Note: Descriptions are shown in the official language in which they were submitted.
1 :1 63938
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DESCRIPTION
BACKGROUND OF THE INVENTION
The characteristics and preparation of the antibiotic lincomycin
are disclosed in U.S. Patent 3,086,912. Clindamycin is disclosed in
U.S. Patent 3,496,163. These antibiotics have been extensively used
as medicines in humans and animals. A number of patents world-wide
have issued concerning these antibiotics and a variety of derivatives
thereof.
The structural formulas for lincomycin ~1) and clindamycin (2
are shown in Chart 1.
Lincomycin and clindamycin 3-nucleotides are disclosed and
claimed in U.S. Patent 3,671,647. All of the lincomycin and clinda-
mycin compounds disclosed in U.S. 3,671,647 have the propyl hygric
acid moiety. These 3-nucleotides were found by test against S. aureus
ln vivo to have an activity approximately one-tenth of the parent
compound.
BRIEF SUMM~RY OF THE INVENTION
The subject invention concerns the 3-ribonucleotides of linco-
mycin- and clindamycin-type compounds in which the propyl hygric acid
moiety has been replaced by different cyclic amino acids. Unexpect-
edly, these nucleotides demonstrate in vivo antibacterial activity ashigh as the parent compounds. ~ecause of these highly relevant char-
acteristics, the nucleotides of the subject invention are considered
to be prime candidates for medicinal use. These compounds also are
useful for the prophylactic and therapeutic treatment of sub,iects
hosting a protozoan parasite. For example, when the protozoan is a
malarial parasite, the subject can be animal, e.g., mice infected with
Plasmodium berghei; birds, e.g. ducks infected with P. lophurae and
chic~s infected with P. gallinaceum, and mammals such as primates,
e.g., monkeys infected with P. cynomolgi, and humans infected with P.
falciparunl, P. vivax, and P. malariae.
Mammals hosting a parasitic protozoan of the class Sporazoa,
order Coccidia (a microparasite producing the disease coccidiosis) can
be treated by administration of the compounds of the present inven-
~ 3 63938
3713/3841-2-
tion. For example cattle infected with Eimeria zurnl1, E. bovis~ E.ellipsoidalis; sheep and goats with E. parva, E. faurei; swine with .
debliecki, E. scabra, and Isospora suis; dogs and cats with Isospora
bigemina, I. felis, E. canis, E. felina; poultry with ~. tenella;
rabbits with E. stiedae, E~ perforans; and mink with E. mustelae can
be treated.
The lincomycin- and clindamycin-type compounds which can be
converted to the 3~ribonucleotides are shown in Chart 2. In place of
the hydroxyl at the three position of the lincosaminide moiety, there
is substituted a nucleotide selected from the group consisting of
adenylic acid, guanylic acid, cytidylic acid and uridylic acid.
The 3-ribonucleotides of the subject invention can be prepared by
microbiological transformation procedures. The 3-(5'-ribonucleotides)
obtained by transformation of U-57930 are shown in Chart 3
DETAILED DESCRIPTION
The parent compounds disclosed in ~hart 2 can be prepared by the
procedures disclosed in applicant's U.S. pa~ent No. 4,278,7S9,
issued July 14, 1981.
The 3-~(5'-ribonucleotides) of the compounds of Chart 2 can be
prepared by following the procedures disclosed in U.S. -Patent
3,671,647. Salts of these nucleotides also can be prepared following
the procedures in U.S. 3,671,647.
Formulations of the nucleotides of this invention can be made
following the composition examples in U.S. patent 4,278,789.
The ~ormulations are prepared by substitutin~ a nucleotide
of the subject invention for the active compound in the examples. The
substitution can be on an equimolar basis.
General assay and characterization procedures which can be
employed to determine and characterize the nucleotides of the inven-
tion are as follows:
Assay of 3-~5'-Ribonucleotides)
Since the 3-ribonucleotides of this invention lack in vitro
antibacterial activity, their formation from the antibacterially-
active parent compounds can be followed easily by measuring the lossof such antibiotic activity. To determine the amoun~s of antibac-
terially-act1ve parent compound in culture filtrates or reaction
mixtures, a standard assay with Sarcina lutea ATCC 9341 is employed.
1 ~ 63938
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To assay for the presence of the 3-ribonucleotides in fermentation
beers, extracts, and purified materials, the phosphodiester bond is
first hydrolyzed with crude alkaline phosphatase, or snake venom
phosphodiesterase, by the procedures described below. The
5 antibacterially-active compound in the hydrolysate is determined by
standard assay.
Enzymatic Hydrolyses
Alkaline Phosphatase: Stock solutions (0.5 mg/ml, 0.54 Units/mg)
of pigeon intestine alkaline phosphatase, EC 3.1.3,1 (Sigma) are
10 prepared in Tris (hydroxymethyl) aminomethane hydrochloride buffer7
0.01 M pH 8Ø Samples to be treated are diluted 1:2 with the enzyme
buffer mixture and are incubated at 28C for 18 hours.
Snake Venom Phosphodiesterase: Stock solutions (100 mg/ml, 0.026
Units/mg) of purified snake venom phosphodiesterase LC 3.1.4.1 (Sigma)
15 are prepared in distilled water. Incubation mixtures contain 0.2 ml
of a solution (1 mg/ml) of the sample to be treated in water, 0.6 ml
of 0.01 M Tr7s-hydrochloride buffer, pH s.n, o.l ml of 0.3 M MgCl2,
and 0.1 ml of the enzyme stock solution. Incubation is carried out at
37C for 18 hours.
Spleen Phosphodiesterase: Stock solutions of spleen phosphodi-
esterase EC 3.1.4.18 (Sigma) are prepared (1 mg/ml, 19.6 Units/mg) in
distilled water. Incubation mixtures contain 0.4 ml of a solution
(0.5 mg/ml) of the sample to be treated in water, 0.5 ml of ().(~2 M
Tris buffer, pH 7.0 and 0.1 ml of the enzyme stock solution. Incu-
25 bation is carried out at 37C for 18 hours.
Thin-La~r Chromatographic Analysis of Preparations_and Enzy-
matic Hydrolysates.
The production and purification of the 3-ribonucleotides is
followed by assay against S. lutea (see above) and by TLC using silica
30 gel G and methyl ethyl ketone-acetone-water (186:52:20, v/v) or ethyl
acetate-acetone-water (8:5:1) as the solvent systems. The bioactive
parent compounds are detected by bioautography on agar seeded with S.
lutea.
The products of enzymatic or chemical hydrolysis of the 3-nucleo-
35 tides are separated by the following TLC systems:
A: Silica gel GF plates (Analtech Inc.); water as the solvent
system.
B: Silica gel GF plates; n-propyl alcohol-conc. ammonium
i 1 6393~
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--4--
hydroxide-water (55:10:35, v/v).
C: NM-Polygram Cellulose 300 (Brinkman Instruments Inc.); 1-
butanol-water-formic acid (77:13:10, v/v).
UV absorbing materials are detected by a short wavelength UV
lamp. Bioinactive, UV-nonabsorbing materials are detected by a
permanganate-periodate spray reagent. Bioactive nucleotide materials
are detected by bioautography on agar seeded with S. lutea.
The following example shows the fermentation and purification
procedures for preparing the nucleotide of the compound designated as
U-57930E. The structural formula of U-57930E is shown in Chart 3. Ry
following the procedures of this example, or obvious equivalents
thereof, there can be made the 3-l~ibonucleotides of the other com-
pounds disclosed in Chart 2.
The following example is illustrative of the process and products
of the invention, but are not to be construed as limiting. All per-
centages are by weight and all solvent mixture proportions are by
volume unless otherwise noted.
Exam~le 1
A. Fermentation Procedure
Streptomyces rochei, NRRL 3533, is grown in a medium consisting
of glucose, 10 g/liter; Difco peptone, 4 g/liter; Difco yeast extract,
4 g/liter; MgS04.7H20, 0.5 g/liter, KH2P04, 2.0 g/liter, K2HP04, 4
gtliter for three days at 28C on a rotary shaker. The mycelium from
this growth is used to inoculate a fermentation medium containing the
same ingredients. The fermentation is carried out for 48 hours at
28C on a rotary shaker. At the end of this 48-hour incubation,
U-57930 is added to a final concentration of 50 mg/liter and the
fermentation continued at 32C. After twelve hours, additional
U-57930 is added to make the total concentration 150 mg/liter. After
30 twelve additional hours, the U-57930 concentration is increased to 25n
mg/liter. The fermentation is continued at 32 for 24 hours after the
last addition of U-57930. At this time the culture filtrates are
harvested and Found to contain no more than 1 mg/liter of U-5793
The remaining 249 mg/liter is converted to bioinactive material.
S. rochei, NRRL 3533, is a known microbe which is available to
the public upon request from the NRRL repository. The address of this
repository is as follows: Northern Utilization and Research Division,
~; Agricultural Research Service, U.S. Department of Agriculture, Peoria,
1 1 ~3938
~ 713/3841
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Ill., U.S.A.
B. Isolation and Purification Procedures
Isolation of U-57930 3-Ribonucleotides from Ferm_ntation ~roth
Adsor~tion on Amberlite XAD-2: Fermentation broth (ca 12 liter)
containing 3 g of nactivated U-57930 is filtered at harvest pH (7.7)
by using filter aid. The mycelial cake is washed with 1.2 liter of
water and discarded. The clear filt,ate and wash are combined and
adjusted to pH 6.0 and passed over a column prepared from 600 ml of
Amberlite* XAD-2 (Rohm and Haas Co., Philadelphia, PA), at a flow rate
of 40 ml/minute. The spent is tested for bioactivity before and after
treatment with alkaline phosphatase and is discarded. The column is
washed with 2 liters of water. The aqueous wash is also found bio-
inactive before and after treatment with alkaline phosphatase and is
discarded. The column is then eluted with methanol-water (70:30 v/v).
