ANTI-CANCER COMPOUND AND PHARMACEUTICAL COMPOSITION CONTAINING
THE SAME
The present invention relates to the compound of formula (I):

and to the pharmaceutical composition comprising it. This compound is preferably the
levogyratory compound (1a). This compound can be used as an anti-cancer ingredient. The
invention also relates to a process for preparing the compound (I) or (la) and also to some
of the intermediates in said process.
[Technical problem]
A number of cancer treatment strategies are aimed at inhibiting the Aurora-type kinases,
particularly Aurora A and B, which are involved in the regulation of mitosis; in this regard,
see Nature Reviews 2004, 4, 927-936; Cancer Res. 2002, 94, 1320; Oncogene 2002, 21,
6175; Mol.Cell.Biol. 2009, 29(4), 1059-1071; Expert Opin. Ther. Patents 2005, 75(9),
1169-1182; Clin.CancerRes. 2008, 74(6), 1639.
Some Aurora inhibitor compounds (for example, MLN-8237 from Millennium, AZD-1152
from Astra-Zeneca or SNS-314 from Sunesis) are presently under evaluation in clinical
trials. MLN-8237 is selective for Aurora A while AZD-1152 is selective for Aurora B. Since
both kinases, Aurora A and B, are deregulated in cancer, inhibiting both Aurora A and B
provides an advantage relative to selective inhibition of one kinase or the other. Moreover,
multikinase compounds are in existence, such as the compound AT-9283 from Astex,
which inhibit a number of kinases, including Aurora A and B. For this type of compound it
is difficult to predict that the inhibition of the Aurora kinases might actually be exploited
clinically, since the inhibition of kinases other than Aurora A and B is likely to give rise to
side effects. One technical problem the invention intends to solve is therefore that of
developing a compound which is a potent and selective inhibitor of Aurora A and B.

The cyclic nucleotide phosphodiesterase enzyme PDE3 plays a major part in the signalling
mediated by the cyclic nucleotides cAMP and cGMP that takes place in the myocytes of
the smooth cardiac and vascular muscles. The inhibition of PDE3 by small molecules has
an inotropic and vasodilatory action, which may prove to be useful on a short-term basis
for the treatment of certain cardiomyopathies in which defects in cardiac contraction are a
feature. It has been shown, however, that the long-term use of these molecules increases
mortality among this type of patient. Furthermore, the use of PDE3 inhibitors in patients
who do not present this type of pathology, such as patients affected by cancer, may give
rise to unwanted effects on cardiac rhythm. It is therefore important, in the context of an
anti-cancer therapy, not to inhibit PDE3. In this regard, see Exp.Opin.lnvest.drugs 2002,
11, 1529-1536 "Inhibitors of PDE3 as adjunct therapy for dilated cardiomyopathy"; Eur.
Heart J. supplements 2002, 4(supplement D), D43-D49 "What is wrong with positive
inotropic drugs? Lessons from basic science and clinical trials". Another technical problem
the invention intends to solve is that the Aurora A and B inhibitor compound shall not
inhibit the enzyme PDE3.
It is also important that the anti-cancer ingredient presents a metabolic stability (see
section 10.2.2 of "Chimie pharmaceutique" G.L.Patrick, De Boeck, published 2003, ISBN =
2-7445-0154-9). The reason is that the inadequacy of the pharmacokinetics of
pharmaceutical compounds is one of the primary reasons for failure in their development
(Curr. Pharm. 2005,11, 3545 "Why drugs fail—a study on side effects in new chemical
entities"). Moreover, the metabolism is often a major determinant of clearance, of drug
interactions, of intra-individual variability in pharmacokinetics, and of clinical efficacy and
toxicity (Curr.Drug Metab. 2004, 5(5), 443-462 "Human hepatocytes in primary culture: the
choice to investigate drug metabolism in man"). Another technical problem the invention
intends to solve is that the Aurora A and B inhibitor compound shall exhibit high chemical
and metabolic stability.
[Prior art]
Bioorg.Med.Chem.Lett. 2002, 12, 1481-1484 describes in table II the compound 6A,
which has a different tricyclic structure.
WO 01/36422 describes compounds having a different tricyclic structure.
WO 2004/005323 describes the compound E5A29 of formula (a):


as an EPO receptor having a different tricyclic structure. Furthermore, the compound does
not include a phenyl ring substituted by the group -O-benzimidazolyl at the top of the
tricyclic ring system.
WO 2005/016245 describes anti-cancer compounds having a different tricyclic structure, of
formula (b):
in which R4 may represent a substituted phenyl group. Substitution by the -O-
benzimidazolyl group is neither described nor suggested.
WO 2007/012972 and EP 1746097 describe anti-cancer compounds of formula (c)

and, in one embodiment, of the formula (c'):

