SUBSTITUTED [(5H-PYRROLO[2,l - ][l,4]BENZODIAZEPIN-ll-
YL)PIPERAZIN-l-YL]-2,2-DIMETHYLPROPANOIC ACID COMPOUNDS AS
DUAL ACTIVITY HI INVERSE AGONISTS/5-HT2A ANTAGONISTS
Histamine plays an important role in a variety of physiological processes through
its interaction with at least four different G-protein coupled receptors, the H1-H4
receptors. In the CNS, HI receptors play a key role in the sleep regulation cycle and HI
antagonists/inverse agonists are known to induce somnolence.
Likewise, serotonin plays important roles in a variety of physiological processes
through its interaction with at least fourteen different G-protein coupled receptors.
Modulation of 5-HT2Areceptors in the CNS plays a key role in the sleep regulation cycle
and 5-HT2Aantagonists have been shown to improve slow wave sleep and sleep
maintenance in patients with insomnia.
Compounds having HI or 5-HT2Ainverse agonist or antagonist activity have been
used in the treatment of insomnia (e.g. doxepin and trazodone, respectively) and have
exhibited significant pharmacological effects in animal sleep studies. However, no
selective dual activity H 1/5-HT2Ainverse agonists/antagonists are currently commercially
available.
US 4,192,803 describes certain substituted 4-(5H-pyrrolo[2,l-c][l,4]
benzodiazepin-1 1-yl)piperazinyl compounds for use as antipsychotics and neuroleptics.
The present invention provides a family of substituted (5H-pyrrolo[2,lc][
l,4]benzodiazepin-ll-yl)piperazin-l-yl]-2,2-dimethylpropanoic acid compounds with
high inverse agonist potency for the HI receptor and high antagonist potency for the
5-HT2Areceptor. Certain compounds of the present invention are also selective for the
HI and 5-HT2Areceptors, particularly as against other histamine receptors, serotonin
receptors and other physiologically relevant receptors, particularly as against the 5-HT2c
receptor, GABAA receptor, muscarinic receptors, dopaminergic receptors, adrenergic
receptors, and the hERG channel. Certain compounds have also demonstrated through
animal models that they may be useful for the treatment of sleep disorders characterized
by poor sleep maintenance. As such, the compounds of the invention are believed to be
useful for the treatment of sleep disorders characterized by poor sleep latency or poor
sleep maintenance or both, such as the treatment of insomnia, as for example chronic or
transient primary insomnia, or chronic or transient secondary insomnia, or both.
Examples of secondary insomnia include, but are not limited to insomnia associated with
depressive disorders (e.g. major depressive disorder, dysthymia, and/or cyclothymia),
insomnia associated with anxiety disorders (e.g. generalized anxiety disorder and/or
social phobia), insomnia associated with pain (e.g. fibromyalgia, chronic bone or joint
pain, such as associated with inflammatory arthritis or osteoarthritis, or diabetic
neuropathic pain), insomnia associated with allergic reactions (e.g. allergic asthma,
pruritus, rhinitus, congestion, etc.), insomnia associated lung or airway disorders (e.g.
with obstructive sleep apnea, reactive airway disease, etc.), insomnia associated with
psychiatric disorders, dementia, and/or neurodegenerative diseases, and/or insomnia
associated with circadian rhythm sleep disorders (e.g. shift work sleep disorder, jet lag
disorder, delayed sleep phase disorder, advanced phase sleep disorder, and non-24 hour
sleep-wake syndrome, etc.).
Further, certain of the compounds of the present invention demonstrate
potentiation of their effects on non-rapid eye movement sleep (NREM sleep) and sleep
maintenance when coadministered with selective serotonin reuptake inhibitors.
The present i
I
where R1 is chloro or methyl;
R2 is methyl, ethyl, isopropyl, chloro, bromo, trifluoromethyl, or methylthio; and
R is hydrogen or methoxy;
or a pharmaceutically acceptable salt thereof.
In another aspect of the invention there is provided a pharmaceutical composition
comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, in
combination with at least one pharmaceutically acceptable carrier, diluent, or excipient.
Furthermore, this aspect of the invention provides a pharmaceutical composition adapted
for the treatment of insomnia, as for example insomnia characterized by prolonged sleep
latency or poor sleep maintenance or both, as for example primary insomnia, jet lag, shift
work sleep disorder, delayed sleep phase disorder, advanced phase sleep disorder, and/or
non-24 hour sleep-wake disorders, comprising a compound of Formula I or a
pharmaceutically acceptable salt thereof, in combination with one or more
pharmaceutically acceptable excipients, carriers, or diluents.
A further embodiment of this aspect of the invention provides a pharmaceutical
composition comprising a compound according to Formula I, or pharmaceutically
acceptable salt thereof, in combination with at least one pharmaceutically acceptable
carrier, exciepient or diluent, and optionally other therapeutic ingredients. In a yet further
embodiment of this aspect of the invention, the pharmaceutical composition further
comprises a second therapeutic agent which is a serotonin reuptake inhibitor, as for
example citalopram, paroxetine, fluoxetine and/or fluvoxetine.
The present invention also provides a method of treating insomnia, as for example
insomnia characterized by prolonged sleep latency or poor sleep maintenance or both, as
for example primary insomnia, jet lag, shift work sleep disorder, delayed sleep phase
disorder, advanced phase sleep disorder, and/or non-24 hour sleep-wake disorders, in a
mammal comprising administering to a mammal in need of such treatment an effective
amount of a compound of Formula I or a pharmaceutically acceptable salt thereof. In
another embodiment of this aspect of the invention, the method further comprises
administering in simultaneous, separate or sequential combination, a second therapeutic
agent which is a serotonin reuptake inhibitor, as for example citalopram, paroxetine,
fluoxetine and/or fluvoxetine. In one particular embodiment of these methods of
treatment, the mammal is a human.
This invention also provides a compound of Formula I or a pharmaceutically
acceptable salt thereof for use in therapy. Within this aspect, the invention provides a
compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the
treatment of insomnia. In further embodiments, the insomnia is characterized by
prolonged sleep latency or poor sleep maintenance or both, as for example primary
insomnia, jet lag, shift work sleep disorder, delayed sleep phase disorder, advanced phase
sleep disorder, and/or non-24 hour sleep-wake disorders. In another embodiment of this
aspect, the invention provides a compound according to Formula I, or a pharmaceutically
acceptable salt thereof, for use in simultaneous, separate or sequential combination with a
serotonin reuptake inhibitor, as for example citalopram, paroxetine, fluoxetine and/or
fluvoxetine, in the treatment of insomnia. One particular embodiment of this aspect of
the inventions, the uses are in mammals, particular humans.
Another aspect of this invention provides the use of a compound of Formula I, or
a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the
treatment of insomnia, as for example primary insomnia characterized by prolonged sleep
latency or poor sleep maintenance or both, as for example primary insomnia, jet lag, shift
work sleep disorder, delayed sleep phase disorder, advanced phase sleep disorder, and/or
non-24 hour sleep-wake disorders. Another embodiment of this aspect of the invention
provides the use of a compound of Formula I, or a pharmaceutically acceptable salt
thereof, and a second therapeutic agent which is a serotonin reuptake inhibitor, as for
example citalopram, paroxetine, fluoxetine and/or fluvoxetine, in the manufacture of a
medicament for the treatment of insomnia, as for example, insomnia characterized by
prolonged sleep latency and/or poor sleep maintenance, as for example primary insomnia,
jet lag, shift work sleep disorder, delayed sleep phase disorder, advanced phase sleep
disorder, and/or non-24 hour sleep-wake disorders.
For clarity, the following numbering of the tricyclic ring structure will be used
throughout the application:
3
Compounds of this invention have basic and acidic moieties, and accordingly
react with a number of organic and inorganic acids and bases to form pharmaceutically
acceptable salts. Pharmaceutically acceptable salts of each of the compounds of the
present invention are contemplated within the scope of the present application. The term
"pharmaceutically acceptable salt" as used herein, refers to any salt of a compound of the
invention that is substantially non-toxic to living organisms. Such salts include those
listed in Journal of Pharmaceutical Science, 66, 2-19 (1977), which are known to the
skilled artisan.
Preferred classes of compounds of the present invention are compounds wherein:
1) R3 is hydrogen;
2) R3 is methoxy;
3) R1 is chloro;
4) R1 is methyl;
5) R1 is chloro and R is hydrogen;
6) R1 is methyl and R3 is hydrogen;
7) R2 is methyl, ethyl, or isopropyl;
8) R2 is methyl;
9) R2 is chloro or bromo;
10) R2 is chloro;
11) R2 is trifluoromethyl; and
12) R2 is methylthio.
It will be understood that further preferred compounds are those combining the
above preferred selections for a given substituent or substituents with preferred selections
of other substituents. Examples of such combinations include, but are not limited to the
following preferred classes of compounds:
13) preferred compounds of any one of preferred classes 7-12 (which are
preferred selections for R2) wherein R3 is hydrogen (preferred class 1);
14) preferred compounds of any one of preferred classes 7-12 (which are preferred
selections for R2) wherein R1 is chloro and R3 is hydrogen (preferred class 5); and
15) preferred compounds of preferred class 8 wherein R is methoxy (preferred
class 2).
Specific preferred compounds are those described in the Examples including their
freebases and pharmaceutically acceptable salts thereof.
One certain preferred compound is
3-[4-(7-Chloro-2-methyl-5H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-yl)piperazin- 1-
yl]-2,2-dimethylpropanoic acid, or a pharmaceutically acceptable salt thereof
(i.e. the compound of Example 1 and pharmaceutically acceptable salts thereof).
Abbreviations used herein are defined as follows:
"BSA" means bovine serum albumin.
"DCG IV" means (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine.
"DCM" means dichloromethane.
"DMEM means Dulbecco's Minimum Eagle's Medium.
"DMSO" means dimethyl sulfoxide.
"DPBS" means Dulbecco's Phosphate Buffered Saline.
"DSC" means differential scanning calorimetry.
"EtOAc" means ethyl acetate.
"GC" means gas chromatography.
"HBSS" means Hank's Buffered Salt Solution.
"HEPES" means 4-(2-hydroxyethyl)- 1-piperazine ethanesulfonic acid.
"HPLC" means high pressure liquid chromatography
"nr." or "h" means hour or hours.
"IBMX" means 3-isobutyl-l-methylxanthine
"IC50" means the concentration at which 50% of the maximum inhibition is
achieved.
"i.v." means intravenous or intravenously
"i.p." means intraperitoneal.
"LC-MS" means HPLC-mass spectrography.
"MeOH" means methanol.
"mFST" means mouse forced swim test; an animal model for antidepressant
activity
"min." or "m" means minutes
"mp" means melting point.
"MS" means mass spectroscopy.
"MS (ES+)" means mass spectroscopy using electrospray ionization.
"MTBE" means methyl t-butyl ether.
"NMR" means nuclear magnetic resonance
"p.o." means per os, by mouth.
"SCX-2" means Biotage Isolute Flash SCX-2® strong cation exchange columns.
"THF" means tetrahydrofuran.
General Chemistry
The compounds of the present invention can be prepared according to the
following synthetic schemes by general methods well known and appreciated in the art.
Suitable reaction conditions for the steps of these schemes are well known in the art and
appropriate substitutions of solvents and co-reagents are within the skill of the art.
Likewise, it will be appreciated by those skilled in the art that synthetic intermediates
may be isolated and/or purified by various well known techniques as needed or desired,
and that frequently, it will be possible to use various intermediates directly in subsequent
synthetic steps with little or no purification. Furthermore, the skilled artisan will
appreciate that in some circumstances, the order in which moieties are introduced is not
critical. The particular order of steps required to produce the compounds of of the present
invention is dependent upon the particular compound being synthesized, the starting
compound, and the relative liability of the substituted moieties, as is well appreciated by
the skilled chemist. All substituents, unless otherwise indicated, are as previously
defined, and all reagents are well known and appreciated in the art.
Generally, a compound of formula I may be prepared from a compound of
formula II where Pg is a suitable carboxyl protecting group (Scheme 1). More
specifically, a compound of formula II where Pg is a C -C3 alkyl group is reacted with a
suitable deprotection agent such as aqueous sodium hydroxide in an organic co-solvent
such isopropyl alcohol to provide, following neutralization with an acid, a compound of
formula I .
