Cl
`
`Available on line at www.sciencedirect.com
`
`SCIENCE@.Dl.RECT"
`
`Bioorganic &
`Medicinal
`Chemistry
`Letters
`
`ELSEVIER
`
`Bioorganic & Medicinal Chemistry Letters 16 (2006) 3039- 3042
`
`Discovery of potent and orally active MTP inhibitors as
`potential anti-obesity agents
`Jin Li,* Peter Bertinato, Hengmiao Cheng, Bridget M. Cole, Brian S. Bronk,
`Burton H. Jaynes, Anne Hickman, Michelle L. Haven, Nicole L. Kolosko,
`Chris J. Barry and Tara B. Manion
`Pfizer Inc., Groton, CT06340, USA
`
`Received 20 January 2006; revised 17 February 2006; accepted 20 February 2006
`Available online 10 March 2006
`·
`.
`
`Abstract-We have successfully identified a number of novel MTP inhibitors with single digit nanomolar potency. Analogues lOaq
`and lOdq demonstrated in vivo efficacy in a murine gut retention assay.
`·
`© 2006 Elsevier Ltd. All rights reserved.
`
`As in human health, obesity is a growing health prob(cid:173)
`lem in companion animals, with 25-40(i'o of the pet
`population estimated to be overweight and 5-10%
`considered severely obese. 1 Obesity predisposes ·dogs
`and cats to a number of harmful conditions including
`diabetes, hepatic lipidosis, cancer, osteoarthritis, der(cid:173)
`matitis and musculoskeletal problems such as cruciate
`and inter-vertebral disk rupture. Obesity also nega(cid:173)
`tively impacts . veterinary patients with cardiovascular
`and respiratory disea.se and limits. the efficacy of
`pharmaceutical therapy in these conditions.2 11 Cur(cid:173)
`rent therapy for obesity is based on food restriction
`and/or exercise and affords limited success in most
`patients. The failure of weight loss programs is large(cid:173)
`ly the result of poor owner compliance due to hunger
`and begging of the pet. Because there are . no veteri(cid:173)
`nary drugs currently available for the treatment of
`obesity, there is a major opportunity for a safe, effi(cid:173)
`cacious agent.
`
`Microsomal triglyceride transfer protein (MTP) 12 is
`involved in the assembly of triglyceride-rich chylo(cid:173)
`microns in enterocytes and very low-density lipopro(cid:173)
`teins (VLDL) in hepatocytcs. 12- 14 MTP is located in
`intestinal and liver tissues where it plays a role in
`
`Keywords: MTP; Microsomal triglyceride transfer protein; Obesity.
`*Corresponding author. Tel.: +1 860 715 3552; fax: +l 860 715
`9259; e-mail: jin.li@!pfizer.com
`
`0960-894X/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
`doi: I 0.1016/j.bmcl.2006.02.058
`
`lipid assembly and transport. 12 Inhibition of MTP
`has been shown
`to be an effective method for
`reducing serum cholesterol. 15 Recently we disclosed
`the treatment of
`the use of MTP inhibitors for
`obesity by inhibition of fat absorption. 16
`
`Several potent MTP inhibitors have been disclosed,
`including CP-346086 (l), 17 implitapide (2), 18·19 JNJ-
`(3), 20 diaminohydroindan derivative21
`4506463
`(4)
`and BMS-212122 (5). 22 Starting from I as a lead,
`we successfully identified a new class of potent MTP
`inhibitors,
`represented by
`the
`indole amide 6
`(Fig. l ). 23 In order to further explore the chemical
`space and ADME properties in this series many ana(cid:173)
`logs have been prepared by either replacing the indole
`moiety with other fragments or varying the terminal
`amines. In this paper, we would like to disclose the
`syntheses and SAR of phenyl/substituted phenyl moi(cid:173)
`eties.24 This research effort resulted in the discovery
`of a number of highly potent MTP inhibitors for
`the potential treatment of obesity, highlighted by ana(cid:173)
`log JOdq (entry 35, Table I).
