AU2003262349B2 - Substituted amino acids as erythropoietin mimetics - Google Patents

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Actual Inventors: Address for Service:

CCN:

Invention Title: Ortho-McNeil Pharmaceutical, Inc.

William V. Murray and Peter Connolly Baldwin Shelston Waters MARGARET STREET SYDNEY NSW 2000 3710000352 SUBSTITUTED AMINO ACIDS AS ERYTHROPOIETIN MIMETICS Details of Original Application No. 36540/99 dated 19 April 1999 The following statement is a full description of this invention, including the best method of performing it known to us:- File: 29748AUP01 500261461 1.DOC/5844

I

-1A- TITLE OF THE INVENTION SUBSTITUTED AMINO ACIDS AS ERYTHROPOIETIN MIMETICS The present application is a divisional application of Australian Application No.

36540/99, which is incorporated in its entirety herein by reference.

This invention relates to a series of small molecules which bind to the erythropoietin receptor and compete with the natural ligand for binding to said receptor.

The invention includes pharmaceutical compositions containing these mimetics, their methods of production as well as intermediates used in their synthesis.

Erythropoietin (EPO) is a glycoprotein hormone which is produced in the mammalian kidney has a molecular weight of about 34,000 daltons. Its primary role is stimulation of mitotic cell division and differentiation of erythrocyte precursor cells. As a result this hormone regulates the production of erythrocytes, the hemoglobin contained therein and the blood's ability to carry oxygen. The commercial product Epogen is used in the treatment of anemia. This drug is produced by recombinant techniques and formulated in aqueous isotonic sodium chloride/sodium citrate. Even though it has been used successfully in the treatment of anemia, it is a costly drug that is administered intravenously. This method of administration is both costly and inconvenient for the patient; therefore it would be desirable to find a EPO mimetic which has the potential for oral activity.

A small molecule EPO mimetic has advantages over the natural protein. The immune response associated with large peptides is unlikely to occur with small molecules. In addition, the variety of pharmaceutical formulations that may be used with small molecules are technically unfeasible for proteins. Thus the use of relatively inert formulations for small molecules is possible. The most important advantage of small molecules is their potential for oral activity. Such an agent would ease administration, cost less and facilitate patient compliance.

Although compounds which mimic EPO are useful in stimulating red blood cell synthesis, there are diseases where the overproduction of red blood cells is a problem.

Erythroleukemia and polysythemia vera are examples of such diseases. Since EPO is an agent -2responsible for the maturation of red blood cell precursors, an antagonist of EPO would have utility treating either of those diseases.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

SUMMARY OF THE INVENTION The disclosed invention is drawn to a series of small molecules which demonstrate competitive binding with the natural ligand for the EPO receptor. As such these compounds are potentially useful in the treatment of diseases or conditions associated with this receptor.

In addition, the invention contemplates methods of producing these compounds and intermediates used in their production.

A first aspect of the invention provides a compound of Formula I when used as a medicament

R

2 4

N

R,'N 'Z n 1 wherein: R1 is the side chain of a natural or unnatural o-amino acid, where if said side chain contains a protectable group, that group may be protected with a member of the group consisting of succinyl, glutaryl, 3,3dimethylglutaryl, Ci.-alkyl, C 1 .salkoxycarbonyl, acetyl, N-(9fluorenylmethoxycarbonyl), trifluoroacetyl, omegacarboxyCi.salkylcarbonyl, t-butoxycarbonyl, benzyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido, t-butyl, cinnamoyl, trityl, 4-methyltrityl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and -3substituted phenylureido (where the phenyl substituents are selected from phenoxy, halo, or CIS 5 alkoxycarbonyl) group;

R

2 and R 3 and R 4 and R 5 independently may be taken together to form a six-membered aromatic ring which is fused to the depicted ring, or are independently selected from the group consisting of hydrogen, INDalkyl, CI-5 alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino, phenyl, phenoxy, phenylCI.

5 alkyl, phenyl Ci.salkoxy, substituted phenyl, substituted phenoxy, substituted phenyl Cl-salkyl or substituted C.S 5 alkoxy, (where the substituents are selected from

C

1 salkyl, Cl-s alkoxy, hydroxy, halo, trifluoromethyl, nitro, nitrile, and amino), and substituted amino (where the substituents are selected from one or more members of the group consisting of Cv-salkyl, halosubstitutedC -salkyl, CI-salknyl, Cl-salkenyl, phenyl, phenylCl-salkyl, C 1 -salkylcarbonyl, halo substituted C-s 5 alkylcarbonyl, carboxyCl-.salkyl, Cl-salkoxyCl.salkyl, cinnamoyl, naphthylcarbonyl, furylcarbonyl, pyridylcarbonyl, C 1 .salkylsulfonyl, phenylcarbonyl, phenylC l-salkylcarbonyl, phenylsulfonyl, phenylC 1 -salkylsulfonyl, substituted phenylcarbonyl, substituted phenylC 1 -salkylcarbonyl, substituted phenylsulfonyl, substituted phenylCI.salkylsulfonyl, substituted phenyl, and substituted phenylC 1 -salkyl[where the aromatic phenyl, phenylCI.

5 alkyl, phenylcarbonyl, phenylCi- salkylcarbonyl, phenylsulfonyl, and phenylC salkylsulfonyl substitutents are independently selected from one to five members of the group consisting of CIsalkyl, C 1 .salkoxy, hydroxy, halogen, trifluoromethyl, nitro, nitrile, and amino]); W and Q are independently selected from the group consisting of-CH=CH-, and -CH=N-; X and Y are independently selected from the group consisting of carbonyl,

C

1 .salkyl, Cl 5 alkenyl, C 1

.S

5 alkenylcarbonyl, and (CH 2 where m is n is1, 2, or 3; Z is selected from the group consisting of hydroxy, C 1 5 alkoxy, phenoxy, phenylCI- 5 alkoxy, amino, C 1 5 alkylamino, phenylamino, phenylCI 5 alkylamino, piperidin- l-yl, substituted piperidin- 1l-yl (where the substituents are selected from the group consisting of

C

1 5 alkyl, C 1 5 alkoxy, halo, aminocarbonyl, C 1 5 alkoxycarbonyl, and oxo; substituted phenylCI 5 alkylamino (where the aromatic substitutents are selected from the group consisting Of C 1 5 alkyl, C 1 5 alkoxy, phenylC 1 5 alkenyloxy, hydroxy, halogen, trifluoromethyl, nitro, nitrite, and amino), substituted phenoxy (where the aromatic substitutents are selected from the group consisting Of C 1 5 alkyl, C 1 5 alkoxy, hydroxy, halogen, trifluoromethyl, nitro, nitrite, and amino), substituted phenylC 1 5 alkoxy (where the aromatic substitutents are selected from the group consisting Of C 1 5 alkyl, CI-5alkoxy, hydroxy, halogen, trifluoromethyl, nitro, nitrile, and amino),

-OCH

2

CH

2

(OCH

2

CH

2

)'OCH

2

CH

2

O-,

-NHCH

2

CH

2

(OCH

2

CH

2

),OCH

2

CH

2

NH-,

-NH(CH

2 )pO(CH 2 )qO(CH 2 )pNH-, -NH(CH 2 )qNCH 3

(CH

2 -N1-(CH 2 and (NH(CH 2

)J)

3 N, where s, p, and q are independently selected from 1-7 with the proviso that if n is 2, Z is not hydroxy, C 1 5 alkoxy, amino,

C

1 5 alkylamino, di 1 5 alkylamino, phenylamino, phenylCI- 5 alkylamino, or piperidin- l-yl with the further proviso that if n is 3, Z is (N1-(CH 2 3

N

and salts thereof.

According to a second aspect, the present invention provides a compound when used as a medicament selected from the group consisting of 0 N- NuPN O-CH 2

(CH

2 4

H

H

2 N 2 PhCH 2 0 0 N N

-'CH

2

(CH

2 4

H

HN

HOO 2 O ,and N N- O.CH 2

(CH

2 4

H

O HN HO O 2 and salts thereof.

According to a third aspect, the present invention provides a pharmaceutical composition comprising the compound of the first or second aspect.

According to a fourth aspect, the present invention provides a method for modulating EPO receptor, comprising contacting the EPO receptor with an EPO receptor modulating amount of the compound of the first or second aspect.

According to a fifth aspect, the present invention provides a method for treating a disease or condition mediated by EPO receptor comprising administering an effective amount of the compound of the first or second aspect.

-6- N According to a sixth aspect, the present invention provides use of a compound according to the first or second aspect in the manufacture of a medicament for treating a disease or condition mediated by EPO receptor.

i c n O Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an Sinclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense CK of "including, but not limited to".

SDETAILED DESCRIPTION OF THE INVENTION O The terms used in describing the invention are commonly used and known to those skilled in the art. "Independently" means that when there are more than one substituent, the substituents may be different. The term "alkyl" refers to straight, cyclic and branched-chain alkyl groups and "alkoxy" refers to O-alkyl where alkyl is as defined supra. "Cbz" refers to benzyloxycarbonyl. "Boc" refers to t-butoxycarbonyl and "Ts" refers to toluenesulfonyl. "DCC" refers to 1,3-dicyclohexylcarbodiimide, "DMAP" refers to 4-N',N-dimethylaminopyridine and "HOBT" refers to 1-hydroxybenzotriazole hydrate.

"Fmoc" refers to N-(9-fluorenylmethoxycarbonyl), "DABCO" refers to 1,4- Diazabicyclo[2.2.2]octane, "EDCI" refers to 1-(3-dimethylaminopropyl)-3ethylcarbodiimide, and "Dde" refers to 1-(4,4- 7 dimethyl-2,6-dioxocyclohexylidene)ethyl. The side chains of a-amino acids refer to the substituents of the stereogenic carbon of an ac-amino acid. For example if the amino acid is lysine, the side chain is I-aminobutan-4-yl. The term natural amino acid refers to the 20 aamino acids of the L configuration which are found in natural proteins. Unnatural a-amino acids include synthetic amino acids such as -aminoadipic acid, 4-aminobutanoic acid, 6aminohexanoic acid, -aminosuberic acid, 5 -aminopentanoic acid, p-aminophenylalanine, aminopimelic acid -carboxyglutamic acid, p-carboxyphenylalanine, carnitine, citrulline, -diaminopropionic acid, -diaminobutyric acid, homocitrulline, homoserine, and statine as well as D-configuration amino acids. The term "protectable group" refers to a hydroxy, amino, carboxy, carboxamide, guanidine, amidine or a thiol groups on an amino acid side. Compounds of the invention may be prepared by following general procedures known to those skilled in the art, and those set forth herein.

The compounds of the invention may be prepared by liquid phase organic synthesis techniques or by using amino acids which are bound to a number of known resins. The underlying chemistry, namely, acylation and alkylation reactions, peptide protection and deprotection reactions as well as peptide coupling reactions use similar conditions and reagents. The main distinction between the two methods is in the starting materials. While the starting materials for the liquid phase syntheses are the N-protected amino acids or the lower alkyl ester derivatives of the N-protected amino acids, the starting material for the resin syntheses are amino acids which are bound to resins by their carboxy terminuses.

General Procedure For The Solid-Phase Synthesis Of Symmetrical N ,N -Dis ubstituted Amino Acids Scheme 1.

An equivalent of an N-Fmoc-protected amino acid which is bound to a resin la is suspended in a suitable solvent such as DMF. This solvent is removed and the nitrogen protecting group (Fmoc) is removed by stirring the resin bound amino acid with an organic base, such as piperidine, and an addition portion of the solvent. A solution of -8about two to three equivalents of an appropriately substituted halide, I b, and a suitable base such DIEA is added to the resin bound amino acid and this mixture is shaken for 18- 36 h. The resulting mixture is washed with several portions of a suitable solvent and is suspended and shaken in an acidic solution, such as 50% TFA/CH 2 Cl 2 over several hours to cleave the acid from the resin and give the N-disubstituted amino acid 1c.

By varying the resin bound amino acid l a, one may obtain many of the compounds of the invention. The following resin bound amino acids may be used in Scheme I: alanine, N-g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine, methyltrityl)asparagine, aspartic acid -t-butyl ester), S-(trityl)cysteine, methyltrityl)glutamine, glutamic acid -t-butyl ester), glycine, N-imidazolyl- (trityl)histidine, isoleucine, leucine, N- (2-chlorobenzyloxycarbonyl)lysine, butoxycarbonyl)lysine, methionine, phenylalanine, proline, O-(t-butyl)serine, O-(tbutyl)threonine, N-indolyl-(t-butoxycarbonyl)tryptophan, O-(t-butyl)tyrosine, valine, alanine, -aminoadipic acid, 4-aminobutanoic acid,, 6-aminohexanoic acid, aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine, -aminopimelic acid -carboxyglutamic acid, p-carboxyphenylalanine, carnitine, citrulline, diaminopropionic acid, -diaminobutyric acid, homocitrulline, homoserine, and statine. In addition, the choice of and can be varied by using known halide derivatives of lb. For example using benzylchloride, 2-chloromethylthiophene, or 2chloromethylpyridine gives compounds of the invention where is -CH=CH-, or respectively. For variations in the use of 2-chloroethylphenyl, 3-chloro-lpropenylbenzene, or benzeneacetyl chloride as Ib, give compounds where Y is (CH 2 2

-CH=CH-CH

2 or -CH 2 respectively. Still further, Scheme 1 may be used to produce combinatorial mixtures of products. Using mixtures of resin bound amino acids, la, with only one lb produces said combinatorial mixtures. Alternatively, using one amino acid la with a mixture of lb as well as mixture of la with mixtures of lb gives a large range of combinatorial mixtures.

9 Scheme 1 1. piperidine, DMF 2. DIEA

R

2 R2 F R 3 H W (X)/BrorCI R 2 N lb FmocN Wang lR3 0

R

1 Resin S3. TFA, CHCI 2 W N OH la 1 C

R

Ic General Procedure For The Solid-Phase Synthesis Of Unsymmetrical N ,N -D i substituted Amino Acids.

