CA2251292A1 - N-formyl hydroxylamine containing compounds useful as ace inhibitors and/or nep inhibitors - Google Patents

Abstract

N-formyl hydroxylamines are provided which have structure (I) wherein R and R1 are as defined herein and A is a dipeptide derived from an amino acid or is a conformationally restricted dipeptide mimic.

Description

CA 022~1292 1998-10-09 W097/38705 PCT~S97/05744 N-FORMYL HYDROXYLAMINE CONTAINING COMPOUNDS
USEFU~ AS ACE INHIBITORS AND/OR NF.P INHIBITORS

Summarv 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 cont~in;ng 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~O
J~ I ~A

including a pharmaceutically acceptable salt - thereof where:
x is 0 or l;
R is H, alkyl, alkenyl, aryl-(CH2)p-, heteroaryl-(CH2)p-, cycloheteroalkyl-(CH2)p-~ 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;
Rl is H or -COR2 where R2 is alkyl, aryl-(CH2)p-, cycloheteroalkyl-(CH2)p-~ heteroaryl-(CH2)p-, alkoxy, or cycloalkyl- (CH2)p-;
p is O or an integer from l 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 CA 022~1292 1998-10-09 W O 97/38705 PCT~US97/05744 A(l) ~ 7 ~ R2~
Jr~ ~ N ~ R2b H

O CoR4 where Rla, Rlb, R2a and R2b are independently selected from H, alkyl, aryl-(CH2)p-~ cycloalkyl, S cycloheteroalkyl-(CH2)p-, heteroaryl-(CH2)p-, biphenylmethyl, or Rla and Rlb or R2a and R2b may be joined together to the carbon to which they are attached to form a 3 to 7 membered ring, optionally fused to 1, ';."" R2b a benzene ring; and CoR4 refers to an optional 5 or 6 membered ring contAining a single hetero atom and which may optionally include an R5 substituent (as shown) which is H, alkyl, aryl-(CH2)p or cycloalkyl-(CH2)p~ cycloheteroalkyl-(CH2)p, or cycloheteroaryl-(CH2)p-;
R3 is H, alkyl or aryl -(CH2)p-;
R4 is OH, Oalkyl, O-(CH2)paryl- or NRl(R2) where Rl and R2 are independently H, alkyl, or aryl(CH2)p or heteroaryl-(CH2)p-;
with the proviso that in A(l) at least one of R,3 R5 Rla Rlb I ~ ~
~ N ~ ~ and ~ ~
O CoR4 is other than a natural a-amino acid, and thus must be other than valine, leucine, phenylalanine, t~ro-sine, serine, cysteine, threonine, methionine, aspartic acid, glutamic acid, arginine, lysine or proline.
In addition, A can be a conformationally restricted dipeptide mimic which has the structure W O 97/38705 PCT~US97/05744 A(2 ) N~
H ¦¦ l O CoR4 and is a non-proteinogenic dipeptide.
Thus, the compound of formula I include :rA ~ ~l Rl~ Rlb IR R5 H N--( CH2 ) x~ ~, N'~;~Rab H

S R O CoR4 and IA(2) 1 0 ~
I _(C}~2)x~ N~S

O R O CoR4 The term "conformationally restricted dipeptide mimic" refers to a structural skeleton which has the attributes of a conventional dipeptide R O R O
l 11 1 11 --NH--CEI-C--NEI--CH--C--but 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.

CA 0225l292 l998-lO-09 W 0 97/38705 PCTrUS97/05744 ".5~ f '~ R~

H o CoR4 n O r 1~ CoR4 ~ CoR4 where Y - O S CH2 where X = CH2 and or S(0)o~1~2 '' andX=O,Swhenn=1 ,Xl A~8) X
A(6) ~ A(7) ~ ~

H~\ ~ ~CoR4 where y1 = o, S, NH
where X1 = H, Ph, or S(O)n, NHSo2R5 where X1 = H, Ph, (R5 H) NHSo2R5 (Rs H
A(9) ~ A(10) O~COR~ ~~CO~ COJ~
where Y = O, S, CH2 where Z = O or H, H
or S(0)o,l,2 A(12) ~Z A(13) ~ A(14) H~

N~, R7 IS~ ~ N~ R7 ~ N~J
H o CoR4 ~ CoR4 ~ CoR4 where Z = O or H, H where Y = O, S, CH2 or S(O)0,1,2 A(~ A(1~ 5 A(17)~

H O CoR4 H ~ CoR4 H O CoR4 whereY=O,S, or S(~)0,1,2 A(1Y) ~3 A(19~ ~R 17 ~J\( ~0, --~b' N~ R7 5S~ ~, N~ R7 ~ ~,N~ R7 H ~ CoR4 ~ CoR4 H O CoR4 where Y = O, S, CH2 CA 022~1292 1998-10-09 W097/38705 PCT~S97/05744 A(21) ~ A(22) ~

~N~ R7 ~' N~ ~ R7 H ~ CoR4 H o CoR4 A(23) ~_ whereY=O,S,CH20rs(O)o~1~2 H o CoR4 X =O~S(O)o~1~2~cH2 With respect to A(5), R11 and R12 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl -(CH2)m-, aryl -(CH2)m-, substituted aryl -(CH2)m-, and heteroaryl -(CH2)m-, or R11 and R12 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R11 and R12 taken together with the carbon to which they are attached complete a keto substituent, i.e., /c - o - with respect to A(13) R8, R9 and R7 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl ~(CH2)m~, aryl-(CH2)m~, substituted aryl-(CH2)m-, and heteroaryl-(CH2)m~;
R10 and R6 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl -(CH2)m-, aryl-(CH2)m, substituted aryl -(CH2)m-, and heteroaryl-(CH2)m-, or R6 and R10 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, R6 and R8 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R9 and R10 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons;

CA 022~1292 1998-10-09 W O 97/38705 PCTAUS9710~744 m is zero or an integer from 1 to 6;
R4 is OH, Oalkyl, O-(CH2)m-heteroaryl, Il oJ~o Rl4 , O-(CH2)m-aryl, or -o-c~2 Rl6 NR1(R2);
S where R1 and R2 are independently H, alkyl, aryl(CH2)p, aryl or heteroaryli R14 is hydrogen, lower alkyl, cycloalkyl, or phenyli R15 is hydrogen, lower alkyl, lower alkoxy or phenyli R16 is alkyl or aryl-(CH2)m~; and R17 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl-(CH2)m-, aryl-(CH2)m~, substituted aryl-(CH2)m-, or IS heteroaryl-(CH2)m~-R18 is H, alkyl or alkenyl, and R18 and R17 may be taken together with the carbon and nitrogen to which they are attached to complete a saturated N-cont~; n; ng ring of 5 or 6 ring members.
R19 is H or an alkyl, and in A(4), R19 and X
(which is CH2) together with the carbons to which they are attached may form an aromatic ring of carbons (as in A(15).
The starting compounds H-A(l) 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(l) or H-A(2) wherein A(1) or A(2) is as defined in formulas A(5-), A(13), A(16), A(21), where Y (where present) is CH2 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.

