AU715451B2 - N-formyl hydroxylamine containing compounds useful as ace inhibitors and/or NEP inhibitors - Google Patents

Description

WO 97/38705 PCT/US97/05744 N-FORMYL HYDROXYLAMINE CONTAINING COMPOUNDS USEFUL AS ACE INHIBITORS AND/OR NEP INHIBITORS Summary of the Invention This invention is directed to novel compounds possessing angiotensin converting enzyme (ACE) inhibitory activity and/or neutral endopeptidase (NEP) inhibitory activity and methods of preparing such compounds. This invention is also directed to pharmaceutical compositions containing such ACE and/or NEP inhibiting compounds or pharmaceutically acceptable salts thereof and the method of using such compositions.

The compounds of this invention are those of the formula (I)

R

1 0 Rio H N x A

R

including a pharmaceutically acceptable salt thereof where: x is 0 or 1; R is H, alkyl, alkenyl, aryl-(CH2)p-, heteroaryl-(CH 2 cycloheteroalkyl-(CH 2 or R can be joined together with the carbon to which it is attached to form a 3 to 7 membered ring which may optionally be fused to a benzene ring;

R

1 is H or -COR 2 where R 2 is alkyl, aryl-

(CH

2 cycloheteroalkyl- (CH 2 heteroaryl-

(CH

2 alkoxy, or cycloalkyl-(CH 2 p is 0 or an integer from 1 to 8; and A is a dipeptide derived from one or two non-proteinogenic amino acid or is a conformationally restricted dipeptide mimic as described below.

A is a dipeptide derivative of the structure 1 WO 97/38705 WO 9738705PCTIUS97/05744 A(1) R1ARi R 3

,R

I R 2 a

H

0 COR 4 where R 1 a, Rib, R 2 a and R2b are independently selected from H, alkyl, aryl-(CH 2 cycloalkyl, cycloheteroalkyl-

(CH

2 heteroaryl-

(CH

2 biphenylmethyl, or Rla and Rib or R 2 a and R2b may be joQined together to the carbon to which they are attached to form a 3 to 7 membered ring, optionally fused to a benzene ring; and

COR

4 refers to an optional 5 or 6 membered ring containing a single hetero atom and which may optionally include an R substituent (as shown) which is H, alkyl, aryl-

(CH

2 )p or cycloalkyl-

(CH

2 cycloheteroalkyl-

(CH

2 or cycloheteroaryl-(CH 2

R

3 is H, alkyl or aryl -(CH 2

R

4 is OH, Qalkyl, O-(CH2)paryl- or NRi(R 2 where RI and R 2 are independently H, alkyl, or aryl (CH 2 p or heteroaryl- (CH2)p-; with the proviso that in A(l) at least one of and N K R 2b H 0 COR4 is other than a natural 0x-axnino acid, and thus must be other than valine, leucine, phenylalanine, t~rrcisine, serine, cysteine, threonine, methionine, aspartic acid, glutainic acid, arginine, lysine or proline.

In addition, A can be a conformationally restricted dipeptide mimic which has the structure -2 WO 97/38705 PCT/US97/05744 A(2) 0 COR 4 and is a non-proteinogenic dipeptide.

Thus, the compound of formula I include

RIO

IA(1)

R

a Rib H N CH 2 R2b R o COR 4 and IA(2) R0 S (CH2) 1 H H 0 R 0 COR 4 The term "conformationally restricted dipeptide mimic" refers to a structural skeleton which has the attributes of a conventional dipeptide R O R 0 I II I II H- CH- Cbut having enhanced biological properties due to additional bonds which limit the rotational freedom.

Examples of the A(2) dipeptide mimics include any of the conformationally restricted dipeptide mimics set out below.

3 I I WO 97/38705 WO 9738705PCT/US97/05744 A(3) H 0 COR' where Y 0, S, OH 2 or 001, A(4) 1' 9 H 0 00R 4 n 0O or 1 where X OH 2 and Y 0, S, OH 2 or S(0) 0 1 2 and X 0, S when n ='l1 where Y 0, S, OH 2 or S(0)0,1,2 where Xl H, Ph, NHS0 2

RS

(R 5

H)

where Y' 0, S. NH or S(0), where X' H, Ph, NHS0 2

R

(R 5

H)

A( 1) where Y 0, S, OH 2 or S(0)01,2 where Z 0 or H, H A(14) H tero yle JS )0' H 0 a 4 where Z 0 or H, H where Y 0, S, OH 2 or S(0)0,1,2 A(16) H 00CR 4 H 00CR 4 where Y 0, S, or S(0)0,1,2 A(18 12A(19) R's H 0 C0R 4 H 0 001 where Y 0, S, OH 2 .N yR 7 C0R 4 -4 WO 97/38705 PCT/US97/05744 A(21)

A(

Ri 2 A(22) S COR 4 H 0 COR 4 A(23) J< where Y O, S, CH 2 or S(0),1,2 H O 4 X2 0, S(0) 0

CH

2 H

COR

With respect to R 1 1 and R 1 2 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl aryl substituted aryl and heteroaryl or R 11 and

R

12 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R 1 1 and R 12 taken together with the carbon to which they are attached complete a keto substituent, i.e., =O with respect to A(13) R 8

R

9 and R 7 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl aryl-(CH2)m-, substituted aryl-(CH2)m-, and heteroaryl-(CH2)m-;

R

10 and R 6 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl aryl- (CH2)m, substituted aryl and heteroaryl- (CH2)m-, or R 6 and R 10 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, R 6 and

R

8 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R 9 and R 1 0 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons; 5 I I WO 97/38705 PCTIUS97/05744 m is zero or an integer from 1 to 6;

R

4 is OH, Oalkyl, O-(CH2)m-heteroaryl, Io o ,o o

CH-O-C-R

1 5

R

14 0-(CH2)m-aryl, or -0-CH2 R or

NR

1

(R

2 where R 1 and R 2 are independently H, alkyl, aryl(CH 2 aryl or heteroaryl;

R

14 is hydrogen, lower alkyl, cycloalkyl, or phenyl;

R

15 is hydrogen, lower alkyl, lower alkoxy or phenyl;

R

1 6 is alkyl or aryl-(CH2)m-; and

R

17 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl-(CH2)m-, aryl-(CH2)m-, substituted aryl-(CH2)m-, or heteroaryl-(CH2)m-.

R

1 8 is H, alkyl or alkenyl, and R 1 8 and R 1 7 may be taken together with the carbon and nitrogen to which they are attached to complete a saturated N-containing ring of 5 or 6 ring members.

R

19 is H or an alkyl, and in R 1 9 and X (which is CH 2 together with the carbons to which they are attached may form an aromatic ring of carbons (as in The starting compounds H-A(1) and H-A(2) are described in the literature or are obtained by modifications of known procedures. For example, the starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formulas A(13), A(16), A(21), where Y (where present) is CH 2 are disclosed by Thorsett et al., J. Med.

Chem., 29, p. 251 260 (1988), Harris et al. in U.S. Patents 4,587,050, 4,587,238, 4,629,787 and Yanagisawa et al. in U.S. Patent 4,734,410.

6 WO 97/38705 PCT/US97/05744 The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formulas A(3) and A(13) where Y is S(O)n are disclosed by Yanagisawa et al., Med. Chem., p. 1984 1991 (1987) and 31, p. 422 428 (1988), Karanewsky in U.S. Patent 4,460,579, Cheung et al.

in U.S. Patent 4,594,341, and Yanagisawa et al. in U.S. Patent 4,699,905.

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(5) are disclosed by Karanewsky in U.S.

Patents 4,460,579 and 4,711,884.

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formulas A(3) (Y is -CH2-, and A(21) are disclosed by Watthey et al., J. Med. Chem., 28, p. 1511 1516 (1985) and Watthey in U.S. Patents 4,410,520, 4,470,988, 4,473,575, 4,537,885 and 4,575,503 and also by Parsons et al., Biochemical Biophysical Research Comm., 117, p. 108 113 (1983) and in U.S. Patent 4,873,235.

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(3) and Y is S or O are disclosed by Slade et al., J. Med. Chem., 28, p. 1517 1521 (1985) and in U.S. Patent 4,477,464 and Itoh et al., Chem.

Pharm. Bull., 34, p. 1128 1147 (1986) and 34, p.

2078 2089 (1986) as well as Sugihara et al. in U.S. Patent 4,548,932 (Y is 0) and Katakami et al.

in U.S. Patent 4,539,150 (Y is S).

The starting compounds of formula H-A(l) or H.-A2) wherein A(1) or A(2) is as defined in formula A(16) can be prepared by reduction of the corresponding starting compounds wherein A(1) or A(2) is as defined in formula A(3).

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in 7 WO 97/38705 PCT/US97/05744 formula A(22) are disclosed by Flynn et al in U.S.

Patent 4,973,585.

The starting compounds of formula H-A(1) or H-A(2) wherein A(l) or A(2) is as defined in formula A(10) and Y is S, -SO, or -SO2 are disclosed by Harris et al. and Patchett et al. in U.S. Patents 4,415,496 and 4,617,301.

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(10) and Y is CH2, and is as defined in formula A(23) where X 2 is CH2 is disclosed by Thorsett, Actual. Chim. Ther., 13, p. 257-268 (1986).

The starting compounds of formula H-A(l) or H-A(2) wherein A(1) or A(2) is as defined in formulas A(ll) and A(19) and A(20) are disclosed by Attwood et al., Federation of European Biochemical Studies, 165, p. 201-206 (1984) and in U.S. Patent 4,512,994 and Natoff et al., Drugs Of The Future, 12, p. 475-483 (1987).

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(12) are disclosed by Huang et al. in U.S.

Patent 4,465,679.

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(18) are disclosed by Bolos et al. in Tetrahedron, 48, p. 9567-9576 (1992).

The starting compounds of formula H-A(l) or H-A(2) wherein A(1) or A(2) is as defined in formulas A(4) and A(15) are disclosed in European Patent Application 0629627A2.

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(9) are disclosed in U.S. application Serial No. 100,408 (file HA611a).

8 WO 97/38705 PCT/US97/05744 The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formulas A(7) and A(8) are disclosed in European Patent Application 481,522 (Flynn et al) and European Patent Application 0534363A2 (Warshawsky et al).

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(14) are disclosed in U.S. application Serial No. 153,854 (file HA615).

The starting compounds of formula H-A(1) or H-A(2) wherein A(1) or A(2) is as defined in formula A(17) are disclosed in European Patent Application 0599444A1 (Barrish et al).

15 In addition, in accordance with the present invention, a pharmaceutical composition is provided which includes a therapeutically effective amount of compound I and a pharmaceutically acceptable carrier therefor.

20 The pharmaceutical composition as defined above will be useful in the treatment of cardiovascular diseases such as hypertension and/or congestive heart failure.