R2 represents a substituted aryl or heteroaryl group, X represents N or CR7, R5 and R6
may both represent H or CH3. No example in WO 2007/012972 contains the group

which characterizes the compound of formula (I). Moreover, among
the compounds resolved, WO 2007/012972 teaches that it is dextrogyratory compounds
which are the most active on Aurora A or B (cf. ex.119 and 120 in the table on page 147).
WO 02/062795 describes compounds of formula (d):

in which R4 and R5 may optionally form a 5- or 6-membered ring.
[Brief description of the invention]
The invention relates to the compound of formula (I):

more particularly in its levogyratory form (la), particularly that exhibiting the optical rotation
[α]D= -38.6+0.7 at a concentration of 0.698 mg/ml in methanol. The compound may exist in
the form of a base or an addition salt with an acid, particularly a pharmaceutically
acceptable acid. This compound is a selective inhibitor of Aurora A and B kinases. It can
be used as an anti-cancer ingredient.
The invention also relates to a pharmaceutical composition comprising the compound and
at least one pharmaceutically acceptable excipient, and to the medicament comprising the
compound.

The invention also relates to the process for preparing the compound, comprising:
■ reacting together the three compounds below, PG denoting a protective group for the
NH function of the benzimidazole

to give the compound:
■ deprotecting the NH function of benzimidazole, to give the compound of formula (I);
■ where appropriate, isolating the levogyratory compound.
The reaction between the three compounds is carried out in an alcohol at reflux,
particularly 1-butanol. The following intermediates also form part of the invention:
. PG may be, for example, the group
[Description of the invention]
The invention relates to the compound of formula (I):


This compound may exist in a racemic form or in the form of the two levogyratory (la) and
dextrogyratory (lb) enantiomers. The levogyratory compound (la) has a selective inhibitory
activity on Aurora A and B kinases which is much greater than that of the dextrogyratory
enantiomer (lb). The levogyratory compound (la) also has an anti-proliferative activity
which is greater than that of the dextrogyratory enantiomer (lb) (see Table I).
The three compounds (I), (la) and (lb) may exist in the form of a base or an addition salt of
an acid. The salt is advantageously prepared with a pharmaceutically acceptable acid (see
P.Stahl, C.Wermuth; Handbook of Pharmaceutical Salts; Wiley Ed. ISBN-13: 978-
3906390260, ISBN-10: 3906390268), although the salt of any other type of acid, which
may be used, for example, for a purification or isolation step, also forms part of the
invention.
The compounds (I) and (la) may be used as anti-cancer ingredients or for preparing a
medicament for treating a cancer. The cancer is more particularly a cancer in which Aurora
A and/or B kinase(s) are/is involved.
The compounds (I), (la) and (lb) are obtained according to Scheme I below:

step 1: the NH function of a 2-halo-benzimidazole (Hal= Br or CI) is protected using a
protective group PG, to give A1. PG-X represents a reagent which introduces the

protective group PG. PG may be, more particularly, dihydropyran and, in that
case, PG-X represents 3,4-dihydro-2H-pyran
step 2: A1 is reacted with 3-formyl-phenoi in the presence of a base producing the
corresponding phenolate ion, to give B1. The base may be an alkali metal hydride, such as
NaH, for example. The reaction is carried out in a polar aprotic solvent such as DMF;
step 3: B1, 3-amino-2-ethoxycarbonylpyrrole and 1,3-cyclohexanedione are reacted with
one another, for example in an alcohol (e.g. 1-butanol) at reflux, to give C1;
step 4: the NH function of C1 is deprotected, to give the compound (I). The deprotection
conditions are dependent on the nature of PG. For example, where PG represents
dihydropyran, a strong acid is used;
step 5: using, for example, a chiral chromatography, the two enantiomers (la) and (lb) are
isolated.
For each of steps 1-5, reference may be made to the specific conditions described in
example 1.
[Examples]
Analytical methods
Method LC/MS-A
The products for analysis are separated on an Acquity Beh C18 UPLC column, 1.7 urn
2.1x50 mm (Waters) thermostated at 70°C and eluted at a flow rate of 1 ml/min with a
gradient from acetonitrile containing 0.1% formic acid (solvent B) into water containing
0.1% formic acid (solvent A); elution programmeme: isocratic stage at 5% of solvent B for
0.15 min, gradient from 5% to 100% of solvent B in 3.15 min then return to the initial
conditions over 0.1 min. The products are detected by an Acquity PDA diode-array UV/vis
detector (Waters, wavelength range scanned: 192-400 nm), a Sedex 85 light scattering
detector (Sedere, nebulizing gas: nitrogen, nebulizing temperature: 32°C, nebulizing
pressure 3.8 bar) and an Acquity SQD mass spectrometer (Waters, operating in positive
and negative mode, mass range scanned: 80 to 800 amu).
Method LC/MS-B

