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Novel 4-amino-furo[2,3-d ]pyrimidines as Tie-2and

VEGFR2

dual inhibitors

Yasushi Miyazaki,a,*Shinichiro Matsunaga,a Jun

Tang,a Yutaka Maeda,

Masato Nakano,a Rocher J.Philippe,a, Megumi Shibahara,a Wei Liu,b,àHideyuki Sato,a

Liping Wang b and Robert T.Nolte b

GlaxoSmithKline K.K.,Tsukuba Research Laboratories,43,Wadai,Tsukuba 300-4247,Ibaraki,Japan

GlaxoSmithKline Inc.,Five Moore Drive,Research Triangle Park,NC 27709,USA

Received 19November 2004;revised 4March 2005;accepted 10March 2005

Abstract—A novel class of furo[2,3-d ]pyrimidines has been discovered as potent dual inhibitors of Tie-2and VEGFR2receptor tyrosine kinases (TK)and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes.One of the most active compounds,4-amino-3-(4-((2-?uoro-5-(tri?uoromethyl)phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphen-yl)furo[2,3-d ]pyrimidine (7k )is <3nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure.ó2005Elsevier Ltd.All rights reserved.

Angiogenesis,the formation of new blood vessels by capillary sprouting from pre-existing vasculature,has been shown to be involved in many diseases such as dia-betic retinopathy,psoriasis,rheumatoid arthritis,and cancer.In particular,it is widely accepted that growth and metastasis of solid tumors is dependent on angio-genesis.1Out of the many factors shown to be involved in angiogenesis,vascular endothelial growth factor (VEGF)and angiopoietins are of particular interest as these are speci?c factors for endothelial cells and expres-sion of their receptors is restricted to these cells.2VEGF and angiopoietins have been thought to play comple-mentary and coordinated roles in vascular develop-ment.3During development,VEGF and its receptor VEGFR2play crucial roles in vessel sprouting and new vessel initiation in early stages of angiogenesis through induction of proliferation,migration,and sur-vival of endothelial cells.4Ang1and its receptor Tie-2play an important role in stabilizing the immature endo-thelial cell network,attracting pericytes,and maintain-ing biochemical interactions and vessel integrity,which

are thought to be implemented in secondary stages of blood vessel formation.5

The development of a dual inhibitor of VEGFR2and Tie-2would be expected to demonstrate synergistic ef-fects through inhibition of both critical stages of blood vessel formation and o?ers the potential for new ap-6From the results of a focused screening e?ort,5,6-diaryl-4-amino[2,3-d ]furopyrimidines,such as 1(IC 50=1.25l M and 1l M vs VEGFR2and Tie-2,respectively),were identi?ed as compounds with moderate inhibitory activity against VEGFR2and Tie-2.8According to a pharmacophore model for ATP competitive kinase inhibitors (see Fig.1),we can speculate that (1)the aminopyrimidine moiety of the furopyrimidine core binds to the hinge region through hydrogen-bond nor–acceptor interactions in a similar manner to N1and N6in the adenine base of ATP;(2)the 5-aryl ?ll a hydrophobic region not generally

Keywords :Angiogenesis;Anticancer;Protein kinase;VEGFR2;Tie-2.*Corresponding author.Tel.:+81298645544;fax:+8129645559;e-mail:yasushi.miyazaki@https://www.wendangku.net/doc/189659795.html,

Present address:Addex Pharmaceuticals SA,12,Chemin Des Aulx CH-1228,Plan-les-Ouates,Geneva,Switzerland.à

Present address:Medtronic Vascular,3576Unocal Place,Suite OW19,Santa Rosa,CA 95403,USA.

Bioorganic &Medicinal Chemistry Letters 15(2005)2203–2207

occupied by ATP in most protein kinases;(3)the 6-aryl group would be exposed to the phosphate binding re-gion.Based on these hypotheses,we focused on the derivatization of both the 5-and 6-position,in order to improve the potency.