Furthermore, in accordance with the present invention, a method is provided for treating a cardiovascular disease such as hypertension and/or congestive heart failure, as well as other diseases as set out hereinafter, which includes the step of administering to a mammalian species, including humans, dogs and cats, a therapeutically effective amount of a composition as defined above.

9 Detailed Description of the Invention Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

The term "alkyl" or "lower alkyl" refers to straight or branched chain radicals having up to and including ten carbon atoms, preferably up to and including six carbon atoms, which may o gC 9a C:VWINORO STACYSpCI%54i997.DOC WO 97/38705 PCT/US97/05744 optionally include one, two, or three substituents including a hydroxy, amino, alkyl, cycloalkyl, aryl, halo, trifluoromethyl, cyano, -NH(lower alkyl), -N(lower alkyl) 2 lower alkoxy, lower alkylthio, carboxy or heteroaryl.

The term "alkenyl" refers to straight or branched chain radicals of 3 to 10 carbon atoms having one or two double bonds, preferably straight chain radicals of 3 to 5 carbons having one double bond, which may optionally be substituted with one, two or three substituents including alkyl, aryl, cycloalkyl, hydroxy, amino, halo, trifluoromethyl, cyano, -NH(lower alkyl), -N(lower alkyl)2, lower alkoxy, lower alkylthio, carboxy or heteroaryl.

The terms "alkoxy" or "lower alkoxy" and "alkylthio" or "lower alkylthio" refer to such alkyl groups as defined above attached to an oxygen or sulfur.

The term "cycloalkyl" refers to saturated rings of 3 to 7 carbon atoms.

The term "halo" refers to chloro, bromo, fluoro, and iodo.

The term "aryl" refers to aromatic groups containing 6 to 10 carbons, preferably phenyl, 1naphthyl, and 2-naphthyl, which may optionally contain one, two or three substituents selected from alkyl, alkoxy, alkylthio, halo, hydroxy, trifluoromethyl, -SO 2

NH

2 amino, -NH(lower alkyl), or -N(lower alkyl) 2 di- and tri-substituted phenyl, 1-naphthyl, or 2-naphthyl, wherein said substituents are preferably selected from methyl, methoxy, methylthio, halo, hydroxy, and amino.

The term "heteroaryl" refers to unsaturated rings of 5 or 6 atoms containing one or two O and S atoms and/or one to four N atoms provided that the total number of hetero atoms in the ring is 4 or less, which may optionally be substituted with one, 10 WO 97/38705 PCT/US97/05744 two or three substituents which include alkyl, aryl, cycloalkyl, alkoxy or halo. The heteroaryl ring is attached by way of an available carbon or nitrogen atom. Preferred heteroaryl groups include or 4-pyridyl, 4-imidazolyl, 4-thiazolyl, 2and 3-thienyl, and 2- and 3-furyl. The term heteroaryl also includes bicyclic rings wherein the five or six membered ring containing O, S, and N atoms as defined above is fused to a benzene or pyridyl ring. Preferred bicyclic rings are 2- and 3-indolyl and 4- and 5-quinolinyl. The mono or bicyclic heteroaryl ring can also be additionally substituted at an available carbon atom by a lower alkyl, halo, hydroxy, benzyl, or cyclohexylmethyl.

Also, if the mono or bicyclic ring has an available N-atom such N atom can also be substituted by an Nprotecting group such as

CH

2

CH

2 -S0 2 -CH3 2,4-dinitrophenyl, lower alkyl, benzyl, or benzhydryl.

The compounds of formula I of the invention may be prepared as outlined in Reaction Scheme I set out below (where x is 0 or 1).

11 WO 97/38705 PCT/US97/05744 Reaction Scheme I

SCO,

2 H PG'-O-NH 2 PG1'-CO, CO 2 H optionalchiral PG 1 '-CO CO 2

H

R 2 H F resolution H R 2 H R 3* R 3 (optically pure or (Q is CH 2 where x-1 and enriched) Q is H, Br where x=0) H-A(1) or H-A(2) I Acylation H2 R 4

PG

1 -O H-CO-O-COR (4a) 0 COR 4 -XfrR AYA 0 COR 4 standard peptide coupling R 4 0 P .I removal of one or both PG'-CL Mk protecting groups (PG 1 H Cx..Lr R H 0 COR 4 and/or R 4 R H 0 CC 0 5

IA

As shown in Scheme I, acid 2 may be reacted with a suitably O-protected PG 1 is benzyl, pmethoxybenzyl, tetrahydropyranyl, trityl, benzhydryl, etc.) hydroxylamine to give the adduct 3. Compound 3 may be coupled directly with amine H-A(1) or H-A(2) to give a mixture of diastereomers which may be separated or preferably compound 3 may be optically enriched or purified, employing conventional techniques, to give 3*.

Subsequent coupling with H-A(1) or H-A(2) gives 4 in diastereomerically enriched or pure form.

Reaction of the hydroxylamine nitrogen of 4 with a formylating agent affords 5. At this point one or both protecting groups may be removed, either sequentially or simultaneously, to produce compound of the invention IA. For example, when PG 1 is benzyl and R 4 is Obenzyl, both may be removed by hydrogenolysis. When PG 1 is benzyl and R 4 is -Omethyl or -Oethyl, the PG 1 group may be removed by hydrogenolysis and the ester group may be converted to the acid by base hydrolysis. PG 1 12 1 WO 97/38705 PCT/US97/05744 groups such as THP or trityl may be removed by treatment with strong acid such as hydrogen chloride or trifluoro acetic acid in a protic solvent.

Alternately, compounds of the invention IA may be obtained by the route depicted in Scheme II (where x is 0 or 1).

Reaction Scheme II Acylation PG'-O,,N .C0 2 H H-CO-O-COR

PG

1 C02H optional chiral H R 4a R resolution H

R

3 0 7 H-A(1) or H-A(2)

PCO

2 H H2 COR 4 PG'-O" I i H_ 0 R H COR 4 R standard peptide coupling o 0 7* As seen in Reaction Scheme II, compound 3 may be formylated with an formylating agent 4a to give acid compound 7. This acid may be coupled with A(1) or A(2) directly or optically resolved to give 7* and then coupled to give compound Compound 5 is then converted to compound of the invention IA as described above.

The compounds of formula I of the invention contain one or more asymmetric centers. Thus, these compounds can exist in diastereoisomeric forms or in mixtures thereof and all of such forms are within the scope of this invention. The above described processes can utilize racemates, enantiomers, or diastereomers as starting materials. When diastereomeric compounds are prepared, they can be separated by conventional 13

I

WO97/38705 PCT/US97/05744 chromatographic or fractional crystallization methods.

The compounds of formula I of the invention can be isolated in the form of a pharmaceutically acceptable salt. Suitable salts for this purpose are alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, and salts derived from amino acids such as arginine, lysine, etc. These salts are obtained by reacting the acid form of the compound with an equivalent of base supplying the desired ion in a medium in which the salt precipitates or in aqueous medium and then lyophilizing.

The compounds of formula I of the invention are inhibitors of angiotensin converting enzyme and/or neutral endopeptidase. Thus, the compounds of formula I including their pharmaceutically acceptable salts are useful in the treatment of physiological conditions in which either angiotensin converting enzyme inhibitors or neutral endopeptidase inhibitors have been shown to be useful. Such conditions include cardiovascular diseases, particularly, hypertension, congestive heart failure, renal failure, and hepatic cirrhosis, as well as analgesic activity. The compounds of formula I are also inhibitors of other metalloproteases such as the matrix metalloproteases, for example, gelatinase, collagenase and stromylysin and thus are useful in the treatment of osteroarthritis, rheumatoid arthritis, metastatic tumors, and angiogenesis.

Diuresis, natriuresis, and blood pressure reduction are produced in a mammalian host such as man by the administration of from about 1 mg. to about 100 mg. per kg. of body weight per day, preferably from about 1 mg. to about 50 mg. per kg.

14 WO 97/38705 PCT/US97/05744 of body weight per day, of one or more of the compounds of formula I or a pharmaceutically acceptable salt thereof. The compounds of formula I are preferably administered orally, but parenteral routes such as subcutaneous, intramuscular, and intravenous can also be employed. The daily dose can be administered singly or can be divided into two to four doses administered throughout the day.

The ACE and/or NEP inhibitors of formula I can be administered in combination with human ANF 99 126. Such combination would contain the inhibitor of formula I at from about 1 to about 100 mg. per kg. of body weight and the human ANF 99 126 at from about 0.001 to about 0.1 mg. per kg. of body weight.

The ACE and/or NEP inhibitors of formula I can be administered in combination with other classes of pharmaceutically active compounds. For example, a calcium channel blocker, a potassium channel activator, a cholesterol reducing agent, etc.

The ACE and/or NEP inhibitors of formula I or a pharmaceutically acceptable salt thereof and other pharmaceutically acceptable ingredients can be formulated for the above described pharmacetical uses. Suitable compositions for oral administration include tablets, capsules, and elixirs, and suitable compositions for parenteral administration include sterile solutions and suspensions. About 10 to 500 mg. of active ingredient is compounded with physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring, etc., in a unit dose form as called for by accepted pharmaceutical practice.

15

I

WO 97/38705 PCT/US97/05744 Preferred compounds of the invention are those of formula I wherein

R

1 is H, x is 1, R is alkyl or arylalkyl, and A is preferably Ra Rib where o is preferably a non-proteinogenic amino acid portion wherein, Rla and R 1 b are each independently alkyl such as methyl or ethyl, or arylalkyl such as benzyl, or Rl a and R 1 b together with the carbon to which they are attached form a 3-7 membered ring, preferably a 5-membered ring, or

R

la and/or R 1 b is biphenylmethylene and the other may be H.

Also preferred are compounds where A is R2.

N R 2 b preferably where COR 4 and is a nonproteino-genic amino acid where R 3 is H, alkyl, such as methyl or ethyl, aryl such as phenyl, or arylalkyl, such as benzyl,

R

2a and R 2 b are independently selected from H, alkyl, aryl, arylalkyl (with at least one of R 2a and R 2 b being other than H) or R 2a and R 2 b together with the carbon to which they are attached form a 3-7 membered ring, preferably 5- or 6-membered ring.

Also preferred are compounds where A is A(2) wherein R 4 is OH.

The following Examples represent preferred embodiments of the present invention.

16 I I WO 97/38705 PCT/US97/05744 Example 1 0 OHON N H 0 CO 2

H

A.

