Useful As MOGAT-2 Inhibitors
Ingestion of excess dietaiy fat is a leading cause of diet induced obesity and can
have a profound detrimental effect on a people's health. More than 90% of dietaiy fat
for humans is triacylglyceroi (or triglyceride), which is nearly completely absorbed by the
small intestine. The enzyme acyi CoA:monoacylgiycerol acytransferase-2 (MOGAT-2)
is believed to play an important role in the absorption of dietaiy fat in the small intestines.
It has been demonstrated that MOGAT-2 deficient mice when fed a high fat diet are
protected against developing obesity, glucose intolerance, hypercholesterolemia and
developing a fatty liver. Further, it has also been shown that MOGAT-2 deficient mice
exhibit lower plasma triacylglyceroi levels after a dietary olive oil challenge. (Yen, et al,
Nat. Med. 2009, 15(4), 442-446.)
There is a need for additional drugs for the treatments of hypertriglyceridemia.
There is also a need to for new inhibitors of the MOGAT-2 receptor. The present
invention addresses one or more of these needs by providing alternative compounds and
treatment methods, which may be suitable for the treatment hypertriglyceridemia.
The present invention provides a compound of Formula I
wherein l is selected from: -C and -CF ; R2 is selected from: and -CH ; R3 is
selected from: and -CH ; R4 is selected from: , -OC1-3alkyi, and halogen; R5 is
selected from: H, -CF , -OCH and halogen; R6 is selected from: and halogen;
provided that at least one of R4, R5, and R6 is ; or a pharmaceutically acceptable salt
thereof.
Compounds of the present invention can have one or more chiral centers. In the
compounds of Formula II below, one of the chiral centers is identified with an asterisk
(*). When Rl is -CH3, preferred compounds have the (R) configuration at this chiral
center. When Rl is -CF , preferred compounds have the (S) configuration.
h one embodiment Rl is -CH 3. In another embodiment Rl is -CF 3.
Preferably R2 is .
Preferable R3 is H.
Preferably R4 is selected from: , -OCH(CH3) , and halogen. More preferably
R4 is selected from: H and halogen. Still more preferably R4 is selected from; and
CI.
Preferably R5 is selected from: H, -CF -OCH 3, F, and CI. More preferably
R5 is selected from: , -CF 3, F, and CI. Still more preferably R5 is H.
Preferably R6 is selected from H and F. Preferably R6 is F.
The present invention provides a compound of Formula III below:
or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according either Formulae I or II
wherein: Rl is -CH 3; R2 is selected from and -C ; R3 is selected from and -
C ; R4 is selected from: , -OCH(CH )2, and halogen; R5 is selected from: H -CF3,
-OCH3, CI, and F; and R6 is selected from or F; or a pharmaceutically acceptable salt
thereof.
The present invention provides a compound according to either Formulae I or II
wherein Rl is -CH ; R2 is selected from and -C¾; R3 is selected from and -CH ;
R4 is selected from: H, and halogen; R5 is selected from: , -CF3, F, and CI; and R6
is selected from: and F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formulae I or II
wherein: Rl is -CH3; R2 is selected from: nd -CH ; R3 is selected from: nd
CH3; R4 is selected from: H and halogen; R5 is selected from: H, F, and CI; and
R6 is selected from H or F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formula I or II
wherein: Rl is -CH ; R2 is H; R3 is H; R4 is selected from: H, and CI; R5 is H, CI,
and F; and R6 is selected from H or F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formula I or II
wherein: Rl is -CH3; R2 is H; R3 is H; R4 is selected from: H, and CI; R5 is H; and
R6 is selected from H or F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formula I or II
wherein: Rl is -CH ; each of R2, R3, R4, and R5 is H; and R6 is F.
The present invention provides a compound according either Formulae I or II
wherein: Rl is -CF ; R2 is selected from H and -CH3; R3 is selected from H and -CH ;
R4 is selected from: H, -OCH(CH )2, and halogen; R5 is selected from; H -CF ,
-OCH 3, CI, and F; and R6 is selected from H or F; or a pharmaceutically acceptable salt
thereof.
The present invention provides a compound according to either Formulae I or II
wherein Rl is -CF3; R2 is selected from H and -CH ; R3 is selected from H and -CH ;
R4 is selected from: H, and halogen; R5 is selected from: H, -CF3, F, and CI; and R6
is selected from: H and F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formulae I or II
wherein: Rl is -CF3; R2 is selected from: H and -CH3; R3 is selected from: H and
—CH3: R4 is selected from: H and halogen; R5 is selected from: H, F, and CI; and
R6 is selected from H or F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formula I or II
wherein: Rl is -CF3; R2 is H; R3 is H; R4 is selected from: H, and CI; R5 is H, CI,
and F; and R6 is selected from H or F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formula I or II
wherein: R is -CF ; R2 is ; R3 is H; R4 is selected from: H, and C ; R5 is ;
and R6 is selected from H or F; or a pharmaceutically acceptable salt thereof.
The present invention provides a compound according to either Formula I or II
wherein: Rl is -CF3; each of R2, R3, R4, and R5 is H; and R6 is F.
Preferably the pharmaceutically acceptable salt is selected from: a chloride salt
and a maleate salt. More preferably the pharmaceutically acceptable salt is maleate salt.
Preferred compounds are - ( R - -(4-{[(2S)-2-(4-Fluol phenyί mo holin-4-
yl]methyl}phenyl)ethyl]methanesulfonamide;
N-[( 1R)- -(4- {[(2S)-2-(4-Fluorophenyl)morpholin-4-
yl]methyl}phenyl)ethyl]methanesulfonamide hydrochloride; and
N-[(lR)- l-(4- {[(2S)-2-(4-Fluorophenyl)morpholin-4-yl]methyl}phenyl)ethyl]
methanesulfonamide maleic acid.
The present invention provides a pharmaceutical composition comprising a
compound of Formulae I, II, or III as described above or a pharmaceutically acceptable
salt thereof, and at least one of a pharmaceutically acceptable carrier, diluent, or
excipient.
The present invention also provides a method of treating a patient in need of
treatment for hypertriglyceridemia, the method comprises administering to the patient an
effective amount of a compound, according to Formulae I, II, or III above.
The present invention provides a compound, according Formulae I, II, or III above
for use in the treatment of hypertriglyceridemia.
The present invention provides for the use of a compound according Formulae I,
II, or III above in the manufacture of a medicament to treat hypertriglyceridemia.
The term "pharmaceutically-accep table salt" refers a salt of the compound of the
invention considered to be acceptable for clinical and/or veterinary use. Pharmaceutically
acceptable salts and common methodology for preparing them are well known in the art.
See, e.g., P. Stahl, et a].. Handbook of Pharmaceutical Salts: Properties, Selection and
Use, (VCHA/Wiley-VCH, 2002); S.M. Berge, et ah, "Pharmaceutical Salts," Journal of
Pharmaceutical Sciences, Vol. 66, No. 1, January 977.
Pharmaceutical formulations of the present invention may be prepared by
procedures known in the art using known or readily available additives. The term
"pharmaceutically acceptable carrier, diluent, or excipient" as used herein refers to one or
more carriers, diluents, and excipients that are compatible with the other ingredients of
the formulation and not deleterious to a patient. Pharmaceutical compositions and
processes for their preparation are known in the art and examples can be found in
Remington, "The Science and Practice of Pharmacy" (A. Gennaro, et al. eds. 19th ed.
