ACE inhibitors produced from Nocardia orientalis

N.sup.2 -[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl)-D-.a lpha.-glutamine, N.sup.2 -[N-(N-acetylmuramoyl)-L-alanyl]-N-[5,6-diamino-1-[[1-[(1-carboxyethyl)car bamoyl]ethyl]carbamoyl]-6-oxohexyl]-D-.alpha.-glutamine and EM5556C are obtained as a mixture by cultivating a strain of the microorganism Nocardia orientalis A.T.C.C. No. 39444.

BACKGROUND OF THE INVENTION 
U.S. Pat. No. 4,186,194, issued Jan. 29, 1980, discloses water soluble 
agents which are said to be immunological adjuvants for stimulating in the 
host the immune response to various antigens. The compounds comprise an 
acetyl or glycolyl muramic acid group, and a short peptide chain linked 
thereto, the first amino acid of the peptide chain being alanine, serine 
or glycine and the second amino acid of the peptide chain being glutamic 
acid or aspartic acid. 
SUMMARY OF THE INVENTION 
A mixture of at least four components, designated EM5556, is obtained by 
cultivating a strain of the microorganism Nocardia orientalis which has 
been deposited in the American Type Culture Collection as A.T.C.C. No. 
39444. EM5556 is a mixture of at least four components, two of which have 
been characterized as N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl)-D.al 
pha.-glutamine glutamine which has the structural formula 
##STR1## 
and N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-[5.6-diamino-1-[[1-[(1-carboxyethyl))ca 
rbamoyl]ethyl]-carbamol]-6-oxohexyl]-D-.alpha.-glutamine which has the 
structural formula 
##STR2## 
The third component has been designated EM5556C and is known to be a 
muramylpentapeptide containing N-acetylmuramic acid, alanine, glutamic 
acid, serine and diaminopimelic acid in a 1:2:1:1:1 ratio based on the NMR 
spectrum. The fouth component has not been isolated in pure form. 
Reduction of N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1carboxy-6-oxohexyl) 
-D-.alpha.-glutamine yields 
2-(acetylamino)-3-O-[(R)-2-[(S)-2-[[(R)-1-carboxy-4-[[(1S,5R)-5,6-diamino- 
1-carboxy-6-oxohexyl]amino]-4-oxobutyl]amino]-1-methyl-2-oxoethyl]amino]-1- 
methyl-2-oxoethyl]-2-desoxy-D-glucitol, which has the structural formula 
##STR3## 
The mixture EM5556, the three components which have been isolated in pure 
form, and 
2-(acetylamino)-3-O-[(R)-2-[(S)-2-[[(R)-1-carboxy-4-[[(1S,5R)-5,6-diamino- 
1-carboxy-6-oxohexyl]amino]-4-oxobutyl]amino]-1-methyl-2-oxoethyl]amino]-1- 
methyl-2-oxoethyl]-2-desoxy-D-desoxy-D-glucitol are hypotensive agents. 
They inhibit the conversion of the decapeptide angiotensin I to 
angiotensin II and therefore, are useful in reducing or relieving 
angiotensin related hypertension. The action of the enzyme renin on 
angiotensinogen, a pseduoglobulin in blood plasma, produces angiotensin I. 
Angiotensin I is converted by angiotensin converting enzyme (ACE) to 
angiotensin II. The latter is an active pressor substance which has been 
implicated as the causative agent in several forms of hypertension in 
various mammalian species, e.g., humans. The compounds of this invention 
intervene in the angiotensinogen.fwdarw.(renin).fwdarw. angiotensin 
I.fwdarw.(ACE).fwdarw. angiotensin II sequence by inhibiting angiotensin 
converting enzyme and reducing or eliminating the formation of the pressor 
substance angiotensin II. Thus by the administration of a composition 
containing one (or a combination) of the compounds of this invention, 
angiotensin dependent hypertension in a species of mammal (e.g., humans) 
suffering therefrom is alleviated. A single dose, or preferably two to 
four divided daily doses, provided on a basis of about 0.1 to 100 
milligrams per kilogram of body weight per day, preferably about 1 to 15 
milligrams per kilogram of body weight per day is appropriate to reduce 
blood pressure. The substance is administered parenterally.

DETAILED DESCRIPTION OF THE INVENTION 
The Microorganism 
The microorganism used for the production of EM5556 is a strain of Nocardia 
orientalis isolated from the soil. A subculture of the organism may be 
obtained from the American Type Culture Collection, Rockville, Md. Its 
accession number in the repository is A.T.C.C. No. 39444. 