Scheme 1
II I
Generally, a compound of formula II may be prepared from a compound of
formula III or a compound of formula IV (Scheme 2). More specifically, a compound of
formula III is reacted with phosphoryl chloride in a suitable solvent such as
methoxybenzene or dichloromethane to provide the imino chloride intermediate. The
imino chloride may be isolated or reacted directly with Pg-2,2-dimethyl-3-piperazin-l-yl
propanoate in the presence of a suitable base such as potassium carbonate to provide a
compound of formula II. The reaction is carried out in a suitable solvent such as
acetonitrile. Alternatively, the imino chloride may be reacted with piperazine in the
presence of a suitable base such as cesium carbonate to provide a compound of formula
rv. The reaction is carried out in a suitable solvent such as acetonitrile. A compound of
formula IV is alkylated with Pg-2,2-dimethyl-3-oxopropanoate in the presence of a
suitable reducing agent such as sodium triacetoxyborohydride to provide a compound of
formula II. The reaction is typically carried out in a solvent such as dichloromethane.
Scheme 2
IV
A compound of formula III may be prepared from a compound of formula V
where R is methyl or ethyl (Scheme 3). More specifically, a compound of formula V is
reacted with an agent suitable for reducing the aryl nitro group to the corresponding
aniline. Suitable reducing agents include hydrogen in the presence of a transition metal
catalyst such as platinum; iron in acetic acid; and tin dichloride in hydrochloric acid. The
corresponding aniline may be first isolated or directly reacted under cyclization
conditions to provide a compound of formula III. The cyclization is carried out in the
presence of an acid such as hydrochloric acid or a base such as potassium ?-butoxide. A
compound of formula V where is methyl or ethyl may be prepared as described in the
preparations or by procedures known in the chemical arts for the production of
structurally analogous compounds.
Scheme 3
V III
In the following illustrative preparations and examples, reagents were obtained
from a variety of commercial sources. Solvents are generally removed under reduced
pressure (evaporated). In some procedures indicated yields are representative crude
yields for products which are isolated by evaporation or filtration and used directly
without further purification.
Preparation 1
Synthesis of methyl 2,2-dimethyl-3-oxo-propanoate.
Add methyl 3-hydroxy-2,2-dimethylpropanoate (33 g, 250 mmol) to Dess-Martin
periodinane (106 g, 250 mmol) suspended in DCM (1000 mL) at 0°C and stir at room
temperature for 18 h. Filter the reaction mixture through a celite bed and concentrate the
filtrate. Wash the concentrated filtrate with pentane (2 x 200 mL). Separate the pentane
layer and concentrate in vacuo to give methyl 2,2-dimethyl-3-oxo-propanoate (31.93 g,
quantitative). 'HNMR (d6-DMSO) 9.59 (s, 1H), 3.67 (s, 3H), 1.26 (s, 6H).
Preparation 2
Synthesis of -butyl 4-(3-methoxy-2,2-dimethyl-3-oxopropyl)piperazine-l-carboxylate.
Stir a solution of methyl 2,2-dimethyl-3-oxo-propanoate (30.88 g, 237.31
mmoles) and tert-butyl piperazine-l-carboxylate (34.00 g, 182.55 mmoles) in DCM (500
mL) at room temperature for 20 minutes. Add acetic acid (2 equiv); 20.92 mL, 365.09
mmoles) followed by sodium triacetoxyborohydride (1.4 equiv; 54.17 g, 255.56 mmoles)
over 0.5 h and stir the resulting mixture stirred at room temperature overnight. Carefully
quench with water (250 mL) and transfer the mixture to a separating funnel with DCM
(300 mL). Wash the resulting organic layer with brine. Dry over MgS04, filter and
evaporate to give -butyl 4-(3-methoxy-2,2-dimethyl-3-oxopropyl)piperazine-lcarboxylate.
(58 g, 100%). MS (m/z): 301.2 (M+l).
Preparation 3
Synthesis of methyl 2,2-dimethyl-3-(piperazin-l-yl)propanoate dihydrochloride.
To a solution of tert-butyl 4-(3-methoxy-2,2-dimethyl-3-oxopropyl)piperazine-lcarboxylate
(58.0 g, 193.08 mmoles) in isopropyl alcohol (150 mL) add a 4M dioxane
solution of hydrogen chloride ((4 equiv); 193.08 mL, 772.31 mmoles) over 15 minutes,
observing gas evolution and a fine precipitate. Heat at 55°C for 3h to give a white
precipitate. Cool to 10°C and collect the white solid by filtration, wash with further
isopropyl alcohol (30 mL), then EtOAc. Dry in a vacuum oven at 45°C for 1 h to give
methyl 2,2-dimethyl-3-(piperazin-l-yl)propanoate dihydrochloride (31 g, 59% yield).
MS (m/z): = 201.1 (M+l).
Synthesis of methyl 2,2-dimethyl-3-(piperazin-l-yl)propanoate hydrochloride.
Dissolve methyl 2,2-dimethyl-3-(piperazin-l-yl)propanoate dihydrochloride
( 112.0 g, 409.95 mmoles) in water (250 mL). Add solid sodium bicarbonate to give an
aqueous layer of pH 4 and extract the mixture into a solution of 10% DCM in diethyl
ether (300 mL) to remove some dark solid matter and some color. Discard the organic
layer. Basify the aqueous phase to pH 8 with 2M aqueous sodium hydroxide solution,
then saturate with solid sodium chloride. Extract into a 10% solution of isopropanol in
chloroform. Add further 2M aqueous sodium hydroxide to maintain pH 8. Repeat the
extraction into a 10% solution of isopropanol in chloroform. Continue the extraction
process until 85% of the expected material has been recovered. Wash the isopropanol in
chloroform solution with brine, dry over a2S0 4, filter and evaporate to give methyl 2,2-
dimethyl-3-(piperazin-l-yl)propanoate hydrochloride (82.1 g % yield). MS (m/z): 201.1
(M+l).
Preparation 5
Synthesis of methyl 4-ethenyl-l H-pyrrole-2-carboxylate.
Degas, by purging with nitrogen for 10 minutes, a solution of methyl 4-bromolH-
pyrrole-2-carboxylate (2.0 g, 9.8 mmoles), triethenylboroxin pyridine (1.3 equiv; 3.1
g, 12.7 mmoles), and potassium carbonate (3 equiv; 4.1 g, 29.4 mmoles) in a mixture of
1,4-dioxane (20 mL) and water (10 mL). Add tris(dibenzylideneacetonyl)bis-palladium
(Pd2(dba)3) (0.01 equiv; 0.0925 g, 98.0 ΐ ) and l,l'-bis(di-tertbutylphosphino)
ferrocene (dtbpf) (0.03 equiv; 0.0853 g, 294.1 ) and heat at 95°C
for 3 h. Cool to room temperature then partition between water (50 mL) and a 1:1
mixture of hexanes/diethyl ether (100 mL). The organic layer is washed with water (2 x
50 mL) then brine. Dry over a2S0 4, filter and evaporate to give methyl 4-ethenyl- 1Hpyrrole-
2-carboxylate as a straw colored oil (1.5 g). Use without further purification. 1-
NMR (CDC13), : 8.90 ( 1, br. s), 7.02 ( 1, m), 6.95 ( 1, m), 6.56 ( 1, q) 5.48 ( 1, dd),
5.05 ( 1, dd), 3.86 (3, s).
Preparation 6
Synthesis of methyl 4-ethyl-l H-pyrrole-2-carboxylate.
Charge to a 500 mL Parr bottle, a mixture of 5% palladium on charcoal (0.2 g) in
ethanol (25 mL) and add methyl 4-ethenyl-l H-pyrrole-2-carboxylate (1.4 g, 9.3 mmoles).
Hydrogenate at 206.8 kPa for 3 h to give complete conversion by LC-MS. Filter through
celite and evaporate to give an oil. Pass through a silica gel pad with iso-hexane/ethyl
acetate (100:0 to 75:25) to give the title compound as a pale yellow oil (1.12g). IH-NMR
(CDCI 3), : 8.87 (1H, br. s), 6.73-6.78 (2H, m), 3.83 (3H, s), 2.50 (2H, q, J =7.8Hz) and
1.19 (3H, t, J =7.3Hz).
Preparation 7
Synthesis of methyl 4-(prop-l-en-2-yl)-l H-pyrrole-2-carboxylate.
Mix methyl 4-iodo-l H-pyrrole-2-carboxylate (1.5 g, 5.98 mmoles), 4,4,5,5-
tetramethyl-2-(prop-l-en-2-yl)-l,3,2-dioxaborolane (3.01 g, 17.98 mmoles)
l,l'-bis(di-tert-butylphosphino)ferrocene (dtbpf) (0.3 g, 0.06 mmoles),
tris(dibenzylideneacetonyl)bis-palladium (Pd 2(dba) ) (0.3 g, 0.06 mmoles), tripotassium
phosphate (2.54 g, 11.95 mmoles) and methanol (12 mL). Heat the mixture in a sealed
tube in a microwave at 140°C for 30 minutes. Cool, filter the reaction mixture and wash
sinter with methanol, evaporate the filtrate under reduced pressure and chromatograph on
silica eluting with isohexane/dichloromethane (gradient elution, 100:0 to 0:100).
Evaporate the fractions containing product to give methyl 4-(prop-l-en-2-yl)-l H-pyrrole-
2-carboxylate. (0.679 g, 68.79% yield). MS (m/z): 166.1 (M+l).
Preparation 8
Synthesis of ethyl 3-methoxy-4-methyl-l H- rrole-2-carboxylate.
Add dimethyl sulfate (3 mL, 3.99 g, 3 1.63 mmoles) to a mixture of 3-hydroxy -4-
methyl-l H-pyrrole-2-carboxylic acid ethyl ester (3.5 g, 20.69 mmoles) and 2M sodium
hydroxide (75 mL, 1500 mmoles) and stir the reaction mixture vigorously for 30 mins at
room temp (water cooling bath applied). Collect the resultant precipitate and wash with
water. Add more dimethyl sulfate (3 mL, 3.99 g, 3 1.63 mmoles) to the filtrate and stir for
30 minutes. Collect the resultant precipitate and wash with water. Add more dimethyl
sulfate (3 mL, 3.99 g, 3 1.63 mmoles) to the filtrate and stir for lh. Combine the isolated
precipitates and dry in a vacuum oven at 50°C for 30 minutes to give ethyl 3-methoxy-4-
methyl- lH-pyrrole-2-carboxylate (3.221 g, 84.98% yield). MS (m/z): 184.09 (M+l).
Preparation 9
Synthesis of ethyl l-(5-chloro-2-nitrobenzyl)-4-methyl-l H-pyrrole-2-carboxylate.
Vigorously stir a solution of 2-(bromomethyl)-4-chloro-l-nitro-benzene (25.02 g,
99.88 mmoles) in DCM (200 mL), and to this add a solution of ethyl 4-methyl-l Hpyrrole-
2-carboxylate (15 g, 97.92 mmoles) in DCM (200 mL). Then add 30-hydrated
tetra-n-butylammonium hydroxide (0.508 g) and cool the stirred mixture under nitrogen
to 5°C using an external ice-bath. Add 25% aq. sodium hydroxide (200 mL) dropwise
over 15 minutes observing the internal temperature rise to 10°C. Stir and allow to warm
to room temperature. Stir at room temperature for 3.5 h. Add further 50% aq. sodium
hydroxide (20 mL) and stir at room temperature for a further 1 h. Stop the stirring and
allow the layers to separate. Transfer to a separating funnel and wash the organic layer
with IN hydrochloric acid (200mL), observing the pH of this layer to be acidic using
indicator paper. Next wash the organic layer with water (600 mL) and then brine (600
mL) and finally dry over Na2S0 4. Filter the mixture and remove the solvent in vacuo at
40°C to give an orange oil. Purify by passing through a silica gel pad with isohexane/
ethyl acetate (gradient eluting from 10 to 20%) to give ethyl l-(5-chloro-2-
nitrobenzyl)-4-methyl-l H-pyrrole-2-carboxylate as a yellow oil (33 g, quantitative). MS
(m/z): 322.98 (M+l).