Mack Publishing Co.) Non-limiting examples of pharmaceutically acceptable carriers,
excipients, and diluents are suitable for such formulations include the following: starch,
sugars, mannitol, and silica derivatives; binding agents such as carboxymethyl cellulose
and other cellulose derivatives, alginates, gelatin, and polyvinvl-pyrroiidone; moisturizing
agents such as glycerol; disintegrating agents such as calcium carbonate and sodium
bicarbonate.
As used herein patient refers to an animal in need of treatment, preferably not
exclusively a mammal, which is preferably a human; or alternatively a companion animal
such as a dog or cat; or a fowl.
Unless noted to the contrary, the compounds illustrated herein are named and
numbered using either ACDLABS or Symyx Draw 3.2.
General Chemistry
As used herein, the following terms have the meanings indicated: "ACN" refers to
actonitrile; "DCM" refers to dichloromethane; "DEA" refers to diethylamine; "DMEA"
refers to dimethylethylamine; "DMF" refers to dimethylformamide; "ee" refers to
enantiomeric excess; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethanol; "h"
refers to hour(s); "HPLC" refers to high performance liquid chromatography; "IPA"
refers to isopropyi alcohol; "Isomer 1" refers to the first eluting isomer; "Isomer 2" refers
to the second eluting isomer; "LC/MS" refers to liquid chromatography followed by mass
spectroscopy; "MeOH" refers to methanol; "min" refers to minute(s); "MS" refers to
mass spectroscopy; "NMR" refers to nuclear magnetic resonance; "SFC" refers to
supercritical fluid chromatography; "THF" refers to tetrahydrofuran.
Scheme 1
Scheme 1 illustrates the general synthesis of compound of Formula I .
A substituted morpholine compound , which is either commercially available or
synthesized by known literature methods, reacts with an aldehyde or ketone 2 under
reductive animation conditions to provide the compound of Formula I . ((See: Richard C.
Larock, Comprehensive Organic Transformations: a guide to functional group
preparations . 2nd edition. Page 835-846, Wiley-VCH, (1999)). Preferably, morpholine
compound 1 reacts with compound 2 with the existence of a reducing agent such as
triacetoxyborohydride and an acid such as acetic acid in dichloromethane to provide the
compound of Formula I . which can be converted to a suitable salt with appropriate acids,
for example, HC1 to form the hydrochloride salt.
Preparation 1
(N-Z)-N-[(4-Bromophenyl)methylene]-(R)-2-methyl-piOpane-2-sulfinamide
Add (R)-2-methylpropane-2-sulfmamide (40.5 g, 0.33 mol) portion-wise to a
solution of 4-bromobenzaldehyde (65.57 g, 0.35 mol) in toluene (283 mL). Stir the
mixture at ambient temperature for 5 minutes and then add sodium hydroxide (1.34 g,
0.33 mol). Stir the suspension at ambient temperature for 12 h. Add sodium sulphate (16
g) and Celite® (16 g) and stir the suspension for 15 min. Filter and concentrate the
filtrate under reduced pressure. Purify the residue by silica gel chromatography eluting
with hexane /EtOAc (100% to 70% hexane) to afford the title compound as a white solid
(85.5 g, 88% yield). MS (m/z): 288 (M+l).
Preparation 2
N-[(lS)-l-(4-Bromophenyl)-2,2,2-trifluoro-ethyl]-(R)-2-methyl-propane-2-sulfinamide
Add neat (trifluoromethyl)trimethylsilane (109 mL, 0.74 mo ) at 0 °C to a stirred
solution of tetrabutylammonium acetate (88 g, 0.29 mol) and (N-Z)-N-[(4-
bromophenyl)methylene]-(R)-2-methyl-piOpane-2-sulfinamide (85 g, 0.29 mol) in DMF
( .2 L) at 0 °C. Stir the mixture at 0-5°C for 90 min. Add saturated aqueous ammonium
chloride solution (1.2 L) and extract with EtOAc (4 x 400 mL). Combine the extracts and
sequentially wash the extracts with water then brine (2 x 1 L); dry over magnesium
sulphate; filter; and concentrate the filtrate under reduced pressure. Triturate the residue
with hexane (200 mL) for minutes, filter and dry the filtrate under reduced pressure to
afford the title compound as a yellow solid (8 g, 76% yield, >98 de). MS (m/z): 358
(M+l).
Preparation 3
(lS)-l-(4-Bromophenyl)-2,2,2-trifluoroethanamine
Add HCl (4M in dioxane, 226 mL, 0.9 mol) to a suspension of N-[(lS)-l-(4-
bromophenyl)-2,2,2-trifluoro-ethyl]-(R)-2-methyl-propane-2-sulfinaiTiide (81 g, 0.23 mol)
in MeOH (670 mL). Stir the mixture at ambient temperature for one hour. Remove the
solvent under reduced pressure and triturate the residue with methyl tert-butyl ether (200
mL) for 10 min to give the HCl salt as a brown solid. Dissolve the salt in water (1.2) and
sufficient add 2N NaOH solution to raise the pH of the aqueous solution to a pH of 10.
Extract the mixture with methyl tert-butyl ether (3 x 500 mL). Wash the organic phase
with water then brine (500 mL each); dry over magnesium sulphate; filter; and
concentrate the filtrate under reduced pressure to give the title compound as a yellow
solid (46 g, 80% yield, 98% ee). MS (m z): 358 (M+l).
Preparation 4
N-[(lS)-l-(4-Bromophenyl)-2,2 2-nifluoi -ethyl]methanesulfonainide
Add methanesulfonyl chloride (16.42 mL, 0.21 mol) drop-wise to a mixture of
(lS)-l-(4-biOmophenyi)-2,2,2-trifluoroethanamine (49 g, 0.19 mol), 4-
dimethylaminopyridine (1.18 g, 9.0 mmol), 2,6-iutidine (67 mL, 0.57 mol) in DCM (250
mL) at 0°C. Warm the mixture to ambient temperature and stir at that temperature for 20
hours. Dilute the reaction mixture with DCM (300 mL) and wash it sequentially with 2M
HCl (2 x 200 mL), water (250 mL), then brine (250 mL). Collect the organic phase and
dry over magnesium sulphate; filter; and concentrate the filtrate under reduced pressure.
Triturate the residue with hexane (200 mL) for min; filter; and dry the solid under
reduced pressure to give the title compound as a pale brown solid (60 g, 93% yield, 98%
ee). MS (m/z): 332 (M+l).
Preparation 5
N-[( 1S)-2,2,2-Trifluoro- -(4-formylphenyl)ethyl]methanesulfonamide
Add N-[(lS)-l-(4-bromophenyl)-2,2,2-trifluoro-ethyl]methanesulfonamide (30 g,
90 mmol), palladium(II) acetate (0.8 g, 3.6 mmol), butyidi-l-adamantyiphosphine (3.89
g, 10.84 mmol) and tetramethylethylenediamine (10.50 g, 90 mmol) in toluene (1.5 mL)
to a 2L PARR reactor. Seal the reactor and pressure it with synthesis gas (1:1 CO at
75 psi). Stir the reaction mixture for 1 h while maintaining the temperature at 95 °C.