CA 022~l292 Isss-lo-os W 097/38705 PCT~US97/05744 The starting compounds of formula H-A(l) or H-A(2) wherein A(l) 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., J., Med. Chem., 30, 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(l) or H-A(2) wherein A(l) 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(l) or H-A(2) wherein A(l) 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(l) or H-A(2) wherein A(l) 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 (lg86) and 34, p.
2078 - 2089 (1986) as well as Sugihara et al. in U.S. Patent 4,548,932 (Y is O) and Katakami et al.
in U.S. Patent 4,539,150 (Y is S).
The starting compounds of formula H-A(l) or H-A~2) wherein A(l) or A(2) is as defined in formula A(16) can be prepared by reduction of the corresponding starting compounds wherein A(l) or A(2) is as defined in formula A(3).
The starting compounds of formula H-A(l) or H-A(2) wherein A(l) or A(2) is as defined in CA 022~1292 1998-10-09 W O 97/38705 PCTrUS97/0~744 formula A(22) are disclosed by Flynn et al in U.S.
Patent 4,973,585.
The starting compounds of formula H-A~l) 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(l) or H-A(2) wherein A(l) or A(2) is as defined in formula A(10) and Y is CH2, and is as defined in formula A(23) where x2 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(l) 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(l) or H-A(2) wherein A(l) 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(l) or H-A(2) wherein A(l) 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(l) or A(2) is as defined in forml]lA.s A~4) and A~15) are disclosed in European Patent Application 0629627A2.
The starting compounds of formula H-A~l) or H-A(2) wherein A(l) or A(2) is as defined in formula A(9) are disclosed in U.S. application Serial No. 100,408 (file HA611a).

CA 022~1292 1998-10-09 W O 97/38705 rCT~US97/05744 The starting compounds of formula H-A(l) or H-A(2) wherein A(l) or A(2) is as defined in formulas A(7) and A(8) are disclosed in European Patent Application 481,~22 (Flynn et al) and European Patent Application 0534363A2 (Warshawsky et al).
The starting compounds of formula H-A(l) or H-A(2) wherein A(l) 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(l) or H-A(2) wherein A(l) or A(2) is as defined in formula A(17) are disclosed in European Patent Application 0599444Al (Barrish et al).
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.
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 2S 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 ~m;nistering to a m~mm~lian species, including humans, dogs and cats, a therapeutically effective amount of a composition as defined above.

Detailed Descri~tion Of The Invention The term "alkyl" or "lower alkyl" refers to straight or br~nche~ chain radicals having up to and including ten carbon atoms, preferably up to and including six carbon atoms, which may g CA 022~1292 1998-10-09 W O 97138705 PCTnUS97/05744 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 S 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 contAin;ng 6 to 10 carbons, preferably phenyl, 1-naphthyl, and 2-naphthyl, which may optionally contain one, two or three substituents selected from alkyl, alkoxy, alkylthio, halo, hydroxy, trifluoromethyl, -SO2NH2, amino, -NH(lower alkyl), or -N(lower alkyl)2, di- and tri-substituted phenyl, l-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 cont~;n;ng one or two 0 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, CA 022~1292 1998-10-09 W097/38705 PCT~S97/05744 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 2-, 3-, or 4-pyridyl, 4-imidazolyl, 4-thiazolyl, 2-and 3-thienyl, and 2- and 3-furyl. The term heteroaryl also includes bicyclic rings wherein the five or six membered ring cont~; n; ng 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 N-protecting group such as - CH2 - O- CH2 ~ ' -S02 ~ 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).

W O 97/38705 PCT~US97/05744 Reaction Scheme I

Qq~C02H PG1-O-NH? PG o~ N~C02 reS~Iution HN~

3 (optically pure or (Q is CH2, where x-1 and enriched) Q is H, Br where x=0) H-A(1) or H-A(2) O ~ Acylation o N~oR4 PG1 ~~ . H-C0-0-COR (4a) standard peptide coupling R H ~ CoR4 ~ ~C I removal of one orboth ~ J'C
pG1 Q ~ u~ 9 groups (PG1 HO'~ N~
~0 R 5 ~ coR4 and/or R ) ~ R H o CoR4 IA

As shown in Scheme I, acid 2 may be reacted with a suitably O-protected (e.g. PGl is benzyl, p-methoxybenzyl, tetrahydropyranyl, trityl, benzhydryl, etc.) hydroxylamine to give the adduct 3. Compound 3 may be coupled directly with amine H-A(l) 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(l) 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 se~uentially or simultaneously, to produce compound of the invention IA. For example, when PGl is benzyl and R4 is Obenzyl, both may be removed by hydrogenolysis. When pGl is benzyl and R4 is ~Omethyl or ~Oethyl, the PGl group may be removed by hydrogenolysis and the ester group may be converted to the acid by base hydrolysis. pGl CA 022~1292 1998-10-09 W O 97/3870~ PCTrUS97/05744 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.
- 5 Alternately, compounds of the invention IA
may be obtained by the route depicted in Scheme II
- (where x is 0 or l).

Reaction Scheme II
Acylation PG1 O~,C02H H.co-o-COR PG -Q~CO2H optionalchiral H-A(1 ) or H-A(2) ~C I

PG~,CO2H ~N~C~oR4 PG1-~ ~ CoR4 standard peptide coupling 5 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~l) or A(2) directly or optically resolved to give 7* and then coupled to give compound 5.
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 àre within the scope o~ 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 CA 022~1292 1998-10-09 W O 97~870~ PCTrUS97/05744 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 m~mm~l ian host such as man by the ~m; nl stration 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.

CA 022~1292 1998-10-09 W097~8705 PCT~S97/05744 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 ~m; n; stered 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 ph~rm~ceutically 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 30 ~m; ni stration include sterile solutions and suspensions. About ~0 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 phArm~ceutical practice.

CA 022~1292 1998-10-09 W O 97/38705 PCTrUS97/05744 Preferred compounds of the invention are those of formula I wherein R1 is H, x is 1, S R is alkyl or arylalkyl, and A is A(l), preferably Rl~ Rlb H ~
where ~ is preferably a non-proteinogenic amino acid portion wherein, R1a and R1b are each independently alkyl such as methyl or ethyl, or arylalkyl such as benzyl, or R1a and R1b together with the carbon to which they are attached form a 3-7 membered ring, preferably a 5-membered ring, or R1a and/or R1b is biphenylmethylene and the other may be H.
Also preferred are compounds where A is R13 ,R
N jj~ R2b A(l~, preferably where COR" and is a non-proteino-genic amino acid where R3 is H, alkyl, such as methyl or ethyl, aryl such as phenyl, or arylalkyl, such as benzyl, R2a and R2b are independently selected from H, alkyl, aryl, arylalkyl (with at least one of R2a and R2b being other than H) or R2a and R2b together with the carbon to which they are attached form a 3-7 membered ring, preferably 5- or 6-membered ~ing.
Also preferred are compounds where A is A(2) wherein R4 is OH.
The following Examples represent preferred embodiments of the present invention.