Fractions of 20 ml are collected at a rate of 20 ml/minute. Testing
for bioactivity before (-E) and after (~E) treatment with alkaline
phosphatase shows the following.
Zone (S. lutea)
Fraction No. -E +E
0 0
0 0
11 0 o
13 0 36
33 55
32 54
31 50
29 48
26 39
23 38
21 37
19 27
17 26
17 26
'` *trade mark
.,
1 ~ 63938
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Zone (S. lutea~
Fraction No. - +E
16 26
16 26
16 26
16 26
100 16 ` 26
110 16 21
120 15 21
130 15 21
140 15 21
150 15 21
160 15 21
17Q 15 21
180 15 21
190 15 21
200 15 21
Fractions 12-80 are combined, concentrated to an aqueous solution and
freeze-dried to give prep ADA-34.1, 12.22 g.
In another series of experiments, 6 liters of fermentation broth
containing 2 g of "inactivated" U-57930 is treated as described above~
The methanolic eluates from the Amberlite* XAD-2 column are kept as
ADA-143B. This solution is not concentrated to dryness; instead, it
is purified by Dowex*1 Ohromatography as described below.
r~ The column is prepared from 300 ml of
Dowex*1 (X-4) in the acetate form. The methanolic solution, ADA~143B
pH 8.2 is passed through the column. The spent is collected at a rate
of 2.5 ml/m~nute in 20 ml-fractions. (Fractlons 1-60). The column is
washed with 1.5 liter of water (10 ml/min9 fractions 66-108). The
column is then eluted with 5% acetic acid (rate, 10 ml/minute, ~rac-
tions 109-310). The following pools are made:
Pool 1 Fractions 1-80 1000 ml (ADA-lA)
2 Fractions 81-110 600 ml (ADA-2A)
3 Fractions 111-130 450 ml (ADA-3A)
4 Fractions 131-150 450 ml (ADA-4A)
Fractions 151-190 900 ml (ADA-5A)
6 Fractions 191-230 900 ml (ADA-6A)
7 Frac~ions 231-270 900 ml (ADA-7A)
8 Fractions 271-310 900 ml (ADA-8A)
Testing before (-E) and after (~E) ~reatment with alkaline phosphatase
shows the following:
*trade mark
: 'j
1 31 ~3~3~
3713/3841
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E Zone (S. lutea) F_
_
Pool 1 31 52
2 36 49
3 34 45
4 18 40
6 27 29
7 27 29
8 2g 31
Pools 1 and 2 are combined, concentrated to an aqueous solution
and freeze-dried to give prep ADA-2.1, 1.48 9.
Pools 3 and 4 are also combined and treated similarly to give
ADA-2.2, 2.5 9.
Preparations ADA-2.1 and -2.2 give U-57930 after treatment with
alkaline phosphatase.
Preparations ADA-34.1, -2.1 and -2.2 are combined and purified by
the counter double current distribution procedure described below.
Counter Double Current Distribution: The material obtained by
combination of preparations ADA-34.1, -2.1 and -2.2, 16.20 9, is
dissolved in 25 ml of each phase of the solvent system consisting of
equal volumes of 1-butanol-water (1:1), The solutions are added in
the center tubes of an all-glass counter double current distribution
apparatus (100 tubes, 25 ml/phase). The distribution is analyzed,
after 150 transfers, for bioactivity before (-E) and after (~E) treat-
ment with alkaline phosphatase. Results follow:
1 1 63~38
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-8 -
Zone (S. lutea-sensitive~
Lower Collector -E ~E
29 31
31 33
33
27 33
25- 22 33
17 34
0 34
0 33.5
0 33
o 3~
0 34
0 35
0 36
0 38
0 39
0 40
0 41
0 42
0 43
100 0 43.5
Zone (S. lutea-sensitive)
Lower Machine -E +E
0 45
trace 46
47
17 47.5
17 47
18 47
17.5 48
17 48
16 48
O trace 50
Upper Machine
trace 49
trace 48.5
trace 48
trace 48.5
trace 49
trace 51
52
16 53
21 54
..,
~ . .,
1 1 8 3 9 3 8 3713/3841
Zone (S. lutea-sensitive~
Upper Collector _ +
100 17.5 54
17 53.5
19 53.5
~'0 53.5
~'1 53.5
22 53.5
24 53.5
26 53.5
28 53 5
52 5
32.5 52
~3 52
52
36 52
39 49
41 47
43 48
43 46
43 43
The following pools are made. Each pool is concentrated to an
aqueous solution and freeze-dried to give the corresponding prepara
tions.
Pool I: Lower collector 1-50;
Pool II: Lower collector 51-100, lower machine 50-30;
Pool III: Lower machine 29-0; Upper machine 1-50j Upper
collector 100-30.
25Preparations obtained are:
From pool I, prep. ADA-47.1, 9.78 9
From pool ~I, prep. ADA-47.2, 0.30 9
From pool III, prep. ADA-47.3, 5029 9
Preparations ADA-47.2 and -47.3 are combined and purified by
DEAE-Sephadex chromatography as described below~
DEAE-Sephadex*Ohromatography: Three hundred g of DEAE-Sephadex~
~A-25) are stirred for 1 hour with water and for 2 hours with 0.5 N
~queous sodium hydroxide. The ionic exchanger is washed with water
until the pH is ca 7.5. The material is then stirred for 2 hours with
0.5 N aqueous acetic acid, washed with water to a neutral pH, and
poured into a column and packed under 2 lbs pressure to a constant
height. The column is washed with 4 liter of water, 8 liter of 0.1X
aqueous solution of tris-(hydroxymethyl)aminomethane (THAM), and 3
~3 *trade mark
1 1 63g3~
3713/3841
-10-
liter of 0.03 M THAM acetate buffer pH 8.0 (prepared by dissolving
3.64 g of THAM in 800 ml water, adjusting the pH to 8.0 with glacial
acetic acid and then adjusting the volume to 1 liter).
Starting material, preparations ADA-47.2 and 47.3 ca 5.50 9, is
5 dissolved in 20 ml of 0.03 M THAM acetate pH 8.0 buffer and added on
the top of the column. The column is then eluted downflow with 0.3 M
THAM acetate pH 8.0 buffer. Fractions 1-190 (20 ml) are collected.
At this point elution of the column is continued in an upflow manner.
Fractions A, B, C, D, and E (1 liter each) are collected. Testing for
bioactivity before (-E) and after l+E) treatment with alkaline phos-
phatase shows the following:
Zone (S~ lutea-sensitive)
Fraction No. -E +E
3 0 0
6 0 0
9 O O
12 0 o
0 0
18 0 0
21 oO o
27 0 l 0
0 0
33 0 0
36 38 39
39 43. 5 44
42 36 36
23.5 23
7 1 63938
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-11 -
Zone (S. lutea-sensitive)
Fraction No. -E +E
48 15 16
51 0 0
54 0 0
57 0 0
0 0
6~ 0 0
66 0 o
69 15 16
72 17 20
10 75 21 26
78 23 27
81 23 30
84 23 29
87 22 24
21 23
93 21 24
96 22 26
15 99 21 35
102 22 44
1~5 23 51
108 22.5 54
111 22.5 52.5
114 22 52
117 22 54.5
120 20.5 56
20 123 20 56
The following pools are made:
Pool I Fractions 34-38, 280 ml (ADA-69B)
Pool II Fractions 75-90, 330 ml (ADA-69C)
Pool III Fractions 101-111, 180 ml (ADA-69D)
Pool IV Fractions 114-150, 580 ml (ADA-69E)
Pool V Fractions 151-164, 100 ml (ADA-69F)
Pool VI Fractions 165-186, 125 ml (ADA-69G)
Pool VII Fraction C, 1 liter (ADA-69A)
Pool I (ADA-69B) contains unchanged U-57930 and is discarded.
Pool II (ADA-69C) contains an unknown material which yields
U-57930 by treatment with alkaline phosphatase. UV: ~ max 275 nm.
Pool IIT (ADA-69D) contains U-57930 cytidylate and is treated as
described later. UV: ~ max 270 nm.
Pool IV (ADA-69E) contains U-57930 adenylate and is treated as
described later. UV: ~ max 260 nm.
Pool V (ADA-69F) contains a mixture of U-57930 adenylate, U-57930
- uridylate and U-57930 guanylate. This solution is treated as des-
I ~ 63938 3/1~ 41
cribed later.
Pool VI (ADA-69G) contains U-57930 guanylate and is treated as
described later. UV: ~ max 254; sh at 275.
Pool VII (ADA-69A) contains a mixture of U-57930 guanylate and
U-57930 uridylate. This solution is treated as described later.
Isolation of Essentially Pure U-57930-Cytidylate, U-57930-
Adenylate and U-57930-Guanylate from Po~ls III, IV and VI, Respec-
tively. Removal of THAM Acetate_BuFfer by Amberlite XAD-2 Chroma-
tography: Pools III, IV and VI, obtained as described above, are
passed over columns containing Amberlite XAD-2. The spents are dis-
carded. The columns are washed with water and then eluted with
methanol-water (70:30 v/v). Fractions are analyzed by UV and by
testing for bioactivity before and after treatment with alkaline
phosphatase. Appropriate fractions are combined, concentrated to an
aqueous solution and freeze-dried. Details on the amount of Amberlite
XAD-2 used for each pool, the amount of water wash, the amount of
methanolic eluate and the amount of material obtained are listed in
the following table.
