Note: Descriptions are shown in the official language in which they were submitted.
2037783
- BACRGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel analogs of rebeccamycin which
posses antineoplastic properties.
2. Background Art
U.S. Pat. Nos. 4,487,925 and 4,552,842 disclose the anti-tumor
agent designated rebeccamycin, and the 5'-methyl and 5',2',3'',6''-
tetraacetate derivatives thereof, and a process for producing the
same agent by cultivating a rebeccamycin-producing strain of
Nocardia aerocoloniqenes, preferably Nocardia aerocolonigenes ATCC
39243, or a rebeccamycin-producing mutant thereof in an aqueous
nutrient medium containing assimilable sources of carbon and
nitrogen under submerged aerobic conditions until a substantial
amount of rebeccamycin is produced. Recently, Nocardia
aerocoloniqenes, ATCC 39243 was reclassified as Saccharothrix
aerocolonigenes, ATCC 39243 (Bush et at, J. Antibiotics, 40:668-
678, 1987).
8~MMARY OF TRE lNv~NllON
The present invention provides new analogs of the antitumor
agent designated rebeccamycin (Formula I).
cr
H 20377~3
- o ~
\
Cl ~J H Cl
O ~OH
HO~H
OCH3
Formula I
More specifically, there are provided rebeccamycin analogs of
the Formulas II and III below,
O~
X1~}X2
O~)H H
HO~H
OH
Formula II
2 0 3 7 7 8 3
O~GO
X1 ~H 3x2
O ~ H
HO ~ ~
~ OH
OCH3
Formula III
wherein X1 and X2 are independently fluorine or hydrogen, provided
that Xl and X2 are not simultaneously hydrogen; as well as
pharmaceutically acceptable-acid addition salts of such analogs.
The compounds of the Formulas II and III are produced by
supplementing the cultures of a rebeccamycin producing strain of
Saccharothrix aerocolonigenes with the appropriate tryptophan
analog.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the IR spectrum for the compound of Formula IV.
Figure 2 shows the lH-NMR for the compound of Formula IV.
Figure 3 shows the 13C-NMR for the compound of Formula IV.
Figure 4 shows the IR spectrum for the compound of Formula V.
Figure 5 shows the lH-NMR for the compound of Formula V.
Figure 6 shows the 13C-NMR for the compound of Formula V.
Figure 7 shows the IR spectrum for the compound of Formula VIII.
Figure 8 shows the lH-NMR for the compound of Formula VIII.
2037783
Figure 9 shows the 13C-NMR for the compound of Formula VIII.
Figure 10 shows the mass spectrum of the compound of Formula IV.
Figure 11 shows the W spectrum of the compound of Formula IV.
Figure 12 shows the mass spectrum of the compound of Formula V.
Figure 13 shows the W spectrum of the compound of Formula V.
Figure 14 shows the mass spectrum of the compound of Formula
VIII.
igure 15 shows the W spectrum of the compound of Formula VIII.
DETAILED DESCRIPTION OF THE lNV~N~ ION
U.S. Patent Nos. 4,487,925 and 4,552,842 disclose the
production and isolation of the antitumor agent designated
rebeccamycin (Formula I)
H
0~0
O ~ H
HO ~
OH
OCH3
Formula I
The above-mentioned rebeccamycin compound is the
principal component of the fermentation of the rebeccamycin
producing strain of Saccharothrix aerocoloniqenes.
It has now been found according to the present
invention that the fermentation procedure disclosed in U.S.
Patent Nos. 4,487,925 and 4,552,842 can be carried out in the
presence of certain
-,i ~
, ~ .. .. . . . . . . . . .. .... . . . ... ... .. .. . . . - .
203~783
tryptophan analogs to produce new analogs of rebeccamycin having
_ valuable antitumor properties. The rebeccamycin analogs of the
present invention have the Formulas II and III below.
oyN~o
X1~x2
O~)H H
HO ~H
OH
Formula II
0~
X1~=x2
O~OH H
HO`~?H
OCH3
Formula III
wherein Xl and X2 are fluorine or hydrogen, provided that X1 and X2
are not simultaneously hydrogen; as well as pharmaceutically
acceptable acid addition salts thereof.
A preferred example of the compounds of the present invention
21)37183
is the compound having the structural formula IV below:
oyN~
F~
O~OH H
HO ~H
OH
Formula IV
Another preferred example of the compounds of the present
invention is the compound having the structural formula V below:
0~
F~
O~OH H
HO~H
OH
Formula V
Another preferred example of the compounds of the present
invention is the compound having the structural formula VI below:
2037783
F ~ ~) F
O~OH H
HO ~
r OH
OH
Formula VI
Still another preferred example of the compounds of the
present invention is the compound having the structural formula VII
below:
0 ,~
O ~OH
HO_~
OH
Formula VII
Still another preferred example of the compounds of the
present invention is the compound having the structural formula
VIII below:
--8--
2n:37~3
~` ~
F
O ~ H
HO~H
OCH3
Formula VIII
Another preferred example of the compounds of the present
invention is the compound having the structural formula IX below:
0~0
F
O ~ H
HO ~
OH
OCH3
Formula IX
Another preferred example of the compounds of the present
invention is the compound having the structural formula X below:
2037 ~3
F yN~) F
g~
O~OH
HO~H
OCH3
Formula X
Yet another preferred example of the compounds of the present
invention is the compound having the structural formula XI below:
0~0
O ~)H
HO--~H
OCH3
Formula XI
In the present process a tryptophan analog is added to the
rebeccamycin fermentation medium and is incorporated during
fermentation into the rebeccamycin structure creating a
corresponding rebeccamycin analog. A more extensive description of
the process is given below and in the illustrative examples which
follow.