CA 022~1292 1998-10-09 W097/3870~ PCT~S97/0~744 Exam~le 1 0~

o y N ~ N ~ CO2H
H

A.
0~

Boc~
N

Atl).
o H, ~ C- OC(CH3)3 --~ COOH

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 (0~, ice-salt bath) suspension of 60% NaH (10.1 g, 0.25 mole) in dry dimethylformamide (200 ml) and stirring was continued at 0~ until the frothing subsided (ca. 2.0 hours). The reaction mixture was treated dropwise with 1-fluoro-2-nitrobenzene (14.3 ml, 0.13 mole) over a period of 20 minutes, stirred at 0~ under argon for 4.0 hours then poured into ice-water (750 ml) and extracted with Et2O (2 x 100-ml). The a~ueous phase was brought to pH 1.0 with 6 N HCl (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 (Merc~), eluting the column with CA 022~1292 1998-10-09 WOg7/3870~ PCT~S97/05744 CH2Cl2:CH3OH:HOAc (100:5:0.2) to give title compound as a thick yellow syrup (27.222 g, 70.7%) with consistent lH-NMR and 13C-NMR spectral data.
TLC: Rf 0.27 (Silica gel; CH2C12:CH30H:HOAc-100:5:0.5; W, PMA).

A(2).
o H~ ~,N+C- OC(CH3)3 --COOH

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 CH3OH (5 x 100 ml). The dark filtrate was evaporated to dryness and dried ln vacuo to give a dark solid. The crude product was triturated with CH2Cl2:Hexane (1:4) to give title compound as a light tan solid (17.69 g, 71. %) with consistent 1H-NMR and 13C-NMR spectral data. TLC:
Rf 0.15 (Silica gel; CH2C12:CH30H:HOAc- 20:1:1;
W) .