Scheme 2, Step A An equivalent of an N-Fmoc-protected amino acid which is bound to a resin la is suspended in a suitable solvent such as DMF. This solvent is removed and the nitrogen protecting group (Fmoc) is removed by stirring the resin bound amino acid with an organic base, such as piperidine, and an addition portion of the solvent. Trimethyl orthoformate and an appropriately substituted aldehyde 2a (5 equivalents) is added and the mixture is shaken under N 2 overnight. This mixture is treated with a suspension of NaBH(OAc) 3 (5 equivalents) in CH 2 C1 2 and shaken under N 2 overnight. After filtration and washing with a suitable solvent, the resulting product, resin bound Nmonosubstituted amino acid 2b, is rinsed with a suitable solvent and its identity is confirmed by MS and or HPLC analysis after treatmet of a portion of the resin with

TFA/CH

2

CI

2 Scheme 2, Step B The resin 2b is suspended in an appropriate solvent such as DMF and is filtered.

The appropriately substituted alkyl or arylkyl halide, 2c, and an appropriate base such as DIEA are added with some additional solvent and the mixture is shaken under N 2 for 18- 36 h. The resin bound N ,N -disubstituted amino acid, 2d, is isolated from the suspension and the resin is cleaved with an acidic solution to give the free acid 2e.

10 Scheme 2 1. piperidine, DMF 2

R

2 2. (MeO) 3 CH, 3 H

(X*)-CHO

Fmoc- N Wang 2a

R

1 Resin SResin 3. NaBH(OAc) 3 CH2C1 2 la H

O

Wang

R

1 Resin 2b R 4 DIEA, R Br or CI

R

4 R 5 R(Y) Wang R

R

1 Resin 2d

AC

TFA, CH 2

CI

2

R

4

R

2< 0 R2

O

R

R

2e Scheme 3, Step C A resin bound amine, 2d, where R 4 is nitro, is suspended in a suitable solvent, such as DMF, and is filtered. This mixture is treated with SnCl 2 dihydrate in DMF and shaken under N 2 overnight. The solvent is removed and the resin is washed successive portions of a suitable solvent to give the resin bound compound 3a where R 4 is amino.

The resin is suspended in a suitable solvent and is combined with an organic base, such as pyridine an appropriately substituted carboxylic acid anhydride, acid chloride, or sulfonyl chloride. The mixture is shaken under N 2 overnight and is filtered to give the resin bound amino acid 3b. This material is treated with an acid and a suitable solvent to give the free amino acid 3b.

Scheme 3, Step D 11 The resin bound amine 3a is treated with TMOF and an appropriately substituted aldehyde 3c is added and the mixture is shaken under N 2 overnight. The resulting mixture is drained and treated with a suspension of NaBH(OAc) 3 in an appropriate solvent and this mixture is shaken under N 2 overnight. The resin bound 3-aralkylaminophenyl amino acid is identified my spectral techniques after clevage to give the free acid 3d as previously described.

Scheme 3, Step E Resin bound, 2d, where R' is (CH 2 4 NH(Dde) is mixed with a suitable solvent, such as DMF, and shaken with successive portions of 2% solution of hydrazine hydrate in DMF over about 30 min. The resin is filtered and treated with a suitable solvent and a cyclic anhydride derivative 3e, and a base such as DMAP and pyridine. This mixture is shaken under N 2 overnight and filtered to give the resin bound amine, 3f. This material is identified by spectral techniques after clevage to give the free acid 3f as previously described.

12 Scheme 3 2 0 R3 Wang R R 1 Resin R= NO 2 SnCI 2

DMF

Rl=(CH 2 4 NH(Dde) 1. 2% N 2

H

4

DMF

2. 0 0 3e 3. TFA, CH 2 01 2 1. R 6COCI, (R'CO) 2 0, or R 6 SO0 2 CI, pyridine 2. TEA, CH 2

CI

2 or SO 2 NH2 3 XN 0 Wang R3a

R

1 Resin 1. (MeO)3CH,

R

2. NaBH(OAc:) 3

CH

2

CI

2 3. TFA, CH 2

CI

2 Scheme 4, Step F.

13 Resin bound 2b, where R 2 is nitro is suspended in CH 2 C1 2 and is treated with an organic base, such as pyridine, and 9-fluorenylmethoxy chloride. This mixture is shaken under N 2 overnight, filtered and resuspended in a suitable solvent. This mixture is treated with SnCI 2 dihydrate in DMF and shaken under N 2 overnight. The solvent is removed and the resin is washed successive portions of a suitable solvent and filtered to give the resin bound compound 4a where R 2 is amino. The resin 4a is then suspended in a suitable solvent, such as CH 2 C1 2 and is combined with 0.4 mmol of pyridine and 0.25-0.4 mmol of the appropriately substituted carboxylic acid anhydride, acid chloride, or sulfonyl chloride. The mixture is shaken under N 2 overnight, filtered, and washed successively with three portions each of CH 2 C12 and MeOH. This resin is suspended in DMF, filtered, and shaken under N 2 with 5 mL of a 40% solution of piperidine in DMF. After 1 h, the solvent is drained and the resin was washed successively with three portions each of suitable solvents to give the resin bound 4b. The identity of the compound was confirmed by spectral analysis after cleveage as previously described.

Scheme 4 H 0 R 2 =4-NO 2 H Fmoc 0 R2H H2N X N Wang HWang Wang 1. Fmoc-CI, pyridine, CH 2

CI

2 3 Wang R I Resin 1Resin 2b R Resin 2. SnCI 2 DMF 4a Resin 1. R'COCI, (R 6

CO)

2 0, or R 6

SO

2 CI, pyridine 2. piperidine, DMF

R

6 -(CO or SO 2 HNC-- H S(X) N Wang R R 1 Resin 4b Scheme 14 The resin 2b (0.2 mmol) is suspended in CH 2 C1 2 filtered, and is resuspended in

CH

2

C

2 This suspension is treated with diethyl phosphonoacetic acid and diisopropylcarbodiimide or other suitable carbodiimide reagent, and the mixture is shaken under N 2 overnight. The solvent is drained and the resulting resin 5a was washed successively with three portions each of CH 2 C1 2 and MeOH. The resin is suspended in DMF and filtered. A solution of the appropriately substituted aldehyde 5b (0.6-1.0 mmol) in 3-5 mL of DMF, lithium bromide (0.6-1.0 mmol), and a suitable base such as DIEA or Et 3 N (0.6-1.0 mmol) is added and the mixture is shaken under N 2 overnight. The solvent is removed and the resin is washed successively with three portions each of DMF, CH 2

C

2 and MeOH. The identity of the resin bound substituted amino acid 5c was confirmed spectral techniques. The resin bound material may be treated with 50% TFA/CH 2 C12 over 1-1.5 h, to give the acid Scheme -o (EtO) 2

P'

O

(EtO) 2

POCH

2

CO

2 H, 0 SH diisopropylcarbodiimide R 2 YR H W N RX)N Wang R Wang R Resin Resin 2b

R

4 R4 1. DIEA, LiBr, DMF, R- 1E LCHO 5 2. TFA, CH 2

CI

2 R2 OH R.

RI

Scheme 6 15 To prepare compounds where n is 2 and Z is NH(CH 2 )sNH, products of Schemes may be used in Scheme 6. Treatment of two equivalents of the substituted amino acid Ic with an equivalent of the diamine 6a, in the presence of HOBT and a peptide coupling agent such as EDCI and a base such as DIEA at room temperature over 16 h gives the dimer 6b.

Scheme 6 R 2

RR

R

R

2 3 O

R

3

I

W (X)N OH H 2 N ,NH 2 '1

(CH

2 )s c

R

6a 6b H General Procedure For The Solution-Phase Synthesis Of Symmetrical N ,N -D i substituted Amino Acids Scheme 7, Step A A solution of of amino acid ester 7a, an appropriately substituted halide derivitive Ib, and an appropriate base such as DIEA, Na 2

CO

3 or Cs 2

CO

3 in a suitable solvent, such as DMF, is heated at 50-100 °C under N 2 overnight, or until the starting material is exhausted, to give a mixture of the di and mono-substituted amines, 7b and 7c respectively. If the side chains of R'contain acid cleavable protecting groups, those groups may be cleaved by treatment with 30-80% TFA/CH 2 C1 2 Esters 7b and 7c may be 16 independently converted to the corresponding acids 7d and 7e by hydrolysis with an appropriate base such as aqueous NaOH.

Scheme 7

O

H

2 N

OR

9

R'

DIEA,

R

2 R3 (X)B r lb R3 R3

W

aq. NaOH

R

2

R

2 W R3_^rn

O

W -"OH R1 7d

R

2 R3- I H W OR'

R

1 7c aq. NaOH

R

2 R 3" 0 (x W

OH

7e 7e General Procedure For The Solution-Phase Synthesis Of Unsymmetrical N ,N -D i substituted Amino Acids Scheme 8, Step A 17 A solution of 1 mmol of amino acid ester 8a (or the corresponding HCI salt and 1.1 mmol of DIEA) and 1-1.5 mmol of the appropriately substituted aldehyde 2a in 3-5 mL of trimethyl orthoformate was stirred at room temperature under N 2 overnight. The solution was either concentrated and used directly for the next reaction, or was partitioned between EtOAc and water, washed with brine, dried over Na 2

SO

4 and concentrated to give crude product, which was purified by MPLC to give mono-substituted product 8b.

Scheme 8, Step B Amino ester 8b was dissolved in DMF, combined with 1.1-1.5 mmol of the appropriately substituted chloride or bromide 2c, and heated at 50-100 °C overnight. The reaction mixture was cooled and partitioned between water and EtOAc. The organic layer was washed three times with water and once with brine, dried over Na 2

SO

4 and concentrated. The crude product was purified by MPLC to give pure 8c. For examples of 8c wherein the side chain R' contained an acid-cleavable protecting group such as tbutylcarbamate, t-butyl ester, or t-butyl ether, 8e was stirred in 30-80% TFA/CH 2

CI

2 for 1-3 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected form of 8c. For examples of 8c where R 9 was equal to t-butyl, 8c was stirred in 30-80% TFA/CH 2

C

2 for 1-3 h and treated as described above to give acid 8d. For examples of 8c where R 9 was equal to methyl, ethyl, or other primary or secondary alkyl esters, 8c was stirred with with 1-2 mmol of aqueous LiOH, NaOH, or KOH in MeOH, EtOH, or THF at 20-80 °C until TLC indicated the absence of 8c. The solution was acidified to pH 4-5 with aqueous citric acid or HCI and was extracted with

CH

2

C

2 or EtOAc. The organic solution was washed with brine, dried over Na 2

SO

4 and concentrated to give 8d.

Scheme 8, Step C For examples of amino acid ester 8c where R' (CH 2 4 NHBoc, 8c (1 mmol) was stirred in 30-80% TFA/CH 2

CI

2 for 1-3 h. The reaction mixture was concentrated to provide 8e as the TFA salt. Optionally, the TFA salt was dissolved in CH 2

CL

2 or EtOAc 18 and washed with aqueous NaOH or Na 2

CO

3 dried over Na 2

SO

4 and concentrated to give 8e as the free base.

Scheme 8, Step D A solution of 1 mmol of Se, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride 3e was stirred in CH 2

CI

2 or DMF under N 2 overnight. The resulting mixture was diluted with CH 2 C12 or EtOAc and washed with aqueous HC1, water, and brine, was dried over Na 2

SO

4 and concentrated to provide 8f. Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted carboxylic acid anhydride (R"CO) 2 0 or acid chloride R"CO C I was stirred in CH 2 C1 2 or DMF under N 2 overnight and worked up as above to provide 8g. Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted isocyanate R12NCO was stirred in CH 2 C2 or DMF under N 2 overnight and worked up as above to provide 8h.

Scheme 8.

19 1. piperidine, 1 2. (MeO) 3

CH,

H

0 Fmoc") OR9 3. NaBH(OAc) 3

CH

2

CI

2 DIEA, R 5 rX 2 0 R

R

3 N l 8c

R

RK~ 4 TFA, CH 2

CI

2

(R

9 t-Bu) NaOH or KOH (R 9 Me, Et) J R' (CH 2 4 NHBoc TFA, CH 2

CI

2 R

OR

3 (XN

O

NH

2 o- 0 0

(RO

3e R11COCI or

(R

11 C0) 2 0 R Y NH 8g R 12NCO R 12

I

0 20 Scheme 9, Step A For examples of 8c where R 5

NO

2 a solution of 1 mmol of 8c (where R 2

R

3

R

4 or) and 10-12 mmol of SnCl2 dihydrate was stirred in MeOH, EtOH, or DMF at 20-80 °C for 0.5-24 h under N 2 The solution was taken to room temperature and poured into aqueous Na 2

CO

3 with rapid stirring. The resulting mixture was extracted with EtOAc or

CH

2 C1 2 and the organic extracts were washed with brine, dried over Na 2

SO

4 and concentrated to give the aminophenyl product 9a, which was purified by MPLC or used without further purification.

Scheme 9, Step B A solution of 1 mmol of aminophenyl compound 9a and 1-1.5 mmol of the appropriately substituted aldehyde 2a in 3-5 mL of trimethyl orthoformate was stirred at room temperature under N 2 overnight. The solution was either concentrated and used directly for the next reaction, or was partitioned between EtOAc and water, washed with brine, dried over Na 2

SO

4 and concentrated to give crude product, which was purified by MPLC to give 9b. For examples of 9b wherein the side chain R' or R 9 contained an acidcleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, 9b was stirred in 30-80% TFA/CH 2 CI2 for 1-3 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected form of 9b.

