Bioorganic & Medicinal Chemistry 10 (2002) 3787 _3805

Highly Potent Inhibitors of TNF- Production.Part II:

Metabolic Stabilization of a Newly Found Chemical Lead and

Conformational Analysis of an Active Diastereoisomer

Toshiaki Matsui,a,*Takashi Kondo,b Yoshitaka Nishita,a Satoshi Itadani,b

Hiroshi Tsuruta,b Setsuko Fujita,b Nagashige Omawari,c Masaru Sakai,b

Shuichi Nakazawa,c Akihito Ogata,b Hideaki Mori,a Wataru Kamoshima,b

Kouichiro Terai,b Hiroyuki Ohno,b Takaaki Obata,b Hisao Nakai b and Masaaki Toda b

a Fukui Research Institute,Ono Pharmaceutical Co.,Ltd.,Technoport,Yamagishi,Mikuni,Sakai,Fukui 913-8638,Japan

b Minase Research Institute,Ono Pharmaceutical Co.,Ltd.,Shimamoto,Mishima,Osaka 618-8585,Japan

c Headquarter,Ono Pharmaceutical Co.,Ltd.,Doshoumachi,Chuou,Osaka 541-8526,Japan

Received 25June 2002;accepted 5August 2002

Abstract—Design and synthesis of metabolically stabilized inhibitors of TNF-a production,which could be new drug candidates,are reported.Conformational analysis of an active diastereoisomer was performed based on biological evaluations of the con-formationally ?xed indane derivatives 17and 18.Structure–activity relationships (SARs)based on biological evaluations of the optically active derivatives are also discussed.Full details including chemistry are reported.

#2002Elsevier Science Ltd.All rights reserved.

Introduction

TNF-a plays a central role in the initiation and main-

tenance of many in?ammatory and autoimmune dis-

eases such as rheumatoid arthritis (RA),multiple

sclerosis,sepsis,ulcerative colitis,congestive heart fail-

ure,in?ammatory bowel disease and Crohn’s dis-

ease.1a,b In the preceding paper,2a àc we reported on the

discovery of a new chemical lead followed by its opti-

mization.Since the ?nal goal of our project is to identify

and develop an inhibitor of TNF-a production which is

a clinically useful drug candidate,we next focused our

attention on the metabolic stabilization of the newly

discovered chemical leads without loss of their highly

potent activity because one of the crucial drawbacks of

them as a drug candidate was found to be the rapid

metabolic hydrolysis of their phosphate moiety as found

in our in vitro study (Fig.1and Scheme 1).Reported in

this publication are the full details of the discovery

0968-0896/02/$-see front matter #2002Elsevier Science Ltd.All rights reserved.P I I :S 0968-0896(02)00380-

2Bioorganic &Medicinal Chemistry 10(2002)

3787–3805

Figure 1.Design of the metabolically stabilized molecules.*Corresponding author.Tel.:+81-756-82-6161;fax:+81-756-82-

8420;e-mail:to.matsui@ono.co.jp

Scheme 1.Metabolic hydrolysis of the phosphates 12and 21

.

Scheme 2.Synthesis of compounds 2,5,10and 11.Reagents and conditions;(a)see ref 3a,b (b)n -C 7H 15COCl,NaHCO 3aq,dioxane;Method A(c,e,f):(c)(i)n -BuLi,THF;(ii)(BnO)2P(O)Cl;Method B (d–f):(d)(i)i Pr 2NP(OBn)2,tetrazole,CH 3CN;(ii)m -CPBA,CH 2Cl 2;(e)H 2,Pd–C,MeOH;(f)NaOHaq,EtOH;(g)(Boc)2O,1N-NaOH,dioxane;Method C (h–k,f):(h)(Cl 3CCH 2O)2P(O)Cl,DMAP,Py;(i)4N-HCl,dioxane;(j)n -C 7H 15COCl,Py,CH 2Cl 2;(k)Zn,Py,AcOH;(l)l -lysine,EtOH,H 2

O.

Scheme 3.Synthesis of compounds 8and 6.Reagents and conditions:(a)(Boc)2O,1N-NaOH,dioxane;(b)PhCOOH,DEAD,Ph 3P,THF;(c)NaOMe,MeOH;Method C (d–h):(d)(Cl 3CCH 2O)2P(O)Cl,DMAP,Py;(e)4N-HCl,dioxane;(f)n -C 7H 15COCl,Py,CH 2Cl 2;(g)Zn,Py,AcOH;(h)NaOHaq,EtOH.3788T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

Scheme 4.Synthesis of compounds 9and 7.Reagents and conditions:(a)PhCOOH,DEAD,Ph 3P,THF;(b)NaOHaq,THF,MeOH;(c)4N-HCl,dioxane;(d)n -C 7H 15COCl,NaHCO 3aq,THF;Method B (e–g):(e)(i)i -Pr 2NP(OBn)2,tetrazole,CH 3CN;(ii)m -CPBA,CH 2Cl 2;(f)H 2,Pd–C,MeOH;(g)NaOHaq,

EtOH.

Scheme 5.Synthesis of compounds 3and 4.Reagents and conditions;(a)n -C 7H 15COCl,NaHCO 3aq,THF;(b)i -PrI,K 2CO 3,acetone;(c)n -C 6H 13OCOCl,NaHCO 3aq,THF ;Method A(d,f,g):(d)(i)n -BuLi,THF;(ii)(BnO)2P(O)Cl;Method B (e–g):(e)(i)i -Pr 2NP(OBn)2,tetrazole,CH 3CN;(ii)m -CPBA,CH 2Cl 2;(f)H 2,Pd-C,MeOH;(g)NaOHaq,EtOH;(h)tris(hydroxymethyl)aminomethane,EtOH,H 2

O.Scheme 6.Synthesis of compound 13.Reagents and conditions:(a)(Boc)2O,CHCl 3;(b)MeI,K 2CO 3,DMF;(c)MeMgBr,THF;(d)4N-HCl/dioxane;(e)n -C 7H 15COCl,NaHCO 3aq,dioxane;Method B (f–i):(f)i -Pr 2NP(OBn)2,tetrazole,CH 3CN;(g)m -CPBA,CH 2Cl 2;(h)H 2,Pd–C,MeOH;(i)NaOHaq,

EtOH.

Scheme 7.Synthesis of compound 14.Reagents and conditions:(a)TMSCN,TMSOTf,CH 2Cl 2;(b)PhMgI,Et 2O;(c)NaBH 4,MeOH;(d)(Boc)2O;Method C (e–h):(e)(Cl 3CCH 2O)2P(O)Cl,DMAP,LiI,Py;(f)4N-HCl/dioxane;(g)n -C 7H 15COCl,Py,CH 2Cl 2;(h)Zn,Py,AcOH.T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053789

process for the metabolically stabilized derivatives as

well as conformational analysis of an active diastereoi-

somer which was based on the SARs from the con-

formationally ?xed indane derivatives that possessed

spatially ?xed functional groups.Chemistry Synthesis of all the compounds listed in the Tables 2–4are described in Schemes 2–11.As outlined in Scheme 2,compounds 5and 10were prepared from 22a ,

while Scheme 8.Synthesis of compound 15.Reagents and conditions:(a)NaN 3,NH 4Cl,dioxane,H 2O;(b)H 2,Pd–C,6N-HCl,EtOH;(c)n -C 7H 15COCl,NaHCO 3aq,THF;(d)TBDPSCl,imidazole,DMF;Method B (e–i):(e)i -Pr 2NP(OBn)2,tetrazole,CH 3CN;(f)m -CBPA,CH 2Cl 2;(g)H 2,Pd–C,MeOH;(h)TBAF,THF;(i)NaOHaq,

EtOH.

Scheme 9.Synthesis of compound 16.Reagents and conditions:(a)n -C 7H 15COCl,NaHCO 3aq,dioxane;Method D (b–e):(b)i -Pr 2NP(OCH 2CH 2CN)2,tetrazole,CH 3CN,CH 2Cl 2;(c)m -CPBA,CH 2Cl 2;(d)50%Me 2NHaq,EtOH;(e)NaOHaq,

EtOH.

Scheme 10.Synthesis of compounds 17and 18.Reagents and conditions:(a)n -C 7H 15COCl,NaHCO 3aq,dioxane;(b)(Cl 3CCH 2O)2P(O)Cl,DMAP,Py;(c)Zn,AcOH,Py;(d)NaOHaq,EtOH;Method E (e–d):(e)(BnO)2P(O)OH,DEAD,Ph 3P,THF;(f)H 2,Pd–C,

MeOH.Scheme 11.Synthesis of compound 19.Reagents and conditions:(a)EtOCOCl,NaHCO 3aq,dioxane;(b)LiALH 4,THF;(c)4N-HCl,dioxane;(d)n -C 7H 15COCl,NaHCO 3aq,dioxane;Method A(e–g):(e)(i)n -BuLi,THF;(ii)(BnO)2P(O)Cl;(f)H 2,Pd–C,MeOH;(g)NaOHaq,EtOH.3790T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

2and11were prepared from22b.Styrene derivatives 22a,b were converted to23a,b according to the reported procedure.3a,b N-Acylation of23a,b a?orded 24a,b,which were converted to25a,b,respectively according to the following successive procedures:(1) phosphorylation;4aàc and(2)deprotection by bde86c05caaedd3383c4d3d3pounds25a,b were converted to their corresponding disodium salts5and2,respectively according to the usual bde86c05caaedd3383c4d3d3pounds26a,b were also prepared from22a,b according to the reported procedure.3a,b Compound26a could be con-verted to10via29and30according to the following successive procedures:(1)N-tert-butoxycarbonylation;

(2)phosphorylation;5,6(3)acidic deprotection;(4) N-acylation;(5)reductive deprotection;5,6and6)treat-ment with NaOH bde86c05caaedd3383c4d3d3pound26b was transformed to11via27and28according to the same procedures as described above.The l-lysine salt of25b was pro-duced as the crystalline compound25b.Lys.As descri-bed in Scheme3,compound31and33were prepared from23a and26a,respectively according to the following successive reactions:(1)N-tert-butox-ycarbonylation;(2)Mitsunobu reaction;7and(3)alka-line bde86c05caaedd3383c4d3d3pounds8and6were prepared from 31and33,respectively according to the same proce-dures as described for the preparation of10from29via 30.As described in Scheme4,compounds9and7were prepared from36and38respectively according to the same procedures as described for the preparation of2 bde86c05caaedd3383c4d3d3pounds36and38were prepared from35 and37,respectively according to the following succes-sive reactions:(1)Mitsunobu reaction;7(2)alkaline hydrolysis;(3)acidic deprotection;and(4)N-acylation. As described in Scheme5,compounds3and4were also prepared from23c and23b,respectively according to essentially the same procedures as described for the preparation of5and2from23a and23b,respectively. The diammonium salt of40b was produced as the crystalline bis[tris(hydroxymethyl)aminomethane]salt (40b.TRIS).Synthesis of compound13is described in Scheme6.Protection of the amino group of418fol-lowed by O-alkylation provided42,dimethylation of which with methyl magnesium bromide a?orded43. Deprotection followed by N-acylation provided44, which was transformed to13by the above-described method.Synthesis of14is described in Scheme7. Cyclobutanone45was converted to46by the conventional method.9Compound46was converted to 47by the following successive reactions:(1)treatment with phenyl magnesium iodide followed by sodium borohydride reduction;10and(2)protection of the amino group with tert-butoxycarbonyl bde86c05caaedd3383c4d3d3-pound47was converted to14via48according to the usual procedures.5,6Synthesis of the hydroxymethyl derivative15is outlined in Scheme8.Aring opening reaction of the epoxide49with sodium azide a?orded 50,catalytic hydrogenation of which gave the amino-alcohol51.N-Acylation of51followed by the selective monosilylation of the diol52provided53,which was converted to54by the usual procedure.Catalytic hydrogenation of54followed by the treatment with TBAF provided55,which was converted to15by the usual method.As described in Scheme9,compound16was prepared from the aminoalcohol56via57.N-Acy-lation of56gave57,which was converted to the dis-odium salt16according to the usual procedure. Synthesis of conformationally?xed isomers17and18is outlined in Scheme10.N-Acylation of the aminoalcohol 58with octanoyl chloride a?orded59,bis(2-tri-chloroethoxy)phosphorylation5,6of which provided60. Reductive deprotection5,6of60followed by the treat-ment with sodium hydroxide a?orded17.Dibenzyl phosphorylation of the hydroxy group of59with a S N2inversion gave61,11deprotection of which a?or-ded62.The free acid form62was converted to18by the usual method.The N-methyl derivative19was prepared as described in bde86c05caaedd3383c4d3d3pound64was prepared from(2R)-2-amino-2-phenylethanol63by the following successive reactions:(1)N-ethoxycarbonyla-tion;(2)reduction with LiAlH4;(3)treatment with acid; and(4)N-acylation with octanoyl bde86c05caaedd3383c4d3d3pound19 was prepared from64according to the usual procedure.