A(3)-~0~

N~
H o A solution of Part A(2) compound (16.69 g, 56.3 mmoles) in dry dimethyformamide (121 ml) was treated with 1-ethyl-3-(3-dimethylaminopropyl)-30 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) CA 022~1292 1998-10-09 and 1.0 N NaHCO3 (492 ml), and the combined organic extracts were washed with H2O (3 x 492 ml), brine (492 ml), dried (anhydrous MgSO4), 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 (1:4; 1:2; 1:1) to give title compound as off-white crystals (10.5 g, 72.4%) with consistent 1H-NMR and 13C-NMR spectral data. TLC:
Rf 0.40 (Silica gel; EtOAc:Hexane- 1:4i W).

~~~_D

B~N ~ ~
H o Co2~n 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 ,uL, 687 mg, 3.0 mmol). After 25 minutes, the mixture was quenched with saturated NH4Cl, diluted with H2O, and extracted with EtOAc.
The EtOAc extract was washed with H2O and brine, then dried (Na2SO4), filtered and stripped to give a yellow oil. Flash chromatography (Merck SiO2, 3/7-EtOAc/hexanes as eluant) provided title compound (967 mg, 98%) as a colorless oil/foam.

~0~

HCI-H2N~ ~
~ CO2Bn CA 022~1292 1998-10-09 WO 97/3870~ PCT/US97/05744 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 HCl in 1,4-dioxane (6 mL) at room temperature. After 3 hours, the mixture was 5 concentrated in vacuo, triturated with Et2O to give a solid and stripped to afford title compound (858 mg, 105% of theory).
m.p. 152-155~C.

I0 D.
O ~~

~~ ~ H ~ CO2Bn 0¢~

D(l).
~3 ~ coo~
A solution of benzylmalonic acid (23.06 g, 0.12 mole) in H2O (200 mL) was treated with 37%
CH20 solution (278.4 mL) and 40% aqueous (CH3)2NH
(35 mL, 0 31 mole) then stirred overnight at room 20 temperature under argon. The clear solution was heated to an internal temperature of 90~C for 2.0 hours (at which time gas evolution had ceased), cooled and acidified to pH 1.0 with 12 N HCl (20 mL). The white precipitates were filtered off, 25 washed with H2O (3 x 25 mL) and dried in vacuo to give title compound as a white solid (12.85 g, 66.6%) with consistent lH-N~ and 13C-N~. spectral data. TLC: Rf 0.63 (Silica geli CH2C12:MeOH- 9:1;
W). m.p. 66-68~C.

CA 022~1292 1998-10-09 W097/38705 PCT~S9710~744 D(2). H
~ , COOH

(J. Med. Chem. 28, 1985, 1167) s A solution of Part D(l) 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 _ NaOH ~55 ml), stirred for 15 minutes then extracted with EtOAc (4x 18 ml). The organic phase was washed with H2O (3 x 10 ml) and the a~ueous extracts were combined and acidified to pH 2.0 with 1.0 N HCl (62 ml). The acidic aqueous phase was then extracted with EtOAc (5 x 75 ml) and the combined organic extracts washed with H2O (2 x - 30 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo. The crude product (3.93 g, 25.1%) was triturated with Et2O:Hexane (1:4; 2 x 25 ml) and all solids obtained were dissolved in CH2Cl2 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 lH-NMR and 13C-NMR
spectral data.
TLC: Rf 0.33 (Silica gel; CH2C12:MeOH- 9:1; W, ~MA) .
M.p. 69-71~C.

.

CA 022~1292 1998-10-09 W O 97/38705 PCT~US97/05744 D(3)-OHC
~ ~, COOH

A cooled (0~C, ice-salt bath~ mixture of HCOOH (17.5ml) and acetic anhydride (AC2O) (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 Et2O (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 1H-NMR and 13C-NMR spectral data. TLC: Rf 0.23 (Silica geli CH2C12:MeOH- 9:1; W, PMA).

D(4)-~~~_D
~ --~ NH~ N~
0~

A solution of Part D~3) compound (366 mg, 1.19 mmol) in CH2Cl2 (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 ,uL, 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 5%
- KHS04. The EtOAc extract was washed successively .

CA 022~1292 1998-10-09 with H2O, 50% saturated NaHCO3 and brine, then dried (Na2SO4), filtered and stripped. Flash chromatography (Merck SiO2, 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.

O (~

~/ _ H O CO2H
H ~
W 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
o ~~
0~ N I H~
H ~
Isomer B
~ . Trituration of a solution of the residue in MeOH with Et2O provided 350 mg of the diastereomeric mixture. Approximately 255 mg of this mixture was separated by preparative HPLC
('YM~ 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%H20-10% MeOH-0.1%
TFAi solvent B: 10% H2O-90% MeOH-0.1% TFA); title Isomer A tR = 14.4 min; separation performed in three runs). The desired fractions were stripped, W O 97/38705 PCTnUS97/05744 azetroped with EtOAc, re-dissolved in EtOAc and triturated with Et2O to give title Isomer A (105.5 mg) as an off-white solid.

S MS: (M+NH4)+ 459; (M-H)- 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% H2O-10% MeOH-0.2%
H3PO4; solvent B:0% H2O-90% MeOH-0.2% H3PO4); flow rate 1.5 mL/min detecting at 220 nm; tR=9.67 min (96.0%).
Anal. Calc'd for C22H23N3O7-1.6H2O-0.lEtOAc-0.1Et2O
C, 56.29; H, 5.80; N, 8.64 Found: C, 56.21; H, 5.15; N, 8.29.

CA 022~1292 1998-10-09 W O 97/38705 PCT~US97/OS744 Exam~le 2 O ~~

~ ¦ H o CO2H
H ~

S 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 Et2O 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%H20-10% MeOH-0.1% TFA ; solvent B: 10% H2O-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 Et2O to give Isomer B
(88.0 mg) as an off-white solid.

MS: (M+NH4)+ 459; (M-H)- 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%H20-10% MeOH-0.2%
H3PO4; solvent B:0% H2O-90% MeOH-0.2% H3PO4); flow rate 1.5 mL/min detecting at 220 nm; tR = 13.8 min (94.0%).

CA 022~1292 1998-10-09 W O 97138705 PCTnUS97/05744 Anal. Calc'd for C22H23N3O7-1.5H2O-0 2Et2O
C, 56.66; H, 5.84i N, 8.69 Found: C, 56.84; H, 5.22; N, 8.42.

Exam~le 3 ~ ~Me y _ O CO2H
H ~

~ CO2H
-- t1R. 2S)~ eph~d~i"e ¢~/ salt A solution of Example 1 Part D(l) compound ' ~, CO2H ~

(2.563 gm, 8.98 mmol) in CH3CN (20 mL) was treated with (lR,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 Et2O (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 r,insed with 1:1 Et2O:hexanes and dried to afford 2.101 gm of white crystals ([a]D = -16.4~ (c 0.6, CH2C12)). The solid (2.087 gm) was dissolved in CH2C12, concentrated and diluted with Et2O (18 mL) and hexane (8 mL) and seeded. The precipitate was collected by filtration and washed with 1:1-Et2O:hexanes followed by hexanes to give title CA 022~1292 1998-10-09 W 0 97~8705 PCT~US97/05744 compound (1.995 gm) which was diastereomerically enriched in one isomer but not diastereomerically pure ([a]D = -17.0~ (c 0.6, CH2C12)).

mp 110-114~C

Material suitable for x-ray crystallographic analysis was obtained by repeated recrystallization of the solid from CH3CN. mp 117-119~Ci ([a]D = -19.7~ (c 0.4, CH2C12)).

O
CO2Et B(1) .
r OH

(Pht is phthaloyl) Pht=N CO2H

To a stirred solution of L-(+)-hydroxynor-leucine (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-ethoxy-carbonyl-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 25 was cooled in an ice bath and carefully acidified to pH=3 with 6N HCl solution. The white solid which had precipitated was filtered and dried over P2Os ln vacuo to afford Compound 1 (124. 5 g) in 88.1% yield.
M.P. 162~C