`
`Two factors were considered in replacing the indole
`fragment in 6: (1) the rigidity and (2) the size of the
`new fragments. A parallel synthesis approach was em(cid:173)
`ployed in order to quickly explore the SAR of the
`new templates. As depicted in Figure 2, the acid deriv(cid:173)
`atives Sa-Si were chosen to replace the indole moiety
`in 6 based on the considerations mentioned above.
`
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`.; .
`
`.. •
`~- .
`
`~.· '
`
`.•'.'".
`
`.. ~
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`: ·;~:
`
`..
`'•" ..
`
`..
`
`,.
`
`..
`
`.. _,.
`
`3040
`
`J. Li et al. I Bioorg. Med. Chem. Leu." 16 (2006! 3039-3042
`
`Table 1. In vitro canine MTP inhibition data
`
`.gCF3
`
`~·"'°
`O
`Jl
`,···-X N
`H O
`h
`
`.6- R2
`N_
`R1
`
`r
`,
`
`2 implitapide
`
`Ently Compound
`
`MTP inhibition
`IC50 (nM)
`
`1 CP-346086
`
`\ S~GI
`
`N-{
`t.,,, ,N
`N
`
`O~
`~NJ
`l._..,N~ 0 r
`~N)lN)..___
`'=N
`
`3 JNJ-4506463
`
`4 dlamlnolndanes
`
`on_~:o
`x({H
`->=o
`
`R2-N
`R"
`
`5 BMS-212122
`
`Figure 1. Selected MTP inhibitors.
`
`A diverse set of amines was selected in order to quick(cid:173)
`ly explore SAR (Fig. 3).
`
`in
`is outlined
`The preparation of the analogues
`Scheme 1. The 4' -(triftuoromethyl)-2-biphenylcarboxy(cid:173)
`lic acid (7) was reacted with Sa-8i to provide the es-
`. ter intermediak:>, which were then hydrolyzed under
`the acids (9a-9i), The
`basic conditions to furnish
`phenylglycine . derivatives 13j-13r were prepared by
`coupling Boe-protected phenylglycine 11 with amines
`12j-12r. Several standard amide coupling reaction
`conditions were screened m order to avoid epimer(cid:173)
`ization of the chiral center of the phenylglycine.
`The
`coupling
`condition,
`PyBroP/DlPEA/DCM,
`proved .to be the most robust for the coupling pro(cid:173)
`cess without epimerization as monitored by chiral
`HPLC. Subsequently .. mpling the acid 9a-9i with
`the
`the phenylglycine dcn\atives 13j-13r provided
`final analogues represented by 10 for biology screen(cid:173)
`ing Table 2.
`
`All analogues were tested in a canine MTP in vitro
`binding assay. 25 The results arc summarized in Table
`1. In general, analogues prepared from the mono-aryl
`templates (Sa-Sd, entries 1-36) demonstrated good
`in vitro potency despite their decreased size compared
`to the indole analog 6. The analogues derived from
`3-methoxy Sc and 3-mcthyl Sd templates showed the
`most potent inhibition toward MTP, suggesting ·a
`lipophilic binding pocket for these substituents. An
`clecfron-donating group on
`the 2-position of the
`phenyl rmg was tolerated (entries 19-27). Electron
`deficient pyridyl acid template Sa showed good but
`decreased potency compared to analogues 10bj-10dr
`
`2
`3
`4 ·
`5
`6
`7
`8
`9
`
`10
`II
`12
`13
`14
`15
`16
`17
`18
`
`19
`20
`21
`22
`23
`24
`25
`26
`27
`
`28
`29
`30
`31
`32
`33
`34
`35
`36
`
`37
`38
`39
`40
`41
`42
`43
`44
`45
`
`46
`47
`48
`49
`50
`51
`52
`53
`54
`
`toaj
`IOak
`IO al
`IOam
`IOan
`IOao
`I0a11
`IOaq
`IOar
`
`IObj
`IO bk
`IO bl
`lObm
`IObn
`IObo
`lOb11
`IObq
`IObr
`
`lOcj
`IO ck
`IOcl
`lOcm
`Hin
`l(k.