Scheme 9, Step C A solution of 1 mmol of 3-aminophenyl compound 9a, 1.1-2 mmol of pyridine, and 1-1.5 mmol of the appropriately substituted acid chloride, acid anhydride, or sulfonyl chloride in 3-5 mL of CH 2

C

2 or CICH 2

CH

2 C1 was stirred at room temperature under N 2 overnight. The solution was partitioned between EtOAc and water, washed with water, saturated aqueous NaHCO 3 and brine, dried over Na 2

SO

4 and concentrated to give crude product which was optionally purified by MPLC to give amide or sulfonamide 9c. For examples of 9c wherein the side chain R' or R 9 contained an acid-cleavable protecting group such as t-butylcarbamate, t-butyl ester, or t-butyl ether, 9c was stirred in 30-80% 21

TFAICH

2

CI

2 for 1-3 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give the deprotected formn of 9c.

Scheme 9.

0 3 TXY

OR

9 Rc R 5

NO

2 SnCI 2

DMF

2 2 0

R

9a

R

1. (MeO) 3 CH, RZ 7

CHO

3c 2. NaBH(OAc) 3

CH

2

C'

2

HN

r OR 9

R

6

COCI,

(R

6

CO)

2 0, or R6 SO 2

CI,

pyridine R6 HN J6 or SO 2 X(Y) 0 R 3 7 (XN rI

OR

9

RR

9C General Procedure For The Solution-Phase Synthesis Of Symmetrical N ,N -D i substituted Amino Amides And Their Dimers and Trimers Scheme 10, Step A A solution of I mmol of N-Cbz-protected amino acid 10Oa and the appropriate amnine (ZH, 1 mmnol), diamine (ZH 2 0.5 mmol), or triamine (ZH 3 0.33 mmol), was treated with 1. 1 mmol of HOBt, 1. 1 mmol of DIEA, and 2 1, mniol of EDCI in 3-6 mL of C14 2 0 2 22 or DMF. [Alternatively, 1 mmol of the pentafluorophenyl ester or N-hydroxysuccinimide ester of 10a was mixed with the appropriate portion of amine diamine (ZH 2 or triamine (ZH 3 in 3-6 mL of DMF.] The solution was stirred at room temperature under

N

2 for 12-24 h, and EtOAc was added. The organic solution was washed with aqueous citric acid, water, saturated NaHCO 3 and brine, dried over Na 2

SO

4 and concentrated. The crude product was optionally purified by MPLC to afford amide Compound 10b was stirred in 30-80% TFA/CH 2 CI2 for 1-3 h. The reaction mixture was concentrated to provide the TFA salt which was dissolved in CH 2 C12 or EtOAc and washed with aqueous NaOH or Na 2

CO

3 dried over Na 2

SO

4 and concentrated to give as the free base.

Scheme 10, Step B A solution of 1 mmol of amino acid ester 10c (n 2.5-3 mmol of the appropriately substituted chloride or bromide 2c, and 2.5-3 mmol of an appropriate base such as DIEA, Na 2

CO

3 or Cs 2

CO

3 in 3-5 mL of DMF was heated at 50-100 OC under N 2 for 18-24 h. (For examples of 10c where n 2 or 3, the amounts of 2c and base were increased by two- or three-fold, respectively.) The reaction mixture was cooled and partitioned between water and EtOAc. The organic layer was washed three times with water and once with brine, dried over Na 2

SO

4 and concentrated. The crude product was purified by MPLC to give pure amide Alternatively, a solution of 1 mmol of amino acid ester 10c (n 2.5-3 mmol of the appropriately substituted aldehyde 2a, and 2.5-3 mmol of borane-pyridine complex in mL of DMF or EtOH was stirred at room temperature under N 2 for 3-5 days. (For examples of 10c where n 2 or 3, the amounts of 2c and borane-pyridine complex were increased by two- or three-fold, respectively.) The mixture was concentrated to dryness and was partitioned between water and CH 2 C1 2 washed with brine, dried over Na 2

SO

4 and concentrated. The crude product was purified by MPLC to give pure amide Scheme 10, Step C 23 For examples of 10d where R' CH 2

CH

2

CO

2 -t-Bu or CH 2

CO

2 -t-Bu, 10d was stirred in 30-80% TFA/CH 2 C1 2 for 1-24 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give acid Scheme 10, Step D For examples of 10d where R' is equal to (CH 2 4 NHBoc, 10d was stirred in

TFA/CH

2

CI

2 for 1-24 h. The reaction mixture was concentrated and optionally dissolved in HOAc and freeze-dried to give amine 10f as the TFA salt which was optionally dissolved in CH 2 C12 or EtOAc, washed with aqueous NaOH or Na 2 CO3, dried over Na 2

SO

4 and concentrated to give 1Of as the free base.

Scheme 10, Step E A solution of 1 mmol of 1Of, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride 3e was stirred in CH 2 C12 or DMF under N 2 overnight. The resulting mixture was diluted with CH 2

C

2 or EtOAc and washed with aqueous HCI, water, and brine, was dried over Na 2

SO

4 and concentrated to provide acid 10g. Alternatively, 1 mmol of 10f, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted carboxylic acid anhydride (R ICO) 2 0 or acid chloride R"COCI was stirred in CH 2 C2 or DMF under N 2 overnight and worked up as above to provide 10h. Alternatively, I mmol of 8e, 1-4 mmol of an appropriate base such as DIEA, and 1-2 mmol of the appropriately substituted isocyanate

R'

2 NCO was stirred in CH 2 C1 2 or DMF under N 2 overnight and worked up as above to provide Scheme 10. -2 24 H 0 Cbz', l OH ZH,, EDO!, HOBt, DIEA H 0 Pd-C, NH 4

HCO

2 Cbz'N, 11- or Pd-C, H 2

[H

2 N il zo DIEA,

R

3 NX Br or CI r BH 3 -Pyridine,

:-)CHO

Ri=CH 2

CO

2 -t-BU,

CH

2

CH

2 C0 2 -t-Bu TFA, CH 2

CI

2 R 2J R- 0 R/.3

.N

R

CH

2 )m H0 2 C Rl= (CH 2 4 NHBoc TFA, CH 2

CI

2 3e

R

1 'COCl or (Rl C0)20

R

1

NH

0 R 12NCO H I R1N

H

0 25 Although the claimed compounds are useful as competitive binders to the EPO receptor, -some- compounds are more active than others and are either preferred or particularly preferred.

26 The prefer-red compounds of the invention incl ude:

F

Br 0 0

F

NH

0 N

H

2 N 1 '0 HN l NH 6H 3

H

3 CO, IN0 2

H

H

3

C.

Iand 27 The particularly preferred "s are the side chain of lysine, ornithine, arginine, aspartic acid, glutamic acid, glutamine, cysteine, methionine, serine, and threonine.

The particularly preferred "R 2 and R 3 s are phenoxy, substituted phenoxy, benzyloxy, and substituted benzyloxy.

The particularly preferred "R 4 and R 5 s are phenoxy, substituted phenoxy, benzyloxy, and substituted benzyloxy.

The particularly preferred is -CH=CH- The particularly preferred is -CH=CH- The particularly preferred are Ci.salkenyl and CH 2 The particularly preferred are Ci 1 salkenyl and CH 2 The particularly preferred are 1 and 2.

The particularly preferred are hydroxy, methoxy, phenethylamino, substituted phenethylamino, and -NH(CH 2 2 0(CH 2 2 0(CH 2 2

NH-.

28 Pharmaceutically useful compositions the compounds of the present invention, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the compound of the present invention.

Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose'disorders in which modulation of EPO receptor-related activity is indicated. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, transdermal, oral and parenteral.

The term "chemical derivative" describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule.

Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the EPO receptor or its activity while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.

The present invention also has the objective of providing suitable topical, transdermal, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds according to this invention as the active ingredient for use in the 29 modulation of EPO receptors can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds or modulators can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by transdermal delivery or injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, transdermal, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. The compounds of the present invention may be delivered by a wide variety of mechanisms, including but not limited to, transdermal delivery, or injection by needle or needle-less injection means. An effective but non-toxic amount of the compound desired can be employed as an EPO receptor modulating agent.

The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per patient, per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. The dosages of the EPO receptor modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

Advantageously, compounds or modulators of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds or 30 modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

The dosage regimen utilizing the compounds or modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds or modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when 31 desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds or modulators of the present invention may also be coupled 32 with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds or modulators of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels, and other suitable polymers known to those skilled in the art.

For oral administration, the compounds or modulators may be administered in capsule, tablet, or bolus form or alternatively they can be mixed in the animals feed. The capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. These unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained. An inert ingredient is one that will not react with the compounds or modulators and which is non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection. The active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed. Alternatively the active ingredient may be mixed with an inert carrier and the resulting composition may then either be mixed with the feed or fed directly to the animal. Suitable inert carriers include corn meal, citrus meal, fermentation residues, soya grits, dried

I

33 grains and the like. The active ingredients are intimately mixed with these inert carriers by grinding, stirring, milling; or tumbling such that the final composition contains from 0.001 to 5% by weight of the active ingredient.

The compounds or modulators may alternatively be administered parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraruminal, intratracheal, or subcutaneous, either by needle or needle-less means.

The injectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier. Acceptable liquid carriers include the vegetable oils such as peanut oil, cotton seed oil, sesame oil and the like as well as organic solvents such as solketal, glycerol formal and the like. As an alternative, aqueous parenteral formulations may also be used. The vegetable oils are the preferred liquid carriers. The formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.

Topical application of the compounds or modulators is possible through the use of a liquid drench or a shampoo containing the instant compounds or modulators as an aqueous solution or suspension. These formulations generally contain a suspending agent such as bentonite and normally will also contain an antifoaming agent. Formulations containing from 0.005 to 10% by weight of the active ingredient are acceptable. Preferred formulations are those containing from 0.01 to 5% by weight of the instant compounds or modulators.

The compounds of Formula I may be used in pharmaceutical compositions to treat patients (humans and other mammals) with disorders or conditions associated with the production of erythropoietin or modulated by the EPO receptor. The compounds can be administered in the manner of the commercially available product or by any oral or 34 parenteral route (including but not limited to, intravenous, intraperitoneal, intramuscular, subcutaneous, dermal patch), where the preferred route is by injection. When the method of administration is intravenous infusion, compound of Formula I may be administered in a dose range of about 0.01 to 1 mg/kg/min. For oral administration, the dose range is about 0.1 to 100 mg/kg.

The pharmaceutical compositions can be prepared using conventional pharmaceutical excipients and compounding techniques. Oral dosage forms may be used and are elixirs, syrups, capsules, tablets and the like. Where the typical solid carrier is an inert substance such as lactose, starch, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, mannitol and the like; and typical liquid oral excipients include ethanol, glycerol, water and the like. All excipients may be mixed as needed with disintegrants, diluents, granulating agents, lubricants, binders and the like using conventional techniques known to those skilled in the art of preparing dosage forms.

Parenteral dosage forms may be prepared using water or another sterile carrier.

Typically the compounds of Formula I are isolated as the free base, however when possible pharmaceutically acceptable salts can be prepared. Examples of such salts include hydrobromic, hydroiodic, hydrochloric, perchloric, sulfuric, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic and saccharic.

In order to illustrate the invention the following examples are included. These examples do not limit the invention. They are only meant to suggest a method of practicing the invention. Those knowledgeable in chemical synthesis and the treatment of EPO related disorders may find other methods of practicing the invention. However those methods are deemed to be within the scope of this invention.

BIOLOGICAL EXAMPLES 35 The compounds of the invention were evaluated for the ability to compete with EPO in the following immobilized EPO receptor preparation (EBP-Ig, EPO binding protein-Ig).

EBP-Ig fusion protein (as disclosed in W097/27219 which is herein incorporated by reference) was purified by affinity chromatography from the conditioned media of NSO cells engineered to express a recombinant gene construct which functionally joined the N-terminal 225 amino acids of the human EPO receptor and an Ig heavy chain as described herein. The interaction of biotin and streptavidin is frequently employed to capture and effectively immobilize reagents useful in assay protocols and has been employed here as a simple method to capture and immobilize EBP-Ig. EBP-Ig is initially randomly modified with an amine reactive derivative of biotin to produce biotinylated- EBP-Ig. Use of streptavidin coated plates allows the capture of the biotinylated EBP-Ig on the surface of a scintillant impregnated coated well (Flash plates, NEN-DuPont). Upon binding of 25 I]EPO to the ligand binding domain, specific distance requirements are satisfied and the scintillant is induced to emit light in response to the energy emitted by the radioligand. Unbound radioligand does not produce a measurable signal because the energy from the radioactive decay is too distant from the scintillant. The amount of light produced was quantified to estimate the amount of ligand binding. The specific assay format was suitable for the multi-well plate capacity of a Packard TopCount Microplate Scintillation counter. Compounds which were capable of reducing the amount of detected signal through competitive binding with the radioligand were identified.

Biotinylated EBP-Ig was prepared as follows. EBP-Ig (3 mL, OD 28 0 2.9) was exchanged into 50 mM sodium bicarbonate, pH 8.5 using a Centriprep 10 ultrafiltration device. The final volume of the exchanged protein was 1.2 mL (OD 2 8 0 2.6, representing about 2 mg total protein). 10 L of a 4 mg/ml solution of NHS-LC-Biotin (Pierce) was added and the reaction mixture placed on ice in the dark for two hours. Unreacted biotin was removed by exchange of the reaction buffer into PBS in a Centriprep 10 device and the protein reagent aliquoted and stored at -70 oC.

36 Each individual binding well (200 L) contained final concentrations of 1 g/mL of biotinylated EBP-Ig, 0.5 nM of 25 I]EPO (NEN Research Products, Boston, 100 C i/ g) and 0-500 M of test compound (from a 10-50 mM stock in 100% DMSO).