Amberlite XAD-2 Water Methanolic Isolated
20 PoolUsed (ml) Wash (ml) Fluate tml)_ Material (m~
III 50 200 300 150
IV 200 800 600 3510
VI - 50 200 300 470
The material obtained from pool III is kept as ADA-73.1; from
poo~ IV as ADA-74.1; and ~rom pool VI as ADA-75.1.
Removal of THAM Acetate Buffer from Pool V (ADA-69F) and Pool VII
(ADA-69A) by Amberlite*XAD-2 Chromato~raphy: The column is prepared
from 300 ml of Amberlite XAD-2. Pools V and VII containing a mixture
30 of U-57930 adenylate; U-57930-uridylate and U-57930 guanylate are
passed through the column. The spent is discarded. The column is
washed with 600 ml of water. The spent is discarded. The column is
eluted with methanol-water (70:30). Fractions yielding bioactive
material after treatment with alkaline phosphatase are combined, 300
ml, concentrated to an aqueous solution and freeze-dried to give prep
ADA-71.1, 670 mg. Prep -71.1 is treated as described below.
Separation of U-57930 Uridylate from U-5?930-Adenylate and U-
57930-Guanylate DEAE-Sephadex Chromatography. Six hundred ml of
, ~ ,,
*trade mark
1 1 6 3 9 3 8 3/~ L
-13-
DEAE-Sephadex in the acetate form, prepared as described earlier, are
washed with 0.03 M THAM acetate pH 8.0 buffer and packed into a glass
column (ID, 4.5 cm; height, 40 cm) under hydrostatic pressure.
Prep ADA-71.1 (see above) is dissolved in 10 ml of 0O03 M THAM
acetate pH 8.0 buffer and added on the top of the column. The column
is eluted with:
1) 0.03 M THAM acetate, pH 8.0 (Fractions 1-79)
2) 0.12 M THAM acetate, pH 8.0 (Fractions 80-395)
3) 0.25 M THAM acetate, pH 8.0 (Fractions 396-750)
Fractions of 20 ml are collectecl and analyzed by UV and by test-
ing for bioactivity before and after treatment with alkaline phospha-
tase. Fractions 51-60 contain U-57930 adenylate; fractions 62-73
(ADA-94.B) contain U-57930 uridylate; fractions 75-110 contain U-57930
guanylate).
Isolation of Essentially Pure U-57930 Uridylate. _Removal of
THAM-Acetate Buffer by Amberlite XAD-2 Chromatography. The column is
prepared from 50 ml of Amberlite XAD-2. Rool ADA-94~, containing
U-57930 uridylate, is passed over the column at a rate of 2 ml/minute.
The spent is discarded. The column is washed with 200 ml of water.
The wash is discarded. The column is eluted with methanol-water
(70:30 v/v). Fractions containing (by UV) U-57930 uridylate are
combined (200 ml), concentrated to an aqueous solution, and freeze-
dried to give ADA-95.1, 60 mg.
~` *trade mark
:
1 ~ ~3~3~
* 1
-14-
CHARACTERIZATION OF U-57~9~ Lll9
1. IR Tabulation
Tables listing the IR absorptions (Nujol and KBr) are as
follows:
Band Band
Freq. _Inten. Type __ Freq. Inten. Type _
3417.3 24 SH 1249.0 33 SH
3341.1 19 BRD 1214.3 21 AVG
3211.8 19 BRD 1146.8 42 SH
3108.6 25 BRD 1089.9 15 ~RD
2951.4 2 BRD M 1070.6 12 AVG
2326.3 1 BRD M 1056.1 14 SH
2854.9 2 BRD M 992.4 39 AVG
2729.6 48 BRD M 972.2 39 AVG
2693.9 51 SH 955.8 49 SH
2535.7 65 SH 930.7 51 AVG
1649.3 8 AVG 889.2 36 AVG
1610.7 26 AVG 860.3 52 AVG
1575.0 35 AVG 849.7 53 AVG
1528.7 31 AVG 804.4 46 SH
1489.2 23 AVG 788.9 40 AVG
1462.2 9 AVG M 721.4 44 AVG M
1404.3 41 BRD 705.0 47 BRD
1377.3 18 AVG M 654.9 45 SH
1368.6 31 SH M 632.7 38 AVG
1286.6 34 AVG
Band Freq.: Band Frequencies in Wavenumbers (cm-1
Inten.: Intensity in percent transmittance (YoT )
Data Type in Local Peak Region: BRD - Broad; AVG - Average; SHP
- Sharp; SH - Shoulder
Peak list edited. * Indicates peaks added.
M: Possible interference from mineral oil.
*trade mark
1 3 6.~93~
3713/3841
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25 Strongest Peaks
%T Freq. %T Freq.
1 2926.2 24 3417.2
2 2951.3 25 3108.5
2 2854.8 26 1610.6
8 1649.2 31 152~.6
9 1462.1 31 1368.5
12 1070.5 33 1249.0
14 1056.0 34 1286.5
1089.8 35 1575.0
18 1377.2 36 889.1
19 3341.0 38 632.6
19 3211.7 39 992.3
21 1214.2 39 972.1
23 1489.1
Prep: Mineral Oil Mull
Max %T: 87 @ 1848.0
%T at 3800 (cm-1): 83
Density (cm-1/pt): 0.964
Band Band
Freq. Inten. Type Freq. Inten. Type
3408.610 BRD 1088.9 12 BRD
3102.826 SH 1071.5 9 AVG
2963.928 AVG 1057.1 11 SH
2930.227 BRD 992.4 35 AVG
2878.137 AVG 972.2 36 AVG
2862.739 SH 956.8 45 SH
2768.151 SH 928.8 48 AVG
2511.665 BRD 889.2 32 AVG
1649.3 4 AVG 859.3 47 AVG
1614.620 SH 851.6 47 SH
1576.030 AVG 804.4 39 SH
1528.728 AVG 788.9 34 AVG
1491.121 AVG 743.6 47 SH
1462.233 AVG 705O0 40 AVG
1450.635 SH 654.9 39 SH
1404.337 AVG 634.6 35 AVG
1384.033 AVG 595.1 33 AVG
1360.942 SH 57209 33 AVG
1286.632 AVG 525.6 32 AVG
1251.930 SH 447.5 36 AVG
1215.218 AVG
Band Freq.: Band Frequencies in wavenumbers (cm-1
Inten.: Intensity in Percent Transmittance (Y~T)
1 3 63938
3713/3841
-16-
Data Type in Local Peak Region: BRD - Broad; AVG - Average; SHP
- Sharp; SH - Shoulder
Peak List Edited. *Indicates peaks added.
25 Strongest Peaks
%T Freq. %T Freq.
4 1649.2 30 1251.8
9 1071.5 32 1286.5
3408.5 32 889.1
11 1057.0 32 525.5
12 1088.8 33 1462.1
18 1215.1 33 1384.0
1614.5 33 595.0
21 1491.0 33 572.8
26 3102.7 34 788.8
27 2930.1 35 1450.5
28 2963.8 35 992.3
28 1528.6 35 634.5
1576.0
Prep: KBR Pellet
Max %T: 100 @ 403.1
%T at 4000 (cm-1): 78
Density (cm-llpt): 0.964
2. UY Absorption Spectrum [~ max (a~]
In water at:
pH 200, 279 nm (6.5)
pH 7.0, 270 nm (9.9)
pH 11.0, 271 (9.6)
3. Elemental Composition
Mol. formula: C26H43NsO~2 SClP. Molecular Weight, 715.
Calcd: C, 43.64; H, 6.01; N, 9.79; O, 26.88; S, 4.47; Cl,
4.89, P, 4.33.
4. Optical Rotation
~]D2s, +107 (C, 0.854, water)
5. Solubilities
Highly soluble in water, methanol and ethanol. Slightly
soluble in acetone and other ketones, ethyl acetate and other
esters, chloroform, methylene chloride. Insoluble in
saturated hydrocarbon solvents.
`' .:
~ 1 6 ~ 9 3 8
-17-
6. Antibacterial Activit~
U-57930 3-(5'-cytidylate) is not active in vitro. However,
treatment with alkaline phosphatase or phosphodiesterase I
yields U-57930 which is highly active against a variety of G+
organisms, both in vitro and in vivo.
7. Melting point: 205-207 (w~th decomposition).
CHARACT~RIZATION OF U-57930 3'(5'-ADENYLATE)
1. IR Tabulation
Tables listing the IR absorptions (Nujol and KBr) are as follows:
Band Band
Freq. lnten. Type Freq. Inten. _ T~pe_
3335.316 BRD 1245.1 24 SH
3267.817 BRD 1213.3 17 AVG
3210.817 BRD 1175.7 43 SH
2954.3 3 BRD M 1146.8 40 SH
2924.4 2 BRD M 1089.9 13 AVG
2868.4 6 SH M 1069.6 10 AVG
2854.9 4 AVG M 1055.1 13 SH
2727.646 BRD M 991.5 35 AVG
2520.261 BRD 972.2 36 AVG
1684.022 SH 957.7 45 SH
1641.614 AVG 930.7 48 AVG
1600.128 AVG 889.2 32 AVG
1576.031 AVG 861.3 47 AVG
1550.943 SH 848.7 49 AVG
1509.453 SH 818.8 45 AVG
1463.115 AVG M 798.6 40 AVG
2 1420.735 AVG 722.4 36 AVG M
1377.324 AVG M 708.9 40 SH
1367.635 SH M 647.1 33 SH
1332.036 AVG 635.6 31 AVG
1299.234 AVG
Band Freq.: Band Frequencies in Wavenumbers (cm-l)
Inten. Intensity in percent transmittance (ZT)
Data Type in Local Peak Region: BRD Broad; AVG - Average; SHP
- Sharp; SH - Shoulder
Peak list edited. *Indicates peaks added.