`lOq>
`lOcq
`IO er
`
`lOdj
`lOdk
`lOdl
`IOdm
`lOdn
`lOdo
`t0d11
`lOdq
`lOdr
`
`IOej
`lOek
`lOel
`IO em
`HIP!>
`H•· ...
`h
`1 ·•·.:
`Hlh
`
`IOfj
`lOfk
`lOfl
`IOfm
`IOfn
`lOfo
`10f11
`IOfq
`IO fr
`
`12j
`12k
`121
`12m
`12n
`120
`1211
`12q
`12r
`
`12j
`12k
`121
`12m
`12n
`120
`1211
`12q
`12r
`
`12j
`12k
`121
`12m
`12n
`120
`1211
`12q
`12r
`
`12j
`12k
`121
`12m
`12n
`120
`1211
`12q
`12r
`
`12j
`12k
`121
`12m
`12n
`120
`1211
`12q
`12r
`
`12j
`12k
`121
`12m
`12n
`120
`1211
`12q
`12r
`
`Ba
`
`Sb
`
`~OH
`H:.>My
`
`OMe
`Sc
`
`0
`
`.H•ifOH
`
`Eld
`
`n00H Ha
`
`Se
`
`~OH
`H2N~
`Sf
`
`16.93
`2.53
`22.46
`29.51
`69.39
`35.40
`12.97
`17.38
`13.29
`
`20.51
`ND
`13.27
`6.23
`14.78
`I.OJ
`23.13
`6.47
`5.28
`
`2.0
`3.85
`6.38
`ND
`1.68
`7.14
`1.75
`2.34
`
`ND
`1.64
`3.50
`ND
`ND
`1.78
`4.62
`3.37
`
`85.5
`. 5.53
`26.8
`29
`·n.7
`ND
`26.6
`14.9
`18.3
`
`15.6
`61.15
`2.97
`5.67
`11.78
`16.94
`4.11
`8.15
`5.05
`
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`J. Li el al. I Bioorg. Med. Chem. Lei/. 16 (2006) 3039-3042
`
`3041
`
`Table 1 (continued)
`
`Entry Compound NHrx-COOH
`
`NR 1R2 MTP inhibition
`IC00 (nM)
`17.27
`35.92
`>JOO
`55.84
`>JOO
`69.84
`7.33
`8.27
`ND
`
`12j
`12k
`121
`12m
`12n
`120
`1211
`12q
`12r
`
`0
`II
`
`s
`ii} .__,.........OH
`_.v
`
`H,N
`
`Bg
`
`12j
`12k
`121
`~N"j(OH
`12m
`JV H
`12n
`120
`Bh
`1211
`12q
`12r
`
`0
`
`H,,N
`
`~OH
`H~J
`Bi
`
`12j
`12k
`121
`12m
`12n
`120
`12p
`12q
`i2r
`
`>JOO
`>JOO
`>JOO
`>JOO
`>JOO
`>100
`>JOO
`>JOO
`>JOO
`
`23.56
`96.71
`69.52
`95.89
`>JOO
`>JOO
`76.5'2
`>100
`9.43
`
`55
`56
`57
`58
`59
`60
`61
`62
`63
`
`64
`65
`66
`67
`68
`69
`70
`71
`72
`
`73
`74
`75
`76
`77
`78
`79
`80
`81
`
`lOgj
`lOgk
`lOgl
`IOgm
`lOgn
`togo
`10g11
`lOgq
`lOgr
`
`IOhj
`lOhk
`tohl
`tohm
`tohn
`10ho
`t0h11
`lOhq
`tOhr
`
`. toij
`tOik
`toil
`toim
`toin
`lOio
`toip
`toiq
`lOir
`
`a
`
`7
`
`Ba-i
`
`9a-i
`
`10
`
`"''-~(»; +
`
`H
`
`0
`
`d -
`
`11
`
`12j-r
`
`13j-r
`
`Scheme 1. Reagents and conditions: (a) i-PyBroP, DIPEA, DCM,
`0 °C to rt, ii-LiOH, THF/H 2, reflux, >95%; (b) EDC, HOBT,
`DIPEA, DCM, rt, >85%; (c) LiOH, THF/H20, reflux, >98%;
`(d) i-PyBroP, DIPEA, DCM, 0 °C to rt, ii--4 N HCl/dioxane, 100%.