All wells were adjusted to a final DMSO concentration of All assay points were performed in triplicate and with each experiment a standard curve for unlabelled EPO was performed at final concentration of 2000, 62, 15, 8, 4, and 0 nM. After all additions were made, the plate was covered with an adhesive top seal and placed in the dark at room temperature overnight. The next day all liquid was aspirated from the wells to limit analyte dependent quench of the signal, and the plates were counted on a Packard TOPCOUNT Microplate Scintillation Counter. Non-specific binding (NSB) was calculated as the mean CPM of the 2000 nM EPO wells and total binding (TB) as the mean of the wells with no added unlabelled EPO. Corrected total binding (CTB) was calculated as: TB NSB CTB. The concentration of test compound which reduced CTB to 50% was reported as the IC 50 Typically the ICso value for unlabelled EPO was ca. 2-7 nM and EMP1 was 0.1 M. Table 1 lists the average inhibition, and if determined the

IC

5 o and IC 30 values for compounds of Formula I, where the compound numbers refer to the compounds in the tables accompanying the preparative examples.

Inhibition of EPO binding to EBP-Ig Table 1 cpd %inh@ 50 M ic30, M* ic50, M* 11 70 nd nd 12 59 nd nd 14 30 nd nd 48 nd nd 77 52 30 82 32 nd nd 86 37 nd nd 100 34 nd nd 101 32 nd nd 104 78 10 37 105 70 25 107 78 30 42 108 81 23 36 110 54 6 112 59 2 114 37 10 nd 115 35 nd nd 116 32 nd nd 117 34 nd nd 118 36 2 119 34 nd nd 120 35 nd nd 121 45 6 nd 137 60 5 139 46 2 178 36 nd nd 179 30 nd nd 183 36 nd nd 184 53 10 nd 203 37 50 nd 211 62 20 220 45 30 221 48 10 222 56 5 nd 224 51 25 227 48 20 230 42 nd nd 231 36 nd nd 235 49 20 237 55 30 238 39 nd nd 239 46 8 243 75 2 18 244 66 1 28 246 79 10 247 47 7 18 248 56 7 249 72 7 250 78 7 38 251 49 10 261 51 1.5 2 262 93 1 263 88 1 264 89 1.5 8 265 65 1 6 266 82 1 4 267 83 2 6 268 40 nd nd 269 55 8 270 56 7 100 271 77 2 7 272 78 5 285 41 nd nd 286 46 35 287 36 nd nd *nd not determined PREPARATIVE EXAMPLES Unless otherwise noted, materials used in the examples were obtained from commercial suppliers and were used without further purification. Melting points were determined on a Thomas Hoover apparatus and are uncorrected. Proton nuclear magnetic resonance NMR) spectra were measured in the indicated solvent with tetramethylsilane (TMS) as the internal standard using a Bruker AC-300 NMR spectrometer. NMR chemical shifts are expressed in parts per million (ppm) downfield from internal TMS using the d scale. 1H NMR data are tabulated in order: multiplicity, (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), number of protons, coupling constant in Hertz). Electrospray (ES) mass spectra (MS) were determined on a Hewlett Packard Series 1090 LCMS Engine. Elemental analyses were performed by Quantitative Technologies, Inc. (QTI), PO Box 470, Salem Industrial Park, Bldg Whitehouse, NJ 08888-0470. Analytical thin layer chromatography (TLC) was done with Merck Silica 39 Gel 60 F 2 54 plates (250 micron). Medium pressure liquid chromatography (MPLC) was done with Merck Silica Gel 60 (230-400 mesh).

Example 1 N,N-bis(3-Phenoxycinnamyl)Glu(O-t-Bu)-OMe and N-(3-phenoxycinnamyl)Glu(O-t-Bu)- OMe A solution of 500 mg (1.97 mmol) of H-Glu(O-t-Bu)OMe-HC1, 997 mg (3.45 mmol) of 3-phenoxycinnamyl bromide (Jackson, W. Islip, P. Kneen, Pugh, A.; Wates, P. J. J.Med.Chem. 31 1988; 499-500), and 0.89 mL (5.1 mmol, 660 mg) of DIEA in 5 mL of DMF was stirred under N 2 at room temperature for 40 h. The mixture was partitioned between EtOAc and water and the organic layer was washed with water and brine. After drying over Na 2

SO

4 the organic solution was concentrated to give 1.24 g of orange oil. The crude residue was purified by MPLC using a solvent gradient ranging from 10-30% EtOAc/hexanes to give two products. The less polar product (235 mg, 19% based on starting amino acid), cpd 96, was isolated as a pale yellow oil; 'H NMR (CDC1 3 300 MHz) 1.39 9H), 2.0 2H), 2.33 (dt, 2H, J 2, 7 Hz), 3.24 (dd, 2H, J 8, 15 Hz), 3H), 3.69 3H), 6.13 2H), 6.47 2H, J 16 Hz), 6.86 (dd, 2H, J 1.5, 8 Hz), 7.0-7.4 (complex, 16H); MS m/z 634 The more polar product (422 mg, 50% based on starting amino acid), N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe, was isolated as a pale yellow oil; 'H NMR (CDC1 3 300 MHz) 1.42 9H), 1.9 2H), 2.35 2H, J 7.5 Hz), 3.2-3.4 (complex, 3H), 3.71 3H), 6.17 (dt, 1H, J 16, 6 Hz), 6.46 1H, J 16 Hz), 6.87 (dd, 1H, J 1.5, 8 Hz), 7.01 3H), 7.10 2H, J 7.5 Hz), 7.2-7.4 (complex, 3H); MS m/z 426 Anal. Calcd for C 25

H

31 N0 5

C,

70.57; H, 7.34; N, 3.29. Found: C, 70.29; H, 7.14; N, 3.08.

Example 2 N-(3-Phenoxycinnamyl)Glu-OMe 40 A solution of 95 mg (0.22 mmol) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe in 3 mL of 50% TFA/CH 2 C2 was stirred for 2 h at room temperature. The mixture was concentrated and the residue was dissolve in acetic acid and freeze-dried to give 117 mg of N-(3-phenoxycinnamyl)Glu-OMe as an off-white solid; 'H NMR (CD30D, 300 MHz) 2.3-2.7 (complex, 4H), 3.78 3H), 3.81 2H, J 7 Hz), 4.09 1H, J 5 Hz), 6.17 (dt, 1H, J 16, 7 Hz), 6.55 1H, J 16 Hz), 6.9 4H), 7.11 2H, J 7.5 Hz), 7.3 4H); MS m/z 370 209. Anal. Calcd for C 21

H

23 N0 5

-C

2

HF

3 0 2

C,

57.14; H, 5.00; N, 2.90. Found: C, 57.07; H, 5.02; N, 2.73.

Example 3 N,N-bis(3-Phenoxycinnamyl)Asp(O-t-Bu)-O-t-Bu cpd 106 A solution of 1.00 g (3.55 mmol) of Asp(O-t-Bu)-O-t-Bu-HCI, 2.05 g (7.1 mmol) of 3-phenoxycinnamyl bromide, and 1.86 mL (10.7 mmol, 1.38 g) of DIEA in 15 mL of DMF was heated under N 2 at 60 oC overnight. Additional 3-phenoxycinnamyl bromide g, 3.4 mmol) and DIEA (0.95 mL, 0.705 g, 5.4 mmol) were added and heating was continued for an additional 14 h. The mixture was cooled and partitioned between EtOAc and water. The organic layer was washed twice with water, once with brine, and was dried over Na2S0 4 The solution was concentrated to give 3.5 g of an amber oil which was purified by MPLC using a solvent gradient ranging from 2.5-3% EtOAc/hexanes to afford 1.21 g of cpd 106 as a pale yellow oil; 'H NMR (CDCl 3 300 MHz) 1.41 9H), 1.48 9H), 2.49 (dd, 1H, J 7.5, 15.5 Hz), 2.70 (dd, 1H, J 15.5 Hz), 3.26 (dd, 2H, J 7.5, 14.5 Hz), 3.47 (dd, 2H, J 4, 14.5 Hz), 3.88 1H, J 6.13 2H), 6.48 (d, 2H, J 16 Hz), 6.86 (dd, 2H, J 2, 8 Hz), 7.0 6H), 7.1 4H), 7.2-7.4 (complex, 6H); MS m/z 662 Example 4 N,N-bis(3-Phenoxycinnamyl)Asp-OH 41 cpd 107 A solution of 1.14 g 1.62 mmol) of cpd 106 in 16 mL of 50% TFA/CH 2

C

2 was stirred at room temperature for 24 h. The solution was concentrated and pumped to give 1.37 g cpd 107 as an amber oil; 'H NMR (CD 3 OD, 300 MHz) 3.1 2H), (dd, 2H, J 8, 14 Hz), 4.27 (dd, 2H, J 8, 16 Hz), 4.70 1H, J 4 Hz), 6.38 (2H, dt, J= 16, 8 Hz), 6.7-7.4 (complex, 20H); MS m/z 562 Example N,N-bis(4-Benzyloxybenzyl)Lys(Boc)-OMe (cpd 111) and N-(4-Benzyloxybenzyl)Lys(Boc)-OMe A solution of 594 mg (2.0 mmol) of Lys(Boc)-OMe*HCI, 524 mg (2.25 mmol) of 4-benzyloxybenzyl chloride, 75 mg (0.5 mmol), of NaI, and 0.61 mL (3.5 mol, 452 mg) of DIEA was warmed at 50-70 °C under N 2 overnight. The mixture was cooled and partioned between EtOAc and water. The organic layer was washed twice with water, once with brine, and was dried over Na 2

SO

4 The organic solution was concentrated to give 0.83 g of amber oil which was purified by MPLC using a solvent gradient ranging from 15-40% EtOAc/hexanes to give two products. The less polar product (296 mg), cpd 111, was isolated as a pale yellow oil; 'H NMR (CDC1 3 300 MHz) 1.28 4H), 1.43 (s, 9H), 1.70 2H), 3.03 2H), 3.28 1H, J 7 Hz), 3.40 2H, J 13.5 Hz), 3.74 (s, 3H), 3.81 2H, J 13.5 Hz), 5.05 4H), 6.92 4H, J 7.23 4H, J 7.25-7.5 (complex, 10H); MS m/z 653 The more polar product (406 mg), N-(4-Benzyloxybenzyl)Lys(Boc)-OMe, was isolated as a white solid; 'H NMR (CDC 3 300 MHz) 1.4 4H), 1.43 9H), 1.65 3H), 3.08 2H), 3.23 1H, J 6.5 Hz), 3.54 1H, J 12.5 Hz), 3.71 3H), 3.73 1H, J 12.5 Hz), 5.05 2H), 6.92 2H, J 8.5 Hz), 7.23 2H, J 8.5 Hz), 7.25-7.5 (complex, 5H); MS m/z 457 Example 6 N-(4-Benzyloxybenzyl)-N-(3-nitrobenzyl)Lys(Boc)-OMe 42 cpd 113 A solution of 374 mg (0.82 mmol) of N-(4-Benzyloxybenzyl)Lys(Boc)-OMe, 221 mg (1.03 mmol) of 4-nitrobenzyl bromide, and 197 L (1.13 mmol, 146 mg) of DIEA was warmed at 50-70 °C for 4 h, then at 40-50 OC overnight. After the addition of 0.2 mL of 1N aqueous HCI, the mixture was partioned between EtOAc and water. The organic layer was washed twice with water, once with brine, and was dried over Na 2

SO

4 The organic solution was concentrated to give 610 mg of an amber oil which was purified by MPLC 1:3 EtOAc/hexanes to afford 436 mg cpd 113 as a pale yellow oil; 'H NMR (CDC1 3 300 MHz) 1.35 4H), 1.42 9H), 1.75 (broad q, 2H, J 8 Hz), 3.06 (broad q, 2H, J 6 Hz), 3.28 1H, J 7.5 Hz), 3.48 1H, J 13.5 Hz), 3.66 1H, J 14.5 Hz), 3.76 3H), 3.79 1H, J 13.5 Hz), 3.97 1H, J 14.5 Hz), 4.47 (broad s, 1H), 5.05 2H), 6.93 2H, J 8.5 Hz), 7.22 2H, J 8.5 Hz), 7.3-7.5 (complex, 6H), 7.65 1H, J 7.5 Hz), 8.09 1H, J 8 Hz), 8.22 1H); MS m/z 592 Example 7 N-(3-Aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe A solution of 361 mg (0.61 mmol) of cpd 113 and 835 mg (3.7 mmol) of SnCI 2 dihydrate was stirred under N 2 at room temperature for 6 h. The slightly cloudy mixture was poured into 200 mL of 5% aqueous Na 2

CO

3 with rapid stirring. The resulting milky suspension was extracted with three 75 mL portions of CH 2 C12 and the combined organic layers were washed with brine and dried over Na 2

SO

4 The extracts were concentrated to give 344 mg of colorless oil which was purified by MPLC using 1:2 EtOAc/hexanes to provide 291 mg of N-(3-aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe as a yellow oil; 'H NMR (CDC13, 300 MHz) 1.25 4H), 1.44 9H), 1.70 2H), 3.31 (dd, 1H, J 6, 9 Hz), 3.38 1H, J 14 Hz), 3.40 1H, J 13.5 Hz), 3.74 3H), 3.81 1H, J 14 Hz), 3.83 1H, J 13.5 Hz), 4.52 (broad s, 1H), 5.05 2H), 6.50 (broad d, 1H, J 8 Hz), 6.70 2H), 6.92 2H, J 8.5 Hz), 7.08 1H, J 7.5 Hz), 7.2-7.5 (complex, 7H); MS m/z 562 (base, 506.