Synthesis of 5,6-diaryl-furo-4-amino[2,3-d ]pyrimidines 7for exploring the structure–activity relationship (SAR)of the 5-and 6-positions was

accomplished using proce-dures illustrated

in Scheme 1.9In the procedure to deri-vatize the 6-position,1-aryl-2-bromoethanone (2)was converted to 1-aryl-2-hydroxyethanone (3)using potas-sium formate and aqueous sodium bicarbonate,which was treated with malononitrile in the presence of dieth-ylamine to give furan 4.Cyclization with formamide led to furo[2,3-d ]pyrimidine 5.Bromination with NBS gave 6and a palladium mediated coupling with a variety of boronic acids resulted in derivatives of 7.In another procedure focused on derivatizing the 5-position,1-aryl-3,3-dicyanopropan-1-one (8)was prepared from 2and malononitrile using NaOEt as the base.A double cyclization,?rst under acidic conditions followed by

pyrimidine formation using formamide a?orded 10.The 5-bromo intermediate 11was prepared by bromin-ation of 6-aryl-furo[2,3-d ]pyrimidine 10with NBS.Intermediates 11were coupled under palladium cata-lyzed conditions with aryl boronic acids leading to 7.The enzyme inhibitory activities are summarized in Table 1.As indicated in the data,mono aryl derivatives 5a and 10a show reduced activity compared to that of 1.Apparently,5,6-diaryl moieties are important for po-tent enzyme activity.With R1retained as the 4-meth-oxyphenyl substituent,changes to determine the SAR at the 6-position were https://www.wendangku.net/doc/189659795.html,pounds with hydrogen-bond donor–acceptors such as 3-carboxamide 7b modestly improved the enzyme inhibitory activity compared with that of the initial hit compound 1.How-ever,a meta -methanesulfonamide 7d introduced a mod-est 3-fold improvement in Tie-2activity and a more dramatic 20-fold improvement in VEGFR2activity.When R2is retained as the 4-methoxyphenyl group and the 3-methanesulfonamide substituent,the 5-posi-tion N ,N -dimethylaminophenyl and biphenyl derivatives

2204Y.Miyazaki et al./Bioorg.Med.Chem.Lett.15(2005)2203–2207

(7e and f ,respectively),increased enzyme potency.We envision that this is due to hydrophobic interactions of the biphenyl group at the 5-position,which is expected to project into the hydrophobic region that is not fully occupied by the 4-methoxyphenyl group.Additionally,interactions of the dimethylamino moiety with Lys866(see Fig.3)may have also improved the activity.Based on the assumption that a large hydrophobic pocket exists into which the 5-position of the furopyrimidine core projects and that interactions with Lys866would be expected,additional 5-position functionalized ana-logues were evaluated and are listed in Table 2.Based on this hypothesis,compound 7j wherein arylurea is substituted at the para position was prepared and it had remarkably enhanced Tie-2and VEGFR2enzyme activities.The 2-?uoro-5-tri?uoromethyl phenyl-urea 7k is the most active analogue and enhanced potency by approximately 15-fold against Tie-2and 20-fold against VEGFR2,compared with the unsubstituted phenyl-urea 7j .Amide and sulfonamide derivatives (7h and i )decreased potency.

The crystal structure of VEGFR2

with 7k was deter-mined at high resolution,as shown in Figure 2.10The NH and CO motifs of the urea form interactions with

the backbone of Asp1044and the carboxylic acid resi-due of Glu883,respectively.The NH 2and nitrogen of

Table 1.Tie-2and VEGFR2kinase enzyme inhibition of 4-NH 2furo[2,3-d ]pyrimidines 5

N N

O NH 2R2

Compound R1

Tie-2(l M)VEGFR2(l M)14-OMe-phenyl 4-OMe-phenyl 1.0 1.125a 4-OMe-phenyl H

>20 5.510a H

4-OMe-phenyl >20>177a 4-OMe-phenyl 3,4-Cl 2-phenyl 1.78>207b 4-OMe-phenyl 3-CONH 2-phenyl 0.540.187c 4-OMe-phenyl 3-CONMe 2-phenyl 1.48ND 7d 4-OMe-phenyl 3-NHSO 2Me-phenyl 0.300.0657e 4-NMe 2-phenyl 4-OMe-phenyl

0.600.357f 4-Biphenyl

3-NHSO 2Me-phenyl 0.190.0367g

3-NHAc-phenyl

4-OMe-phenyl

>20

IC 50value are generated by measuring inhibition of peptide substrate added to enzyme reaction in homologous time-resolved ?uorescence format (HTRF).ND =not determined.

