Inhibitors of nitric oxide biosynthesis

The present invention is a method of treating hypotension and shock using select ornithine or N.sup.G -arginine derivatives.

The present invention is directed to a new class of arginine derivatives 
which are useful in inhibiting the biosynthesis of nitric oxide from 
L-arginine. Another aspect of this invention is directed to the treatment 
of a number of disease states such as, for example, conditions associated 
with low blood pressure and to the treatment of inflammatory diseases and 
stroke. A further aspect of the invention is directed to compositions 
containing these arginine derivatives. 
BACKGROUND OF THE INVENTION 
It has recently been discovered that nitric oxide mediates a number of 
physiological processes. It serves as a signal transduction mechanism in 
the central nervous system, is intimately involved with the regulation of 
blood pressure, and is produced by activated inflammatory cells thereby 
playing a role in pathological inflammatory conditions. 
Garthwaite et al. and Stevens reported that stimulation of the glutamate 
site of the NMDA receptor complex leads to the release of nitric oxide. 
The nitric oxide stimulates the guanylate cyclase present in the CNS which 
in turns leads to an increase in cyclic GMP levels. A great deal of 
attention has been focused upon these excitatory amino acid receptors 
because it is believed that they are associated with learning and 
development. It is also believed that over-stimulation of these receptors 
is associated with a number of disease states such as, for example, 
epilepsy, stroke, and neurodegenerative diseases such as senile dementia, 
etc. Nature, 336, pages 308-309 and 385-388 (November 1988). 
Knowles et al. reported the mechanism by which the nitric oxide is formed. 
An enzymatic reaction occurs in which L-arginine is converted into nitric 
oxide and citrulline. Knowles et al. also reported that the presence of 
N-monomethyl-L-arginine inhibited this enzymatic transformation. Proc. 
Natl. Acad Sci. USA, 86, pages 5159-5161 (July 1989). 
Moncada et al. provided a review of the information which had been 
published to date regarding the physiological role of nitric oxide. 
Moncada et al. described the work of Knowles and Garthwaite regarding the 
role of nitric oxide in the CNS and postulated that nitric oxide is 
involved in seizures and other disease states associated with excessive 
excitatory neurotransmission. Biochemical Pharmacology, 38, No. 11, pages 
1709-1715, (1989). 
Moncada et al. reported that nitric oxide is associated with the regulation 
of blood pressure. These authors reported that an enzymatic pathway exists 
in the vasculature by which L-arginine is converted into nitric oxide. 
Nature, 333, pages 664-666 (1988). The nitric oxide relaxes the smooth 
muscles of the vasculature, producing dilation of these vessels which 
results in a lowering of blood pressure. Inhibition of nitric oxide 
synthesis by the administration of N-monomethyl-L-arginine produces a 
marked rise in blood pressure in test animals. Proc. Natl. Acad. Sci, USA, 
86, pages 3375-3378 (1989). 
Moncada et al. also reviewed earlier literature which reported that nitric 
oxide can be produced in large amounts by macrophages and 
polymorphonuclear leucocytes (PMN) and is associated with the effector 
functions of the cells. N-monomethyl-L-arginine inhibits the synthesis of 
nitric oxide by these cells. 
The discovery of compounds capable of inhibiting the biosynthesis of nitric 
oxide would provide a new treatment for a number of disease states. They 
could be utilized in the treatment of conditions associated with low blood 
pressure such as, for example, shock. They could also be used to treat 
conditions caused by over-activity of the NMDA receptor complex such as 
epilepsy, stroke, neurodegenerative diseases, etc. They could also be used 
to treat conditions associated with excessive production of nitric oxide 
by macrophages and polymorphonuclear leucocytes such as arthritis, 
cirrhosis, transplant rejections, etc.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention it has been discovered that the 
biosynthesis of nitric oxide can be inhibited by the following class of 
arginine derivatives: 
##STR1## 
in which: a) in Formula Ia, A is represented by --(CH.sub.2).sub.2 --, 
--(CH.sub.2).sub.3 -- or --CH.dbd.CH--; X is represented by cyano, 
cyclopropyl, 2-propyne, 2,3-butadiene or NHR.sub.2 in which R.sub.2 is 
represented by H, --CF.sub.3, --CH.sub.2 CF.sub.3, or C.sub.1-6 alkyl; and 
R is represented by an amino acid or OM, in which M is represented by H, 
C.sub.1-6 alkyl, benzyl, phenyl, pivoyl methyl ether, and the 
pharmaceutically acceptable salts thereof; 
b) in Formula Ib, R and X are as defined as above, Z is represented by O or 
NR.sub.1 in which R.sub.1 is represented by H, --CF.sub.3, --CH.sub.2 
CF.sub.3, or C.sub.1-6 alkyl; and the pharmaceutically acceptable salts 
thereof and; 
c) in Formula Ic, W is represented by a substituent selected from the group 
consisting of --CH.sub.2 --NH--, --(CH.sub.2).sub.2 --NH--, --CH.sub.2 
--NH--NH--, --CH.sub.2 --O--NH--, --NH--NH--, and --O--NH--; and R, Z, and 
X are as defined above; and the pharmaceutically acceptable salts thereof; 
with the following proviso's: 
1) that when W is --NH--NH or --CH.sub.2 --NH--NH-- then Z must be 
represented by O; 
2) when Z is represented by O and X is represented by NR.sub.1, then W must 
be --CH.sub.2 --NH or --(CH.sub.2).sub.2 --NH-- and; 
3) that when W is represented by CH.sub.2 --NH, Z is represented NH.sub.2, 
and X is NHR.sub.2, then R.sub.2 is not represented by H. 
The compounds encompassed by Formulae Ia-c inhibit the biosynthesis of 
nitric oxide. They prevent the bioconversion of L-arginine into nitric 
oxide and citrulline. Thus these compounds will be useful in the treatment 
of a number of disease states in which excessive levels of nitric oxide is 
implicated. 
As used in this application: the term, 
a) "cyano" refers to the following substituent: --CN; 
b) "cyclopropyl" refers to the following substituent: 
##STR2## 
c) "2-propyne" refers to the following substituent: CH.sub.2 --C.tbd.CH; 
d) "2,3-butadiene" refers to the following substituent: --CH.sub.2 
--CH.dbd.C.dbd.CH.sub.2 ; 
e) "C.sub.1-6 alkyl" refers to a straight chain, branched, or cyclic alkyl 
group containing up to 6 carbon atoms. Representative examples of suitable 
alkyl groups include, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, 
pentyl, hexyl, cyclopropyl, and cyclopentyl; 
f) "pivoyl methyl ether" refers to the following substituent: --CH.sub.2 
--O--CO--C--(CH.sub.3).sub.3 ; 
g) "amino acid" refers to one of the naturally occurring amino acids, and 
more preferably either aspartic acid or glutamic acid. The amino acids 
encompassed by the present invention are listed below in Table I. It is 
preferred that these amino acids be in their L-configuration Their 
structures are disclosed in the well known text Lehninger's Biochemistry. 
The amine function of these amino acids is bonded to the carbonyl group 
adjacent to R in the compounds of Formula Ia-c. 
TABLE I 
______________________________________ 
AMINO ACID SYMBOL 
______________________________________ 
Alanine Ala 
Arginine Arg 
Aspargine Asn 
Aspartic acid Asp 
Cysteine Cys 
Glutamine Gln 
Glutamic acid Glu 
Glycine Gly 
Histidine His 
Isoleucine Ile 
Leucine Leu 
Lysine Lys 
Methionine Met 
Phenylalanine Phe 
Proline Pro 
Serine Ser 
Threonine Thr 
Tryptophan Trp 
Tyrosine Tyr 
Valine Val 
______________________________________ 
Since the compounds of Formula I are derivatives of the amino acid 
arginine, they can exist as acid addition salts, basic addition salts, as 
zwitterions, or as their free base. Thus the term "pharmaceutically 
acceptable salts" should be construed as encompassing pharmaceutically 
acceptable acid additions salts, pharmaceutically acceptable basic 
addition salts or the zwitterion form of the molecule. 
The expression "pharmaceutically acceptable acid addition salts" is 
intended to apply to any non-toxic organic or inorganic acid addition salt 
of the base compounds represented by Formula Ia-c or any of its 
intermediates. Illustrative inorganic acids which form suitable salts 
include hydrochloric, hydrobromic, sulphuric, and phosphoric acid and acid 
metal salts such as sodium monohydrogen orthophosphate, and potassium 
hydrogen sulfate. Illustrative organic acids which form suitable salts 
include the mono-, di-, and tricarboxylic acids. Illustrative of such 
acids are for example, acetic, glycolic, lactic, pyruvic, malonic, 
succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, 
hydroxymaleic, benzoic, hydroxy-benzoic, phenylacetic, cinnamic, 
salicyclic, 2-phenoxy-benzoic, p-toluenesulfonic acid, and sulfonic acids 
such as methane sulfonic acid and 2-hydroxyethane sulfonic acid. Such 
salts can exist in either a hydrated or substantially anhydrous form. 
The expression "pharmaceutically acceptable basic addition salts" is 
intended to apply to any non-toxic organic or inorganic basic addition 
salts of the compounds represented by Formula Ia-c or any of its 
intermediates. Illustrative bases which form suitable salts include alkali 
metal or alkaline-earth metal hydroxides such as sodium, potassium, 
calcium, magnesium, or barium hydroxides; ammonia, and aliphatic, 
alicyclic, or aromatic organic amines such as methylamine, dimethylamine, 
trimethylamine, and picoline. 
All of the compounds of Formula Ia-c exist as optical isomers. Those 
compounds of Formula Ia-c in which X is represented by a butadiene will 
exist as stereoisomers as well. Those compounds of Formula Ia-c which are 
in the L-configuration are preferred due to their superior potency. 
However, any reference to the compounds of Formula Ia-c or to any of their 
intermediates should be construed as referring to either an optical 
isomer, an enantiomer, or to a racemic mixture. The specific optical 
isomers and enantiomers can be separated and recovered by techniques known 
in the art such as chromatography on chiral stationary phases or 
resolution via chiral salt formation and subsequent separation by 
selective crystallization. Alternatively utilization of a specific optical 
isomer or enantiomer as the starting material will produce the 
corresponding isomer or enantiomer as the final product. 
Those compounds of Formula Ia, in which A is represented by an ethlylene 
moiety, and all of the compounds of Formula Ib will exist as geometrical 
isomers. Any reference to the compounds of Formula Ia or Ib should be 
construed as encompassing either the cis or trans isomers. 
Those compound of Formula Ib in which Z is represented by NR.sub.2, as well 
as those compounds of Formula Ic in which Z is NR.sub.2 while W is 
--CH.sub.2 --NH or --(CH.sub.2).sub.2 --NH-- will exist as tautomers. The 
geminal nitrogen atoms will exist in a state of equilibrium in which they 
share a pair of electrons. Any reference to the compounds of Formula Ib or 
Ic should be construed as encompassing any of these tautomers. 