--10--
20~7783
PreParation of the Antibiotics
The compounds Formulas IV - XI are produced by cultivating a
rebeccamycin producing strain of Saccharothrix aerocoloniqenes,
with DL-4-fluorotryptophan, DL-5-fluorotryptophan, DL-6-
fluorotryptophan, or DL-7-fluorotryptophan. The preferred
producing organism is a novel strain of Saccharothrix
aerocoloniqenes, previously designated as Nocardia aerocolonigenes
strain C38,383-RK2 (ATCC 39243) in United States Patent 4,487,925.
Recently, this strain was reclassified as Saccharothrix
aerocolonigenes (Bush et al., J. Antibiotics 40:668-678, 1987) and
is designated herein as Saccharothrix aerocolonigenes strain
C38,383-RK2 (ATCC 39243). This strain was isolated from a soil
sample collected in Panama. A biologically pure culture of strain
C38,383-RK2 has been deposited with the American Type Culture
Collection, Rockville, Maryland, and added to their permanent
collection of microorganisms as ATCC 39243. This culture,
designated as C38,383-RK2, is also maintained as a dormant culture
in lyophile tubes and cryogenic vials in the Bristol-Myers Squibb
Co. Pharmaceutical Research and Development Division Culture
Collection, 5 Research Parkway, Wallingford, Connecticut 06492.
The taxonomic studies on strain C38,383-RK2 (ATCC 39243) have
been described in detail in United States Patent 4,487,925 and in
J. Antibiotics 40:668-678, 1987. The strain has been classified as
a novel strain of Saccharothrix aerocoloniqenes.
It is to be understood that the present invention is not
limited to use of the particular preferred strain ATCC 39243 or to
organisms fully answering its description. It is especially
intended to include c~her rebeccamycin producing strains or mutants
--11--
2~37783
of the described organism which can be produced by conventional
means such as x-radiation, ultraviolet radiation, treatment with
nitrogen mustard, phage exposure and the like.
In practicing the present process, a rebeccamycin-producing
strain of Saccharothrix aerocolonigenes, having the identifying
characteristics of strain C38,383-RK2 (ATCC 39243), or a mutant or
variant thereof, is cultivated in a conventional aqueous nutrient
medium supplemented with the appropriate tryptophan analog. For
optimal production of the compound of Formulas VI and X, the medium
should be supplemented with DL-4-fluorotryptophan. For optimal
production of the compounds of Formulas IV and IX, the medium
should be supplemented with DL-5-fluorotryptophan. For optimal
production of the compounds of Formula V and VIII, the medium
should be supplemented with DL-6-fluorotryptophan. For optimal
production of the compounds of Formulas VII and XI, the medium
should be supplemented with DL-7-fluorotryptophan. The organism is
grown in a nutrient medium containing known nutritional sources for
actinomycetes. Thus, the organism is grown in a nutrient medium
containing an assimilable carbon source such as sucrose, lactose,
glucose, rhamnose, fructose, glycerol or soluble starch. The
medium should also contain an assimilable nitrogen source such as
fishmeal, peptone, peanut meal, cottonseed meal, corn steep liquor,
amino acids or ammonium salts. Nutrient inorganic salts can also
be incorporated in the medium so as to provide sodium, potassium,
ammonium, calcium, phosphate, sulfate, nitrate, carbonate and like
ions. Trace elements such as copper, manganese, iron zinc, etc.
are added to the medium if desired, or they may be present as
impurities of other constituents of the media. Submerged aerobic
20377~3
conditions are preferably employed for the production of large
quantities of antibiotic, although for production of limited
amounts, surface cultures and bottles may also be used. The
general procedures used for the cultivation of other actinomycetes
are applicable to the present invention.
Production of the antibiotics of the present invention can be
effected by any temperature conducive to satisfactory growth of the
producing organism, e.g. 18 to 39 C and is conveniently carried
out at a temperature of about 28 C. The fermentation may be
carried out in flasks or in laboratory or industrial fermentors of
varlous capaclty.