The spectra were obtained on a Waters UPLC-SQD instrument in positive and/or negative
electrospray ionization mode (ES+/-), under the following liquid chromatography conditions:
column: ACQUITY BEH C18 1.7 urn, 2.1x50 mm; TColUmn: 50°C; flow rate: 1 ml/min;
solvents: A: H20 (0.1% formic acid); B: CH3CN (0.1% formic acid); gradient (2 min): 5% to
50% B in 0.8 min; 1.2 min: 100% B; 1.85 min 100% B; 1.95 min 5% B.
1HNMR
The spectra are recorded on a Bruker spectrometer, the product being dissolved in DMSO-
d6. The chemical shifts 8 are expressed in ppm.
IR
The infrared spectrum is recorded on a Nicolet Nexus spectrometer, on a KBr disc, with a
resolution of 2 cm-1.
Measurement of the optical rotation
The optical rotations were recorded on a Perkin-Elmer 341 polarimeter.
Elemental analysis
The elemental analyses were made on a Thermo EA1108 analyser.
Measurement of the activity on Aurora A and B
The capacity to inhibit the kinase activity of the enzyme is estimated by measuring the
residual kinase activity of the enzyme in the presence of different concentrations of the test
compound (generally from 0.17 to 10 000 nM). A dose-response curve is produced, which
allows an IC50 (50% inhibitory concentration) to be determined. The kinase activity is
measured by a radioactive assay of the amount of radioactive phosphate (33P)
incorporated into a fragment of the protein NuMA (Nuclear Mitotic Apparatus protein) after
30 minutes of incubation at 37°C. The test compound is first dissolved at different
concentrations in dimethyl sulphoxide (DMSO). Reaction takes place in the wells of a
FlashPlate microtiter plate (Nickel Chelate FlashPlate-96, PerkinElmer). Each well (100 μl)
contains 10 nM Aurora A, 500 nM NuMA, 1 uM ATP and 0.2 uCi ATP-y-33P in a buffer of
50 mM Tris-HCI, pH=7.5; 10 mM MgCI2; 50 mM NaCI, 1 mM dithiothreitol. The final
percentage of DMSO is 3%. After homogenization by stirring, the plate is incubated at
37°C for 30 minutes. The contents of the wells are then removed and the wells are washed
with PBS buffer. The radioactivity is then measured using a TRILUX I450 Microbeta
counter (WALLAC). In each plate, there are eight control wells: four positive controls

(maximum kinase activity), for which measurement is made in the presence of enzyme and
substrate and in the absence of compound of the invention, and four negative controls
(background) for which measurement is made in the absence of enzyme, substrate and
test compound. The measurements are given in Table I.
Aurora A
The recombinant human enzyme Aurora A used is expressed in entire form with a poly-
Histidine tag in N-terminal position and is produced in E. coli. A fragment (amino acids
1701-2115) of the human protein NuMA, with a poly-Histidine tag in C-terminal position, is
expressed in recombinant form in E. coli.
Aurora B/lncenp
The entire human enzyme Aurora B is coexpressed with a fragment of the human protein
Incenp (aa 821-918) in a baculovirus system and is expressed in insect cells. Aurora B has
a poly-Histidine tag in N-terminal position, while the Incenp fragment possesses a
Glutathione-S-Transferase (GST) tag in N-terminal position. The two proteins form a
complex which is called Aurora B/lncenp. A fragment (aa1701-2115) of the human protein
NuMA with a poly-Histidine tag in C-terminal position is expressed in recombinant form in
E. coli. This fragment is used as substrate.
Measurement of the cell proliferation
Cells (tumour cell line HeLa - ref.: ATCC CCL-2 and HCT116 ref.: ATCC CCL-247) are
contacted with the test compound for 96 hours, with 14C-thymidine added during the last 24
hours. The cell proliferation is estimated by the amount of 14C-thymidine incorporated in
the cells.
The test compound is dissolved to form a stock solution at 10 mM in DMSO, and this stock
solution is used to produce a range of serial dilutions, generally from 10 000 uM to 0.3 uM,
these serial dilutions being themselves diluted 1/50 in the cell culture medium (20x solution)
which will be used for 1/20 dilution in the cell culture plates. The final concentrations of the
test compound will generally be between 10 000 and 0.3 nM.
DO: the cells are seeded in 96-well Cytostar plates in 180 ul_ of culture medium. The plates
are then placed in an incubator at 37°C, 5% C02 for four hours. The test products are then
added in a volume of 10 uL per well, starting from a 20x solution. This solution contains