Boc, N N H O A(1) 0

II

H M+C- OC(CH 3 3

COOH

NO

2 A solution of BOC-L-serine (24.3 g, 0.118 mole) in dry dimethylformamide (25 ml) was added dropwise over a period of 1.0 hour to a cooled (00, ice-salt bath) suspension of 60% NaH (10.1 g, 0.25 mole) in dry dimethylformamide (200 ml) and stirring was continued at 00 until the frothing subsided (ca. 2.0 hours). The reaction mixture was treated dropwise with l-fluoro-2-nitrobenzene (14.3 ml, 0.13 mole) over a period of 20 minutes, stirred at 00 under argon for 4.0 hours then poured into ice-water (750 ml) and extracted with Et20 (2 x 100-ml). The aqueous phase was brought to pH with 6 N HC1 (70 ml), extracted with EtOAc (3 x 500 ml) and the combined organic extracts were washed with brine (100 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo.

The crude product mixture was chromatographed on a silica gel column (Merck), eluting the column with 17 WO 97/38705 PCT/US97/05744 CH2Cl2:CH 3 0H:HOAc (100:5:0.2) to give title compound as a thick yellow syrup (27.222 g, 70.7%) with consistent 1 H-NMR and 13 C-NMR spectral data.

TLC: Rf 0.27 (Silica gel; CH2C12:CH30H:HOAc- 100:5:0.5; UV, PMA).

A(2).

0

II

H M+C- OC(CH 3 3

COOH

NH

2 A solution of Part A(1) compound (27.1 g, 83 mmoles) in dry methanol (500 ml) was treated with 10% Pd/C (900 mg) and hydrogenated at 40 psi for 2.0 hours. The reaction mixture was filtered through a Celite® pad in a millipore unit, washing the pad well with CH30H (5 x 100 ml). The dark filtrate was evaporated to dryness and dried in vacuo to give a dark solid. The crude product was triturated with CH2Cl2:Hexane to give title compound as a light tan solid (17.69 g, 71. with consistent 1H-NMR and 13 C-NMR spectral data. TLC: Rf 0.15 (Silica gel; CH2Cl2:CH30H:HOAc- 20:1:1;

UV).

A(3).

BocA H O A solution of Part A(2) compound (16.69 g, 56.3 mmoles) in dry dimethyformamide (121 ml) was treated with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (10.64 g, 55.5 mmoles) and stirred at room temperature for 3.0 hours. The reaction mixture was partitioned between EtOAc (2 x 492 ml) 18 WO 97/38705 PCT/US97/05744 and 1.0 N NaHC03 (492 ml), and the combined organic extracts were washed with H20 (3 x 492 ml), brine (492 ml), dried (anhydrous MgS04), filtered, evaporated to dryness and dried in vacuo. The crude product was chromatographed on a silica gel column (Merck), eluting the column with EtOAc:Hexane mixtures 1:2; 1:1) to give title compound as off-white crystals (10.5 g, 72.4%) with consistent 1H-NMR and 13 C-NMR spectral data. TLC: Rf 0.40 (Silica gel; EtOAc:Hexane- 1:4; UV).

B.

H 0 CO 2 Bn A solution of Part A compound (640 mg, 2.30 mmol) in dry THF (12 mL) at 0°C was treated with LiN(TMS) 2 (1.0 M in THF, 2.60 mL, 2.60 mmol) followed approximately 30 seconds later with benzyl bromoacetate (475 4L, 687 mg, 3.0 mmol). After minutes, the mixture was quenched with saturated NH4C1, diluted with H20, and extracted with EtOAc.

The EtOAc extract was washed with H 2 0 and brine, then dried (Na2SO4), filtered and stripped to give a yellow oil. Flash chromatography (Merck SiO 2 3 /7-EtOAc/hexanes as eluant) provided title compound (967 mg, 98%) as a colorless oil/foam.

C.

HCI'H

2 N 1 0 CO 2 Bn 19 WO 97/38705 PCTIUS97/05744 A solution of Part B compound (960 mg, 2.25 mmol) in 1,4-dioxane (4 mL) was treated with a solution of 4.0 M HC1 in 1,4-dioxane (6 mL) at room temperature. After 3 hours, the mixture was concentrated in vacuo, triturated with Et20 to give a solid and stripped to afford title compound (858 mg, 105% of theory).

m.p. 152-155 0

C.

D.

0 N

N

H O C02Bn 0

I-

D(1).

COOH

A solution of benzylmalonic acid (23.06 g, 0.12 mole) in H 2 0 (200 mL) was treated with 37% solution (278.4 mL) and 40% aqueous (CH 3 2

NH

mL, 0.31 mole) then stirred overnight at room temperature under argon. The clear solution was heated to an internal temperature of 90 0 C for hours (at which time gas evolution had ceased), cooled and acidified to pH 1.0 with 12 N HCl mL). The white precipitates were filtered off, washed with H 2 0 (3 x 25 mL) and dried in vacuo to give title compound as a white solid (12.85 g, 66.6%) with consistent 1 H-NMR and 13 C-NMR spectral data. TLC: Rf 0.63 (Silica gel; CH 2 Cl 2 :MeOH- 9:1; UV). m.p. 66-68 0

C.

20 WO 97/38705 PCT/US97/05744 D(2).

H

COOH

00^ Med. Chem. 28, 1985, 1167) A solution of Part D(1) compound (8.9 g, 54.9 mmoles) and O-benzylhydroxylamine (26.7 g, 0.23 mole) in absolute EtOH (9.0 ml) was refluxed for 7 days, cooled to room temperature and evaporated to dryness. The residual syrup was dissolved in 1.0 N NaOH (55 ml), stirred for minutes then extracted with EtOAc (4x 18 ml). The organic phase was washed with H20 (3 x 10 ml) and the aqueous extracts were combined and acidified to pH 2.0 with 1.0 N HC1 (62 ml). The acidic aqueous phase was then extracted with EtOAc (5 x 75 ml) and the combined organic extracts washed with H20 (2 x ml), dried (anhydrous Na2S04), filtered, evaporated to dryness and dried in vacuo. The crude product (3.93 g, 25.1%) was triturated with 2 x 25 ml) and all solids obtained were dissolved in CH2C12 and filtered, washing the insoluble precipitates with CH2C12.

The clear filtrate was evaporated and dried in vacuo to give title compound as an opaque colorless solid with consistent 1 H-NMR and 13

C-NMR

spectral data.

TLC: Rf 0.33 (Silica gel; CH2C12:MeOH- 9:1; UV, PMA) M.p. 69-71 0

C.

21 WO 97/38705 PCTfUS97/05744 D(3).

OHC

COOH

0O A cooled ice-salt bath) mixture of HCOOH (17.5ml) and acetic anhydride (Ac20) (1.75 ml) was stirred for 20 minutes, treated with Part D(2) compound (1.0 g, 3.5 mmoles) and stirring was continued at 0°C for another 3.0 hours. The reaction mixture was stripped to dryness, evaporated from Et20 (2 x 25 ml), toluene (20 ml) and hexane (2 x 50 ml) then dried in vacuo to give title compound as a thick syrup (1.096 g, 100% crude yield) with consistent 1 H-NMR and 13 C-NMR spectral data. TLC: Rf 0.23 (Silica gel; CH2Cl2:MeOH- 9:1; UV, PMA).

D(4).

o r NU H 0 C02Bn A solution of Part D(3) compound (366 mg, 1.19 mmol) in CH 2 C1 2 (9 mL) at 0°C was treated with HOBT hydrate (210 mg) followed by EDAC (230 mg, 1.20 mmol). After 20 minutes, the mixture was treated with Part C amine hydrochloride 3 (390 mg, 1.07 mmol) followed by 4-methylmorpholine (200 pL, 184 mg, 1.8 mmol). The mixture was stirred at 0 C for 1 hour and at room temperature for 2 hours.

The reaction was partitioned between EtOAc and

KHSO

4 The EtOAc extract was washed successively 22 WO 97/38705 PCTIUS97/05744 with H 2 0, 50% saturated NaHC0 3 and brine, then dried (Na 2

SO

4 filtered and stripped. Flash chromatography (Merck Si0 2 50% to 60% EtOAc in hexanes as eluant) provided title compound (550 mg, 84%) as a white foam which was shown by NMR and HPLC to be a 1:1 mixture of diastereomers.

E.

O HO No SN H o CO 2

H

H Isomer

A

A solution of Part D compound (535 mg, 0.87 mmol) in MeOH (10 mL) was hydrogenated (balloon) over 10% Pd/C (123 mg) at room temperature for 2.75 hours. The solvent was filtered through Celite and the filtrate was stripped to give a diastereomeric mixture of title Isomer A and Isomer B SHON

N

O H O CO 2

H

H

SIsomer B Trituration of a solution of the residue in MeOH with Et20 provided 350 mg of the diastereomeric mixture. Approximately 255 mg of this mixture was separated by preparative HPLC ('YMC S5 ODS 30 x 250 mm column; flow rate 25 mL/min detecting at 220 nm; 40 to 100% B over a 30 minute linear gradient (solvent A: 90%H 2 0-10% MeOH-0.1% TFA; solvent B: 10% H 2 0-90% MeOH-0.1% TFA); title Isomer A tR 14.4 min; separation performed in three runs). The desired fractions were stripped, 23 WO 97/38705 PTU9/54 PCTIUS97/05744 azetroped with EtOAc, re-dissolved in EtOAc and triturated with Et 2 O to give title Isomer A (105.5 mg) as an off-white solid.

MS: (M+NHi 4 459; 440 HPLC YMC S3 ODS column (6.0 x 150 mm); eluted with B:A solvent mixture, 40 to 100% B over a 20 minute linear gradient (solvent A: 90% H 2 0-10% MeOH-0.2%

H

3 P0 4 solvent B:0% H 2 0-90% MeOH-0.2% H 3 P0 4 flow rate 1.5 mL/min detecting at 220 tR= 9 6 7 min Anal. Calc d for C 2 2

H

2 3

N

3 0 7 6H 2 0.0. lEtOAc.0 .lEt 2

O

C, 56.29; H, 5.80; N, 8.64 Found: C, 56.21; H, 5.15; N, 8.29.

24 WO 97/38705 PCT/US97/05744 Example 2

HO

0 HON N ON H o CO 2

H

H

A solution of Example 1 Part E Isomers A and B (1:1 mixture of diastereomers, 535 mg, 0.87 mmol) in MeOH (10 mL) was hydrogenated (balloon) over 10% Pd/C (123 mg) at room temperature for 2.75 hours. The solvent was filtered through Celite and the filtrate was stripped to give a diastereomeric mixture of Isomers A and B. Trituration of a solution of the residue in MeOH with Et20 provided 350 mg of the diastereomeric mixture.

Approximately 255 mg of this mixture was separated by preparative HPLC (YMC S5 ODS 30 x 250 mm column; flow rate 25 mL/min detecting at 220 nm; 40 to 100% B over a 30 minute linear gradient (solvent A: 2 0-10% MeOH-0.1% TFA solvent B: 10% H 2 0-90% MeOH-0.1% TFA); Isomer B tR 18.6 min; separation performed in three runs). The desired fractions were stripped, azetroped with EtOAc, re-dissolved in EtOAc and triturated with Et20 to give Isomer B (88.0 mg) as an off-white solid.