43 Example 8 N-(4-Benzyloxybenzyl)-N-(3-((2-furancarbonyl)amino)benzyl)Lys-Ome cpd 117 A solution of 42 mg (0.075 mmol) of N-(3-aminobenzyl)-N-(4benzyloxybenzyl)Lys(Boc)-OMe and 12 L (12 mg, 0.15 mmol) ofpyridine in 0.5 mL of 1,2-dichloroethane was combined with 8.1 L (11 mg, 0.083 mmol) and stirred under N 2 overnight. EtOAc (3 mL) was added and the solution was washed twice with 2 mL of water and 2 mL of saturated aqueous NaHCO 3 The EtOAc solution was filtered through a pad of Na 2

SO

4 and concentrated to give 44 mg of N-(4-benzyloxybenzyl)-N-(3-((2furancarbonyl)amino)benzyl)Lys(Boc)-OMe; MS m/z 356 The Bocprotected intermediate was stirred in 2 mL of 50% TFA/CH 2

CI

2 for 2 h and was concentrated and pumped at high vacuum to provide 66 mg of cpd 117 as the bis-TFA salt; 'H NMR (CD 3 OD, 300 MHz) 1.55 2H), 1.65 2H), 2.10 2H), 2.93 2H, J 7 Hz), 3.68 1H, J 7 Hz), 3.78 3H), 4.20 4H), 5.09 2H), 6.66 (dd, 1H, J 1.5, 3.5 Hz), 7.03 2H, J 8.5 Hz), 7.1-7.6 (complex, 11H), 7.76 8.07 1H); MS m/z 556 (base, 360, 197.

Example 9 N,N-bis(3-Nitrobenzyl)Asp(O-t-Bu)-O-t-Bu cpd 62 A solution of 0.50 mg (1.77 mmol) of Asp(O-t-Bu)-O-t-Bu*HC1, 1.17 g (5.42 mmol) of 3-nitrobenzyl bromide, and 1.25 mL (0.93 g, 7.2 mmol) of DIEA in 6 mL of DMF was stirred at room temperature under N 2 for 24 h and was heated at 70-80 °C overnight. The reaction mixture was partitioned between EtOAc and water and the organic layer was washed twice with water and once with brine. After drying over Na 2

SO

4 the organic solution was concentrated to give 0.86 g of a yellow oil which was purified by MPLC using 1:9 EtOAc/hexanes to afford 0.849 g cpd 62 as a pale 44 yellow oil; 'H NMR (CDCI 3 300 MHz) 1.43 9H), 1.57 9H), 2.59 (dd, 1H, J 16 Hz), 2.76 (dd, 1H, J 7, 16 Hz), 3.72 1H, J 7.5 Hz), 3.78 2H, J 14 Hz), 3.92 2H, J 14 Hz), 7.47 2H, J 8 Hz), 7.67 2H, J 7.5 Hz), 8.09 (broad d, 2H J 8 Hz), 8.16 (broad s, 2H); MS m/z 538 516 (base, 460, 404, 237.

Example N,N-bis(3-Aminobenzyl)Asp(O-t-Bu)-O-t-Bu A solution of 0.644 g (1.25 mmol) of cpd 62 and 2.82 g (12.5 mmol) of SnC12*2

H

2 0 in 12 mL of absolute EtOH was refluxed for 1.5 h. The mixture was cooled and poured into 300 mL of 5% aqueous Na 2

CO

3 with rapid stirring. The cloudy mixture was extracted with three 150 mL portions of CH 2 C2 and the organic extracts were washed with brine and dried over Na 2

SO

4 The CH 2 Cl solution was concentrated to afford 210 mg of N,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a cloudy yellow oil which was used without purification; 'H NMR (CDC13, 300 MHz) 1.40 9H), 1.52 9H), 2.48 (dd, 1H, J 7, 16 Hz), 2.76 (dd, 1H, J 8, 16 Hz), 3.48 2H, J 14 Hz), 3.55 IH), 3.73 2H, J 14 Hz), 6.56 (broad d, 2H J 8 Hz), 6.70 (broad s, 2H), 6.77 2H, J Hz), 7.08 2H, J 8 Hz); MS m/z 478 456 (base, 400, 344.

Example 11 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu To a solution of 109 mg (0.24 mmol) of N,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu, 29 mg (0.24 mmol) of DMAP, 125 L (93 mg, 0.72 mmol) of DIEA in 1 mL of CH 2

C

2 was added 95 L (111 mg, 0.72 mmol) of 4-methylbenzoyl chloride. The solution was stirred under N2 overnight and was then partitioned between EtOAc and water. The organic layer was washed with saturated aqueous NaHCO 3 and brine, dried over Na 2

SO

4 and concentrated to give 177 mg of yellow oil. The crude material was purified by MPLC using a solvent gradient ranging from 20-25% EtOAc/hexanes to provide 87 mg of N,Nbis(3-(4-methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a pale yellow oil; 'H NMR

I

45 (CDC1 3 300 MHz) 1.36 9H), 1.55 9H), 2.35 6H), 2.53 (dd, IH, J 6, 16 Hz), 2.76 (dd, 1H,J J=9, 16 Hz), 3.69 2H, J 14), 3.77 (dd, 1H, J 6, 9 Hz), 3.83 2H, J 14), 7.01 (in, 6H), 7.26 2H, J 8 Hz), 7.59 (in, 6H), 8.11 2H), 8.49 2H); MS miz 714 692 (base, 636, 580.

Example 12 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp-OH cpd 64 A solution of 87 mng (0.13 inmol) of N,N-bis(3-(4-methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu in 1 mL of 50% TFAICH- 2

CI

2 was stirred overnight. The mixture was concentrated and the residue was dissolved in HOAc and freeze-dried to afford 77 mng cpd 64 as a white solid; 1H NMR (CD 3 OD, 300 MHz) 2.40 6H), 2.85 (dd, 1H, J 6, 16.5 Hz), 2.98 (dd, I1H, J 8, 16.5 Hz), 4.02 2H, J =13.5 Hz), 4.08 4H, J 13.5 Hz), 4. 10 I H, J 6.5 Hz), 7.22 (in, 6H), 7.34 2H, J =7.5 Hz), 7.60 (broad d, 2H, J 9 Hz), 7.76 4H, J 8 Hz), 7.88 (broad s, 2H1); MS ml/z 580 (base, MH+).

Example 13 [N-Cbz-Glu(O-t-Bu)-NIICH 2

CH

2

OCH

2 2 To a solution of 1.69 g (5.0 minol) of N-Cbz-Glu(O-t-Bu)-OH, 0.365 niL (0.371 g, 2.5 mmol) of 1,8-diamino-3,6-dioxaoctane, 0.743 g (5.5 minol) of HOBT, and 1.05 mL (0.776 g, 6.0 inmol) of DIEA in 15 mL of CH 2 C1 2 was added 1.05 g (5.5 mnmol) of EDCI in one portion. After stirring at room temperature under N 2 overnight, the mixture was partitioned between EtOAc and 10% aqueous citric acid. The organic layer was washed with water, saturated NaHCO 3 and brine, dried over Na 2

SO

4 and concentrated to give 1.87 g of (N-Cbz-Glu(O-t-Bu)-NHCH 2

CH

2

OCH

2 2 as a colorless oil; 'H NMR (CD 3

OD,

300 MHz) 1.43 18H), 1.85 (in, 2H), 2.05 (in, 2H), 2.31 4H, J 8 Hz), 3.37 4H, J 5 Hz), 3.52 4H, J 5 Hz), 3.58 4H), 4.15 (in, 2H), 5.09 (dd, 4H, J 12, 16 Hz), 7.30 (mn, 1OH); MS inlz 809 (base, MNa+), 787 46 Example 14 [Glu(O-t-Bu)-NHCH 2

CH

2 0CH 2 2 Ammonium formate (0.78 g, 12.4 mmol) and 0.16 g of 10% palladium on carbon were added to a solution of (N-Cbz-Glu(O-t-Bu)-NHCH 2 CH20CH 2 2 in 12 mL of MeOH and the resulting suspension was stirred under N 2 at room temperature overnight. The mixture was diluted with CH 2

CI

2 and filtered through a Celite pad. The solids were washed thoroughly with CH 2 C2 and the combined organic filtrates were concentrated to dryness. The residue was partitioned between CH 2

CI

2 and saturated aqueous NaHCO 3 washed with brine, dried over Na 2

SO

4 and concentrated to give 1.13 g of (Glu(O-t-Bu)-

NHCH

2

CH

2 0CH 2 2 as a colorless oil; 1.44 18H), 1.81 2H), 2.08 2H), 2.35 (m, 4H), 3.39 (dd, 2H, J 5, 7.5 Hz), 3.47 4H, J 5 Hz), 3.58 4H, J 5 Hz), 7.53 (m, 2H).

Example [N,N-bis(4-Benzyloxybenzyl)Glu(O-t-Bu)-NHCH 2 CH20CH 2 2 cpd 245 A solution of 199 mg (0.384 mmol) of [Glu(O-t-Bu)-NHCH 2

CH

2 0CH 2 2 403 mg (1.73 mmol) of 4-benzyloxybenzyl chloride, 30 mg (0.2 mmol) of Nal, and 334 L (248 mg, 1.92 mmol) of DIEA was stirred under N 2 at room temperature for several days. The solution was partitioned between EtOAc and water and the organic layer was washed three times with water and once with brine. After drying over Na 2

SO

4 the solution was concentrated to give 528 mg of yellow oil which was purified by MPLC using a solvent gradient ranging from 42-50% EtOAc/hexanes to afford 318 mg of cpd 245 as a white foam; 'H NMR (CDC3, 300 MHz) 1.42 18H), 2.01 4H), 2.38 2H), 2.55 2H), 3.03 (dd, 2H, J 5, 8 Hz), 3.31 2H), 3.4-3.6 (complex, 18H), 4.99 8H), 6.89 8H, J 7.1-7.4 (complex, 47 Example 16 [N,N-bis(4-Benzyloxybenzyl)GluNHCH 2

CH

2

OCH

2 2 cpd 246 A solution of 219 mg (0.168 mmol) of cpd 245 in 2 mL of 33% TFA/CH 2 C1 2 was stirred ad room temperature overnight. The mixture was concentrated to give a crude product which was dissolved in HOAc and freeze-dried to afford 251 mg of cpd 246 as an amber oil; 'H NMR (CD 3 OD, 300 MHz) 2.1-2.6 (complex, 8H), 3.3-3.6 (complex, 8H), 3.57 4H), 3.78 2H), 4.25 (broad d, 4H, J 14 Hz), 4.36 (broad d, 4H, J 14 Hz), 5.09 8H), 7.03 8H, J 8 Hz), 7.3-7.5 (complex, 28H); MS m/z 1192 995, 596, 197 (base).

Example 17 [N-(3-Phenoxybenzyl)Glu(O-t-Bu)-NHCH 2 CH20CH 2 2 A solution of 680 mg (0.76 mmol) of [Glu(O-t-Bu)-NHCH 2

CH

2 0CH 2 2 and 278 L (317 mg, 1.6 mmol) of 3-phenoxybenzaldehyde in 3 mL of TMOF was stirred overnight at room temperature under N 2 The mixture was concentrated and pumped at high vacuum to give a colorless oil which was dissolved in 3 mL of CH 2 C1 2 and treated with 678 mg (3.2 mmol) of NaBH(OAc)3. After stirring under N 2 for 2 days, 50 mL of saturated aqueous NaHCO 3 was added and the mixture was extracted with CH 2 C1 2 The organic layers were combined, dried over Na 2

SO

4 and concentrated and the crude product (1.01 g) was purified by MPLC using a solvent gradient ranging from 2-4% MeOH/CH 2

C

2 to afford 490 mg of [N-(3-phenoxybenzyl)Glu(O-t-Bu)-

NHCH

2

CH

2

OCH

2 2 as a colorless oil; 'H NMR (CDCI 3 300 MHz) 1.41 18H), 1.89 (m, 4H), 2.31 4H), 3.12 2H, J 6 Hz), 3.45 8H), 3.55 4H), 3.60 2H, J 13.5 Hz), 3.73 2H, J 13.5 Hz), 6.86 (dd, 2H, J 1.5, 8 Hz), 7.00 8H), 7.2-7.4 (complex, 8H); MS m/z 883 589, 442, 414, 386 (base), 183.

Example 18 48 -Nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NIICH 2

CH

2

OCH

2 2 DIEA (269 L, 199 mg, 1.54 mmol), 3-nitrobenzyl bromide (322 mg, 1.49 mmol), and [N-(3-phenoxybenzyl)Glu(O-t-Bu)-NHCH 2

CH

2

OCH

2 2 (482 mg, 0.546 mmol) were combined in 2 mL of DMF and heated at 60-70 'C under N 2 for 2 days. The reaction mixture was cooled and partitioned between 100 mL of EtOAc and water. The organic layer was washed with three times with water and once with brine, dried over Na 2

SO

4 and concentrated to give 661 mg of [N-(3-nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(Ot-Bu)-NHCH 2

CH

2

OCH

2 2 which was used without purification; MS miz 1154 577, 130 (base).

Example 19 [N-(3-Aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NIICH 2

CH

2 oCH 2 2 A solution of 661 mg (0.54 mmol) of crude [N-(3-nitrobenzyl)-N-(3-phenoxy-benzyl)- Glu(O-t-Bu)-NHCH 2

CH

2

OCH

2 2 and 2.71 g (12.0 mmol) of SnCI 2 2 H 2 0 in 20 mL of absolute EtOH was refluxed under N 2 for 30 min. The cooled solution was poured into 500 mL of 2.5% aqueous Na 2

CO

3 with rapid stirring and the resulting cloudy mixture was extracted thoroughly with EtOAc. The slightly cloudy organic extracts were washed twice with brine, dried over Na 2

SO

4 anc concentrated to give 604 mg of yellow oil which was purified by MPLC using 3% MeOICH 2

CI

2 to provide 350 mg of aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH 2

CH

2 oCH 2 2 as a pale yellow oil; 'HNMvR (CDCl 3 300 MHz) 1.41 18H), 1.97 (in, 4H), 2.25 (mn, 4H), 2.48 (mn, 4H), 3.03 (dd, 2H, J 5, 8 Hz), 3.30 (in, 2H), 3.4-3.8 (complex, 24H), 6.47 2H, J 7.5 Hz), 6.65 (mn, 4H), 6.85 2H, J 9.5 Hz), 6.9-7.15 (complex, 12H), 7.2-7.4 (complex, 8H); MS ni/z 1094 547 (base).