Cool the mixture; vent; and open the reactor. Filter the mixture through Celite® and
concentrate the filtrate under reduced pressure. Purify the crude residue by silica gel
chromatography eluting with hexane/EtOAc (8:2 to 1:1) to afford the title compound
(22.8 g, 90 %, 80% ee). Emich the chiral purity of the compound by eiuting it through a
chiral column: Chiralpak AS-H (2.1x25cm, 5 uM) C0 EtOH (9:1) to provide the title
compound (19 g, 75 % yield, 98 % ee). MS (m/z): 282 (M+l).
Preparation 6
N-[( 1R)- l-(4-Bromophenyl)ethyl]methanesulfonamide
Add methanesulfonyl chloride (13.44 mL, 0.17 mmol) to a mixture of (lR)-l-(4-
bromophenyl)ethanamine (25 g, 0.12 mol) and triethylamine (51 mL, 0.36 mol) in DCM
(250 mL) at 0 °C. Warm to ambient temperature and stir for 2.5 h. Wash reaction
mixture with 2M aqueous HCl (100 ml). Then sequentially wash the organic phase with
water then brine (2 x 100 mL). Dry the organic phase over anhydrous sodium sulphate;
filter; and concentrate the filtrate under reduced pressure to give a residue. Triturate the
residue with hexane (150 mL); filter; and dry under reduced pressure to afford the title
compound as a yellow solid (33.24 g, 96 %, ee > 98%). MS (m/z): 278 (M+l).
Preparation 7
N-[( IR)- l-(4-Formylphenyl)ethyl]methanesulfonamide
Combine N-[(lR)-l-(4-bromophenyl)ethyl]-methanesulfonamide (10 g, 35
mmol), ( l -bis(diphenyiphosphino)-ferrocene)palladium(II) chloride (733 mg, 0.9
mmol), sodium carbonate (3.81 g, 35 mmol) and DMF (50 mL) in a 300mL PARR
reactor. Add triethylsilane ( .6 mL, 0.72 mmol) and purge the reactor with carbon
monoxide three times. Charge the reactor with carbon monoxide (50 psi) and stir the
mixture at 90°C for 5 h. Cool the reactor to ambient temperature; open; filter mixture
through a Celite® pad; and wash the pad with DCM (150 mL). Sequentially wash the
filtrate with water then brine (2 x 80 mL). Concentrate the organic phase under reduced
pressure to obtain the residue as an orange oil. Purify the orange oil by silica gel flash
chromatography eluting with hexane/EtOAc (0 to 30% EtOAc) to provide the title
compound (5.6 g, 70%, ee> 98%). MS (m/z): 228 (M+l).
Preparation 8
(1S)-2-Bromo- 1-(4-fluoropheny Methanol
Under a nitrogen atmosphere, charge a 30 liter round bottom flask with a solution of
(S)-l-methyl-3,3-diphenyl-3a,4,5,6-tetrahydropyrrolo[l,2-c][l,3,2]oxazaborole ( 1 M in
toluene; 44 niL; 44 mmol) at 22 °C. Add a solution of borane-N,N-diethylaniline
complex (1230 g; 7540 mmol) in methyl t-butyl ether (4.5 L). Heat and maintain the
mixture at 40°C for 30 min. Add a solution of bromo-4-fluoroacetophenone (1640 g;
7530 mmol) in methyl t-butyl ether (4.5 L) drop-wise over 30 min. Stir the mixture at 40
°C for 2 hours. Cool to °C using an ice water bath; then slowly add MeOH (590 niL)
to quench the reaction. Stir the mixture for 30 min while maintaining it at 10-20 °C. Add
hydrochloric acid (3.0 M, 7.5 L) to the mixture while it is at 10 °C. Stir for one hour and
filter. Collect the filtrate. Separate the layers in the filtrate; extract the aqueous phase
with methyl tert-butyl ether (lx 3 L); combine the organic phases and wash with brine;
dry over a S0 and filter; and remove the volatiles from the filtrate under reduced
pressure to give the title compound as a pale yellow oil (1650 g 99%). MS (m/z): 201
(M-OH); ee value: 97.5% (AD-H 250 mm x 4.6 mm x 5 column using 99:1
hexanes:EtOH at 25 °C with a flow rate of 1.0 mL/min).
Preparation 9
(S)-2-(4-Fluorophenyi)oxirane
Dissolve (lS)-2-bromo-l-(4-fluorophenyl)ethanol (1650 g, 7.53 mol) in 6.8 L of
methyl tert-butyl ether. Add NaOH (2M, in H 0 ; 4.93 L) while the mixture is at 20 °C.
Stir the mixture for 3 h while maintaining it at 20-22 °C. Separate the layers and extract
the aqueous layer with methyl tert-butyl ether ( x 2 L). Combine the organic phases;
wash the organic phases with brine ( 2 L); dry over a S0 4; and filter; concentrate the
filtrate to give a residue. Purify via silica gel flash column chromatography using a 50:
mixture of petroleum ether.EtOAce to elute the product. Concentrate the product
fractions to give the title compound as a pale yellow oil (880 g, 84%). NMR (300
MHz, CDC1 ) : 7.27-7.21 (m, 2H), 7.06-7.01 (m, 2H), 3.86 (dd, J= 2.6, 4.0 Hz, 1H), 3.16
(dd, J= 4.1, 5.5 Hz, 1H), 2.79 (dd, J= 2.6, 5.4 Hz, 1H); ee value : 97.5% (AD-H 250 mm x
4.6 mm x 5 column using 99:5 hexanes:ethyl alcohol at 25 °C with a flow rate of 1.0
mL/min).
Preparation 10
(lS)-2-(Benzylamino)-l-(4-fluorophenyl)ethanol
Charge a 10 L round bottom flask with (S)-2-(4-fluoiOphenyl)oxirane (880 g, 6.38
mo ) under a nitrogen atmosphere. Add benzylamine (2047 g, 19.13 mol) while the
mixture is maintained at 20 °C. Heat the mixture to 80 °C and stir it at that temperature
for 5 h. Cool to 22 °C and stir for 6 h. Add H20 (3 L) to quench the reaction. Filter and
wash the filter cake with water (2 x 1 L). Slurry the solid obtained with heptane (2 L) and
filter to give the title compound (1216 g, 77%). MS (m/z): 246 (M+l); ee value: 99.0 %
(AD-H 250 mm x 4.6 mm x 5 column using 90: 0 hexanes (with 0.02%
diethylamine):EtOH at 25 °C with a flow rate of .0 mL/min).
Preparation
N-Benzyl-2-chloro-N-[(2S)-2-(4-fluorophenyl)-2-hydroxyethyl]acetamide
Dissolve (lS)-2-(benzylamino)-l-(4-fluorophenyl)ethanol (1215 g, 4.96 mol) in
12.15 L DCM and cool the mixture to 0 °C. AddNaOH (1M in H20 , 5.46 L, 5.46 mol)
dropwise over 30 min. Stir the mixture vigorously for 0 min while maintaining it at 0-3
°C; then add a solution of chloroacetyl chloride (616.5 g, 5.46 mol) in 4.86 L of DCM
dropwise over 1 h keeping the temperature below 6 °C. Stir the mixture for 1 h at 0 °C;
separate the layers; and extract the aqueous phase with DCM ( 1 x 2 L). Combine the
organic layer and extracts; wash them with 10% hydrochloric acid (1.5 L), water (1.5 L)
and M NaOH ( 1 L). Dry over Na2S0 4 and filter. Collect the filtrate and remove the
solvent under reduced pressure to provide the title compound as a colorless oil (1440 g,
90%). MS (m/z): 322(M+1).