The characteristics of Nocardia orientalis A.T.C.C. No. 39444 are: 
The organism producing EM5556 is identified as a strain of Nocardia 
orientalis found to be identical with the type strain of this species 
ISP-5040 which is A.T.C.C. No. 19795. 
The vegetative mycelium exhibits characteristic fragmentation, and a 
rudimentary aerial mycelium is produced. Cell wall acid hydrolysates 
contain meso-DAP, with galactose and arabinose as the major sugar 
components. This provides the basis for placement in the genus Nocardia. 
The organism produces positive test responses with respect to the following 
biochemical characteristics: 
(1) decomposition of casein, testosterone, tyrosine and xanthine; 
(2) acid production from adonitol, cellobiose, meso-erythritol, lactose, 
maltose, .alpha.-methyl-D-glucoside; 
(3) urease. 
The following characteristics give negative responses: 
(1) decomposition to adenine; 
(2) acid from melezitose; 
(3) resistance to lysozyme and rifampin. 
All tests were run according to the methods described in the following 
references: 
(1) Gordon, R. E., and Mihm, J. M. (1957) & J. Bacteriol. 73:15-27 
(2) Gordon, R. E., et al. (1978) J. Gen. Microbiol. 109:69-79 
(3) Goodfellow, M., Schaal, K. P. (1979) Identification Methods for 
Nocardia, Actinomadura and Rhodococcus, pp. 261-276. In: Loveluck, D. W., 
Skinner, F. A. (eds.), Identification Methods for Microbiologists, 2nd ed. 
Soc. App. Bacteriol. Tech. Ser. London: Academic Press. 
For isolating the organism, a portion of the soil sample (obtained in 
Hamilton Township, N.J.) is stamped onto an agar of the following 
composition: 
______________________________________ 
Glycerol 12.6 ml 
Citric Acid 1.2 gm 
(NH.sub.4).sub.2 HPO.sub.4 
0.4 gm 
KCl 0.08 gm 
MgCl.sub.2.6H.sub.2 O 0.418 gm 
MnCl.sub.2.4H.sub.2 O 0.036 gm 
FeSO.sub.4.7H.sub.2 O 0.023 gm 
ZnCl.sub.2.6H.sub.2 O 0.021 gm 
CoCl.sub.2.6H.sub.2 O 0.004 gm 
Agar 15.0 gm 
______________________________________ 
The medium is adjusted to pH about 7.2 and sterilized in an autoclave at 
121.degree. C. for 30 minutes. After 3 to 5 days incubation at 25.degree. 
C., the colonies of Nocardia orientalis A.T.C.C. No. 39444 are isolated 
from the plated soil. The isolated colonies are picked off and maintained 
on an agar medium composed of: 
______________________________________ 
Oatmeal 20 gm 
Tomato paste 20 gm 
Agar 15 gm 
Tap water to 1000 ml 
______________________________________ 
The medium is adjusted to pH about 7 and sterilized in an autoclave at 
121.degree. C. for 15 minutes. 
Production of EM5556 
Nocardia orientalis A.T.C.C. No. 39444 produces EM5556 which possesses 
angiotensin converting enzyme inhibitory activity. To form EM5556 
according to the preferred methodology of this invention, Nocardia 
orientalis A.T.C.C. No. 39444 is grown at, or near, room temperature 
(25.degree. C.) under submerged aerobic conditions in an aqueous nutrient 
medium containing an assimilable carbohydrate and nitrogen source. The 
fermentation is carried out until substantial activity is imparted to the 
medium, usually about 120 to 144 hours. 
After the fermentation is complete, cells are removed by centrifugation and 
the broth supernate is adjusted to pH 3.0 (if needed) and loaded onto a 
column of Darco granular charcoal. Eluting with acetone-water (1:1) the 
active fractions are determined by assaying for ACE inhibition using a 
spectrophotometer with captopril as the positive standard and 
p-nitrobenzyloxycarbonylglycyl(S-4-nitrobenzo-2-oxa-1,3-diazole)-L-cystein 
ylglycine as the chromogenic substrate and partially purified rabbit lung 
ACE as enzyme (see A. V. Persson et al., A New Chromogenic Substrate for 
Angiotensin-Converting Enzyme, Analytical Biochemistry, 91:674 (1978)). 
The active fractions are purified by anion exchange chromatography. 