Example [N-(3-Phenoxybenzyl)-N-(3 -(pentanoylainino)benzyl)-Glu-NHCH 2

CH

2

OCH

2 2 cpd 247 49 Pentanoyl chloride (16 uL, 16 mg, 0.136 mmol) was added dropwise to a solution of 68 mg (0.062 mmol) of [N-(3-aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-

NHCH

2

CH

2 0CH 2 2 20 L (20 mg, 0.25 mmol) of pyridine in 0.3 mL of 1,2dichloroethane. The mixture was shaken under N 2 overnight and was then partitioned between EtOAc and water. The organic layer was washed with saturated aqueous NaHCO 3 dried over Na 2

SO

4 and concentrated to give 77 mg of [N-(3-phenoxybenzyl)-N- (3-(pentanoylamino)benzyl)-Glu(O-t-Bu)-NHCH 2

CH

2 0CH2] 2 MS m/z 1073, 575 (base, The crude product was dissolved in 1 mL of 50% TFA/CH 2 C1 2 and allows to stand overnight. The solution was concentrated and the resulting oil was dissolved in HOAc and freeze-dried to provide 82 mg of cpd 247; H NMR (CD3OD, 300 MHz) 3.98 6H, J 7.5 Hz), 1.39 (sextet, 4H, J 7.5 Hz), 1.66 (quintet, 4H, J 7.5 Hz), 1.65 (m, 2H), 1.78 2H), 2.35 4H, J 7.5 Hz), 2.45 4H), 3.38 4H), 3.50 2H, J 3.57 4H), 4.10 (broad s, 8H), 6.9-7.25 (complex, 14H), 7.25-7.4 (complex, 10H), 7.71 2H); MS m/z 1150 575 (base).

Example 21 [N-Cbz-Lys(Boc)-NHCH 2

CH

2 3

N

A solution of 1.0 g (2.63 mmol) of N-Cbz-Lys(Boc)OH, 0.131 mL (0.128 g, 0.876 mmol) of tris(2-aminoethyl)amine, 0.391 g (2.98 mmol) of HOBt, 0.555 g (2.89 mmol) of EDCI, and 0.55 mL (0.408 g, 3.16 mmol) of DIEA in 5 mL of CH 2 C2 was stirred under

N

2 at room temperature overnight. The mixture was diluted with EtOAc and washed with aqueous citric acid, saturated aqueous NaHCO 3 and brine. The solution was dried over Na 2

SO

4 and concentrated to give 0.872 g of [N-Cbz-Lys(Boc)-NHCH 2

CH

2 3 N as an off-white solid; 'H NMR (CD30D, 300 MHz) 135 12H), 1.40 27H), 1.60 3H), 1.72 3H), 2.51 6H), 2.99 6H), 3.10 3H), 3.21 3H), 4.12 3H), 5.00 3H, J 12.5 Hz), 5.08 3H, J 12.5 Hz), 7.29 15H); MS m/z 1243 (base, 567, 467.

50 Example 22 [Lys(Boc)-NHCH 2

CH

2 ]3N A solution of 0.841 g (0.682 mmol) [N-Cbz-Lys(Boc)-NHCH 2

CH

2 3 N, 0.252 g of Pd-C, and 0.774 g (12.3 mmol) of ammonium formate in 10 mL of MeOH was stirred for 5 h at room temperature under N 2 The mixture was filtered through a Celite pad, the solids were washed with CH 2 C1 2 and the reslulting solution was concentrated to dryness. The residue was partitioned between CH 2 C1 2 and brine; the organic layer was dried over Na 2

SO

4 and concentrated to provide 0.191 g of [Lys(Boc)-NHCH 2

CH

2 3 N as an off-white solid; 'H NMR (CD30D, 300 MHz) 1.40 27H), 1.45 12H), 1.75 6H), 2.62 6H), 3.01 6H), 3.28 6H), 3.64 3H); MS m/z 853 831 266 (base).

Example 23 [N,N-bis(3-Phenoxybenzyl)Lys(Boc)-NHCH 2

CH

2 3

N

A solution of 65 mg (0.078 mmol) of[Lys(Boc)-NHCH 2 CH2] 3 N, 120 L (140 mg, 0.70 mmol) of 3-phenoxybenzaldehyde, and 71 L (65 mg, 0.70 mmol) of boranepyridine complexin 3 mL of absolute EtOH was stirred for 4 days at room temperature under N 2 The mixture was concentrated to dryness and partitioned between water and

CH

2 C1 2 The organic layer was concentrated to give a yellow oil which was purified by MPLC using 2.5% MeOH/CH 2

CI

2 to give 78 mg of [N,N-bis(3-phenoxybenzyl)Lys(Boc)-

NHCH

2

CH

2 3 N as a yellow oil; MS m/z 872 (base, [M-Ci 3

HI

2 611, 443.

Example 24 [N,N-bis(3-Phenoxybenzyl)Lys-NHCH 2

CH

2 3

N

cpd 277 A solution of 78 mg (0.048 mmol) of [N,N-bis(3-phenoxybenzyl)Lys(Boc)-

NHCH

2

CH

2 3 N in 2 mL of 50% TFA/CH 2

CI

2 was stirred for 2 h at room temperature.

The mixture was diluted with CH 2 C1 2 washed with water and 5% Na 2

CO

3 and 51 concentrated to give 57 mg of cpd 277 as an off-white foam; 'H NMR (CD 3 OD, 300 Mffz) 1.35 (in, 6H), 1.52 (in, 6H), 1.76 (in, 2.75 (mn, 6H), 3.19 (mn, 6H), 3.40 (in, 6H), 3.60 (in, 9H), 3.77 (in, 6H), 6.79 6H, J 8 Hz), 6.93 (in, 24H), 7.05 (mn, 6H), 7.19 (in, 6H), 7.29 (in, 12H); MS m/z 813 ([MH 2 721, 542 (base, R 2 Table 2 cpd inh R' (amino acid side chain) [R2 R 3 11 70 Asn, Asp, Gin, Glu 3-PhO CH=CH 12 59 Cys, Met, Ser, Thr 3-PhO ___CH=CH 13 nd Arg, Gly, His, Pro 3-PhO ___CH=CH 14 30 Lys(2-Cl-Cbz), Phe, Trp, Tyr 3-PhO ___CH=CH 48 Ala, le, Leu, Val 3-PhO 16 nd Glu, Asp 2,3-benzo CH=CH 17 nd Cys, Met 2,3-benzo ___CH=CH 18. nd Ser, Thr 2,3-benzo 19 nd His, Arg(Mtr) 2,3-benzo _CH CH nd Pro, Gly 2,3-benzo 21 nd Phe, Tyr 2,3-benzo ___CH=CH 22 nd Trp, Lys(2-Cl-Cbz) 2,3-benzo 23 nd Ile, Ala 2,3-benzo 24 nd Val,Leu 2,3-benzo nd Asn, Lys 2,3-benzo 26 nd Ala, le 3,4-benzo CH=CH 27 nd Arg(Mtr), Lys(2-C1-Cbz) 3,4-benzo CH=CH 28 nd Asp, Glu _____3,4-benzo CH=CH 29 nd Cys, Met 3,4-benzo CH=CH nd Gly, Pro _____3,4-benzo CH=CH 31 nd -His, Lys _____3,4-benzo CH=CH 52 32 nd Leu, Val _____3,4-benzo ]CH=C 133 nd Lys(2-Cl-Cbz), Phe jCH=CH 134 nd Ser, Thr ICH=C nd Trp 3,4-benzo jCH=C Table 3 EPO /EBP-Ig 2 9MS cpd %inh @50 M R' R IR 9 WQ MH+ 36 nd CH 3 4-CF 3 H CH=CH 458 37 19 H 4-CF 3 H CH=CH 430 38 Ind (CH 2 4 NH(2-Cl-Cbz) 4-F H ICH=CH 448 nd CH 3 4-F H CH=CH 223 41 nd CH 2

CO

2 H 4-F H CH=CH 266 42 nd CH 2

CH

2

CO

2 H 4-F -H CH=CH 281 43 nd (CH 2 3

NIIC(=NH)NH

2 4-F H CH=CH 308 Ind PhCH 2 4-F H CH=CH 299 46 nd 4-HO-PhCH 2 4-F IH CH=CH 315 47 nd CH 2 0H 4-F H CH=CH 238 48 nd CH(OH)CH 3 4-F H CH=CH 253 49 1 (CH 2 3 iNHC(=NH)NH- 2 H H is 419 1-6 (CH 2 4 N-1 2 H H S 391 51 nd CH(CH 3

)CH

2

CH

3 H H S 376 52 21 CH 2

CH

2

CO

2 H H H S 392 53 14 CH 2

CO

2 H H H S 378 54 18 GCl 3 H H S 334 14 CH 2

CH

2

CONH

2 H H S 391 56 nd (CH 2 4 NHCbz H Me S 539 57 0 (CH 2 4 NHCbz H CH 2 Ph S 615 58 nd CH 2 (indol-3-yl) H Me Is 463 59 26 CH 2

CH

2

CO

2 t-Bu H Me is 462 53 160 19 ICH 2

CH

2

CO

2 Et IH IMe IS 1434 61 14 ICH 2

CH

2

CO

2 H IH Me IS 1406 54 Table 4 EPO EBP-Ig

MS

cpd %inh @50 M R a R' R 4

R

9

IM±

62 nd t-Bu NO 2

NO

2 t-Bu 516 63 20 H PhCH 2 NII PhO H 511 64 -4 H 4-MePhCONI 14-MePhCONH H 580 -7 H 4-MePhSO 2 NH 14-MePhSO 2 NHi H 652 66 -16 H 3-CIPhCH 2 NH- PhO H 1546 67 -8 H 3-BrPhCH 2 NH PhO H 590 68 -13 H 2-FPhCH 2 NI{ PhO H 529 69 -13 H 2-MePhCH 2 N- PhO H 525 -8 H 4-FPhCH 2 PhO IH 529 71 -6 H 3-C1PhCH 2 NH 4-Me-PhO H 560 72 -14 H F 5 -PhCH 2 NII 4-Me-PhO H 615 73 -13 H 2-FPhCH 2 NH 4-Me-PhO H 543 74 1-7 H 3-CNPhCH 2 NH 4-Me-PhO H 550 175 _-III II PhCH 2 NH 4-Me-PhO -H 525 55 O (CH 2 RaO Table EPO/ EBP-Ig MS cpd %inh 50Ra R R' R R R 9 n MH+

M

76 25 t-Bu PhO H PhO H t-Bu 1 636 77 52 H PhO H PhO H H 1 524 78 nd H H 4-MePhCONII H PhCH 2 H 2 593 0_ 79 nd H H n-BuCONH H PhCH 2 H 2 559 0 nd H H 2-naphthyl CONH H PhCH 2 H 2 629 81 nd H H 2-furyl CONH H PhCH 2 H 2 569 82 32 H H 4-MeO-PhCONH H PhCH 2 H 2 609 83 18 H H HO 2

C(CH

2 3 CONH H PhCH 2 H 2 589 0 84 14 H H C 2

F

5 CONH H PhCH 2 H 2 621 8520 H H CF 3 CONI H PhCH 2 H 2571 0 86 37 H H 4-pyridyl-CONI H PhCH 2 H 2 580 0 87 23 H H 4-MePhSO 2 NII H PhCH 2 H 2 629 0 88 10.3 H H H0 2

CCH

2 H PhCH 2 H 2 643 cyclopentyl) 0

CH

2 C0NH 89 22 H H PhOCONH H PhCH 2 H 2595 56 29 H H 4-Ph-PhCONH HPhCH 2 H 2 655 91 19 H H 4-N0 2 -PhCONII H PhCH 2 H 2 24 0 57 -r OR 9 0 (C H 2 )n Ra o Table 6 EPO /EBP-lg

MS

cpd %inh 50OR2 R 2 R 3 R 4 R' R' R' MH M 92 20 H H H H H 2 Me394 93 120 t-Bu H H H H 2 Me 450 94 25 Et H H H H 2 Me422 15 t-Bu 2,3-benzo 12,3-benzo 12 Me 1550 96 -5 t-Bu lPhO H PhO H 2 Me 634 97 14 t-Bu 13,4-benzo ___3,4-benzo H 536 98 12 t-Bu IH Ph H Ph '2 Me 602 99 13 t-Bu 3,4-di-CI- H 3,4-di-CI- H 2 Me 772 PhO ___PhOI I 100 34 H HI Ph H Ph12 Me 546 101 32 H 3,4-di-Ci- H 3,4-di-CI- H 2 Me 716 PhO ___PhO 102 5 t-Bu 4-t-Bu-PhO H 4-t-Bu-PhO H 2 t- 789 ___Bu 103 17 t-Bu 3-CF3-PhO H 3-CF3-PhO H 2 t- 812 Bu 1 104 78 H 4-t-Bu-PhO H 4-t-Bu-PhO IH 2 H 1676 105 70 H 3-CF3-PhO H 3-CF3-PhO H 2 H1 700 106 20 t-Bu PhO H PhO H 1 t- 662 Bu 107 78 H PhO H 2 H 562* 108 181 11 1 PhO IH jPhO JH 1 IH 1550_ EM-Hf- 58 Table 7

EPOI

EBP-

ig

MS

cpd %inh R R R 3 R4R 5

R

9

MH

50 M 109 7 BoC PhCH 2 O H PhCH 2 O H Me 653 110 54 H H PhCH2 H PhCH 2 Me 553 0 111 5 Boc H PhCH 2 H PhCH 2 Me 653 _0 0 112 59 H PhCH 2 O H PhCH 2 O H Me 553 113 24 Boc H PhCH 2