Results and Discussion

To identify an inhibitor of TNF-a production which could be a clinically useful drug candidate,the design and synthesis of metabolically stable derivatives was carried out and their ability to inhibit TNF-a produc-tion was evaluated biologically.Plasma TNF-a produc-tion and the potency of the test compounds were evaluated according to the same procedures as described in the preceding papers.2aàc Apharmacological evalua-tion of the optimized derivatives2–4in the animal dis-ease models was also carried out.

Conformational analysis of the optically active diaster-eoisomers2and9was performed based on the informa-tion obtained from the SARs of the conformationally ?xed indane derivatives17and18.

According to our in vitro experimental data regarding the metabolism of the test compounds,the newly dis-covered inhibitor12was very sensitive to metabolic hydrolysis with tissue homogenates as described in Scheme1and Table1.The formed metabolite20did not show any inhibitory activity as shown in Table4.The metabolic inactivation of12with tissue homogenates from the small intestine and liver was very fast as descri-bed in Table1while with the plasma it was relatively slow.As such,it was thought that the metabolic stabili-zation of the chemical lead1could be accomplished by blocking the rapid hydrolysis of the phosphate moiety in1.In fact,1-methyl-2-octanoylamino-2-phenylethyl disodium phosphate21consisting of four isomers5,6,8 and10was found to be stable after its treatment with tis-sue homogenates as described in Scheme1and Table1. Based on the above-described results,the design and synthesis of the metabolically stabilized inhibitors was started with the introduction of a methyl group into the optically active backbone.As shown in Table2,two classes of four isomers5,6,8and10as well as2,7,9 and11were synthesized and biologically evaluated inpidually.According to our intact data,the meta-

T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053791

bolically stabilized compound 5exhibited more potent activity than 12although their ID 50values in rats appeared to be close to each other (5:4.5mg/kg,iv;12:3.0mg/kg,iv).For example,intravenous administration of 5(3mg/kg)demonstrated a more than 50%inhibi-tion of TNF-a production (58%)while that of 12(3mg/kg)demonstrated less than 50%inhibition (43%).Also in their evaluation in mice,5exhibited a more potent ID 50value (0.4mg/kg,iv)than 12(0.8mg/kg,iv).Thus,metabolic stabilization of 5was found to be e?ective at increasing its activity.

This successful results of the metabolic stabilization was thought to be bene?cial for the discovery of the highly potent inhibitors 2–4described in Table 5.

As shown in Table 2,the (S )-con?guration of the newly introduced methyl group and (R )-con?guration of the N -acyl moiety were needed for potent inhibitory activity as illustrated in compounds 5,2and 7.Introduction of a meta -methoxy group into the phenyl moiety of 5a?or-ded 2with a marked increase in activity.The same ten-dency was observed upon the chemical modi?cation of 6

Table 1.Results of the in vitro metabolism of 12and 21d

Remaining%after incubation (30min)a

Tissue homogenates 1221Mouse liver

N.D.b 93Mouse small intestine N.D.b 97Mouse plasma 8995Rat liver

N.D.b 95Rat small intestine N.T.c 97Rat plasma

74

91

a

In vitro stability of compounds 12and 21was analyzed by HPLC after treatment with tissue homogenates.To a solution of the test compound in PBS (à)(15m g/100m L)1%of tissue homogenate was added.The mixture was incubated at 37 C for 30min,and the reaction quenched with MeOH (2mL).[iso -Butyl paraben (10m g/mL)was used as an internal standard for 12.iso -Amyl paraben (10m g/mL)was used as an internal stan-dard for 21].After centrifugation at 3000rpm for 10min,the organic layer was evaporated.A solution of the residue in CH 3CN/H 2O (1/1,200m L)was analyzed by HPLC [column:Inertsil ODS-3,4.6?150mm (GL Science Inc.);detection:UV at 210nm;Eluent:A=20mM KH 2PO 4(pH2)/B=CH 3CN,(gradient)d ;Flow rate:1.0mL/min;25 C;Sample:50m L was injected].b

ND:Not Detected.c

NT :Not Tested.d

Gradient for analyzing 12.gradiant for analyzing 21.time (min)A/B Time (min)A/B 060/40060/4020.060/401060/4020.520/8010.150/5035.520/8025.050/5036.060/4025.160/4046.0

60/40

35.0

60/40

Table 2.Biological evaluation of the metabolically stabilized

derivatives

Compd R 1R 2R 3

R 4

R 5Inhibition of TNF-a production a

ID 50(mg/kg,iv)rats

5Me H –NHCO–n -C 7H 15H H 4.52Me H –NHCO-n -C 7H 15

H

OMe 0.036Me H H –NHCO–n -C 7H 15H (17)c 7Me H H

–NHCO–n -C 7H 15

OMe 3.78H Me –NHCO–n -C 7H 15H H (23)c 9H Me –NHCO–n-C 7H 15

H

OMe (à13)b 10H Me H –NHCO–n -C 7H 15H (5)c 11H Me H

–NHCO–n -C 7H 15

OMe (38)b 3Me H –NHCO–n -C 7H 15H O i Pr 0.054

Me

H

–NHCOO–n -C 6H 13

H

OMe

0.02

a See Experimental.

b

Inhibition (%)at 10mg/kg,iv.c

Inhibition (%)at 30mg/kg,iv.

3792T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

to 7while 6exhibited relatively weak inhibitory activity.Amarked reduction of inhibitory activity was observed in all the (R )-methyl derivatives 8–11.Therefore,the con?guration of the methyl group was thought to have a dominant e?ect on the ability to inhibit TNF-a pro-duction.The con?guration of the N -acyl moiety also plays an important role,although it dose not seem to be as critical as that of the methyl group (R 1),as illustrated in 5and 7.Introduction of a meta -methoxy group also increased the inhibitory activity as observed upon the chemical modi?cation of 5to 2and 6to 7.Replacement of the meta -methoxy group of 2with a meta -iso-propyloxy group provided 3which retained the highly potent activity.Replacement of the N -octanoyl moiety of 2with a N -hexyloxycarbonyl moiety a?orded 4also with strong activity to inhibit the TNF-a production.Thus,the (1S ,2R )-con?guration was found to be a structural requirement for the potent inhibitory activity in this series of compounds.

As shown in Table 3,further chemical modi?cation was continued to identify another substituent which was

acceptable instead of the (1S )-methyl group.The syn-thesized compounds 13–16were evaluated biologically

and their potentials compared with the chemical leads 12and 5.Introduction of another methyl group at the geminal position of 2a?orded 13with a marked reduc-tion in activity presumably because of the bulkiness of the newly constructed 2,2-dimethyl moiety,while the less hindered 2,2-trimethylene derivative 14,which was biologically evaluated as a dl -mixture,had an ID 50value of 9.5mg/kg,iv in mice.Replacement of the methyl group of 12with a hydroxymethyl group pro-vided 15with a decrease in activity.Introduction of a phenyl moiety to 5instead of a methyl group a?orded 16with a marked reduction in activity.Thus,it was concluded that (1S )-methyl is the optimized partial structure.

To elucidate the three dimensional active conformation,compounds 17–19,in which free rotations are restricted and/or blocked,were synthesized as the optically active forms and evaluated biologically as described in Table 4.Interestingly,the trans -isomer 18was much more potent than the cis -isomer 17.The trans -isomer 18was estimated to occupy a three dimensional structure close to the real active conformer.This SAR strongly suggested an active conformation of the optimized compounds 2–4.As illustrated in Fig.2,the more active compound 2is able to occupy the favored conformation more easily than the less active compound 9because of a less hin-dered intramolecular repulsion between the methyl group and the ortho -hydrogen of the phenyl moiety.The N -methyl derivative 19retained quite a good activity as shown in Table 4.The N -methyl group did not appear to prevent 19from occupying the favored conformation.As shown in Fig.3,increased production of the plasma TNF-a after the intravenous administration of LPS (30m g/kg)was signi?cantly suppressed by administration of compounds 2–4in a dose-dependent manner.The ID 50values of 2–4are 0.03,0.05and 0.02,respectively.The e?cacy in disease models 12a àe and safety of 2–4were evaluated.

Table 3.Attempted study to search for a substitute for the (1S )-methyl

group

Inhibition of TNF-a production a

Compd R 1R 2R 3

ID 50(mg/kg,iv)rats

12H H H 3.05

Me H H 4.513(dl )Me Me OMe (12)b 14(dl )–CH 2-CH 2-CH 2—H 9.5c 15CH 2OH H H

(42)b

16

H

H

(3)b

a

See Experimental.b

Inhibition (%)at 10mg/kg,iv.c

Tested in mice.

Table 4.Analysis of the structural requirements for the inhibitory

activity

Compd R Inhibition of TNF-a production a

ID 50(mg/kg,iv)rats

17C 7H 15(34)b 18C 7H 15 2.419C 7H 159.820

C 7H 15

(14)c

a

See Experimental.

b Inhibition (%)at 10mg/kg,iv.c

Inhibition (%)at 10mg/kg,iv in mice.

T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

3793

Figure 2.Proposed active conformation based on the more active trans-isomer 18

.

Figure 3.E?ect of compound 2,3,and 4on LPS-induced TNF-a production in rats.Test compound was administered intravenously just before an intravenous injection of LPS.After 90min of LPS injection,heparinized blood was obtained.Plasma TNF-a concentration was determined by ELISA.The data were expressed as the mean ?SEM (n =5;***,signi?cantly di?erent from LPS control,P <0.001,ANOVA-Dunnett’s t -test).

Table 5.Biological evaluation of 2–4Compd

LPS-induced shock model in mice (n =18or 20)a MED c mg/kg,iv (survival rate)

d -(+)-Galactosamine/LPS-induced

hepatitis model in rats

(n =18)b

MED c mg/kg,iv (survival rate)

Safety in rats (n =3)MLD d

mg/kg,iv (mortality rate)20.1(10/18)0.3(7/18)>100(0/3)30.3(10/18)0.3(8/18)100(1/3)4

0.1(14/20)

0.1(6/18)

>100(0/3)

a See Experimental.b

See Experimental.c

MED (minimum e?ective dose):survival rates are described in parentheses.d

MLD (minimum lethal dose):at least one of the tested animals died at this dose.Mortality rates are described in parentheses.

3794T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

Figure 4.E?ect of compound 4on LPS-induced shock model in mice.BALB/c mice were injected intravenously with compound 4and then immediately given an intraperitoneal injection of LPS (20mg/kg).The survival rate of the mice was evaluated after 96h (n =5–20;*,signi?cantly di?erent from LPS control,P <0.01;N.S.,not signi?cant,Mantel–Cox

test).