2% of DMSO in the culture medium. The final concentration of DMSO is therefore 0.1%.
The plates are then placed in an incubator at 37°C/5% CO2 for 72 hours.
D3: after 72 hours, 10 μL per well of 14C-thymidine at 10 uCi/mL in the culture medium are
added. The plates are then placed in an incubator at 37°C, 5% CO2 for 24 hours.
D4: The incorporation of 14C thymidine is measured on a Micro-Beta radioactivity counter
(Perkin-Elmer) after this 24-hour, "pulse" period. The total time of treatment of the cells
with the test product is 96 hours.
The percentage inhibitions IC50 are calculated in Excel using the following formula:

X = Measurement for the sample
CC = Cell Control
Blank = Measurement in the wells without cells
IC50 is calculated using the XLfit software (IDBS, UK) with the aid of formula 205, with the
parameter D (Hill number) locked to a value of 1. The results are given in Table I.
Evaluation of the effect of the compounds of the invention on the activity of the
enzyme PDE3
The effect of the compounds of the invention on the activity of the enzyme PDE3 was
evaluated by the company CEREP (Le bois I'Eveque, 86600 Celle I'Evescault, France;
http://www.cerep.fr) in accordance with its standard protocol (see Bender, A.T., Beavo, J.A.
Pharmacol Rev. 2006, 58, 488-520: the enzyme PDE3A in recombinant form is expressed
in Sf9 cells, the substrate is cAMP and the residual AMPc is measured by HTRF. The
reference inhibitor in the test is milrinone, whose IC50 is 270 nM. The residual activity %
are related to the control without inhibitor). The results are expressed either as the
concentration which induces inhibition by 50% (IC50) or as a percentage inhibition
measured at a set concentration of the compound. The results are given in Table I.
Measurement of the chemical stability of the compounds
The chemical stability of the compounds was measured in various media: 0.05 N
hydrochloric acid in a 50/50 (v/v) water/acetonitriie mixture; 0.05 N sodium hydroxide in a
50/50 (v/v) water/acetonitrile mixture; sodium phosphate buffer, 25 mM, pH=7.4, in a 50/50
(v/v) water/acetonitrile mixture; sodium phosphate buffer 25 mM, pH=7.4, in a 50/50 (v/v)
water/acetonitrile mixture containing 1% (w/v) of benzylamine hydrochloride; sodium
phosphate buffer, 25 mM, pH=7.4 in a 50/50 (v/v) water/acetonitrile mixture containing 1%

(v/v) of 2-mercaptoethanol. The compounds are diluted in the media under study at a final
concentration of 100 uM, by dilution of a 10 mM stock solution in DMSO. The solutions are
stored at 20°C for a total time of 48 hours, and the concentration of the compounds under
study is measured over time (t=0, 1, 6, 12, 24 and 48 hours) by HPLC. HPLC analysis is
carried out with an Agilent system 1100 instrument equipped with a diode array detector
on a Luna C18 column, 30x4.6 mm, 3 urn (Phenomenex) which is eluted with a gradient
from acetonitrile (solvent B) into water containing 0.5% (v/v) of formic acid (solvent A) at a
flow rate of 1.5 ml/min and a temperature of 25°C. The elution programme consists of a
gradient from 10 to 90% solvent B in 5 minutes, followed by an isocratic stage of one
minute at 90% of solvent B, and return to the initial conditions over one minute. The
concentration of the products under study is estimated from the height and area of the
characteristic peak of the product under study on a chromatogram at the maximum
wavelength of each product. The area and the height measured at each time of sampling
are related to the area and height obtained for the sample at time 0. When degradation is
observed, a half-life is measured from the resulting time-concentration curve. The results
are given in Table II.
Evaluation of the metabolism in the presence of microsomal preparations of human
and murine livers.
Whereas microsome preparations remain important in determining the metabolic stability
of a pharmaceutical compound, primary hepatocyte culture allows a more detailed
evaluation of its intrinsic clearance, and better, vitro-vivo correlations suggest hepatic
clearance in humans.
The compounds of the invention (5 uM) are incubated at physiological temperature over
human and murine microsomal liver fractions (1 mg/ml of proteins), diluted in a phosphate
buffer, in the presence of bovine serum albumin (1 mg/ml BSA), and the reduced form of
nicotinamide adenine dinucieotide phosphate (1 mM NADPH). To terminate incubation,
four volumes of acetonitrile containing corticosterone as internal standard (IS) are added.
The samples are centrifuged and the supematants are analysed by liquid
chromatography/tandem mass spectrometry coupling (LC/MS-MS). The LC/MS-MS
analysis is performed on a QTRAP API4000 mass spectrometer (Sciex) equipped with an
1100 series chromatography system (Agilent) and a Pal CTC automatic injector. The data
are acquired and analysed using Analyst 1.4.1 software. The samples are separated on a
3 um C18 Polaris column, eluted at a flow rate of 0.7 ml/min with a gradient from
acetonitrile (solvent B) into water containing 0.1% formic acid (solvent A). The elution