M: Possible interference from mineral oil.
L~ * trade mark
~ ~ ~3938
3713/3841
-18-
25 Strongest Peaks
%T _ Freq. %T Freq.
2 2924.3 22 1684.0
3 2954.2 24 1377.2
~ 2854.8 24 1245.0
6 2868.3 28 1600.0
1069.5 31 1576.0
13 1089.8 31 635.5
13 1055.0 32 889.1
14 1641.5 33 647.0
1463.0 34 1299.1
16 3335.2 35 1420.6
17 3267.7 35 1367.5
17 3210.7 35 991.5
17 1213.2
Prep: Mineral Oil Mull
Max %T: 85 @ 1864.4
%T at 3800 (cm-1): 81
Density (cm-1/pt): 0.964
Band Band
Freq. Inten. Type Freq.Inten. Type
3375.8 7 BRD 1090.8 8 AVG
3223.410 BRD 1069.6 5 AVG
3124.017 SH 1050.3 8 SH
2963.022 AVG 990.5 27 AVG
2929.222 BRD 972.2 29 AVG
2878.131 AVG 956.8 38 SH
2863.633 SH 929.8 42 AVG
2756.645 BRD 889.2 24 AVG
2521.260 BRD 861.3 38 AVG
2188.575 BRD 851.6 40 SH
1678.215 SH 818.8 36 AVG
1643.5 7 AVG 807.3 36 BRD
1602.020 AVG 798.6 30 AVG
1576.023 AVG 768.7 41 BRD
1553.835 SH 721.4 30 AVG
1511.449 SH 706.9 31 BRD
1475.727 AVG 648.1 26 AVG
1421.729 AVG 636.5 26 AVG
1384.032 AVG 584.5 28 SH
1332.031 AVG 571.9 26 AVG
1301.128 AVG 533.3 27 SH
1246.119 SH 522.7 25 AVG
1215.212 AVG 503.4 25 AVG
1176.736 SH
.
~ .
~ ~ ~393~ 37l3/384l
-19-
Band Freq.: Band Frequencies in Wavenumbers (cm-1)
Inten.: Intensity in percent transmittance (~T)
Data Type in Local Peak Region: BRD - Broad; AVG - Average; SHP
- Sharp, SH - Shoulder
Peak list edited. *Indicates peaks added.
25 Strongest Peaks
%T Freq~ ~oT _Freq.
1069.5 23 1576.0
7 3375.7 24 889.1
7 1643.5 25 522.6
8 1090.7 25 503.3
8 1050.2 26 648.0
3223.3 26 636.5
12 1215.1 26 571.8
1678.1 27 1475.6
17 3124.0 27 990.5
19 1246.0 27 533.2
1602.0 28 1301.0
22 2963.0 28 584.5
22 2929.1
20Prep: KBR Pellet
Max %T: 95 @ 405.0
%T at 4000 (cm-1): 77
Density (cm-1/pt): 0.964
2. UV Absorption Spectrum [~ max (a)~
In water at:
pH 2.0, 258 (16.0)
pH 7.0, 261 (16.5~
pH 11.0, 261 (16.0
3. Elemental Composition
Molecular formula: C27H~3N701o SClP. Molecular Weight, 723.
Calcd C, 44.81; H, 5.94; N, 13.55; O, 22.13; S, 4.42; Cl,
4.84; P, 4.28. Found N, 12.87; S, 5.39; Cl, 4.76; P, 3.83.
4. Optical Rotation
[~]D2s~ +94 (C, 0.887, water).
5~ Solubilities
Highly soluble in water, methanol and ethanol. Slightly
~ ~ ~3938
371
-20-
soluble in acetone and other ketones, ethyl acetate and other
esters, chloroform and methylene chloride. Insoluble in
saturated hydrocarbon solvents.
6. Antibacterial Activit~
U-57930 [3-(5'-adenylate)~ is not active in vitro. However,
treatment with alkaline phosphatase or phosphodiester I
yields U-57930, which is highly active against a variety of
G+ organisms b~th in vitro and in vivo. U-57930 3-(5'-aden-
ylate) was found active in vivo (subcutaneously, mice) ~ith a
CDso of 0.62 (0.48-0.79) mg/kg. against S. pyogenes.
7. Melting Point: 203.5-205 (with decomposition)
CHARACTERIZATION OF U-57930 3-(5'-URIDYLATE)
1. IR Tabulation
Tables listing the IR absorptions (Nujol and K~r) are as
follows:
Band Band
Freq. Inten. Type _ Freq. Inten. Type _
3330.4 19 BRD 1332.9 44 BRD
3224.4 21 BRD 1296.3 40 SH
2952.4 1 BRD M 1251.9 28 RRD
2924.4 0 BRD M 1215.2 19 AVG
2867.5 4 SH M 1089.9 13 AVG
2854.0 3 AVG M 1071.5 9 AVG
2733.4 49 SH M 1056.1 13 SH
2695.B 53 SH 991.5 39 AVG
2532.8 67 SH 973.2 39 AVG
1757.3 73 SH 957.7 48 SH
1685.9 8 AVG 931.7 49 AVG
1647.4 21 SH 890.2 34 AVG
1602.0 41 AVG 858.4 50 AVG
1574.1 42 AVG 813.0 43 AVG
1555.7 43 BRD 798.6 47 AVG
146202 12 AVG M 767.7 SO AVG
1425.5 37 SH 721.4 42 AVG M
1378.3 22 AVG M 634.6 35 AVG
1367.6 37 SH M
Band Freq.: Band Frequencies in Wavenumbers (cm-1)
Inten.: Intensity in percent ~ransmittance (%T)
Data Type in Local Peak Region: BRD - Broad, AVG - Average; SHP
- Sharp, SH - Shoulder
This peak list is unedited.
F~
i!~i,,, ~ j~
il~ * trade mark
- 1 ~ 6~938
3713/3841
-21-
M: Possible interference from mineral oil.
25 Stron~est Peaks
%T Fre~1~ %T Freq.
0 2924.3 22 1378.2
1 295203 28 1251.8
3 2854.0 34 890.1
4 2~67.5 35 634.5
8 1685.8 37 1425.5
9 1071.5 37 1367.5
12 1462.1 39 991.5
13 1089.8 39 973.1
13 1056.0 40 1296.2
19 3330.3 41 1602.0
19 1215~1 42 1574.0
21 322403 42 721.3
21 1647.3
Prep: Mineral Oil Mull
Max %T: 86 ~ 3764.5
%T at 3800 (cm-1): 85
Density (cm-1/pt): 0.964
Band Band
Freq. Inten. Type Freg.Inten. Ty~e
3387.412 BRD 1055.1 10 SH
3114.425 SH 992.4 35 AVG
2962.026 AVG 973.2 36 AVG
2931.126 BRD 956.8 45 SH
2879.136 AVG 929.8 48 AVG
2863.638 SH 889.2 31 AVG
2833.743 SH 859.3 46 AVG
2509.664 BRD 813.0 40 AVG
1685.0 6 BRD 811.1 40 SH
1647.417 SH 798.6 43 AVG
1605.935 SH 782.2 48 RRD
1576.037 AVG 768.7 48 AVG
1556.739 BRD 707.9 43 AVG
1463.131 AVG 669.3 43 SH
1423.635 AVG 649.1 40 SH
1384.032 AVG 634.6 37 AVG
1331.043 AVG 585.4 38 SH
1297.238 SH 567.1 34 AVG
1255.824 AVG 523.7 35 AVG
1214.317 AVG 447.5 39 AVG
1090.810 AVG
1070.6 7 AVG
.,
Band Freq.: Band Frequencies in Wavenumbers (cm-1)
~ 1 B~938
3713/3841
-22-
Inten.: Intensity in percent transmittance (q~T)
Data Type in Local Peak Region: BRD - Broad; AVG - Average; SHP
- Sharp; SH - Shoulder
Peak List Edited. *Indicates peaks added.
25 Stronge.st Peaks
%T _ _ Freq. %T Freq.
6 1685.0 32 1384.0
7 1070.5 34 567.0
1090.7 35 1605.8
1055.0 35 1423.5
12 3387.3 35 992.3
17 1647.3 35 523.6
17 1214.2 36 2879.0
24 1255.7 36 973.1
3114~3 37 1576.0
26 2962.0 37 634.5
26 2931.0 38 2863.5
31 1463.0 38 1297.1
31 &89.1
Prep: K~R Pellet
Max %T: 101 @ 405.0
%T at 4000 (cm-1): 76
Density (cm-1/pt): 0.964
2. UV Absorption Spectrum [~max (a~
In water at:
pH 2.0, 261 (11.5)
pH 7.0, 262 (10.7)
pH 11.0, 262 (11.5)
3. Elemental Composition
Molecular formula: C26H42N4013 SClP. Molecular Weight, 716.
Calcd C, 43.57; H, 5.86; N, 7.82; O, 29.05; S, 4.46; Cl,
4.89; P, 4.33.
4. Op~ical Rotation
~]D25 +105~ (C, 0.94, water)
5. Solubilities
Highly soluble in water, methanol and ethanol. Slightly
soluble in acetone and other ketones, ethyl acetate and other
--~ esters, chloroform and methylene chloride. Insoluble in
~ ~ 6~938 3713/3841
-23-
saturated hydrocarbon solvents.