MS: (M+NH 4 459; 440 HPLC YMC S3 ODS column (6.0 x 150 mm); eluted with B:A solvent mixture, 40 to 100% B over a 20 minute linear gradient (solvent A: 90%H 2 0-10% MeOH-0.2%

H

3 P0 4 solvent B:0% H 2 0-90% MeOH-0.2% H 3

PO

4 flow rate 1.5 mL/min detecting at 220 nm; tR 13.8 min 25 WO 97/38705 PCT/IUS97/05744 Anal. Calc'd for C 22

H

23

N

3 0 7 -1.5H 2 0.0.2Et 2 0 C, 56.66; H, 5.84; N, 8.69 Found: C, 56.84; H, 5.22; N, 8.42.

Example 3 Me 0 HO N Me H 0 CO2H

H

(f

A.

BnO H,

,CO

2

H

(1R, 2S)-(-)-ephedrine salt A solution of Example 1 Part D(1) compound BnO C02H (2.563 gm, 8.98 mmol) in CH 3 CN mL) was treated with (1R,2S)-(-)-ephedrine (1.522 gm, 9.2 mmol) and stirred until homogeneous. Most of the solvent was removed by rotary evaporation and the residue was dissolved in Et20 (25 mL) and treated with hexane (16 mL) in portions until the mixture was slightly turbid. The solution was seeded and let stand overnight at room temperature.

The precipitate was collected by filtration and rinsed with 1:1 Et20:hexanes and dried to afford 2.101 gm of white crystals ([aID -16.40 (c 0.6,

CH

2 C1 2 The solid (2.087 gm) was dissolved in

CH

2 C12, concentrated and diluted with Et20 (18 mL) and hexane (8 mL) and seeded. The precipitate was collected by filtration and washed with 1:1followed by hexanes to give title 26 WO 97/38705 PCT/US97/05744 compound (1.995 gm) which was diastereomerically enriched in one isomer but not diastereomerically pure -17.0° (c 0.6, CH 2 C1 2 mp 110-114 0

C

Material suitable for x-ray crystallographic analysis was obtained by repeated recrystallization of the solid from CH 3 CN. mp 117-119 0

C;

-19.70 (c 0.4, CH 2 C1 2

B.

H

2

N

CO

2 Et B(1).

OH

(Pht is phthaloyl) Pht=N C0 2

H

To a stirred solution of L-(+)-hydroxynorleucine (75 g, 509.6 mmole) and sodium carbonate (54 g, 509.6 mmole) in water (900 ml) at room temperature under argon was treated with N-ethoxycarbonyl-phthalimide (111.7 g, 509.6 mmole). After being stirred for 2.0 hours, the resulting solution was filtered through a pad of celite. The filtrate was cooled in an ice bath and carefully acidified to pH=3 with 6N HC1 solution. The white solid which had precipitated was filtered and dried over

P

2 0 5 in vacuo to afford Compound 1 (124.5 g) in 88.1% yield.

M.P. 162 0

C

H

1 -NMR (DMSO): d 1.32 6H), 2.13 2H), 4.38 OH), 5.75 1H), 7.92 4H) ppm 27

I

WO 97/38705 PCT/US97/05744 B(2).

OH

Pht=N CO 2 Bn To a stirred slurry of Part B(1) compound (124.5 g, 0.449 mole) and cesium carbonate (73.2 g, 0.225 mole) in DMF (1.25 L) at room temperature under argon was added benzyl bromide (98.4 g, 0.575 mole). After 2.5 hours, the resulting solution was poured into EtOAc (3.0 washed with water (3X), LiCI solution and brine, dried over anhydrous Mg 2 S04 and evaporated in vacuo to afford title compound (142 g) as an oil in 86.1% yield.

H1-NMR (CDCl 3 d 1.50 4H), 2.32 2H), 3.62 2H), 4.91 (dd, 1H), 5.22 2H), 7.31 (m, 7.77 2H), 7.86 2H) ppm

C

1 3 -NMR (CDC13): 22.62, 28.46, 31.91, 52.32, 62.32, 67.46, 123.55, 128.06, 128.31, 128.53, 131.77, 134.23, 135.28, 167.76, 169.25 ppm B(3).

H

Pht=N CO 2 Bn S To a stirred and chilled (-78 0 C, Dry ice- IPA bath) oxalyl chloride solution (2.0 M solution in CH 2 C12, 16.3 ml, 32.6 mmole) under argon was added dropwise a solution of dimethyl sulfoxide (4.64 ml, 65.32 mmole) in dry CH 2 C1 2 (10 ml).

After the addition was complete, the solution was 28 WO 97/38705 PCT/US97/05744 stirred at -780 for 15 minutes, then treated with a solution of Part B(2) compound (10g, 27.22 mmole) in dry CH 2 C12 (70 ml), stirred at -780 for another minutes and slowly treated with triethylamine (16 ml). The resulting solution was stirred at -780 for 15 minutes, gradually warmed up to 00, poured into 1:1 EtOAc-Et2O (500 ml), washed with N HC1 solution, water and brine, dried over anhydrous Mg 2

SO

4 and evaporated in vacuo to afford title compound (10 g) as a light yellow oil in 100% yield.

H

1 -NMR (CDC1 3 d 1.66 2H), 2.40 4H), 4.90 (dd, 1H), 5.18 2H), 7.35 5H), 7.74 (m, 2H), 7.86 2H), 9.72 1H) ppm

CI

3 -NMR (CDC13): 18.66, 27.99, 42.87, 51.83, 67.47, 123.50, 128.00,128.26, 128.44, 131.58, 134.21, 135.04, 167.55, 168.80, 201.31 ppm B(4).

OH

Pht=N CO0Bn A stirred and chilled ice bath) solution of Part B(3) compound (10.1 g, 27.64 mmole) in dry CH 2 C1 2 (100 ml) under argon was treated with a solution of trimethylaluminum (2.0 M solution in hexane, 23.4 ml, 46.8 mmole). The resulting solution was stirred for 45 minutes, quenched with 100 ml of a saturated NH 4 C1 solution (foaming) and partitioned between 1:1 (400 ml). The organic layer was separated and the aqueous layer was.re-extracted with EtOAc (2x150 ml). The organic extracts were combined, washed 29 WO 97/38705 PCTIUS97/05744 with brine, dried over anhydrous Mg2SO4 and evaporated in vacuo to afford title compound (10.3 g) as a gum in 98.7% yield.

TLC: Silica gel, 6:4 EtOAc-hexane, Rf 0.42, UV and PMA.

H1-NMR (CDC13): d 1.12 3H), 1.43 4H), 3.73 2H), 4.90 (dd, 1H), 5.19 2H), 7.30 5H), 7.76 2H), 7.86 2H) ppm

C

13 -NMR (CDC13): 22.5, 23.40, 28.47, 28.59, 38.20, 38.34, 52.20.67.35, 67.51, 123.43, 127.94, 128.19, 128.41, 131.65, 134.11, 135.16, 167.62, 167.67, 169.13 ppm Pht=N

CO

2 Bn To a stirred and chilled (-780C, Dry ice- IPA bath) oxalyl chloride solution (2.0 M solution in CH 2 C12, 257.3 ml, 514.6 mmole) under argon was added CH 2 C12 (300ml). To this solution, a solution of dimethyl sulfoxide (80.4 g, 1.03 mole) in dry

CH

2 C1 2 (30 ml) was added dropwise. After the addition was complete, the reaction mixture was stirred at -780 for 20 minutes, treated with a solution of Part B(4) compound (151 g, 395.88 mmole) in dry CH 2 C1 2 (700 ml), stirred at -780C for another 20 minutes and slowly treated with triethylamine (300 ml). The resulting solution was stirred at -780 for 15 minutes, gradually warmed up to 00, poured into 1:1 EtOAc-Et20 (3 washed with 1.0 N HC1 solution, water and brine, dried over anhydrous Mg2S0 4 and evaporated in vacuo to 30 WO 97/38705 PCT/US97/05744 afford title compound (149.4 g) as a yellow oil in 99.5% yield.

TLC: Silica gel, 6:4 EtOAc-hexane, Rf=0.5, UV and

PMA.

H

1 -NMR (CDC1 3 d 1.60 2H), 2.10 3H), 2.26 2H), 2.47 4.90 (dd, 1H), 5.19 2H), 7.30 5H), 7.74 2H), 7.84 2H) ppm C1 3 -NMR (CDC1 3 20.15, 27.93, 29.84, 42.47, 51.89, 67.40, 123.46, 127.97, 128.23, 128.43, 131,61, 134.17, 135.10, 167.57, 168.93, 207.80 ppm B(6).

OH

Pht=N CO 2 Bn A chilled (-78 0 C, Dry ice-IPA Bath) and stirred solution of titanium(IV) chloride (112.05 g, 590.65 mmole) in CH 2 C12 (1.5 L) under argon was treated with methylmagnesium chloride (3 M solution in THF, 196.9 ml, 590.65 mmole). The black solution was allowed to warm up to -35 0 C and a solution of Part B(5) compound (149.4g, 393.77 mmole) was added dropwise. After the addition was complete, the resulting solution was allowed to warm up to 0°C, stirred at 0°C for 2 hours and quenched with saturated NH 4 Cl solution. The CH 2 C12 layer was separated. The aqueous layer was extracted with CH 2 C1 2 (2x700 ml). The CH 2 C12 extracts were combined, washed with brine, dried over anhydrous Mg 2 SO4 and evaporated in vacuo. The black residue was passed through a pad of silica 31 WO 97/38705 PCTIUIS97/05744 gel Merck, 230-400 mesh, 900 g) eluting with EtOAc-hexane to afford a tlc-homogeneous title compound (144.8 g) as a yellow oil in 93% in yield.

TLC: Silica gel, 1:1 EtOAc-hexane, Rf=0.4, UV and

PMA.

H

1 -NMR (CDC1 3 d=1.14 6H), 1.45 4H), 2.30 2H), 4.90 (dd, 1H), 5.19 2H), 7.30 7.74 2H), 7.86 2H) ppm

C

13 -NMR (CDC1 3 20.88, 29.00, 29.17, 42.78, 52.13, 67.35, 70.47, 123.44,127.95, 128.19, 128.41, 131.66, 134.11, 167.66, 169.14 ppm B(7).

Pht=N C02Bn A stirred solution of Part B(6) compound (44.3 g, 364.89 mmole) and azidotrimethylsilane (63.06 g, 547.34 mmole) in dry CH 2 C12 (2.2 L) at room temperature under argon was treated with boron trifluoride diethyl etherate (67.32 g, 474.36 mmole). After being stirred for 5 days, the resulting solution was quenched with water (1.5 L).