Preparation 9 alternative
Synthesis of ethyl l-(5-chloro-2-nitrobenzyl)-4-methyl-l H-pyrrole-2-carboxylate.
Under a nitrogen atmosphere with stirring add cesium carbonate (96 g, 295
mmoles) to a solution of ethyl 4-methyl-l H-pyrrole-2-carboxylate (30 g, 196 mmoles) in
DMF (240 mL) and heat the reaction mixture to 40 °C to 45 °C for 1 hour. Cool the
reaction mixture to room temperature and add a solution of 2-(bromomethyl)-4-chloro-lnitro-
benzene (54 g, 216 mmoles) in DMF (120 mL) drop-wise. Stir the resulting
suspension for 1.5 h to 2.0 h, filter the solids and rinse the filtercake with DMF (45 mL).
Transfer the solids to a clean reaction vessel, add water (200 mL) and cool the suspension
to 10 °C to 15 °C with stirring for 1 h to 2 h. Filter the suspension, rinse with water (75
niL) and transfer the solids to another reaction vessel. Add ethanol (125 mL) and stir the
suspension at 5 °C to 10 °C for 1 h to 2 h. Filter the solids, rinse the filtercake with
ethanol (20 mL), and dry the solids in an oven at less than 50 °C under reduced pressure
to give ethyl l-(5-chloro-2-nitrobenzyl)-4-methyl-l H -pyrrole-2-carboxylate as a yellow
solid (54 g, 94.1% purity, 73.3% yield).
Preparation 10
Synthesis of ethyl ethyl 4-bromo-l-(5-chloro-2-nitrobenzyl)-l H-pyrrole-2-carboxylate.
Wash 60% mineral oil suspended sodium hydride (1.2 equiv; 1.1 g, 27.52
mmoles) with a small amount of iso-hexane (x 2). Suspend in N,N-dimethylacetamide
(15 mL) and chill in an ice-bath. Add ethyl 4-bromo-l H-pyrrole-2-carboxylate (5.0 g,
22.93 mmoles) portionwise over 15 minutes allowing gas evolution to subside between
additions. Stir at room temperature for 15 minutes to give a brown solution. Add 2-
(bromomethyl)-4-chloro-l-nitro-benzene (1.15 equiv; 6.61 g, 26.37 mmoles) in portions
over 15 minutes and stir the resulting purple solution at room temperature for 2.5 h. Cool
in an ice-bath and quench with water (10 mL). Partition between 1M hydrochloric acid
(100 mL) and EtOAc (300 mL). Wash the organic layer with water (2 x 100 mL) then
brine. Dry over a2S0 4 and decolorize with activated charcoal. Filter through celite and
evaporate to give ethyl ethyl 4-bromo-l-(5-chloro-2-nitrobenzyl)-l H-pyrrole-2-
carboxylate as a an orange oil (9. lg, quantitative). 1H-NMR (CDCI3) : 8.12 (1H, d),
7.40 (1H, dd), 7.07 (1H, d), 6.92 (1H, d), 6.53 (1H, d), 5.87 (2H, s), 4.17 (2H, q), 1.25
(3H, t).
The following compounds are prepared essentially by the method of Preparation
10.
:
(1H,
s), 5.87
q),
(3H, t)
:
(1H,
6.88
d),
(3H, s)
(M+l)

Preparation
Name Structure Physical Data
No.
Ethyl 3-methoxy-4-methyl-l-(5-
2 1 methyl-2-nitrobenzyl)- IH- MS (m/z): 333. 18 (M+l)
pyrrole-2-carboxylate
Preparation 22
Synthesis of ethyl l-(2-amino-5-chlorobenzyl)-4-methyl-l H-pyrrole-2-carboxylate.
Charge 5% platinum(S)/charcoal (2.5 g), and ethyl l-(5-chloro-2-nitrobenzyl)-4-
methyl-l H-pyrrole-2-carboxylate (16.0 g, 49.57 mmoles) to a 500 mL Parr bottle. Add
ethanol (200 mL) then zinc dibromide (0.22 equiv; 2.46 g, 10.91 mmoles) and place the
mixture under hydrogen at 275.8 kPa and hydrogenate at room temperature overnight,
monitoring formation of a partially hydrogenated intermediate. Remove the Parr bottle,
heat gently in a water bath to dissolve the crystallized material, and filter through celite.
Combine with the filtrate from an identical run and evaporate to give ethyl l-(2-amino-5
chlorobenzyl)-4-methyl-l H-pyrrole-2-carboxylate an off-white solid. Place under high
vacuum to remove residual ethanol to give the material (35 g, quantitative). MS (m/z):
293.1 (M+l).
Preparation 22 alternative
Synthesis of ethyl l-(2-amino-5-chlorobenzyl)-4-methyl-l H-pyrrole-2-carboxylate.
In a reaction vessel under nitrogen charge a solution of ethyl l-(5-chloro-2-
nitrobenzyl)-4-methyl-l H-pyrrole-2-carboxylate (100.0 g, 310 mmoles) in THF (800
mL) and add zinc dibromide (0.22 equiv; 15.4 g, 68.4 mmoles) with stirring at room
temperature. Charge a slurry of 5% platinum(S)/charcoal (13.3 g) in THF (25 mL), and
place the vessel under hydrogen at 380 kPa. Hydrogenate at room temperature for 30 hr.
to 40 hr., monitoring formation of a partially hydrogenated intermediate and filter over
diatomite. Rinse the filter aid with THF (300 mL) and concentrate the filtrate, at below
40 °C, to arrive at a volume of approximately 200 mL. Add DCM (250 mL), concentrate
the solution, at below 40 °C, to arrive at a volume of approximately 200 mL, and add
additional DCM (600 mL). This process can be repeated as necessary to remove
undesired levels of THF from the reaction mixture. Charge water (500 mL), separate the
layers, and wash the organic layer with water (300 mL), followed by 25% aqueous
solution of sodium chloride (250 mL). Concentrate the solution, 40 °C, to arrive at a
volume of approximately 200 mL, add heptane (400 mL), and concentrate the solution, at
below 40 °C, to arrive at a volume of approximately 200 mL. Add heptane (400 mL) and
heat the reaction mixture to 40 - 45 °C with stirring for 2 - 3 hr. Cool the reaction
mixture to 5 - 10 °C and continue stirring at this temperature for 1 - 2 hr. Filter the
resulting solids and dry in an oven at less than 50 °C under reduced pressure to give ethyl
l-(2-amino-5-chlorobenzyl)-4-methyl-l H-pyrrole-2-carboxylate (81.4 g, 95.8% purity,
85.9% yield) as a yellow solid.
The following compounds are prepared essentially by the method of Preparation
22.
Preparation
Name Structure MS (m/z): (M+l).
No.
Ethyl l-(2-amino-5-
23 methylbenzyl)-4-methyl- IH- 273.09
pyrrole-2-carboxylate
Preparation 26
Synthesis of ethyl l -(2-amino-5-chlorobenzyl)-4-bromo-lH -pyrrole -2-carboxylate.
To a well-stirred solution of ethyl 4-bromo- l -(5-chloro-2-nitrobenzyl)-lH -
pyrrole -2-carboxylate (9.1 g, 23.48 mmoles) in acetic acid (60.00 mL) at 70°C, add iron
(5 equiv; 6.56 g, 117.38 mmoles) in portions over 0.5 h. Halfway through the addition,
observe an exotherm to maintain a temperature of 85°C with the oil-bath removed. The
reaction mixture will get thicker as the exotherm dissipates and the rest of the iron can be
added. Stir at 85°C for 0.5 h. Cool to room temperature and pour onto water (200 mL).
Extract into chloroform (2 x 200 mL). Combine organic layers and wash with water (2 x
100 mL) then saturated aqueous aHC0 solution. Dry over a2S04, filter and
evaporate to the title compound as an orange oil (7.7 g, 92% yield). MS (m/z): 358.98
(M+l).
The following compounds are prepared essentially by the method of Preparation
26.
Preparation 32
Synthesis of 7-chloro-2-methyl-5,10-dihydro-l lH-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-
one.
To a solution of ethyl l-[(2-amino-5-chloro-phenyl)methyl]-4-methyl-pyrrole-2-
carboxylate (35 g, 119.55 mmoles) in dimethyl sulfoxide (120 mL) add potassium tbutoxide
(14.76 g, 131.5 mmoles) over 10 minutes. Heat at 80°C (oil-bath temperature)
for 1 h. Allow to cool then pour onto water (400 mL). Collect the resulting brown
powdery solid by filtration, washing well with water. Suck as dry as possible on the
sinter then transfer to a dish and dry in a vacuum oven at 55°C over phosphorus pentoxide
overnight, to give 7-chloro-2-methyl-5,10-dihydro-l lH-pyrrolo[2,lc][
l,4]benzodiazepin-l l-one. (22 g, 91%). MS (m/z): 247.1 (M+l).
Preparation 32 alternative
Synthesis of 7-chloro-2-methyl-5,10-dihydro-l lH-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-
one.
To a solution of ethyl l-[(2-amino-5-chloro-phenyl)methyl]-4-methyl-pyrrole-2-
carboxylate (20 g, 68.3 mmoles) in dimethyl sulfoxide (60 mL) add potassium ?-butoxide
(8.4 g, 74.9 mmoles) in DMSO (40 mL) and heat the reaction mixture to 75 - 80 °C for 1
- 2 hr. Add water (200 mL) slowly, cool to room temperature, and stir for 1 - 2 hr. Filter
the solids, wash the filtercake with water (75 mL), and transfer the solids to a clean
reaction vessel. Add THF (100 mL) and heat the reaction mixture to 30 - 35 °C until the
solids dissolve. Add DCM (350 mL) and continue to stir at 30 - 35 °C for 1 - 2 hr. Add
IN hydrochloric acid (250 mL) and continue to stir at 30 - 35 °C for 1 - 2 hr. Separate the
layers, wash the organic layer with IN hydrochloric acid (250 mL) followed by a 25%
aqueous solution of sodium chloride (50 mL), and concentrate the solution, at below 50
°C, to arrive at a volume of approximately 25 mL. Add ethanol (50 mL), concentrate the
solution, at below 50 °C, to arrive at a volume of approximately 25 mL, and add
additional ethanol (50 mL). Concentrate the solution, at below 50 °C, to arrive at a
volume of approximately 25 mL, heat the reaction mixture to 45 - 50 °C, and stir for 1 - 2
hr. Cool the reaction mixture to 5 - 10 °C with stirring for 2 - 3 hr., and filter the resulting
solids. Rinse the filtercake with ethanol (25 mL) and dry in an oven at less than 50 °C
under reduced pressure to give 7-chloro-2-methyl-5,10-dihydro-l lH-pyrrolo[2,lc][
l,4]benzodiazepin-l 1-one as an off-white solid (14.9 g, 99.9% purity, 88.5% yield).
The following compounds are prepared essentially by the method of Preparation

Preparation 43
Alternative synthesis of 7-chloro-2-(trifluoromethyl)-5,10-dihydro-l lH-pyrrolo[2,lc]
[1,4]benzodiazepin- 11-one.
Add tin dichloride (3 equiv; 3.02 g, 15.77 mmol) in 5M hydrochloric acid (20 mL,
100 mmol) to ethyl l-(5-chloro-2-nitrobenzyl)-4-(trifluoromethyl)-l H-pyrrole-2-
carboxylate ( 1 equiv; 1.98 g, 5.26 mmoles) and ethanol (100 mL) at 50°C. Heat
overnight and then for a further 26 h, remove most of the ethanol in vacuo and dilute the
resultant solution with water and collect the resultant precipitate by filtration, wash well
with water and dry in vacuo at 40°C. Absorb the solid onto silica, chromatograph eluting
with DCM/methanol (5:95) to give uncyclised amino ester (0.260 g) and 7-chloro-2-
(trifluoromethyl)-5,10-dihydro-l lH-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-one (0.613 g).