Preparation 12
(6S)-4-Benzyl-6-(4-fiuorophenyl) morpholin-3-one
Add N-benzyl-2-chloro-N-[(2S)-2-(4-fluorophenyl)-2-hydroxyethyl]acetamide
(1440 g, 4.48 mol) to tert-butyl alcohol (14.5 L) and add potassium tert-butoxide (753 g,
6.73 mol) portion-wise while maintaining the mixture at 22 °C. Maintain the mixture at
22 °C and stir for 1.5 hours. Add a saturated aqueous solution of ammonium chloride
(1306 g) to quench the reaction. Stir for an additional 1 hour and then add 20 (2 L).
Extract with EtOAc (2 x 0 L); combine the extracts; and concentrate the extracts under
reduced pressure provide a residue. Re-dissolve the residue in EtOAc (20 L) and wash
with 20 (10 L). Diy the EtOAc solution over Na S0 4 and filter. Collect the filtrate and
remove the solvent under reduced pressure to provide the title compound as a colorless oil
(1250 g, 98%). MS (m/z): 286 (M+l).
Preparation 13
(2S)-4-Benzyl-2-(4-fluorophenyl)morpholine
Under a nitrogen atmosphere, drop-wise add a solution of (6S)-4-benzyl-6-(4-
fluorophenyl) morpholin-3-one (625 g, 2.19 mo ) in THF (22 L) to lithium aluminum
hydride in THF (1M in THF, 5.0 L, 5 mol) while maintaining the temperature at 20 °C.
Heat the mixture to 70 °C and stir for 1.5 h . Cool the mixture to 0 °C and add H 0 (200
mL) to quench the reaction, followed by aqueous NaOH (4 M, .25 L), then add more
¾ 0 (600 mL). Stir the resulting mixture for 30 min; filter; and rinse the solid with
EtOAc (10 L). Collect the filtrate and concentrate it under reduced pressure to provide
the title compound (561 g, 94%). MS (m/z): 272 (M+l).
Preparation 14
-(4-fSuorophenyl)morpholine hydrochloride
Dissolve (2S)-4-benzyl-2-(4-fiuoiOphenyl)morpholine (948 g,
dichloroethane (17 L). Heat the mixture to 70 °C and add -chloroethyl chloroformate
( 500 g, 10.5 mol) drop-wise while heating. Stir the mixture at 70 °C for 3 h and then
concentrate the mixture to give a residue. Dissolve the residue in MeOH (10 L) and heat
to 70 °C while stirring for 1 h. Concentrate the solution under reduced pressure to give a
residue. Slurry the residue with EtOAc (5 L); filter; and wash the solid with EtOAc ( 1 L)
to provide an off white solid. Slurry the solid with 10:1 EtOAc/MeOH (10:1. 3 L); filter;
collect the solid to provide the title compound as a white solid (300 g). Concentrate the
mother liquor to provide additional material. Slurry the this material with a mixture of
EtOAc/MeOH (2:1; 1L) and filter to give an additional 105 g of the title compound as a
white solid. Mix the product batches to give the title compound (405 g, 53%). MS (m/z):
182 (M-Cl). ee value 100 % (AD-H 250 mm x 4.6 mm x 5 urn column using 90: 0
hexanes (with 0.02% DEA):ethyl alcohol at 25 °C with a flow rate of .0 mL/min).
Preparation
2-Bromo- -(2-isopropoxyphenyl)ethanone
Dissolve 1-(2-isopropoxyphenyl)ethanone (1.0 g, 6 mmol) in EtoO (25 mL) and
add bromine (0.3 mL, 6 mmol) drop-wise while stirring stir the mixture in the dark at
ambient temperature. Wash the reaction mixture with a saturated aqueous a C ,
solution. Dry it over MgS0 4; filter; collect the filtrate; removed the voiatiles under
reduced pressure to provide the title compound (1.5 g, 83%). MS (m/z): 258 (M+l).
Preparation 6
4-Benzyl-2-(2-chloro-4-fluoro-phenyl)mo o i e
Combine formic acid (98-100%; 0.30 mL, 8 mmol) and 2-benzylaminoethanol
( .2 1 g, 8 mmol) and cool the resulting mixture with an ice bath. Add 2-bromo-l-(2-
chloro-4-fluoro-phenyl)ethanone (1.0 g, 4 mmol); heat the mixture to reflux; and stir at
that temperature for 20 h. Dilute the mixture with DCM and wash with saturated aqueous
Na CC 3 solution. Diy organic phases over MgS0 ; filter; collect the filtrate; and
concentrate under reduced pressure. Purify via flash column chromatography eluting
with a gradient of 0-20% methyl tertiary-butyl ether in hexanes. Combine the product
fractions and remove the solvents under reduced pressure to give the title compound as a
yellow oil (1.22 g, 43%). MS (m/z): 306 (M+l).
Preparation 17
4-Benzyl-2-(2-isopropoxyphenyl)morpholine
Prepare 4-benzyi-2-(2-isopiOpox)'phenyl)moiphoiine essentially by the method of
Preparation 16. MS m z 312 (M+l)
Preparation 8
2-(6-Chloro-4-fiuoro-cyclohexa-2 ,4-dien- 1-yl)morphohne
Dissolve 4-benzyl-2-(2-chioro-4-fluoro-phenyi)morpholine (529 mg, 1.73 mmol)
in DCM (2.5 mL). Add -chloroethyl chloroformate (1.25 g, 8.65 mmol); heat to 80 °C;
and stir overnight. Add MeOH (2.5 mL) and stir at 65 °C for 3 hours. Concentrate under
reduced pressure and purify via SCX chromatography eiuting with a gradient of 0-100%
of (2N NHs/MeOH) in MeOH. Combine the product fractions and remove the solvents
under reduced pressure to provide the title compound as a white solid (345 mg, 92%).
MS ( /z): 216(M+1).
Preparation 19
2-(2-Isopropoxyphenyl)morpholine
Under a nitrogen atmosphere combine 4-benzyl-2-(2-
isopropoxyphenyl)morpholine (0.51 g, 2 mmol), 10% PdOH/Carbon (0.51 g, 10 mol%)
and anhydrous ammonium formate (0.53 g, 10 mmol). Heat the resulting mixture to
reflux and stir. Monitor the progress of the reaction via thin layer chromatography. After
completion, filter reaction mixture through a pad of Celite ®; collect the filtrate; and
remove the solvent under reduced pressure to give the title compound as an oil (0.25 g,
63%). MS (m/z): 433 (M+l).
Preparation 20
N-[2-[2-Bromo-l-(3-methoxyphenyl)ethoxy]ethyl]-4-nitro-benzenesulfonamide
Add ethylene oxide ( mL, 220 mmol) all at once to DCM cooled to 0 °C; then
add l-methoxy-3-vinylbenzene (7.09 g, 52.82 mmol) via a syringe. Stir the mixture
while maintaining it 0 °C. Add N-bromosuccinimide (9.4 g, 52.82 mmol) and 4-
nitrobenzenesulfonamide (8.9 g, 44.02 mmol). Wrap flask in foil and stir the reaction
mixture for 20 h while maintaining it at ambient temperature. Concentrate under reduced
pressure; filter; and concentrate the filtrate under reduced pressure to provide a residue.