NO

2 H Me 592 114 37 H H PhCH 2

NO

2 H Me 492 1 0 115135 H H PhCH 2

NH

2 H Me 462 116 32 H H PhCH 2 n-BuCONH H Me 546 0 117 34 H H PhCH 2 2-ftirylCONH H Me 556 1 1 ~0 1 118136 IH H PhCH, 4-MePhCONH H Me 580 0 119 34 IH H PhCH 2 i-Pr-CONH H Me 532 0 120 35 H H PhCH 2 4-pyridyl- H Me 567 O CONHi___ 121 45 H H PhCH 2 2-naphthyl- H Me 616 0 ICONH I__I 59 122 nd Boc PhCH 2 NH H PhCH 2 NH H Me 651 123 nd Boc 2- H 2-MePhCH 2 NH H Me 679 MePhCH 2

NII

124 nd Boc 4-MeO- H 4-MeO- H Me 711 PhCH 2 NI lPhCH 2

NH

125 nd Boc 3,4-di-MeO- H 3,4-di-MeO- H Me 771 2 NH PhCH 2

NH

126 nd Boc -NH 2 H [-NI-b H Me 471 127 nd H PhCH 2 NH H IPhCH 2 NH H Me 551 128 nd H 2- H 2-MePhCH 2 NH H Me 579 MePhCH 2 NH 129 nd H 4-MeO- H 4-MeO- H Me 611 PhCH 2 N}I PhCH 2 NII 130 nd H 3,4-di-MeO- H 3,4-di-MeO- H Me 671 1 1PhCH 2 NH jPhCH 2 NH 131 nd H PhCH 2

CH

2 N H PhCH 2

CH

2 NH H Me 579 H 132 nd HO 2

CCH

2

CH

2 PhCH 2 NH H PhCH 2 NII H Me 651 co 133 nd HO 2

CCH

2

CH

2 2- H 2-MePhCH 2 NH H Me 679 1 CO MePhCH 2

NII

134 nd HO 2

CCH

2

CH

2 4-MeO- H 4-MeO- H Me 711 MO PhCH 2 NH PhCH 2

NH

135 nd HO 2

CCH

2

CH

2 3,4-di-MeO- H 3,4-di-MeO- H Me 771 CO PhCH 2 NH PhCH 2

NH

136 nd HO 2

CCH

2

CH

2 PhCH 2

CH

2 N H PhCH 2

CH

2 NII H Me67 I I CO H Table 8 I EPO EBP-Ig MS II

I

60 cpd %inh 50 Ra

R

5 R9MH+

__M

137 nd H PhO PhO H Me 551 138 nd Boc 4-t-Bu-PhO PhCH 2 O H Me 721 139 nd H 4-t-Bu-PhO PhCH 2 O H Me 621 140 nd H (CF3CO)2N PhGH 2 O H H 666 141lnd H PhCONH PhCH 2 O H H 578 142 nd H 4-pyridyl-CONH PhCH 2 O H H .579 143 nd H (CF 3

CO)

2 N PhO H H 652 144 nd H PhCONH PhO H H 564 145 nd H 4-pyridyl-CONH PhO H H 565 146 Ind H (CF 3

CO)

2 N MeO MeO H 620 147 nd H PhCONII IMeO MeO H 532 148 nd H 4-pyridyl-CON-H MeO MeO H .533 149 nd H (CFICO) 2 N H PhO H 652 150 nd H PhCON- H PhO H 564 151 nd H 4-pyvridvl-CON'H H PhO H 565 152 nd H PhCONH H PhCH 2 H 578 153 nd H 4-pyridyl-CONH H PhCH 2 H 579 0 154 nd H (CF 3

CO)

2 N H PhCH 2 H 666 0 155 nd HO 2

CCH

2

CH

2 C 4-MeO- PhO H H 694 o PhCONHT 156 nd H0 2

CCH

2

CH

2 C PhCONH PhO H H 664 1 0 157 nd HO 2

CCH

2

CH

2 C 2-naphthyl- PhO H H 714 o CONH 158 nd HO 2

CCH

2

CH

2 C 4-Me-PhSO 2 NHi PhO H H 714 0 159 nd HO 2

CCH

2

CH

2 C 4-MeG- 2,3-benzo H 652 o PIICONH__ 160 nd HO 2

CCHICH

2 C PhCONH 2,3-benzo H 622 0__ 161 nd HO 2

CCH

2

CH

2 C 2-naphthyl- 2,3-benzo H 672 o CONE 162 nd H0 2

CCHCH

2 C 4-Me-PhSO 2 NH 2,3-benzo H 672 10_ [163 Id IH02CCH 2

CH

2 C 4-MeO- JH IF H 620 61 o PhCONH 164 nd HO 2

CCH

2

CH

2 C PhCONH- H F H 590 0 165 nd HO 2

CCH

2

CH

2 C 2-naphthyl- H F H 640 o CONH 166 nd HO 2

CCH

2

CH

2 C 4-Me-PhSO 2 NH H F H 640 167 nd HO 2

CCH

2

CH

2 C 4-MeO- PhCH 2 O H H 708 o PhCONH- 168 nd HO 2

CCH

2

CH

2 C PhCONH PhCH 2 O H H 678 0 169 nd HO 2

CCH

2

CH

2 C 2-naphthyl- PhCH 2 O H H 1728 o CONE 170 nd HO 2

CCH

2

CH

2 C 4-Me-PhSO 2 NIH [PhCH 2 0 H H 728 Table 9

EPO/

EBP-

Ig3 %inh R MIS cpd 5 ORa R 2 R 4 R 5

R

9

ME

M

I+

171 b Cbz H H H H Me 527 172 15 Cbz H H H H H 513 173 5 Cbz H H H H t- 569 1 Bu 1 174 23 Cbz H MeO H MeO Me 587 175 1 Cbz 3,4- 3,4- Me 627 benzo benzo 176 -4 Cbz PhO HI __PhO IH Me 711

I

62 177 nd Cbz 2,3-benzo 12,3-benzo Me 627 178 36 Boc H NO 2 H INO 2 Me 58-3 179 30 Boc H NO 2 H NO 2 H 569 180,-4 *BoclPhO H PhO H Me 677 1811-9 Boc 14-t-Bu-PhO H 4-t-Bu-Pho H IMe 7901 182118 H 14-t-Bu-PhO H 4-t-Bu-PhO H Me 689 183136 Boc N0 2 H NO 2 H Me 583 184 53 H INO 2 H NO 2 H Me 483 185 29 H NH 2 H NH 2 H IMe 423 186 nd H n-Bu-CONH H n-Bu-CONH H [Me 591 187 nd H 2-furyl-CONH H 2-fuiryl-CONH H ~Me 611 188 Ind Hi PhCONH H PhCONH H tMe 1631 189 nd H 4-Me-PhCONH H 4-Me-PhCONH H IMe 659 190 nd H 4-N0 2 -PhCONH H 4-N0 2 -PhCON-H H Me 721 191 nd H 4-Me-PhSO 2 NH H 4-Me-PhSO 2 NH H Me 731 192 rid H Cbz-N}I H Cbz-NH- H Me 691 193 Ind H 4-Br-PhCON}1 H 4-Br-PhCO H Me 789 194 Ind H 2-MeO-PhCONH H 2-MeO-PhCONI{ H Me 1691 195 rid H 3-MeO-PhCONH H 3-MeO-PhCONH H Me 691 196 nd H 4-MeO-PhCONH H 4-MeO-PhCONII H Me 691 197 nd H CH 3 CH=CHCON H CH 3 CH=CHCON H Me 559 SH __H 198 nd H C 2

F

5 CONH H C 2

F

5 CONH H Me 715 199 nd H 2-naphthyl-CONH H 2-naphthyl-CON'H H Me 731 200 nd H EtO 2

CCH

2

CH

2 CO H EtO 2

CCH

2

CH

2 CO H Me 679 NH Ni 201 Ind H CF 3 CONH H ICF 3 CONH 1H Me 615 202 Ind H MeSO 2 NH H IMeSO 2 NH IH Me 1579 63

R

(C H 2 4

HN'

Table

EPO/

EBP-

Ig 2 3 4 cpd %inh R( R( R 3 R( R 5

MH

50 M 203 37 Boc H H H H 4-(MeCOCH 2

CH

2 )-PhN 640 204 -6 H H H H H 4-(MeCOCH 2

CH

2 )-PhaI 540 205 26 H H H H H n-Bu-NH 434 206 17 2-MeO-PhCO H H 1H H n-Bu-NIL 568 207 20 4-MeO-PhCO H H H1 H In-Bu-NH 568 208 22 PhCO H H IH H n-Bu-N~f 538 209 25 2-MeO-PhCO H H H H n-Bu-NH 568 210 nd Boc H H H H 4-MeO-PhCH 2

CH

2 NH 612 211 162 H H H H H j4-MeO-PhCH 2

CH

2 N~i 512 212 1-10 H H H H H n-Pr-NH 420 214 Ind Boc H H H H 3,4-di-MeO- 642 1 PhCH 2

CH

2

NH

215 nd Boc H H JH H 3-MeO-PhCH 2

CH

2 NII 612 216 10 Boc H H IH H 4-(PhCH=CHCH 2 700 PhCH 2

NH

217 ud Boc H H IH H 14-HO-PhCH 2 NII 584 218 nd Boc H 1H IH H 219 nd Boc H -H H H MeNH 492 220 45 H H H IH H 4-(PhCH=CHCH 2 600 ___PhCH 2

NH

221 48 H H H IH H 3,4-di-MeO- 542 PhCH 2

CH

2

NII__

1222 156 JH H H IH IH 3-McQ-PhCH 2

CH

2 NH 15121 2-23 nd B1oc H H IH HI 12-MeO-PhCH 2

CH

2 NH 16121 64 224 51 H H H H H 2-MeO-PhCH 2

CH

2 N{ 1512 225 10 Boc PhO H PhO H 4-MeO-PhCH 2

CH

2 NH 1797 226 rid Boc H H H H PhCH 2

CH

2 NH 582 227 48 H H H H H PhCH 2

CH

2 NH- 482 228 121 PhNH-CO PhO H PhO Hf 4-MeO-PhCH 2

CH

2 Ni 1816 229 122 4-.PhO-PhNHCO H H H H 4-MeO-PhCH 2

CH

2 NH 723 230 142 3,4-di-CI- H H H H 4-MeO-PhCH 2

CH

2 NH 700 PhNHCO 231 36 4-EtO2C- H H H H 4-MeO-PhCH 2

CH

2 NH 703 PhNHCO I_1_ 232 14 4-PhO-PhNHCO PhO H PhO H 4-MeO-PhCH 2

CH

2 N- 1908 233 18 H H NO H NO 3-MeO-PhCH 2

CH

2 NH 602 1? 2 234 rid Boc H H IH H PhCH-)NH 568 235 149 H H H H H IPhCH 2 NH 468 236 rid Boc H Ph H Ph 14-MeO-PhCH 2

CH

2 NI{ 765 237 55 HO2CCH 2

CH

2 CO H H H H 13-MeO-PhCH7CH 2 NII 612 238 39 H H Ph H Ph J4-MeO-PhCH 2

CH

2 NE 664 239 46 H PhO IH IPhO H PhCH 2

CH

2 )NH 666 240 nd H0 2

CCH

2

CH

2 CH PhO H PhO H PhCH 2

CH

2 NH 780 2 C0 285 40 H H H jH -H 4- N2CO)Plperidin-1-yl 1489

R

R

N

R3 0 (CH 2 R a 2 Table 11I EPO!-_

EBP-

Ig 3 cpd %inh R' R R R Ri Z r [MH 2 /2

I

m I 241 2 t- H. PhCH 2 H PhCH 2

NH(CH

2 3 0(CH 2 4 0(CH 2 3 1 666 __Bu 0 _0 NH 242 1 t- H PhCH 2 H PhCH 2

NH(CH

2 )30(CH 2

CH

2 0) 2 (C 1 674 0 0 H 2 3 NH1__ 243 75 H H PhCH 2 H PhCH 2

NH(CH

2 3 0(CH 2 4 0(CH 2 3 1 610 1 10o 0 INH1 244 66 H H PhCH 2 H PhCH 2

NH(CH

2 3 0(CH 2

CH

2 0) 2 (C 1 618 o 0 H 2 3 NH 245 0 t- H PhCH 2 H PhCH 2

NH(CH

2 2 0(CH 2 2 0(CH 2 2 2 652 Bu 1 0 0 INH_ 246 79 H H PhCH 2 H PhCH 2

NH(CH

2 2 0(CH 2 2 0(CH 2 2 2 596 0 0 NI- 247 47 H n-Bu- H Ph H NH(CH 2 2 0(CH 2 2 0(CH 2 2 2 575 1CONHE 0 NH 248 56 H 2-fuiryl- H Ph H NH(CH1 2 2 0(CH 2 2 0(CH 2 2 2 585 ICONH 0 NHl__ 249 72 H 4-Me- H Ph H NH(CH 2 2 0(CH 2 2 0(CH 2 2 2 609 MOCN 0 NH S H 250 78 H 4-Me- H Ph H NH(CH 2 2 0(CH 2 2 0(CH 2 2 2 645 PhSO 2 N 0 NEl I H I_ R4 0 0 It(CH 2 )rZ LRaO 12 Table 12 EPO EBP-- MS cpd Ig R a R 2 R 4 Z r [MIH 2 12 %inh 50 1251149 IH HI H INH(CH 2 2 0(CH 2 2 0(CH 2 2 12 1436 66 NHl 252 -4 t-Bu 4-t-Bu- 4-t-Bu- NH(CH 2 3 0(CH 2 4 0(CH 2 3 1 803 PhO PhO NH 253 -5 [t-Bu 4-t-Bu- 4-t-Bu- NH(CH 2 3 0(CH 2