Figure 5.E?ect of compound 4on d -(+)-galactosamine/LPS-induced hepatitis model in rats.SD rats were injected intravenously with compound 4and then immediately given an intraperitoneal injection of d -(+)-galactosamine/LPS (1g/7.5mg/kg).The survival rate was evaluated after 96h (n =18/group;*,signi?cantly di?erent from d -(+)-galactosamine/LPS control,P <0.01;N.S.,not signi?cant,Mantel–Cox test).T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053795

As described in Table5,the minimum e?ective doses (MEDs)of compounds2–4in the LPS-induced shock model of mice were0.1,0.3and0.1mg/kg,iv,respec-tively.The minimum e?ective doses(MEDs)of com-pounds2–4in the d-(+)-galactosamine/LPS-induced hepatitis model of rats were0.3,0.3and0.1mg/kg,iv, respectively.Among the tested,compound4was the most hopeful one.As shown in Figs4and5,compound 4demonstrated an e?cacy equivalent to predonisolone (10mg/kg,iv),which was used as the positive control,at the dose of0.3mg/kg,iv,signi?cantly13in both of the above described disease models.

With respect to the safety concern,the minimum lethal dose(MLD)of3in rats was100mg/kg,iv,while the MLDs of2and4were more than100mg/kg,iv, respectively.All three compounds,2–4,demonstrated a su?cient margin of safety for pharmacological evaluation.

The representative compounds12and2–4were also evaluated for their oral activity.As shown in Table6, much higher dosages than expected were needed to reproduce the same extent of ID50values as those obtained after their intravenous administration.These results were ascribed to their presumed poor oral absorption.Three to seven-fold ID50values were obtained in fed rats compared to fasted rats.Their site of action was also estimated to be in the liver and spleen according to the experimental results reported in the preceding paper.2c

Conclusion

In summary,through the design and synthesis of meta-bolically stabilized inhibitors of TNF-a production,we have discovered the drug candidates2–4for use in the treatment of diseases caused by the overexpression of TNF-a.These three compounds demonstrated e?cacy in animal models of disease and are expected to be clinically useful although no specialized uses are yet intended.

Experimental

General directions

Analytical samples were homogeneous as con?rmed by TLC,and the spectroscopic results obtained were consistent with the assigned structures.All1H NMR spectra were taken on a Varian Gemini-200,VXR-200s or Mercury300spectrometer.MS spectra were obtained on a Hitachi M1200H,JMS-DX303HF or PerSeptive Voyager Elite spectrometer.The matrix assisted laser desorption ionization-time of?ight high-resolution mass spectra(MALDI-TOF,HRMS)were obtained on a PerSeptive Voyager Elite spectrometer.IR spectra were measured on a Perkin–Elmer FT-IR1760X or Jasco FT/IR-430spectrometer.Elemental analyses for carbon,hydrogen and nitrogen were carried out by the Analytical Section of ONO Pharmaceutical Co.,Ltd.on a Perkin–Elmer PE2400SeriesII CHNS/O analyzer. Melting points were uncorrected.Optical rotations were measured on a Jasco DIP-1000polarimeter.Column chromatography was carried out on silica gel(Merck silica gel60(0.063–0.200mm)or Fuji Silysia FL60D). Thin layer chromatography was performed on silica gel (Merck TLC plate,silica gel60F254).The following abbreviations for solvents and reagents are used:THF, tetrahydrofuran;EtOAc,ethyl acetate;MeOH,methanol; EtOH,ethanol;DMF,dimethylformamide;CH2Cl2,di-chloromethane;CHCl3,chloroform;EDC.HCl,1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydro-chloride;HOBt.H2O,N-hydroxybenzotriazole hydrate; m-CPBA,meta-chloroperbenzoic acid;DEAD,diethyl azodicarboxylate;TBDPSCl,tert-butylchlorodiphenyl-silane;DBU,1,8-diazabicyclo[5.4.0]undec-7-ene;TBAF, tetrabutylammonium?uoride;DMAP,N,N-dimethyl-pyridin-4-amine

N-[(1R,2S)-2-Hydroxy-1-(3-methoxyphenyl)propyl]octa-namide(24b).(1R,2S)-1-Amino-1-(3-methoxyphenyl) propan-2-ol hydrochloride23b was prepared from 1-methoxy-3-[(1E)-prop-1-enyl]benzene22b by follow-ing the known procedure:3a,b23b:white solid;TLC R f=0.47(CHCl3/MeOH/H2O,10/5/1);MS(MALDI-TOF,Pos.)m/z182(M+H)+;1H NMR(300MHz, CDCl3)d8.53(brs,3H),7.23(t,J=8.1Hz,1H),7.06(s, 1H),6.97(d,J=7.8Hz,1H),6.83(dd,J=8.4,2.4Hz, 1H),4.85–4.60(br,1H),4.46–4.32(m,2H),3.70(s,3H), 0.99(d,J=6.0Hz,3H).To a stirred mixture of23b (20.0g,92.0mmol)and NaHCO3(23.2g,275.9mmol) in THF(92mL)–H2O(92mL)was added dropwise octanoyl chloride(15.9mL,92.9mmol)at0 C and stirring was continued at that temperature for7h.The reaction mixture was poured into H2O and extracted with EtOAc.The organic layer was successively washed with H2O and brine before being dried over MgSO4. Removal of the solvent by evaporation a?orded a white solid,which was washed with n-hexane and dried under reduced pressure to obtain24b as a white powder(24.7 g,87%):TLC R f=0.32(EtOAc/toluene,2/1);MS (APCI,Pos.20eV)m/z308(M+H)+,290;IR(KBr) 3308,2927,1645,1604,1543,1261cmà1;1H NMR (300MHz,CDCl3)d7.27(t,J=7.8Hz,1H),6.89–6.82 (m,3H),6.33(brd,J=7.8Hz,1H),4.90(dd,J=7.8,3.9 Hz,1H),4.11–4.03(m,1H),3.80(s,3H),2.24–2.19(m, 2H),1.90(brs,1H),1.68–1.57(m,2H),1.31–1.24(m, 8H),1.09(d,J=6.3Hz,3H),0.89–0.85(m,3H).

N-[(1R,2S)-2-Hydroxy-1-phenylpropyl]octanamide(24a). (1R,2S)-1-Amino-1-phenylpropan-2-ol hydrochloride 23a was prepared from(1E)-prop-1-enylbenzene22a

Table6.Oral activity of the representative compounds

Compd Inhibition of TNF-a production

ID50(mg/kg,rats,n=5)

iv po

Fed Fasted

12 3.0>300104

20.035316

30.05639

40.02204

3796T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

according to the known procedure.3a,b The title com-pound24a was prepared from23a according to essen-tially the same procedure as described for the preparation of24b from23b:white powder;TLC R f=0.40(n-hexane/EtOAc,1/2);MS(MALDI,Pos.)m/ z300(M+Na)+,278(M+H)+;IR(KBr)3305,1644, 1549,1454,1123cmà1;1H NMR(300MHz,CDCl3)d 7.40–7.23(m,5H),6.28(d,J=8.0Hz,1H),4.95(dd, J=8.0,3.6Hz,1H),4.09(m,1H),2.23(t,J=6.6Hz, 2H),1.95(d,J=7.5Hz,1H),1.63(m,2H),1.27(m, 8H),1.09(d,J=6.3Hz,3H),0.87(t,J=6.6Hz,3H). Hexyl(1R,2S)-2-hydroxy-1-(3-methoxyphenyl)propyl-carbamate(39b).To a stirred mixture of23b(14.5g, 66.7mmol)and NaHCO3(16.8g,200mmol)in THF/ H2O(70mL/70mL)was added hexyl chlor-idocarbonate(11.0mL,67.3mmol)at0 C.Stirring was continued at that temperature for30min and at room temperature for30min.The reaction mixture was poured into ice-water and extracted with EtOAc.After the organic layer was successively washed with H2O and brine,it was dried over MgSO4.Removal of the solvent by evaporation gave a solid which was washed with n-hexane to a?ord39b as a white solid(19.7g,96%yield): TLC R f=0.28(n-hexane/EtOAc,1/1);MS(MALDI-TOF,Pos.)m/z310(M+H)+;1H NMR(300MHz, CDCl3)d7.32–7.22(m,1H),6.92–6.81(m,3H),5.50 (brd,J=6.9Hz,1H),4.67–4.56(br,1H),4.13–3.95(m, 3H),3.81(s,3H),1.80–1.47(m,2H),1.42–1.20(m,6H), 1.11(d,J=6.3Hz,3H),0.88(t,J=6.9Hz,3H).

N-[(1R,2S)-2-Hydroxy-1-(3-isopropoxyphenyl)propyl]oc-tanamide(39c).3-[(1R,2S)-1-Amino-2-hydroxy-propyl]phenol hydrochloride23c was prepared from1-(benzyloxy)-3-[(1E)-prop-1-enyl]benzene by following the known procedure.3a,b23c:TLC R f=0.45(CHCl3/ MeOH/H2O,65/35/8);1H NMR(300MHz,CDCl3)d 7.23(t,J=8.4Hz,1H),6.92–6.88(m,2H),6.84–6.81 (m,1H),4.20–4.12(m,2H),1.04(d,J=6.0Hz,3H). N-[(1R,2S)-2-hydroxy-1-(3-hydroxyphenyl)propyl]octan-amide was prepared from23c according to essentially the same procedure as described for the preparation of 24b from23b.Puri?cation was performed by column chromatography on silica gel(Merck7734,n-hexane/ EtOAc,1/1)to give N-[(1R,2S)-2-Hydroxy-1-(3-hydroxy-phenyl)propyl]octanamide as a colorless viscous oil (80%yield):TLC R f=0.43(EtOAc/toluene,2/1);1H NMR(300MHz,CDCl3)d8.05(brs,1H),7.14(t, J=7.5Hz,1H),6.78–6.70(m,3H),6.65(d,J=8.1Hz, 1H),4.81(dd,J=8.1,4.2Hz,1H),4.08–3.98(m,1H), 2.85(brs,1H),2.20–2.15(m,2H),1.65–1.52(m,2H), 1.30–1.20(m,8H),1.06(d,J=6.6Hz,3H),0.90–0.80 (m,3H).To a stirred mixture of N-[(1R,2S)-2-hydroxy-1-(3-hydroxyphenyl)propyl]octanamide(5.28g,18.0 mmol)and K2CO3(6.22g,45.0mmol)in acetone(40 mL)was added2-iodopropane(2.70mL,27.0mmol). Stirring was continued under re?ux for12h.The reac-tion mixture was poured into ice–water and extracted with Et2O.The organic layer was successively washed with H2O and brine before being dried over Na2SO4. Removal of the solvent by evaporation gave a residue which was puri?ed by column chromatography on silica gel(Merck7734,n-hexane/EtOAc,1/2)to give39c as a colorless oil(4.82g,80%yield):TLC R f=0.56(EtOAc/

toluene,2/1);MS(APCI,Pos.20eV)m/z336(M+H)+;

IR(KBr)3307,2928,1641,1544,1258,1118cmà1;1H

NMR;(300MHz,CDCl3)d7.24(dd,J=8.7,7.8Hz, 1H),6.85–6.80(m,3H),6.29(d,J=7.8Hz,1H),4.90

(dd,J=7.8,3.9Hz,1H),4.58–4.50(m,1H),4.12–4.01

(m,1H),2.24–2.19(m,2H),2.13(brd,J=6.6Hz,1H), 1.68–1.58(m,2H),1.33(d,J=6.0Hz,3H),1.33(d,

J=6.0Hz,3H),1.32–1.23(m,8H),1.10(d,J=6.3Hz, 3H),0.89–0.85(m,3H).

General method A

(1S,2R)-2-(3-Isopropyloxyphenyl)-1-methyl-2-(octanoyla-mino)ethyl disodium phosphate(3).To a stirred solution

of39c(4.0g,11.9mmol)in THF(100mL)was added

dropwise n-butyllithium in n-hexane(1.53M,21.0mL, 32.1mmol)atà78 C under an argon atmosphere.