Purify the residue via flash column chromatography eluting with a 5-40% gradient of
EtOAc in hexanes. Combine the product fractions, and remove the solvents under
reduced pressure to provide the title compound as a dark yellow oil (14.22 g, 70.3%). MS
(m/z): 459(M+1).
Preparation 2
2-(3-Methoxyphenyl)-4-(4-nitrophenyl)sulfonyl-morpholine
Dissolve N-[2-[2-bromo-l-(3-methoxyphenyl)ethoxy]ethyl]-4-nitrobenzenesulfonamide
(14.22 g, 30.96 moles) in ACN (200 mL); add potassium carbonate
(6.42 g, 46.44 mmol); and heat the mixture to reflux. Stir the mixture for 3 h while
refiuxing. Cool the resulting mixture to ambient temperature and dilute it with EtOAc.
Filter through Celite ®; concentrate the filtrate under reduced pressure to provide a
residue. Purify the residue via flash column chromatography eluting with a 50-80%
gradient of EtOAc in hexanes. Combine the product fractions and remove the solvents
under reduced pressure to provide the title compound as an orange solid ( 11.2 g, 95.6%).
MS (m/z): 379(M+1).
Preparation 22
2-(3-Methoxyphenyl)morpholine
Dissolve 2-(3-methoxyphenyl)-4-(4-nitrophenyl)sulfonyl-morpholine ( 11.2 g,
29.6 mmol) in ACN (150 mL) and water (2.67 mL). Add LiOH (6.21 g, 147.99 mmol)
while stirring the mixture and followed by 1-propanethiol (13.42 mL, 147.99 mmol). Stir
the mixture for 25 h at ambient temperature. Dilute the mixture with EtOAc and add
brine. Extract twice with EtOAc. Collect and concentrate the extracts to 200 mL under
reduced pressure, then wash three times with N HCl. Combine the aqueous acid extracts
and add N CO until the mixture is basic. Extract the basic solution three times with
EtOAc; combine the extracts; wash the extracts with brine; and dry over a S0 . Filter;
collect the filtrate; and remove the solvents under reduced pressure to provide a residue.
Purify the residue via flash column chromatography eluting with EtOAc, followed by a 5-
100% gradient of (10% 2M NH3 in MeOH)/DCM. Combine the product fractions, and
remove the solvents under reduced pressure to provide the title compound as a yellow oil
(3.09 g, 15.99 mmol). MS (m/z): 194(M+1).
Preparation 23
N-[(lR)-l-(4-Acerylphenyl)ethyl]methanesulfonamide
Charge a tube with N-[(lR)-l-(4-bromophenyl)ethyl]methanesulfonamide (29 g,
104 mmol), butyl vinyl ether (34.23 mL, 261 mmol), pailadium(II) acetate (14.04 g, 63
mmol), bis-(l,3-diphenylphosphino)propane (52.7 g, 125 mmol) and potassium carbonate
(17.3 g, 125 mmol). Degas the tube with nitrogen for 2 minutes, and then add H 0 (69.5
mL) and DMF (69.5 mL). Seal the tube and stir at 0 °C for 20 h. Cool the reaction
mixture to ambient temperature and add HC1 (2N, 60 mL). Stir the mixture at ambient
temperature for 0 min. Adjust the p of the mixture to a pH of 7 using NaOH pellets;
dilute with DCM (220 mL); filter through a Celite ® pad; and sequentially wash the
filtrate with aqueous K2C0 3 (2x 20 mL), brine (2x 100 mL), and ¾ 0 (100 mL). Dry
the mixture over MgSC>4; filter; and concentrate the filtrate under reduced pressure to
provide a residue. Purify the residue via flash column chromatography eluting with
EtOAc in hexanes (step gradient of 0, 5, 10, 20, 30 and finally 40% EtOAc). Combine
the product fractions and remove the solvents under reduced pressure to give the title
compound (17.6 g, 70.0%) as a yellow oil. MS ( n z): 242 (M+l).
Preparation 24
N-[( 1R)- 1-[4-( 1-HydiOxyethyl)phenyl]ethyl]methanesulfonamide, isomer 2
Dissolve N-[(lR)-l-(4-acetylphenyl)ethyl]methanesulfonamide ( 5 g, 62 mmol)
in EtOH ( 155.4 mL) and cool with an ice bath. Add sodium borohydride ( 1.2 g, 31.
mmol) and stir the resulting mixture in the ice bath for 2 h . Quench the reaction with
H 0 (20 mL) and concentrate under reduced pressure. Dilute the residue with EtOAc (90
mL) and 20 (50 mL). Separate the layers and wash the organic layer with brine (2x 50
mL), dry over MgSC^; filter; and concentrate the filtrate under reduced pressure to
provide a residue. Purify and separate isomers using chromatography conditions K (see
below) collecting the second eluting isomer as the title compound (2.01 g, 13%). MS
(m/z): 261 (M+l 8).
Example 1
N-[( 1R)- -(4- {[(2S)-2-(4-Fluorophenyl)morpholin-4-
yl]methyl}phenyl)ethyl]methanesulfonamide
Under a nitrogen atmosphere, suspend (2S)-2-(4-fluorophenyl)morpholine
hydrochloride (29.5 g, 128.8 mmol) in DCM (17 L) at 22 °C and add triethylamine (35.89
mL, 257.5 mmol). Add N-[(lR)-l-(4-formyiphenyi)ethyl]methanesulfonamide (29.26 g,
128.8 mmol) and stir the resulting solution for 30 min. Add acetic acid (8.85 mL, 154.5
mmol), then sodium triacetoxyborohydride (86.17 g, 386.3 mmol) portion-wise in 3
batches. Stir for 3 h; monitor the reaction via LCMS until completion. Quench the
reaction via the slow addition of a saturated aqueous solution of sodium bicarbonate
(259.3 mL) to give a solution with a pH of 8. Separate the layers, and extract the
aqueous layer with 200 mL DCM. Combine the organic layers, wash with saturated
sodium bicarbonate, water, brine, and then dry over MgSC^. Filter; collect the filtrate;
and concentrate to give a residue. Purify the residue via silica gel flash column
chromatography, using a gradient of 100% DCM to DCM:MeOH (95:5). Combine the
product fractions, and concentrate to provide the title product as a yellow thick oil (41 g,
81.13 %). MS (m/z): 393 (M+l)
Example 2
-[(1 )-1-(4-{[(28)-2-(4- 1 Ό1 ) 1-4-
yljmethyl }phenyl)ethyl]methanesulfonamide maleate
Method 1:
Dissolve N-[( 1R)-l -(4- {[(2S)-2-(4-fluorophenyl)n^holin-4-
yl]methyl}phenyl)ethyl]methanesulfonamide (250.47 mg) in EtOAc (10 mL). Add
maleic acid (85 mg) dissolved in EtOAc (2 mL) at 60 °C. Cool to ambient temperature
and stir the mixture for 30 min. Filter the slurry and rinse with EtOAc (5 mL). Collect
and dry the filter cake under reduced pressure to provide the title compound as a solid
(280 mg, 97.7%). MS (m/z): 393 (M-maleic acid+1).