CH

2

O)

2 (C 1 811 I PhO PhO H 2 3

NH

254 -9 t-Bu 4-t-Bu- 4-t-Bu- NH(CH 2 1 ONH 1 787 1 PhO PhO___ 255 0 t-Bu 4-t-Bu- 4-t-Bu- NH(CH 2 12 NH 1 801 PhO PhO 256 10 t-Bu 4-t-Bu- 4-t-Bu- NH(C11 2 2 0(CH 2 2 0(CH 2 2 1 789 PhO IPhO INH I___I PhCH 2

O

PhGH 2 O 0

N

Ra (CH 2 4 H Table 13 EPO /EBP-Ig MIS cpd %inh 50ORa Z [ml1 2 /2] M 257 -26 Boc NH(CH 2 3 0(CH 2

CH

2

O)

2

(CH

2 731 3

N'H

258 -24 Boc NH(CH 2 3 0(CH 2 4 0(CH 2 3 N 723 259 -13 Boc NH(CH 2 12 NH 721 260 -12 Boc N-H(CH 2 2 0(CH 2 2 0(CH 2 2 N 695

H

261 51 H NH(CH 2 2 0(CH 2 2 0(CH 2 2 N 595 262 93 HO 2

CCH

2

CH

2 CO N"H(CH 2 2 0(CH 2 2 0(CH 2 2 N 695 263 88 HO 2

C(CH

2 3 C0 ~NE(CH 2 2

O(CB

2 2

O(CH

2 2 N 709 jH 264 89 H0 2 CCH CMe 2

CH

2

INI(CH

2 2 0(CH 2 2 0(CH 2 2 N 737 CH11 67 265 65 HO 2

CCH

2

CH

2 CO NII(CH 2 3 0(CH 2 4 0(CH 2 3 N 723

H

266 82 HO 2

C(CH

2 3 C0 N-H(CH 2 3 0(CH 2 4 0(CH 2 3 N 737

H

267 83 HO 2

CCH

2 CMe 2

CH

2

NH(CH,)

3 0(CH 2 4 0(CH 2 3 N 765

H

268 40 HO 2

CCH

2 CMe2CH 2

NH(CH

2 12 N11 764 Co 269 55 HO 2

CCH

2

CH

2

CH

2 C NII(CH 2 12 NH 735 0 270 56 HO 2

CCH

2

CH

2 CO NII(CH 2 12 NH 721 271 77 HO 2

CCH

2

CH

2 CO NH(CH 2 3 0(CH 2

CH

2

O)

2

(CH

2 731 3

NH

272 78 HO 2

CCH

2

CH

2

CH

2 C NH(CH 2 3 0(CH 2

CH

2

O)

2

(CH

2 745 0o 3 NH I_ 68 Table 14

EPO/

EBP-

Ig %inh MS cpd 50 R' R 2 R4Z n [MH 2 /2 M 273 nd HO 2

CCH

2

CH

2 4-Me- 4-Me-PhO NH-(CH 2 2 0(CH 2 2 0(C 2 695 CO PhO 2 2

NH

274 nd HO 2

CCH

2

CH

2 PhO PhO NH(CH 2 2 0(CH 2 2 0(C 2 667 iCO H 2 2 NH 1 275 nd HO 2

CCH

2

CH

2 4-MeO- 4-MeO- NH(CH 2 2 0(CH 2 2 0(C 2 727 CO PhO PhO H 2 2 N11 276 nd HO 2

CCH

2

CH

2 4-t-Bu- 4-t-Bu- NII(CH 2 2 0(CH 2 2 O(C 2 780 1 CO PhO jPhO H 2 2

NH

277 nd H PhO PhO (tTCH 2

CH

2 3 N 3 813 278 nd H 4-Me- 4-Me-PhO (NHCH 2

CH

2 3 N 3 855 ___PhO 279 nd H 4-MeG- 4-MeG- (NIHCH 2

CH

2 3 N 3 903 PhO 280 nd HO 2

CCH

2

CH

2 4-MeG- 4-MeG- (NHCH 2

CH

2 3 N 3 1053 1_ CO PhO PhO 281 Ind HO 2

CCH

2

CH

2 4-Me- 4-Me-PhO (NHCH 2

CH

2 3 N 3 1005 CO PhO 282 Ind HO 2

CCH

2

CH

2 PhO PhO (NHCH 2

CH

2 3 N 3 963

CO

283 Ind Boc PhO PhO NTH(CH 2 3 NMe 2 .666

KCH

2 3 N11 2841 nd Boc 4-Me- 14-Me-PhO jNH(CH 2 )3NMe 2 694 69 I PhO I-(CH 2 3 NH I1 Table 14 EPO EBP-Ig 21 cpd I%inh@_50 M R 1 R{ R( MS, MH+ 285 -28 Me H H 473 286 46 H PhCH 2 O H 1565 287 36 H 4-MePhO H 565 1288 127 H 4-tBuPhO H 607 1289 120 H H PhO 551

Claims (3)

1. A compound of Formula I when used as a medicament R 2 3 R'-W 0 4Y N R R (y,N Y k Z I wherein: R is the side chain of a natural or unnatural a-amino acid, where if said side chain contains a protectable group, that group may be protected with a member of the group consisting of succinyl, glutaryl, 3,3- dimethylglutaryl, C 1 .salkyl, CI.salkoxycarbonyl, acetyl, N-(9- fluorenylmethoxycarbonyl), trifluoroacetyl, omega- carboxyCl 5 alkylcarbonyl, t-butoxycarbonyl, benzyl, benzyloxycarbonyl,

2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido, t-butyl, cinnamoyl, trityl, 4-methyltrityl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substituted phenylureido (where the phenyl substituents are selected from phenoxy, halo, or CI.salkoxycarbonyl) group; R2 and R and R 4 and R 5 independently may be taken together to form a six-membered aromatic ring which is fused to the depicted ring, or are independently selected from the group consisting of hydrogen, C 1 alkyl, C1s5 alkoxy, hydroxy, halo, trifluoromethyl, nitro, amino, phenyl, phenoxy, phenylC 1 s 5 alkyl, phenyl Cl 1 salkoxy, substituted phenyl, substituted phenoxy, substituted phenyl C 1 .salkyl or substituted Ci-salkoxy, (where the substituents are selected from Cls 5 alkyl, C 1 5 -s alkoxy, hydroxy, halo, trifluoromethyl, nitro, nitrile, and amino), and 71 substituted amino (where the substituents are selected from one or more members of the group consisting of Cv 5 alkyl, halosubstitutedC 5 alkyl, C 1 5 alknyl, C 1 5 alkenyl, phenyl, phenylC 1 5 alkyl, C 1 5 alkylcarbo nyl, halo substituted C 1 salkylcarbonyl, carbo xyCI .salkyl, C 1 5 alkoxyCI 5 alkyl, cinnamoyl, naphthylcarbonyl, furylcarbonyl, pyridylcarbonyl, C 1 5 alkylsulfonyl, phenylcarbonyl, phenylC 1 5 alkylcarbonyl, phenylsulfonyl, phenylC 1 5 alkylsulfonyl, substituted phenylcarbonyl, substituted phenylC 1 5 alkylcarbonyl, substituted phenylsulfonyl, substituted phenylCI 5 alkylsulfonyl, substituted phenyl, and substituted phenylC I 5 alkyl [where the aromatic phenyl, phenylC 1 5 alkyl, phenylcarbonyl, phenylC 1 5 alkylcarbonyl, phenylsulfonyl, and phenylC 1 5 alkylsulfonyl substitutents are independently selected from one to five members of the group consisting Of C 1 5 alkyl, CI- 5 alkoxy, hydroxy, halogen, trifluoromethyl, nitro, nitrile, and amino]); W and Q are independently selected from the group consisting of -CH=CH-, and -CH=N-; X and Y are independently selected from the group consisting of carbonyl, C 1 5 alkyl, CI-5alkenyl, C 1 5 alkenylcarbonyl, and (CH 2 where m is n is1, 2, or 3; Z is selected from the group consisting of hydroxy, C 1 5 alkoxy, phenoxy, phenylCI 5 alkoxy, amino, C 1 5 alkylamino, diC 1 5 alkylamino, phenylamino, phenylC I 5 alkylamino, piperidin- Il-yl, substituted piperidin- 1l-yl (where the substituents are selected from the group consisting of C 1 I 5 alkyl, C 1 5 alkoxy, halo, aminocarbonyl, C I 5 alkoxycarbonyl, and oxo; substituted phenylC 1 5 alkylamino (where the aromatic substitutents are selected from the group consisting Of CI-5alkyl, C 1 .salkoxy, hydroxy, halogen, trifluoromethyl, nitro, nitrile, and amino), substituted phenoxy (where the aromatic substitutents are selected from the group consisting Of C I 5 alkyl, C 1 -alkoxy, hydroxy, halogen, trifluoromethyl, nitro, nitrile, and amino), substituted

24. Apr. 2007 9:51 Shelston IP No. 2910 P. 4 -72- 0 phenylCi.salkoxy (where the aromatic substitutents are selected from the Sgroup consisting of Ci.salkyl, C 1 5 alkoxy, hydroxy, halogen, trifluoromethyl, nitro, nitrile, and amino), -OCCH 2 (CH2(OCH 2 CH2)OCH2C20-, -NHCH2CH(OCH 2 CH 2 CH ),OCH 2 CH 2 NH-, -NH(CH2)pOO(H2)qO(CH2N-, -NH(CH 2 )qNCH 3 (CH 2 )NH-, ci -NH(CH 2 )sNH-, and (NH(CH 2 )s) 3 N, where s, p, and q are independently selected from 1-7 Swith the proviso that ifn is 2, Z is not hydroxy, C1-. alkoxy, amino, 0 10 C 1 .alkylamino, diCt-salkylamino, phenylamino, phenylCl-salkylamino, or piperidin-1-yl with the further proviso that ifn is 3, Z is (NH(CH2),)3N and salts thereof. 2. A compound when used as a medicament according to claim 1, wherein R' is the side chain of lysine, ornithine, arginine, aspartic acid, glutamic acid, glutamine, cysteine, methionine, serine or threonine. 3. A compound when used as a medicament according to claim 1, wherein R and R 3 are independently phenoxy, substituted phenoxy, benzyloxy or substituted benzyloxy. 4. A compound when used as a medicament according to claim 1, wherein R 4 and R 5 are independently phenoxy, substituted phenoxy, benzyloxy or substituted benzyloxy. A compound when used as a medicament according to claim 1, wherein W is -CH=CH-. 6. A compound when used as a medicament according to claim 1, wherein Q is -CH-CH-. 7. A compound when used as a medicament according to claim 1, wherein X is Cl.salkenyl or -CH2-. 8. A compound when used as a medicament according to claim 1, wherein Y is CI. 5 alkenyl or -CH 2 COMS ID No: SBMI-07107784 Received by IP Australia: Time 09:53 Date 2007-04-24 24, Apr. 2007 9:51 Shelston IP No. 2910 P. 0 0 (N c (N \O Ci 0 0 -73- 9. A compound when used as a medicament according to claim 1, wherein n is 1 or 2. A compound when used as a medicament according to claim 1, wherein Z is hydroxy, methoxy, phenethylamino, substituted phenethylamino or -NH(CH2)20(OH2)20(CH2)2NH-. 11. A compound when used as a medicament selected from the group consisting of PhCI 2 2 ,and PhCH! and salts thereof. 12. A compound of Formula I as defined in any one of claims 1 to 11, provided that the compound is not: COMS ID No: SBMI-07107784 Received by IP Australia: Time 09:53 Date 2007-04-24 -74- 8 /C R N -CH----OH wherein R 8 is benzyl, 9 R10 O R9 N-CH--C -R R 9 wherein R 9 is benzyl, R' 1 is isobutyl, benzyl, phenethyl, cyclohexylmethyl, cyclohexyl, isopropyl, secondary butyl or methyl and is hydroxy or methoxy, or 0 R12 11 14 R--CH2-CH-C-O-R 4 N R13/ \R13 wherein R 1 2 is 4-methoxy phenyl, R 13 is benzyl and R' 4 is hydrogen or benzyl. 13. The compound of any one of the preceding claims when used as a medicament wherein said compound binds to the EPO receptor. 14. A pharmaceutical composition comprising the compound of Formula I as defined in any one of the preceding claims and a pharmaceutically acceptable carrier. A method for making a pharmaceutical composition which comprises mixing a compound according to Formula I as defined in any one of claims 1 to 13 and a pharmaceutically acceptable carrier. 16. A method for modulating an EPO receptor, comprising contacting the EPO receptor with an EPO receptor modulating amount of the compound of Formula I as defined in any one of claims I to 13. S17. A method for treating a disease or condition mediated by EPO receptor comprising administering an effective amount of the compound of Formula I as defined in anyone of claims 1 to 13. 18. Use of a compound of Formula I according to any one of claims 1 to 13 in the manufacture of a medicament for treating a disease or condition mediated by an EPO q receptor. 19. A compound of Formula I when used as a medicament, substantially as herein described with reference to any one of the examples but excluding comparative examples. 20. A compound of Formula I according to claim 12, substantially as herein described with reference to any one of the examples but excluding comparative examples. 21. A pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, substantially as herein described with reference to any one of the examples but excluding comparative examples. 22. A method for modulating EPO receptor comprising administering a compound of Formula I or a pharmaceutically acceptable salt thereof, substantially as herein described with reference to any one of the examples but excluding comparative examples. 23. A method for treating a disease or condition mediated by EPO receptor comprising administering a compound of Formula 1 or a pharmaceutically acceptable salt thereof, substantially as herein described with reference to any one of the examples but excluding comparative examples. 24. Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament, substantially as herein described with reference to any one of the examples but excluding comparative examples.