programme is composed of the gradient from 20 to 90% of solvent B in 2 minutes, an
isocratic stage at 90% of solvent B, for 0.9 minute, and a return to the initial conditions in
0.1 minute. The area of the chromatographic peaks for the compound and for the internal
standard are integrated using the Analyst-Classic algorithm. The metabolic stability of the
products of the invention is estimated by comparing the integration ratios (ion currents of
the compounds/ion current IS) measured after 0 minute (tO) and 20 minutes (t20) of
incubation. The metabolic stability is then expressed as a percentage disappearance in
accordance with the following formula:
Metabolism% = (ratio of peaks at tO - ratio of peaks at t20) / ratio of peaks at tO
The results are given in Table III.
Evaluation of the clearance in the presence of human hepatocvtes.
The compounds of the invention (0.5 or 5 uM) are incubated for 24 hours in 48-well plates
covered with collagen in the presence of fresh or cryopreserved human hepatocytes
(-200 000 cells/well) obtained from specific donors, in an incubator at a physiological
temperature. The incubations are carried out with a culture medium (HAM F12 -William E).
At various times (0; 0.5; 1; 2; 4; 6; 8 and 24 hours), 100 ul are sampled from each well,
and the kinetics are halted by addition of 700 ul of a 70/30 (v/v) acetonitrile/water mixture
containing corticosterone as internal standard (IS). The cells are then dissociated and the
intracellular and extracellular media are mixed and stored in frozen form at -20°C prior to
their analysis. Following thawing, the samples are centrifuged at 300 g for 20 minutes and
the supematants are analysed by liquid chromatography/tandem mass spectrometry
coupling (LC/MS-MS). The LC/MS-MS analysis is performed on a QTRAP API4000 mass
spectrometer (Sciex) equipped with an 1100 series chromatography system (Agilent) and
a Pal CTC automatic injector. The data are acquired and analysed using Analyst 1.4.1
software. The samples are separated on a 3 urn C18 Polaris column, eluted at a flow rate
of 0.7 ml/min with a gradient from acetonitrile (solvent B) into water containing 0.1% formic
acid (solvent A). The elution programme is composed of the gradient from 20 to 90% of
solvent B in 2 minutes, an isocratic stage at 90% of solvent B, for 0.9 minute, and a return
to the initial conditions in 0.1 minute. The concentration of the products of the invention is
measured by integrating the ion current of the characteristic ions of the products, relative
to the internal standard (IS). The compound/IS ratios obtained are related to calibration
standards of known concentrations, thereby allowing the concentration of the products of
the invention to be ascertained. The intrinsic clearance (expressed in ml.h~1.10"6 cells) is

then determined from the kinetic profiles (concentration/time), using the WinNonLin
software (5.0). The results are given in Table IV.
Example 1: preparation of ethyl 8-oxo-9-r3-(1H-benzimidazol-2-yloxv)phenvn-
4,5,6,7,8,9-hexahvdro-2H-pyrrolor3,4-b1quinoline-3-carboxvlate(l)

A1. 2-Chloro-1-aetrahydro-pyran-2-vl)-1H-benzimidazole (CAS Na 208398-29-2)