6. Antibacterial Activity
U-57930 3-(5'-uridylate) is not active in vitro. However,
treatment with alkaline phosphatase or phosphodiesterase I
yields U-57930 which is highly active against a variety of G
organisms both in vitro and in vivo.
7. Melting Point: 202-203 (with decomposition)
CHARACTERIZATION OF U-57930 3-(5'-GUANYLATE)
1. Tables l1sting the IR absorptions (Nujol and KBr) are as
follows:
Band Band
Freq. Inten. Type Freq. Inten. Type
3335.316 BRD 1250.0 36 SH
3227.219 BRD 1213.3 21 AVG
2953.3 2 AVG M 1173.8 39 AVG
2925.3 1 BRD M 1149.7 41 AVG
2 2868.4 6 SH M 1087.9 14 S~
2855.9 4 AVG M 1071.5 9 AVG
2737.351 BRD M 991.5 42 AVG
2521.273 BRD 972.2 42 AVG
1684.0 6 AVG 956.8 52 SH
1635.811 AVG 929.8 51 AVG
- 1598.221 AVG 890.2 36 AVG
1572.126 AVG 860.3 51 AVG
1534.534 AVG 800.5 46 AVG
1462.218 AVG M 783.1 42 SHP
1414.944 AVG 720.4 43 AVG M
1377.324 AVG M 707.9 45 BRD
1365.731 AVG 681.9 4n AVG
1312.744 AVG 635.6 34 AVG
Band Freq.: Band Frequencies in Wavenumbers (cm-1)
Inten.: Intensity in Percent Transmittance (%T~
Data Type in Local Peak Region: BRD - Broad; AVG - Average; SHP
- Sharp; SH - Shoulder
Peak List Edited. *Indicates Peaks Added.
M: Possible interference from Mineral Oil
*trade mark
::: . .J
1 ~ 6 3 9 3 ~ 3713/3841
-24-
25 Strongest Peaks
%T Freq. %T _req.
1 2925.2 24 1377.~
2 2953.2 26 1572.0
4 2855.8 31 1365.6
6 2868.3 34 1534.5
6 1684.0 34 635.5
9 1071.5 36 1250.0
11 1635.7 36 890.1
14 1087.8 39 1173.7
16 3335.2 40 681.8
18 1462.1 41 1149.6
19 3227.1 42 991.5
21 1598.1 42 972.1
21 1213.2
Prep: Mineral Oil Mull
Max %T: 97 @ 3762.6
%T at 3800~cm-1): 97
Density (cm-1/pt): 0.964
Band Band
Freq. Inten. Type Freq. Inten. Type
3380.6 9 BRD 1174.7 31 AVG
3234.0 13 BRD 1147.7 33 BRD
2963.0 22 AVG 1088.9 9 BRD
2929.2 21 BRD 1070.6 6 AVG
2878.1 31 AVG 991.5 33 AVG
2862.7 33 SH 972.2 34 AVG
2744.0 44 BRD 956.8 43 SH
2522.2 61 BRD 929.8 44 AVG
1683.0 4 BRD 889.2 28 AVG
1634.8 6 AVG 860.3 41 AVG
1598.2 14 AVG 800.5 36 AVG
1571.2 19 AVG 783.1 32 AVG
1534.5 26 AVG 715.6 38 SH
1482.4 38 AVG 705.0 38 SH
1461.2 36 AVG 679.9 33 AVG
1448.7 36 BRD 635.6 31 AVG
1413.9 34 AVG 584.5 34 SH
1384.0 29 AVG 571.9 32 AVG
1359.9 30 AVG 523.7 31 AVG
1312.7 37 AVG 502.5 30 AVG
1250.9 29 SH 447.5 34 AVG
1213.3 16 AVG
~, Band Freq.: Band Frequencies in Wavenumbers (cm-1)
..~
1 ~ ~393~
3713/3841
-25-
Inten.: Intensity in Percent Transmittance (%T)
Data Type in Local Peak Region: BRD - Broad; AVG - Average; SHP
- Sharp, SH - Shoulder
Peak List Edited. *Indicates Peaks Added.
25 Strongest Peaks
%T Freq. %T Freq.
4 1683.0 29 1384.0
6 1634.7 29 1250.8
6 1070.5 30 1359.8
9 3380.5 30 502.5
9 lQ88.8 31 2878.0
13 3234.0 31 1174.6
14 1598.1 31 635.5
16 1213.2 31 523.6
19 1571.1 32 783.0
21 2929.1 32 571.8
22 2963.0 33 2862.6
26 1534.5 33 1147.6
28 889.1
Prep: KBR Pellet
Max %T: 97 @ 405.0
%T at 4000(cm-1): 77
Density (cm-1/pt): 0.964
2. UV Absorption Spectrum ~Amax (a)]
In water at:
pH 2.0, 256 (13.4); 280 (8.4) sh
pH 7.0, 254 (14.5~; 273 (9.7) sh
pH 11.0, 259 (12.6); 266 (12.4) sh
3. Elemental Composition
Molecular formula: C27H43N701l SClP. Molecular Weight 739.
Calcd C, 43.84; H, 5.81; N, 13.26; O, 23.27; S, 4.33; Cl,
4.73; P, 4.19. Found N, 13.32; S, 4.86; Cl, 4.49; P, 3.25.
4. Optical Rotation
[~D25~ +97 (C, 0.855, water)
5. Solubilities
Highly soluble in water, methanol and ethanol. Slightly
soluble in acetone and other ketones, ethyl acetate and other
esters, chloroform and methylene chloride. Insoluble in
~ 3 ~3938
3713/3841
-26-
saturated hydrocarbon solvents.
6. Antibacterial Activit~
U-57930 3-(5'-guanylate) is not active ln vitro. However,
treatment with alkaline phosphatase or phosphodiesterase I
yields U-57930 which is highly active against a variety of G
organisms both in vitro and in vivo.
7. Melting Point: 219-220 (with decomposition)
Since the compounds of the subject invention are active against
various Gram-positive and Gram-negative microbes, they can be used in
various environments to inh;bit such microbes. For example, they can
be used as disinfectants to inhibit S. aureus on washed and stacked
food utensils contaminated with this bacterium. They also can be used
as disinfectants on various dental and medical equipment contaminated
with S. aureus. Further, the compounds of the invention can be used
as bacteriostatic rinses for laundered clothes, and for impregnating
papers and fabrics; and, they are also useful for suppressing the
growth of sensitive organisms in plate assays and other microbio-
logical media.
The compounds of the present invention are also useful in the
treatment of diseases caused by members of the genus Mycoplasma, the
most commonly known forms are PPL0 (pleuropneumonia-like organisms)
such as M. hominis, M. salivarium, M. mycoides, M. hyopneumonia, M.
hyorhinis, M. gallisepticum, M. arthriditis and other species in man
and animals, including domestic animals such as sheep, dogs, cattle,
swine, and poultry (e.g., chickens, turkeys9 ducks, and geese) and
laboratory animals (e.g., rats and mice).
The U-57930 3-(5'-ribonucleotides) can be used in the treatment
of kidney and other infections when L forms of gram-negative and gram-
positive bacteria are present, for example, L forms of P. mirabilis.
Since the compounds of the subject invention are amphotericsubstances, they can form salts with both acids and bases by using
standard procedures. Examples of inorganic acids which can be used to
form salts are hydrochloric, sulfuric, phosphoric, and the like.
Examples of inorganic bases are sodium, potassium, calcium, lithium,
and the like. Salts of the compounds can be used for the same pur-
poses as the parent compounds.
The compounds of the subject invention are useful as antibac-
1 ~ 6393~3
3713/3841-27-
terial agents in suitable compositions. These compositions arepreferably presented for administration to humans and animals in unit
dosage forms, such as tablets, capsules, pills, powders, granules,
sterile parenteral solutions or suspensions, and oral solutions or
suspensions, and oil-water emulsions containing suitable quantities of
the active compound in the form of the free base, or its pharmacolog-
ically acceptable salts.
For oral administration, either solid or fluid unit dosage forms
can be prepared. For preparing solid compositions such as tablets,
the principal active ingredient is mixed with conventional ingredients
such as talc, magnesium stearate, dicalcium phosphate, magnesium
aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl-
cellulose, and functionally similar materials as pharmaceutical dilu-
ents or carriers. The tablets can be laminated or otherwise com-
pounded to provide a dosage form affording the advantage of prolongedor delayed action or predetermined successive action of the enclosed
medication. For example, the tablet can comprise an inner dosage and
an outer dosage component, the latter being in the form of an envelope
over the former. The two components can be separated by an enteric
layer which serves to resist disintegration in the stomach and permits
the inner component to pass intact into the duodenum or to be delayed
in release. A variety of materials can be used for such enteric
layers or coatings, such materials including a number of polymeric
acids or mixture of polymeric acids with such materials as shellac,
cetyl alcohol, cellulose acetate phthalate, styrene maleic acid co-
polymer and the like. Alternatively, the two component system can be
utilized for preparing tablets containing two or more incompatible
active ingredients. Wafers are prepared in the same manner as tab-
lets, differing only in shape and the inclusion of sucrose or other
sweetener and flavor. In their simplest embodiment, capsules, like
tablets, are prepared by mixing the compound of the formulation with
an inert pharmaceutical diluent and filling the mixture into a hard
gelatin capsule of appropriate size. In another embodiment, capsules
are prepared by filling hard gelatin capsules with polymeric acid
coated beads containing a compound of the formulas. Soft gelatin
capsules are prepared by machine encapsulation of a slurry of a com-
pound of the formulas with an acceptable vegetable oil, light liquid
petrolatum or other inert oil.
l ~ ~ 3 g 3 8 3713/3841
-28-
Fluid unit dosage forms for oral administration such as syrups,elixirs, and suspensions can be prepared. The water-soluble forms of
a compound of the formulas can be dissolved in an~aqueous vehicle
together with sugar, aromatic flavoring agents and preservatives to
form a syrup. An elixir is prepared by using a hydroalcoholic (ethan-
ol) vehicle with suitable sweeteners such as sucrose together with an
aromatic flavoring agent. Suspensions can be prepared of the insol-
uble forms with a syrup vehicle with the aid of a suspending agent
such as acacia, tragacanth, methylcellulose, and the like.