Heat the uncyclised ester in a mixture of 5M hydrochloric acid (10 mL, 50 mmol) for 80
h. Remove the ethanol in vacuo and collect the resultant precipitated solid by filtration
and wash with water to give 7-chloro-2-(trifluoromethyl)-5,10-dihydro-l lH-pyrrolo[2,lc]
[1,4]benzodiazepin- 11-one (0. 17 g). Combine with earlier 7-chloro-2-
(trifluoromethyl)-5,10-dihydro-l lH-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-one to give
(0.794 g, 50% yield). MS (m/z): 300.99 (M+l).
The following compound is prepared essentially by the method of Preparation 43.
MS
Preparation Name Intermediate (m/z):
No. (M+l).
7-Chloro- 1-methoxy-2-methyl-5,10-
44 dihydro-1 li/-pyrrolo[2,l- 277.09
c][1,4]benzodiazepin- 11-one
Preparation 45
Synthesis of 7,11-dichloro-2-methyl-5 H-pyrrolo[2, 1-c] [1,4]benzodiazepine.
To a suspension of 7-chloro-2-methyl-5,10-dihydro-l lH-pyrrolo[2,lc][
l,4]benzodiazepin-l 1-one (40.6 g, 164.58 mmoles) in methoxybenzene (250 mL) at
70°C add N,N-dimethylaniline (2.8 equiv; 58.59 mL, 460.8 mmoles) in one portion
followed by phosphoryl chloride (2.3 equiv (molar); 35.18 mL, 378.52 mmoles) over 20
minutes, controlling the exotherm by addition. Heat the resultant dark solution at 90°C
for 1.5 h. Add additional phosphoryl chloride (0.33 equiv; 5.05 mL, 54.3 1 mmoles) and
heat the mixture at 90°C for a further lh to give complete conversion. Evaporate to near
dryness and partition the residue between water (500 mL) and ethyl acetate (2 x 500 mL).
Combine organic layers and wash with water (500 mL) then brine. Dry over a2S0 4,
filter and evaporate onto silica. Pass through a silica gel pad with iso-hexane/ethyl
acetate (gradient elution 5 to 25%). Combine product fractions and evaporate to a small
amount of solvent. Add iso-hexane (150 mL) and collect the resulting yellow powder by
filtration, to give 7,1 l-dichloro-2-methyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepine (34.0 g,
78% yield). MS (m/z): 265.1 (M+l).
Alternative synthesis:
Under a 2 atmosphere add phosphoryl chloride (124 g, 819 mmol) to a mixture
of 7-chloro-2-methyl-5,10-dihydro-l lH-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-one (100 g,
405 mmol), N N-dimethylaniline (138 g, 1.14 mol) and anisole (550 mL). Heat to 80 - 85
°C and stir for 4 hr. to 6 hr. Concentrate to a total of 1.5 volumes to 2.5 volumes while
maintaining the temperature below 75 °C. Cool to 15 - 25 °C. Add dichloromethane
(600 mL) dropwise and stir for 1 hr. Add the resulting mixture to water (600 mL) while
maintaining the temperature between 10 °C and 35 °C. Stir for 1 hr. and then separate the
layers. Extract the aqueous layer with dichloromethane (600 mL). Wash the combined
organic layers with 1.0 HC1 (600 g) followed by 7% NaHC0 3 (600 g). Filter the
organic layer through diatomite (20 g to 30 g) and silica gel (60 g to 100 g). Concentrate
the resulting filtrate to a total of 1.5 volumes to 2.5 volumes while maintaining the
temperature below 45 °C. Add heptane (270 g to 410 g) and concentrate the resulting
filtrate to a total of 1.5 volumes to 2.5 volumes while maintaining the temperature below
45 °C. Add heptane (150 g to 210 g), cool to 5 °C to 10 °C, and stir for 2 hr. to 3 hr.
Filter, rinse the filter the cake with heptane, and dry under vacuum, at below 45 °C, to
obtain the title compound as a pale yellow solid (90% to 95% yield)
The following compounds are prepared essentially by the method of Preparation
Preparation 50
Synthesis of methyl 3-[4-(7-chloro-2-methyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-
yl)piperazin-l-yl]-2,2-dimethylpropanoate.
Add phosphoryl chloride (1.47 mL, 2.43 g, 15.85 mmoles) to 7-chloro-2-methyl-
5,10-dihydro-ll H-pyrrolo[2,l-c][l,4]benzodiazepin-l l-one (1.1 17 g, 4.53 mmoles) in
DCM (50 mL) and stir at room temperature over a weekend. Add ice water to the
reaction mixture then add DCM, separate the aqueous layer and wash the DCM layer with
water and sodium hydrogen carbonate solution. Dry the DCM solution with MgS0 4,
filter and evaporate the solvent in vacuo to give 7,1 l-dichloro-2-methyl-5 H-pyrrolo[2,lc][
l,4]benzodiazepine (1.167 g).
Dissolve methyl 2,2-dimethyl-3-(piperazin-l-yl)propanoate dihydrochloride (1.61
g, 5.89 mmoles) in water and load on to two SCX-2 (10 g) cartridges. Wash the
cartridges with methanol and elute with 2M ammonia in methanol. Concentrate in vacuo,
then dissolve the resultant oil in acetonitrile (40 mL). Add 7,1 l-dichloro-2-methyl-5 Hpyrrolo[
2,l-c][l,4]benzodiazepine (1.167 g, 4.4 mmoles) to the acetonitrile solution.
Divide the acetonitrile solution and place in to 2 microwave tubes, add potassium
carbonate (0.89 g, 6.79 mmoles) to each microwave tube and heat and stir at 140°C in the
microwave for 3.5 hr. Allow the reaction mixture to cool, filter, wash the solid collected
on the sinter with acetonitrile then concentrate the filtrate in vacuo, add methanol and
then evaporate to dryness. Treat with more methanol and collect the resultant precipitate,
wash the precipitate with methanol to give methyl 3-[4-(7-chloro-2-methyl-5 Hpyrrolo[
2,l-c][l,4]benzodiazepin-l l-yl)piperazin-l-yl]-2,2-dimethylpropanoate as a
crystalline solid (1.1 g). MS (m/z): 429.18 (M+l).
Alternative synthesis of methyl 3-[4-(7-chloro-2-methyl-5 H-pyrrolo[2,lc]
[1,4]benzodiazepin- 11-yl)piperazin- 1-yl]-2,2-dimethylpropanoate.
To a suspension of 7,ll-dichloro-2-methyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepine
(34.0 g, 128.23 mmoles) in acetonitrile (300 mL) add methyl 2,2-dimethyl-3-(piperazinl-
yl)-propionate hydrochloride (2.1 equiv; 63.75 g, 269.29 mmoles) and potassium
carbonate (4 equiv; 70.89 g, 512.93 mmoles). Heat the mixture at reflux overnight.
Evaporate to dryness. Partition the residue between water (500 mL) and EtOAc (2 x 500
mL). Combine the organic layers and wash with water (500 mL) then brine. Dry over
sodium sulfate, filter and evaporate to a brown oil. Add iso-hexane (~150mL) and a seed
crystal of Preparation 50. Allow to crystallize over 2 h, then transfer the flask to a fridge
and allow to stand. Collect the resulting heavy crystalline solid by filtration, washing
with cold iso-hexane. Dry in a vacuum oven at 40°C for 1 h, to give methyl 3-[4-(7-
chloro-2-methyl-5 H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-yl)piperazin- 1-yl]-2,2-
dimethylpropanoate (53.6 g, 97% yield). MS (m/z): 429.18 (M+l).
Second alternative synthesis of methyl 3-[4-(7-chloro-2-methyl-5 H-pyrrolo[2,lc]
[1,4]benzodiazepin- 11-yl)piperazin- 1-yl]-2,2-dimethylpropanoate.
Heat a mixture of 7,1 l-dichloro-2-methyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepine
(10.0 g, 37.7 mmol), methyl 2,2-dimethyl-3-(piperazin-l-yl)-propionate dihydrochloride
(19.4 g, 71.0 mmol), diisopropylamine (22.9 g, 226 mmol), and acetonitrile (80 g) at 80
°C to 85 °C for 22 hr. to 26 hr. Cool to 30 °C to 40 °C and add ethyl acetate (80 g). Add
water (80 g) dropwise. Stir 40 min. to 60 min. and separate the layers. Extract the
aqueous layer with ethyl acetate (60 g to 80 g). Wash the combined organic layers with
25% aqueous sodium chloride (2 X 40 g). Concentrate the organic layer to a total of 1.5
volumes and 3.5 volumes while maintaining the temperature below 50 °C. Add heptane
(4 1 g to 55 g) at 40 °C to 50 °C)\ and stir for 2 hr. to 3 hr. Concentrate to a total of 1.5
volumes to 3.0 volumes while maintaining the temperature below 50 °C. Cool to 0 °C to
10 °C and stir for 2 hr. to 3 hr. Filter, wash the filtercake with heptane (3.0 g to 10.0 g),
and dry under vacuum, at below 60 °C, to afford the title compound (16.0 g, 94.7%
w/w% assay, 94% yield) as a light, yellow solid.
Preparation 51
Synthesis of methyl 3-[4-(2,7-dichloro-5 H-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-
yl)piperazin-l-yl]-2,2-dimethylpropanoate.
Add phosphoryl chloride (5 equiv; 4.36 mL, 7.19 g, 46.89 mmoles) to
2,7-dichloro-5,10-dihydro-ll H-pyrrolo[2,l-c][l,4]benzodiazepin-ll-one ( 1 equiv; 2.505
g, 9.38 mmoles) in chloroform (80 mL) and heat and stir at 50°C overnight. Decant the
reaction solution and evaporate in vacuo to an oil. Dissolve the oil in DCM, wash with
saturated aqueous sodium hydrogen carbonate solution. Dry the DCM solution over
Na2S0 4, filter and concentrate to dryness to give 2,7,1 l-trichloro-5 H-pyrrolo[2, 1-
c][l ^benzodiazepine as a cream colored solid. In parallel desalt methyl 2,2-dimethyl-3-
(piperazin-l-yl)propanoate hydrochloride (0.837 g, 3.54 mmoles) by dissolving in
methanol, add the methanolic solution to an SCX-2 column (10 g) wash with methanol
and then elute with 2.5M ammonia in methanol solution, evaporate the methanol
ammonia fraction to give methyl 2,2-dimethyl-3-(piperazin-l-yl)propanoate (0.665 g) as
an oil. Mix this oil with 2,7,1 l-trichloro-5 H-pyrrolo[2,l-c][l,4]benzodiazepine (0.505 g
1.77 mmoles), potassium carbonate (0.733 g, 5.31 mmoles) and acetonitrile (20 mL) and
heat at reflux overnight. Cool the reaction mixture to room temperature and filter,
washing the sinter with EtOAc, then evaporate the filtrate in vacuo to a solid.
Chromatograph on silica eluting with methanol/DCM (gradient elution 2:98 to 8:92).
Evaporate the fractions containing product to give methyl 3-[4-(2,7-dichloro-5Hpyrrolo[
2,l-c][l,4]benzodiazepin-l l-yl)piperazin-l-yl]-2,2-dimethylpropanoate (0.69 g).
MS (m/z): 449.13/451.07 (M+l).
The following compounds are prepared essentially by the method of Preparation
Preparation 60
Synthesis of methyl 2,2-dimethyl-3-[4-(7-methyl-2-(methylsulfanyl)-5 H-pyrrolo[2, 1-
c] [1,4]benzodiazepin- 11-yl)piperazin- 1-yl] -propanoate.
Treat methyl 1-(5-methyl-2-nitrobenzyl)-4-(methylsulfanyl)- lH-pyrrole-2-
carboxylate (3.49 mmoles; 1.12 g) with acetic acid (15 mL) then treat with iron, powder
(10.5 mmoles; 585 mg) then slowly heat in an oil bath at 80°C. After a few hours
increase the oil bath temperature to 90°C. Then leave the reaction at 60°C for 2 days.
Then concentrate to dryness to remove acetic acid then treat with EtOAc and wash
through a pad of silica with more EtOAc until the orange colour stops coming off.