The organic layer was separated, washed with saturated NaHCO 3 solution, water and brine, dried over anhydrous Mg 2

SO

4 and evaporated in vacuo. The residue was chromatographed on a column of silica gel Merck, 230-400 mesh, 700 g) eluting with EtOAc-hexane to afford a tic-homogeneous title compound (124.9 g) as a light yellow oil in 81.3% yield.

32 WO 97/38705 PCT/US97/05744 TLC: Silica gel, 3:7 EtOAc-hexane, Rf=0.5, UV and

PMA.

HI-NMR (CDC13): d=1.20 6H), 1.45 4H), 2.30 2H), 4.90 (dd, 1H), 5.19 2H), 7.30 7.74 2H), 7.86 2H) ppm

C

13 -NMR (CDC1 3 20.97, 25.67, 25.92, 28.80, 40.53, 52.02, 61.16, 67.40, 123.47, 127.97, 128.23, 128.43, 131.66, 134.14, 135.12, 167.60, 169.01 ppm B(8).

Pht=N N H 0 A solution of Part B(7) compound (124.8 g, 296.81 mmole) and 10% Pd/C (32g) in dry DMF (2.0 L) was hydrogenated for 24 hours. After completion, argon was bubbled through the reaction mixture to remove excess hydrogen and methyl sulfide (2.6 ml) was added to poison the palladium. To this solution l-hydroxybenzotriazole hydrate (46.74 g) was added and followed by ethyl-3(3-dimethylamino)propylcarbodiimide hydrochloride salt (68.74 g).

The resulting solution was stirred at room temperature under argon for 3.5 hours, diluted with EtOAc (2 L) and filtered through a pad of celite.

The filtrate was washed with 0.5 N HC1 solution, saturated NaHCO 3 solution, and brine, dried over anhydrous Mg 2 S04 and evaporated in vacuo to give a gum. This was triturated with Et20-hexane to afford a tlc-homogeneous title compound (74.5 g) as a white solid in 87.7% yield.

TLC: Silica gel, 3:7 EtOAc-CH 2 C12, Rf=0.35, UV and

PMA.

33 WO 97/38705 PCT/US97/05744

H

1 -NMR (CDC1 3 d=1.30 3H), 1.45 3H), 1.74 2H), 1.96 3H), 2.74 1H), 4.98 1H), 6.00 1H), 7.20 2H), 7.85 2H) ppm

C

13 -NMR (CDC1 3 23.89, 26.65, 29.58, 33.32, 40.68, 52.69, 54.51, 123.34, 123.15, 133.87, 168.06, 171.03 ppm B(9).

Ph 3

C,

H 0 A stirred solution of Part B(8) compound (74.5 g, 260.19 mmole) in a mixture of CH30H (900 ml) and CH 2 C1 2 (250 ml) at room temperature under argon was treated with hydrazine monohydrate (18.24 g, 364.26 mmole). After 48 hours, the solid was filtered off and the filtrate was evaporated in vacuo to give a solid (41 g).

To a stirred solution of the above solid (41 g) in CH 2 C1 2 (2 L) at room temperature under argon was added triethylamine (50 ml) and triphenylmethyl chloride (83.41 After hours, the resulting slurry was diluted with EtOAc, washed with water and brine, dried over anhydrous Mg 2 So 4 and evaporated in vacuo to give a gum. This was triturated with Et20-pentane to give title compound (100.1 g) as a white solid in 96.5% yield.

TLC-: Silica gel, 6:4 EtOAc-hexane, Rf=0.53, UV and

PMA.

H

1 -NMR (CDC1 3 d=1.00 3H), 1.10 3H), 1.46 6H), 3.36 IH), 4.03 1H), 5.20 1H), 6.00 1H), 7.20 2H), 7.85 2H) ppm 34 WO 97/38705 PCT/US97/05744

C

13 -NMR (CDC1 3 22.86, 25.81, 33.50, 34.23, 40.16, 51.97, 55.60, 71.89, 126.22, 127.61, 128.96, 146.48, 176.71 ppm

H

2

N

0 CO 2 Et To a stirred solution of Part B(9) compound (50 g, 125 mmole) in dry THF (1020 ml) at room temperature under argon was added simultaneously (at same rate) a solution of lithium bis(trimethylsily)amide (1.0 M solution in THF, 627.3 ml, 627.3 mmole) and a solution of ethyl bromoacetate (104.8 g, 627.3 mmole) in THF (523 ml) over the period of 1.0 hour. After the addition was complete, the solution was stirred for hours, quenched with saturated NH 4 C1 solution liter) and extracted with EtOAc (3x700 ml). The EtOAc extracts were combined, washed with saturated NaHC0 3 solution and brine, dried over anhydrous Mg 2

SO

4 and evaporated in vacuo to afford a black oil. The experiment was repeated on the same scale to give a similar result. The combined black oils was chromatographed on a column of silica gel (E.

Merck, 230-400 mesh, 1.6 kg) eluting with EtOAchexane to give a light yellow oil. This was dissolved in dry CH 2 C12 (2 L) and treated with trifluoroacetic acid (78 ml). The solution was stirred at room temperature under argon for hour and then evaporated in vacuo at 300. The residue was diluted with 1.0 N HC1 solution (400 ml) and washed with Et20 (2x400 ml). The aqueous was carefully neutralized to pH=7-8 with solid NaHC0 3 (foaming) and extracted with CH 2 C1 2 (3x1.2 35 WO 97/38705 PCT/US97/05744 The CH 2 C1 2 extracts were combined, dried over anhydrous Na 2 SO4 and evaporated in vacuo to afford a tlc homogeneous title compound (51.5 g) as a light brown oil in 84.7% yield.

TLC: Silica gel, 8:1:1 CH 2 Cl 2

-CH

3 OH-AcOH, Rf=0.3, PMA and Ninhydrin.

H

1 -NMR (CDC1 3 d=1.28 3H), 1.36 3H), 1.38 3H) 1.60 1H), 1.90 5H), 3.75 1H), 4.00 1H), 4.22 2H), 4.28 2H) ppm

C

13 -NMR (CDC1 3 14.00, 20.06, 28.19, 30.07, 32.29, 39.98, 46.87, 53.20, 58.38, 60.73, 170.35, 177,06 ppm

C.

Me 0 Me H 0 C02Et Part A compound (641 mg, 1.42 mmol) was partitioned between EtOAc and 5% KH 2

PO

4 (adjusted to pH 2.5 with H 3

PO

4 The layers were separated and the aqueous layer was back-extracted with EtOAc. The pooled EtOAc extracts were washed with brine, dried (Na 2

SO

4 filtered and stripped to give an oil (assume 1.42 mg). The oil was dissolved in CH 2 C1 2 (10 mL) and the resulting solution was treated with Part B amine (364 mg, 1.50 mmol) in CH 2 C1 2 (2 mL) and cooled to 0°C. The mixture was subsequently treated with HOBT hydrate (195 mg) followed by EDAC (285 mg, 1.48 mmol).

After stirring at 0°C for 45 minutes and at room temperature for 45 minutes, the mixture was 36 WO 97/38705 PCT/US97/05744 partitioned between EtOAc and 5% KH 2

PO

4 (adjusted to pH 2.5 with H 3

PO

4 The EtOAc extract was washed successively with H 2 0, 50% saturated NaHCO 3 and brine, then dried (Na 2 SO4), filtered and stripped. The residue was flash chromatographed (Merck SiO 2 7/3-EtOAc/hexanes as eluant) to obtain title compound (427 mg, 59%, TLC Rf 0.37 (8/2- EtOAc/hexanes)) as a diastereomerically pure compound. In addition, the minor diastereomer was isolated from the column (66 mg, TLC Rf 0.27 (8/2-EtOAc/hexanes)). NMR of this material was consistant with an isomer of the title compound.

D.

Me ON 0 Me yNH O CO 2 Et fm Acetic anhydride (500 pL) was added to formic acid (5.0 mL) at 0°C and the mixture was stirred for 30 minutes. Approximately 2.6 mL of this solution was added to a solution of Part C compound (208 mg, 0.413 mmol) in THF (1.1 mL) at 0°C. After 30 minutes, most of the solvent was removed by rotary evaporation and the residue was partitioned between EtOAc and saturated NaHC0 3 The EtOAc extract was washed with brine, dried (Na2SO 4 filtered and stripped to give title compound (216 mg, 97%) as an oily foam which was used directly in the next reaction without futher purification.

TLC Rf 0.37 (EtOAc) HPLC YMC S3 ODS column (6.0 x 150 mm); eluted with B:A solvent mixture, 40 to 100% B over a 20 minute 37 WO 97/38705 PCT/US97/05744 linear gradient (solvent A: 90%H 2 0-10% MeOH-0.2%

H

3 P0 4 solvent B:0% H 2 0-90% MeOH-0.2% H 3

PO

4 flow rate 1.5 mL/min detecting at 220 nm; tR 17.2 min 100%).

E.

Me HON 0 Me ON 7 H o CO 2 Et A solution of Part D compound (216 mg, 0.402 mmol) in absolute EtOH (5 mL) was hydrogenated (balloon) over 10% Pd/C (33 mg) at room temperature for 2 hours. The mixture was filtered through Celite, stripped, and azeotroped twice with EtOAc/Et20/hexanes to give title compound (174 mg, 97%) as an off-white foam.

TLC Rf 0.33 (5/95-HOAc/EtOAc) HPLC YMC S3 ODS column (6.0 x 150 mm); eluted with B:A solvent mixture, 40 to 100% B over a 20 minute linear gradient (solvent A: 90%H20-10% MeOH-0.2%

H

3 P0 4 solvent B:0% H 2 0-90% MeOH-0.2% H 3 P0 4 flow rate 1.5 mL/min detecting at 220 nm; tR 12.8 min 100%).

F.

Me -HO Me 7

H

O COgH 38 WO 97/38705 PCT/US97/05744 A stirred solution of Part E compound (168 mg, 0.376 mmol) in MeOH (3 mL) at room temperature was treated with aqueous 1 N NaOH (3 mL). An additional portion of aqueous 1 N NaOH (3 mL) was added after 3.5 hours. After a total of 6 hours, the mixture was made acidic with 5% KHS0 4 and extracted twice with EtOAc. The EtOAc extract was washed with brine, dried (Na 2

SO

4 filtered and stripped. The residue was dissolved in a small amount of MeOH and EtOAc and triturated with to give title compound (134 mg, 86%) as an off-white solid/foam +18.00 (c 0.5, CH 2 C12)).