Examples of compounds encompassed by Formula Ia-c include: 
1) 2-Amino-5-[(hydrazinoiminomethyl)amino]-3-pentenoic acid 
2) N.sup.5 -[(cyclopropylamino)iminomethyl]-L-Ornithine 
3) N.sup.5 -[Imino(2-propynylamino)methyl]-L-ornithine 
4) 5-(Hydrozinoiminomethyl)norvaline 
5) 2-Amino-5-[[imino(2-propynylamino)methyl]amino]-3-pentenoic acid 
6) 2-Amino-5-[(hydrazinocarbonyl)amino]-3-pentenoic acid 
7) N.sup.5 -(Hydrazinocarbonyl)-L-ornithine 
8) 2-Amino-5-[(hydrazinocarbonyl)amino]-3-pentenoic acid 
9) N-[3,4-Didehydro-N.sup.5 -(hydrazinoiminomethyl)-L-ornithyl]-L-aspartic 
acid 
10) N-[3,5-Didehydro-N.sup.5 -(hydrazinoiminomethyl)-L-ornithyl]-L-Glutamic 
acid 
11) N-[N.sup.5 -[(Cyclopropylamino)iminomethyl]-L-ornithyl]-L-glutamic acid 
12) N-[N.sup.5 -[(Cyclopropylamino)iminomethyl]-L-ornithyl]-L-aspartic acid 
13) N-[N.sup.5 -[Imino(2-propynylamino)methyl]-L-ornithyl]-L-glutamic acid 
14) N-[N.sup.5 -[Imino(2-propynylamino)methyl]-L-ornithyl]-L-aspartic acid 
15) N-[3,4-Didehydro-N.sup.5 
-[imino(2-propynylamino)methyl]-L-ornithyl]-L-aspartic acid 
16) N-[3,4-Didehydro-N.sup.5 
-[imino(2-propynylamino)methyl]-L-ornithyl-L-glutamic acid 
17) N.sup.5 -[(Cyclopropylamino)iminomethyl]-L-ornithine, methyl ester 
18) 2-Amino-5-[[imino(2-propynylamino)methyl]amino]-3-pentenoic acid, 
methyl ester 
19) Hydrazinecarboxylic acid, 4-amino-4-carboxybutyl ester 
20) 6-[[(Cyclopropylamino)carbonyl]oxy]-norleucine 
21) Hydrazinecarboxylic acid, 4-amino-4-carboxy-2-butenyl ester 
22) N.sup.5 -[Imino(2-methylhydrazino)methyl]-L-ornithine 
23) 2-Amino-4-[2-(hydrazinoiminomethyl)hydrazino]-butanoic acid 
24) N.sup.5 -(Hydrazinoiminomethyl)-L-ornithine 
25) O-[(Hydrazinoiminomethyl)amino]-L-homoserine, flavinate 
26) N.sup.6 -(Hydrazinocarbonyl)-L-lysine 
27) 2-Amino-4-[2-[(cyclopropylamino)carbonyl]hydrazino]-butanoic acid 
28) 5-[2-[(Cyclopropylamino)carbonyl]hydrazino]-norvaline 
It has been discovered that the substituent which is present at the X 
position can affect the relative activity of the compounds. Those 
compounds in which X is bulky substituent such as 2-propyne, cyclopropyl, 
or 2,3-butadiene show superior activity for inhibiting the production of 
nitric oxide within the CNS. 
The compounds of Formula Ia-c can be made using techniques well known in 
the art. Those compounds which can be described by Formula Ia can be made 
using the synthesis outlined below in Reaction Scheme I: 
##STR3## 
In Step A of Reaction Scheme I, a displacement reaction is carried out 
between phosgene (2, Y.dbd.Cl) or triphosgene (2, Y.dbd.OCCl.sub.3) and a 
functionalized amino acid as described by structure (1) in which A is as 
in Formula I and Pr is a t-butyloxycarbonyl group, while Pr' is a t-butyl 
group. This displacement reaction produces a chloroformate or a 
trichloromethylurethane as described by structure (3) in which Pr, Pr' and 
A are as defined above. In Step B, the chloroformate of structure (3, 
Y.dbd.Cl) or the trichloromethylurethane (3, Y.dbd.OCCl.sub.3) is 
subjected to an amidation reaction with an amino-derivative as described 
by structure (4) in which X is as defined in Formula Ia, thereby producing 
the protected compound of Formula Ia'. In Step C, the protecting groups Pr 
and Pr' are removed thus producing a compound of Formula Ia. In Step D, an 
optional functionalization reaction can be carried out in order to 
introduce the appropriate R substituent into the molecule at the position 
indicated. 
The displacement reaction of Step A can be carried out using techniques 
well known in the art. The particular functionalized amino acid of 
structure (1) which is utilized as a starting material should have the 
same function at the A position as is desired in the final product of 
Formula Ia. Typically approximately equivalent amounts of the amino acid 
of structure (1) and the phosgene or triphosgene are contacted in an 
organic solvent such as benzene, toluene or methylene dichloride at a 
temperature range of from about -10.degree. C. to about 60.degree. C. The 
displacement reaction is typically allowed to proceed for a period of time 
ranging from about 0.5 to 2 hours. 
A molar excess of the amino derivative of structure (4) is then added to 
the reaction medium in which the displacement was carried out. The 
amidation reaction of Step B is then allowed to proceed for a period of 
time ranging from about 0.5 to 8 hours. The amidation reaction is carried 
out at a temperature range of from about 0.degree.-40.degree. C. Upon 
completion of the amidation reaction, the desired product can be recovered 
from the reaction medium by extraction or concentration. It can optionally 
be purified by flash chromatography as is known in the art. The particular 
amino-derivative of structure (4) which is utilized as a reactant should 
have the same substituent at the X position as is desired in the final 
product of Formula I. 
The deprotection reaction of Step C can be carried out using techniques 
well known in the art. This deprotection reaction produces a compound of 
Formula Ia in which R is represented by a hydroxyl group as is depicted. 
The protected compound of Formula Ia' is typically subjected to a mildly 
acidic hydrolysis with a mineral acid such as hydrochloric acid. The 
concentration of acid which is utilized is not critical and typically is 
in the range of from about 1-4M. The deprotection reaction is typically 
carried out for a period of time ranging from about 0.25-4 hours at a 
temperature range of from 0.degree.-60.degree. C. The compound of Formula 
Ia can recovered from the reaction medium by either concentration or 
extraction as is known in the art. It can then be purified by either ion 
exchange chromatography or by preparative C.sub.18 reverse phase 
chromatography as is known in the art. 
If R is to be represented by other than a hydroxyl group, then it is 
necessary carry out the optional functionalization reaction of Step D. The 
various ester derivatives of Formula Ia can be prepared utilizing 
techniques well known in the art. For example if an ester is desired, the 
deprotected compound of Formula Ia can be contacted with an alcohol 
corresponding to the desired substituent in the presence of a mineral 
acid. If R is to be represented by an amino acid, then it can be added to 
the compounds of Formula I utilizing peptide coupling techniques known in 
the art. For example, see Meldal et al. Synthesis of a Proposed Antigenic 
Hexapeptide from Escheria coli K88 Protein Fimbriae--Acta Chem. Scand. 
B40: 235-241. 
Methods for producing the functionalized amino acids of structure (1) are 
well known in the art. For example, the preparation of 5-hydroxynorvaline 
is described in J. Chem. Soc., Chem. Commun., 20, 1583-4 (1987) and the 
preparation of 6-hydroxynorleucine is described in Agric. Biol. Chem., 
42(6), 1275-8 (1979). Methods for producing the amino derivatives of 
structure (4) are also well known in the art. 
The following examples represent typical syntheses of compounds of formula 
(Ia). These examples are understood to be illustrative only and are not 
intended to limit the scope of the present invention in any way. 
EXAMPLE 1 
Preparation of Hydrazinecarboxylic acid, 4-amino-4-carboxybutyl ester 
Step A: 
2-(N-t-Butyloxycarbonylamino)-5-(O-chloroformate)-5-hydroxynorvaline, 
t-butyl ester 
Dissolve 5-hydroxynorvaline (13.3 g, 0.1 mol) in 1N sodium hydroxide (200 
mL, 0.2 mol). Add a solution of CuSO.sub.4.5H.sub.2 O (12.5 g, 0.05 mol) 
in water (50 mL). Stir the mixture until solution is complete. Treat with 
benzyl bromide (17.1 g, 0.1 mol) and stir at room temperature until 
reaction is complete. Suction filter, wash the residue with 1:3.5 
methanol-water and dry at 60.degree. C. to give the copper complex. 
Suspend the copper complex in water, treat with excess hydrogen sulfide, 
heat to boiling, then cool to room temperature. Mix with 1N sodium 
hydroxide and filter. Stir the filtrate with a slight excess of 1N 
hydrochloric acid and filter. Wash the residue with water and dry to give 
O-benzyl-5-hydroxynorvaline. 
Suspend O-benzyl-5-hydroxynorvaline (67 g, 0.3 mol) in t-butyl acetate (250 
mL) and cool to 10.degree. C. Add p-toluenesulfonic acid (57 g, 0.3 mol) 
and add, by dropwise addition, concentrated sulfuric acid (80 mL). Stir at 
10.degree. C. until the reaction is complete, basify to pH 8 by adding a 
suspension of sodium bicarbonate in water. Extract with ethyl acetate, dry 
(MgSO.sub.4), and evaporate to give O-benzyl-5-hydroxynorvaline, t-butyl 
ester. Dissolve O-benzyl-5-hydroxynorvaline, t-butyl ester (2.79 g, 10 
mmol) in 50/50 dioxane/water (25 mL) and buffer to pH 10 with 1N sodium 
hydroxide. Add, by dropwise addition, an ether solution of t-butyl 
azidoformate (1.58 g, 11 mmol) at 10.degree. C. Allow to warm to room 
temperature and buffer occasionally to retain pH 10. Acidify with a sodium 
citrate/citric acid buffer to pH 5, extract with ether (3X), dry 
(MgSO.sub.4) and blow to a residue with a stream of nitrogen to give 
N-t-butyloxycarbonyl-O-benzyl-5-hydroxynorvaline, t-butyl ester. 
Mix N-t-butyloxycarbonyl-O-benzyl-5-hydroxynorvaline, t-butyl ester (2.0 g, 
5.3 mmol) and 10% palladium/carbon (0.5 g) in 95% ethanol (40 mL). 
Hydrogenate at room temperature at atmospheric pressure. Filter and 
evaporate the filtrate in vacuo to give N-t-butyloxy-5-hydroxynorvaline, 
t-butyl ester. 
Dissolve N-t-butyloxy-5-hydroxynorvaline, t-butyl ester (2.89 g, 10 mmol) 
in methylene dichloride (50 mL), place under a nitrogen atmosphere and 
cool to 0.degree. C. Add phosgene (1.0 g, 10 mmol) and allow to stir at 
room temperature for 2 hours to give the crude title compound. 
Step B: N-t-Butyl-5-(hydazinoamide)-5-hydroxynorvaline, t-butyl ester 
Add hydrazine (0.5 g, 15 mmol) to 
2-(N-t-butyloxycarbonylamino)-5-(O-chloroformate)-5-hydroxynorvaline, 
t-butyl ester (3.51 g, 10 mmol) and stir at room temperature for 8 hours. 
Pour the reaction mixture into water and separate the organic phase. 
Extract the aqueous phase with ethyl acetate (2X), wash the combined 
organic phases with saturated sodium chloride and dry (MgSO.sub.4). 
Evaporate the solvent in vacuo and purify by silica gel chromatography to 
give the title compound. 
Step C: Hydrazinecarboxylic acid, 4-amino-4-carboxybutyl ester 
Mix N-t-butyl-5-hydrazinoamide)-5-hydroxynorvaline, t-butyl ester (3.47 g, 
10 mmol) and 1N hydrochloric acid (50 mL) and stir under a nitrogen 
atmosphere at room temperature for 4 hours. Pour the reaction mixture into 
ethyl ether and separate the aqueous phase. Wash the aqueous phase with 
ethyl ether (2X), the neutralize with 5N sodium hydroxide. Evaporate the 
water in vacuo then purify by ion-exchange chromatography to give the 
title compound. 