Concentrate the red eluant to dryness then treat with methanol then re-concentrate and
dissolve in DCM (20 mL) and treat with phosphoryl chloride (1.0 mL). Heat the reaction
in an oil bath at 50°C overnight. Then cool the reaction to RT and treat with ice then
wash with water twice and then aHC0 (aq). Dry the organic layer over MgSC^, filter
and concentrate to a tar. Meanwhile dissolve methyl 2,2-dimethyl-3-(piperazin-l-yl)-
propanoate dihydrochloride (1.60 g, 5.86 mmoles) in water then load on to a SCX-2
cartridge (2 x 10 g) and wash with methanol and then elute with ammonia in
methanol. Concentrate the basic solution to dryness to an oil. Then dissolve this oil in
acetonitrile (50 mL) and add to a mixture of the tar and potassium carbonate (1.0 g) then
heat to reflux. After 2 hours, microwave the reaction to 140°C for 2 hours. Then filter
the reaction and extract with acetonitrile and acetone. Combine and concentrate the
mother liquors to dryness onto silica then purify by flash chromatography silica (40 g)
(10-50% EtOAc in hexane). Take the fractions which contain clean product, combine
and concentrate to yield methyl 2,2-dimethyl-3-[4-(7-methyl-2-(methylsulfanyl)-5 Hpyrrolo[
2,l-c][l,4]benzodiazepin-l l-yl)piperazin-l-yl]-propanoate (418 mg; 27% yield).
MS (m/z): 441.18 (M+l).
Preparation 61
Synthesis of 7-chloro-2-(methylsulfanyl)- 11-(piperazin- 1-yl)-5H-pyrrolo[2, 1-
c][l,4]benzodiazepine.
Add phosphoryl chloride (5 equiv; 2.21 mL, 3.64 g, 23.76 mmoles) to
7-chloro-2-(methylsulfanyl)-5 ,10-dihydro- 1lH-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-one
(1.38 g, 4.75 mmoles) in chloroform (20 mL) and heat and stir at 45°C for 2 h. Raise the
temperature of the reaction to 60°C and stir for a further 2 h. Remove the chloroform in
vacuo and dissolve the residue in DCM (100 mL) and wash with saturated sodium
bicarbonate solution (100 mL). Filter the DCM layer through a phase separation frit and
evaporate the solvent in vacuo to give a viscous oil. Dissolve the oil in acetonitrile (10
mL) and add cesium carbonate (3 equiv; 4.65 g, 14.26 mmoles). Add to this mixture
piperazine (10 equiv; 4.09 g, 47.52 mmoles) in dry acetonitrile (10 mL) and heat and stir
the mixture under reflux overnight. Cool the reaction mixture and add saturated
ammonium chloride solution (100 mL) and extract into EtOAc (2 x 50 mL). Combine the
organic layers and wash with water (3 x 100 mL), dry over MgSC^, filter and evaporate
the solvent in vacuo to give 7-chloro-2-(methylsulfanyl)-l l-(piperazin-l-yl)-5 Hpyrrolo[
2,l-c][l,4]benzodiazepine (1.0 g, 60%). MS (m/z): 347.13 (M+l).
Preparation 62
Synthesis of methyl 3-[4-(7-chloro-2-(methylsulfanyl)-5 H-pyrrolo[2,lc]
[1,4]benzodiazepin- 11-yl)piperazin- 1-yl]-2,2-dimethylpropanoate.
Add methyl 2,2-dimethyl-3-oxo-propanoate (0.928 g, 7.13 mmoles) in DCM (5
mL) to 7-chloro-2-(methylsulfanyl)- 1l-(piperazin-l-yl)-5 H-pyrrolo[2,lc][
l,4]benzodiazepine (1.0 g, 2.85 mmoles) in DCM (10 mL) and stir under nitrogen for
30 minutes at room temperature. Add sodium triacetoxyborohydride (3 equiv; 1.89 g,
8.56 mmoles) to the reaction mixture and stir under nitrogen overnight at room
temperature. Add methanol and apply to an SCX-2 column, wash with methanol and
elute with 2M ammonia in methanol. Evaporate the methanol solution to give methyl 3-
[4-(7-chloro-2-(methylsulfanyl)-5 H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-yl)piperazin- 1-
yl]-2,2-dimethylpropanoate (1.23 g, 84%). MS (m/z): 461.12 (M+l).
Preparation 63
Synthesis of methyl 2,2-dimethyl-3-{4-[7-methyl-2-(propan-2-yl)-5 H-pyrrolo[2,lc]
[1,4]benzodiazepin- 11-yljpiperazin- 1-yl} propanoate.
Mix methyl 3-[4-(2-bromo-7-methyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-
yl)piperazin-l-yl]-2,2-dimethylpropanoate (1.00 equiv; 600.00 mg, 1.27 mmoles),
4,4,5, 5-tetramethyl-2-(prop-l-en-2-yl)-l,3,2-dioxaborolane (3.00 equiv; 638.93 mg, 3.80
mmoles) in methanol (15 mL). Add a pre-blended mixture of 1.09 wt%
tris(dibenzylideneacetonyl)bis-palladium (Pd 2(dba) ), 1.16 wt% l,l'-bis(di-tertbutylphosphino)
ferrocene (dtbpf) and 97.75 wt% tripotassium phosphate (1.06 g) and
heat at 140°C for 25 mins in a microwave. Filter the reaction mixture through celite and
dilute to 25 mL volume with methanol. Hydrogenate the methanolic solution by passing
at lmL/min. through a H-Cube® flow hydrogenator using a 10%Pd/C catalytic cartridge
at 50°C. Evaporate the solvent in vacuo to give methyl 2,2-dimethyl-3-{4-[7-methyl-2-
(propan-2-yl)-5 H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-yl]piperazin- 1-yljpropanoate.MS
(m/z): 437.28 (M+l).
Preparation 64
Synthesis of methyl 3-[4-(2-ethyl-7-methyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-
yl)piperazin-l-yl]-2,2-dimethylpropanoate.
Mix methyl 3-[4-(2-bromo-7-methyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepin-l 1-
yl)piperazin-l-yl]-2,2-dimethylpropanoate (1.00 equiv; ; 620.00 mg, 1.31 mmoles),
triethenylboroxin pyridine (1.5 equiv; 472.79 mg, 1.96 mmoles;) in methanol (15 mL).
Add a pre-blended mixture of 1.09 wt% tris(dibenzylideneacetonyl)bis-palladium
(Pd2(dba)3), 1.16 wt% l,l'-bis(di-tert-butylphosphino)ferrocene (dtbpf) and 97.75 wt%
tripotassium phosphate (1.10 g) and heat at 140°C for 25 mins in a microwave. Filter the
reaction mixture through celite and dilute to 25 mL volume with methanol. Hydrogenate
the methanolic solution by passing at lmL/min. through a H-Cube® flow hydrogenator
using a 10%Pd/C catalytic cartridge at 50°C. Evaporate the solvent in vacuo to give
methyl 3-[4-(2-ethyl-7-methyl-5H-pyrrolo[2,l-c][l,4]benzodiazepin-l l-yl)piperazin-lyl]-
2,2-dimethylpropanoate. MS (m/z): 423.18 (M+l).
Example 1
Synthesis of 3-[4-(7-chloro-2-methyl-5H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-
yl)piperazin-l-yl]-2,2-dimethylpropanoic acid.
To a suspension of methyl 3-[4-(7-chloro-2-methyl-5 H-pyrrolo[2,lc][
l,4]benzodiazepin-ll-yl)piperazin-l-yl]-2,2-dimethylpropanoate (28.0 g, 65.27
mmoles) in a mixture of isopropyl alcohol (150 mL) and water (150 mL) add sodium
hydroxide (3 equiv; 7.83 g, 195.82 mmoles) and heat at 70°C for 4 h to give a clear
solution. Heat at 70°C for a further 1 h then cool slightly. Add 5M hydrochloric acid to
give pH 8 and precipitate a white solid. Add further 5M hydrochloric acid to give a pH
between 6.5 and 7. Reduce the volume of solvent by half and then chill the flask in a
fridge for 0.5 h. Collect the resulting white solid by filtration and dry overnight in a
vacuum oven at 40°C over phosphorus pentoxide to give 3-[4-(7-chloro-2-methyl-5 Hpyrrolo[
2,l-c][l,4]benzodiazepin-l l-yl)piperazin-l-yl]-2,2-dimethylpropanoic acid (26.5
g, 98% yield). MS (m/z): 415.3 (M+l). DSC melting point = 246.5°C (onset).
The following compounds are prepared essentially by the method of Example 1.

Example 13
Synthesis of 3-[4-(7-Chloro-2-methyl-5H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-
yl)piperazin-l-yl]-2,2-dimethylpropanoic acid dihydrochloride.
To a suspension of 3-[4-(7-chloro-2-methyl-5H-pyrrolo[2,l-c][l,4]benzodiazepinll-
yl)piperazin-l-yl]-2,2-dimethylpropanoic acid (24.25 g, 58.44 mmoles) in isopropyl
alcohol (250 mL) at 60°C, add a 4M dioxane solution of hydrogen chloride (2.4 equiv;
35.07 mL, 140.26 mmoles) over 10 minutes to give a clear solution. Allow to cool
slightly then evaporate to an off-white solid. Triturate with a small amount of diethyl
ether and collect the powdery cream solid by filtration. Dry in a vacuum oven at 40°C
overnight. Grind to a fine powder and dry in a vacuum oven at 60°C for 6 h. Monitor
levels of residual isopropyl alcohol by 1NMR. Dissolve in warm ethanol (350 mL) and
evaporate to dryness. Triturate with ethanol (50 mL) and evaporate to dryness again.
Triturate with dry diethyl ether (200 mL) and collect the resulting solid by filtration. Dry
for 6 h in a vacuum oven at 50°C to give 3-[4-(7-chloro-2-methyl-5H -pyrrolo[2,lc][
l,4]benzodiazepin-l l-yl)piperazin-l-yl]-2,2-dimethylpropanoic acid dihydrochloride
(26.9 g, 94% yield) MS (m/z): 415.2 (M+l).
The following compounds are prepared essentially by the method of Example 13.

Example 25
Synthesis of 3-[4-(2-chloro-7-methyl-5 H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-
yl)piperazin-l-yl]-2,2-dimethylpropanoic acid dihydrochloride.
Dissolve methyl 2,2-dimethyl-3-(piperazin-l-yl)-propanoate dihydrochloride
(1.49 g, 5.47 mmoles) in water and absorb onto an SCX2 column. Wash the column with
methanol and elute methyl 2,2-dimethyl-3-piperazin-l-yl-propanoate with 2M ammonia
in methanol. Remove the methanol in vacuo and add the methyl 2,2-dimethyl-3-
(piperazin-l-yl)propanoate to acetonitrile (12 mL). Add 2,1 1-dichloro-7-methyl-5 Hpyrrolo[
2,l-c][l,4]benzodiazepine (0.85 g, 3.22 mmoles) and sodium bicarbonate (0.405
g, 4.83 mmoles) to the acetonitrile solution. Heat and stir in the microwave for 30
minutes at 140°C. Cool to room temperature, absorb the reaction mixture onto silica and
purify by chromatography (gradient elution with EtOAc/isohexane 0:100% to 100:0%).
Collect the fractions containing product and evaporate the solvent in vacuo and dissolve
the residue in methanol (10 mL) and add lithium hydroxide (0.235 g, 9.65 mmoles). Heat
and stir the methanolic solution in the microwave for 12.5 minutes at 140°C. Cool to
room temperature and acidify with acetic acid and then evaporate the solvent under
reduced pressure. Dissolve the residue in excess 2M HCI (aq) and then evaporated to
dryness. Dissolve the residue in water and immobilise onto a macroporous polystyrene
hydrogen carbonate (PL-HC03) resin. Wash the resin with water and elute from the resin
with 2M HCl (aq). Evaporate the solution to dryness to give 3-[4-(2-chloro-7-methyl-5H -
pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-yl)piperazin- 1-yl]-2,2-dimethylpropanoic acid
dihydrochloride (0. 177 g; 11.28% yield). MS (m/z): 415. 18 (M+l).
The following compounds are prepared essentially by the method of Example 25.
Example 29
Synthesis of Sodium 3-[4-(7-chloro-2-ethyl-5 H-pyrrolo[2,l-c][l,4]benzodiazepin-llyl)
piperazin-l-yl]-2,2-dimethylpropanoate.