Hl-NMR (DMSO): d = 1.32 (m, 6H), 2.13 (m, 2H), 4.38 (s, OH), 5.75 (m, lH), 7.92 (m, 4H) ppm CA 022~l292 l998-lO-09 W 097t38705 PCT~US97/05744 B(2).
rOH

Pht=N CO2Bn S To a stirred slurry of Part B(l) 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 L), washed with water (3X), 5% LiCl solution and brine, dried over anhydrous Mg2SO4 and evaporated i vacuo to afford title compound (142 g) as an oil in 86.1% yield.

H1-NMR (CDC13): d = 1.50 (m, 4H), 2.32 (m, 2H), 3.62 (m, 2H), 4.91 (dd, lH), 5.22 (d, 2H), 7.31 (m, 5H), 7.77 (m, 2H), 7.86 (m, 2H) ppm C13-N~. (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)-Pht=N CO2Bn - To a stirred and chilled (-78~C, Dry ice-IPA bath) oxalyl chloride solution (2.0 M solution in CH2C12, 16.3 ml, 32.6 mmole) under argon was added dropwise a solution of dimethyl sulfoxide (4.64 ml, 65.32 mmole) in dry CH2Cl2 (10 ml).
After the addition was complete, the solution was CA 022~1292 1998-10-09 W O 97/38705 rCTnUS97/05744 stirred at -78~ for 15 minutes, then treated with a solution of Part B(2) compound (lOg, 27.22 mmole) in dry CH2Cl2 (70 ml), stirred at -78~ for another 15 minutes and slowly treated with triethylamine (16 ml). The resulting solution was stirred at -78~ for 15 minutes, gradually warmed up to 0~, poured into 1:1 EtOAc-Et2O t500 ml), washed with 1.0 N HC1 solution, water and brine, dried over anhydrous Mg2SO4 and evaporated in vacuo to afford title compound (10 g) as a light yellow oil in 100%
yield.

H1-NMR (CDCl3): d = 1.66 (m, 2H), 2.40 (m, 4H), 4.90 (dd, lH), 5.18 (d, 2H), 7.35 (m, 5H), 7.74 (m, IS 2H), 7.86 (m, 2H), 9.72 (s, lH) ppm C13-NMR (CDCl3): 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 CO2Bn A stirred and chilled (0~C, ice bath) solution of Part B(3) compound (10.1 g, 27.64 mmole) in dry CH2C12 (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~Cl solution (foaming) and partitioned between 1:1 Et2O-water (400 ml). The organic layer was separated and the aqueous layer was.re-extracted with EtOAc (2x150 ml). The organic extracts were combined, washed CA 022~1292 1998-10-09 W097/38705 PCT~S97tO5744 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, W
and PMA.

H1-NMR (CDCl3): d = 1.12 (d, 3H), 1.43 (m, 4H), 3.73 (m, 2H), 4.90 (dd, lH), 5.19 (d, 2H), 7.30 IO (m, 5H), 7.76 (m, 2H), 7.86 (m, 2H) ppm C13-NMR (CDC13): 22.5, 23.40, 28.47, 28.59, 38.20, 38.34, 52.20.67.35, 67.S1, 123.43, 127.94, 128.19, 128.41, 131.65, 134.11, 135.16, 167.62, 167.67, 15 169.13 ppm B(5)-,.~
- Pht=N CO2Bn To a stirred and chilled (-78~C, Dry ice-IPA bath) oxalyl chloride solution (2.0 M solution in CH2C12, 257.3 ml, 514.6 mmole) under argon was added CH2C12 (300ml). To this solution, a solution of dimethyl sulfoxide (80.4 g, 1.03 mole) in dry 25 CH2C12 (30 ml) was added dropwise. After the addition was complete, the reaction mixture was stirred at -78~ for 20 minutes, treated with a solution of Part B(4) compound (151 g, 395.88 ~mmo~le) in dry CH2Cl2 (700 ml), stirred at -78~C for another 20 minutes and slowly treated with triethylamine (300 ml). The resulting solution was stirred at -78~ for 15 minutes, gradually warmed up to 0~, poured into 1:1 EtOAc-Et2O (3 L), washed with l.O N HCl solution, water and brine, dried over anhydrous Mg2SO4 and evaporated ln vacuo to CA 022~1292 1998-10-09 W O 97/38705 rCTrUS97/05744 afford title compound (149.4 g) as a yellow oil in 99.5% yield.

TLC: Silica gel, 6:4 EtOAc-hexane, Rf=0.5, W and PMA.

Hl-NMR ~CDCl3): d = 1.60 (m, 2H), 2.10 (s, 3H), 2.26 (m, 2H), 2.47 (m, 2H),, 4.90 (dd, lH), 5.19 (d, 2H), 7.30 (m, 5H), 7.74 (m, 2H), 7.84 (m, 2H) ppm C13-NMR (CDCl3): 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 C02~n A chilled (-78~C, Dry ice-IPA Bath) and stirred solution of titanium(IV) chloride (112.05 g, 590.65 mmole) in CH2C12 (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~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 NH4Cl solution. The CH2Cl2 layer was separated. The aqueous layer was extracted with CH2C12 (2x700 ml). The CH2C12 extracts were combined, washed with brine, dried over anhydrous Mg2SO4 and evaporated ln vacuo. The black residue was passed through a pad of silica CA 022~1292 1998-10-09 W097l38705 PCT~S97/05744 gel (E. Merck, 230-400 mesh, 900 g) eluting with EtOAc-hexane (1:1) to afford a tlc-homogeneous title compound ~144.8 g) as a yellow oil in 93~ in yield.
s TLC: Silica gel, 1:1 EtOAc-hexane, Rf=0.4, W and PMA.

Hl-NMR (CDCl3): d=1.14 (s, 6H), 1.45 (m, 4H), 2.30 (m, 2H), 4.90 (dd, lH), 5.19 (d, 2H), 7.30 (m, 5H), 7.74 (m, 2H), 7.86 (m, 2H) ppm C13-NMR (CDCl3): 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 CO2Bn A stirred solution of Part B(6) compound (44.3 g, 364.89 mmole) and azidotrimethylsilane (63.06 g, 547.34 mmole) in dry CH2Cl2 (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 NaHCO3 solution, water and brine, dried aver anhydrous Mg2SO4 and evaporated in vacuo. The residue was chromatographed on a column of silica gel (E. Merck, 230-400 mesh, 700 g) eluting with EtOAc-hexane (1:3) to afford a tlc-homogeneous title compound (124.9 g) as a light yellow oil in 81.3% yield.

CA 022~1292 1998-10-09 W O 97/38705 PCT~US97/05744 TLC: Silica gel, 3:7 EtOAc-hexane, Rf=0.5, UV and PMA.

S H1-MMR (CDC13): d=1.20 (s, 6H), 1.45 (m, 4H), 2.30 (m, 2H), 4.90 (dd, lH), 5.19 (d, 2H), 7.30 (m, 5H), 7.74 (m, 2H), 7.86 (m, 2H) ppm C13-NMR (CDC13): 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 ~ I~

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 1-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 HCl solution, saturated NaHCO3 solution, and brine, dried over anhydrous Mg2SO4 and evaporated in vacuo to give a gum. This was triturated with Et2O-hexane (2:1) to afford a tlc-homogeneous title compound (74.5 g) as a white solid in 87.7% yield.

TLC: Silica gel, 3:7 EtOAc-CH2C12, Rf=0.35, W and PMA.

CA 022~1292 1998-10-09 W O 97/38705 PCT~US97/05744 H1-NMR (CDC13): d=1.30 (s, 3H), 1.45 (s, 3H), 1.74 (m, 2H), 1.96 (m, 3H), 2.74 (m, lH), 4.98 (d, lH), 6.00 (s, lH), 7.20 (m, 2H), 7.85 (m, 2H) ppm s C13-NMR (CDC13): 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).

Ph3C~ N~(~

A stirred solution of Part B(8) compound (74.5 g, 260.19 mmole) in a mixture of CH30H (900 ml) and CH2C12 (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 ln vacuo to give a solid (41 g).
To a stirred solution of the above solid (41 g) in CH2C12 (2 L) at room temperature under argon was added triethylamine (50 ml) and triphenylmethyl chloride (83.41 g). After 1.5 hours, the resulting slurry was diluted with EtOAc, washed with water and brine, dried over anhydrous Mg2SO4 and evaporated in vacuo to give a gum. This was triturated with Et2O-pentane to give title compound (100.1 g) as a white solid in 96.5% yield.

~LC: Silica gel, 6:4 EtOAc-hexane, Rf=0.53, W and PMA.

H1-NMR (CDCl3): d=1.00 (s, 3H), 1.10 (s, 3H), 1.46 (m, 6H), 3.36 (m, lH), 4.03 (m, lH), 5.20 (d, lH), 6.00 (s, lH), 7.20 (m, 2H), 7.85 (m, 2H) ppm ~ , . . .