Dibenzylphosphorochloridate in THF4a(1M,35.7mL,

35.7mmol)was added to the resulting mixture at that temperature and stirring was continued for10min at

à20 C.The reaction was quenched with1M NaOH and the resulting mixture was warmed to room tem-perature with stirring.After the reaction mixture was

diluted with EtOAc,the organic layer was successively

washed with saturated NaHCO3aq and brine,it was

dried over MgSO4.Removal of the solvent by evapora-tion gave a residue which was puri?ed by column chro-matography on silica gel(Merck7734,n-hexane/EtOAc, 4/1)to a?ord dibenzyl(1S,2R)-2-(3-isopropyloxyphe-nyl)-1-methyl-2-(octanoylamino)ethyl phosphate as a colorless oil(4.54g,64%yield):TLC R f=0.58 (EtOAc/toluene,1/1).A mixture of dibenzyl(1S,2R)-2-(3-isopropyloxyphenyl)-1-methyl-2-(octanoylamino)ethyl phosphate(4.54g)and10%Pd-C(400mg)in MeOH was stirred at room temperature under an atmospheric pressure of hydrogen for1h.Removal of the catalyst by ?ltration through a pad of Celite followed by evapora-tion gave an oily residue which was dissolved in satu-rated NaHCO3aq and extracted with EtOAc.The aqueous layer was acidi?ed by1M HCl and then extracted with EtOAc.The organic layer was succes-sively washed with H2O and brine before being dried over MgSO4.Removal of the solvent by evaporation gave40c as a white amorphous powder(2.56g,81%). To a stirred solution of40c(2.24g,5.4mmol)in EtOH (50mL)was added1M NaOH(10.8mL,10.8mmol)at room temperature.Removal of the solvent by evapora-tion followed by the dissolution of the residue in EtOH was repeated several times to remove the H2O azeo-tropically.The addition of Et2O followed by evapora-tion a?orded3as a white amorphous powder:TLC R f=0.33(CHCl3/MeOH/H2O,65/35/8);IR(KBr)3423, 1637,1089,984cmà1;1H NMR(300MHz,CD3OD) 7.12(t,J=7.8Hz,1H),6.99(brd,J=7.8Hz,1H),6.96 (brs,1H),6.73–6.69(m,1H),4.64–4.54(m,3H),2.35–2.19(m,2H),1.61–1.49(m,2H),1.29(d,J=6.0Hz, 3H),1.26(d,J=6.0Hz,3H),1.30–1.20(m,8H),1.06 (d,J=6.3Hz,3H),0.89–0.84(m,3H);optical rotation [a]24Dà53.3(c1.05,MeOH);MS(FAB,Pos.)m/z460 (M+H)+,438,416;HRMS(MALDI-TOF,Pos.) calcd for C20H32NO6P.2Na+H+:460.1841;found: 460.1805.

T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053797

(1S,2R)-1-Methyl-2-octanoylamino-2-phenylethyl diso-

diumphosphate(5).The title compound5was prepared

from24a according to the general method A:TLC

R f=0.24(CHCl3/MeOH/H2O,65/25/4);IR(KBr)3336, 1637,1535,1454,1385,1089cmà1;1H NMR(300MHz,

CD3OD)d7.42(brd,J=6.6Hz,2H),7.25–7.14(m,

3H),4.62–4.57(m,2H),2.33–2.18(m,2H),1.60–1.50 (m,2H),1.35–1.15(m,8H),1.03(d,J=6.6Hz,3H),

0.86(brt,J=6.6Hz,3H);optical rotation[a]D25à64.5(c 0.96,MeOH);MS(FAB,Pos.)m/z424(M+Na)+,402 (M+H)+;HRMS(MALDI-TOF,Pos.)calcd for the

free acid form C17H28NO5P+Na+:380.1603;found:

380.1599.

General method B

(1S,2R)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-no)ethyl disodiumphosphate(2).To a stirred mixture of 24b(25.5g,83mmol)and tetrazole(11.64g,166mmol) in CH3CN(330mL)was added dibenzyl diisopropyl-phosphoramidite(33.5mL,99.7mmol)at room tem-perature and stirring was continued at that temperature for3h.The reaction mixture was poured into satu-rated NaHCO3aq and the resultant precipitates were removed by?ltration.The?ltrate was then diluted with EtOAc and washed with brine before being dried over Na2SO4.Removal of the solvent by evaporation gave dibenzyl(1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl phosphite which was used for the next reaction without further puri?cation(52.0g, >100%,pale yellow oil):TLC R f=0.60(n-hexane/ EtOAc,1/2).To a stirred solution of dibenzyl(1S,2R)-1 -methyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl phosphite(52.0g)in CH2Cl2(415mL)was added m-CPBA(77%,20.5g,91.4mmol)at0 C and stirring was continued at that temperature for1h.The reaction mixture was treated with saturated Na2S2O3aq and extracted with CH2Cl2.The organic layer was succes-sively washed with H2O,saturated NaHCO3aq and brine before being dried over Na2SO4.The m-chlor-obenzoic acid was removed by passing the mixture through a pad of Al2O3(100g).Removal of the solvent by evaporation gave dibenzyl(1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl phosphate which was used for the next reaction without further puri?cation(50.6g,>100%,colorless oil):TLC R f=0.35(n-hexane/EtOAc,1/2).A mixture of dibenzyl (1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylamino) ethyl phosphate(50.6g)and10%Pd-C(50%wet,10g) in MeOH(850mL)was vigorously stirred at room temperature under an atmospheric pressure of hydrogen for5.5h.Removal of the catalyst by?ltration through a pad of Celite followed by evaporation a?orded an oily residue which was dissolved in saturated NaHCO3aq and extracted with EtOAc.The aqueous layer was acidi?ed by1M HCl and extracted with EtOAc.The organic layer was washed with brine and dried over Na2SO4. Removal of the solvent by evaporation gave25b(23.5g, 73%in3steps,beige amorphous powder):TLC R f=0.28(CHCl3/MeOH/H2O,65/35/8);1H NMR (300MHz,CD3OD)d7.22(t,J=8.1Hz,1H),6.96–6.90 (m,2H),6.84–6.81(m,1H),5.01(d,J=4.2Hz,1H), 4.71–4.60(m,1H),3.78(s,3H),2.26(t,J=7.2Hz,2H),1.65–1.55(m,2H),1.30–1.25(m,8H),1.21(d,J=6.3

Hz,3H),0.90–0.85(m,3H).To a stirred solution of25b

(2.13g,5.5mmol)in EtOH(50mL)was added1M

NaOH(11mL,11mmol)at0 C.Removal of the sol-vent by evaporation followed by the dissolution of the

residue in EtOH was repeated several times to remove

the H2O azeotropically.Then addition of Et2O followed by evaporation a?orded2as a white amorphous pow-

der(5.18g,98%):TLC R f=0.28(CHCl3/MeOH/H2O,

65/35/8);IR(KBr)3389,1638,1534,1087,984cmà1; 1H NMR(300MHz,CD3OD)d7.14(t,J=8.4Hz,1H), 7.01–6.99(m,2H),6.74(ddd,J=8.4,2.7,1.2Hz,1H),

4.65–4.53(m,2H), 3.78(s,3H), 2.35–2.19(m,2H),

1.61–.49(m,2H),1.30–1.20(m,8H),1.05(d,J=6.3Hz,

3H),0.88–0.84(m,3H);optical rotation[a]24

D

à63.3(c 1.02,MeOH);MS(FAB,Pos.)m/z432(M+H)+,454, 410;HRMS(MALDI-TOF,Pos.)calcd for C18H28NO6P.2Na+H+:432.1528;found:432.1524.

L-Lysine salt of compound2(25.Lys).Compound2was one of the best inhibitors for TNF-a production,so an alternative salt was prepared.To a stirred solution of 25b(15.6g,40.2mml)in EtOH/H2O(20/1,130mL) was added l-lysine(5.94g,40.6mmol).Stirring was continued under re?ux until the reaction mixture chan-ged from a suspension to a clear solution.After cooling to room temperature,the resultant precipitates were collected by?ltration and dried under reduced pressure to give25.Lys as a white powder(17.7g,82%):TLC R f=0.22(CHCl3/MeOH/H2O,10/5/1);MS(FAB,Pos.) m/z534(M+H)+,426,410,388,290,147;IR(KBr) 3296,2927,2856,1643,1602,1545,1467,1416,1379, 1346,1258,1165,1124,1049cmà1;1H NMR(300MHz, CD3OD)d7.18(t,J=8.1Hz,1H),6.99–6.92(m,2H), 6.82–6.76(m,1H),4.84(d,J=3.3Hz,1H),4.64–4.51 (m,1H),3.78(s,3H),3.56(t,J=6.0Hz,1H),2.92(t, J=7.5Hz,2H),2.34–2.17(m,2H),1.95–1.75(m,2H), 1.75–1.41(m,6H),1.38–1.17(m,8H),1.12(d,J=6.6 Hz,3H),0.87(t,J=6.9Hz,3H);mp194.5–196.5 C;

optical rotation[a]26

D

à45.99(c1.0,MeOH);Anal calcd for C18H30NO6P.C6H14N2:C,54.02;H,8.31;N,7.87; Found:C,54.04;H,8.37;N,7.79.

(1S,2R)-1-Methyl-2-[(hexyloxycarbonyl)amino]-2-(3-methoxyphenyl)ethyl disodium phosphate(4).The title compound4was prepared from39b according to the general method B:TLC R f=0.30(CHCl3/MeOH/H2O, 65/35/4);IR(KBr)3239,2932,1699,1602,1491,1456, 1436,1343,1255,1105cmà1;1H NMR(300MHz, CD3OD)d7.12(t,J=8Hz,1H),7.04–6.94(m,2H), 6.78–6.72(m,1H),4.66–4.52(m,1H),4.49(m)and4.38 (brs)total1H,3.93(t,J=6.6Hz,2H),3.78(s,3H), 1.64–1.48(m,2H),1.40–1.20(m,6H),1.05(d,J=6.6

Hz,3H),0.88(m,3H);optical rotation[a]26

D

à48.83(c 1.2,MeOH);mp>300 C(decomposed);MS(FAB, Pos.)m/z434(M+H)+,412,390;HRMS(MALDI-TOF,Pos.)calcd for C17H26NO7P.2Na+H+:434.1321; found:434.1363.

Bis[tris(hydroxymethyl)aminomethane]salt of compound 40b(40b.TRIS).Compound4was one of the best inhi-bitors for TNF-a production,so an alternative salt was prepared.Amixture of40b(21.8g,56.2mmol)and

3798T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

Tris(hydroxymethyl)aminomethane(13.6g,112mmol) in EtOH/H2O(20/1,200mL)was heated under re?ux until the reaction mixture changed from a suspension to a clear solution.After cooling to room temperature,the resultant precipitates were collected by?ltration and dried under reduced pressure to give40b.TRIS as a white powder(24.7g,70%yield):TLC R f=0.20 (CHCl3/MeOH/H2O,65/25/4);MS(FAB,Pos.)m/z 511,412,390,122;IR(KBr)2931,1691,1611,1541, 1491,1466,1077cmà1;1H NMR(300MHz,CD3OD)d 7.18(t,J=8.0Hz,1H),6.69(s)and6.95(d,J=8.0Hz) total2H,6.80–6.75(m,1H),4.66–4.52(m,2H),3.96(t, J=6.6Hz,2H),3.78(s,3H),3.58(s,12H),1.64–1.50 (m,2H),1.42–1.24(m,6H),1.08(d,J=6.3Hz,3H),

0.94–0.84(m,3H);optical rotation[a]26

D à32.16(c1.0,

MeOH);mp130–133 C;Anal calcd for C17H28NO7 P.2C4H11NO3.H2O:C,46.22;H,8.07;N,6.47;Found: C,46.41;H,8.19;N,6.49.