The active components of EM5556 are separated by reverse phase 
chromatography on a column of MCI gel CHP20P (a styrene and divinylbenzene 
copolymer having a macroreticular structure) eluting with water followed 
by a linear gradient of 0-66% acetone-water. The major activity elutes 
with water and is fractionated on the basis of TLC and HPLC analysis. 
The following examples further illustrate this invention. 
EXAMPLE 1 
The following is a detailed description of the fermentation of Nocardia 
orientalis A.T.C.C. No. 39444, and the isolation of the resulting product. 
Nocardia orientalis A.T.C.C. No. 39444 was maintained on the following 
sterilized agar medium (A): 
______________________________________ 
Grams 
______________________________________ 
Oatmeal 20 
Tomato paste 
20 
Agar 15 
Tap water to 
1 Liter 
______________________________________ 
The pH was adjusted to 7.0 before sterilization at 121.degree. C. for 15 
minutes. A loopful of surface growth from an agar slant (Medium A) of 
Nocardia orientalis was used to inoculate each of eight 500 ml Erlenmeyer 
flasks containing 100 ml each of the following sterilized medium (B): 
______________________________________ 
Grams 
______________________________________ 
Yeast extract 4 
Malt extract 10 
Dextrose 4 
Distilled water to 
1 Liter 
______________________________________ 
The pH was adjusted to 7.3 before sterilization at 121.degree. C. for 15 
minutes. After inoculation, the flasks were then incubated at 25.degree. 
C. on a rotary shaker (300 rpm: 2 inch stroke) for approximately 72 hours. 
After the appropriate incubation as described above, 3.0% (vol./vol.) 
transfers were made from the grown culture flasks to two hundred 500 ml 
Erlenmeyer flasks containing 100 ml each of the following sterilized 
medium (C): 
______________________________________ 
Grams 
______________________________________ 
(NH.sub.4).sub.2 SO.sub.4 
2.64 
K.sub.2 HPO.sub.4 
4.3 
KH.sub.2 PO.sub.4 
2.38 
MgSO.sub.4.7H.sub.2 O 
1.0 
Glucose 10.0 
(Sterilized separately) 
Salt solution* 1.0 ml/liter 
Distilled H.sub.2 O to 
1 Liter 
______________________________________ 
*Salt Solution 
gm/100 ml distilled water: CuSO.sub.4.5H.sub.2 O 0.64; 
FeSO.sub.4.7H.sub.2 O 0.11; 
MnCl.sub.2.4H.sub.2 O 0.79; 
ZnSO.sub.4.7H.sub.2 O 0.15 
The pH was adjusted to 6.8-7.0 before sterilization of 121.degree. C. for 
15 minutes. After inoculation, the flasks were incubated at 25.degree. C. 
on a rotary shaker (300 rpm: 2 each stroke) for approximately 120 hours. 
At this time the contents of the flasks were pooled and the broth was 
centrifuged yielding approximately 19 liters of supernatant broth; the 
cells were discarded. 
The broth supernate (19 L, PH 3.0) was loaded onto a Darco granular 
charcoal** column (5.times.45cm) at a rate of 63 ml/minute. The column was 
washed with water (3 L) and then eluted with four 1 L-portions of acetone- 
water (1:1). The fractions were assayed for ACE inhibition using a 
spectrophotometer with captopril as the positive standard and 
o-nitrobenzyloxycarbonylglycyl 
(S-4-nitrobenzo-2-oxa-1.3-diazole)-L-cysteinylglycine as the chromogenic 
substrate and partially purified rabbit lung ACE as enzyme. The active 
fractions were combined (4 L) and concentrated in vacuo (9.97 g). The 
residue was dissolved in water (10 ml). and the pH of the solution was 
adjusted to 9.0 with 5N NaOH. The solution was applied to a column of 
BioRad AGl.times.2, OAc.sup.- *, 200-400 mesh (5.times.30 cm). The column 
was washed with five 120 ml portions of water and then eluted with a 
linear gradient prepared from water (3 L) and 0.25M pyridine-acetic acid, 
pH 5.1 (3 L). Fractions (25 ml) were collected, a small portion of each 
fraction was diluted with water (1:30 dilution) and assayed for ACE 
inhibition. The active fractions (123-144) were combined (525 ml) and 
concentrated in vacuo to give 1.2 g of a yellow solid which contained 
N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5.6-diamino-1-carboxy-6-oxohexyl)-D-.a 
lpha.-glutamine, N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-[5,6-diamino-1-[[1-[(1-carboxyethyl)car 
bamoyl]ethyl]-carbamoyl]-6-oxohexyl]-D-.alpha.-glutamine and EM5556C. 
