. R.; Davidsen, S. K.; Ulrich,
R. G. Analysis of two matrix metalloproteinase inhibitors and their
metabolites for induction of phospholipidosis in rat and human
hepatocytes. Biochem. Pharmacol. 2001, 62, 1661–1673. (c) Sun, M.;
Zhao, C.; Gfesser, G. A.; Thiffault, C.; Miller, T. R.; Marsh, K.; Wetter,
J.; Curtis, M.; Faghih, R.; Esbenshade, T. A.; Hancock, A. A.; Cowart,
M. D. Synthesis and SAR of 5-Amino- and 5-Aminomethyl-benzo-
furan Histamine H3 Receptor Antagonists with Improved Potency.
J. Med. Chem. 2005, 48, 6482–6490. (d) Schurdak, M. E.; Vernetti,
L. A.; Abel, S. J.; Thiffault, C. Adaptation of an In Vitro Phospho-
lipidosis Assay to an Automated Image Analysis System. Toxicol.
Mech. Methods 2007, 17, 77–86.
(34) Kapnang, H.; Chaeles, G. Direct deamination of tertiary N,N-
dimethylamines. Tetrahedron Lett. 1980, 21, 2951–2954.
(35) Bhutani, K. K.; Vaid, R. M. An unusual oxidative demethylation
reaction of conessine with N-bromosuccinimide. Tetrahedron Lett.
1988, 29, 359–360.
(36) (a) Haworth, R. D. New alkaloid from Holarrhena antidysenterica
seeds. J. Chem. Soc. 1932, 631–634. (b) Zirihi, G. N.; Grellier, P.;
Guede-Guina, F.; Bodo, B.; Mambu, L. Isolation, characterization and
antiplasmodial activity of steroidal alkaloids from Funtumia elastica
(Preuss) Stapf. Bioorg. Med. Chem. Lett. 2005, 15(, 2637–2640.
(37) Dong, Q.; Anderso, C E.; Ciufolini, M. A. Reductive cleavage of
TROC groups under neutral conditions with cadmium-lead couple.
Tetrahedron Lett. 1995, 36, 5681–5682.
(21) Hancock, A. A. The challenge of drug discovery of a GPCR target:
Analysis of preclinical pharmacology of histamine H3 antagonists/
inverse agonists. Biochem. Pharmacol. 2006, 71, 1103–1113.
(22) Howard, H. R. Fused thiazole derivatives having affinity for the
histamine H3 receptor. Expert Opin. Ther. Patents 2007, 17, 129–
131.
(23) (a) Ulrici, F. Conessine. Arch Pharm. 1918, 256, 57–80. (b) Giemsa,
G.; Halberkann, J. Conessine. Arch. Pharm. 1918, 256, 201–222. (c)
McNiven, N. L. Some simple 18-oxygenated steroids: the constitution
of conessine. Chem. Ind. 1957, 1296–1297.
(24) Hermans, A.H.W.M. Conessine, the alkaloid from Holarrhena anti-
dysenterica. Pharmaceutisch. Weekblad 1927, 64, 821–828.
(25) Kavitha, D.; Shilpa, P. N.; Niranjali, D. S. Antibacterial effect of
conessine, an alkaloid from Holarrhena antidysenterica against
enteropathogenic Escherichia coli. Biomedicine 2003, 23, 66–71.
(26) Oberlies, N. H.; Kroll, D. J. Camptothecin and taxol: historic
achievements in natural products research. J. Nat. Prod. 2004, 67,
129–135.
(38) Esbenshade, T. A.; Hancock, A. A. Characterization of Histaminergic
Receptors. In Current Protocols of Pharmacology; John Wiley & Sons:
New York, 2000; Chapter 1.19.1.
(39) Esbenshade, T. A.; Fox, G. B.; Krueger, K. M.; Miller, T. R.; Kang,
C. H.; Denny, L. I.; et al. Pharmacological properties of ABT-239
[4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzoni-
trile]: I. Potent and selective histamine H3 receptor antagonist with
drug-like properties. J. Pharm. Exp. Ther. 2005, 313, 165–175.
(40) Method for genotoxicity test see Hancock, A. A.; Marilyn, S. D.;
Ramin, F.; Bush, E. N.; Krueger, K. M.; Krishna, G.; Miller, T. R.;
Wilcox, D. M.; Nguyen, P.; Pratt, J. K.; Cowart, M. D.; Esbenshade,
T. A.; Jacobson, P. B. In vitro Optimization of Structure-Activity
Relationships of Analogues of A-331440 Combining Radioligand
Receptor Binding Assays and Micronucleus Assays of Potential
Antiobesity Histamine H3 Receptor Antagonists. Basic Clin. Phar-
macol. Toxicol. 2004, 95, 144–152.
(41) Hancock, A. A.; Fox, G. B. Perspectives on cognitive domains, H3
receptor ligands and neurological disease. Expert Opin. InVestig. Drugs
2004, 13, 1237–1248.
(27) Clement, J. A.; Yoder, B. J.; Kingston, D. G. I. Natural products as a
source of CNS-active agents. Mini-ReV. Org. Chem. 2004, 1, 183–
208.
JM8003625