Method 2:
Dissolve N-[( 1R)- -(4- {[(2S)-2-(4-fluorophenyl)morpholin-4-
yl]methyl}phenyl)ethyl]methanesulfonamide (270 g) in EtOAc (10 L). Heat the mixture
to 60 °C and add maleic acid (96 g, 1.1 eq) in EtOAc (2.8L). Allow the mixture to cool to
ambient temperature and stir the resulted mixture for 14 h . Filter the slurry; rinse the
solid with EtOAc (5L); and dry the filter cake under reduced pressure. Dissolve the solid
in 5 volumes of EtOH (1.4L); heat at 90°C; and add water (280 mL). Heat the mixture at
90 °C for Ih, and then cool it to ambient temperature overnight. Filter the precipitate, dry
in a vacuum oven at 40 °C to provide the title compound as a white solid (256 g, 70%).
MS (m/z): 393 (M-maleic acid+1).
Example 3
N-[(lR)-l-(4-{[(2S)-2-(4-fluorophenyl)morpholin-4-
yl]methyl}phenyl)ethyl]methanesulfonamide hydrochloride
Dissolve N-[( 1R)- -(4- {[(2S)-2-(4-fluorophenyl)moholin-4-
yl]methyl}phenyl)ethyl]methanesuifonamide (50 g, 127.39 mmol) in isopropyl alcohol
(200 mL). Add HCl (4M in dioxanes; 63.70 mL, 254.78 mmol) drop-wise to the solution
and stir at ambient temperature for 50 min. Remove the volatiles under reduced pressure;
add H 0 (200 mL); then evaporate the water. Add H 0 (200 mL) and isopropyl alcohol
(100 mL), and concentrate to a total volume of 80 mL. Filter the resulting thick slurry;
wash solid with H 0 ; collect by filtration; and dry the filter cake under reduced pressure
at 55 °C to provide the title compound as a white solid (40.6 g, 75%). MS (m/z): 393 (MCI).
Example 4
N-[( R)- 1-(4- {1-[(2S)2-(4-fluoiOphenyl)morpholin-4-
yl]ethyl}phenyl)ethyl]methanesulfonamide hydrochloride, Isomer 1
ABS
Isomer 1
Prepare N-[(lR)-l-(4-{l-[2-(4-fiuorophenyl)morpholin-4-
yi]ethyl}phenyi)ethyl]methanesulfonamide hydrochloride, isomer 1 essentially by the
method of Example 3. MS (m/z) 407 (M-Cl)
Example 5
N-[(lS)-l-(4-{[2-(2-chiorophenyl)mo holin-4-yl]methyl}phenyi)-2,2,2-
trifluoroethyljmethanesiilfonamide hydrochloride, Isomer 2
Combine 2-(2-chlorophenyl)morphoiine, oxalic acid salt (200 mg, 0.696 mmol),
triethylamine (193 , 1.39 mmol), N-[(lS)-2,2,2-trifluoro-i-(4-formyiphenyi)-ethyl]-
methanesulfonamide (205.3 mg, 0.73 mmol) and DCM (15 niL). Add acetic acid (47.8
\xL, 0.83 mmol) and sodium triacetoxyborohydride (465 mg, 2.09 mmol), and stir for 5 h
at ambient temperature. Adjust the pH of the mixture to 10 with a saturated aqueous
Na C( solution. Stir until gas evolution ceases; separate the layers; and extract the
aqueous layer twice with DCM. Combine the organic extracts; wash them with brine;
and dry over MgS0 4. Filter mixture; collect the filtrate; and concentrate it under reduced
pressure to give a residue. Purify the residue on a 10 g SCX cartridge, wash the cartridge
with DCM, 50% MeOH/DCM, 100% MeOH, then elute with NH3 in MeOH (2N).
Concentrate the product fractions under reduced pressure to provide the crude product as
an oil. Purify the oil via chirai HPLC, using conditions E (see below) to privide the free
base (104 mg, 32.3%) as the second eluting isomer. Dissolve the free base (104 mg,
0.224 mmol) in 1mL DCM and add HC1 in Et 0 (2 M, 561.6 L 1.12 mmol) drop-wise.
Stir at ambient temperature for 5 minutes and then remove the solvents under reduced
pressure to give the title compound (99 mg, 88.2%). MS (m/z): 463(M-C1).
The following compounds are prepared essentially by the method of Example 5 .
All the following Examples in Table 1 are isolated as single isomers either starting from
chirai starting materials and/or using the chromatographic columns and conditions
identified below. The separation can be performed with the free base or with its salt form.
Table 1
MS Chrom
Ex # Chemical name Structure
(m/z): Cond.
N-[(lR)-I-(4-{[2-(3-
FluoiOphenyl)morpholin-
4-
393
6 yl]methyl}phenyl)ethyl] 0"0 A
Isomer 1 (M-Cl)
methanesulfonamide *HC1 salt was used for
hydrochloride, separation.
Isomer 1
N-[(lR)-l-(4-{[2-(3-
C H
Methoxyphenyi)morpholi
n-4-
405
7 yljmethyl }phenyl)ethyl] J
(M-Cl)
methanesulfonamide I
hydrochloride,
Isomer 2 isomer 2

MS Chrom
Ex # Chemical name Structure
(m/z): Cond.
N-[(lR)-l-(4-{[2-(3-
Chlorophenyl)morpholin-
4-yljmethy 1}phenyl) 409
12
ethyl]methanesulfonamid y D
(M-Cl)
e hydrochloride, isomer 2
Isomer 2
N-[(lR)-l-(4-{[(2R,6S)-
2-Methyl-6-
° , o
phenyhnorpholin-4- 389
13
yljmethyl }phenyl)ethyl] (M-Cl)
methanesulfonamide
hydrochloride
N-[(lS)-2,2,2-Trifluorol-(
4-{[2-(4-
fluorophenyl)morpholin-
4- 447
14 F
yl]methyl}phenyl)ethyl] (M-Cl)
methanesulfonamide Isomer 1 F
hydrochloride.
Isomer 1
N-[(lR)-l-(4-{[2-(2- HCI
ChloiOphenyl)morpholin-
4-yl]methyl}phenyl) 409
15 G
ethyljmethanesulfonamid (M-Cl)
e hydrochloride.
Isomer 2 Isomer 2
Example 7
N-[(lR)-I-[4-[l-[(2S) 2-(4-FluoiOphenyl)morpholin-4-
yl]ethyl]phenyl]ethyl]methanesulfonamide, Isomer 1
ABS
Combine N-[( 1R)- -[4-( 1-hydiOxyethyl)phenyl]ethyl]-methanesulfonamide
isomer 2 (420 mg, .73 mmol) and DCM (5 mL). Cool the mixture to 0 °C and it purge
with nitrogen. Add acetyl bromide (295.8 , 3.45 mmol) and stir the reaction for 10
min while maintaining it at 0 °C. Add an additional amount of acetyl bromide (519.5 ,
6.90 mmol) and stir for an additional 10 min. Dilute the reaction with DCM, and
evaporate solvents under reduced pressure to provide a residue. Dissolve the residue in
DMF (2 mL) and add (2S)-2-(4-fluorophenyl)morpholine hydrochloride (71.1 mg, 0.327
mmol), K2CO3 (135.4 mg, 0.980 mmol) and stir at ambient temperature overnight. Filter
the reaction, and purify the filtrate using SCX chromatography with an elution order of
DCM, DCM/MeOH (1:1), MeOH, and finally 2M N /MeOH. Combine the product
fractions and remove the solvents under reduced pressure to provide a residue. Purify the
residue via reverse phase HPLC (XTerra MS CI8 column, p 8) collecting the first
eluting isomer (isomer I) as the title compound (9.6 mg, 7.2%). MS (m/z): 407 (M+l).