EXAMPLE 2 
Preparation of 6-[[(Cyclopropylamino)carbonyl]oxy]-norleucine 
Step A: 
2-(N-t-Butyloxycarbonylamino)-6-(O-chloroformate)-6-hydroxynorleucine, 
t-butyl ester 
Dissolve 6-hydroxynorleucine (14.7 g, 0.1 mol) in 1N sodium hydroxide (200 
mL, 0.2 mol). Add a solution of CuSO.sub.4.5H.sub.2 O (12.5 g, 0.05 mol) 
in water (50 mL). Stir the mixture until solution is complete. Treat with 
benzyl bromide (17.1 g, 0.1 mol) and stir at room temperature until 
reaction is complete. Suction filter, wash the residue with 1:3.5 
methanol-water and dry at 60.degree. C. to give the copper complex. 
Suspend the copper complex in water, treat with excess hydrogen sulfide, 
heat to boiling, then cool to room temperature. Mix with 1N sodium 
hydroxide and filter. Stir the filtrate with a slight excess of 1N 
hydrochloric acid and filter. Wash the residue with water and dry to give 
O-benzyl-6-hydroxynorleucine. 
Suspend O-benzyl-6-hydroxynorleucine (71.2 g, 0.3 mol) in t-butyl acetate 
(250 mL) and cool to 10.degree. C. Add p-toluenesulfonic acid (57 g, 0.3 
mol) and add, by dropwise addition, concentrated sulfuric acid (80 mL). 
Stir at 10.degree. C. until the reaction is complete, basify to pH 8 by 
adding a suspension of sodium bicarbonate in water. Extract with ethyl 
acetate, dry (MgSO.sub.4), and evaporate to give 
O-benzyl-6-hydroxynorleucine, t-butyl ester. 
Dissolve O-benzyl-6-hydroxynorleucine, t-butyl ester (2.93 g, 10 mmol) in 
50/50 dioxane/water (25 mL) and buffer to pH 10 with 1N sodium hydroxide. 
Add, by dropwise addition, an ether solution of t-butyl azidoformate (1.58 
g, 11 mmol) at 10.degree. C. Allow to warm to room temperature and buffer 
occasionally to retain pH 10. Acidify with a sodium citrate/citric acid 
buffer to pH 5, extract with ether (3X), dry (MgSO.sub.4) and blow to a 
residue with a stream of nitrogen to give 
N-t-butyloxycarbonyl-O-benzyl-6-hydroxynorleucine, t-butyl ester. 
Mix N-t-butyloxycarbonyl-O-benzyl-6-hydroxynorleucine, t-butyl ester (2.0 
g, 5.1 mmol) and 10% palladium/carbon (0.5 g) in 95% ethanol (40 mL). 
Hydrogenate at room temperature at atmospheric pressure. Filter and 
evaporate the filtrate in vacuo to give N-t-butyloxy-6-hydroxynorleucine, 
t-butyl ester. 
Dissolve N-t-butyloxy-6-hydroxynorleucine, t-butyl ester (3.03 g, 10 mmol) 
in methylene dichloride (50 mL), place under a nitrogen atmosphere and 
cool to 0.degree. C. Add phosgene (1.0 g, 10 mmol) and allow to stir at 
room temperature for 2 hours to give the crude title compound. 
Step B: N-t-Butyl-6-(cyclopropylamide)-6-hydroxynorleucine, t-butyl ester 
Add cyclopropylamine (0.86 g, 15 mmol) to 
2-(N-t-butyloxycarbonylamino)-6-(O-chloroformate)-6-hydroxynorleucine, 
t-butyl ester (3.65 g, 10 mmol) and stir at room temperature for 8 hours. 
Pour the reaction mixture into water and separate the organic phase. 
Extract the aqueous phase with ethyl acetate (2X), wash the combined 
organic phases with saturated sodium chloride and dry (MgSO.sub.4). 
Evaporate the solvent in vacuo and purify the by silica gel chromatography 
to give the title compound. 
Step C: 6-[[(Cyclopropylamino)carbonyl]oxy]-norleucine 
Mix N-t-butyl-6-(cyclopropylamide)-6-hydroxynorleucine, t-butyl ester (3.86 
g, 10 mmol) and 1N hydrochloric acid (50 mL) and stir under a nitrogen 
atmosphere at room temperature for 4 hours. Pour the reaction mixture into 
ethyl ether and separate the aqeous phase. Wash the aqueous phase with 
ethyl ether (2X), the neutralize with 5N sodium hydroxide. Evaporate the 
water in vacuo then purify by ion-exchange chromatography to give the 
title compound. 
EXAMPLE 3 
Hydrazinecarboxylic acid, 4-amino-4-carboxy-2-butenyl ester 
Step A: 2-(N-t-Butyloxycarbonylamino)-5-(O-chloroformate)-3-pentenoic acid, 
t-butyl ester 
Dissolve dimethyl 2-acetimido-2-(3-hydroxy-1-propene) malonate 
(Tetrahedron. Lett., 29(47), 6183-4 (1988) (2.45 g, 10 mmol) in 
tetrahydrofuran (30 mL). Treat with 1N sodium hydroxide and stir at room 
temperature until hydrolysis of the esters is complete. Acidify with 5N 
hydrochloric acid and stir at 50.degree. C. until decarboxylation is 
complete. Cool to room temperature pour into methylene chloride and 
separate the organic phase. Extract the aqueous phase with methylene 
chloride (2X), dry (MgSO.sub.4) and evaporate the solvent in vacuo to give 
5-hydroxy-2-(acetamido)-3-pentenoic acid. 
Mix 5-hydroxy-2-(acetamido)-3-pentenoic acid (1.73 g, 10 mmol) and acylase 
(I) (Merck) in pH 7.2 buffer and stir at 37.degree. C. until hydrolysis is 
complete. Purify by ion exchange chromatography to give 
5-hydroxy-2-amino-3-pentenoic acid. 
Dissolve 5-hydroxy-2-amino-3-pentenoic acid (13.1 g, 0.1 mol) in 1N sodium 
hydroxide (200 mL, 0.2 mol). Add a solution of CuSO.sub.4.5H.sub.2 O (12.5 
g, 0.05 mol) in water (50 mL). Stir the mixture until solution is 
complete. Treat with benzyl bromide (17.1 g, 0.1 mol) and stir at room 
temperature until reaction is complete. Suction filter, wash the residue 
with 1:3.5 methanol-water and dry at 60.degree. C. to give the copper 
complex. Suspend the copper complex in water, treat with excess hydrogen 
sulfide, heat to boiling, then cool to room temperature. Mix with 1N 
sodium hydroxide and filter. Stir the filtrate with a slight excess of 1N 
hydrochloric acid and filter. Wash the residue with water and dry to give 
O-benzyl-5-hydroxy-2-amino-3-pentenoic acid. 
Suspend O-benzyl-5-hydroxy-2-amino-3-pentenoic acid (66.4 g, 0.3 mol) in 
t-butyl acetate (250 mL) and cool to 10.degree. C. Add p-toluenesulfonic 
acid (57 g, 0.3 mol) and add, by dropwise addition, concentrated sulfuric 
acid (80 mL). Stir at 10.degree. C. until the reaction is complete, basify 
to pH 8 by adding a suspension of sodium bicarbonate in water. Extract 
with ethyl acetate, dry (MgSO.sub.4), and evaporate to give 
O-benzyl-5-hydroxy-2-amino-3-pentenoic acid, t-butyl ester. 
Dissolve O-benzyl-5-hydroxy-2-amino-3-pentenoic acid, t-butyl ester (2.77 
g, 10 mmol) in 50/50 dioxane/water (25 mL) and buffer to pH 10 with 1N 
sodium hydroxide. Add, by dropwise addition, an ether solution of t-butyl 
azidoformate (1.58 g, 11 mmol) at 10.degree. C. Allow to warm to room 
temperature and buffer occasionally to retain pH 10. Acidify with a sodium 
citrate/citric acid buffer to pH 5, extract with ether (3X), dry 
(MgSO.sub.4) and blow to a residue with a stream of nitrogen to give 
2-(N-t-butyloxycarbonylamino)-5-(O-benzyloxy)-3-pentenoic acid, t-butyl 
ester. 
Mix 2-(N-t-butyloxycarbonylamino)-5-(O-benzyloxy)-3-pentenoic acid, t-butyl 
ester (2.0 g, 5.3 mmol) and 10% palladium/carbon (0.5 g) in 95% ethanol 
(40 mL). Hydrogenate at room temperature at atmospheric pressure. Filter 
and evaporate the filtrate in vacuo to give 
2-(N-t-butyloxycarbonylamino)-5-hydroxy-3-pentenoic acid, t-butyl ester. 
Dissolve 2-(N-t-butyloxycarbonylamino)-5-hydroxy-3-pentenoic acid, t-butyl 
ester (2.87 g, 10 mmol) in methylene dichloride (50 mL), place under a 
nitrogen atmosphere and cool to 0.degree. C. Add phosgene (1.0 g, 10 mmol) 
and allow to stir at room temperature for 2 hours to give the title 
compound. 
Step B: 5-(O-Hydrazinoamide)-2-(N-t-butyloxyamino)-5-hydroxy-3-pentenoic 
acid, t-butyl ester 
Add hydrazine (0.5 g, 15 mmol) to 
2-(N-tert-butyloxycarbonylamino)-5-(O-chloroformate)-3-pentenoic acid, 
t-butyl ester (3.5 g, 10 mmol) and stir at room temperature for 8 hours. 
Pour the reaction mixture into water and separate the organic phase. 
Extract the aqueous phase with ethyl acetate (2X), wash the combined 
organic phases with saturated sodium chloride and dry (MgSO.sub.4). 
Evaporate the solvent in vacuo and purify the by silica gel chromatography 
to give the title compound. 
Step C: O-[Hydrazino-carbonyl]-2-amino-5-hydroxy-3-pentenoic acid 
Mix 5-(O-hydrazinoamide)-2-(N-t-butyloxyamino)-5-hydroxy-3-pentenoic acid, 
t-butyl ester (3.45 g, 10 mmol) and 1N hydrochloric acid (50 mL) and stir 
under a nitrogen atmosphere at room temperature for 4 hours. Pour the 
reaction mixture into ethyl ether and separate the aqueous phase. Wash the 
aqueous phase with ethyl ether (2X), the neutralize with 5N sodium 
hydroxide. Evaporate the water in vacuo then purify by ion-exchange 
chromatography to give the title compound. 
Those compounds of Formula Ib in which Z is represented by NR.sub.1 can 
also be made using techniques known in the art. Reaction Scheme II shows 
one method by which these compounds can be produced. 
##STR4## 
In Step A, a displacement reaction is carried out between a functionalized 
amino acid as described by structure (5) and an amidino derivative as 
described by structure (6). In the amidine structure (6), Alk is 
represented by a C.sub.1-4 alkyl, and R.sub.1 and X are as defined in 
Formula Ia. This displacement reaction produces the optionally protected 
compound which has been depicted as Ib'. In step B, the protecting groups, 
if present, are removed via an acidic hydrolysis and in Step C the 
appropriate R substituent may be placed on the molecule as is depicted. 