Table 2.Tie-2and VEGFR2kinase enzyme inhibition of 4-NH 2furo[2,3-d ]pyrimidines

N N

NH 2O

Compound R

Tie-2(l M)VEGFR2(l M)7h –NHCO-(3-?uorophenyl)0.501ND 7i –NHSO 2-(3-chlorophenyl)0.851ND 7j –NHCONH-phenyl

0.0280.0627k –NHCONH-(2-?uoro-5-tri?uorophenyl)0.0020.0037l –NHCONH-(4-chlorophenyl)0.022ND 7m –NHCONH-cyclohexyl 0.0590.1867n

–NHCONH 2

0.275

0.058

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2205

the aminopyrimidine form interactions with Glu915and Cys917.Taking into consideration the fact that the en-zyme potency of 7m and n decreased because of the lack of a terminal aryl group,the binding potency therefore relies on strong interactions of the 2-?uoro-5-tri?uoro-methyl phenyl moiety with the hydrophobic area which exists beyond the hydrophilic region ?anked

by Lys866,Glu883,and the NH of Asp1044.As shown in Figure 3,the terminal aryl ring and its substitutions are accom-modated into this hydrophobic area composed of resi-dues Ile886,Leu887,Ile890,Val896,and Leu1017.Additionally the urea NH and carbonyl groups assisted in stabilizing the molecule by interaction with the Lys866-Glu883salt bridge and the NH of Asp1044.In a manner similar to the binding in VEGFR2,the termi-nal aryl group of the urea moiety in 7k would be ex-pected to reside in the hydrophobic back pocket,and the CO and NH of the urea moiety would be expected to interact with the conserved Glu883and Asp982of Tie-2.

Compound 7k was evaluated for its ability to inhibit the growth of human umbilical vein endothelial cells (HU-VECs)stimulated by VEGF and the autophosphoryl-ation of c-fms-Tie-2kinase chimeric receptor transfected in 3T3cells.Additionally,a cytotoxic cell as-say was conducted using the HFF cell line.11Data are summarized in Table 3,which show a good correlation between enzyme and cellular potency.Importantly,there is >10-fold selectivity between HUVECs and HFF cell lines suggesting that general cytotoxicity is not the mechanism for inhibition of HUVEC growth.In conclusion we have discovered 4-NH 2-furo[2,3-d ]pyr-imidines bearing a diarylurea substituent at the 5-posi-tion as a novel class of highly potent inhibitors of the angiogenesis receptor type tyrosine kinases,VEGFR2and Tie-2.In addition to activity in isolated enzyme

assays,Compound 7k exhibits potent cellular inhibitory activity versus proliferation of HUVEC and autophos-phorylation of the c-fms-Tie-2receptor.The arylurea function plays an important role by means of hydropho-bic and hydrophilic interactions which have been veri-?ed through X-ray analysis.

Acknowledgements

We acknowledge Noriko Tadotsu and Hiroko Toyoda for providing cellular data.We thank Anne Truesdale,Hiroshi Sootome,Joseph H.Chan,Karen https://www.wendangku.net/doc/189659795.html,ckey,and Stephen V.Frye for their guidance and support.

References and notes

1.(a)Carmeliet,P.;Jain,R.K.Nature 2000,407,249;(b)Hanahan,D.;Folkman,J.Cell 1996,86,353;(c)Folk-man,J.Nat.Med.1995,1,27.

2.Yancopoulos,G.D.;Davis,S.;Gale,N.W.;Rudge,J.S.;Wiegand,S.J.;Holash,J.Nature 2000,407,242.

3.Maisonpierre,P.C.;Suri,C.;Jones,P.F.;Bartunkova,S.;Wiegand,S.J.;Radziejewski,C.;Compton,D.;McClain,J.;Aldrich,T.H.;Papadopoulos,N.;Daly,T.J.;Davis,S.;Sato,T.N.;Yancopoulos,G.D.Science 1997,277,55.

4.(a)Shalaby,F.;Rossant,J.;Yamaguchi,T.P.;Gertsen-stein,M.;Wu,X.F.;Breitman,M.L.;Schuh, A. C.Nature 1995,376,62;(b)Carmeliet,P.;Ferreira,V.;Breier,G.;Pollefeyt,S.;Kieckens,L.;Gertsenstein,M.;Fahrig,M.;Vandenhoeck,A.;Harpal,K.;Eberhardt,C.;Declercq,C.;Pawling,J.;Moons,L.;Collen,D.;Risau,W.;Nagy, A.Nature 1996,380,435;(c)Ferrara,N.;Davis-Smyth,T.Endocr.Rev.1997,18,4;(d)For recent review of small molecule of VEGFR2inhibitor,see:Bilodeau,M.T.;Fraley,M.E.;Hartman,G.D.Expert Opin.Investig.Drugs 2002,99,11393;(e)Boyer,S.J.Curr.Top.Med.Chem.2002,2,973.

5.(a)Davis,S.;Aldrich,T.H.;Jones,P.F.;Acheson,A.;Compton, D.L.;Jain,V.;Ryan,T. E.;Bruno,J.;Radziejewski,C.;Maisonpierre,P.C.;Yancopoulos,G.D.Cell 1996,87,1161;(b)Suri,C.;Jones,P.F.;Patan,S.;Bartunkova,S.;Maisonpierre,P.C.;Davis,S.;Sato,T.N.;Yancopoulos,G.D.Cell 1996,87,1171;For review,see:Thurston,G.Cell Tissue Res.2003,314,61.