`
`Table 2. In vivo data for compounds toaq and todq
`ED25 (mg/kg, rat)
`6.59
`3
`
`Entry
`
`8
`35
`
`Compound
`lOaq
`lOdq
`
`(entries 10-36). When a conformationally restricted
`template 8e was used, all analogues prepared showed
`a significant drop in potency toward MTP. Templates
`in which the aniline functionality was replaced with
`more flexible benzylic amines (Sf, Sg, Sh and Si) were
`in general less potent toward MTP.
`
`Several potent analogs were progressed into in vivo
`studies. The murine gut retention assay23 was used to as(cid:173)
`sess a compound's ability to inhibit intestinal MTP. In
`this assay; compounds lOaq and JOdq were potent inhib(cid:173)
`itors of intestinal MTP, with ED25s of6.93 and 3 mg/kg,
`respectively.
`
`In summary, we have successfully identified a number of
`novel and potent MTP inhibitors. Analogues lOaq and
`lOdq also demonstrated in vivo activity when tested in
`a murine gut retention assay.
`
`References and notes
`
`1. Pfizer Animal Health Data File.
`2. Annstrong, P. J.; Lund, E. M. Vet. Clin. Nutr. 1996, 3, 83.
`3. Burkholder, W. J.; Bauer, J.E. J. Am. Vet. Chem. Assoc.
`1998, 212, 658.
`4. Edney, A. T.; Smith, P. M. Vet. Rec. 1986, 118, 391.
`5. Kassab, S.; Patterson, S.; Wilkins, F. C.; Mizelle, H. L.;
`Reinhart, G. A.; Granger, J.P. Hypertension 1994, 23, 997.
`6. Mason, E. Vet. Rec. 1970, 86, 612.
`7. Mattheeuws, D.; Rottiers, R.; Kaneko, J. J.; Venneulen,
`A. Am. J. Vet. Res. 1984, 45, 98.
`
`ND, not determined.
`
`0
`
`OMeO
`
`L (oH
`H2N
`N
`
`0
`
`Ba
`
`H2N f10H
`
`i joH
`H2N
`
`Bb
`
`0
`
`HN
`
`~H
`
`~OH
`
`H2N-._)U .
`
`Bd
`
`Bg
`
`Se
`
`Bh
`
`Bf
`
`~OH
`H~J
`
`Si
`
`Figure 2. Acid derivatives.
`
`HN~ H2N~
`I
`
`12j
`
`OH
`12m
`
`12k
`
`01
`~NH
`12n
`
`HNV
`.o
`I
`
`12p
`
`Ho
`
`12q
`
`Figure 3. Amine derivatives.
`
`H2N v
`
`121
`
`H2Nv
`
`120
`
`l
`
`/'-../NH
`
`12r
`
`3 of 4
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`
`3042
`
`8 .
`
`9 .
`
`IO.
`1 I.
`
`.. .. -·
`
`·i
`
`.•.
`.. ' ;
`
`'• ..
`
`; .
`
`. ":•
`
`._
`
`..
`
`12.
`
`13.
`
`14.
`
`15.
`
`Mattheeuws, D.; Rottiers, R.; Baeyens, D. J. Am. Anim.
`Hosp. Assoc. 1990, 20, 287.
`Panciera, D. L.; Thomas, C. B.; Eicker, S. W.; Atkins, C.
`E. J. Am. Vet. Med. Assoc. 1990, 197, 1504.
`Robertson, I. D. Prev. Vet. Med. 1999, 40, 75.
`Scarlett, J. M.; Donoghue, S. J. Am. Vet. Med. Assoc.
`1998, 212, 1725.
`Wetterau, J. R.; Lin, M. C.; Jamil, H. Biochim. Biophys.
`Acta 1997, 1345, 136.
`Gordon, D. A.; Wetterau, J. R.; Gregg, R. E. Trends Cell
`Biol. 1995, 5, 317.