The displacement reaction of Step A can be carried out using techniques 
well known in the art. The meanings for Pr and Pr' should be complementary 
(i.e. both should be hydrogen, organic residues or copper). The 
appropriate amidine (6) is one in which X is represented by the 
substituent which is desired in the final product. The linear C.sub.1-4 
alkyl represented by Alk is not retained in the final product, and thus 
its identity is immaterial. 
The particular manner in which the displacement reaction is carried out 
depends upon the particular substituent which is present at the Pr and Pr' 
position. If Pr and Pr' are represented by organic protecting groups, then 
the reaction is carried out be contacting approximately equivalent amounts 
of the functionalized amino acid and the amindino-derivative in an aprotic 
solvent such as dimethylformamide at a temperature range of from room 
temperature to about 80.degree. C. The reaction is typically allowed to 
proceed for a period of time ranging from about 8 hours to about 120 
hours. Upon completion of the reaction, the functionalized amino acid of 
Formula Ib' can be recovered by either concentration or extraction as is 
known in the art. It can then be optionally purified by flash 
chromatography. In Step B these protecting groups are removed by an acidic 
hydrolysis. This hydrolysis can be carried out in the same manner as the 
deprotection reaction of Reaction Scheme I. The deprotected compound of 
Formula Ib can be purified by either ion exchange chromatography or by 
preparative C.sub.18 reverse phase chromatography. Likewise, the 
appropriate R substituent may be placed on the compound of Formula Ib 
utilizing the same methods as taught in Reaction Scheme I. 
If Pr and Pr' are represented by either hydrogen atoms or by a copper 
complexm, then the displacement reaction is carried out in the following 
manner. Approximately equivalent amounts of the functionalized amino acid 
of structure (5) and the amidino derivative of structure (6) are contacted 
in a dilute solution of an inorganic base such as sodium hydroxide. The 
displacement reaction is carried out at about 40.degree. C. for a period 
of time ranging from about 8 to 120 hours. The desired product of Formula 
Ib can then be recovered from the reaction by either extraction or 
concentration as is known in the art. This crude product can then be 
purified by ion exchange chromatography or by preparative C.sub.18 reverse 
phase chromatography. 
Methods for preparing the functionalized amino acids of structure (5) are 
well known in the art. For example, 5-amino-2-(N-acetylamino)-3-pentenoic 
acid is described in Tetrahedron Letters, 29(47), 6183-4 (1988). Methods 
for preparing the amidino derivatives of structure (6) are also well known 
in the art. For examples, see G. V. Nair, Ind. J. Chem., 4, 516 (1966). 
The following example presents a typical synthesis as described above in 
Scheme II. This example is understood to be illustrative only and is not 
intended to limit the scope of the present invention in any way. 
EXAMPLE 4 
Preparation of 2-Amino-5-[(hydrazinoiminomethyl)amino]-3-pentenoic acid 
Step A: N.sup.5 
-[Hydrazino-iminomethyl]-2-(acetylamino)-5-amino-3-pentenoic acid 
Mix 5-amino-2-(N-acetylamino)-3-pentenoic acid HCl (4.16 g, 20 mmol) and 
S-methylisothiosemicarbazide (2.11 g, 20 mmol) in 1N NaOH (40 mL) and stir 
under a blanket of N.sub.2 at 40.degree. C. for 48 hours. Cool the 
reaction to room temperature, neutralize with 1N HCl and blow to a residue 
with a stream of N.sub.2 to give the title compound. 
Step B: 2-Amino-5-[(hydrazinoiminomethyl)amino]-3-pentenoic acid 
Mix N.sup.5 -[hydrazino-iminomethyl[-2-(acetylamino)-5-amino-3-pentenoic 
acid (4.6 g, 20 mmol) and acylase (I) (Merck) in pH 7.2 buffer and stir at 
37.degree. C. until hydrolysis is complete. Purify by ion exchange 
chromatography to give the title compound. 
Alternatively, those compounds of Formula Ib in which Z is represented by O 
can be made utilizing the methods disclosed in Reaction Scheme IV. The 
only modification is that the functionalized amino acid that is utilized 
as one of the starting materials should have a double bond connecting the 
.alpha. and .beta. carbon atoms. This starting material may be represented 
by the following formula: 
##STR5## 
in which Pr and Pr' are as above. 
Those compounds of Formula Ic in which Z is represented by NR1 can also be 
made using techniques known in the art. Reaction Scheme III shows one 
method by which these compounds can be produced. 
##STR6## 
In Step A, a displacement reaction is carried out between a functionalized 
amino acid as described by structure (7) and an amidino derivative as 
described by structure (6). In structure (7), W' is represented by 
--CH.sub.2 --NH.sub.2, --NH--NH.sub.2, --CH.sub.2 --O--NH.sub.2, 
--CH.sub.2 --NH--NH.sub.2, --(CH.sub.2).sub.2 --NH.sub.2 or --O--NH.sub.2. 
If W' is represented by --CH.sub.2 --NH then Pr and Pr may both be 
hydrogen or copper. Alternatively Pr' is t-butyl while Pr is 
t-butyloxycarbonyl. If W' is either NH.sub.2 --NH.sub.2 --, --CH.sub.2 
--O--NH.sub.2, --CH.sub.2 --NH--NH.sub.2, or O--NH.sub.2 --, then Pr and 
Pr' may both be copper, or Pr' is ethyl while Pr is a benzyloxycarbonyl. 
In structure (6), Alk is represented by a C.sub.1-4 alkyl, and R.sub.1 and 
X are as defined in Formula Ic. This displacement reaction produces the 
optionally protected compound which has been depicted as Ic'. In step B, 
the protecting groups, if present, are removed via an acidic hydrolysis 
and in Step C the appropriate R substituent may be placed on the molecule 
as is depicted. 
The displacement reaction of Step A can be carried out using techniques 
well known in the art. The appropriate functionalized amino acid of 
structure (7) is one in which W' corresponds to the substituent which is 
desired at this position in the final product (i.e. if W is to be 
represented by --CH.sub.2 --NH--, then W' should be represented by 
--CH.sub.2 --NH.sub.2). The meanings for Pr and Pr' should be 
complementary (i.e. both should be hydrogen, organic residues or copper). 
The appropriate amidine is one in which X is represented by the 
substituent which is desired in the final product. The linear C.sub.1-4 
alkyl represented by Alk is not retained in the final product, and thus 
its identity is immaterial. 
The particular manner in which the displacement reaction is carried out 
depends upon the particular substituent which is present at the Pr and Pr' 
position. If Pr and Pr' are represented by organic protecting groups, then 
the reaction is carried out by contacting approximately equivalent amounts 
of the functionalized amino acid and the amidino-derivative in an aprotic 
solvent such as dimethylformamide at a temperature range of from room 
temperature to about 80.degree. C. The reaction is typically allowed to 
proceed for a period of time ranging from about 8 hours to about 120 
hours. Upon completion of the reaction, the functionalized amino acid of 
formula Ic' can be recovered by either concentration or extraction as is 
known in the art. It can then be optionally be purified by flash 
chromatography. In Step B these protecting groups are removed by an acidic 
hydrolysis. This hydrolysis can be carried out in the same manner as the 
deprotection reaction of Reaction Scheme I. The deprotected compound of 
Formula Ic can be purified by either ion exchange chromatography or by 
preparative C.sub.18 reverse chromatography. Likewise, the appropriate R 
substituent may be placed on the compound of Formula Ic utilizing the same 
methods as taught in Reaction Scheme I. 
If Pr and Pr' are represented by either hydrogen atoms or by a copper 
complex then the displacement reaction is carried out in the following 
manner. Approximately equivalents amounts of the functionalized amino acid 
of structure (7) and the amidino derivative of structure (6) are contacted 
in a dilute solution of an inorganic base such as sodium hydroxide. The 
displacement reaction is carried out at about 40.degree. C. for a period 
of time ranging from about 8 to 120 hours. The desired product of Formula 
Ic can then be recovered from the reaction by either extraction or 
concentration as is known in the art. This crude product can then be 
purified by ion exchange chromatography or by preparative C.sub.18 reverse 
chromatography. 
Methods for producing the functionalized amino acids of structure (7) are 
known in the art. For example, the preparation of canaline is described in 
J. Am. Chem. Soc., 79, 1222 (1957). 
The following examples represent typical syntheses as described above in 
Scheme III. These examples are intended to be illustrative only and are 
not intended to limit the scope of the present invention in any way. 
EXAMPLE 5 
Preparation of N.sup.5 -[Imino(2-methylhydrazino)methyl]-L-ornithine, 
flavinate 
This example demonstrates the preparation of a compound according to 
formula Ic in which Z is N, utilizing the methodology of Reaction Scheme 
III. 
L-Ornithine HCl (3.36 g, 20 mmol) and S-methyl-4-methylisothiosemicarbazide 
hydroiodide (5.00 g, 20 mmol) in 1M NaOH (40 ml) were mixed and stirred 
under a blanket N.sub.2 at 40.degree. C. (for 48 hours. The reaction was 
then neutralized with 1M HCl and blown to a residue with a stream of 
N.sub.2. The residue was applied to a Dowex AG50-X8 column and eluted with 
0.2M NH.sub.4 OH. The fractions containing the desired product were 
lyophilized and the resulting solid taken up in water (20 ml). Flavianic 
acid (3.2 g) in water (20 ml) was added and the solution cooled to 
4.degree. C. for 16 hours. The resulting solid was filtered and 
crystallized from water to yield 1.3 g of the title compound as a yellow 
solid. .sup.1 HNMR (300 MH.sub.2, .delta..sub.6 DMSO) 1.45 (2,m) 1.78 
(2,m) 2.85 (3,s) 3.15 (2 m) 3.85 (1,t) 7.8 (1,m) 8.6-8.8 (2,m) 9.2 (1,s). 
Anal. Cald. for C.sub.17 H.sub.23 N.sub.7 O.sub.10 S: C, 39.46; H, 4.48; 
N, 18.95. Found C, 38.56; H, 4.48; N, 18.95. 
EXAMPLE 6 
Preparation of N.sup.5 -[(Cyclopropylamino)iminomethyl]-L-ornithine, 
flavinate 
This example also demonstrates the methodology of Reaction Scheme III. 
t-Butyl isothiocyanate (11.6 ml, 0.1 mol) and cyclopropylamine (6.9 ml, 0.1 
mol) were mixed in toluene (200 ml) and stirred at room temperature 
overnight. The resulting mixture was evaporated to a residue and 
crystallized from cyclohexane to yield N-cyclopropyl-N-butyl thiourea. 
N-cyclopropyl-N-t-butyl thiourea (5 g) was suspended in 5M HCl (50 ml) and 
heated to 80.degree. C. for 10 minutes. The resulting solution was cooled, 
poured into a saturated sodium bicarbonate (250 ml), and extracted with 
ethyl acetate (2.times.125 ml). The solution was dried (MgSO.sub.4), 
evaporated to a residue and crystallized from ethyl acetate to yield a 
white solid, cyclopropyl thiourea, 110 mg. 
Cyclopropyl thiourea (0.65 g, 0.0056 mol) and iodomethane (1.2 g, 0.0085 
mol) were mixed in acetone, heated to boiling and refluxed for 15 minutes. 
The reaction was cooled and blown to an oil. Ethyl acetate (10 ml) was 
added, and the resulting mixture was blown to a white solid. 