FNT **Darco granular charcoal (MCB Manufacturing Chemists, Inc., Gibbstown, 
N.J.) 
FNT *BioRad AGl.times.2, OAc.sup.- (Bio-Rad Laboratories, Richmond, Calif.) is 
a strongly basic anion exchange resin with quaternary ammonium functional 
groups attached to the styrene divinylbenzene copolymer. 
The 1.2 g of the yellow solid containing N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl)-D-.a 
lpha.-glutamine, N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-[5,6-diamino-1-[[1-[(1-carboxyethyl) 
carbamoyl]ethyl]carbamoyl]-6-oxohexyl]-D-.alpha.-glutamine and EM5556C was 
dissolved in water (1 ml, pH 3.5) and applied to a reverse phase column of 
MCI gel CHP20P* (2.5.times.54cm). The column was eluted with water (600 
ml, 7 ml fractions collected) followed by a linear gradient prepared from 
water (300 ml) and 66% acetone-water (300 ml, 8 ml fractions collected). 
Fractions were assayed for ACE inhibition using the methodology described 
above, and were combined on the basis of TLC (silica gel, ethyl 
acetate-butanol-acetic acid-water, 1:1:1:1, Rydon-visualization, system I) 
and HPLC analysis on a C.sub.18 spherical packed column with Z-module** 
using 10% acetonitrile, 0.1% 1-heptanesulfonic acid, sodium salt, in water 
(isocratic, pH 2.1 with HCl, 1.5 ml/minute, 210 nm). Fractions 44-74 
contained N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl)-D-.a 
lpha.-glutamine by TLC (r.sub.f =0.19, system I) and HPLC (Table 1) 
analysis, these were concentrated in vacuo to give 121 mg of N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy 
-6-oxohexyl)-D-.alpha.-glutamine, the sodium salt, as a white powder. 
FNT *MCI gel CHP 20P (Mitsubishi Chemical Industries, Ltd., Japan) is a styrene 
and divinylbenzene copolymer in a bead form having a macroreticular 
structure. 
FNT **Z-module TM (Waters Assoc.) is a radial compressor system. It is a 
self-contained manually operated unit into which a Radial-pak cartridge is 
inserted. The module utilizes a precision stainless steel chamber which 
fits tightly over the cartridge to cause uniform radial compression 
throughout the cartridge's packed bed. 
Fractions 75-113 contained a mixture of the above N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy 
-6-oxohexyl)-D-.alpha.-glutamine and EM5556C as shown by TLC (r.sub.f 
=0.19 for N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5.6-diamino-1-carboxy -6-oxohexyl)-D-. 
alpha.-glutamine and r.sub.f =0.15 for EM5556C, system I) and HPLC (Table 
1) analysis. These were combined and concentrated to dryness to give 145 
mg of a residue consisting of N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy 
-6-oxohexyl)-D-.alpha.-glutamine and EM5556C. This mixture was further 
purified as described below to give more of N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6diamino-1-carboxy-6-oxohexyl)-D-.al 
pha.-glutamine and EM5556C. 
The residue (145 mg) obtained above was dissolved in water (0.7 ml, pH 3.5) 
and applied to a reverse phase column of MCI Gel CHP20P (2.5.times.50cm). 
The column was eluted with water (1.2 L, 8 ml fractions), and the 
fractions were assayed for ACE inhibition. The active fractions were 
fractionated on the basis of TLC (system I) and HPLC analysis. Fractions 
30-84 contained N.sup.2 -[N-(acetylmuramoyl) 
-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl) -D-.alpha.-glutamine by 
TLC (r.sub.f =0.19, system I) and HPLC (Table 1), these were combined and 
concentrated in vacuo to give 42.2 mg of N.sup.2 -[N-(N-acetylmuramoyl) 
-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl-D-.alpha.-glutamine, sodium 
salt, as a white powder. Fractions 130-145 contained EM5556C by TLC 
(r.sub.f =0.15, system I) and HPLC (Table 1). These were combined and 
concentrated in vacuo to give 5.3 mg of EM5556C, sodium salt as a white 
powder. 
The infrared spectrum of EM5556C as the monosodium salt in potassium 
bromide is shown in FIG. 1. The 400 MHz .sup.1 H-NMR spectrum of EM5556C 
as the monosodium salt in deuterium is shown in FIG. 2. 
A mass spectrum of EM5556C was obtained by the fast atom bombardment (FAB) 
technique, which gave peaks at m/z 823 and 845 in the positive-ion mode, 
indicating a molecular weight of 822 and 844 for the free acid and the 
monosodium salt, respectively. 