Topical ointments can be prepared by dispersing the active com-
pound in a suitable ointment base such as petrolatum, lanolin, poly-
ethylene glycols, mixtures thereof, and the like. Advantageously, the
compound is finely divided by means of a colloid mill utilizing light
liquid petrolatum as a levigating agent prior to dispersing in the
ointment base. Topical creams and lotions are prepared by dispersing
the compound in the oil phase prior to the emulsification of the oil
phase in water.
For parenteral administration, fluid unit dosage forms are pre-
pared utilizing a compound of the formulas and a sterile vehicle,
water being preferred. The compound, depending on the form and con-
centration used, can be either suspended or dissolved in the vehicle.
In preparing solutions, a water-soluble form of a compound of the
formulas can be dissolved in water for injection and filter sterilized
before filling into a suitable vial or ampul and sealing. Advantage-
ously, adjuvants such as a local anesthetic, preservative and buffer-
ing agents can be dissolved in the vehicle. To enhance the stability,
the composition can be frozen after filling into the vial and the
water removed under vacuum. The dry lyophilized powder is then sealed
in the vial and an accompanying vial of water for injection is sup-
plied to reconstitute the powder prior to use. Parenteral suspensionsare prepared in substantially the same manner except that the compound
is suspended in the vehicle instead of being dissolved and steriliza-
tion cannot be accomplished by filtration. The compound can be ster-
ilized by exposure to ethylene oxide before suspending in the sterile
vehicle. For sustained action, an intramuscular suspension is pre-
pared with an insoluble form such as the trimethylsilyl ether or the
pamoate salt. Advantageously, a surfactant or wetting agent is in-
cluded in the composition to facilitate uniform distribution of the
1 1 63938 3713/3~41
-29-
compound.
The term unit dosage form as used in the specification and claimsrefers to physically discrete units suitable as unitary dosages for
human subjects and animals~ each unit containing a predetermined
quantity of active material calculated to produce the desired thera-
peutic effect in association w;th the required pharmaceutical diluent,
carrier or vehicle. The specifications for the novel unit dosage
forms o~ this invention are dictated by and directly dependent on (a)
the unique characteristics of the active material and the particular
therapeutic effect to be achieved, and (b) the limitations inherent in
the art of compounding such an active material for therapeutic use in
humans and animals, as disclosed in detail in this specification,
these being features of the present invention. Examples of suitable
unit dosage forms in accord with this invention are tablets, capsules,
pills, troches, suppositories, powder packets, granules, wafers,
cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampuls, vials,
segregated multiples of any of the foregoing, and other forms as
herein described.
An active compound is compounded with a suitable pharmaceutical
carrier in unit dosage form for convenient and effective administra-
tion. In the preferred embodiments of this invention, the dosage
units contain 10, 25, 50, 100, 250, and 500 mg amounts of a compound
of the formulas for systemic treatment~ 5 to 65 percent w/v for
parenteral treatment. The dosage of compositions containing an active
compound and one or more other active ingredients is to be determined
with reference to the usual dosage of each such ingredient.
The following examples are illustrative of the best mode contem-
plated by the inventor for carrying out his invention and are not to
be construed as limiting.
The examples use the 3-(5'-ribonucleotide) of u-57~93nE or
U-60,970E as the active compound, but it should be understood that
this is only exemplary of the other active compounds of the subject
invention. U-60,970E is the 4-cis-n-butyl-L-pipecolic acid a~ide of
7-Cl-methylthiolincosaminide. Its preparation 1s shown in Example 7
of U.S. patent 4,27~,789,
Reference hereinafter to U-57,930E or U-60~970E means the 3-~5'-
ribonucleotide) of these compounds. The 3-ribonucleotides are those
as disclosed herein.
~ 1 ~393~
3713/3841
-30-
Composition Example 1 - Capsules
One thousand two-piece hard gelatin capsules for oral use, each
containing 250 mg of U-57,930E or U-60,970E, are prepared from the
following types and amounts of materials:
U-57,930E or U-60,970E 250 gm
Corn starch 100 gm
Talc 75 gm
Magnesium stearate 25 gm
The materials are thoroughly mixed and then encapsulated in the
usual manner.
The foregoing capsules are useful for the systemic treatment of
infection in adult humans by oral administration of one capsule every
6 hours.
Using the procedure above, capsules are similarly prepared con-
taining U-57,930E or U-60,970E in 10, 25, 50, 100, and 500 mg amounts
by substituting 10, 25, 50, 100 and 500 gm of U-57,930E or U-6n,97nE
for the 250 gm used above.
Composition Example 2 - Capsules
One thousand two-piece hard gelatin capsules for oral use, each
containing 200 mg of U-57,930E or U-60,970E and 250 mg of tetracy-
cline hydrochloride, are prepared from the following types and amounts
of ingredients:
U-57,930E or U-60,970E 200 gm
Tetracycline hydrochloride250 gm
Talc 75 gm
Magnesium stearate 25 gm
The ingredients are thoroughly mixed and then encapsulated in the
usual manner.
The foregoing capsules are useful for the systemic treatment of
infection in adult humans by the oral administration of one capsule
every 6 hours.
Using the procedure above, capsules are similarly prepared con-
taining U-57,930E or U-60,970E and each of the following antibiotics
in place of tetracycline by substituting 250 gm of such other anti-
biotic for tetracycline: chloramphenicol, oxytetracy~line, chlor-
tetracycline, fumagillin, erythromycin, streptomycin, dihydronovo-
biocin and novobiocin. When a penicillin, such as potassium peni-
cillin G, is to be used in place of tetracycline, 25n,0no units per
i 1 63~38
3713/3841
-31-
capsule is employed.
Such combination products are useful for the systemic treatmentof mixed infections in adult humans by the oral administration of one
capsule every 6 hours.
Composition Example 3 - Tablets
One thousand tablets for oral use, each containing 500 mg of
U-57,930E or U-60,970E, are prepared from the following types and
amounts of materials:
U-57,930E or U-60,970E 500 gm
Lactose 125 gm
Corn starch 65 gm
Magnesium stearate 25 gm
Light liquid petrolatum 3 gm
The ingredients are thoroughly mixed and slugged. The slugs are
broken down by forcing through a number 16 screen. The resulting
granules are then compressed into tablets, each tablet containing 50n
mg of U-57,930E or U-60,970E.
The foregoing tablets are useful for systemic treatment of infec-
tions, including malarial infections, in adult humans by oral admini-
stration of one tablet three times a day.
Using the above procedure, except for reducing the amount of
U-57,930E or U-60,970E to 250 gm, tablets containing 250 mg of
U-57,930E or U-603970E are prepared.
Composition Example 4 - Tablets
One thousand oral tablets, each containing 25n mg of U-57,930E or
U-60,970E and total of 250 mg (83.3 mg each) of sulfadiazine, sulfa-
merazine, and sulfamethazine, are prepared from the following types
and amounts of materials:
U-57,930E or U-60,970E 250 gm
Sulfadiazine 83.3 gm
Sulfamerazine 83.3 gm
Sulfamethazine 83.3 gm
Lactose 50 gm
Corn starch 50 gm
Calcium stearate 25 gm
Light liquid petrolatum 5 gm
The ingredients are thoroughly mixed and slugged. The slugs are
broken down by forcing through a number sixteen screen. The resulting
~ :3 ~393~
3713/3841
-32-
granules are then compressed into tablets, each containing 250 mg ofU-57,930E or U-60,970E and a total of 250 mg (83.3 mg each) of sulfa-
diazine, sulfamerazine, and sulfamethazine.
The foregoing tablets are useful for systemic treatment of infec-
tions by the oral administration of four tablets first and then oneevery six hours.
For the treatment of urinary infections, the triple sulfas in the
above formulation is advantageously replaced by 250 gm of sulfamethyl-
thiadiazole or 250 gm of sulfacetamide.
Composition Example 5 - Oral Syrup
One thousand cc of an aqueous suspension for oral use, containing
in each 5 cc dose 250 mg of U-57,930E or U-60,970E and 500 mg of total
sulfas is prepared from the following types and amounts of ingredi-
ents:
U-57,930E or U-60,970E 50 gm
Sulfadiazine 33.3 gm
Sulfamerazine 33.3 gm
Sulfamethazine 33.3 gm
Citric acid 2 gm
Benzoic acid 1 gm
Sucrose 700 gm
Tragacanth 5 gm
Lemon oil 2 cc
Deionized water, q.s. 1000 cc
The citric aid, benzoic acid, sucrose, tragacanthf and lemon oil
are dispersed in sufficient water to make 850 cc of solution. The
U-57,930E or U-60,970E and finely divided sulfas are stirred into the
syrup until uniformly distributed. Sufficient water is added to make
1000 cc.
The composition so prepared is useful in the systemic treatment
of pneumonia in adult humans at a dose of 1 tablespoonful (10 cc) 4
times a day.