L-Ornithine.HCl (0.94 g, 0.0056 mol) and 1M NaOH (15 ml) were added and 
the resulting reaction warmed to 40.degree. C. and stirred under a blanket 
of N.sub.2 for 16 hours. The reaction mixture was cooled to room 
temperature, acidified with acetic acid and blown with a stream of N.sub.2 
to 1/2 volume. Flavianic acid (1 g) in water (10 ml) was added and the 
resulting solid filtered, dried and recrystallized from boiling water to 
yield 1.4 g of the title compound which appeared as a yellow solid. .sup.1 
HNMR (d.sub.6 DMSO) 0.52 (2,m) 0.82 (2,m) 1.45 (2 m) 1.78 (2,m) 2.5 (1,m) 
3.15 (2,m) 3.85 (1,m) 7.8 (1,m) 8.1-8.8 (2,m) 9.2 (1,s). Anal Calcd. for 
C.sub.9 H.sub.18 N.sub.4 O.sub.2.C.sub.10 H.sub.6 N.sub.2 O.sub.8 
S.1/2H.sub.2 O: C, 41.97; H, 4.64; N, 15.76. Found C, 42.67; H, 4.56; N, 
15.76. 
EXAMPLE 7 
Preparation of 2-Amino-4-[2-(hydrazinoimino-methyl)hydrazino]-butanoic 
acid, flavinate 
Mix canaline (2.65 g, 20 mmol) and S-methylisothiosemicarbazide hydroiodide 
(4.66 g, 20 mmol) in 1M NaOH (40 ml) and stir under a blanket N.sub.2 at 
40.degree. C. for 48 hours. Neutralize the reaction with 1M HCl and blow 
to a residue with a stream of N.sub.2. Apply the residue to a Dowex 
AG50-X8 column and elute with 0.2M NH.sub.4 OH. Lyophilize the fractions 
containing the desired product and take up the resulting solid in water 
(20 ml). Add flavianic acid (3.2 g) in water (20 ml) and cool the solution 
to 4.degree. C. for 16 hours. Filter the resulting solid and crystallize 
to yield the title compound. 
EXAMPLE 8 
Preparation of N.sup.5 -(Hydrazino-iminomethyl)-L-ornithine, flavinate 
Mix lysine HCl (3.65 g, 20 mmol) and S-methylisothiosemicarbazide 
hydroiodide (4.66 g, 20 mmol) and adjust to pH 10 with 1M NaOH. Stir under 
a blanket N.sub.2 at 40.degree. C. (for 48 hours. Neutralize the reaction 
with 1M HCl and blow to a residue with a stream of N.sub.2. Apply the 
residue to a Dowex AG50-X8 column and elute with 0.2M NH.sub.4 OH. 
Lyophilize the fractions containing the desired product and dissolve the 
resulting solid in water (20 ml). Add a solution of flavianic acid (3.2 g) 
in water (20 ml) and cool to 4.degree. C. for 16 hours. Filter the 
resulting solid and crystallize from water to yield 1.2 g of the title 
compound. .sup.1 HNMR (d.sub.6 DMSO) 1.3-1.6 (4,m), 1.85 (2,m), 3.15 
(2,m), 3.95 (1,t), 8.05 (2,m), 8.8 (3,m), 9.2 (1,m); .sup.13 CNMR (d.sub.6 
DMSO) 22.3, 28.7, 30.2, 41.04, 52.0, 57.4, 124.7, 125.2, 129.1, 129.6, 
130.3, 130.7, 132.0, 144.5, 158.8, 168, 172.1, 174.1. 
EXAMPLE 9 
Preparation of O-[(Hydrazinoimino-methyl)amino]-L-homoserine 
Dissolve 5-hydroxynorvaline (20 g, 0.15 mol) in water (90 mL) and add 
anhydrous potassium carbonate (10.4 g, 0.075 mol) with stirring. Heat the 
slightly alkaline solution on a steam-bath for a few minutes, then add a 
solution of potassium cyanate (13.0 g, 0.16 mol) in water (50 mL). Heat 
the final solution on a steam-bath for 2 hours. Treat the solution with 
48% hydrobromic acid (100 mL) and heat for an additional 2 hours on the 
steam-bath. Evaporate the solvent in vacuo and digest the residue with hot 
acetone (150 mL) and filter. Wash the potassium bromide residue with hot 
acetone until white. Evaporate the acetone filtrate and heat the residue 
with additional 48% hydrobromic acid for 2 hours on the steam-bath. 
Evaporate the solvent in vacuo, dissolve the residue in hot water (75 mL), 
bring to pH 5-6 with concentrated ammonia, filter, cool and collect 
5-(3-bromopropyl)-hydantoin. 
Dissolve 85% potassium hydroxide (3.96 g, 60 mmol) in absolute ethanol (60 
mL). Add 5-(3-bromopropyl)hydantoin (6.63 g, 30 mmol) and a solution of 
hydroxyurethane (6.30 g, 60 mmol) in absolute ethanol (40 mL). Reflux for 
3 hours, cool and filter to remove potassium bromide. Evaporate the 
solvent in vacuo, dissolve the residue in water (30 mL), neutralize with 
dilute hydrochloric acid, cool and collect 
5-[3-(carbethoxyaminooxy)-propyl]-hydantoin. 
Mix 5-[3-(carbethoxyaminooxy)-propyl]-hydantoin (2.45 g, 10 mmol), barium 
hydroxide octahydrate (18.15 g, 10 mmol) and water (55 mL). Reflux for 12 
hours, remove the white solid by filtration, extract the filter cake with 
boiling water (25 ml) and finally wash with hot water (25 mL). Combine the 
filtrate and washings, treat with ammonium carbonate (5.7 g) and heat with 
stirring. Filter off the barium carbonate, wash the filter cake with hot 
water and evaporate the filtrate and washings in vacuo to give 
2-amino-5-aminooxy-pentanoic acid. 
Mix 2-amino-5-aminooxy-pentanoic acid (2.97 g, 20 mmol) and 
S-methyl-isothiosemicarbazide hydroiodide (4.66 g, 20 mmol) in 1M NaOH (40 
ml) and stir under a blanket N.sub.2 at 40.degree. C. (for 48 hours. 
Neutralize the reaction with 1M HCl and blow to a residue with a stream of 
N.sub.2. Apply the residue to a Dowex AG50-X8 column and elute with 0.2M 
NH.sub.4 OH. Lyophilize the fractions containing the desired product and 
take up the resulting solid in water (20 ml). Add flavianic acid (3.2 g) 
in water (20 ml) and cool the solution to 4.degree. C. for 16 hours. 
Filter the resulting solid and crystallize to yield the title compound. 
An alternative method for producing the compounds of Formula Ic in which Z 
is represented by NR.sub.1 and R.sub.1 is hydrogen, is to carry out a 
displacement reaction between a functionalized amino acid as described by 
structure (7) in which Pr and Pr' are represented by protecting groups and 
an amino nitrile of the Formula, X-NH-CN, in which X is as defined in 
Formula Ic. This displacement reaction produces the protected compound of 
Formula Ic' disclosed above in Reaction Scheme III. This displacement can 
be carried out by contacting equivalent amounts of the reactants in an 
aprotic solvent such as toluene or benzene at a temperature of about 
-5.degree. C. to reflux for a period of time ranging from about 1 hour to 
16 hours. The compound of Formula Ic' can be recovered by either 
extraction or concentration. It can then be purified by ion exchange 
chromatography or preparation C.sub.18 reverse phase chromatography. The 
desired compounds of Formula Ic can then be produced via the deprotection 
reaction and optional functionalization reactions taught in steps B and C 
of Reaction Scheme II. 
Those compounds of Formula Ic in which Z is represented by O can also be 
produced by methods known in the art. One such method is disclosed below 
in Reaction Scheme IV: 
##STR7## 
In Step A an addition reaction is carried out between a functionalized 
amino acid as described by structure (7) in which Pr, Pr' and W' are as 
defined in Reaction Scheme III, and an isocyanate derivative as described 
by structure (8) in which X is as defined in Formula Ic and Z is O as is 
depicted. This addition reaction produces the protected compound of 
Formula Ic'. The deprotection reaction of Step B produces the desired 
compound of Formula Ic. If desired this compound can be subjected to the 
optional functionalization reaction of Step C. 
The appropriate isocyanate derivative is one in which X is represented by 
the same substituent as is desired in the final product of Formula Ic. The 
addition reaction of Step A can be carried out using techniques well known 
in the art. Typically approximately equivalent amounts of the reactants 
are contacted in an organic solvent such as toluene, benzene, or methylene 
dichloride at a temperature range of from -10.degree. C. to about 
80.degree. C. for a period of time ranging from about 1 to 16 hours. The 
protected compound of Formula Ic' can then be recovered from the reaction 
by either concentration or extraction as is known. It can then be purified 
by flash chromatography. The deprotection reaction of Step B and the 
optional functionalization reaction of Step C can be carried out using the 
methods taught in the previous Reaction Schemes. 
Those compounds of Formula Ic in which Z is represented by O and W is 
represented by either --CH.sub.2 --NH-- or --(CH.sub.2).sub.2 --NH-- can 
also be made by the following alternative method. A displacement reaction 
is carried out between phosgene (previous structure 2 of Reaction Scheme 
I, Y.dbd.Cl) or triphosgene (previous structure 2 of Reaction Scheme I, 
Y.dbd.OCCl.sub.3) and a functionalized amino acid of the formula: in which 
W' is represented by either --CH.sub.2 --NH.sub.2 or --(CH.sub.2).sub.2 
--NH.sub.2 and in which Pr and Pr' are as defined above Reaction Scheme 
##STR8## 
I. This displacement reaction can be carried out using techniques known in 
the art. Typically approximately equivalent amounts of the reactants are 
contacted in an organic solvent such as toluene, benzene or methylene 
dichloride at a temperature range of from -10.degree. C. to 60.degree. C. 
The reaction is usually allowed to proceed for a period of time ranging 
from about 1 to 16 hours. At this point, a molar excess of an amine of the 
formula, NH.sub.2 X, in which X is as defined in Formula Ic, is added to 
the reaction. The reactants are heated to a temperature range of 
-10.degree. C. to 60.degree. C. for a period of time ranging from 1 to 16 
hours. This reaction produces a product which can be described by Formula 
Ic' in Reaction Scheme III in which W is represented by CH.sub.2 --NH, and 
X, Pr and Pr' are as above, and Z is represented by O as is depicted. 
These compounds can then be subjected to a deprotection reaction and an 
optional functionalization reaction in the same manner as disclosed in 
Reaction Scheme III. 
The following examples represent typical syntheses as described above for 
compounds of formula Ic, wherein Z is O, W is --(CH.sub.2).sub.2 NH--, and 
X is NH.sub.2. These examples are intended to be illustrative only and are 
not intended to limit the scope of the present invention in any way. 
EXAMPLE 10 
Preparation of N.sup.5 -(Hydrazinocarbonyl)-L-ornithine 
Step A: N-t-Butyloxycarbonyl-N.sup.5 -(trichlormethylcarbonate)ornithine, 
t-butyl ester 
Dissolve ornithine (13.3 g, 0.1 mol) in 1N sodium hydroxide (200 mL, 0.2 
mol). Add a solution of CuSO.sub.4.5H.sub.2 O (12.5 g, 0.05 mol) in water 
(50 mL). Stir the mixture until solution is complete. Treat with benzyl 
chloroformate (17.1 g, 0.1 mol) and stir at room temperature until 
reaction is complete. Suction filter, wash the residue with 1:3.5 
methanol-water and dry at 60.degree. C. to give the copper complex. 