A 10 I reactor is charged, under argon and with stirring, with 2.5 I of THF, 180 g of 2-
chlorobenzimidazole (1.18 mol) and 325 ml of 3,4-dihydro-2H-pyran (6.56 mol, 3 eq.). The
reactor is heated until dissolution occurs (temperature of the mixture: 40°C). Then 6.3 g of
para-toluenesulphonic acid (0.033 mol, 0.028 eq.) are introduced. The temperature is held
at between 49 and 52°C for 2.5 h. Cooling takes place at 12°C and 7.65 g of sodium
methoxide (0.142 mol, 0.12 eq.) are added, with stirring maintained for a total time of
15 min. The temperature is then taken to 18°C, 5 I of n-heptane are added, and the whole
mixture is filtered on 300 g of Clarcel FLO-M, the retentate being washed with 5 I of n-
heptane. The filtrate is concentrated to dryness under reduced pressure to give 292.6 g of

2-chloro-1-(tetrahydro-pyran-2-yl)-1H-benzimidazole in the form of a slightly yellow oil
(quantitative yield). 1H NMR (400 MHz, DMSO-d6): 1.42 to 2.01 (m, 5H); 2.21 to 2.34 (m,
1H); 3.69 to 3.78 (m, 1H); 4.12 (d, J=11.4 Hz, 1H); 5.72 (dd, J=2.4 and 11.2 Hz, 1H);7.22
to 7.34 (m, 2H); 7.62 (d, J=7.2 Hz, 1H); 7.78 (d, J=7.2 Hz, 1H).
B1: 3-f1-(Tetrahvdro-Pvran-2-vn-1H-benzimidazol-2-vloxv1benzaldehvde

Two 2 I three-necked round-bottomed flasks, each equipped with a condenser, a
thermometer and a stirrer shaft, are charged under argon with N,N-dimethylformamide (0.4
I per flask), and 3-hydroxybenzaldehyde (68.5 g, flask 1; 64.2 g, flask 2; 1.08 mol). Sodium
hydride (60% dispersion in mineral oil) is then added in portions (flask 1: 26 g; flask 2: 24 g;
1.25 mol, 1.2 eq.), the maximum temperature during the addition being 32°C. 2-chloro-1-
(tetrahydro-pyran-2-yl)-1H-benzimidazole (A1), purity estimated at 85%) is then introduced
(flask 1: 151 g in 0.5 I of N,N-dimethylformamide; flask 2: 142 g in 0.5 I of N,N-
dimethylformamide; 1.05 mol, 0.97 eq.). The mixture is then heated at reflux (temperature
140°C, temperature rise time 40 min) and the reflux is maintained for 1 h. Heating is then
stopped and the mixture is allowed to cool over 1.5 h. The contents of the two flasks are
combined. The combined mixture is mixed slowly into 5 I of ice-water. The aqueous phase
obtained is then extracted with 4x2.5 I of ethyl acetate (AcOEt). The organic phases are
then combined, washed with 3 I of water and then with 2 I of saturated NaCI solution, and
finally dried by addition of MgS04 overnight. The organic phase obtained is then filtered on
a glass frit (porosity 4) and concentrated to dryness under reduced pressure to give 385 g
of a brown oil (LC/MS-A, tr (retention time)=1.86 min, MS positive mode: m/z=323.16).
A fraction of 158 g of the crude product obtained above is dissolved hot in 1.5 I of an n-
heptane/AcOEt mixture (8/2 by volume), combined with 500 g of silica (70-30 mesh), and
the mixture is stirred for 45 min. The resulting suspension is filtered on Celite, and washed
with 3 I of an n-heptane/AcOEt mixture (8/2 by volume). The organic phase obtained is
concentrated to dryness under reduced pressure. The residue is resuspended in 200 ml of
isopropyl ether by mechanical stirring and ultrasound treatment, and then filtered on a
glass frit (porosity 3). The resulting solid is washed with 2x40 ml of isopropyl ether and
dried under reduced pressure at 40°C for 16 h to give 68 g of solid. A similar treatment
applied to the remainder of the crude product produces 87.8 g of solid. The solids obtained