`Olofsson, S. O.; Asp, L.; Boren, J. Curr. Opin. Lipidol.
`1999, 10, 341.
`Wetterau, J. R.; Gregg, R. E.; Harrity, T. W.; Arbeeny,
`C.; Cap, M.; Connolly, F.; Chu, C.-H.; George, R. J.;
`Gordon, D. A.; Jamil, H.; Jolibois, K. G.; Kunselman, L.
`K.; Lan, S.-J.; Maccagnan, T. J.; Ricci, B.; Yan, M.;
`Young, D.; Chen, Y.; Fryszman, 0. M.; Logan, J. V. H.;
`Musial, C. L.; Poss, M. A.; Rob!, J. A.; Simpkins, L. M.;
`Slusarchyk, W. A.; Sulsky, R.; Taunk, P.; Magnin, D. R.;
`Tino, J. A.; Lawrence, R. M.; Dickson, J. K.; Biller, S. A.
`Science 1998, 282, 751.
`16. Chandler, C. E.; Hickman, M.A.; Lundy, K. M.; Morgan,
`B. P. EPI099439A2, 2001.
`17. Chandler, C. E.; Wilder, D. E.; Pettini, J. L.; Savoy, Y. E.;
`Petras, S. F.; Chang, G.; Vincent, J.; Harwood, H. J.
`J. Lipid Res. 2003, 44, 1887.
`18. Iglesias, P.; Diez, J. J. Expert Opin. lnvestig. Drugs 2003,
`12, 1777.
`19. Sorbera, L.A.; Martin, L.; Silvestre, J.; Castaner, J. Drugs
`Future 2000, 25, 1138.
`20. Roevens, P.; Heeres, J.; Meerpoel, L.; Dupont, A.;
`Borghys, H. Atherosclerosis 1999, 144(Suppl. !), 38.
`21. Ksander, G. M.; deJesus, R.; Yuan, A.; Fink, C.; Moskal,
`M.; Carlson, E.; Kukkola, P.; Bilci, N.; Wallace, E.;
`Neubert, A.; Feldman, D.; Mogelesky, T.; Poirier, K.;
`Jeune, M.; Steele, R.; Wasvery,.J.; Stephan, Z.; Cahill, E.;
`Webb, R.; Navarrete, A.; Lee, W.; Gilbson, J.; Alexander,
`N.; Sharif, H.; Hospattankar, A. J. Med. Chem. 2001, 44,
`4677.
`22. Robl, J. A.; Sulsky, R.; Sun, C.-Q.; Simpkins, L. M.; Wang,
`T.; Dickson, J. K.; Chen, Y.; Magnin, D. R.; Taunk, P.;
`Slusarchyk, W. A.; Biller, S. A.; Lan, S.-J.; Connolly, F.;
`Kunselman, L. K.; Sabrah, T.; Jamil, H.; Gordon, D.;
`Harrity, T. W.; Wetterau, J. R. J. Med. Chem. 2001, 44, 851.
`23. Bcrtinato, P.; Blizc, A. E.; Bronk, B. S.; Cheng, H.;
`Huatan, H.; Li, J.; Mason, C. P. WO 2003002533, 2003.
`24. Bertinato, P.; Bronk, B. S.; Cheng, H.-M.; Chang, G.;
`Cole, B. M.; Li, J.; Ruggeri, R. B. WO 2004056777, 2004.