Suspend the copper complex in water, treat with excess hydrogen sulfide, 
heat to boiling, then cool to room temperature. Mix with 1N sodium 
hydroxide and filter. Stir the filtrate with a slight excess of 1N 
hydrochloric acid and filter. Wash the residue with water and dry to give 
N.sup.5 -benzyloxycarbonyl-ornithine. 
Suspend N.sup.5 -benzyloxycarbonyl-ornithine (80 g, 0.3 mol) in t-butyl 
acetate (250 mL) and cool to 10.degree. C. Add p-toluenesulfonic acid (57 
g, 0.3 mol) and add, by dropwise addition, concentrated sulfuric acid (80 
mL). Stir at 10.degree. C. until the reaction is complete, basify to pH 8 
by adding a suspension of sodium bicarbonate in water. Extract with ethyl 
acetate, dry (MgSO.sub.4), and evaporate to give N.sup.5 
-benzyloxycarbonyl-ornithine, t-butyl ester. 
Dissolve N.sup.5 -benzyloxycarbonyl-ornithine, t-butyl ester (3.22 g, 10 
mmol) in 50/50 dioxane/water (25 mL) and buffer to pH 10 with 1N sodium 
hydroxide. Add, by dropwise addition, an ether solution of t-butyl 
azidoformate (1.58 g, 11 mmol) at 10.degree. C. Allow to warm to room 
temperature and buffer occasionally to retain pH 10. Acidify with a sodium 
citrate/citric acid buffer to pH 5, extract with ether (3X) dry 
(MgSO.sub.4) and blow to a residue with a stream of nitrogen to give 
N-t-butyloxycarbonyl-N.sup.5 -benzyloxycarbonyl-ornithine, t-butyl ester. 
Mix N-t-butyloxycarbonyl-N.sup.5 -benzyloxycarbonyl-ornithine, t-butyl 
ester (2.0 g, 5.0 mmol) and 10% palladium/carbon (0.5 g) in 95% ethanol 
(40 mL). Hydrogenate at room temperature at atmospheric pressure. Filter 
and evaporate the filtrate in vacuo to give 
N-t-butyloxycarbonyl-ornithine, t-butyl ester. 
Dissolve N-t-butyloxycarbonyl-ornithine, t-butyl ester (2.88 g, 10 mmol) in 
methylene dichloride (30 mL), place under a nitrogen atmosphere and cool 
to 0.degree. C. Add triphosgene (2.81 g, 10 mmol) and allow to stir at 
room temperature for 2 hours to give the crude title compound. 
Step B: N.sup.2 -t-Butyl-N.sup.5 -hydrazinoamide-ornithine, t-butyl ester 
Add hydrazine (0.5 g, 15 mmol) to N-t-butyloxycarbonyl-N.sup.5 
-(trichlormethylcarbonate)-ornithine, t-butyl ester (4.5 g, 10 mmol) and 
stir at room temperature for 8 hours. Pour the reaction mixture into water 
and separate the organic phase. Extract the aqueous phase with ethyl 
acetate (2X), wash the combined organic phases with saturated sodium 
chloride and dry (MgSO.sub.4). Evaporate the solvent in vacuo and purify 
by silica gel chromatography to give title compound. 
Step C: N.sup.5 -(Hydrazinocarbonyl)-L-ornithine 
Mix N.sup.2 -t-butyl-N.sup.5 -hydrazinoamide-ornithine, t-butyl ester (3.46 
g, 10 mmol) and 1N hydrochloric acid (30 mL) and stir under a nitrogen 
atmosphere at room temperature for 4 hours. Pour the reaction mixture into 
ethyl ether and separate the aqueous phase. Wash the aqueous phase with 
ethyl ether (2X), the neutralize with 5N sodium hydroxide. Evaporate the 
water in vacuo then purify by ion-exchange chromatography to give the 
title compound. 
EXAMPLE 11 
Preparation of N.sup.6 -(Hydrazinocarbonyl)-L-lysine 
Step A: N-t-Butyloxycarbonyl-N.sup.6 -(trichloromethylcarbonate)-lysine, 
t-butyl ester 
Dissolve lysine (14.6 g, 0.1 mol) in 1N sodium hydroxide (200 mL, 0.2 mol). 
Add a solution of CuSO.sub.4.5H.sub.2 O (12.5 g, 0.05 mol) in water (50 
mL). Stir the mixture until solution is complete. Treat with benzyl 
chloroformate (17.1 g, 0.1 mol) and stir at room temperature until 
reaction is complete. Suction filter, wash the residue with 1:3.5 
methanol-water and dry at 60.degree. C. to give the copper complex. 
Suspend the copper complex in water, treat with excess hydrogen sulfide, 
heat to boiling, then cool to room temperature. Mix with 1N sodium 
hydroxide and filter. Stir the filtrate with a slight excess of 1N 
hydrochloric acid and filter. Wash the residue with water and dry to give 
N.sup.5 -benzyloxycarbonyl-lysine. 
Suspend N.sup.5 -benzyloxycarbonyl-lysine (84.1 g, 0.3 mol) in t-butyl 
acetate (250 mL) and cool to 10.degree. C. Add p-toluenesulfonic acid (57 
g, 0.3 mol) and add, by dropwise addition, concentrated sulfuric acid (80 
mL). Stir at 10.degree. C. until the reaction is complete, basify to pH 8 
by adding a suspension of sodium bicarbonate in water. Extract with ethyl 
acetate, dry (MgSO.sub.4), and evaporate to give N.sup.5 
-benzyloxycarbonyl-lysine, t-butyl ester. 
Dissolve N.sup.5 -benzyloxycarbonyl-lysine, t-butyl ester (3.36 g, 10 mmol) 
in 50/50 dioxane/water (25 mL) and buffer to pH 10 with 1N sodium 
hydroxide. Add, by dropwise addition, an ether solution of t-butyl 
azidoformate (1.58 g, 11 mmol) at 10.degree. C. Allow to warm to room 
temperature and buffer occasionally to retain pH 10. Acidify with a sodium 
citrate/citric acid buffer to pH 5, extract with ether (3X), dry 
(MgSO.sub.4) and blow to a residue with a stream of nitrogen to give 
N-t-butyloxycarbonyl-N.sup.5 -benzyloxycarbonyl-lysine, t-butyl ester. 
Mix N-t-butyloxycarbonyl-N.sup.5 -benzyloxycarbonyl-lysine, t-butyl ester 
(2.0 g, 4.6 mmol) and 10% palladium/carbon (0.5 g) in 95% ethanol (40 mL). 
Hydrogenate at room temperature at atmospheric pressure. Filter and 
evaporate the filtrate in vacuo to give N-t-butyloxycarbonyl-lysine, 
t-butyl ester. 
Dissolve N-t-butyloxycarbonyl-lysine, t-butyl ester (3.02 g, 10 mmol) in 
methylene dichloride (30 mL), place under a nitrogen atmosphere and cool 
to 0.degree. C. Add triphosgene (2.81 g, 10 mmol) and allow to stir at 
room temperature for 2 hours to give the crude title compound. 
Step B: N.sup.2 -t-Butyl-N.sup.5 -hydrazinoamide-lysine, t-butyl ester 
Add hydrazine (0.5 g, 15 mmol) to N-t-butyloxycarbonyl-N.sup.6 
-(trichloromethyl-carbonate)-lysine, t-butyl ester (4.64 g, 10 mmol) and 
stir at room temperature for 8 hours. Pour the reaction mixture into water 
and separate the organic phase. Extract the aqueous phase with ethyl 
acetate (2X), wash the combined organic phases with saturated sodium 
chloride and dry (MgSO.sub.4). Evaporate the solvent in vacuo and purify 
by silica gel chromatography to give the title compound. 
Step C: N.sup.6 -(Hydrazinocarbonyl)-L-lysine 
Mix N.sup.2 -t-butyl-N.sup.5 -hydrazinoamide-lysine, t-butyl ester (3.60 g, 
10 mmol) and 1N hydrochloric acid (30 mL) and stir under a nitrogen 
atmosphere at room temperature for 4 hours. Pour the reaction mixture into 
ethyl ether and separate the aqueous phase. Wash the aqueous phase with 
ethyl ether (2X), the neutralize with 5N sodium hydroxide. Evaporate the 
water in vacuo then purify by ion-exchange chromatography to give the 
title compound. 
EXAMPLE 12 
Preparation of 
2-Amino-4-[2-[(cyclopropylamino)carbonyl]hydrazino]-hydrazino-butanoic 
acid 
Step A: 
N-t-Butyloxycarbonyl-2-amino-4-[(trichloromethylcarbonate)hydrazino]-butan 
oic acid, t-butyl ester 
Dissolve homoserine (17.9 g, 0.15 mol) in water (90 mL) and add anhydrous 
potassium carbonate (10.4 g, 0.075 mol) with stirring. Heat the slightly 
alkaline solution on a steam-bath for a few minutes, then add a solution 
of potassium cyanate (13.0 g, 0.16 mol) in water (50 mL). Heat the final 
solution on a steam-bath for 2 hours. Treat the solution with 48% 
hydrobromic acid (100 mL) and heat for an additional 2 hours on the 
steam-bath. Evaporate the solvent in vacuo and digest the residue with hot 
acetone (150 mL) and filter. Wash the potassium bromide residue with hot 
acetone until white. Evaporate the acetone filtrate and heat the residue 
with additional 48% hydrobromic acid for 2 hours on the steam-bath. 
Evaporate the solvent in vacuo, dissolve the residue in hot water (75 mL), 
bring to pH 5-6 with concentrated ammonia, filter, cool and collect 
5-(2-bromoethyl)-hydantoin. 
Dissolve 85% potassium hydroxide (3.96 g, 60 mmol) in absolute ethanol (60 
mL). Add 5-(2-bromoethyl)hydantoin (6.21 g, 30 mmol) and a solution of 
ethyl carbazate (6.3 g, 60 mmol) in absolute ethanol (40 mL). Reflux for 3 
hours, cool and filter to remove potassium bromide. Evaporate the solvent 
in vacuo, dissolve the residue in water (30 mL), neutralize with dilute 
hydrochloric acid, cool and collect 
5-(2-(carbethoxyhydrazino)-ethyl)-hydantoin. 
Mix 5-[(2-(carbethoxyhydrazino)-ethyl)]-hydantoin (2.3 g, 10 mmol), barium 
hydroxide octahydrate (18.15 g, 10 mmol) and water (55 mL). Reflux for 12 
hours, remove the white solid by filtration, extract the filter cake with 
boiling water (25 ml) and finally wash with hot water (25 mL). Combine the 
filtrate and washings, treat with ammonium carbonate (5.7 g) and heat with 
stirring. Filter off the barium carbonate, wash the filter cake with hot 
water and evaporate the filtrate and washings in vacuo to give 
2-amino-4-hydrazino-butanoic acid. 
Dissolve 2-amino-4-hydrazino-butanoic acid (13.3 g, 0.1 mol) in 1N sodium 
hydroxide (200 mL, 0.2 mol). Add a solution of CuSO.sub.4.5H.sub.2 O (12.5 
g, 0.05 mol) in water (50 mL). Stir the mixture until solution is 
complete. Treat with benzyl chloroformate (17.1 g, 0.1 mol) and stir at 
room temperature until reaction is complete. Suction filter, wash the 
residue with 1:3.5 methanol-water and dry at 60.degree. C. to give the 
copper complex. Suspend the copper complex in water, treat with excess 
hydrogen sulfide, heat to boiling, then cool to room temperature. Mix with 
1N sodium hydroxide and filter. Stir the filtrate with a slight excess of 
1N hydrochloric acid and filter. Wash the residue with water and dry to 
give N.sup.5 -benzyloxycarbonyl-2-amino-4-hydrazino-butanoic acid. 