6.Holash,J.;Maisonpierre,P.C.;Compton,D.;Boland,P.;Alexander, C.R.;Zagzag, D.;Yancopoulos,G. D.;Wiegand,S.J.Science 1999,284,1994.

7.Adams,J.J.;Bryan,D.D.;Feng,Y.;Matsunaga,S.;Miyazaki,Y.;Nakano,M.;Rocher,J.-P.;Sato,H.;Semones,M.;Silva, D.J.;Tang,J.PCT Application,WO03022852,2003.Analytical data of compound 7k :1H NMR (DMSO-d 6)ppm 3.75(s,3H),6.95(d,J =9.1Hz,2H),7.41(d,J =9.1Hz,2H),7.43(d,J =8.8Hz,2H),7.53(dd,J =8.8Hz,10.9Hz,1H),7.66(d,J =8.6Hz,2H),8.64(d,J =7.1Hz,1H),9.04(s,1H),9.46(s,1H).MS (ESI)m /z 538(M+H)+.The yields for the preparation of 7k (R2=4-OMe in Scheme 1)are as follows:step (f)

Figure 3.Angle from phosphate binding region.Protein surface was added and colored by atom (C,gray;N,purple;O,red;S,yellow;F,cyan).

Table 3.Cellular inhibitory activity of compound 7k expressed as IC 50value in micromolars Compound Tie-2autophosphorylation (l M)HUVECv (l M)HFF (l M)7k

0.0063

0.045

0.79

2206Y.Miyazaki et al./Bioorg.Med.Chem.Lett.15(2005)2203–2207

89%,(g)56%,(c)69%,(d)88%,(e)31%,and the reaction with corresponding isocyanate is60%.

8.The enzyme assay was performed by HTRF(homoge-

neous time-resolved?uorescence)method using baculo-virus-expressed recombinant protein.HTRF is based on the proximity of a donor label(europium chelate)and acceptor label(allophycocyanin,APC)which have been brought together by a speci?c binding reaction.When the two entities come into close proximity and upon excita-tion,energy transfer occurs and APC re-emits a speci?c long-lived?uorescence at665nm.The kinases were puri?ed as the intracellular domain of human Tie-2or VEGFR2fused by GST tag.The catalytic activity of each kinases was detected by a biotinylated synthetic peptide as a substrate,biontin-C6-LEARLVAYEGWVAGKKK-amide,and biotin-aminohexyl-EEEEYFELVAKKKK-NH2for TIE-2and VEGFR2,respectively.Phosphoph-orylated substrate is measured by streptavidin linked-APC (Molecular Probes)and europium-labeled anti-phosphor-ylated tyrosine antibody(Perkin Elmer).Assay conditions are as follows.Tie-2:pre-activated GST-TIE-2with2mM ATP,5mM MgCl2and12.5mM DDT was incubated for 30min with1l M peptide,80l M ATP,10mM MgCl2,

0.1mg/ml BSA and test compound in1mM HEPES.

VEGFR2:GST-VEGFR2was incubated for40–60min with360nM peptide,75l M ATP,5mM MgCl2,0.1mM DTT,0.1mg/ml BSA and test compound in100mM HEPES.

9.(a)Temnikova,T.I.;Sharanin,Y.U.A.;Karavan,V.S.

https://www.wendangku.net/doc/189659795.html,SR(Engl.Trans.)1967,651;(b) Matsuda,T.;Yamagata,K.;Tomioka,Y.;Yamazaki, M.Chem.Pharm.Bull.1985,33,937;(c)Maeda,Y.;

Nakano,M.;Sato,H.;Miyazaki,Y.;Schweiker,S.L.;

Smith,J.L.;Truesdale,A.T.Bioorg.Med.Chem.Lett.

2004,14,3907.

10.The PDB deposition code is1YWN for this structure.

11.Tie-2-autophosphorylation at the cellular level was mea-

sured by ELISA using recombinant mouse3T3cells(TIE-2/c-fms)that stably overexpress the chimeric protein of c-fms extracellular domain and Tie-2intracellular domain.

The test compound was incubated with TIE-2/c-fms cells for1h followed by the activation of TIE-2-c-fms receptor using c-fms ligand,MCSF(macrophage colony stimulat-ing factor).For capture ELISA with anti-c-fms antibody, phosphorylation was detected using phosphotyrosine antibody and colorimetric substrate.

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