TLC Rf 0.10 (5/95-HOAc/EtOAc) HPLC YMC S3 ODS column (6.0 x 150 mm); eluted with B:A solvent mixture, 40 to 100% B over a 20 minute linear gradient (solvent A: 90%H 2 0-10% MeOH-0.2%

H

3 P0 4 solvent B:0% H20-90% MeOH-0.2% H 3 P0 4 flow rate 1.5 mL/min detecting at 220 nm; tR 9.00 min Anal. Calc'd for C 2 1H 2 9

N

3 06-0.75H 2 0-0.3Et 2 0 C, 58.57; H, 7.42; N, 9.23 Found C, 58.31; H, 7.20; N, 8.99.

Example 4 [S-(R*,R*)]-3-[[3-(Formylhydroxyamino)-l-oxo-2- (phenylmethyl)propyl]amino]-2,3,4,5-tetrahydro-2oxo-lH-benzazepine-l-acetic acid HO H O COOH 39 WO 97/38705 PCTIUS97/05744

A.

N. COOC 2 Hs

H

2

N

0 A(1) 1 0

H

Solid sodium azide (26.0 0.2 mole) was introduced into a 3-neck round-bottom flask with an overhead stirrer, made into a paste with warm water (26 ml), layered with chloroform (160 ml) and cooled down to 00 (ice-salt bath). The mixture was treated dropwise with concentrated sulfuric acid (11.2 ml, 0.5 eq.) over a period of 10 minutes, stirred for an additional 10 minutes then decanted into a flask containing anhydrous sodium sulfate.

The dried solution was filtered through a glass wool plug in a funnel into a 500-ml round-bottom flask. Titration of an aliquot (1.0 ml) with 1.0 N NaOH using phenolphthalein as an indicator gave a normalitity of 1.7 N for the hydrazoic acid.

Tetralone (15.94 g, 0.108 mole) was added to the hydrazoic acid solution (0.136 mole or 1.25 heated to 40-450 (oil bath) then treated dropwise with 36.0 N H2S04 (28.7 ml, 5 eq.) over a period of 1.0 hour. (Intense bubbling took place with each drop added for the first 30 minutes).

The reaction mixture was cooled down to room temperature, poured into H20 (720 ml) and stirred for 5 minutes. The solution was then extracted with EtOAc (3 x 250 ml) and the combined organic extracts were washed with brine (100 ml), dried (anhydrous MgSO4), filtered, evaporated to dryness and dried in vacuo. The crude product (17.819 g) 40 WO 97/38705 PCT/S97/05744 was recrystallized from CH2C12 (70 ml) and Hexane (400 ml) to give title compound as off-white precipitates (10.017 g, m. pt. 138-140 0 C) with consistent 1 H-NMR and 13 C-NMR spectral data.

The mother liquor was chromatographed on a silica gel column (Merck, 240 eluting the column with EtOAc:Hexane to give an additional amount of 5.058 g (total yield= 15.075 g, 85.6 TLC: Rf 0.37 (Silica gel; EtOAc:Hexane-l:l; UV).

A(2).

Br I 0

H

A solution of Part A(1) compound (1.0 g, 6.20 mmoles) in dry CHC13 (15 ml) was cooled down to 0°C (ice-salt bath), treated with PC15 (1.5 g, 7.20 mmoles) followed by 12 (15 mg) then stirred at 0°C under argon for 30 minutes. The yellow solution was treated with Br2 (0.39 ml or 1.2 g, 7.51 mmoles), warmed up to room temperature and refluxed under argon for 4.0 hours. The mixture was then poured into ice-water (20 stirred and the phases were separated, washing the aqueous phase with CHC13 (25 ml). The combined organic extracts were washed with H20 (5.0 ml), dried (anhydrous MgS04), filtered, evaporated to dryness and dried in vacuo. The crude product mixture was chromatographed on a silica gel column (Merck, g),-eluting the column with EtOAc:Hexane to give title compound as off-white precipitates (1.137 m.pt. 170-1720, 70.1 with consistent 1 H-NMR and 13C-NMR spectral data. TLC: Rf 0.13 (Silica gel; EtOAc:Hexane UV).

41

I

WO 97/38705 PCTIUS97/05744 A(3).

N 0

O

H

A solution of Part A(2) compound (936 mg, 3.9 mmoles) and NaN3 (300 mg, 4.6 mmoles) in dry dimethylsulfoxide (20 ml) was stirred at 600 (oil bath) under argon for 6.0 hours. The reaction mixture was cooled down to room temperature, poured into cold water (125 ml), stirred for 15 minutes and filtered, washing the solids formed with water.

The crude product was dried in vacuo at 600 over drierite for 24 hours to give title compound (725 mg, m.pt. 150-1520, 91.9 as an off-white solid with consistent 1 H-NMR and 13 C-NMR spectral data.

TLC: Rf 0.58 (Silica gel; EtOAc:Hexane- 1:4 then 1:1; UV).

A(4).

N

3

NN

COOC

2

H

A solution of Part A(3) compound (10.858 g, 53.7 mmoles) in dry tetrahydrofuran (100 ml) was treated with Bu4NBr (1.791 g, 5.56 mmoles) and powdered KOH (3.937 g, 70.2 mmoles) followed by ethyl bromoacetate (6.8 ml, 61.3 mmoles). The reaction mixture was stirred at room temperature under argon for 1.5 hours then partitioned between (196 ml) and CH2C12 (2 x 375 ml). The combined organic extracts were washed with H20 (2 x 196 ml) and brine (100 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo.

The crude product was combined with the crude product mixture from a previous run (2.936 g, 12.86 42 WO 97/38705 PCT/US97/05744 mmole scale) and chromatographed on a silica gel column (Merck), eluting the column with Toluene:EtOAc (98.2) and EtOAc:Hexane to give title compound as a solid (15.48 g, 93.5%) 1 with consistent 1H-NMR and 13 C-NMR spectral data.

TLC: Rf 0.63 (Silica gel; EtOAc:Hexane- 1:2; UV).

SNH 2

COOC

2 Hs A solution of Part A(4) compound (8.95 g, 31.0 mmoles) in absolute ethanol (50 ml) was treated with 10% Pd/C (443 mg) and hydrogenated at psi for 3.5 hours, venting the Parr bottle every 30 minutes for the first 1.5 hours. The mixture was filtered through a Celite® pad in a millipore unit, washing the pad well with absolute ethanol (3 x 50 ml). The clear filtrate was evaporated to dryness and dried in vacuo to give title compound as a thick yellow syrup (7.929 g, 97.5%) with consistent 1 H-NMR and 13 C-NMR spectral data. TLC: Rf 0.45 (Silica gel; CH2C12:CH30H- 9:1; UV).

A(6).

COOC

2

H

0 A solution of Part A(5) compound (14.8 g, 56.4 mmoles) and L-tartaric acid (8.50 g) in hot absolute ethanol (118 ml) was kept overnight at 00, at room temperature for 3 days and then at 00 for another 2 days. The solid that formed was recrystallized from absolute ethanol (118 ml) two 43 I WO 97/38705 PCT/US97/05744 more times until a consistent specific rotation was obtained. The precipitates (6.319 g) from the second recrystallization was then suspended in EtOAc (100 ml), treated with 10% NH40H (12 ml) and stirred for 5 minutes. The organic phase was separated, washed with 10% NH40H (10 ml) and brine ml), dried (anhydrous Na2S04), filtered, evaporated to dryness and dried in vacuo to give title compound as a white solid (3.927 g, m.pt.

105-1070, 26.5%) with consistent 1 H-NMR and 13

C-NMR

spectral data.

[a]D -2770 (c 0.99, EtOH). TLC Rf 0.45 (Silica gel; CH2C12:CH30H- 9:1; UV).

B.

BnO i HN

N

rI O 0 B H Example 3 Part A ephedrine salt (414 mg, 0.93 mmole), was partitioned between 5 KH2P04 (adjusted to pH 2.5; 4.0 ml) and EtOAc 2 x 20 ml) and the combined organic extracts were washed with brine (4.0 ml), dried (anhydrous Na2S04), filtered, evaporated to dryness and dried in vacuo to give the free acid of the Example 4 Part A compound as a clear syrup (286.6 mg, 100 crude yield).

A solution of the above free acid (286.6 mg, 0.93 mmole) in dry CH2C12 (6.0 ml) was cooled to 0 0 C (ice-salt bath) and treated sequentially with a solution of the above free amine (271 mg) in dry CH2C12, HOBT-H20 (126.1 mg, 0.93 mmole) and EDAC (185.4 mg, 0.97 mmole). The reaction mixture was stirred at 0°C for 1.0 hour, at room 44 WO 97/38705 PCTIS97/05744 temperature for 2.0 hours, then partitioned between EtOAc (2 x 20 ml) and H20 (4.0 ml). The organic extracts were washed with 5% KH2PO4 (adjusted to pH 4.0 ml), H20 (4.0 ml), saturated NaHC03 ml) and brine (4.0 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo.

The crude product was chromatographed on a silica gel column (Merck, 70 eluting the column with EtOAc:Hexane mixtures 1:1) to give pure title compound (202 mg) and impure product. A second chromatography gave title compound as a syrup (total of 292.1 mg, 59.3%) with consistent 1

H-NMR

and 13C-NMR spectral data. TLC: Rf 0.32 (Silica gel; EtOAc:Hexane

UV).

C.

0 OHC N. COOC 2

H

Br/ 0

H

A cooled solution of HCOOH (5.0 ml) was treated with acetic anhydride (Ac20) (0.5 ml) and stirred at 0 C for 30 minutes. A solution of Part B compound (288 mg, 0.54 mmole) in dry THF (1.5 ml) was cooled to 0°C (ice-salt bath), treated with the above Ac20/HCOOH mixture (3.4 ml) and stirred at 0°C for 1.0 hour. The reaction mixture was evaporated to dryness and the residual syrup was dissolved in EtOAc (40 ml), washed with saturated NaHC03 (5.0 ml) and brine (5.0 ml), dried (anhydrous Na2S04), filtered, evaporated to dryness, evaporated from toluene and dried in vacuo to give title compound as a syrup (311.3 mg, 100 45 WO 97/38705 PCT/US97/05744 crude) with consistent 1H-NMR and 13C-NMR spectral data. TLC: Rf 0.18 (Silica gel; EtOAc:Hexane (1:1;

UV).

D.

OHC W, COOC 2

H

,N N HO I 0

H

A solution of Part C compound (311 mg) in (10 ml) was treated with 10% Pd/C (53 mg) and hydrogenated (balloon) at room temperature for hours. The reaction mixture was diluted with ml) and filtered through a Celite® pad in a millipore unit, washing the pad well with CH30H (3 x 10 ml). The clear filtrate was evaporated to dryness and dried in vacuo to give title compound as a syrup (256.7 mg, 100% crude) with consistent 1 H-NMR and 13 C-NMR data. TLC: Rf 0.25 (Silica gel; CH2Cl2:MeOH- 9:1; UV).