Suspend N.sup.5 -benzyloxycarbonyl-2-amino-4-hydrazino-butanoic acid (80.2 
g, 0.3 mol) in t-butyl acetate (250 mL) and cool to 10.degree. C. Add 
p-toluenesulfonic acid (57 g, 0.3 mol) and add, by dropwise addition, 
concentrated sulfuric acid (80 mL). Stir at 10.degree. C. until the 
reaction is complete, basify to pH 8 by adding a suspension of sodium 
bicarbonate in water. Extract with ethyl acetate, dry (MgSO.sub.4), and 
evaporate to give N.sup.5 -benzyloxycarbonyl-2-amino-4-hydrazino-butanoic 
acid, t-butyl ester. 
Dissolve N.sup.5 -benzyloxycarbonyl-2-amino-4-hydrazino-butanoic acid, 
t-butyl ester (3.23 g, 10 mmol) in 50/50 dioxane/water (25 mL) and buffer 
to pH 10 with 1N sodium hydroxide. Add, by dropwise addition, an ether 
solution of t-butyl azidoformate (1.58 g, 11 mmol) at 10.degree. C. Allow 
to warm to room temperature and buffer occasionally to retain pH 10. 
Acidify with a sodium citrate/citric acid buffer to pH 5, extract with 
ether (3X), dry (MgSO.sub.4) and blow to a residue with a stream of 
nitrogen to give N-tert-butyloxycarbonyl-N.sup.5 
-benzyloxycarbonyl-2-amino-4-hydrazino-butanoic acid, t-butyl ester. 
Mix N-t-butyloxycarbonyl-N.sup.5 
-benzyloxycarbonyl-2-amino-4-hydrazino-butanoic acid, t-butyl ester (2.11 
g, 5.0 mmol) and 10% palladium/carbon (0.5 g) in 95% ethanol (40 mL). 
Hydrogenate at room temperature at atmospheric pressure. Filter and 
evaporate the filtrate in vacuo to give 
N-t-butyloxycarbonyl-2-amino-4-hydrazino-butanoic acid, t-butyl ester. 
Dissolve N-t-butyloxycarbonyl-2-amino-4-hydrazino-butanoic acid, t-butyl 
ester (2.88 g, 10 mmol) in methylene chloride (30 mL), place under a 
nitrogen atmosphere and cool to 0.degree. C. Add triphosgene (2.81 g, 10 
mmol) and allow to stir at room temperature for 2 hours to give the crude 
title compound. 
Step B: N.sup.2 -t-Butyloxycarbonyl-N.sup.6 
-(cyclopropylaminocarbonyl)-2-amino-4-hydrazino-butanoic acid, t-butyl 
ester 
Add cyclopropylamine (0.86 g, 15 mmol) to 
N-t-butyloxycarbonyl-2-amino-4-[(trichloromethylcarbonate)hydrazino]butano 
ic acid, t-butyl ester (4.5 g, 10 mmol) and stir at room temperature for 8 
hours. Pour the reaction mixture into water and separate the organic 
phase. Extract the aqueous phase with ethyl acetate (2X), wash the 
combined organic phases with saturated sodium chloride and dry 
(MgSO.sub.4). Evaporate the solvent in vacuo and purify by silica gel 
chromatography to give the title compound. 
Step C: 2-Amino-4-[2[cyclopropylamino)carbonyl]hydrazino]-butanoic acid 
Mix N.sup.2 -t-butyloxycarbonyl-N.sup.6 
-(cyclopropylamino-carbonyl)-2-amino-4-hydrazino-butanoic acid, t-butyl 
ester (3.72 g, 10 mmol) and 1N hydrochloric acid (30 mL) and stir under a 
nitrogen atmosphere at room temperature for 4 hours. Pour the reaction 
mixture into ethyl ether and separate the aqueous phase. Wash the aqueous 
phase with ethyl ether (2X), the neutralize with 5N sodium hydroxide. 
Evaporate the water in vacuo then purify by ion-exchange chromatography to 
give the title compound. 
EXAMPLE 13 
Preparation of 5-[2-[(Cyclopropylamino)carbonyl]hydrazino]-norvaline 
Step A: 
N-t-Butyloxycarbonyl-2-amino-5[(trichlormethylcarbonate)hydrazino]-pentano 
ic acid, t-butyl ester 
Dissolve 85% potassium hydroxide (3.96 g, 60 mmol) in absolute ethanol (60 
mL). Add 5-(3-bromopropyl)hydantoin (see Example 9) (6.63 g, 30 mmol) and 
a solution of ethyl carbazate (6.3 g, 60 mmol) in absolute ethanol (40 
mL). Reflux for 3 hours, cool and filter to remove potassium bromide. 
Evaporate the solvent in vacuo, dissolve the residue in water (30 mL), 
neutralize with dilute hydrochloric acid, cool and collect 
5-[3-(carbethoxyaminooxy)-propyl]-hydantoin. 
Mix 5-[3-(carbethoxyhydrazino)-propyl]-hydantoin (2.44 g, 10 mmol), barium 
hydroxide octahydrate (18.15 g, 10 mmol) and water (55 mL). Reflux for 12 
hours, remove the white solid by filtration and extract the filter cake 
with boiling water (25 ml) and finally wash with hot water (25 mL). 
Combine the filtrate and washings, treat with ammonium carbonate (5.7 g) 
and heat with stirring. Filter off the barium carbonate, wash the filter 
cake with hot water and evaporate the filtrate and washings in vacuo to 
give 2-amino-5-hydrazino-pentanoic acid. 
Dissolve 2-amino-5-hydrazino-pentanoic acid (14.7 g, 0.1 mol) in 1N sodium 
hydroxide (200 mL, 0.2 mol). Add a solution of CuSO.sub.4.5H.sub.2 O (12.5 
g, 0.05 mol) in water (50 mL). Stir the mixture until solution is 
complete. Treat with benzyl chloroformate (17.1 g, 0.1 mol) and stir at 
room temperature until reaction is complete. Suction filter, wash the 
residue with 1:3.5 methanol-water and dry at 60.degree. C. to give the 
copper complex. Suspend the copper complex in water, treat with excess 
hydrogen sulfide, heat to boiling, then cool to room temperature. Mix with 
1N sodium hydroxide and filter. Stir the filtrate with a slight excess of 
1N hydrochloric acid and filter. Wash the residue with water and dry to 
give N.sup.7 -benzyloxycarbonyl-2-amino-5-hydrazino-pentanoic acid. 
Suspend N.sup.7 -benzyloxycarbonyl-2-amino-5-hydrazino-pentanoic acid (84.4 
g, 0.3 mol) in t-butyl acetate (250 mL) and cool to 10.degree. C. Add 
p-toluenesulfonic acid (57 g, 0.3 mol) and add, by dropwise addition, 
concentrated sulfuric acid (80 mL). Stir at 10.degree. C. until the 
reaction is complete, basify to pH 8 by adding a suspension of sodium 
bicarbonate in water. Extract with ethyl acetate, dry (MgSO.sub.4), and 
evaporate to give N.sup.7 -benzyloxycarbonyl-2-amino-5-hydrazino-pentanoic 
acid, t-butyl ester. 
Dissolve N.sup.7 -benzyloxycarbonyl-2-amino-5-hydrazino-pentanoic acid, 
t-butyl ester (3.37 g, 10 mmol) in 50/50 dioxane/water (25 mL) and buffer 
to pH 10 with 1N sodium hydroxide. Add, by dropwise addition, an ether 
solution of t-butyl azidoformate (1.58 g, 11 mmol) at 10.degree. C. Allow 
to warm to room temperature and buffer occasionally to retain pH 10. 
Acidify with a sodium citrate/citric acid buffer to pH 5, extract with 
ether (3X), dry (MgSO.sub.4) and blow to a residue with a stream of 
nitrogen to give N-tert-butyloxycarbonyl-N.sup.7 
-benzyloxycarbonyl-2-amino-5-hydrazino-pentanoic acid, t-butyl ester. 
Mix N-tert-butyloxycarbonyl-N.sup.7 
-benzyloxycarbonyl-2-amino-5-hydrazino-pentanoic acid, t-butyl ester (2.18 
g, 5.0 mmol) and 10% palladium/carbon (0.5 g) in 95% ethanol (40 mL). 
Hydrogenate at room temperature at atmospheric pressure. Filter and 
evaporate the filtrate in vacuo to give 
N-t-butyloxycarbonyl-2-amino-5-hydrazino-pentanoic acid, t-butyl ester. 
Dissolve N-t-butyloxycarbonyl-2-amino-5-hydrazino-pentanoic acid, t-butyl 
ester (2.91 g, 10 mmol) in methylene dichloride (30 mL), place under a 
nitrogen atmosphere and cool to 0.degree. C. Add triphosgene (2.81 g, 10 
mmol) and allow to stir at room temperature for 2 hours to give the crude 
title compound. 
Step B: N.sup.2 -t-Butyloxycarbonyl-N.sup.7 
-(cyclopropylaminocarbonyl)-2-amino-5-hydrazino-pentanoic acid, t-butyl 
ester 
Add cyclopropylamine (0.86 g, 15 mmol) to 
N-t-butyloxycarbonyl-2-amino-5-[(trichlormethylcarbonate)hydrazino]-pentan 
oic acid, t-butyl ester (4.65 g, 10 mmol) and stir at room temperature for 
8 hours. Pour the reaction mixture into water and separate the organic 
phase. Extract the aqueous phase with ethyl acetate (2X), wash the 
combined organic phases with saturated sodium chloride and dry 
(MgSO.sub.4). Evaporate the solvent in vacuo and purify by silica gel 
chromatography to give the title compound. 
Step C: 5-[2-[(Cyclopropylamino)carbonyl]hydrazino]-norvaline pentanoic 
acid 
Mix N.sup.2 -t-butyloxycarbonyl-N.sup.7 
-(cyclopropylamino-carbonyl)-2-amino-5-hydrazino-pentanoic acid, t-butyl 
ester (3.86 g, 10 mmol) and 1N hydrochloric acid (30 mL) and stir under a 
nitrogen atmosphere at room temperature for 4 hours. Pour the reaction 
mixture into ethyl ether and separate the aqueous phase. Wash the aqueous 
phase with ethyl ether (2X), the neutralize with 5N sodium hydroxide. 
Evaporate the water in vacuo then purify by ion-exchange chromatography to 
give the title compound. 
Those compounds of Formula Ia-c in which X is represented by a 2-propyne 
residue are typically made in the following manner. Initially one of the 
protected compounds according to Formula Ia'-c' is made in which X is 
represented by hydrogen. This can be done utilizing the methods taught in 
Reaction Schemes I-IV. The only modification is that X should be 
represented by hydrogen in the amino compound of structure (4), the 
amidino compound of structure (6) and the isocyanate of structure (8). 