Chromatography conditions:
Table 2
MOGAT-2 Inhibitory Assay
The i vitro inhibitoiy activity of compounds against human MOGAT-2 is
evaluated in this assay. MOGAT-2 transfers an oleoyl group to monooieoyl-glycerol
("MAG") from oleoyl-CoA to form dioleoyl-giycerol ("DAG") in the intestinal
triglyceride resynthesis pathway. The assay takes advantage of Microscint E extraction,
which extracts hydrophobic molecules selectively over hydrophilic ones to separate the
C-oleoyl-CoA from 4C-DAG.
Genetically engineered insect SF9 cells express human MOGAT-2. Prepare the
cell lysate in 20 mM of NaC with protease inhibitor (Roche Cat# 1873580001).
Homogenize the SF9 cells expressing human MOGAT-2 at 15,000 rpm for 20 x2 seconds
(PT-3 100 Polytrone). Centrifuge the homogenate at 1000 g for 10 minutes at 4 °C.
Collect the supernatant into a separate tube for protein quantification and activity testing.
Purify the glycerol monooleate substrate (Spectrum Chemical, CAS#25496-72-4)
chromatographically. Prepare the monoacylglyerol (MAG) substrate in phospholipid
vescicles (dioleoyl phosphatidylcholine "DOPC")- Prepare the MAG/DOPC vesicles at
20 mM concentration of total lipids (MAG and DOPC). Prepare different molar ratios of
MAG to total lipids for either compound screening (8.9%) or compound kinetic studies
(2.6-40%). Mix the appropriate amount of purified MAG and DOPC (Avanti Po ar
Lipids # 850375C) in chloroform in a glass tube. Subsequently, evaporate chloroform
under stream of N2 gas and then dry under reduced pressure for 30 minutes. Add an
appropriate amount of buffer (Tris-Cl pH 7.4, 250 mM sucrose, 1mM EDTA) to the
dried MAG/DOPC mixture for the desired total lipid concentration. Sonicate the
MAG/DOPC solution until the solution is clear. Measure the vesicle size using dynamic
light scattering to confirm uniformity.
The assay buffer consists of 100 mM Tris, pH 7.5 (Invitrogen 15567-022), 11%
DMSO, 250 mM sucrose (Sigma S-0389), 1mM, EDTA, and Complete Protease
Inhibitor cocktail (Roche Diagnostic 12454800). Add the test compounds to the buffer
together with the substrates and enzymes. The final concentration for the reaction is 0.016
mg/mL SF9 cell extract, 20 oieoyl-CoA (3.5 C-oleoyi-CoA), 1.26 mM total
lipid in the form of sonicated vesicles, composed of 8.9:91 . (molar ratio) MAG.DOPC.
Stop the reaction after 90 minutes of incubation at room temperature by adding AESSM
(12.5% of 100% denatured EtOH; % DI H20; 2.5% 1.0N NaOH; 59% Isopropanol
(Mallinckrodt 3031-08); 15% Heptane (Omni Solv HX0078)), by volume. Add
Microscint E and then seal the plates and count on a scintillation counter after at least 4
hours of equilibration at room temperature. Calculate the IC (concentration to reach half
maximum inhibition) using Excel Fit software (version 4; Data analyzing using a 4-
parameter nonlinear logistic equation (ABase Equation 205)) by plotting concentration vs
relative MOGAT-2 activity.
All the compounds exemplified herein have an IC of less than 100 nM, and
example 2 exhibits an IC50 of 12 nM in this MOGAT-2 in vitro assay.
Inhibitory Activity in MOGAT-2 Cell Assay
The inhibitory activity of compounds against human MOGAT-2 in a cell
environment is evaluated in this assay. Caco-2 is a human colon carcinoma cell line and
is often used as a model for intestinal epithelial cells. Caco-2 does not express MOGAT-
2, and, thus, human MOGAT-2 is engineered into the cell line through a stable
transfection. A MAG analogue, 2-O-Hexadecylglycerol (HDG), is utilized to detect
cellular MOGAT-2 activity, because HDG is not hydroiyzed and the resulting product is
readily monitored by mass spectrometry. The substrate is delivered to cells using as a
mixture with DOPC in the form of sonicated vesicles.
Seed the Caco2 cells onto 100 mm dishes to be 80% confluent after 24 hours in
complete media (3/1 DMEM: F12 + 10% FBS + 20mM HEPES + gentamicm). Transfect
the cells with hMOGAT-2 plasmid (MOGAT-2-pCDNA3.1-Hygro) using Lipofectamine
2000 (Invitrogen). After a 6 hour exposure to the transfection mixture, wash the cells
three times in PBS and then add media. Incubate the cells for an additional 18 hours
incubation, trypsinize the cells and serially dilute them into 100 mm dishes. Add
complete media + 400 g ml hygromycin and incubate until clones appear. Isolate and
transfer the clones into 24 well dishes and grow to confluency. Prepare the RNAs from
these clones using a Qiagen RNAeasy kit. Perform Taqman analysis using an ABI
inventoried assay (HS00228262) on a 7900 Sequence Detection System (ABI). Analyze
the lysates from these clones by Western blot analysis using a goat polyclonal antibody
(Santa Cruz, SC-32392 to confirm human MOGAT-2 expression of a 38 kD protein
corresponding to MOGAT-2.
Mix 2-O-hexadecylglycerol ("HDG", Biosynth Chemistry & Biology, # H-1806,
562.7 ί of 20 mg/ml ) and DOPC (14.3 ml of 20 mg/mi) in chloroform in a glass tube;
dry first under N2 gas; and then under reduced pressure for additional 30 minutes. Add
20 ml of buffer ( 50 mM Tris-CI pH 7.4, 250 mM sucrose, 1mM EDTA) to the dried
HDG/DOPC mixture while sonicating until the solution becomes clear. Plate the Caco2
cells into a poly-D-lysine coated 96-well plate (the "Cell Plate") at 37 °C, 5% CO
overnight. Remove the growth media and pretreat the cells with the test compound in
DMEMF12 (3:1) media (GIBCO 93-0152DK) containing 2% BSA (Sigma) for 30
minutes. Treat the cells with one test compound in 2% BSA DMEMF12 (3:1) media
containing 40 of oleic acid and 800 of 8.9:91.9 (molar ratio) HDG/DOPC for 4
hours. Trypsinize the ceils with 50 ΐ of trypsin solution and add 50 ΐ of PBS.
Immediately freeze the cells on dry ice and store at -20°C for LC-MS analysis. Extract
the cells with chloroform/methanol as follows: transfer the cells to a 2 ml plate; wash the
cell plate with 200 methanol and then transfer the methanol wash to the 2 ml plate;
wash the cell plate again with 200 ΐ PBS and transfer the PBS wash to the 2 ml plate.
Add chloroform (400 ΐ ) with internal standard (19.52 ng/mL) DAG (15:0,15:0
(Sigma)), D5-TAG (39.03 ng mL) CDN (16,16,16) to the 2 mL Plate. Turn the sealed 2
mL Plate up and down (lOx), then vortex and spin. Remove 400 of the lower layer
from the 2 mL plate and add to the wells of another plate the "Final Plate". Add
CHCl^MeOH (400 2: ) to the 2 mL Plate. Again turn the sealed 2 mL Plate up and
down (lOx), vortex and spin. Remove 220 ΐ of the lower layer from the 2 mL Plate and
add to the Final Plate. Dry the Final Plate and reconstitute with 500 mL of IPA. Seal the
Final Plate and shake for 5 min. Inject ΐ of a sample from the Final Plate onto a Halo
C8 column (2. 1 x 50, 2.7 uL particle size) held at 60 °C using a Leap auto sampler with a
loop, interfaced to a Shimadzu solvent delivery system. Monitor the channels to
collect data for the D5 C16 TAG internal standard as well as the ether TAG, and C52 and
C54 natural TAGs. Solvent A is 80/20 H20/Methanol with 20 ammonium acetate.