E. [S-(R*,R*)]-3-[[3-(Formylhydroxyamino)l-oxo-2-(phenylmethyl)propyllamino]- 2,3,4,5-tetrahydro-2-oxo-lH-benzazepine-lacetic acid A solution of Part D compound (256.7 mg) in CH30H (3.5 ml) was treated with 1.0 N NaOH (2.17 ml, 4 eq) and stirred at room temperature for hour under argon. The reaction mixture was brought to pH 1.0 with 5% KHS04 (9.45 ml), extracted with EtOAc (40 ml) and the organic extract washed with brine (5.0 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo. The crude product was triturated with CH2C12:Hexane 46 WO 97/38705 PTU9/54 PCTIUS97/05744 (1:4-25 ml) and hexane (20 ml) then dried in vacuo to give title compound as an amorphous off-white solid (215.6 mg, 90.4%) with consistent MS, IR, 1

H-

NMR and analytical data. TLC: Rf 0.30 (Silica gel; EtOAc:HOAC- 95:5; UV).

[cL]D -332.80 0.558, CH3OH) HPLC: tR= 5.21 min (95.8% R isomer); tR =9.58 min (3.59% S isomer); YMC S3 ODS-A 150 x 6 mm; 220 rim, flow rate =1.5 mi/mmn; 56% (10% H20- 90% CH3OH- 0.2% H3P04)/44% (90% H20- 10% CH30H-0.2% H3P04), isocratic.

Anal. Calc'd for C 2 3

H

2 5

N

3 0 6 C, 62.86; H, 5.73; N, 9.56 Found: C, 62.88; H, 5.98; N, 9.20.

ExamT~le

H

0

N

HO'

N

H 0 COOH

A.

OH

H

N

Pht=N 0 COOCH 3 A solution of L-hydroxynorleucine (2.0 g, 13.6 mmoles) in dry methanol (70 ml) was saturated with lid gas until a clear yellow solution was obtained. The reaction mixture was cooled to room temperature, stirred for 2.0 hours, evaporated to 47 WO 97/38705 PCT/US97/05744 dryness, evaporating the syrup once from toluene (100 ml) then evaporated in vacuo to give the ester as a yellow oil. The crude ester was dissolved in dry CH2C12 (50 ml) and dry DMF (15 ml), treated with NMM (2.5 ml, 22.7 mmoles) and cooled to 00C (ice-salt bath). The mixture was treated with Nphthaloyl-L-phenylalanine (4.0 g, 13.6 mmoles), HOBt-H 2 0 (1.89 g, 13.99 mmoles) and EDAC (2.87 g, 14.98 mmoles), stirred at 0°C for 25 minutes and at room temperature for 2.0 hours.

The reaction mixture was partitioned between EtOAc (2 x 200 ml) and H20 (60 ml) and the combined organic extracts were washed sequentially with 0.5 N HC1 (60 ml), H20 (60 ml), 1/2 saturated NaHC03 (60 ml) and brine (60 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo. The crude product mixture was chromatographed on a silica gel column (Merck, 200 eluting the column with EtOAc to give the desired product as a syrup (4.0 An additional 321 mg was obtained on re-chromatography of the impure fractions to give title compound (4.32 g, 73%) with consistent IH-NMR and 13C-NMR spectral data.

TLC: Rf 0.43 (Silica gel; EtOAc; UV).

B.

0

H

Pht=N 0 COOCH 3 A solution of oxalyl chloride (1.02 ml,11.7 mmoles) in dry CH2C12 (56 ml), was cooled to -780C (dry-ice-acetone bath), treated with a solution of dry DMSO (1.67 ml, 21.6 mmoles) in CH 2 C1 2 (2.0 ml) 48 WO 97/38705 PCT/US97/05744 and stirred at -78 0 C for 20 minutes. The mixture was treated with a solution of Part A compound (4.29 g, 9.78 mmoles) in dry CH2C12 (22 ml), stirred at -78 0 C for another 15 minutes, then treated with triethyl-amine (8.4 ml). The reaction mixture was stirred at -78 0 C for 5.0 minutes, allowed to come to room temperature over a period of 45 minutes, then partitioned between EtOAc (200 ml) and 0.5 N HC1 (2 x 20 ml). The organic phase was washed with brine (40 ml), dried (anhydrous 4 filtered, evaporated to dryness and dried in vacuo to give title compound as a thick syrup (4.428 g, 100% crude yield), with consistent 1

H-NMR

and 13 C-NMR spectral data.

TLC: Rf 0.73 (Silica gel; EtOAc; UV).

C.

Pht= i 0 COOCH 3 A mixture of Part B compound (4.428 g, 9.78 mmoles) and TFA (0.20 ml, 2.6 mmoles) in dry CH2C12 (62 ml) was refluxed under argon for 2.0 hours.

The reaction mixture was cooled to room temperature, washed with 1/2 saturated NaHC03 ml) and brine (20 ml), dried (anhydrous Na2S04), filtered, evaporated to dryness and dried in vacuo.

The crude product mixture was chromatographed on a silica gel column (Merck, 200 eluting the column with CH2Cl2:EtOAc to give the desired product as a syrup. The syrup was triturated with (2:1-60 ml) to give title compound as a white precipitate (2.92 g, 72%; m.p. 141-143 0

C)

with consistent 1H-NMR and 13 C-NMR spectral data.

49 WO 97/38705 PCT/US97/05744 TLC: Rf 0.67 (Silica gel; CH2C12:EtOAc-9:1;

UV).

D.

H

Pht=

N

0

COOH

A solution of Part C compound (2.923 g, 6.99 mmoles) in dry CH2C12 (14 ml) was treated with triflic acid (4.15 ml, 6.7 eq) and the resulting yellow solution was stirred at room temperature for 20 hours. The reaction mixture was then poured into ice-water (100 ml), extracted with EtOAc (3 x 100 ml) and the combined organic extracts washed with H20 (2 x 25 ml) and brine (25 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo. The crude product mixture was chromatographed on a silica gel column (Merck), eluting the column with EtOAc:Hexane mixtures (1:1; 2:1) and EtOAc:HOAc (100:1). The desired fractions were combined, evaporated to dryness and dried in vacuo to give impure title compound as a solid foam (1.238 g, 42%) with consistent 1 H-NMR and 13

C-

NMR spectral data. TLC Rf 0.73 (Silica gel; EtOAc:HOAc-95:5;

UV).

E.

H

N

Pht= N 0 COOBn A solution of Part D compound (1.238 g, 3.06 mmoles) in dry DMF (3.5 ml) was treated 50 WO 97/38705 PCT/US97/05744 sequentially with benzyl bromide (0.35 ml, 2.94 mmoles) and Cs2CO3 (450 mg, 1.38 mmoles) then stirred at room temperature for 3.0 hours. The mixture was diluted with EtOAc (50 ml), washed with H20 (5.0 ml), 0.5 N HC1 (5.0 ml) and brine ml), dried (anhydrous Na2S04), filtered, evaporated to dryness and dried in vacuo. The crude product (1.63 g) was chromatographed on a silica gel column (Merck), eluting the column with EtOAc:Hexane (1:3) to give title compound as a syrup (586.4 mg, 39%) with consistent 1 H-NMR and 13C-NMR spectral data.

TLC: Rf 0.45 (Silica gel; EtOAc:Hexane-l:l; UV).

F.

H

N

H

2

N

COOBn A solution of Part E compound (586 mg, 1.18 mmoles) in dry methanol (15 ml) was treated with NH2NH2-H20 (66 ul, 1.2 eq) and stirred at room temperature for 48 hours. The reaction mixture was diluted with Et20 (50 ml) and filtered through a millipore unit, washing the solids well with ml). The clear solution was evaporated to dryness and the solids obtained were suspended in CH2C12 (90 ml) and the solution filtered through a millipore unit, washing the solids well with CH2C12 ml). The combined organic extracts were washed with brine (15 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo to give title compound as a thick syrup (351 mg, 82 with a consistent 1H-NMR spectrum.

TLC: Rf 0.42 (CH2C12:MeOH-9:1; UV, Ninhydrin) 51 WO 97/38705 PCT/US97/05744

G.

H

0 H, N BnO I SH 0 COOBn Example 3 Part A ephedrine salt (538 mg, 1.2 mmoles), was partitioned between 5% KH2PO4 (adjusted to pH 2.5; 5.4 ml) and EtOAc (2 x 22 ml) and the combined organic extracts were washed with brine (5.4 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo to give the free acid of the ephedrine salt as a clear syrup (323 mg, 100% crude yield).

A solution of the free acid in dry CH2C12 (8.0 ml) was cooled to 0 C (ice-salt bath) and treated sequentially with a solution of Part F compound (351 mg, 0.96 mmole) in dry CH2C12 ml), HOBT-H20 (163 mg, 1.2 mmoles) and EDAC 240 mg, 1.25 mmoles). The reaction mixture was stirred at 0°C for 1.0 hour, at room temperature for hours, then partitioned between EtOAc (40 ml) and H20 (5.0 ml). The organic extracts were washed with 5 KH2P04 (adjusted to pH 2.5; 5.0 ml), ml), saturated NaHCO3 (5.0 ml) and brine ml), dried (anhydrous Na2S04), filtered, evaporated to dryness and dried in vacuo. The crude product (810 mg) was chromato-graphed on a silica gel column (Merck), eluting the column with EtOAc:Hexane to give pure title compound (494 mg, 65%) as a solid foam with consistent 1H- NMR and 1 3 C-NMR spectral data.

TLC: Rf 0.45 (Silica gel; EtOAc:Hexane UV).

52 WO 97/38705 PCT/US97/05744

H.

H

0 OHC

N

BnO B H 0 COOBn A cooled solution ice-salt bath) of HCOOH (5.0 ml) was treated with Ac20 (0.5 ml) and stirred at 0°C for 30 minutes. A solution of Part G compound (493 mg, 0.78 mmole) in dry THF (2.2 ml) was cooled to 0°C (ice-salt bath), treated with the above Ac20/HCOOH mixture (4.9 ml) and stirred at 0°C for 1.5 hours. The reaction mixture was evaporated to dryness, evaporated from Et20 (50 ml) and the residual syrup was dissolved in EtOAc ml), washed with saturated NaHCO3 (7.0 ml) and brine (7.0 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness, evaporated from toluene and dried in vacuo to give title compound as a syrup (558.3 mg, 100 crude) with consistent 1 H-NMR and 1 3 C-NMR spectral data.

TLC: Rf 0.2 (Silica gel; EtOAc:Hexane-l:l; UV).

I.