`25. Canine in vitro MTP assays. (A) Canine hepatic microsoinc
`isolation: canine microsomes are first isolated from canine
`liver by thawing frozen liver on ice and rinsing several times
`with 0.25 M sucrose. A 50% liver homogenate (w/v) is made
`in 0.25 M sucrose. The hon1ogenatc is diluted 1: I with
`0.25 M sucrose, and centrifuged at I O,OOOg at 4 °C for
`20 min. The supernatant is saved. The pellet is re-suspended
`
`J. Li et al. I Bioorg. Med. Chem. Lett. 16 (2006) 3039--3042
`in a minimal volume of0.25 M sucrose and re-centrifuged at
`I O,OOOg for 20 min at 4 °C. The supernatants arc combined
`and centrifuged at I 05,000g for 75 min at 4 °C. The
`supernatant is discarded and the resulting microsomal
`pellet is saved. The microsomal pellet is re-suspended in a
`minimum volume of 0.25 M sucrose and diluted to J ml/g
`liver weight in 0.15 M Tris--HCI, pH 8.0. The resulting
`suspension is divided into 12 tubes and centrifuged at
`105,000g for 75 min. The resulting microsomal 1>:;kts are
`. stored at -80 °C until needed. M'TP is isolated by thawing
`the microsomal pellet tube_ and suspending in 12 ml/tube of
`cold 50 mM Tris-HCI, 50 mM KCI, 2 mM MgCI, pH 7.4,
`and sfowly addini,r 1.2 ml of a 0.54% deoxycholatc. pH 7.4
`solution. Aftc .:: ,. i
`utiation on ice with gentle mixing,
`at I 05,000g for 75 min at 4 °C.
`the solution ;
`. •:c.c
`The supernataEc .. ~,,
`. :g solubic"MTP, is dialyzed for 2-
`3 days with 5 change. 01 assay buffer (15.0 mM Tris-HCI,
`40mM NaCl, I mM EDTA, 0.0'.?" NaN,, pH 7.4). (B)
`MTP activity assay reagents: dor..
`ios'omes are created
`by adding 447 mM egg phosr
`;choline (68/20 mil,
`· .'.O ml) and 0.91 mM
`83 mM bovine heart eardiolipir
`14C]triolein (I 10 Ci/mol) (20/2t':
`fhe lipids are avail-
`[
`able in chlorofo1111 and are first dncd under nitwgen and
`then hydrated · ·
`buffer to the volume nccoed. To
`create liposomc~. "1'· · .. ffc sonicated for,.._, 7 min. Lipids are
`centrifuged at I 05,0llVg for 2 hand liposomcs are harvested
`by removing the top rv80% of supernatant into separate
`tube. Acceptor liposomes are created by adding 1.33 mM
`egg phosphatidylcholine (404/40 ml), 2.6 mM triokin (JOO/
`40 ml) and 0.5 nM [3H]egg phosphatidylcholinc (50 Ci/mo!)
`(I 0/40 ml). The lipids are available in chloroform and are
`first dried under nitrogen and then hydrated in assay buffer
`to the volume needed. To create liposomes, lipids arc
`sonicated for rv20 min. Lipids are centrifuged at 105,000g
`·"' l :•~ by removing the top rv80% of
`for 2 h and :1
`supernatan•.
`rnbe. (C) MTP in vitro lipid
`transfer inhi:
`. f'propriately diluted drug or
`control sampk~'"
`:_,,ay buffer containing 5% BSA
`are added to reaction n :: <'- containing assay buffer, 50 ml
`donor liposomes, 100 ml acceptor liposomes, and partially
`purified liver MTP. The tubes are vortexed and incubated
`on a tube shaker for I h at ;? °C to allow lipid transfer
`reaction to occur. Donor liposomes are precipitated by
`adding 300 ml of a 50'Yo (w/v) DEAE cellulose suspension in
`assay buffer to each tube, followed by gentle/repeated
`inversion for5 min at room temperature. Tubes arc then
`centrifuged at ,.._,I 000 rpm to pellet resin. Four hundred
`milliliters of supernatant is transferred into a scintillation
`vial with scintillation fluid and DPM counts for both [3H]
`and C4CJ arc determined. Triolcin transfer is calculated by
`comparing the amount of [14C] and ["'HJ remaining in the
`supernatant to (',· ' wd [3H] in the original donor and
`',•1i•1cly. 0/c, Triolcin transfer =
`acceptor lip<"'·
`14C]supcm<ll'' ..
`ix (["'H]acceptorl[3H]superna-
`([
`tant) x I 00 IC 50 V<l,..
`· ,c;iincd using standard methods
`and first order kineti<: ~·":::ulations.
`
`4 of 4
`
`PENN EX. 2262
`CFAD V. UPENN
`IPR2015-01836