Once the appropriate compound of Formula Ia'-c' has been prepared, it is 
subjected to an N-alkylation reaction with 1-bromo-2-propyne, (i.e. a 
compound of the formula, Br--CH.sub.2 --C.tbd.CH). This N-alkylation can 
be carried out using techniques well known in the art. Approximately 
equivalent amounts of the reactants are contacted in an aprotic solvent 
such as toluene, benzene, or dimethylformamide at a temperature ranging 
from room temperature to reflux. The reaction is allowed to proceed for a 
period of time ranging from 1 to 16 hours. The desired product may then be 
recovered by extraction or concentration. It may then be purified by ion 
exchange chromatography, preparative C.sub.18 reverse phase 
chromatography, or by flash chromatography depending upon the particular 
protecting group being utilized. This compound may then be subjected to 
the deprotection and optional functionalizations discussed above in 
Reactions Schemes I-IV. 
The following example is typical of a compounds of formula Ic, wherein W is 
CH.sub.2 NH.sub.2, X is 2-propyne, and Z is NH. This example is 
illustrative only and is not intended to limit the scope of the present 
invention in any way. 
EXAMPLE 14 
Preparation of N.sup.5 -[Imino(2-propynylamino)methyl]-L-ornithine 
Step A: N.sup.2 -t-Butyloxycarbonyl-N.sup.5 
-[Imino-(2-propyneamino)methyl]-ornithine 
Mix N.sup.2 -t-butyloxycarbonyl-L-arginine HCl (3.10 g, 10 mmol), anhydrous 
potassium carbonate (1.38 g), dimethylformamide (50 mL) and propargyl 
bromide (1.1 mL). Stir at room temperature to 24 hours. Heat to 60.degree. 
C. for 8 hours, then heat to 90.degree. C. for 16 hours. Acidify, blow to 
a residue, apply the residue to a Dowex AG50-X8 column and elute with 0.2M 
NH.sub.4 OH and lyophlylize to give the title compound. 
Step B: N.sup.5 -[Imino(2-propynylamino)methyl]-L-ornithine 
Mix N.sup.2 -t-butyloxycarbonyl-N.sup.5 
-[Imino-(2-propyneamino)methyl]ornithine (1.44 g, 4.6 mmol) and 10% 
palladium/carbon (0.5 g) in 95% ethanol (40 mL). Hydrogenate at room 
temperature at atmospheric pressure. Filter and evaporate the filtrate in 
vacuo to give the title compound. 
As noted above, the compounds of Formula Ia-c inhibit the biosynthesis of 
nitric oxide and are therefore useful in the treatment of a number of 
disease states in which excess levels of nitric oxide play a detrimental 
role. These include conditions associated with low blood pressure, 
inflammatory diseases and CNS disorders. 
As noted above it has been demonstrated that nitric oxide relaxes the 
smooth muscles of the vasculature, producing a dilatory effect upon these 
vessels which results in a reduction in blood pressure. Since the 
compounds of Formula Ia-c inhibit the biosynthesis of nitric oxide, the 
administration of the compounds of Formula Ia-c will produce a rise in 
blood pressure and thus can be used to treat conditions associated with 
low blood pressure. Representative examples of such conditions include 
shock, endotoxic shock, hypovolemic shock, cardiogenic shock. 
In order to exhibit this hypertensive effect it is necessary that the 
compounds be administered in an amount sufficient to inhibit the 
biosynthesis of nitric oxide. The dosage range at which these compounds 
exhibit this hypertensive effect can vary widely depending upon the 
particular disease being treated, the severity of the patient's disease, 
the patient, the particular compound being administered, the route of 
administration, and the presence of other underlying disease states within 
the patient, etc. Typically the compounds exhibit their hypertensive 
effects at a dosage range of from about 0.1 mg/kg to 500 mg/kg/day. 
Repetitive daily administration may be desirable and will vary according 
to the conditions outlined above. Typically, the compounds will be 
administered from 1-4 times daily. 
The ability of these compounds to inhibit the biosynthesis of nitric oxide 
can be demonstrated by in vitro assays known in the art. Igengar et al. 
Proc. Nat'l. Acad. Sci., 84, pages 6369-6373 (September 1987). In this 
assay macrophages are exposed to bacterial lipopolysaccharides and 
Interferongamma, which stimulates the macrophages to produce nitric oxide. 
The compounds of Formula Ia-c will either inhibit or decrease production 
of nitric oxide by the macrophages. 
It is well known in the art that excessive activity by macrophages is 
implicated in a number of inflammatory diseases such as, for example, 
rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, 
cirrhosis of the liver and lungs, sarcoidosis, and granulomatous lesions. 
In a recent review, Collier et al. stated that it is currently believed 
that the cytotoxic activity of macrophages is due their ability to produce 
nitric oxide. Trends in Pharmacological Sciences, 10(11), page 123, 
(1989). Thus the compounds of the instant invention will either inhibit or 
decrease the amount of nitric oxide which is produced by the macrophages 
and thus will be useful in the treatment of these disease states 
associated with excessive activity of macrophages. The compounds will also 
be useful in preventing transplant rejection by macrophages. 
In order to exhibit this beneficial effect in inflammatory diseases, it is 
necessary that the compounds of Formula Ia-c be administered in a quantity 
sufficient to inhibit the production of nitric oxide by macrophages. The 
dosage range at which these compounds exhibit this effect can vary widely 
depending upon the particular disease being treated, the severity of the 
patient's disease, the patient, the particular compound being 
administered, the route of administration, and the presence of other 
underlying disease states within the patient, etc. Typically the compounds 
exhibit their therapeutic effect at a dosage range of from about 0.1 
mg/kg/day to about 500 mg/kg/day for any of the diseases or conditions 
listed above. Repetitive daily administration may be desirable and will 
vary according to the conditions outlined above. 
The ability of these compounds to inhibit the generation of cyclic GMP 
within the central nervous system can be demonstrated by procedure's known 
in the art such as Baron et al. Journal of Pharmacol. and Exp. Therap., 
250, pages 162-169 (1989). In this test, mice are administered harmaline 
which produces a rise in cyclic GMP levels with the cerebella of the CNS. 
The pre-administration of one of the compounds of Formula Ia-c will block 
the rise in cyclic GMP levels that is typically associated with harmaline 
administration. 
It has been demonstrated that stimulation of the NMDA receptor complex 
leads to the release of nitric oxide which in turn stimulates guanylate 
cyclase which in turn produces a rise in cyclic GMP levels. Since the 
compounds of Formula Ia-c will inhibit the production of cyclic GMP and 
thus will negate the physiological effects typically associated with 
stimulation of the NMDA receptor complex, these compounds will be useful 
in the treatment of disease states associated with overstimulation of 
these excitatory neurons. Overstimulation of the NMDA receptor complex has 
been associated with seizures and thus the compounds of Formula Ia-c 
exhibit anti-convulsant properties and are useful in the treatment of 
epilepsy. They are useful in the treatment of grand mal seizures, petit 
mal seizures, psychomotor seizures, and autonomic seizures. 
Overstimulation of the NMDA receptor complex has also been associated with 
the neurotoxicity associated with ischemic, hypoxic, traumatic, or 
hypoglycemic conditions. Representative examples of such ischemic, 
hypoxic, or hypoglycemic conditions include strokes or cerebrovascular 
accidents, carbon monoxide poisoning, hyperinsulinemia, cardiac arrest, 
drownings, suffocation, and neonatal anoxic trauma. The compounds should 
be administered to the patient within 24 hours of the onset of the 
hypoxic, ischemic, or hypoglycemic condition in order for the compounds to 
effectively minimize the CNS damage which the patient will experience. 
The compounds of Formula Ia-c are also useful in the treatment of 
neurodegenerative diseases such as Huntington's disease, Alzheimer's 
disease, senile dementia, glutaric acidaemia type I, multi-infarct 
dementia, and neuronal damage associated with uncontrolled seizures. The 
administration of these compounds to a patient experiencing such a 
condition will serve to either prevent the patient from experiencing 
further neurodegeneration or it will decrease the rate at which the 
neurodegeneration occurs. 
As is apparent to those skilled in the art, the compounds will not correct 
any CNS damage that has already occurred as the result of either disease 
or a lack of oxygen or sugar. As used in this application, the term 
"treat" refers to the ability of the compounds to prevent further damage 
or delay the rate at which any further damage occurs. 
The dosage range at which these compounds will exhibit their effect upon 
excitatory neurotransmission can vary widely depending upon the particular 
disease being treated, the severity of the patient's disease, the patient, 
the particular compound being administered, the route of administration, 
and the presence of other underlying disease states within the patient, 
etc. Typically the compounds exhibit their therapeutic effect at a dosage 
range of from about 0.1 mg/kg/day to about 500 mg/kg/day for any of the 
diseases or conditions listed above. Repetitive daily administration may 
be desirable and will vary according to the conditions outlined above. 
As used in this application: 
a) the term "patient" refers to warm blooded animals such as, for example, 
guinea pigs, mice, rats, cats, rabbits, dogs, monkeys, chimpanzees, and 
humans; 
b) the term "treat" refers to the ability of the compounds to either 
relieve, alleviate, or slow the progression of the patient's disease; 
c) the term "neurodegeneration" refers to a progressive death and 
disappearance of a population of nerve cells occurring in a manner 
characteristic of a particular disease state and leading to brain damage. 
d) the term "inflammatory disease" refers to a condition in which 
activation of leukocytes leads to an impairment of normal physiologic 
function. Representative examples of such conditions include rheumatoid 
arthritis, inflammatory bowel disease, sepsis, and adult respiratory 
distress syndrome. 
e) the term "hypotensive condition" refers to a condition in which blood 
pressure is lowered to a point to which normal physiologic functioning is 
impaired. 
The compounds of the present invention may be administered by a variety of 
routes. They are effective if administered orally. The compounds may also 
be administered parenterally (i.e., subcutaneously, intravenously, 
intramuscularly, intraperitoneally, or intrathecally). 
Pharmaceutical compositions can be manufactured utilizing techniques known 
in the art. Typically an inhibitory amount of the compound will be admixed 
with a pharmaceutically acceptable carrier. 
For oral administration, the compounds can be formulated into solid or 
liquid preparations such as capsules, pills, tablets, lozenges, melts, 
powders, suspensions, or emulsions. Solid unit dosage forms can be 
capsules of the ordinary gelatin type containing, for example, 
surfactants, lubricants and inert fillers such as lactose, sucrose, and 
cornstarch or they can be sustained release preparations. In another 
embodiment, the compounds of Formula Ia-c can be tableted with 
conventional tablet bases such as lactose, sucrose, and cornstarch in 
combination with binders, such as acacia, cornstarch, or gelatin, 
disintegrating agents such as potato starch or alginic acid, and a 
lubricant such as stearic acid or magnesium stearate. Liquid preparations 
are prepared by dissolving the active ingredient in an aqueous or 
non-aqueous pharmaceutically acceptable solvent which may also contain 
suspending agents, sweetening agents, flavoring agents, and preservative 
agents as are known in the art. 
For parenteral administration the compounds may be dissolved in a 
physiologically acceptable pharmaceutical carrier and administered as 
either a solution or a suspension. Illustrative of suitable pharmaceutical 
carriers are water, saline, dextrose solutions, fructose solutions, 
ethanol, or oils of animal, vegetative, or synthetic origin. The 
pharmaceutical carrier may also contain preservatives, buffers, etc., as 
are known in the art. When the compounds are being administered 
intrathecally, they may also be dissolved in cerebrospinal fluid as is 
known in the art. 
The compounds may also be admixed with any inert carrier and utilized in 
laboratory assays in order to determine the concentration of the compounds 
within the serum, urine, etc., of the patient as is known in the art.