H

OHCN N

HO

S H 0 COOH A solution of Part H compound (535 mg, 0.78 mmole) in CH30H (15 ml) was treated with 10 Pd/C (83 mg) and hydrogenated (balloon) at room 53 WO 97/38705 PCT/US97/05744 temperature for 4.0 hours. The reaction mixture was diluted with CH30H (15 ml) and filtered through a celite pad in a millipore unit, washing the pad well with CH30H (3 x 15 ml). The clear filtrate was evaporated to dryness and dried in vacuo to give a syrup (354.8 mg) which was triturated with CH2Cl2:Hexane (1:5-30 ml) and hexane (25 ml) then dried in vacuo. Title compound was obtained as an off-white solid foam (348.5 mg, TLC: Rf 0.38 (Silica gel; CH2Cl2:MeOH- 9:1; UV).

MS (M+H) 480 [a]D +44.60 (c 0.52, HPLC tR= 11.72 min (95.9% YMC S3 ODS-A 150 x 6 mm; 220 nm, flow rate 1.5 ml/min; 55% (10% CH30H- 0.2% H3P04)/ 45% (90% H20- 10% 0.2% H3P04), isocratic.

Anal. Calc'd for C26H29N306-0.4 H20*0.14 Hexane (Eff. Mol. Wt. 497.08): C, 64.63; H, 6.83; N, 8.46 Found: C, 64.24; H, 6.43; N, 8.12 The following are examples of additional compounds of the invention which may be prepared employing procedures set out hereinbefore and in the working Examples.

54 WO 97/38705 WO 9738705PCTIUS97/05744 H uA Examnple No.

H1 H 1

R

CH

2 Ph

CH

2 Ph H 1 CH 2

CH(CH

3 2 H 1

CH

2 Ph H 1 CH 2

CH(CH

3 2 H 1

CH

2 Ph H 1

CH

2 Ph 0

N

S-N

H 0 C0 2

H

55 WO 97/38705 WO 9738705PCTIUS97/05744 13 H 1 (CH 2 h3-K& OMe 14 H 1 CH(CH 3 2 J:Ph

H

C0 2

H

0 H 1 CH(CH 3 2 5 2 01

N

H 1 CH(CH 3 2 H 1 CH 2 Ph Me Me

COH

00

OH

H

Nl--,C 2

H

H

0 0 Ph 56

Claims (16)

1. A compound of the formula R 1 O o I o H A R including a pharmaceutically acceptable salt thereof wherein x is 0 or 1, R is H, alkyl, alkenyl, aryl-(CH2)p-, heteroaryl-(CH 2 cycloheteroalkyl-(CH 2 or 10 R can be joined together with the carbon to which it is attached to form a 3 to 7 membered ring which may optionally be fused to a benzene ring; R 1 is H or -COR 2 where R 2 is alkyl, aryl- (CH 2 cycloheteroalkyl-(CH 2 heteroaryl- (CH 2 alkoxy or cycloalkyl-(CH2)p-; p is 0 or an integer from 1 to 8; and A is a dipeptide derived from one or two Snon-proteinogenic amino acids or is a conformationally restricted dipeptide mimic.

2. The compound as defined in Claim 1 wherein A is a dipeptide derivative of the structure R 3 R A(1) R 1 R R2 N N R2b O COR 4 wherein R 1a Rib, R 2a and R 2 b are independently selected from H, alkyl, aryl-(CH2)p-, cycloalkyl, cycloheteroalkyl-(CH 2 heteroaryl-(CH 2 biphenylmethyl, or R l a and R 1 b or R 2a and R 2 b may be joined together to the carbon to which it is attached to form a 3 to 7 memebered ring, optionally fused to a 57 WO 97/38705 WO 9738705PCT/US97/05744 benzene ring; and R~ refers to an optional 5 or 6 memnbered ring containing a single hetero atom and which may optionally include an R substituent which is H, alkyl, aryl-(CH 2 )p, cycloalkyl- (CH 2 cycloheteroalkyl- (CH 2 )p or cycloheteroaryl- (CH 2 R 3 is H, alkyl or aryl (CH 2 R 4 is OH, Qalkyl, oaryl-(CH2)p- or NRi(R 2 where R 1 and R 2 are independently H, alkyl, aryl, aryl(CH 2 )p or heteroaryl(CH 2 )p; with the proviso that in A(l) at least one of TO R R~a Rlb a ~NS K and NjRb H 0 COR' is other than a natural ax-arnino acid.

3. The compound as defined in Claim 1 wherein A is a conforinationally restricted dipeptide mimic.

4. The compound as defined in Claim 3 wherein the conformnationally restricted dipeptide mimic has the structure A(2) N 0 CR

5. The compound as defined in Claim 3 wherein A has the formula 58 WO 97/38705 WO 9738705PCTIUS97/05744 Ry H CaR 4 where Y 0, S, OH 2 or S(0) 0 12 H 0 COR 4 )O'i H 0 OR 4 n is 0 or 1 where X OH 2 and Y 0, S, OH 2 or S(C and X Swhen n )n N "yNY)R 7 H 0 COR 4 where Y 0, 8, OH 2 )012 orS00,2 1 H 0 YCR where S, NH or S(0)01,2 where X' H, Ph, NHSO 2 R (R 5 H) where X' H, Ph, NHS0 2 R 5 (R 5 H) where YV 0, S, OH 2 orS00,, where Z 0 or H, H ro .yleS 0 where Z 0 or H, H where Y 0, S, OH 2 or 0012 where Y 0, S, NH or S(0) 0 12 59 WO 97/38705 PCT/US97/05744 where Y O, S, CH 2 12 )1,2Y R 7 yR 7 H O COR 4 COR 4 YR where Y O, S, CH 2 or S(0) 0 ,1,2 H COR 4 S(0) 0 1 2 CH 2 with respect to R11 and R 1 2 are independently selected from hydrogen, alkyl, alkenyl, cycloalkyl aryl and heteroaryl or R 1 1 and R 1 2 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R 11 and R 12 taken together with the carbon to which they are attached complete a keto substituent, with respect to A(13), R 8 R 9 and R 7 are independently selected from hydrogen, alkyl, alkenyl, cycloalkyl aryl-(CH2)m-, and heteroaryl-(CH2)m-; R 10 and R 6 are independently selected from hydrogen, alkyl, alkenyl, cycloalkyl -(CH2)p-, aryl-(CH2)p, and heteroaryl-(CH2)p-, or R 6 and R 10 taken together with the carbons to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, R 6 and R 8 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R 9 and R 10 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons; R 4 is OH, Oalkyl, 0-(CH2)p-heteroaryl, 60 41 WO 97/38705 PCT/US97/05744 o A CH-0-C-R5 R (CH2)p-aryl or CH 2 R 1 6 or NR 1 (R 2 where R 1 and R 2 are independently H, alkyl, aryl, aryl-(CH 2 )p or heteroaryl; R 14 is hydrogen, alkyl, cycloalkyl, or phenyl; R 15 is hydrogen, alkyl, alkoxy or phenyl; R 1 6 is alkyl or aryl-(CH2)m-; and R 17 is hydrogen, alkyl, substituted alkyl, alkenyl, cycloalkyl-(CH2)m-, aryl-(CH2)m-, or heteroaryl-(CH2)m-. R 1 8 is H or alkyl or alkenyl, and R 1 8 and R 1 7 may be taken together with the carbon and nitrogen to which they are attached to complete a saturated N-containing ring of 5 or 6 ring members. R 1 9 is H or an alkyl, and in R19 and X (which is CH 2 together with the carbons to which they are attached may form an aromatic ring of carbons (as in

6. The compound as defined in Claim 1 wherein A is R19 R7 H O COR 4 SS-j 4 where n is 0 or 1 H O COR 4 where X CH 2 and M N R Y O0, S, CH 2 or S(0) 0 ,1, 2 where Y O, S, CH 2 ,S(0)0,1 2 and X O, S when n 1 H COR 4 X 1 H H O C O R 4 £o "R where X' H, Ph, H 0 COR 4 NHSO2R s h R 5 H where Y=0,S,CH 2 ,S(0)o, 1 (where R 5 H) H COR 4 where Z 0 or H, H 61 WO 97/38705 0COR 4 where Y 0, S, CH 2 ,S(0) 0 12 PCTIUS97/05744 S. S *5 H 0 00R 4 where Y 0, S, NH, S(0)01,2 H 0 CaR 4 H 0 NO 4COR 4

7. The compound as defined in Claim 6 wherein A is 2 AR11 H 0 OR where Y 0, S, OH 2 S(0)01.2 Mi 0 bOR 4 where Y 0, S, CH 2 ,S(O) 0 1 2 where Y S, CH 2 ,S(O) 0 1 2

8. The compound as defined in any one of claims 1-7 wherein R' is H, R is alkyl or arylalkyl, R 4 is OH.

9. The compound as defined in Claim 2 where in A(l) is a non-proteinogenic amino acid portion. 62 WO 97/38705 PCT/US97/05744 The compound as defined in claims 2 or 9 wherein R 1a and R 1 b are independently alkyl or arylalkyl, or R 1a and Rib together with the carbon to which they are attached form a 3 to 7 membered ring; or one of R 1a and R 1 b is biphenylmethylene and the other is biohenvlmethvlene or H.

11. The compound as defined in any one of claims 2, 9 or where in A(1), SR I R2 /N R 2 b .COR 4 10 is a non-proteinogenic amino acid where R 3 is H, alkyl or arylalkyl, S• R 2 a and R 2 b are independently selected from Si H, alkyl, aryl or arylalkyl, with at least one of R 2 a and R 2 b being other than H, or R 2a and R 2 b together with the carbon to which they are attached form a 3 to 7 membered ring.

12. A pharmaeutical composition comprising a therapeutically effective amount of a compound as defined in any one of claims 1-11 and a pharmaceutically 20 acceptable carrier therefor.

13. The pharmaceutical composition as defined in Claim 12 useful in the treatment of cardiovascular diseases such as hypertension and/or congestive heart failure.

14. A method of treating a cardivascular disease such as hypertension and/or congestive heart failure, which comprises administering to a mammalian species a therapeutically effective amount of a composition as defined in Claim 12.

15. The compound as defined in Claim 1 which is 63 WO 97/38705 PCT/US97/05744 y i o CO 2 H H r^Y^ HO H H CO 2 H Me HO~ AN Me S H O CO 2 H H 0 1 OHC i HO H HO -H COOH 0/ COOH or a pharmaceutically acceptable salt thereof. 64

16. Use of a compound as defined in any one of claims 1-11 or 15 in the preparation of a medicament for the treatment of cardiovascular diseases such as hypertension and/or congestive heart failure.

17. A compound as defined in claim 1 substantially as hereinbefore described with reference to any one of the examples. S 0* 0 30 C:\WINWORD\STACY\SPECIZ41g 9 7.DOC