tert-Butyl(1S,2S)-2-hydroxy-1-phenylpropylcarbamate (33).(1S,2R)-1-Amino-1-phenylpropan-2-ol hydrochlo-ride26a was prepared from(1E)-prop-1-enylbenzene 22a according to the known procedure:3a,b26a:white powder;TLC R f=0.50(EtOAc/AcOH/H2O3/1/1);MS (APCI,Pos,20eV)m/z152,135,106;1H NMR (200MHz,CDCl3+CD3OD)d7.50–7.30(m,5H),4.43–4.28(m,1H),4.21(d,J=3.6Hz,1H),1.04(d,J=6.6 Hz,3H).To a stirred solution of26a(1.26g, 6.72 mmol)in dioxane(35mL)were added2M NaOH(6.8 mL,13.4mmol)and di-tert-butyl dicarbonate(1.76g, 8.06mmol)at0 C and stirring was continued at that temperature for3h.The reaction mixture was diluted with EtOAc.After the organic layer was successively washed with H2O and brine,it was dried over Na2SO4. Removal of the solvent by evaporation gave a residue which was solidi?ed with n-hexane to a?ord tert-butyl (1S,2R)-2-hydroxy-1-phenylpropylcarbamate29as a white powder(92%yield):TLC R f=0.53(n-hexane/ EtOAc,1/1);1H NMR(300MHz,CDCl3)d7.40–7.25 (m,5H),5.45–5.30(br,1H),4.70–4.52(br,1H),4.15–4.00(m,1H),1.90–1.70(br,1H),1.41(s,9H),1.09(d, J=6.3Hz,3H).To a stirred mixture of29(1.54g,6.12 mmol),PPh3(1.60g,6.12mmol)and benzoic acid(747 mg,6.12mmol)in THF(60mL)was added DEAD (2.3M in toluene,2.7mL,6.12mmol)at0 C and stir-ring was continued at that temperature for2h. Removal of the solvent by evaporation gave a residue which was puri?ed by column chromatography on silica gel(Merck7734,n-hexane/EtOAc,3/1)to a?ord an oil, which was solidi?ed by n-hexane to obtain(1S,2S)-2-[(tert-butyloxycarbonyl)amino]-1-methyl-2-phenylethyl benzoate as a white powder(83%yield):TLC R f=0.58 (n-hexane/EtOAc,3/1);1H NMR(200MHz,CDCl3)d 8.08–7.98(m,2H),7.62–7.20(m,8H), 5.50–5.35(m, 1H),5.20(d,J=9.2Hz,1H),4.98–4.80(m,1H),1.40–1.20(m,12H).To a stirred solution of(1S,2S)-2-[(tert-butyloxycarbonyl)amino]-1-methyl-2-phenylethyl benzoate(1.74g,4.9mmol)in MeOH(50mL)was added NaOMe(53mg,0.98mmol)at room temperature and stirring was continued at that temperature for20h. Removal of the solvent by evaporation gave a residue which was puri?ed by column chromatography on silica gel(Merck7734,n-hexane/EtOAc,3/1–1/1)and recrys-tallization from EtOAc/n-hexane to a?ord33as a white crystal(1.09g,88%yield):TLC R f=0.33(n-hexane/ EtOAc,2/1);1H NMR(300MHz,CDCl3)d7.40–7.25 (m,5H),5.32(d,J=8.1Hz,1H),4.62–4.50(m,1H), 4.10–3.95(m,1H),2.10–1.95(br,1H),1.43(s,9H),1.23 (d,J=6.3Hz,3H).

General method C

(1S,2S)-1-Methyl-2-octanoylamino-2-phenylethyl diso-diumphosphate(6).To a stirred mixture of33(873mg, 3.48mmol)and DMAP(636mg,5.22mmol)in pyri-dine(10mL)was added bis(2,2,2-trichloroethyl)chlor-idophosphate(1.98g,5.22mmol)at0 C.Stirring was continued at room temperature for18h.The reaction mixture was diluted with EtOAc.The organic layer was successively washed with1M HCl,H2O and brine before being dried over Na2SO4.Removal of the solvent by evaporation gave34as a colorless oil(3.15g,quant) which was used for the next reaction without further puri?cation:TLC R f=0.50(n-hexane/EtOAc,2/1).A solution of34(3.15g,3.48mmol)in4M HCl/dioxane (20mL)was stirred at room temperature for2h. Removal of the solvent by evaporation gave a residue which was solidi?ed by Et2O to a?ord(1S,2S)-2-amino-1-methyl-2-phenylethyl bis(2,2,2-trichloroethyl)phos-phate hydrochloride as a white powder(1.73g,94% yield):TLC R f=0.50(CHCl3/MeOH);1H NMR (200MHz,CDCl3+CD3OD)d7.62–7.50(m,2H),7.50–7.38(m,3H),5.25–5.05(m,1H),4.82–4.55(m,4H),4.40 (d,J=8.8Hz,1H),1.40(d,J=6.2Hz,3H).To a stirred suspension of(1S,2S)-2-amino-1-methyl-2-phenylethyl bis(2,2,2-trichloroethyl)phosphate hydrochloride(1.72 g,3.24mmol)in CH2Cl2(16mL)were added octanoyl chloride(631mg,3.89mmol)and pyridine(1.31mL, 16.2mmol)at0 C.Stirring was continued at room temperature for45min.Removal of the solvent by evaporation gave(1S,2S)-1-methyl-2-phenyl-2-(octa-noylamino)ethyl bis(2,2,2-trichloroethyl)phosphate which was used for the next reaction without further puri?cation.To a stirred solution of(1S,2S)-1-methyl-2-phenyl-2-(octanoylamino)ethyl bis(2,2,2-trichloroethyl) phosphate(1.96g,3.24mmol)in pyridine(15mL)/ AcOH(3mL)was added Zn powder(1.90g,29.2 mmol)at0 C.Stirring was continued at room tem-perature for2h.Removal of the Zn powder by?ltration through a pad of Celite followed by evaporation a?or-ded an oily residue which was dissolved in5M NaOH and extracted with EtOAc.The aqueous layer was acidi?ed by2M HCl and extracted with EtOAc.The organic layer was successively washed with H2O and brine before being dried over Na2SO4.Removal of the solvent by evaporation gave a residue,which was pur-i?ed by column chromatography on silica gel(Merck 7734,CHCl3/MeOH/NH4OH,65/25/2-CHCl3/MeOH/ H2O,65/25/4)to a?ord a solid.The solid was dissolved in2M HCl/EtOAc.After the organic layer was succes-sively washed with H2O and brine,it was dried over Na2SO4.Removal of the solvent by evaporation gave a residue,which was solidi?ed by n-hexane to obtain (1S,2S)-1-methyl-2-octanoylamino-2-phenylethyl dihy-drogen phosphate as a white powder(697mg,60% yield from34).The title compound6was prepared from

T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053799

(1S,2S)-1-methyl-2-octanoylamino-2-phenylethyl dihy-drogen phosphate according to the same procedure as described for the preparation of2from25b:white powder;TLC R f=0.43(CHCl3/MeOH/H2O,65/35/8); IR(KBr)3399,2928,2857,2359,1637,1549,1496, 1455,1378,1301,1204,1090cmà1;1H NMR(300MHz, CD3OD)d7.40–7.30(m,2H),7.30–7.15(m,3H),4.41–4.25(m,2H),2.38–2.15(m,2H),1.63–1.45(m,2H), 1.38–1.12(m,8H),1.06(d,J=6.0Hz,3H),0.87(t,

J=6.8Hz,3H);optical rotation[a]25

D +17.3(c 1.0,

MeOH);MS(FAB,Pos.)m/z424(M+Na)+,402 (M+H)+,380;HRMS(MALDI-TOF,Pos.)calcd for C17H26NO5P.2Na+H+:402.1422;found:402.1437. (1R,2R)-1-Methyl-2-octanoylamino-2-phenylethyl diso-diumphosphate(8).The title compound8was prepared from23a according to the general method C;TLC R f=0.24(CHCl3/MeOH/H2O,65/25/4);IR(KBr)3408, 1636,1455,1091,985cmà1;1H NMR(300MHz, CD3OD)d7.35(brd,J=7.2Hz,2H),7.26(brt,J=7.2 Hz,2H),7.18(m,1H),4.40–4.24(m,2H),2.38–2.10(m, 2H),1.55(m,2H),1.35–1.10(m,8H),1.05(d,J=5.7Hz,

3H),0.87(t,J=6.6Hz,3H);optical rotation[a]24

D à14.9

(c1.0,MeOH);MS(FAB,Pos.)m/z424(M+Na)+,402 (M+H)+,380;HRMS(MALDI-TOF,Pos.)calcd for C17H26NO5P.2Na+H+:402.1422;found:402.1428. (1R,2S)-1-Methyl-2-octanoylamino-2-phenylethyl diso-diumphosphate(10).The title compound10was pre-pared from29according to the general method C;o?-white powder;TLC R f=0.23(CHCl3/MeOH/H2O,65/ 25/4);IR(KBr)3346,1639,1538,1454,1093cmà1;1H NMR(300MHz,CD3OD)d7.44(d,J=6.9Hz,2H), 7.28–7.12(m,3H),4.61(m,2H),2.35–2.18(m,2H),1.55 (m,2H),1.25(m,8H),1.04(d,J=6.3Hz,3H),0.87(t,

J=6.3Hz,3H);optical rotation[a]25

D +60.9(c1.25,

MeOH);MS(FAB,Pos.)m/z402(M+H)+,380,358; HRMS(MALDI-TOF,Pos.)calcd for C17H26NO5P.2-Na+H+:402.1422;found:402.1416.

N-[(1S,2S)-2-Hydroxy-1-(3-methoxyphenyl)propyl]octa-namide(38).tert-Butyl(1S,2R)-2-hydroxy-1-(3-methox-yphenyl)propylcarbamate37was prepared from26b according to the same procedure as described for the preparation of tert-butyl(1S,2R)-2-hydroxy-1-phenyl-propylcarbamate from26a.(1S,2R)-2-[(tert-Butyloxy-carbonyl)amino]-1-methyl-2-(3-methoxyphenyl)ethyl benzoate was prepared from37according to the same procedure as described for the preparation of(1S,2S)-2-[(tert-butyloxycarbonyl)amino]-1-methyl-2-phenylethyl benzoate from tert-butyl(1S,2R)-2-hydroxy-1-phenyl-propylcarbamate(96%yield).To a stirred solution of (1S,2R)-2-[(tert-butyloxycarbonyl)amino]-1-methyl-2-(3-methoxyphenyl)ethyl benzoate(3.48g,9.5mmol)in THF(50mL)/MeOH(20mL)was added1M NaOH (20mL,20mmol)at room temperature and stirring was continued at that temperature for20h.Removal of the solvent by evaporation gave a reside,which was extrac-ted with EtOAc.The organic layer was successively washed with1M NaOH and brine before being dried over MgSO4.Removal of the solvent by evaporation a?orded a residue which was puri?ed by column chro-matography on silica gel(Merck7734,n-hexane/EtOAc,2/1)to obtain tert-butyl(1S,2S)-2-hydroxy-1-(3-meth-oxyphenyl)propylcarbamate(1.36g,51%yield):TLC R f=0.52(n-hexane/EtOAc,1/1);1H NMR(300MHz, CD3OD)d7.28(dd,J=9.0,7.2Hz,1H),6.87–6.79(m, 3H),5.30(brs,1H),4.53(brs,1H),4.03(brs,1H),3.80 (s,3H),1.43(brs,9H),1.22(d,J=6.3Hz,3H).Asolu-tion of tert-butyl(1S,2S)-2-hydroxy-1-(3-methox-yphenyl)propylcarbamate(1.34g,6.72mmol)in4M HCl/dioxane(20mL)was stirred at0 C for2h. Removal of the solvent by evaporation followed by the dissolution of the residue in toluene was repeated sev-eral times to give(1S,2S)-1-amino-1-(3-methox-yphenyl)propan-2-ol hydrochloride as an oil.To a stirred mixture of(1S,2S)-1-amino-1-(3-methox-yphenyl)propan-2-ol hydrochloride in THF(50mL) and0.5M NaHCO3(50mL)was added octanoyl chlo-ride(1.1g,6.79mmol)in dioxane(5mL)at0 C and stirring was continued at that temperature for4h.The reaction mixture was diluted with EtOAc.The organic layer was successively washed with1M HCl and brine before being dried over MgSO4.Removal of the solvent by evaporation a?orded a residue,which was puri?ed by column chromatography on silica gel(Merck7734, n-hexane/EtOAc,2/1–1/1)to a?ord38(1.53g,100% yield):TLC R f=0.22(n-hexane/EtOAc,1/1);1H NMR (200MHz,CD3OD)d7.26(dd,J=8.4,8.4Hz,1H), 6.87–6.78(m,3H),6.29(brd,J=8.4Hz,1H),4.87(dd, J=8.4,4.0Hz,1H),4.09(m,1H),3.80(s,3H),2.26 (brt,J=8.2Hz,2H),1.70–1.60(m,2H),1.40–1.25(m, 8H),1.20(d,J=6.6Hz,3H),0.87(brt,J=6.6Hz,3H).