CA 022~1292 1998-10-09 W O 97~870S PCTrUS97/05744 C13-NMR (CDCl3): 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 B(10).
Me Me H2N~ N~l ~ CO2Et To a stirred solution of Part B(9) compound l0 (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 30 hours, quenched with saturated NH4Cl solution (1.0 - liter) and extracted with EtOAc (3x700 ml). The EtOAc extracts were combined, washed with saturated NaHCO3 solution and brine, dried over anhydrous Mg2SO4 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 EtOAc-hexane (1:4) to give a light yellow oil. This was dissolved in dry CH2Cl2 (2 L) and treated with trifluoroacetic acid (78 ml). The solution was stirred at room temperature under argon for 1.0 hour and then evaporated in vacuo at 30~. The residue was diluted with 1.0 N HCl solution (400 ml) and washed with Et2O (2x400 ml). The a~ueous was carefully neutralized to pH=7-8 with solid NaHCO3 (foaming) and extracted with CH2Cl2 (3x1.2 CA 022~1292 1998-10-09 W O 97138705 PCTrUS97/05744 L). The CH2C12 extracts were combined, dried over anhydrous Na2SO4 and evaporated in vacuo to afford a tlc homogeneous title compound (51.5 g) as a light brown oil in 84.7% yield.
s TLC: Silica gel, 8:1:1 CH2C12-CH3OH-AcOH, Rf=0.3, PMA and Ninhydrin.

Hl-NM~ (CDC13): d=1.28 (t, 3H), 1.36 (s, 3H), 1.38 (s, 3H) 1.60 (m, lH), 1.90 (m, 5H), 3.75 (m, lH), 4.00 (d, lH), 4.22 (q, 2H), 4.28 (d, 2H) ppm C13-NMR (CDCl3): 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 ~ Me HN ~ N ~ ~
H o C02Et Part A compound (641 mg, 1.42 mmol) was partitioned between EtOAc and 5% KH2PO4 (adjusted to pH 2.5 with H3PO4). The layers were separated and the aqueous layer was back-extracted with EtOAc. The pooled EtOAc extracts were washed with brine, dried (Na2SO4), filtered and stripped to give an oil (assume 1.42 mg). The oil was dissolved in CH2Cl2 (10 mL) and the resulting solution was treated with Part B amine (364 mg, 1.50 mmol) in CH2Cl2 (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 , . ..... ...

CA 022~1292 1998-10-09 W O 97~8705 PCTrUS97/05744 partitioned between EtOAc and 5% KH2PO4 (adjusted to pH 2.5 with H3PO4). The EtOAc extract was washed successively with H2O, 50% saturated NaHCO3 and brine, then dried (Na2SO4), filtered and stripped. The residue was flash chromatographed (Merck SiO2, 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, 9%, TLC Rf O . 27 (8/2-EtOAc/hexanes)). NMR of this material was consistant with an isomer of the title compound.

~M e ~ ~ M e y - H O CO2Et ~
Acetic anhydride (500 ~L) 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 NaHCO3.
The EtOAc extract was washed with brine, dried (Na2SO4), 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 W O 97/38705 PCTrUS97/05744 linear gradient (solvent A: 90%H20-10% MeOH-0.2%
H3PO4; solvent B:0% H2O-90% MeOH-0.2% H3PO4); flow rate 1.5 mL/min detecting at 220 nm; tR = 17.2 min 100%).

E.
~M e ~ ~'b' Me y _ O CO2Et H ~

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/Et2O/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%
H3PO4; solvent B:0% H2O-90% MeOH-0.2% H3PO4); flow rate 1.5 mL/min detecting at 220 nm; tR = 12.8 min ( 100%).

F.

~' ~ OyN~~cMMOeeH
H ~

CA 022~1292 1998-10-09 W097/38705 PCT~S97/05744 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% KHSO4 and extracted twice with EtOAc. The EtOAc extract was washed with brine, dried (Na2SO4), filtered and stripped. The residue was dissolved in a small amount of MeOH and EtOAc and triturated with Et2O/hexanes to give title compound (134 mg, 86%~
as an off-white solid/foam ([a]D = +18.0~ (c 0.5, CH2Cl2)).

I5 TLC Rf 0.10 (5/95-HOAc/EtOAc) HPLC YMC S3 ODS column (6.0 x 150 mm)i eluted with B:A solvent mixture, 40 to 100% B over a 20 minute linear gradient (solvent A: 90%H20-10% MeOH-0.2%
H3PO4; solvent B:0% H2O-90% MeOH-0.2% H3PO4); flow rate 1.5 mL/min detecting at 220 nm; tR = 9~~~
min (>97.4%).

Anal. Calc'd for C21H29N3O6-0.75H2O-0-3Et2O
C, 58.57; H, 7.42; N, 9.23 Found C, 58.31i H, 7.20i N, 8.99.

Exam~le 4 ~S-(R*,R*)]-3-[[3-(Formylhydroxyamino)-l-oxo-2-(phenylmethyl)propyl]amino]-2,3,4,5-tetrahydro-2-oxo-lH-benzaze~ine-l-acetic acid N~~ N~ N~
HO - H ~ COOH
13' CA 022~1292 1998-10-09 W O 97/3870~ PCTrUS97/05744 ~ N~, COOC2H5 H2N ll A~l).
Z~o H
Solid sodium azide (26.0 g., 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 0~ (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 cont~lnlng 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 eq.), heated to 40-45~ (oil bath) then treated dropwise with 36.0 N H2SO4 (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 H2O (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) CA 022~1292 1998-10-09 W097/38705 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~C) with consistent lH-NMR and 13C-NMR spectral data.
The mother liquor was chromatographed on a silica gel column (Merck, 240 g), eluting the column with EtOAc:Hexane (1:4) to give an additional amount of 5.058 g (total yield= 15.075 g, 85.6 %).
TLC: Rf 0.37 (Silica geli EtOAc:Hexane-l:li W).

A(2).

~ ~ Br A solution of Part A(l) compound (1.0 g, 6.20 mmoles) in dry CHC13 (15 ml) was cooled down to 0~C (ice-salt bath), treated with PCls (1.5 g, 7.20 mmoles) followed by I2 (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 g), stirred and the phases were separated, washing the a~ueous phase with CHC13 (25 ml). The combined organic extracts were washed with H2O (5.0 ml), dried (anhydrous MgSO4), filtered, evaporated to dryness and dried in vacuo. The crude product mixture was chromatographed on a silica gel column (Merck, 70 g),-eluting the column with EtOAc:Hexane (1:9) to give title compound as off-white precipitates (1.137 g., m.pt. 170-172~, 70.1 %) with consistent H-NMR and 13C-NMR spectral data. TLC: Rf 0.13 (Silica geli EtOAc:Hexane -1:4; W).

CA 022~1292 1998-10-09 W O 97/38705 PCT~US97/05744 A(3)-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 60~ (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 60~ over drierite for 24 hours to give title compound (725 mg, m.pt. 150-152~, 91.9 %) as an off-white solid with consistent lH-NMR and 13C-NMR spectral data.
TLC: Rf 0.58 (Silica gel; EtOAc:Hexane- 1:4 then 1:1; W).

A(4).
~ ~ ~ N3 cooC2H5 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 H2O (196 ml) and CH2C12 (2 x 375 ml). The combined organic extracts were washed with H2O (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 CA 022~1292 1998-10-09 W O 97138705 PCT~US97/05744 mmole scale) and chromatographed on a silica gel column (Merck), eluting the column with Toluene:EtOAc (98.2) and EtOAc:Hexane (1:9) to give title compound as a solid (15.48 g, 93.5%)1 with consistent lH-NMR and 13C-NMR spectral data.
TLC: Rf 0.63 (Silica geli EtOAc:Hexane- 1:2; W).

A(5)-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 45 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, w~.~h;ng 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 1H-NMR and 13C-NMR spectral data. TLC:
Rf 0.45 (Silica gel; CH2Cl2:CH30H- 9:1; W).

A(6)-H2N~ N~COOC2H5 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 0~, at room temperature for 3 days and then at 0~ for another 2 days. The solid that formed was recrystallized from absolute ethanol (118 ml~ two CA 022~1292 1998-10-09 W097/38705 PCT~S97/OS744 more times until a consistent specific rotation was obt~'ne~. 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 (15 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.
105-107~, 26.5%) with consistent lH-NMR and 13C-NMR
spectral data.
[a]D = -277~ (c 0.99, EtOH). TLC : Rf 0.45 (Silica gel; CH2C12:CH30H- 9:1; W).

I5 B.

,N ~ N
~~ -- 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 ~g,~0.93 mmole) in dry CH2C12 (6.0 ml) was cooled to 0~C (ice-salt bath) and treated sequentially with a solution of the above free amine (271 mg) in dry CH2Cl2, 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 CA 022~1292 1998-10-09 W097/38705 PCT~S97/057 temperature for 2.0 hours, then partitioned between EtOAc (2 x 20 ml) and H2O (4.0 ml). The organic extracts were washed with 5% KH2PO4 (adjusted to pH
2.5; 4.0 ml), H2O (4.0 ml), saturated NaHCO3 (4.0 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 g.), eluting the column with EtOAc:Hexane mixtures (1:3i 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 lH-NMR
and 13C-NMR spectral data. TLC: Rf 0.32 (Silica gel;
EtOAc:Hexane -1:1; W).

OHC N~J~ , N~ COOC2H5 A cooled solution of HCOOH (5.0 ml) was treated with acetic anhydride (Ac2O) (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 Ac2O/HCOOH mixture (3.4 ml) and stirred at 0~C for 1.0 hour. The reaction mixture was èvaporated to dryness and the residual syrup was dissolved in EtOAc (40 ml), washed with saturated NaHCO3 (5.0 ml) and brine (5.0 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness, evaporated from toluene and dried in vacuo to give title compound as a syrup (311.3 mg, 100 %

CA 022~1292 1998-10-09 W O 97/38705 PCTrUS97/05744 crude) with consistent lH-NMR and 13C-NMR spectral data. TLC: Rf 0.18 (Silica gel; EtOAc:Hexane (1:1;
W) .

S D.

N--J~ N$~CooC2H5 HO - H ~

A solution of Part C compound (311 mg) in CH30H (10 ml) was treated with 10% Pd/C (53 mg) and hydrogenated (balloon) at room temperature for 2.0 hours. The reaction mixture was diluted with CH30H
(10 ml) and filtered through a Celite~ pad in a millipore unit, washing the pad well with CH30H (3 x 10 ml). The clear riltrate was evaporated to dryness and dried in vacuo to give title compound as a syrup (256.7 mg, 100% crude) with consistent lH-NMR and 13C-NMR data. TLC: Rf 0.25 (Silica gel;
CH2Cl2:MeOH- 9:1; W).

E. [S-(R*,R*)]-3-[[3-(Formylhydroxyamino)-l-oxo-2-~phenylmethyl)propyl]amino]-2,3,4,5-tetrahydro-2-oxo-lH-benzazepine-l-acetic acid A solution of Part D compound (256.7 mg) in CH30H (3.5 ml) was treated with 1.0 _ NaOH (2.17 ml, 4 e~) and stirred at room temperature for 1.0 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 Na2S04), filtered, evaporated to dryness and dried in vacuo. The crude product was triturated with CH2Cl2 Hexane W O 97/38705 PCT~US97/05744 ~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, lH-NMR and analytical data. TLC: Rf O.30 (Silica gel EtOAc:HOAc- 95:5i W).

[a] D = -332.8~ (c 0.558, CH30H) 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 nm, flow rate = 1.5 ml/min; 56% (10% H20- 90% CH30H-0.2% H3P04)/44% (90% H20- 10% CH30H-0.2% H3P04), isocratic.

Anal. Calc'd for C23H25N306:
C, 62.86i H, 5.73; N, 9.56 Found: C, 62.88i H, 5.98; N, 9.20.

ExamDle 5 O ~U

OHC ~ N ~ll' N~ ~J
H ~ COOH

A.

OH
H f Pht=N ~ ~~

A solution of L-hydroxynorleucine (2.0 g, 13.6 mmoles) in dry methanol (70 ml) was saturated with HCl gas until a clear yellow solution was obt~'ne~. The reaction mixture was cooled to room temperature, stirred for 2.0 hours, evaporated to CA 022~1292 1998-10-09 W097l38705 PCT~S97/05744 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 0~C
(ice-salt bath). The mixture was treated with N-phthaloyl-L-phenylalanine (4.0 g, 13.6 mmoles), HOBt-H2O (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 H2O (60 ml) and the combined organic extracts were washed sequentially with 0.5 N HCl (60 ml), H2O (60 ml), 1/2 saturated NaHCO3 (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 g), eluting the column with EtOAc to give the desired product as a syrup (4.0 g). An additional 321 mg was obtained on re-chromatography of the impure fractions to give title compound (4.32 g, 73%) with consistent 1H-NMR and 13C-NMR spectral data.
TLC: Rf 0.43 (Silica gel; EtOAci W).

O
H //
Pht=N ~ ~~

A solution of oxalyl chloride (1.02 ml,11.7 mmoles) in dry CH2C12 (56 ml), was cooled to -78~C
(dry-ice-acetone bath), treated with a solution of dry DMSO (1.67 ml, 21.6 mmoles) in CH2Cl2 (2.0 ml) .. . , . ~

CA 022~1292 1998-10-09 W O 97/38705 PCTrUS97/05744 and stirred at -78~C for 20 minutes. The mixture was treated with a solution of Part A compound (4.29 g, 9.78 mmoles) in dry CH2Cl2 (22 ml), stirred at -78~C for another 15 minutes, then treated with triethyl-amine (8.4 ml). The reaction mixture was stirred at -78~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 HCl (2 x 20 ml). The organic phase was washed with brine (40 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo to give title compound as a thick syrup (4.428 g, 100% crude yield), with consistent lH-NMR
and 13C-NMR spectral data.
TLC: Rf 0.73 (Silica gel; EtOAc; W).
¢~

Pht= ~ ~
o COOCH3 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 NaHCO3 (20 ml) and brine (20 ml), dried (anhydrous Na2SO4), filtered, evaporated to dryness and dried in vacuo.
The crude product mixture was chromatographed on a sil~ica gel column (Merck, 200 g), eluting the column with CH2C12:EtOAc (9:1) to give the desired product as a syrup. The syrup was triturated with Et2O:Hexane ~2:1-60 ml) to give title compound as a white precipitate (2.92 g, 72%; m.p. 141-143~C) with consistent lH-NMR and 13C-NMR spectral data.

CA 022~1292 1998-10-09 W097/38705 PCT~S97/OS744 TLC: Rf 0.67 (Silica gel; CH2C12:EtOAc-9:li W).

Pht= N~ ll 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 H2O (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 20 vacuo to give impure title compound as a solid foam (1.238 g, 42%) with consistent lH-NMR and 13C-NMR spectral data. TLC : Rf 0.73 (Silica gel;
EtOAc:HOAc-95:5; W).

E.

Pht=N~
o COOBn A solution of Part D compound (1.238 g, 3.06 mmoles) in dry DMF (3.5 ml) was treated CA 022~1292 1998-10-09 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 H2O (5.0 ml), 0.5 _ HCl (5.0 ml) and brine (5.0 ml), dried (anhydrous Na2SO4), 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 lH-NMR and 13C-NMR spectral data.
TLC: Rf 0.45 (Silica geli EtOAc:Hexane-l:l; W).

H2N O cooen A solution of Part E compound (586 mg, 1.18 mmoles) in dry methanol (15 ml) was treated with NH2NH2-H2O (66 ,ul, 1.2 eq) and stirred at room temperature for 48 hours. The reaction mixture was diluted with Et2O (50 ml) and filtered through a millipore unit, washing the solids well with Et2O