Dissolve 3-[4-(7-chloro-2-ethyl-5H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-
yl)piperazin-l-yl]-2,2-dimethylpropanoic acid dihydrochloride (201 mg, 400 in
water and methanol (5 mL) then load on to a SCX-2 cartridge (2 g). Wash with methanol
and then elute with ammonia in methanol. Concentrate the basic solution to dryness to
give 3-[4-(7-chloro-2-ethyl-5H-pyrrolo[2,l-c][l,4]benzodiazepin-l l-yl)piperazin-l-yl]-
2,2-dimethylpropanoic acid (160 mg, 373 ^). Then treat with 2N sodium hydroxide
(187 , (373 ) and water (3 mL) to cause solution. Then freeze-dry to yield
sodium 3-[4-(7-chloro-2-ethyl-5H-pyrrolo [2, 1-c] [1,4]benzodiazepin- 11-yl)piperazin- 1-
yl]-2,2-dimethylpropanoate (164 mg, 98% yield). MS (m/z): 429.18 (M+l).
Example 30
Synthesis of 3-[4-(7-Chloro-2-methyl-5H-pyrrolo [2, 1-c] [1,4]benzodiazepin- 11-
yl)piperazin- 1-yl]-2,2-dimethylpropanoic acid di-methanesulfonate
Add acetonitrile (5 niL) to 3-[4-(7-chloro-2-methyl-5 H-pyrrolo[2,l-c][l,4]
benzodiazepine-ll-yl)piperazin-l-yl]-2,2-dimethylpropanoic acid (0.106 g, 0.255 mmol)
to create a slurry. Add methanesulfonic acid (0.050 ml, 0.075 g, 0.76 mmol) to the
stirring slurry (1000 rpm) and stir the resultant solution at 60°C until a white solid
precipitates. Cool this slurry, and isolate the solid by filtration to obtain 3-[4-(7-chloro-2-
methyl-5H-pyrrolo[2, 1-c] [1,4]benzodiazepin- 11-yl)piperazin- 1-yl]-2,2-
dimethylpropanoic acid di-methanesulfonate (0.15 g).
Literature data (Morairty SR, Hedley L, Flores J, Martin R, Kilduff TS. (2008)
Selective 5-HT2A and 5-HTe receptor antagonists promote sleep in rats. Sleep 31, 34-44.;
and Barbier, A.J., and Bradbury, M.J., Histaminergic Control of Sleep-Wake Cycles:
Recent Therapeutic Advances for Sleep and WakeDisorders, CNS & Neurological
Disorders - Drug Targets, vol 6, pg. 3 1-43 (2007)) and data generated in non-clinical
animal studies support a role for dual activity HI inverse agonists / 5-HT2A antagonists in
the treatment of insomnia and in the symptomatic treatment of insomnia associated with
other disorders such as depressive disorders, anxiety disorders, pain, allergies, lung or
airway disorders, psychiatric disorders, dementia, and/or neurodegenerative diseases,
and/or circadian rhythm sleep disorders. Specifically it is found that certain dual activity
HI inverse agonists / 5-HT2A antagonists are effective in increasing total sleep time using
EEG monitored rodents without disproportionate or clinically relevant hypoactivity,
decrease in REM sleep, or hypersomnolence.
To further demonstrate the characteristics of the present compounds, compounds
may be run in the following in vitro and in vivo assays:
In vitro binding and activity assays:
HI competition binding assay
[ H]-Pyrilamine binding experiments are carried out in SPA (scintillation
proximity assay) 96-well format. Membranes used in this assay are prepared from HEK-
293 cells stably expressing recombinant HI receptor (human). The incubation is initiated
by the addition of a mixture of WGA PVT SPA beads (lmg/well, Perkin Elmer (MA,
USA) RPNQOOOl) and 3 g membranes to assay buffer (67 mM Tris; pH 7.6) containing
3.5 nM [ H]-Pyrilamine and varying concentrations of the test compound (10 point
concentration response curves). Non-specific binding is determined in the presence of 10
Triprolidine. Samples are incubated for four hours at room temperature (22° C) and
then read in a Microbeta Trilux.
5-HT competition binding assay
[ H]-Ketanserin binding experiments are carried out in SPA 96-well format.
Membranes used in this assay are prepared from AV-12 cells stably expressing
recombinant 5-HT2A receptor (human). The incubation is initiated by the addition of a
mixture of WGA YSi SPA beads (lmg/well, Perkin Elmer (MA, USA), RPNQ001 1) and
2 g membranes to assay buffer (67 mM Tris, 0.5 mM EDTA; pH 7.6) containing 3.1 nM
[ H]-Ketanserin and varying concentrations of the test compound (10 point concentration
response curves). Non-specific binding is determined in the presence of 20 1-(1-
Naphthyl) piperazine. Samples were incubated for four hours at room temperature (22°
C) and then read in a Microbeta Trilux.
5-HTV competition binding assay
[1 I]-(±)DOI binding experiments are carried out in SPA 96-well format.
Membranes used in this assay are prepared from AV-12 cells stably expressing
recombinant 5-HT2C receptor (human). The incubation is initiated by the addition of a
mixture of WGA PVT SPA beads (0.5 mg/well, Perkin Elmer (MA, USA), RPNQ0001)
and 2.5 g membranes to assay buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM EDTA,
10 pargyline, 0.1% ascorbic acid, pH7.4) containing 0.2 nM [[1 I]-(±)DOI and
varying concentrations of the test compound (10 point concentration response curves).
Non-specific binding is determined in the presence of 20 l-(l-Naphthyl) piperazine.
Samples are incubated for four hours at room temperature (22° C) and then read in a
Microbeta Trilux.
Binding data analysis
Curves are evaluated using a 4-parameter logistic nonlinear equation to obtain the
concentration of competitor causing 50% inhibition of radioligand binding (IC50) .
Equilibrium dissociation constants (¾) are calculated according to the equation ¾ =
IC5o/(l+L /Kd), where L equals the concentration of radioligand used in the experiment
and K equals the equilibrium dissociation constant of the radioligand for the receptor,
determined from standard saturation analysis or homologous competition experiments.
Reported values for ¾ , where n values are indicated, are shown as geometric mean ± the
standard error of the mean (SEM), with the number of replicate determinations indicated
by n. Geometric means are calculated by the equation GeoMean = 10 (Average (log ¾ 1
+ log ¾ 2 +. ..log ¾ n)/sqrt n).
GABAA antagonism using native receptors in primary neuronal cultures
Activity of compounds on native GABAA receptors is evaluated by monitoring
calcium fluxes using a 96 well format FLIPR® system (Fluorometric Imaging Plate
Reader (FLIPR®, Molecular Devices). Briefly, cortical embryonic neurons are
dissociated from El 8 rat embryos and plated at optimum density into black-walled,
transparent bottom poly-D-lysine coated 96-well FLIPR® plates. After loading the cells
with a calcium sensitive dye (Fluo4-AM, Molecular Devices), the cells are bathed in a
solution containing low chloride (chloride replaced by gluconate). Under these
conditions activation of GABAA receptors causes an efflux of chloride ions (in the
direction of the chemical gradient), which results in membrane depolarization and
consequently activation of voltage gated calcium channels (VGCCs). Calcium influx
through VGCCs is recorded and analysed offline using the FLIPR® system. For a
pharmacological validation of the assay, concentration response curves (CRC) are
recorded for the standard agonist (GABA) and standard antagonist (Gabazine). Any
effects are determined in CRC mode against a fixed concentration of agonist GABA at 10
(equivalent to an EC90 GABA response).
Methods:
The antagonist effects of compounds are quantified using 10-point dose response
curves by comparing the peak fluorescent responses to the agonist GABA in the presence
and absence of compound. The assay window is defined as the maximal response
obtained by GABA at its predetermined EC90 concentration minus the response obtained
by a fully inhibiting concentration of gabazine (50 ) . Antagonist effects are calculated
as a percent of the assay window. All data are calculated as relative IC50 values using a
four-parameter logistic curve fitting program (Prism Graphpad® 3.01). Antagonist
potencies for all compounds are compared to gabazine with three replicates in each assay
run.
Exemplified compounds or exemplified salts thereof are tested essentially as
described above and are found to have high affinity for the HI and 5-HT2A receptors and
selectivity over the 5-HT 2c receptor. K ' s for the HI and 5-HT2Areceptors for the
exemplified compounds are found to be less than 100 nM and 200 nM, respectively,
while the K ' s for the 5-HT2c receptor are found to be greater then 1000 nM.
Further, the compounds of the invention may be tested in binding assays and
functional activity assays by well known methods for other physiologically important
receptors such as, but not limited to, the hERG channel, other serotonin receptors
(specifically 5-HT1A, 5-HTi B, 5-HTi D, 5-HTi E, 5-HTi F receptors, lack of agonist activity
at 5-HT2B receptors, 5-HT2C, 5-HT4, 5-HT , 5-HT , and 5-HT7 receptors), muscarinic
receptors, dopaminergic receptors (specifically Dl, D2, and D3), GABAA receptors,
adrenergic receptors and monoamine transporters. Certain exemplified compounds are
tested at these receptors and are shown to lack significant activity.
The compounds of examples 1 and 13 are tested essentially as described above
and are found to have activity profiles as shown in Table 1.
Table 1. Selectivity data
Example 1 Example 13
HI ¾ (nM) 27.3 16.8
5-HT2A Ki (nM) 57.5 63.2
5-HT2B Agonist EC 0 (nM) > 10000 >10000
5-HT2CKi (nM) 3810 2370
GABAA ICJO () - >50
hERG Channel () >100 >100
Dopamine Di ¾ (nM) 3790 1860
Dopamine D2 ¾ (nM) >4500 3420
Dopamine D ¾ (nM) >5680 >4 110
5-HT1A Ki (nM) >6940 -
5-HTIB (nM) >5580 >5580
5-HT1D Ki (nM) >3980 >8550
5-HTIE (nM) - >5370
5-HTIF ¾ (nM) - >8250
5-HT5 ¾ (nM) >8830 >9090
5-HT6 Ki (nM) 2690 >5830
5-HT7 Ki (nM) >2060 >3970
Adrenergic alphaiA i (nM) >89 10 >89 10
Adrenergic alphas ¾ (nM) >10900 >10900
Adrenergic alpha2A i (nM) >9030 >8470
Adrenergic alpha2B¾ (nM) >4980 >4800
Adrenergic alpha2c ¾ (nM) >4020 >5330
Serotonin Transporter >64 1 >5 15
Norepinephrine Transport >682 >526
Dopamine Transporter >879 >880
Therefore, physiologically relevant doses of the compounds of the invention are
expected to provide substantial inhibition of H I and 5-HT 2Areceptors in vivo, while not
substantially interacting with other physiologically relevant receptors, and thus are
expected to provide the desired pharmacology while avoiding undesired effects associated
with off-target activity. Such undesired effects include, but are not limited to the
following: 5-HT2C antagonist activity associated with treatment emergent weight gain, 5-
HT2B agonist activity associated with valvulopathy, hERG channel modulation associated
with QT prolongation, and GABAA activity associated with seizure activity.
Furthermore, interference with sleep/wake physiology is avoided by the selectivity over
dopamine receptors, other serotonin receptors, adrenergic receptors, and monoamine
transporters.
5-HTTA Receptor Occupancy: Receptor occupancy is assayed to demonstrate the extent
of interaction with the 5-HT2A Receptor in vivo. Briefly, male Sprague-Dawley rats
(Harlan Sprague-Dawley, Indianapolis, ) weighing approximately 230-280 grams are
given ad lib access to food and water until the beginning of the 3-hour experimental
protocol. 1 mg/kg ketanserin (non-selective 5-HT2A antagonist) is used as a positive
control to establish assay validity. Test compounds or control are administered by oral
gavage in a vehicle comprised of 20% hydroxypropyl beta-cyclodextrin. MDL 100907 (
(R)-(+)-a-(2,3-Dimethoxyphenyl)-l-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol), a
selective 5-HT2A antagonist, is used as a tracer. MDL 100907 is suspended in water with
5 ΐ dilute lactic acid ( 1 mg/ml), diluted to 6 g/ml with saline, and administered in a
volume of 1 mL/kg intravenously via the lateral tail vein to yield a tracer dose of 3 g kg.