are combined and homogenized to give 155.8 g of 3-[1-(tetrahydro-pyran-2-yl)-1H-
benzimidazol-2-yloxy]benzaldehyde in the form of pale beige crystals (LC/MS-A, tr=1.87
min, MS positive mode m/z=323.13). MS(LC/MS-B): tr=1.00 min; [M+H]+: m/z 323; 1H
NMR (400 MHz, DMSO-d6): 1.54 to 1.62 (m, 1H); 1.63 to 1.84 (m, 2H); 1.92 to 2.03 (m,
2H); 2.30 to 2.42 (m, 1H); 3.70 to 3.79 (m, 1H); 4.10 (d, J=11.5 Hz, 1H); 5.74 (dd, >2.1
and 11.1 Hz, 1H); 7.13 to 7.22 (m, 2H); 7.43 (d, J=7.3 Hz, 1H); 7.65 (d, J=7.3 Hz, 1H);
7.73 (t, J=7.8 Hz, 1H); 7.78 to 7.83 (m, 1H); 7.87 (d, J=7.8 Hz, 1H); 7.96 (s, 1H); 10.05 (s,
1H).
Washing of the silica phases used above with 2 I of an n-heptane/AcOEt mixture (1/1 by
volume) produces 67 g of product following concentration to dryness under reduced
pressure. This product is taken up in 2 I of an n-heptane/AcOEt mixture (9/1 by volume),
combined with 285 g of silica (70-30 mesh), stirred and treated with ultrasound for 1 h. The
suspension is then filtered on Celite and the solid phase is washed with 2 I of an n-
heptane/AcOEt mixture (9/1 by volume). The filtrate is concentrated to dryness under
reduced pressure and the residue is triturated in 400 ml of an n-heptane/ethanol mixture
(95/5 by volume), filtered on a glass frit (porosity 3) and dried under reduced pressure to
give 35 g of 3-[1-(tetrahydro-pyran-2-yl)-1H-benzimidazol-2-yloxy]benzaldehyde in the
form of pale beige crystals (LC/MS-A, tr=1.93 min, MS positive mode m/z=323.16).
C1: Ethyl 8-oxo-9-f3-H -(tetrahvdro-pyran-2-v0-1 H-benzimidazol-2-vloxvlphenyl)-
4,5,6,7,8,9-hexahydro-2H-pyrrolof3.4-b1quinoline-3-carboxvlate

A 2 I conical flask is charged, with magnetic stirring, with 50 g of 3-amino-2-
ethoxycarbonylpyrrole hydrochloride and 0.204 I of 2N sodium hydroxide solution. The
mixture is stirred for 15 minutes at ambient temperature (AT), and then extracted with
3x0.3 I of dichloromethane. The organic phases are combined, dried over MgS04 and
concentrated to dryness under reduced pressure. The residue is triturated with n-pentane,
filtered and dried under reduced pressure to a constant weight, to give 36.4 g of 3-amino-
2-ethoxycarbonylpyrrole in the form of a brown solid.

A 2 I three-necked round-bottomed flask equipped with a stirrer shaft, a thermometer and a
condenser is charged with 1.2 I of 1-butanol, 145 g of 3-[1-(tetrahydro-pyran-2-yl)-1H-
benzimidazol-2-yloxy]benzaldehyde (0.405 mol, B1), 62.4 g of 3-amino-2-
ethoxycarbonylpyrrole (1 eq., 0.405 mol), 46.8 g of 1,3-cyclohexanedione in 97% form
(1 eq., 0.405 mol) and 70.5 ml of N,N-diisopropylethylamine (1 eq.) and the mixture is
taken to reflux (temperature rise time 55 min, reflux maintained for 30 min, temperature
114°C). The mixture is then cooled to AT and concentrated to dryness under reduced
pressure to give 290 g of a brown oil containing ethyl 8-oxo-9-{3-[1-(tetrahydro-pyran-2-yl)-
1H-benzimidazol-2-yloxy]phenyl}-4.5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-
carboxylate (LC/MS-A, tr=1.96 min, MS positive mode m/z=553.33). A similar operation
carried out with 35 g of 3-[1-(tetrahydro-pyran-2-yl)-1H-benzimidazol-2-yloxy]-
benzaidehyde (0.098 mol, example B1) produces 72 g of a brown oil containing ethyl 8-
oxo-9-{3-[1-(tetrahydro-pyran-2-yl)-1H-benzimidazol-2-yloxy]phenyl}-4,5,6,7,8,9-
hexahydro-2H-pyrrolo[3,4-b]quinoline-3-carboxylate (LC/MS-A, tr=1.96 min, MS positive
mode m/z=553.35).
D1: Ethyl 8-oxo-9-r3-(1 H-benzimidazol-2-vloxv)phenvn-4.5,6,7,8,9-hexahvdro-2H-
pyrrolof3,4-blquinoline-3-carboxylate (compound I)