(1S,2S)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-no)ethyl disodiumphosphate(7).The title compound7 was prepared from compound38according to the Gen-eral Method B:94%yield;TLC R f=0.22(CHCl3/MeOH/ H2O,65/35/4);IR(KBr)3412,2929,1638,1457,1266, 1092,984,801cmà1;1H NMR(200MHz,CD3OD)d7.16 (dd,J=8.0,8.0Hz,1H),6.92(m,2H),6.74(ddd,J=8.0, 2.4,1.2Hz,1H),4.35–4.29(m,2H),3.76(s,3H),2.38–2.12 (m,2H),1.53(m,2H),1.30–1.20(m,8H),1.05(d,J=6.0 Hz,3H),0.86(brt,J=6.6Hz,3H);optical rotation[a]25

D +17.0(c 1.0,MeOH);MS(FAB,Pos.)m/z454 (M+Na)+,432(M+H)+;HRMS(MA LDI-TOF,Pos.) calcd for C18H28NO6P.2Na+H+:432.1528;found: 432.1524.

(1R,2R)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-no)ethyl disodiumphosphate(9).The title compound9 was prepared from36according to the general method B:white powder;TLC R f=0.28(CHCl3/MeOH/H2O, 65/25/4);IR(KBr)3328,2929,1638,1457,1265,1091, 801cmà1;1H NMR(200MHz,CD3OD)d7.16(dd, J=8.0,8.0Hz,1H),6.92(m,2H),6.74(ddd,J=8.0,2.4, 1.2Hz,1H),4.35–4.29(m,2H),3.76(s,3H),2.38–2.12(m, 2H),1.53(m,2H),1.30–1.20(m,8H),1.05(d,J=6.0Hz,

3H),0.86(brt,J=6.6Hz,3H);optical rotation[a]25

D

à16.6 (c1.0,MeOH);MS(FAB,Pos.)m/z454(M+Na)+,432 (M+H)+;HRMS(MALDI-TOF,Pos.)calcd for C18H28NO6P.2Na+H+:432.1528;found:432.1568.

(1R,2S)-1-Methyl-2-(3-methoxyphenyl)-2-(octanoylami-no)ethyl disodiumphosphate(11).The title compound11 was prepared from27according to the General Method

3800T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

B:white powder;TLC R f=0.20(CHCl3/MeOH/H2O, 65/25/4);IR(KBr)3333,2928,1638,1535,1256,1088, 984cmà1;1H NMR(300MHz,CD3OD)d7.13(dd, J=8.1,8.1Hz,1H),7.01–6.99(m,2H), 6.73(ddd, J=8.1,2.4,1.2Hz,1H),4.63–4.57(m,2H),3.77(s,3H), 2.36–2.18(m,2H),1.55(m,2H),1.35–1.20(m,8H),1.05 (d,J=6.3Hz,3H),0.86(brt,J=6.6Hz,3H);optical

rotation[a]25

D +65.4(c1.0,MeOH);MS(FAB,Pos.)

m/z432(M+H)+;HRMS(MALDI-TOF,Pos.)calcd for C18H28NO6P.2Na+H+:432.1528;found:432.1505. Methyl[(tert-butoxycarbonyl)amino](3-methoxypheny-l)acetate(42).Methyl amino(3-hydroxyphenyl)acetate 41was prepared from3-(benzyloxy)benzaldehyde according to the same procedure as described in the preceding paper.2c,8To a stirred solution of41(5.0g, 27.6mmol)in CHCl3(150mL)was added dropwise a solution of di-tert-butyl dicarbonate(5.96g,27.3mmol) in CHCl3(20mL)at room temperature and stirring was continued at that temperature for5h.The reaction was quenched with NH4OH and extracted with CHCl3.The organic layer was successively washed with saturated NaHCO3,H2O and brine before being dried over Na2SO4.Removal of the solvent by evaporation gave methyl[(tert-butoxycarbonyl)amino](3-hydroxyphenyl)-acetate which was used for the next reaction without further puri?cation:TLC R f=0.39(n-hexane/EtOAc,1/1). To a stirred mixture of methyl[(tert-butoxy-carbonyl)amino](3-hydroxyphenyl)acetate(27.6mmol) and K2CO3(11.5g,82.9mmol)in DMF(54mL)was added iodomethane(5.16mL,82.9mol)at room tem-perature and stirring was continued at that temperature for2h.The reaction mixture was treated with H2O and then extracted with EtOAc.After the organic layer was successively washed with1M HCl,H2O and brine,it was dried over Na2SO4.Removal of the solvent by evaporation gave42(7.91g,97%yield in2steps):TLC R f=0.63(n-hexane/EtOAc,1/1);1H NMR(200MHz, CDCl3)d7.31–7.22(m,1H),6.95–6.82(m,3H),5.52–5.48(m,1H),5.28(d,J=7.0Hz,1H),3.79(s,3H),3.71 (s,3H),1.42(s,9H).

tert-Butyl2-hydroxy-1-(3-methoxyphenyl)-2-methylpro-pylcarbamate(43).To a stirred solution of42(4.07g, 13.8mmol)in THF(64mL)was added dropwise methylmagnesium bromide(0.84M in Et2O63.9mL, 53.4mmol)at0 C.Stirring was continued at room temperature for2h.The reaction was quenched with 1M HCl and extracted with EtOAc,and the organic layer was washed with brine before being dried over Na2SO4.Removal of the solvent by evaporation gave 43:TLC R f=0.50(n-hexane/EtOAc,1/1);1H NMR (300MHz,CDCl3)d7.27–7.22(m,1H),7.11–6.79(m, 3H),5.50(d,J=6.9Hz,1H),4.48(d,J=6.9Hz,1H), 3.83(s,3H),1.40(s,9H),1.32(s,3H),1.06(s,3H).

N-[2-hydroxy-1-(3-methoxyphenyl)-2-methylpropyl]oc-ta namide(44).The title compound44was prepared from43according to essentially the same procedures as described for the preparation of38from tert-butyl (1S,2S)-2-hydroxy-1-(3-methoxyphenyl)propylcarbamate: TLC R f=0.48(n-hexane/EtOAc,1/2);1H NMR (300MHz,CDCl3)d7.24(t,J=7.2Hz,1H),6.88–6.79(m,3H),6.43(d,J=8.7Hz,1H),3.79(s,3H),2.24–2.19 (m,2H),1.68–1.58(m,2H),1.30–1.07(m,8H),0.86(t, J=6.6Hz,3H).

1,1-Dimethyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl disodiumphosphate(13).The title compound13was prepared from44according to the general method B: TLC R f=0.31(CHCl3/MeOH/H2O,65/25/4);IR(KBr) 3321,2928,1621,1538,1490,1455,1388,1368,1243, 1088cmà1;1H NMR(300MHz,CD3OD)d7.13(t, J=7.8Hz,1H),7.09–7.05(m,2H),6.73–6.69(m,1H), 4.49(d,J=1.5Hz,1H),3.78(s,3H),2.27(t,J=7.2Hz, 2H),1.66(s,3H),1.62–1.53(m,2H),1.32–1.23(m,8H), 1.16(s,3H),0.86(t,J=6.3Hz,3H);MS(FAB,Pos.)m/ z446(M+H)+;HRMS(MALDI-TOF,Pos.)calcd for C19H30NO6P.2Na+H+:446.1684;found:446.1651. tert-Butyl(1-hydroxycyclobutyl)(phenyl)methylcarbamate (47).To a stirred mixture of cyclobutanone(5.0g,71 mmol)and trimethylsilyl cyanide(7.03g,71mmol)in CH2Cl2(100mL)was added dropwise a solution of tri-methylsilyl tri?uoromethanesulfonate(1.27mL,7.1 mmol)in CH2Cl2(50mL)atà78 C and stirring was continued at that temperature for2.5h.The reaction was quenched with a few drops of pyridine.The result-ing mixture was poured into saturated NaHCO3aq and extracted with CH2Cl2.The organic layer was succes-sively washed with H2O and brine before being dried over MgSO4.Removal of the solvent by evaporation gave46as a colorless oil(7.08g,59%yield):1H NMR (300MHz,CDCl3)d3.09(t,J=8.1Hz,2H),2.63–2.58 (m,1H),2.40–2.23(m,1H),2.03–1.80(m,2H),0.22(s, 6H),0.15(s,3H).To a stirred solution of PhMgI in Et2O(2M,23.1mL,46.1mmol)was added dropwise a solution of46(7.08g,41.9mmol)in Et2O(50mL)at 0 C.Stirring was continued at room temperature for2 days.To this resulting mixture a solution of NaBH4 (1.71g,45.3mmol)in MeOH(50mL)was added at 0 C and stirring was continued at room temperature for 4.5h.Next H2O(25mL)and1M HCl(100mL)were added to the stirred reaction mixture.Stirring was con-tinued at room temperature for1h.After removal of the organic layer,the aqueous layer was adjusted to pH 9by1M NaOH and extracted with CHCl3.The organic layer was dried over K2CO3.Removal of the solvent by evaporation gave1-[amino(phenyl)methyl]cyclobutanol which was used for the next reaction without further puri?cation.The title compound47was prepared from 1-[amino(phenyl)methyl]cyclobutanol according to essentially the same procedure as described for the pre-paration of methyl[(tert-butoxycarbonyl)amino](3-hydroxyphenyl)acetate from41:13%yield in3steps; o?-white solid;TLC R f=0.37(n-hexane/EtOAc,3/1), MS(APCI,Pos.40eV)m/z278(M+H)+,178,161;1H NMR(300MHz,CDCl3)d7.40–7.20(m,5H),5.51(br, 1H),4.76(d,J=8.4Hz,1H),2.40–2.23(m,1H),2.20–2.00(m,2H),1.93–1.63(m,3H),1.43(s,9H).

1-(Phenylmethyl)-1-(octanoylamino)cyclobutyl dihydrogen phosphate(14).The title compound14was prepared from47according to the general method C:TLC R f=0.44(CHCl3/MeOH/H2O,65/25/4);IR(KBr)3252, 2930,2858,1557,1456,1010cmà1;1H NMR(300MHz,

T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053801

CD3OD)d7.45–7.38(m,2H),7.35–7.20(m,3H),5.15 (d,J=2.4Hz,1H),2.88(q,J=12Hz,1H),2.45(q, J=12Hz,1H),2.32–2.14(m)and2.27(t,J=6Hz)total 4H,1.94–1.80(m,1H),1.78–1.52(m,3H),1.40–1.16(m, 8H),0.88(t,J=6.6Hz,3H);MS(FAB,Pos.)m/z406 (M+Na)+,384(M+H)+;HRMS(MALDI-TOF, Pos.)calcd for C19H30NO5P+Na+:406.1759;found: 406.1731.