Composition Example 6 - Parenteral Solution
A sterile aqueous solution for intramuscular use, containing 20n
35 mg of U-57,930E or U-60,970E in 1 cc is prepared from the following
types and amounts of materials:
U-57~930E or U-60,970E 200 gm
- Lidocaine hydrochloride 4 gm
3 ~
3713/3841
-33-
Methylparaben 2.5 gm
Propylparaben 0.17 gm
Water for injection, q.s.19000 cc
The ingredients are dissolved in the water and the solution
sterilized by filtration. The sterile solution is filled into vials
and the vials sealed.
Composition Example 7 - Parenteral Preparation
A sterile aqueous composition for intramuscular use, containing
in 1 cc 200 mg of U-57,930E or U-60,970E and ~00 mg of spectinomycin
sulfate is prepared from the following types and amounts of ingre-
dients:
U-57,930E or U-60,970E 200 gm
Spectinomycin sulfate 400 gm
Lactose 50 gm
Water for injection, q.s.1,000 cc
The U-57,930E or U-60,970E, spectinomycin sulfate, and lactose
are dispersed in the water and sterilized. The sterile composition,
in the amount of 2 cc, is filled aseptically into sterile vials.
Composition Example 8 - Top cal Ointment
One thousand gm of 0.25% ointment is prepared from the following
types and amounts of ingredients:
U-57,930E or U-60,970E 2.5 gm
Zinc oxide 50 gm
Calamine 50 gm
Liquid petrolatum (heavy)250 gm
Wool fat 200 gm
White petrolatum, q.s. 1,000 gm
The white petrolatum and wool fat are melted and lOn gm of liquid
petrolatum added thereto. The U-57,93nE or U-60,970E, zinc oxide and
calamine are added to the remaining liquid petrolatum and the mixture
milled until the powders are finely divided and uniformly dispersed.
The powder mixture is stirred into the white petrolatum mixture and
stirring continued until the ointment congeals.
The foregoing ointment is usefully applied topically to the skin
of mammals for the treatment of infection.
The foregoing composition can be prepared by omitting the zinc
oxide and calamine.
Following the procedure above, ointments are similarly prepared
.
3938
3713/3841
-34-
containing U-57,930E or U-60,970E in 0.5, l, 2, and 57~ amounts by
substituting 5, 10, 20 and 50 gm of U-57,930E or U-60,970E for the 2.5
gm used above.
Composition Example 9 - Cream
One thousand gm of a vaginal cream are prepared from the follow-
ing types and amounts of ingredients:
U-57,930E or U-60,970E 50 gm
Tegacid Regularl 150 gm
Spermaceti 100 gm
Propylene glycol 50 gm
Polysorbate 80 5 gm
Methylparaben 1 gm
Deionized water, q.s. 1,000 gm
1Self-emulsifying glyceryl monostearate from Goldschmidt Chemical
Corporation, New York, N.Y.
The Tegacid and spermaceti are melted together at a temperature
of 70-80C. The methylparaben is dissolved in about 500 gm of water
and the propylene glycol, Polysorbate ~30, and U-57,930E or U-60,970E
are added in turn, maintaining a temperature of 75-80C. The methyl-
paraben mixture is added slowly to the -Tegacid and spermaceti melt,
with constant stirring. The addition is continued for at least 30
minutes with continued stirring until the temperature has dropped to
40-45C. The pH of the final cream is adjusted to 3.5 by incorpor-
ating 2.5 gm of citric acid and 0.2 g of dibasic sodium phosphate
dissolved in about 50 gm of water. Finally, sufficient water is added
to bring the final weight to 1,000 gm and the preparation stirred to
maintain homogeneity until cooled and congealed.
The foregoing composition is useful for the treatment of vaginal
infections in humans.
Composition Example 10 - Ointment, Ophthalmic
One thousand gm of an ophthalmic ointment containing 0.5~ U-
57,930E or U-60,970E are prepared from the following types and amounts
of ingredients:
U-57,930E or U-60,97nE 5 gm
Bacitracin 12~2 gm
Polymyxin B sulfate (10,000
units/mg 1 gm
Light liquid petrolatum250 gm
~ ~ ~3g3~
3713/3841
-35-
Wool fat 200 gm
White petrolatum, q.s. 1,000 gm
The solid ingredients are finely divided by means of an airmicronizer and added to the ligh liquid petrolatum. The mixture is
passed through a colloid mill to uniformly distribute the micronized
particles. The wool fat and white petrolatum are melted together,
strained, and the temperature adjusted to 45-50C. The liquid petro-
latum slurry is added and the ointment stirred until congealed.
Suitably the ointment is packaged in one dram ophthalmic tubes.
The foregoing ointment is usefully applied to the eye for treat-
ment of localized infection in humans and other animals.
Advantageously the foregoing composition can contain 5 gm (0.5~0)
of methylprednisolone for the treatment of inflammation, and, alter-
natively, the bacitracin and polymyxin B sulfate can be omitted.
Compos tion Example 11 - Eye-Ear Drops
One thousand cc of a sterile aqueous solution for eye or ear use
containing 10 mg of U-57,930E or U-60,970E and 5 mg of methylpred-
nisolone in each cc is prepared from the following types and amounts
of ingredients:
U-57,930E or U-60,970E 10 gm
Methylprednisolone phosphate sodium 5 gm
Sodium citrate 4.5 gm
Sodium bisulfite 1 gm
Polyethylene glycol 400n120 gm
Myristyl-y-picolinium chloride0.2 gm
Polyvinylpyrrolidone 1 gm
Deionized water, q.s. ad 1000 cc
The ingredients are dissolved in the water and the resulting
solution is sterilized by filtration. The solution is aseptically
filled into sterile dropper containers.
The composition so prepared is useful in the topical treatment of
inflammation and infection of the eye and ear as well as other sensi-
tive tissues of the animal body.
Composition Example 12 - Troches
Ten thousand troches are prepared from the following types and
amounts of ingredients:
U-57~930E or U-60,970E 100 gm
Neomycin sulfate 50 gm
1 ~ 63938
3713/3841
-36-
Polymyxin ~ sulfate (10,000
units/mg) 1 gm
Ethyl aminobenzoate 50 gm
Calcium stearate 150 gm
Powdered sucrose, q.s. 5,000 gm
The powdered materials are mixed thoroughly and then compressed
into half gram troches following the usual techniques for the prep-
aration of compressed tablets.
The troches are held in the mouth and allowed to dissolve slowly
to provide treatment for the mouth and throat of humans.
Composition Example 13 - Suppository, Rectal
One thousand suppositories, each weighing 2.5 gm and containing
100 mg of U-57,930E or U-60,970E are prepared from the following types
and amounts of ingredients:
U-57,930E or U-60,970E lOO gm
Polymyxin B sulfate (10,000
units/mg) 1.25 gm
Methylprednisolone 1 gm
Ethyl aminobenzoate 75 gm
Zinc oxide 62.5 gm
Propylene glycol 162.5 gm
Polyethylene glycol 4,000 q.s. 2,500 gm
The U-57,930E or U-60,970E, polymyxin B sulfate, methylpred-
nisolone, ethyl aminobenzoate, and zinc oxide are added to the propyl-
ene glycol and the mixture milled until the powders are finely dividedand uniformly dispersed. The polyethylene glycol 4000 is melted and
the propylene glycol dispersion added slowly with stirring. The
suspension is poured into unchilled molds at 40C.
The composition is allowed to cool and solidify and then removed
from the mold and each suppository foil wrapped.
The foregoing suppositories are inserted rectally for local
treatment of inflammation and infection.
Alternatively, the foregoing composition can be prepared omitting
the steroid.
Composition Example 14 - Mastitis Ointment
One thousand gm of an ointment for the treatment of mastitis in
dairy cattle is prepared from the following types and amounts o,
- ingredients:
~ 1 6393~
3713/3841
-37-
U-57,930E or U-60,970E25 gm
Methylprednisolone acetate 0.5 gm
Light liquid petrolatum 300 gm
Chlorobutanol, anhydrous 5 gm
Polysorbate 80 5 gm
2% Aluminum monostearate-peanut
oil gel 400 gm
White petrolatum, q.s. 1000 gm
The U-57,930E or U-60,970E and methylprednisolone acetate are
milled with the light liquid petrolatum until finely divided and
uniformly dispersed. The chlorobutanol, polysorbate 80, peanut oil
gel and white petrolatum are heated to 12nF to form a melt and the
liquid petrolatum dispersion stirred in. With continued stirring, the
dispersion is allowed to cool (and congeal) to room temperature and is
filled into disposable mastitis syringes in 10 gm doses.
Composition Example 15 - Animal Feed
One thousand gm of a feed mix is prepared from the following
types and amounts of ingredients:
U-57,930E or U-60,970E10 gm
Soybean meal 400 gm
Fish meal 400 gm
Wheat germ oil 50 gm
Sorghum molasses140 gm
The ingredients are mixed together and pressed into pellets~ The
composition can be fed to laboratory animals, i.e., rats, mice, guinea
pigs, and hamsters for prophylaxis during shipping.
For other animals such as poultry, e.g., chickens, ducks, tur-
keys, and geese, the composition can be added to the animal's regular
feed in an amount calculated to give the desired dose of U-57,93nE or
U-60,970E.
Composition Example 16
Following the procedure of each of the preceding Composition
Examples 1-15, inclusive, each antibacterially-active compound of the
subject invention is substituted in an equivalent amount for the
U-57,930E or U-60,970E shown in the example to provide therapeutic
properties.
Similarly, each of the above free base compounds can be used in
the form of a pharmaceutically (or pharmacologically) acceptable salt,
~ g 3 8 3713/3841
-38-
e.g., hydrochloride, sulfate, phosphoric, sodium, potassium, calcium,and lithium.