(40 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 (40 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 lH-NMR spectrum.
TLC: Rf 0.42 (CH2Cl2 MeOH-9:1; W, Ninhydrin) CA 022~1292 1998-10-09 W O 97/38705 PCTnUS97105744 ~' BnO _ H O COOBn Example 3 Part A ephedrine salt (538 mg, 5 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 10 the free acid of the ephedrine salt as a clear syrup (323 mg, 100% crude yield).
A solution of the free acid in dry CH2Cl2 (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 (2.0 ml), HOBT-H2O (163 mg, 1.2 mmoles) and EDAC t 240 mg, 1.25 mmoles). The reaction mixture was stirred at 0~C for 1.0 hour, at room temperature for 1.5 hours, then partitioned between EtOAc (40 ml) and H2O (5.0 ml). The organic extracts were washed with 5 % KH2PO4 (adjusted to pH 2.5i 5.0 ml), H2O
(-5.0 ml), saturated NaHCO3 (5.0 ml) and brine (5.0 ml), dried (anhydrous Na2SO4), 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 (1:3) to give pure title compound (494 mg, 65%) as a solid foam with consistent lH-N~ and 13C-NMR spectral data.
TLC: Rf 0.45 (Silica gel; EtOAc:Hexane -1:1; W).

CA 022~1292 1998-10-09 W O 97/38705 PCTrUS97105744 O ~

BnO H O COOBn A cooled solution (0~C, 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 (60 ml), washed with saturated NaHC03 (7.0 ml) and brine (7.0 ml), dried (anhydrous Na2S04), 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 lH-NMR and 13C-NMR spectral data.
TLC: Rf 0.2 (Silica gel; EtOAc:Hexane-l:l; W).
I.

OHC~N,J~ N~
~ H ~ 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 CA 022~1292 1998-10-09 W O 97/38705 PCTnUS97tO5744 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 CH2C12: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, 90%).
TLC: Rf 0.38 (Silica geli CH2Cl2:MeOH- 9:1; W).
MS (M+H)+ = 480 [a]D = +44.6~ (c 0.52, CH30H) 15 HPLC : tR= 11.72 min (95.9% ); YMC S3 ODS-A 150 x 6 mm; 220 nm, flow rate = 1.5 ml/mini 55% (10% H20-90% CH30H- 0.2% H3P04)/ 45% (90% H20- 10% CH30H-0.2% H3P04), isocratic.

20 Anal. Calc~d for C26H2gN306-0.4 H20-0.14 Hexane - (Eff. Mol. Wt. = 497.08):
C, 64.63; H, 6.83; N, 8.46 Found: C, 64.24i 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.

PCT~US97/05744 W O 97~870S

~R ~ A

Example No. R-- _ ~ A

6 H 1CH2Ph r )~

~ CO2H

7 H 1 CH2Ph 5--N$~

~ CO2H
~ H

8 H 1CH2CH(CH3)2~--N$~) ~ CO2H

9 H 1 CH2Ph ~ $

~ CO2H
~--N
H 1CH2CH(CH3)25--N~N~
~ CO2H

11 H 1 CH2Ph ~--N~N~Me ~ CO2H

12 H 1 CH2Ph 5--N~ ~--J
~ CO2H

CA ~2251z92 1998 1o og PcTlus97/o5744 13 H I ~

~Nle "f~ Ph 14 H 1 CH(CH3)2 5--~ ,, C~2H

H 1 CH(CH3)2 ~--~N~--c02H
H O 2=

16 H 1 CH(cH3)2 HN$~ N~ Co2H
,.~

~OH
17 H 1 CH2Ph HN~ CO2H

\~ Ph

Claims (15)

What is claimed is:

1. A compound of the formula including a pharmaceutically acceptable salt thereof wherein x is 0 or 1, R is H, alkyl, alkenyl, aryl-(CH2)p-heteroaryl-(CH2)p-, cycloheteroalkyl-(CH2)p-, 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;
R1 is H or -COR2 where R2 is alkyl, aryl-(CH2)p-, cycloheteroalkyl-(CH2)p-, heteroaryl-(CH2)p-, 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 non-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 wherein R1a, R1b, R2a and R2b are independently selected from H, alkyl, aryl-(CH2)p-, cycloalkyl, cycloheteroalkyl-(CH2)p-, heteroaryl-(CH2)p-, biphenylmethyl, or R1a and R1b or R2a and R2b may be joined together to the carbon to which it is attached to form a 3 to 7 memebered ring, optionally fused to a benzene ring; and refers to an optional 5 or 6 membered ring containing a single hetero atom and which may optionally include an R5 substituent which is H, alkyl, aryl-(CH2)p, cycloalkyl-(CH2)p, cycloheteroalkyl-(CH2)p or cycloheteroaryl-(CH2)p-;
R3 is H, alkyl or aryl -(CH2)p-;
R4 is OH, Oalkyl, Oaryl-(CH2)p- or NR1(R2) where R1 and R2 are independently H, alkyl, aryl, aryl(CH2)p or heteroaryl(CH2)p;
with the proviso that in A(1) at least one of and is other than a natural .alpha.-amino acid.

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

4. The compound as defined in Claim 3 wherein the conformationally restricted dipeptide mimic has the structure .

5. The compound as defined in Claim 3 wherein A has the formula ;

with respect to A(5), R11 and R12 are independently selected from hydrogen, alkyl, alkenyl, cycloalkyl -(CH2)p-, aryl -(CH2)p-, and heteroaryl -(CH2)p-, or R11 and R12 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R11 and R12 taken together with the carbon to which they are attached complete a keto substituent, with respect to A(13), R8, R9 and R7 are independently selected from hydrogen, alkyl, alkenyl, cycloalkyl -(CH2)m-, aryl-(CH2)m-, and heteroaryl-(CH2)m-;
R10 and R6 are independently selected from hydrogen, alkyl, alkenyl, cycloalkyl -(CH2)p-, aryl-(CH2)p, and heteroaryl-(CH2)p-, or R6 and R10 taken together with the carbons to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, R6 and R8 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons, or R9 and R10 taken together with the carbon to which they are attached complete a saturated cycloalkyl ring of 3 to 7 carbons;
R4 is OH, Oalkyl, O-(CH2)p-heteroaryl, , -O-(CH2)p-aryl or or NR1(R2) where R1 and R2 are independently H, alkyl, aryl, aryl-(CH2)p or heteroaryl;
R14 is hydrogen, alkyl, cycloalkyl, or phenyl;
R15 is hydrogen, alkyl, alkoxy or phenyl;
R16 is alkyl or aryl-(CH2)m-; and R17 is hydrogen, alkyl, substituted alkyl, alkenyl, cycloalkyl-(CH2)m-, aryl-(CH2)m-, or heteroaryl-(CH2)m-.
R18 is H or alkyl or alkenyl, and R18 and R17 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.
R19 is H or an alkyl, and in A(4), R19 and X
(which is CH2) together with the carbons to which they are attached may form an aromatic ring of carbons (as in A(15).

6. The compound as defined in Claim 1 wherein A is .

7. The compound as defined in Claim 6 wherein A is , or .

8. The compound as defined in Claim 1 wherein R1 is H, R is alkyl or arylalkyl, R4 is OH.

9. The compound as defined in Claim 2 where in A(l) is a non-proteinogenic amino acid portion.

10. The compound as defined in Claim 9 wherein R1a and R1b are independently alkyl or arylalkyl, or R1a and R1b together with the carbon to which they are attached form a 3 to 7 membered ring; or one of R1a and R1b is biphenylmethylene and the other is biphenylmethylene or H.

11. The compound as defined in Claim 9 where in A(l), is a non-proteinogenic amino acid where R3 is H, alkyl or arylalkyl, R2a and R2b are independently selected from H, alkyl, aryl or arylalkyl, with at least one of R2a and R2b being other than H, or R2a and R2b together with the carbon to which they are attached form a 3 to 7 membered ring.

12. A pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in Claim 1 and a pharmaceutically 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 or a pharmaceutically acceptable salt thereof.