Rats are administered test compound, ketanserin, or vehicle (N = 4), followed one hour
later with an intravenous, 3 g/kg tracer dose of MDL 100907. It is at the time of tracer
administration that receptor occupancy (RO) is considered to be measured. Fifteen
minutes after tracer administration, rats are sacrificed by cervical dislocation. Plasma
samples are collected and samples of the frontal cortex and cerebellum are removed. The
level of MDL 100907 tracer is measured in each cortical and cerebellar sample. RO is
calculated using the well-established ratio method which employs a region of high
receptor density representative of total binding (frontal cortex) normalized by an area
without or with very low levels of receptor (cerebellum). This region, referred to as the
null region, represents nonspecific binding of the ligand probe. Vehicle ratio of the tracer
levels in cortex relative to cerebellum represents 0% occupancy. A ratio of 1 represents
100% occupancy and is achieved when all specific binding to the 5-HT2Areceptor of the
MDL 100907 tracer is blocked. The intermediate ratios of cortical to cerebellar tracer
from the test compound pretreated group are interpolated linearly between the ratio of
tracer levels in the vehicle-treated animals (0% occupancy) and a ratio of 1 (100%
occupancy) in order to determine the percent 5-HT2ARO.
MDL 100907 Analysis: Cortex and cerebellar samples are weighed and placed in conical
centrifuge tubes on ice. Four volumes (w/v) of acetonitrile containing 0.1% formic acid
is added to each tube. The samples are then homogenized and centrifuged at 14,000 RPM
(21,920 x g) for 16 minutes. Supernatant is diluted by adding 100 - 900 ΐ sterile water
in HPLC injection vials for LC/MS/MS analysis. Analysis of MDL 100907 is carried out
using an Agilent model 1200 HPLC (Agilent Technologies, Palo Alto, CA) and an API
4000 mass spectrometer. The chromatographic separation is on a 2.1 X 50 mm C18
column (Agilent part number 971700-907) with a mobile phase consisting of 60%
acetonitrile in water with an overall 0.1% formic acid content. Detection of MDL 100907
is accomplished by monitoring the precursor to product ion transition with a mass to
charge ratio (m/z) of 374.2 to 123.0. Standards are prepared by adding known quantities
of analyte to brain tissue samples from non-treated rats and processing as described
above.
Statistical Methods : Curves for each study are fitted to a 4 parameter logistic function
with the bottom fixed at 0% using JMP® version 8.0 (SAS Institute Inc, Cary NC) and
the absolute ED50 is calculated by the software. Values are given as means, standard
errors and 95% confidence intervals. The compound of Example 13 is tested essentially
as described and is found to achieve high 5-HT2Areceptor occupancy with an EC50 of
0.27 mg/kg (SE=0.069, 95% CI = 0.16-0.48 mg/kg).
Histamine HI Receptor Occupancy : HI RO is assayed to demonstrate the extent of
interaction with the HI receptor in vivo. Briefly, male Sprague-Dawley rats (Harlan
Sprague-Dawley, Indianapolis, IN) weighing approximately 230-280 grams are given ad
lib access to food and water until the beginning of the 3-hour experimental protocol. 3-
[4-(8-Fluorodibenzo[b,f] [ 1,4]oxazepin- 11-yl)piperazin-l -yl]-2,2-dimethylpropanoic acid
dihydrochloride is used as a positive control and doxepin is used as a tracer. Test
compounds and control are administered by oral gavage in a vehicle comprised of 20%
hydroxypropyl beta-cyclodextrin. Doxepin is dissolved in sterile water (100 g/ml),
diluted to 2 g/ml with saline, and administered in a volume of 0.5 mL/kg intravenously
via the lateral tail vein to yield a tracer dose of 1 g kg. Rats are administered test
compound, 15 mg/kg positive control, or vehicle, followed 1 hour later by an intravenous
"tracer" dose of doxepin (N = 4). It is at the time of tracer administration that RO is
considered to be measured. 40 mins after tracer administration, rats are sacrificed by
cervical dislocation. Plasma samples are collected and the frontal cortex is removed. The
level of doxepin tracer is measured in each cortical sample. RO is calculated using a
within tissue comparison of tracer levels under vehicle conditions (0% occupancy) and
positive control conditions (100% occupancy). 15 mg/kg of positive control administered
intravenously with a 30 minute pretreatment is predetermined to represent full blockade
of specific binding of the tracer to HI receptors. Additional treatment groups with doses
of test compound are linearly interpolated between the vehicle and positive control groups
to calculate percent HI RO.
Doxepin Analysis : Cortical samples are weighed and placed in conical centrifuge tubes
on ice. Four volumes (w/v) of acetonitrile containing 0.1% formic acid is added to each
tube. The samples are homogenized and centrifuged using a at 14,000 RPM (21,920 x g)
for 16 minutes. Supernatant is diluted with 100 - 900 sterile water in HPLC injection
vials for LC/MS/MS analysis. LC/MS/MS analysis of doxepin was carried out using an
Agilent model 1200 HPLC (Agilent Technologies, Palo Alto, CA) and an API 4000 mass
spectrometer. The chromatographic separation employed a 2.1 X 50 mm CI 8 column
(Agilent part number 971700-907) and a mobile phase using a gradient method consisting
of initial conditions of 10% acetonitrile (ACN) in water with an overall 0.1% formic acid
content, after lmin. - 10% ACN, 2 min. - 90% ACN, 2.9 min. - 90% ACN, 3.1 min. -
10% ACN, 5.5 min. - Stop. Detection of doxepin was accomplished by monitoring the
precursor to product ion transition with a mass to charge ratio (m/z) of 280 to 107. 1.
Standards are prepared by adding known quantities of analyte to brain tissue samples
from non-treated rats and processing as described above.
Statistical Methods : Curves are fitted to a 4 parameter logistic (none held constant) using
Prism® version 3.02 (GraphPad Software, San Diego, CA) and the relative ED50 is
calculated by the software. Values are given as means ± the standard error of the mean.
The compound of Example 13 is tested essentially as described and is found to achieve
high HI RO with an EC 0 of 0.3 mg/kg.
HI Inverse Agonism: To determine the inverse agonist nature of compounds ot the
present invention, their effects on the levels of Myo-Inositol 1 phosphate (IP1) in
HEK293 cells transfected with the human recombinant HI receptor (HEK293/hm HI
clone R-40) are measured. Briefly, HEK293/hm HI cells (clone R-40) are grown to
-90% confluency (3:1 DMEM/F12, 5% FBS, 20 mM HEPES, G418 500 ΐ , 1%
Pen/Strep/Glutamine) and harvested on the day of the assay using l x Trypsin/EDTA
(PAA Pasching, Austria LI 1-003). 35 ΐ cells (300K) are seeded into 96W half area
white solid bottom plates (Corning, UK 3688) in stimulation buffer ( aCl 146 mM,
CaCl2 1mM, KC1 4.2 mM, MgCl2 0.5 mM, Glucose 5.5 mM, HEPES 10 mM and LiCl
50 mM). Test compounds are initially dissolved in 100% DMSO at 1 mM, and serially
diluted (half log) in 100% DMSO to give 10 point dose response curves (Biomek 2000,
Beckman Coulter UK). These are further diluted to x2 final assay concentration in
stimulation buffer using the Cybiwell (CiBio Jena, Germany) and 35 ΐ added to the cells
in the assay plate (10 maximum final concentration). Cells plus compound are
incubated for 1 hour 30 mins at 37 C / 5% C0 2 before addition of 15 ΐ of each of the
HTRF IP1 detection kit reagents (CisBio 62P1APEC). The cell plate is incubated for a
further hour at room temperature before measuring IP1 accumulation (Envision plate
reader, Perkin Elmer). IP 1 accumulation (nM) is calculated by extrapolation from the
standard IP1 curve run on the day of the assay. EC50 values are calculated using a 4-
parameter curve fit (Graph Pad Prism v3.02). Negative efficacy values are expressed
relative to the positive control Tripelennamine (10 , Sigma, UK P5514).
Representative compounds of the present invention are assayed essentially as described
and are found to be inverse agonists at the HI receptor. Compound 13 is tested
essentially as described and is found to fully suppress constitutive activity (126 ± 6%)
with an IC 0 of 53.9 ± 37 nM.
Inhibition of DPI Induced Headshake Activity: The in vivo 5-HT2A receptor antagonist
activity of the compounds of the present invention is demonstrated by their ability to
block head shaking activity induced by the 5-HT2Areceptor agonist 2,5-dimethoxy-4-
iodoamphetamine (DOI). (see for example Bartoszyk GD, van Amsterdam C, B5ttcher
H, Seyfried CA. EMD 281014, a new selective serotonin 5-HT2A receptor antagonist.
Eur J Pharmacol. 2003 473: 229-230.) Briefly, male C57BL/6J mice (20-25 g, Charles
River) are housed in standard housing conditions (32 mice in a large IVC cage, 07.00 to
19.00 light phase, constant temperature (19-23°C) and humidity (50% +/-10), ad lib food
and water). Mice received either vehicle (0.25% Methyl cellulose), DOI (3 mg/kg in
saline) or test compound at lOmg/kg PO plus DOI (3 mg/kg in saline). Test compounds
are individually evaluated in groups of four per experiment with n=4 for each compound,
together with vehicle and DOI+vehicle (n=8). After a test compound pre-treatment time
of 60 minutes the mice receive either vehicle (saline) or 3 mg/kg DOI dosed
subcutaneously, and are then placed into clear perspex observation chambers. Five
minutes after DOI or vehicle administration the number of visually scored head shakes
exhibited by each individual mouse is counted for 15 minutes. The data is analyzed using
an ANOVA and post-hoc Dunnet's Test. Exemplified compounds are tested essentially
as described and are found to inhibit the DOI induced headshake response at greater than
90% at 10 mg/kg. The compound of example 13 is tested essentially as described and is
found to inhibit the DOI induced headshake response at 100% at 10 mg/kg.
Sleep and behavioral monitoring in rats: Representative compounds of the present
invention are tested in rats for their ability to increase the amount of sleep or decrease
sleep interruption or both without undesired effects such as inhibition of REM sleep,
waking motor impairment, and/or rebound insomnia. Test animals are continuously
monitored by electro-encephalograms (EEG), electromyograms (EMG), and motion to
measure cumulative nonREM sleep, cumulative total sleep, average sleep bout duration,
longest sleep bout duration, rebound insomnia, REM sleep inhibition and locomotor
activity intensity during wakefulness. Methods for such studies are known in the art (see
for example methods described in Edgar DM, Seidel WF. Modafinil induces wakefulness
without intensifying motor activity or subsequent rebound hypersomnolence in the rat.
J Pharmacology &Experimental Therapeutics 1997; 283: 75 '-769; van Gelder RN,
Edgar DM, Dement WC. Real-time automated sleep scoring: validation of a
microcomputer-based system for mice. Sleep 1991, 14: 48-55; and Gross BA, Walsh
CM, Turakhia AA, Booth V, Mashour GA, Poe GR. Open-source logic-based automated
sleep scoring software using electrophysiological recordings in rats. JNeurosci Methods.
2009; 184(1): 10-8.) Studies are conducted as follows:
Animal preparation. Adult, male Wistar rats (approximately 270-300 g at time of
surgery) are surgically fitted for chronic recording of EEG, EMG, and motion as follows:
Rats are surgically prepared with a cranial implant consisting of four stainless steel
screws for EEG recording (two frontal [3.9 mm anterior from bregma, and ±2.0 mm
mediolaterally] and two occipital [6.4 mm posterior from bregma, ±5.5 mm
mediolaterally]), and with two Teflon-coated stainless steel wires for EMG recording
(positioned under the nuchal trapezoid muscles). All leads are soldered to a miniature
connector (Microtech, Boothwyn, PA) prior to surgery. The implant assembly is affixed
to the skull by the combination of the stainless steel EEG recording screws, cyanoacrylate
applied between the implant connector and skull, and dental acrylic. Locomotor activity
is monitored via a miniature transmitter (Minimitter PDT4000G, Philips Respironics,
Bend, OR) surgically placed into the abdomen. At least 3 weeks are allowed for
recovery.