(2R,3R)-3-Amino-3-phenylpropane-1,2-diol hydrochloride (51).Amixture of[(2S,3S)-3-phenyloxiran-2-yl]metha-nol49(5.6g,37.3mmol),NaN3(4.86g,74.6mmol)and NH4Cl(991mg,17.8mmol)in dioxane(50mL)/H2O(5 mL)was heated at80 C for2days with stirring.After cooling,the reaction mixture was diluted with EtOAc. The organic layer was successively washed with H2O and brine before being dried over Na2SO4.Removal of the solvent by evaporation gave a residue which was puri?ed by column chromatography on silica gel (Merck7734,n-hexane/EtOAc,3/1–1/1)to a?ord50as a colorless oil(4.28g,59%yield):TLC R f=0.38(n-hexane/EtOAc,1/1);1H NMR(300MHz,CDCl3)d 7.50–7.35(m,5H),4.65(d,J=6.9Hz,1H),3.60(m, 1H),3.80–3.65(m,2H),2.19(br,1H),1.97(br,1H).A mixture of50(4.28g,22mmol)and10%Pd-C(430mg) in EtOH(60mL)/6M HCl(3.7mL)was stirred at room temperature under an atmospheric pressure of hydrogen for14h.Removal of the catalyst by?ltration through a pad of Celite followed by evaporation a?orded51as a white solid:1H NMR(300MHz,CD3OD)d7.54–7.46 (m,2H),7.45–7.39(m,3H),4.44(d,J=3.9Hz,1H), 4.08–4.00(m,1H),3.40(dd,J=11.0,5.5Hz,1H),3.28 (dd,J=11.0,6.0Hz,1H).

N-[(1R,2R)-2,3-dihydroxy-1-phenylpropyl]octanamide (52).The title compound52was prepared from51 according to essentially the same procedure as described for the preparation of24b from23b:white solid;83% yield;TLC R f=0.55(EtOAc);1H NMR(300MHz, CDCl3) 7.43–7.25(m,5H),6.16(d,J=7.2Hz,1H), 5.01(t,J=7.8Hz,1H),3.86(dt,J=7.8,3.0Hz,1H), 3.66(dq,J=12.6,3.0Hz,2H),2.21(t,J=7.5Hz,2H), 1.62(m,2H),1.40–1.10(m,8H),0.87(m,3H).

(1R,2R)-1-Hydroxymethyl-2-octanolyamino-2-phenylethyl disodiumphosphate(15).The title compound15was prepared from55according to the general method B:to a stirred mixture of52(1.3g,4.3mmol)and imidazole (589mg,8.7mmol)in DMF(20mL)was added TBDPSCl(1.4g,5.2mmol)at room temperature and stirring was continued at that temperature for18h.The reaction mixture was diluted with Et2O.The organic layer was successively washed with saturated NaHCO3 aq,H2O and brine before being dried over Na2SO4. Removal of the solvent by evaporation gave53quanti-tatively as a colorless oil(2.3g):TLC R f=0.35(n-hex-ane/EtOAc,2/1);1H NMR(300MHz,CDCl3)d7.73 (m,1H),7.60(m,3H),7.45–7.30(m,6H),7.28–7.20(m, 5H),6.70(d,J=8.1Hz,1H),5.19(dd,J=8.1,4.2Hz, 1H),3.99(dd,J=9.6,4.2Hz,1H),3.59(dd,J=10.8, 4.2Hz,1H),3.48(dd,J=10.8,5.7Hz,1H),2.18(t, J=7.2Hz,2H),1.60(m,2H),1.38–1.15(m,8H),1.09 (s,9H),0.87(t,J=6.6Hz,3H).The compound54was prepared from53according to the same procedure as described for the preparation of dibenzyl(1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl phosphate from24b:colorless oil;70%yield from53. 54:1H NMR(300MHz,CDCl3)d7.63–7.58(m,5H), 7.40–7.20(m,20H),5.49(dd,J=8.1,3.0Hz,1H),5.20–4.85(m,5H),4.65–4.55(m,1H),3.65–3.0(m,2H),2.20–2.10(m,2H),1.65–1.50(m,2H),1.38–1.20(m,8H),1.07 (s,9H),0.85(m,3H).(1R,2R)-1-tert-Butyldiphenylsily-loxymethyl-2-octanonyamino-2-phenylethyl dihydrogen phosphate was prepared from54according to the same procedure as described for the preparation of25b from dibenzyl(1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylamino)ethyl phosphate:TLC R f=0.50 (CHCl3/MeOH/H2O,65/25/4);1H NMR(300MHz, CDCl3)d7.70–7.60(m,4H),7.45–7.20(m,1H),5.50(d, J=3.3Hz,1H),4.63–4.58(m,1H),3.74(dd,J=10.5, 4.2Hz,1H),3.42(dd,J=10.5,7.8Hz,1H),2.24(t, J=7.2Hz,2H),1.65–1.53(m,2H),1.38–1.20(m,8H), 1.09(s,9H),0.89(t,J=6.3Hz,3H).To a stirred solu-tion of(1R,2R)-1-tert-butyldiphenylsilyloxymethyl-2-octanolyamino-2-phenylethyl dihydrogen phosphate (1.8g,3mmol)in THF(20mL)was added TBAF(1M in THF,7.5mL,7.5mmol)at room temperature and stirring was continued at that temperature for15min. The reaction was quenched with H2O and the pH adjusted to12with2M NaOH before the organic layer was extracted with Et2O.The aqueous layer was acid-i?ed with2M HCl and extracted with EtOAc.Next,the organic layer was successively washed with H2O and brine before being dried over Na2SO4.Removal of the solvent by evaporation gave55as an amorphous pow-der(700mg,63%yield):TLC R f=0.23(CHCl3/MeOH/ H2O,65/25/4);1H NMR(300MHz,CDCl3)d7.24–7.20 (m,5H),5.28(d,J=5.0Hz,1H),4.60–4.43(m,1H), 3.60–3.40(m,2H),2.28(t,J=7.5Hz,2H),1.60(m,2H), 1.40–1.20(m,8H),0.88(m,3H).15:TLC R f=0.23 (CHCl3/MeOH/H2O,65/25/4);IR(KBr)3418,2928, 2856,1644,1539,1455,1094cmà1;1H NMR(300MHz, CD3OD)d7.44(d,J=7.2Hz,2H),7.26–7.14(m,3H), 4.93(d,J=2.7Hz,1H),4.48(m,1H),3.38–3.25(m, 2H),2.36–2.18(m,2H),1.55(m,2H),1.38–1.18(m, 8H),0.86(t,J=6.3Hz,3H);MS(FAB,Pos.)m/z418 (M+H)+,396,374;HRMS(MALDI-TOF,Pos.)calcd for C17H26NO6P.2Na+H+:418.1371;found:418.1407. N-[(1R,2S)-2-Hydroxy-1,2-dipenylethyl]octanamide(57). The title compound57was prepared from(1S,2R)-2-amino-1,2-diphenylethanol56according to essentially the same procedure as described for the preparation of 24b from23b:white powder;84%yield;1H NMR (300MHz,CDCl3)d8.11(d,J=9.6Hz,1H),7.35–7.10 (m,10H),5.35(d,J=4.8Hz,1H),4.94(dd,J=9.4,7.8 Hz,1H),4.70(m,1H),1.93(t,J=7.0Hz,2H),1.34–0.90(m,10H),0.85(t,J=6.2Hz,3H).

Method D

(1S,2R)-1,2-Diphenyl-2-octanoylaminoethyl disodium phos-phate(16).To a stirred mixture of57(770mg, 2.3 mmol)and tetrazole(318mg,4.5mmol)in CH3CN(25 mL)–CH2Cl2(25mL)was added a solution of bis(2-cyanoethyl)diisopropylamidophosphite(678mg, 2.5

3802T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

mmol)in CH3CN(5mL)at room temperature and stirring was continued at that temperature for1.5h.The reaction mixture was poured into saturated NaHCO3aq and extracted with EtOAc.The organic layer was washed with brine and dried over Na2SO4.Removal of the solvent by evaporation gave bis(2-cyanoethyl) (1S,2R)-1,2-diphenyl-2-(octanoylamino)ethyl phosphite as a white powder which was used for the next reaction without further puri?cation(1.2g,quant):TLC R f=0.43(n-hexane/EtOAc,1/1).To a stirred solution of bis(2-cyanoethyl)(1S,2R)-1,2-diphenyl-2-(octanoyl-amino)ethyl phosphite(1.2g)in CH2Cl2(30mL)was added m-CPBA(57%,685mg,2.3mmol)at0 C and stirring was continued at that temperature for30min. To the reaction mixture saturated Na2S2O3aq was added and extracted with CH2Cl2.The organic layer was successively washed with H2O,saturated NaHCO3 aq and brine before being dried over Na2SO4.The m-chlorobenzoic acid was removed by passing the solution through a pad of Al2O3.Removal of the solvent by evaporation gave bis(2-cyanoethyl)(1S,2R)-1,2-diphe-nyl-2-(octanoylamino)ethyl phosphate as a white pow-der which was used for the next reaction without further puri?cation(1.0g,84%yield):TLC R f=0.20(CH2Cl2/ EtOAc,1/1).To a stirred solution of bis(2-cyanoethyl) (1S,2R)-1,2-diphenyl-2-(octanoylamino)ethyl phosphate (1.0g,1.9mmol)in EtOH(20mL)was added50% Me2NH aq and stirring was continued under re?ux for8 h.After cooling,removal of the solvent by evaporation gave a residue,which was dissolved in1M NaOH and extracted with Et2O.The aqueous layer was acidi?ed with1M HCl and extracted with EtOAc.The organic layer was successively washed with H2O and brine before being dried over Na2SO4.Removal of the solvent by evaporation gave(1S,2R)-1,2-diphenyl-2-(octanoyla-mino)ethyl dihydrogen phosphate as a white solid(641 mg,80%yield),which was converted to the disodium salt16according to the same procedure as descried for the preparation of2from25b:TLC R f=0.41(CHCl3/ MeOH/H2O,65/25/4);IR(KBr)3062,2927,1652,1520, 1453,1114cmà1;1H NMR(300MHz,CD3OD)d7.20–7.05(m)and7.00(m)total10H,5.68(dd,J=10.2,2.4 Hz,1H),4.96(d,J=2.4Hz,1H),2.40–2.30(m,2H), 1.65–1.55(m,2H),1.40–1.20(m,8H),0.88(t,J=6.3 Hz,3H);MS(FAB,Pos.)m/z486(M+Na)+,464 (M+H)+,442;HRMS(MALDI-TOF,Pos.)calcd for C22H28NO5P.2Na+H+:464.1579;found:464.1557.

N-[(1R,2S)-2-Hydroxy-2,3-dihydro-1H-inden-1-yl]octa na-mide(59).The title compound59was prepared from (1R,2S)-1-aminoindan-2-ol58according to essentially the same procedure as described for the preparation of 24b from23b:86%yield;TLC R f=0.13(n-hexane/ EtOAc,3/2);1H NMR(300MHz,CDCl3)d7.30–7.10 (m,4H),6.20(brd,J=8.4Hz,1H),5.37(dd,J=8.4,5.1 Hz,1H),4.61(ddd,J=5.1,5.1,2.4Hz,1H),3.16(dd, J=16.5,5.1Hz,1H),2.93(dd,J=16.5,2.4Hz,1H), 2.31–2.26(m,2H), 1.70–1.50(m,2H), 1.40–1.20(m, 8H),0.88(brt,J=6.9Hz,3H).

(1R,2S)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl bis(2,2,2-trichloroethyl)phosphate(60).The title com-pound60was prepared from59according to essentially the same procedure as described for the preparation of 34from33:50%yield;1H NMR(200MHz,CDCl3)d 7.30–7.20(m,4H),6.06(brd,J=9.0Hz,1H),5.75–5.67 (m,1H),5.40–5.33(m,1H),4.61(d.J=7.0Hz,2H), 4.59(d,J=7.0Hz,2H),3.38(dd,J=17.6,1.6Hz,1H), 3.24(dd,J=17.6,4.4Hz,1H),2.32(dd,J=7.8,7.2Hz, 2H), 1.80–1.65(m,2H), 1.50–1.20(m,8H),0.89(t, J=6.6Hz,3H).