Composition Example 17 - Capsules
One thousand two-piece hard gelatin capsules for oral use, each
containing 200 mg of U-57,930E or U-60,970E and 200mg of hydroxy-
chloroquine sulfate, are prepared from the following types and amounts
of ingredients:
U-57,930E or U-60,970E 200 gm
Hydroxychloroquine sulfate200 gm
Talc 75 gm
Magnesium stearate 25 gm
The ingredients are thoroughly mixed and then encapsulated in the
usual manner.
The foregoing capsules are useful to prevent recurrent attacks of
P. vivax in adult humans by the oral administration of 1 capsule
weekly.
Composition Example 18 - Tablets
One thousand oral tablets, each containing 125 mg of U-579930E or
U-60,970E and 325 mg of quinine sulfate, are prepared from the follow-
ing types and amounts of materials:
U-579930E or U-60,970E 125 gm
Quinine sulfate 325 gm
Lactose 50 gm
Corn starch 50 gm
Calcium stearate 25 gm
Light liquid petrolatum 5 gm
The ingredients are thoroughly mixed and slugged. The slugs are
broken down by forcing through a number 16 screen. The resulting
granules are then compressed into tablets, each containing 125 mg of
U-57,930E or U-60,970E and 325 mg of quinine sulfate.
The foregoing tablets are useful for treatment of malaria by the
oral administration of two tablets every 8 hours for 7 days, then one
tablet three times a day for 7 days.
Composition Example 19 - Oral Syrup
One thousand cc of an aqueous suspension for oral use, containing
in each 10 cc dose 25 mg of pyrimethamine, 250 mg of U-57,93nE or
U-60,970E and 500 mg of sulfadiazine is prepared from the following
types and amounts of ingredients:
'~ 1 63g38
, 3713/3841
-39-
U-57,930E or U-60,970E 25 gm
Pyrimethamine 2.5 gm
Sulfadiazine 50 gm
Citric acid 2 gm
~enzoic acid 1 gm
Sucrose 700 gm
Tragacanth 5 gm
Lemon oil 2 cc
Deionized water, q~s. 1000 cc
The citric acid, benzoic acid, sucrose, tragacanth, and lemon oil
are dispersed in sufficient water to make 850 cc of solution. The
U-57,930E or U-60,970E pyrimethamine and sulfadiazine are stirred into
the syrup until uniformly distributed. Sufficient water is added to
make 1000 cc.
The composition so prepared is useful in the prophylactic treat-
ment of malaria in adult humans at a dose of 1 tablespoonful (10 cc)
weekly.
Composition Example 20 - Parenteral Preparation
A sterile aqueous composition for intramuscular use, containing
in 1 cc 200 mg of U-57,930E or U-60,970E is prepared from the follow-
ing types and amounts of ingredients:
U-57,930E or U-60,970E 200 gm
Lactose 50 gm
Water for injection, q.s. 1000 cc
The U-57,930E or U-60,970E and lactose are dispersed in the water
and sterilized. The sterile composition, in the amount of 2 cc, is
filled aseptically into sterile vials.
Composition Example 21
Following the procedure of each of the preceding examples, each
antimalarially-active compound of the subject invention is substituted
in an equivalent amount for the U-57,930E or U-60,97nE shown in the
example to provide therapeutic properties. Similarly, each of the
above compounds can be used in the form of a pharmaceutically (or
pharmacologically) acceptable salt, e.g., hydrochloride, sulfate,
phosphoric, sodium, potassium, calcium, and lithium.
~ 3 63~38
3713/3841
-40-
In Vivo Results vs. P. ber~hei
MEDI(mg/kg) ~so2(mg/k9)
COMPOUND Sub~ Q3 OI Sub n _ OI
U-57,g30E 0.16 1.6 16 (12-22) >50
U-21,251F <20 __ 53 (46-61) --
U-24,729A < 1.25 -- 4.7 (3.2-6.9) --
U-8,284 Chloroquine < 5-10 12.5 11.5 (808-15) 14
10 Chloroquine (PO4)2 < 5 ~~ <20 --
MED = Dosage at which median survival time (STso) was increased
signiFicantly (p=0.05) over ST50 of untreated controls.
2 CDso = Median protective dose in mg/kg 95~0 limits.
3 Route of administration
Anti-Malarial_Test (P. berghei~
Test method. Male, CF-1 mice (18-20 g) were housed in groups of
and were infected intraperitoneally with whole blood from mice
infected with P. berghei 3 days prior to bleeding. A n.2-ml amount of
heparinized blood, diluted 1:10 with saline, served as the inoculum.
This volume contained approximately 106 parasites.
At 4 hr postinfection, each group of 10 mice was treated, either
subcutaneously with 0.2 ml or orally by gavage, with n.5 ml of the
desired drug concentration. Treatment was continued once each day for
4 days. The animals were observed for 28 days and deaths were
recorded. Deaths prior to the 6th day were considered traumatic.
Evaluation for efficacy of the various analogues and drug con-
centrations of individual analogues was based on the median survival
time of animals at each treatment level and the median protective dose
of the individual analogue. Calculations were computed on an IBM 370
digital computer. Results based on the treated groups were compared
with those of untreated groups or groups treated with chloroquine.
Other protozoans within the concept of the subject invention
process are intracellular parasites, for example~ the species of
Plasmodia, Toxoplasma, and Leishmania; protozoa that digest the red
blood cells (RBC's) of treated patients, for example, Entamoeba
histolytica and certain Tryp nosoma; and other helminths which ingest
~ ~ 63938
3713/3841
-41-
RBC's during the disease processes, for example the Schistosomes.
~ ~ ~3938
3713 /3841
-42-
CHART 1
ÇH3
LH,N H Ho_lCH3H
H~--N--- C-H
o Hl~ O\H
H~SCH3
H OH
Cl H3 CH3
HV N \IH H-C-Cl
H~--N--C- H ( 2 )
H H O HIOk9\lH
H~SCH3
H OH
1 ~ 63938 3713/3841
-43
C~ART 2
~1 .
l ~---R2
N
wherein Rl, which can be singly or multiply substituted in any posi-
tion of the pyridine ring not alreaqy substituted by R2, is selected
from the group consisting of hydrogen, alkyl and substltuted alkyl
wherein the alkyl portion is from 1 to 8 carbon atoms, inclus1ve, and
isomeric forms thereof, cycloalkyl and substituted cycloalkyl, sub-
stituted oxygen, substituted nitrogen, halogen, phenyl, and substi-
tuted phenyl, -(CH2)m-OH, -(CH2~ -NR4R5, and isomeric forms l:hereof,
wherein m is an integer of from 1 to 8, inclusive, R4 and R5 are H or
alkyl of from 1 to 8 carbon atoms, inclusive, and isomeric forms
thereof, wherein R2, which can be singly substituted in any position
of the pyridine ring not already substituted by Rl, is
101
. -C-X
and X is the amino function of a compound selected from the group
7(R)-hydroxy-methyl l-thio--lincosaminide, 7(S)-hydroxy-methyl 1-
thio-a-lincosaminide~ 7(S)-halo-methyl 1-th1o-a-lincosaminide~ 7(R)-
halo-methyl l-thiO-a-lincosaminide~ 7(S)-methoxy-methyl l-thio-a-
lincosaminide, 7-deoxy-7(S)-(methylthio)-methyl 1-thio-a-lincos-
aminide, 7-deoxy-7(S)-(2-hydroxyethylthio)-methyl 1-thio-a-lincos-
aminide and 7-deoxy-7(S)-(3-hydroxypropylthio)-methyl l-thio-a-lincos-
aminide, and the pharmaceutically acceptable salts thereof.
~CHa) n R1
~R2
N ~
113
1 ,,
,~,
i ~ 6~938
3713/3841
-44 -
CHART 2 (cont.)
wherein Rl and R2, which can be in the 2, 3, 4, 5, 6, 7, 8, or 9
position of the ring, are as defined above; wherein R3 is selected
from the group consisting of H, CH3, C2Hs, and -CH2-CH2-OH; wherein n
is an integer of from 1 to 4, inclusive; and the pharmaceutically
acceptable salts thereof.
R ~ ~ R
E / A
B
wherein A, B and E are selected from the group consisting of nitrogen,
oxygen, sulfur and CRlRl; Rl and R2 are as defined above, and can be
attached to any ring carbon or nitrogen atom; Rl can be multiply
attached to any ring carbon atom, and the pharmaceutically acceptable
salts thereof.
R2
E ~ B
wherein A, B, D and E are selected ~rom the group consisting of nitro-
gen, oxygen, sulfur and CRlRl; Rl and R2 are as defined above and can
be attached to any ring carbon or nitrogen atom; Rl can be multiply
attached to any ring carbon atom, and the pharmaceutically acceptable
salts thereof.
i ~ S3g3~ 3713/3g41
-45-
CHART 3
c. ~
CH2CH3 CH3
5 ~3l H-C-Cl
6 N CONH - CH
10 1: R= H
OH
4NH2 NH2
15 ~3 6 ~N'~ 8
1l 51 o 5~
20OH ~ 1 OH ~ 2
OH OH OH OH
OH ~H
N ~ NH 1 ~ ~ N
4: R= -P-O- CH~ 5: R= -P-O-CH2
30OH ~ 2l OH
OH OH OH OH
1: U-57930
2: U-57930 3-(5'-cytidylate)
3: U-57930 3-(5'-adenylate)
4: U-57930 3-(5'-uridylate)
_: U-57930 3-(5'-guanylate)