Recording environment. Each rat is housed individually within a microisolator cage
modified with an inserted polycarbonate filter-top riser to allow more vertical headroom.
A flexible cable that minimally restricts movement is connected at one end to a
commutator afixed to the cage top and at the other end to the animal's cranial implant.
Each cage is located within separate, ventilated compartments of a stainless steel sleepwake
recording chamber. Food and water are available ad libitum and the ambient
temperature is maintained at about 23±1°C. A 24-hr. light-dark cycle (LD 12:12) using
fluorescent light is maintained throughout the study. Relative humidity averages
approximately 50%. Animals are undisturbed for at least 30 hrs before and after each
treatment.
Study design and dosing. The vehicle (placebo, methylcellulose 15 centipoise 0.25% in
water) or one of the test compound dose levels is administered orally at 1 mL/kg pseudorandomly
such that no rat receives the same treatment twice, and no rat receives more
than two of the 8 treatments in any one study. Each rat is removed from its cage for about
a minute to be weighed and treated. At least 6 days "washout" period precede and follow
each treatment.
Data collection. Sleep and wakefulness discrimination may be automated (e.g., Van
Gelder et al. 1991 (above); Edgar et al. 1997 (above); Winrow CJ, et al,
Neuropharmacology 2010; 58(1): 185-94.; and Gross et al, 2009 (above). EEG is
amplified and filtered ( I0,000, bandpass 1-30 Hz), EMG is amplified and integrated
(bandpass 10-100 Hz, RMS integration), and non-specific locomotor activity (LMA) is
monitored simultaneously. Arousal states are classified in 10 second epochs as non-REM
sleep, REM sleep, wakefulness, or theta-dominated wakefulness. Locomotor activity
(LMA) is recorded as counts per minute and is detected by commerically available
telemetry receivers (ER4000, Minimitter, Bend, OR).
Statistical Analysis. All animals having at least one outcome are included in the summary
results (for example, we include appropriate data from an animal treatment for which
telemetry data is usable but EEG data is not). The post-treatment observation period is
divided into post-dosing intervals appropriate to each Outcome, where the time of dosing
is defined as the start of Hour = 0, and outcomes are summarized in the observation
period by computing either the mean hourly or the cumulative value across each period
(see legend of Table 1 for precise definition of each Outcome). Sleep bouts are analyzed
on the log scale to stabilize the variation, all other variates are analyzed on the linear
scale. Each outcome in each period is analyzed by analysis of covariance using treatment
group and treatment date as factors and the corresponding pre-treatment interval, 24 hrs
earlier, as the covariate. Adjusted means and the change from vehicle means and their
corresponding standard errors are summarized for each treatment group. Outcomes
analyzed on the log scale are back-transformed to report geometric means and mean ratioto-
vehicle results.
The compounds of Examples 13-28 are tested essentially as described. The
compounds of Examples 13, 15, 16, 18 and 19 are found to significantly increase
cumulative NREM sleep time and cumulative total sleep time without significant rebound
insomnia, REM sleep inhibition or inhibition of locomotor intensity (LMI) at 3 mg/kg.
The compound of Example 13 is tested essentially as described and is found to have the
sleep profile and locomotor activity intensity as shown in Table 2.
Table 2. Compound of Example 13.
Efficacy variables Undesired effect variables
Cumulative NREM sleep Rebound Insomnia
Dose (mg/kg PO) N Adj. Mean SE N Adj. Mean LCL
10 10 5 1.8 6.5 10 3.1 -6.1
3 14 39.5 5.5 14 -7.8 -15.8
1 14 28.0 5.5 14 -9.7 - 17.7
0.50 15 22.2 5.3 15 -7.2 - 15 .1
0.25 15 15.2 5.4 15 - 1.9 -9.7
0.10 5 5.9 8 .1 5 -5.4 - 17.2
0.03 9 5 .1 6.4 9 -0.7 - 10 .1
Cumulative Total sleep REM inhibition
Dose (mg/kg PO) N Adj.Mean SE N Adj.Mean LCL
10 10 52.8 7.3 10 2.9 -4.6
3 14 42.3 6.2 14 7.0 0.7
1 14 30.6 6 .1 14 2.6 -3.7
0.50 15 25. 1 6.0 15 5.8 -0.5
0.25 15 17.7 6 .1 15 4.7 - 1.7
0.10 5 5.9 9.0 5 1.0 -8.0
0.03 9 7.3 7.2 9 1.8 -5.8
Average Sleep Bout Locomotor Activity Intensity
Dose (mg/kg PO) N Adj.Mean SE N Adj.Mean LCL
10 10 2.3 0.3 10 -0. 11 -3.09
3 14 1.8 0.2 13 - 1.49 -4. 19
1 14 1.6 0.2 10 -2.94 -5.87
0.50 15 1.4 0 .1 14 -0. 14 -2.75
0.25 15 1.2 0 .1 12 -0.58 -3.30
0.10 5 1.1 0.2 5 -0.35 -4.29
0.03 9 1.1 0 .1 9 -0.97 -4.07
Longest Sleep Bout
Table 2. Outcome statistics: Abbreviations: N = sample size; Adj.Mean = adjusted
group mean value relative to vehicle controls; SE = standard error of
the mean; LCL = lower 95% confidence limit, NREM = non-REM, i.e.,
all sleep other than REM sleep.
Definitions and units — means are adjusted differences from vehicle controls:
Cumulative sleep: across the first 6 hours post-treatment, in minutes ('Total sleep'
denotes NREM sleep + REM sleep).
Average sleep bout: average of hourly-averaged sleep bouts, across the first 6
hours post-treatment, expressed as w-fold increase over vehicle controls.
Longest sleep bout: the longest sleep bout in the first 6 hours post-treatment,
expressed as w-fold increase over vehicle controls.
Rebound insomnia: cumulative minutes of NREM+REM sleep during the first 3
hours of the lights on period, i.e., 7th, 8th and 9th hours post-treatment.
REM inhibition: cumulative minutes of REM sleep during the first 12 hours posttreatment.
Locomotor Activity (LMA) Intensity: expressed as LMA counts per minute of
EEG-defined wakefulness, averaged across the first 6 hours post-treatment.
Determining efficacy. The threshold efficacy for each of the four efficacy variables is
calculated by plotting the increase in each variable relative to vehicle controls during the
6 hour period after treatment against log(dose). The threshold efficacy for each variable
is that dose which gives the defined efficacy threshold value; +30 min. of additional
accumulated non-REM sleep, +25 min. of additional accumulated total sleep, 1.75x
increase in average sleep bout duration, and 1.5x increase in longest sleep bout duration.
The compound of example 13 is found to have threshold efficacious doses as shown in
Table 3.
Determining undesired effects. Each 'undesired effect' outcome variable (see Table 4
legend for definitions), is plotted against log(dose).
The threshhold value for REM inhibition is defined as a cumulative reduction of REM
sleep of -10 min. The threshold value for rebound insomnia is defined as -20 min. The
threshold value for reduced LMI is defined as -5 locomotor activity counts per minute of
EEG-defined wakefulness. A significant undesired effect is defined to occur when the
lower confidence limit goes below the threshold value^ for all doses at or above the
average efficacious dose. For Exemplified compounds, no undesired occurances of REM
inhibition, rebound insomnia, or reduction in LMI are observed at doses up to at least 10
mg/Kg. (Negative value indicate REM inhibition, rebound insomnia and reduced LMI,
respectively).
While it is possible to administer compounds employed in the methods of this
invention directly without any formulation, the compounds are usually administered in
the form of pharmaceutical compositions comprising at least one compound of Formula I,
or a pharmaceutically acceptable salt thereof, as an active ingredient and at least one
pharmaceutically acceptable carrier, diluent and/or excipient. These compositions can be
administered by a variety of routes including oral, sublingual, nasal, subcutaneous,
intravenous, and intramuscular. Such pharmaceutical compositions and processes for
preparing them are well known in the art. See, e.g., Remington: The Science and Practice
of Pharmacy (University of the Sciences in Philadelphia, ed., 1st ed., Lippincott
Williams & Wilkins Co., 2005).
The compositions are preferably formulated in a unit dosage form, each dosage
containing from about 0.1 to about 60 mg, more usually about 1 to about 30 mg, as for
example between about 2 and about 10 mg of the active ingredient. The term "unit
dosage form" refers to physically discrete units suitable as unitary dosages for human
subjects and other mammals, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect, in association with at least
one suitable pharmaceutically acceptable carrier, diluent and/or excipient.
The compounds of Formula I are generally effective over a wide dosage range.
For example, dosages per day normally fall within the range of about 0.002 to about 1.0
mg/kg, more usually from about 0.015 to 0.5 mg/kg, and as for example between 0.03 and
0.15 mg/kg of body weight. In some instances dosage levels below the lower limit of the
aforesaid range may be more than adequate, while in other cases still larger doses may be
employed without causing any harmful side effect, and therefore the above dosage ranges
are not intended to limit the scope of the invention in any way. It will be understood that
the amount of the compound actually administered will be determined by a physician, in
the light of the relevant circumstances, including the condition to be treated, the chosen
route of administration, the actual compound or compounds administered, the age,
weight, and response of the individual patient, and the severity of the patient's symptoms.
We Claim:
1. A compound of the formula
where R 1 is chloro or methyl;
R2 is methyl, ethyl, isopropyl, chloro, bromo, trifluoromethyl, or methylthio; and
R is hydrogen or methoxy;
or a pharmaceutically acceptable salt thereof.
2. A compound according to Claim 1 where R is hydrogen, or a pharmaceutically
acceptable salt thereof.
3. A compound according to either Claim 1 or Claim 2 where R 1 is chloro, or a
pharmaceutically acceptable salt thereof.
4. A compound according to Claim 1 which is 3-[4-(7-Chloro-2-methyl-5H-pyrrolo[2,lc][
l,4]benzodiazepin-l l-yl)piperazin-l-yl]-2,2-dimethylpropanoic acid, or a
pharmaceutically acceptable salt thereof.
5. A compound according to Claim 1 which is 3-[4-(7-Chloro-2-methyl-5H-pyrrolo[2,lc][
l,4]benzodiazepin-l l-yl)piperazin-l-yl]-2,2-dimethyl-propanoic acid dihydrochloride.
6. A pharmaceutical composition comprising a compound according to any one of
Claims 1 to 5, or a pharmaceutically acceptable salt thereof, in combination with at least
one pharmaceutically acceptable carrier, diluent, or excipient.
7. A method of treating insomnia in a mammal comprising administering to the mammal
in need of such treatment an effective amount of a compound according to Claim 1, or a
pharmaceutically acceptable salt thereof.
8. The method of Claim 7 where the mammal is a human.
9. The method of Claim 7 where the insomnia is characterized by difficulties in sleep
onset or sleep maintenance or both.
10. The method of Claim 9 where the mammal is a human.
11. A compound according to any one of Claims 1to 5, or a pharmaceutically acceptable
salt thereof, for use in therapy.
12. A compound according to any one of Claims 1to 5, or a pharmaceutically acceptable
salt thereof, for use in the treatment of insomnia.
13. A compound according to any one of Claims 1to 5, or a pharmaceutically acceptable
salt thereof, for use in the treatment of insomnia where the insomnia is characterized by
difficulties in sleep onset or sleep maintenance or both.
14. A compound for the use according to either Claim 12 or 13 in a human.
15. The use of a compound according to any one of Claims 1to 5, or a pharmaceutically
acceptable salt thereof, in the manufacture of a medicament for the treatment of insomnia.
16. The use of a compound according to any one of Claims 1to 5, or a pharmaceutically
acceptable salt thereof, in the manufacture of a medicament for the treatment of insomnia
where the insomnia is characterized by difficulties in sleep onset or sleep maintenance or
both in a mammal.
17. A pharmaceutical composition comprising a compound according to any one of
claims 1to 5, or a pharmaceutically acceptable salt thereof, in combination with at least
one pharmaceutically acceptable carrier, excipient or diluent, and optionally other
therapeutic ingredients.
18. The pharmaceutical composition of Claim 17 where the other therapeutic ingredient
comprises a selective serotonin reuptake inhibitor.