(1R,2S)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl disodiumphosphate(17).The title compound17was prepared from60according to the same procedures as described for the preparation of6from(1S,2S)-1-methyl-2-phenyl-2-(octanoylamino)ethyl bis(2,2,2-tri-chloroethyl)phosphate:60%yield;TLC R f=0.29 (CHCl3/MeOH/H2O,65/25/4);IR(KBr)3305,1626, 1542,1459,1106,986cmà1;1H NMR(300MHz, CD3OD)d7.25–7.05(m,4H),5.27(d,J=4.8Hz,1H), 5.01(m,1H),3.35(m,1H),3.03(dd,J=16.4,4.4Hz, 1H),2.50–2.25(m,2H),1.70(m,2H),1.50–1.20(m,8H), 0.90(m,3H).;MS(FAB,Pos.)m/z422(M+Na)+,400 (M+H)+;HRMS(MALDI-TOF,Pos.)calcd for C17H24NO5P.2Na+H+:400.1266;found:400.1301. Method E

Dibenzyl(1S,2R)-1-(octanoylamino)-2,3-dihydro-1H-in-den-2-yl phosphate(61).To a stirred mixture of59(1.1 g, 4.0mmol),Ph3P(1.3g, 4.8mmol)and dibenzyl hydrogen phosphate(1.3g,4.8mmol)in THF(12mL) was added DEAD(40%in toluene,2.1mL,4.8mmol) at0 C.Stirring was continued at room temperature for 24h.Removal of the solvent by evaporation gave a residue which was puri?ed by column chromatography on silica gel(FL60D,n-hexane/EtOAc,5/1–4/1)twice to a?ord61(260mg,12%):TLC R f=0.55(CH2Cl2/ MeOH,19/1);1H NMR(300MHz,CDCl3)d7.40–7.15 (m,14H),5.89(d,J=8.1Hz,1H),5.53(dd,J=8.1,7.5 Hz,1H),5.10–5.01(m,4H),4.92(m,1H),3.22(dd, J=15.6,6.9Hz,1H),3.01(dd,J=15.6,7.8Hz,1H), 2.17(dd,J=8.1,7.2Hz,2H),1.80–1.55(m,2H),1.40–1.20(m,8H),0.87(t,J=6.9Hz,3H).

(1S,2R)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl dihydrogen phosphate(62).The title compound62was prepared from61according to the same procedure as described for the preparation of25b from dibenzyl (1S,2R)-1-methyl-2-(3-methoxyphenyl)-2-(octanoylami-no)ethyl phosphate:83%yield;1H NMR(200MHz, CD3OD)d7.30–7.10(m,4H),5.39(brd,J=5.8Hz, 1H),4.85(m,1H),3.40(dd,J=16.0,7.0Hz,1H),3.04(dd, J=16.0,6.6Hz,1H),2.26(brt,J=7.4Hz,2H),1.70–1.55 (m,2H),1.45–1.20(m,8H),0.90(brt,J=6.6Hz,3H). (1S,2R)-1-(Octanoylamino)-2,3-dihydro-1H-inden-2-yl disodiumphosphate(18).The title compound18was prepared from62according to the same procedure as described for the preparation of2from25b:87%yield; TLC R f=0.29(CHCl3/MeOH/H2O,65/25/4);IR(KBr) 3430,1631,1543,1461,1096,987cmà1;1H NMR (200MHz,CD3OD)d7.14(m,4H),5.16(d,J=7.6Hz, 1H), 4.67(ddd,J=8.0,7.6,7.0Hz,1H), 3.41(dd, J=15.8,7.0Hz,1H),2.94(dd,J=15.8,8.0Hz,1H),

T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053803

2.25(m,2H),1.68(m,2H),1.45–1.20(m,8H),0.89(m, 3H);MS(FAB,Pos.)m/z422(M+Na)+,400 (M+H)+;HRMS(MALDI-TOF,Pos.)calcd for C17H24NO5P.2Na+H+:400.1266;found:400.1275.

N-[(1R)-2-hydroxy-1-phenylethyl]-N-methyloctanamide (64).Ethyl(1R)-2-hydroxy-1-phenylethylcarbamate was prepared from(2R)-2-amino-2-phenylethanol63 according to essentially the same procedures as descri-bed for the preparation of24b from23b.To the stirred suspension of LiAlH4(760mg,20mmol)in THF(50 mL)was added dropwise a solution of ethyl(1R)-2-hydroxy-1-phenylethylcarbamate(2.1g,10mmol)in THF(10mL)under re?ux.Stirring was continued for 11h under re?ux.To this stirred reaction mixture H2O (0.76mL),15%NaOH aq(0.76mL)and H2O(2.2mL) were successively added at0 C and the vigorous stirring was continued.The precipitates were removed by?ltra-tion and the?ltrate was concentrated to give a residue, which was dissolved in dioxane(5mL).To the mixture was added4M HCl in dioxane(3mL)and removal of the solvent by evaporation gave a residue,which was solidi?ed by Et2O/EtOH to a?ord(2R)-2-(methylamino)-2-phenyl-ethanol(665mg,36%yield):TLC R f=0.19(CH2Cl2/ MeOH/AcOH,3/1/1).The title compound64was pre-pared from(2R)-2-(methylamino)-2-phenylethanol according to essentially the same procedure as described for the preparation of24b from23b:67%yield:TLC R f=0.26(n-hexane/EtOAc,1/1);1H NMR(200MHz, CDCl3)d7.40–7.15(m,5H),5.85,5.14(dd,J=9.4,5.0 Hz,1H),4.23–4.00(m,2H),2.75(s,3H),2.56–2.35(m, 3H),1.75–1.60(m,2H),1.45–1.20(m,8H),0.85(m,3H). (2R)-2-[hexanoyl(methyl)amino]-2-phenylethyl dihydrogen phosphate(19).The title compound19was prepared from64according to the general method A:17%yield; TLC R f=0.23(CHCl3/MeOH/H2O,65/25/4);IR(KBr) 3399,1618,1451,1406,1096,987cmà1;1H NMR (300MHz,CD3OD,2rotamers)d7.34–7.20(m,5H), 5.94(dd,J=9.0,6.0Hz,0.55H),5.32(dd,J=9.0,5.1 Hz,0.45H),4.41–4.13(m,2H),2.93(s,1.65H),2.88(s, 1.35H),2.60–2.35(m,2H),1.60(m,2H),1.45–1.20(m, 8H),0.89(m,3H);MS(FAB,Pos.)m/z402(M+H)+; HRMS(MALDI-TOF,Pos.)calcd for C17H26NO5P.2-Na+H+:402.1422;found:402.1430.

Biological assay method

Inhibition of LPS-induced plasma TNF- production in rats(iv).LPS from the Escherichia coli strain055B5 (Difco laboratories)and the test compounds were all dissolved in saline.Male Sprague–Dawley(CD)/IGS rats(Charles River Inc.,Japan)aged6–8weeks(n=5) were injected intravenously with the test compounds (0.01–0.1mg/10mL/kg),and then immediately given an intraperitoneal injection of LPS(30m g/kg).Plasma TNF-a production was determined by ELISA90min after the LPS challenge using a commercial kit(R&D Systems).ID50Values were determined by log-linear regression analysis(3–4doses per compound). ID50=The dosage required to inhibit plasma TNF-a production by50%.The data were expressed as the mean?SEM of5animals per group or ID50values.

%Inhibition=100à(CàS)/(LàS)?100

C:Plasma TNF-a concentration of LPS-treated ani-

mals pretreated with a test compound.

L:Plasma TNF-a concentration of LPS-treated ani-mals pretreated with saline.

S:Plasma TNF-a concentration of saline-treated ani-

mals also pretreated with saline.

Inhibition of LPS-induced plasma TNF- production in rats(po).The experiments were performed using male

Sprague–Dawley(CD(IGS))rats,6–8weeks of age,

purchased from Charles River Breeding Laboratories

(Shizuoka,Japan).The animals were given access to food and water ad libitum and were maintained on12h

light/dark cycle at22–23 C.All experimental proce-

dures were conformed to the Animal Care and Use Committee protocols?led at ONO Pharmaceutical Co.,

Ltd.(Osaka,Japan).In experiments under fasted con-

dition,rats were fasted for about16h.Test compounds and LPS from Escherichia coli strain055B5(DIFCO

LABORATORIES,Detroit,MI,USA)were dissolved

in bde86c05caaedd3383c4d3d3pounds were administrated orally(1–100 mg/kg)to rats30min prior to intravenous injection of

LPS at the dose of30m g/kg.After90min of LPS

injection,heparinized blood was obtained.Blood was

centrifuged and plasma samples were kept frozen at à80 C.Plasma TNF-a concentration was determined by enzyme-linked immunosorbent assay using a com-

mercial kit(BIOSOURCE international inc.,CA,USA) according to the manufacturer’s instructions.The data

were expressed as the mean?SEM of5animals per

group or ID50values.ID50values,which describe the e?ective dose with50%inhibition of TNF-a produc-

tion,were determined by log-linear regression analysis

(3–4doses per compound).

Inhibition of LPS-induced plasma TNF- production in

mice(iv).Male BALB/c mice aged8weeks(n=5)were

injected intravenously with the test compounds(0.01–0.1mg/10mL/kg),and then immediately given an intraperitoneal injection of LPS(5mg/10mL/kg). Plasma TNF-a production was determined by ELISA 90min after the LPS challenge using a commercial kit (GENZYME).ID50=The dosage required to inhibit plasma TNF-a production by50%.The data were expressed as the mean?SEM of5animals per group or ID50values.

%Inhibition=100à(CàS)/(LàS)?100

C:Plasma TNF-a concentration of LPS-treated ani-

mals pretreated with a test compound.

L:Plasma TNF-a concentration of LPS-treated ani-mals pretreated with saline.

S:Plasma TNF-a concentration of saline-treated ani-

mals also pretreated with saline.

LPS-induced shock model in mice.LPS from Escherichia

coli strain055B5(Difco Laboratories,Detroit,MI, USA)and the test compounds were all dissolved in saline. Female BALB/c mice(Charles River Inc.,Shizuoka, Japan),7weeks of age,were injected intravenously with the test compounds and then immediately given an

3804T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–3805

intraperitoneal injection of LPS(20mg/kg).The survi-val rate of the mice was evaluated after96h.Pre-donisolone(Shionogi Pharmaceutical Co.,Ltd.,10mg/ kg,iv),which was used as the positive control,demon-strated an e?cacy(survival rate:13/20)equivalent to4 (survival rate15/20at0.3mg/kg,iv)in this model.The survival rate of the controls which were given saline was 2/20.Results are expressed as the mean?SEM.Para-meters were analyzed with ANOVA-Dunnett’s t-test, but survival was analyzed with the Mantel-Cox test.

D-(+)-Galactosamine/LPS-induced hepatitis model in rats.d-(+)-Galactosamine(SIGMA)/LPS(Difco Laboratories,Detroit,MI,USA)and the test com-pounds were all dissolved in saline.Male Sprague–Dawley rats(Charles River Inc.,Shizuoka,Japan),6 weeks of age,were injected intravenously with the test compounds and then immediately given an intraper-itoneal injection of d-(+)-galactosamine/LPS(1g/7.5 m g/5mL/kg).The survival rate of the rats was eval-uated after96h.Predonisolone(Shionogi Pharmaceu-tical Co.Ltd.,10mg/kg,iv),which was used as the positive control,demonstrated an e?cacy(survival rate: 13/18)equivalent to4(survival rate:12/18at0.3mg/kg, iv)in this model.The survival rate of the controls which were given saline was0/18.Results are expressed as the mean?SEM.Parameters were analyzed with ANOVA-Dunnett’s t-test,but survival was analyzed with the Mantel-Cox test.

References and Notes

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2.(a)Matsui,T.;Kondo,T.;Nishita,Y.;Itadani,S.;Naka-tani,S.;Omawari,N.;Sakai,M.;Nakazawa,S.;Ogata,A.; Ohno,H.;Obata,T.;Nakai,H.;Toda,M.Bioorg.Med. Chem.Lett.2002,12,90

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13.See Experimental.

T.Matsui et al./Bioorg.Med.Chem.10(2002)3787–38053805

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