Solvent B is 50/50 IPA/THF with 20 ammonium acetate. Flow rate is 0.4 mL/min.
Wash solvents were H 0 /MeOH and DCM. Using Xcalibur software extract the areas of
the peaks of interest, and export the data to Excel which uses the following formula: (area
of ether TAG/area of C54 natural TAG)/ Area of IS. This ratio effectively accounts for
variance of cell number in each well. The results for this MOGAT-2 cell based assay are
provided below in Table 3. The results of the MOGAT-2cell based assay demonstrate that the
Examples listed in Table 6 inhibit the human MOGAT-2in the cell environment.
Table 3
Example IC 0 nM (Std Dev., n*)
2 44 (27, 4)
3 274 (261, 17)
5 16 (1, 2)
11 98 (34, 2)
14 40 (21, 3)
16 94 (94, 4)
Pharmacological Effects in a Dog Oi Bolus Model
Inhibiting MOGAT-2 found in the small intestine may be useful for treating
hypertriglyceridemia caused by excessive fat intake. To assess the ability of the
exemplified compounds to inhibit TAG absorption, twenty one male beagles (n=7 per
treatment group) are enrolled for each study, each dog selected to have a body weight
between 9 - 3 kg. House the dogs in cages with a standard light cycle (12 hours light and
12 hours dark); at room temperature: 72 ± 8°F; and at 30% - 70% relative humidity. Fast
the dogs for 16 hours prior to the start of the study, then dose the fasted dogs with vehicle
(1% HEC, 0.25%, Tween 80, Antifoam) or one of the test compounds in that vehicle.
Bleed the dogs one hour after dosing, (0.5 ml from the jugular vein) for a time 0 sample.
Dose the dogs with olive oil (Sigma Catalog#: 0 - 5 4, 5 ml/kg) immediately after
collection of the time 0 sample. Collect samples into an EDTA tube on ice at 1.5, 2, 3, 5,
7, and 9 hrs post compound / vehicle dosing.. Centrifuge the samples at 9000 cpm for 5
min and analyze (Roche Cat no. 1877771 ) for plasma total triglyceride using a Roche
Hitachi 917. For plasma TAG .1 18.1 18.1 measurement, extract the samples and
perform LC MS MS analysis similarly to that described above in MOGAT-2 Cell Assay
using 0 of plasma/.
The analyte is the [M+NH4]÷ ion of TAG 18:1 18:1 18:1, which has a mass of 902.8 m/z;
the internal standard is D5 TAG 16:0 16:0 16:0, which has amass of 829.8 m/z. Report
the ratio of the 603.5 m/z daughter ion of 902.8 m z (TAG 18:1 18:1 18:1) and the 556.5
m/z daughter ion of 829.8 m/z (D5 TAG 16:0 16:0 16:0 internal standard) changes in
TAG 18:1 18:1 18:1 relative amount. Calculate the net plasma TAG AUC from total
TAG AUC minus baseline TAG AUC using Graphpad Prism4: (Net AUC TAG= AUC TAG
post oil bolus - AUC TAG at 0 hour). The percent inhibition of plasma triglyceride is
calculated as follows: the (oil bolus group mean of net TAG AUC - oil bolus group mean
of net TAG AUC with compound treatment / oil bolus group mean of net TAG AUC) *
100. The final statistic analysis uses Dunnett's method of One way Anova for
comparison with the control. All Net TAG AUC values are transformed to ranked
averaged AUC for comparison to limit the variability within the studies. The ability of
exemplified compounds of the present invention to inhibit MOGAT-2 activity and reduce
TAG absorption in vivo can be further evaluated according to this assay.
Example 2 was evaluated in this model in three studies at 30 mg/kg dose and in
two studies at the 75 mg/kg dose. Combination of results from those studies
demonstrated statistically significant (p< 0.05) reduction in excursion of postprandial
triglycerides. Results were as follows: 43% inhibition of TAG absorption (45% of 18.1
TAG) at 30 mg/kg PO and 64% inhibition of TAG absorption (63% of 18.1 TAG) at 75
mg/kg PO.
The exemplified compound of the present invention can be readily formulated into
pharmaceutical compositions in accordance within accepted practices such as found in
Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co. Easton Pa.
90. A treating physician or other medical person will be able to determine an effective
amount of the compound for treatment of a person in need, particularly for the treatment
of hypertriglyceridemia. Preferred pharmaceutical compositions can be formulated as a
tablet or capsule for oral administration. The tablet or capsule can include a compound of
the present invention in an effective amount for treating a patient in need of treatment.
>claimed is:
1. A compound of the formula below:
wherein
Rl is selected from: -C¾ and -CF ;
R2 is selected from: and -C¾;
R3 is selected from: and -C¾;
R4 is selected from: H, -OC - a ky , and halogen;
R5 is selected from: , -CF , -OCH 3 and halogen;
R6 is selected from: and halogen;
provided that at least one of R4, R5, and R6 is ;
or a pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 wherein Rl is -C¾.
3. A compound according to claim 1 wherein Rl is -CF .
4 . A compound according to any one of claims 1 to 3 wherein R2 is H.
5. A compound according to any one of claims 1 to 4, wherein R3 is H.
6. A compound according to any one of claims 1 to 5 wherein R4 is selected
from and halogen.
7. A compound according to any one of claims 1 to 6 wherein R5 is selected
from: H, -CF , F, and CI.
8. A compound according to any one of claims 1 to 6 wherein R5 is H.
9 . A compound according to any one of claims 1 to 8 where R6 is F.
10. A compound of the formula below:
or a pharmaceutically acceptable salt thereof.
1 . A compound of according to any one of claims 1 to 10 wherein the
pharmaceutically acceptable salt is selected from: a chloride salt and a maleate salt.
12. A compound of according to claim 11 wherein the pharmaceutically
acceptable salt is a maleate salt.
13. A compound which is N-[(lR)-l-(4-{[(2S)-2-(4-Fluorophenyl)morpholin-
4-yl]methyl}phenyl)ethyl]methanesulfonamide hydrochloride.
14. A compound which is N-[(lR)-l-(4-{[(2S)-2-(4-Fluorophenyl)morpholin-
4-yl]methyl}phenyl)ethyl] methanesulfonamide maleic acid.
15. A pharmaceutical composition comprising a compound according to any
one of claims 1 to 14 and at least one of a pharmaceutically acceptable carrier, diluent, or
excipient.
16. A method of treating a patient in need of treatment for
hypertriglyceridemia, the method comprises administering to the patient an effective
amount of a compound, according to any one of claims 1 to 4 .
17. A compound according to any one of claims 1 to 14 for use in therapy.
8. A compound, according to any one of claims 1 to 14 for use in the
treatment of hypertriglyceridemia.
19. Use of a compound according to any one of claims 1 to 14 in the
manufacture of a medicament to treat hypertriglyceridemia.