A 2 I round-bottomed flask is charged with 224 g of the brown oil containing ethyl 8-oxo-9-
{3-[1-(tetrahydro-pyran-2-yl)-1H-benzimidazol-2-yloxy]phenyl}-4,5,6,7,8,9-hexahydro-2H-
pyrrolo[3,4-b]quinoline-3-carboxylate (example 1.3), 0.7 I of ethanol and 0.243 I of 2N
hydrochloric acid. The mixture is stirred at AT for 16 h and then filtered on a glass frit
(porosity 4). The filtrate is concentrated to dryness under reduced pressure and the
residue is triturated with 0.5 I of isopropyl ether. The solid obtained is dried under reduced
pressure at a constant weight to give 253 g of a brown solid containing ethyl 8-oxo-9-[3-
(1H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-
carboxylate (LC/MS-A, tr=1.46 min, MS positive mode m/z=469.29). A similar operation
carried out with 54 g of the brown oil containing ethyl 8-oxo-9-{3-[1-(tetrahydro-pyran-2-yl)-
1H-benzimidazol-2-yloxy]phenyl}-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-

carboxylate (C1) produces 60 g of a brown solid containing ethyl 8-oxo-9-[3-(1 H-
benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-b]quinoline-3-
carboxylate (LC/MS-A, tr=1.48 min. MS positive mode m/z=469.29).
An aliquot fraction of 0.8 g of the product obtained may be purified by chromatography on
a 50 g silica cartridge (10-90 urn) (Biotage SNAP, KP-Sil) eluted with an isocratic stage of
dichloromethane of 20 min, then a gradient from 0 to 1% by volume of isopropanol in
dichloromethane over 1 h, and, finally, an isocratic stage of dichloromethane/isopropanol
(99/1 by volume) of 20 min. The fractions containing the expected product are combined to
give 0.21 g of a yellow solid. The products of two similar chromatographic separations
carried out on the same scale are crystallized from acetonitrile to give a total of 0.16 g of
ethyl 8-oxo-9-[3-(1H-benzimidazol-2-yloxy)phenyl]-4,5,6,7,8,9-hexahydro-2H-pyrrolo[3,4-
b]quinoline-3-carboxylate in the form of beige crystals (LC/MS-A, tr=1.61 min, MS positive
mode m/z=469.28). 1H NMR (400 MHz, DMSO-d6): 1.29 (t, J=7.0 Hz, 3H); 1.80 to 1.97 (m,
2H); 2.19 to 2.27 (m, 2H); 2.55 to 2.69 (m, 1H); 2.81 (dt, J=4.8 and 17.2 Hz, 1H); 4.26 (q,
J=7.0Hz, 2H);5.11 (s, 1H); 6.73 (d, J=Z.3 Hz, 1H); 7.02 to 7.16 (m, 5H); 7.25 (t, J=7.9 Hz,
1H); 7.31 to 7.38 (m, 2H); 8.34 (s, 1H); 11.33 (broads, 1H); 12.26 (broad s, 1H). Elemental
analysis: C= 68.72%; H= 5.10%; N= 11.82%; H20= 0.38%.
Example 2: Levogyratory enantiomer (la) of ethyl 8-oxo-9-r3-(1H-benzimidazol-2-
vloxv)phenvn-4.5,6.7.8.9-hexahvdro-2H-pyrrolor3.4-b1quinoline-3-carboxylate
The levogyratory enantiomer is purified from the crude product of example D1 on a Welk-
01RR chiral column, 10 uM, 80x350 mm (Regis, USA) eluted with an n-
heptane/dichloromethane/ethanol/triethylamine mixture (50/47.5/2.5/0.1 by volume). The
elution of the products is detected by UV spectroscopy at 265 nm. Amounts of 10 g of the
crude product described in example D1 are injected in each operation. Under these
conditions, the peak corresponding to the levogyratory enantiomer is eluted with a tr of
between 50 and 80 min. The fractions of purified levogyratory enantiomer corresponding to
the operations needed to purify 310 g of the crude product described in example D1, are
combined, homogenized and concentrated to dryness under reduced pressure to give 50 g
of a beige solid. Mass spectrum (LC/MS-B): tr=0.77 min; [M+H]+: m/z 469; [M-H]-: m/z 467.
1H NMR (400 MHz, DMSO-d6): 1.29 (t, J=7.1 Hz, 3H); 1.79 to 1.97 (m, 2H); 2.19 to 2.27
(m, 2H); 2.55 to 2.66 (m, 1H); 2.81 (dt, J=4.9 and 17.1 Hz, 1H); 4.26 (q, J=7.1 Hz, 2H);
5.12 (s, 1H); 6.73 (d, J=3.4 Hz, 1H); 7.02 to 7.16 (m, 5H); 7.25 (t, J=8.3 Hz, 1H); 7.29 to
7.41 (m, 2H); 8.32 (s, 1H); 11.31 (broad s, 1H); 12.26 (broad s, 1H). IR: principal bands:
1678; 1578; 1525; 1442; 1188; 1043 and 743 cm"1. Optical rotation: [