The elemental composition of EM5556C was obtained by high resolution FAB 
mass spectrometry. The observed mass for C.sub.32 H.sub.55 N.sub.8 
O.sub.17 (M+H).sup.+ was 823.sup.. 373 (theory 823.sup.. 368). 
The 1.2 g of the yellow solid containing N.sup.2 
[N-(N-acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy 
-6-oxohexyl)-D-.alpha.-glutamine, N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-[5,6-diamino 
-1[[1-[(1-carboxyethyl)carbamoyl]ethyl]carbamoyl]-6-oxohexyl]-D-.alpha.-gl 
utamine and EM5556C was dissolved in water (1 ml, the pH 3.5) and applied 
to a reverse phase column of MCI gel CHP20P (2.5.times.54cm). The column 
was eluted with water (600 ml, 7 ml fractions collected) followed by a 
linear gradient prepared from water (300 ml) and 66% acetone-water (300 
ml, 8 ml fractions collected). Fractions were assayed for ACE inhibition. 
The major activity eluted with water which contained N.sup.2 
-[N-(N-acetylmuramoyl-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl)-D-.al 
pha.-glutamine and EM5556C (described earlier). 
A less active band was eluted with .about.54% acetone-water (fractions 
162-165); this contained N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-[5.6-diamino-1-[[1-[(1-carboxyethyl)car 
bamoyl]ethyl]carbamoyl]-6-oxohexyl]-D-.alpha.-glutamine by TLC (r.sub.f 
=0.19, system I) and HPLC (Table 1) analysis. Fractions 162-165 were 
combined and concentrated in vacuo to give 410 mg of N.sup.2 
-[N-(N-acetylmuramoyl)-L-alanyl]-N-[5,6-diamino-1-[[1-[(1-carboxyethyl)car 
bamoyl]ethyl]-carbamoyl]-6-oxohexyl]-D-.alpha.-glutamine, sodium salt, as a 
white powder. 
TABLE 1 
______________________________________ 
HPLC Retention Times of Components of EM5556 
Retention Time (Min.) 
Compound .alpha.-anomer 
.beta.-anomer 
______________________________________ 
N.sup.2 --[N--(N--acetylmuramoyl)-L- 
6.2 6.9 
alanyl]-N--(5,6-diamino-1- 
carboxy-6-oxohexyl)-D- 
.alpha.-glutamine 
N.sup.2 --[N--(N--acetylmuramoyl)-L- 
9.7 11.9 
alanyl]-N--[5,6-diamino-1-[[1- 
[(1-carboxyethyl)carbomyl]- 
ethyl]carbomyl]-6-oxohexyl]- 
D-.alpha.-glutamine 
EM5556C 8.5 10.0 
Conditions: 
C.sub.18 spherical packed column (8 mm .times. 
10 cm) with Z-module using 10% 
acetonitrile, 0.1% 1-heptanesulfonic 
acid, sodium salt in water (iso- 
chratic, pH 2.1 with HCl), flow rate 
1.5 ml/minute at 210 nm 
______________________________________ 
EXAMPLE 2 2-(Acetylamino)-3-O-[(R)-2-[(S)-2-[[(R)-1-carboxy-4-[[(1S,5R) 
-5,6-diamino-1-carboxy-6-oxoethyl]amino]-4-oxobutyl]amino]-1-methyl-2-oxoe 
thyl]-2-desoxy-D-glucitol. 
A suspension of N.sup.2 
-[N-(acetylmuramoyl)-L-alanyl]-N-(5,6-diamino-1-carboxy-6-oxohexyl)-D-.alp 
ha.-glutamine (41.2 mg, 62 .mu.mol) in a mixture of water (1 ml) and 
tetrahydrofuran (9 ml) was stirred with sodium borohydride (2.6 mg, 68 
.mu.mol) at room temperature for 15 minutes. The TLC analysis (silica, 
ethyl acetate:n-butanol:acetic acid:water, 1:1:1:1, Rydon-detection) 
showed that the reaction was incomplete. An additional 2 mg (52 .mu.mol) 
of sodium borohydride was added and the reaction mixture was stirred for 
3.5 hours. The solvents were removed in vacuo, and the residue was 
dissolved in water (3 ml) and the pH adjusted to 3.0 with 1N HCl. This was 
purified by reverse-phase chromatography on MCI gel CHP20P (37-75.mu.). 
eluting with water to yield 45.2 mg of the title compound as a white 
solid.