When tank fermentation is to be used it is desirable to
produce a vegetative inoculum in a nutrient broth by inoculating a
small volume of the culture medium with a slant, a cryopreservative
culture or a lyophilized culture of the producing organism. After
obtaining a viable and active inoculum in this manner, it is
transferred aseptically to the fermentation tank charged with
production medium for large scale production of the antibiotic of
the present invention. The medium in which the vegetative inoculum
is grown can be the same as, or different from, that utilized in
the tank as long as it is such that a good growth of the producing
organism is obtained and supplemented with the appropriate
fluorotryptophan. Further agitation may be provided by a
mechanical impeller. Antifoam agents such as lard oil or silicone
oil may also be added if needed. Antibiotic production is
monitored by high performance liquid chromatography assay or by
conventional biological assay. In general, optimum production of
the antibiotics of the present invention is achieved after
2~37 ~ 83
incubation of about 6 days.
- Isolation and purification of the so-obtained derivatives may
be carried out by conventional chromatographic procedures.
PhYsical and Chemical Properties:
The compounds of Formulas IV, V and VIII have the following
Physical and Chemical Properties:
Compound of Formula IV
DescriPtion: Bright yellow amorphous solid
Molecular Formula: C26H1gF2N3O7
Molecular weiqht: 523.454
Mass Spectrum: Kratos MS 25 Mass Spectrometer. FABMS 524 (M +
H)+, 361 (M-162, loss of glucose).
Ultraviolet Spectrum: Hewlett Packard 845A Diode Array
Spectrophotometer. Concentration 1.0 mg/100 ml MeOH. Neutral Amax
nm (E 1%/lcm): 405, 322(457), 288, 277, 259, 230(348).
Infrared sPectrum: Perkin-Elmer 1800 FTIR Spectrometer. KBr
Pellet (cm~l): 3340, 2915, 1752, 1708, 1628, 1591, 1484, 1462,
1392, 1331, 1292, 1247, 1191, 1112, 1081, 1052, 947, 904 795, 762,
752, 752, 511.
360 MHz 1H-NMR: Bruker Model AM-3000 Spectrometer. Duel
carbon-proton probe, 5mm. Solvent d6-DMSO. Observed chemical
shifts (ppm): 11.76 (s,lH), 11.23(s,1H), 8.85(dd,1H), 8.77(dd,1H),
8.01(dd,1H), 7.68(dd,1H), 7.46(m,2H), 6.29(d,1H), 6.12(t,1H),
5.45(d,lH), 5.17(d,lH), 4.95(d,lH), 4.07(d,lH), 3.95(m,2H),
3.81(d,1H), 3.59(m,2H).
90 MHzl3C-NMR: Bruker Model AM-3000 Spectrometer. Proton
2~37783
decoupled spectrum. Duel carbon-proton probe, 5mm. Solvent d6-
DMSO. Observed chemical shifts (ppm): 170.9, 170.8, 157.0(d),
157.0(d), 138.6, 137.2, 130.7, 129.2, 121.7(d), 121.4~d), 121.3,
119.6, 116.6, 115.1, 115.0(d), 114.6(d), 113.3(d), 113.2(d),
109.1(d), 109.1(d), 84.8, 78.7, 76.5, 73.2, 67.6, 58.3.
SolubilitY: Soluble in DMSO, DMF, THF, acetone, EtOAc,
MeOH.
Thin Layer Chromatography (Rf values): Normal phase (silica
gel 60); EtoAc: 0.10. EtoAc-MeOH (9:1 v/v):0.41. Reversed
phase (C18); 0.lM NH4OAc-MeOH-CH3CN (2:4:4: v/v): 0.67
Compound of Formula V
DescriPtion: Bright yellow amorphous solid
Molecular Formula: C26HlgF2N3O7
Molecular Weight: 523.454
Mass Spectrum: Kratos MS 25 Mass Spectrometer. FABMS 524
(M + H)+, 361 (M-162, loss of glucose).
Ultraviolet Spectrum: Hewlett Packard 8452A Diode Array
Spectrophotometer. Concentration 1.0 mg/100 ml MeOH. Neutral Amax
nm (E 1%/lcm): 398(60), 316(640), 280(259), 256(313), 226(526),
204(497).
Infrared Spectrum: Perkin-Elmer 1800 FTIR Spectrometer. KBr
Pellet (cm-1): 3324, 2927, 1745, 1701, 1623, 1580, 1491, 1471,
1452, 1412, 1384, 1330, 1233, 1172, 1116, 1075, 1048, 1016, 963,
916, 829, 764, 745, 646, 635, 617, 498, 490.
360 MHz 1H-NMR: Bruker Model AM-3000 Spectrometer. Duel
carbon-proton probe, 5mm. Solvent d6-DMSO. Observed chemical
shifts (ppm): 11.77 (s,lH), 11.18(s,lH), 9.12(dd,lH), 9.05~dd,lH),
20~7783
7.85(dd,1H), 7.43(dd,1H), 7.23(t,2H), 6.26(d,1H), 6.24(s,1H),
5.31(d,1H), 5.04(d,1H), 3.98(m,2H), 3.87(m,1H), 3.66(s,ZH).
90 MHz 13C-NMR: Bruker Model AM-3000 Spectrometer. Proton
decoupled spectrum. Duel carbon-proton probe, 5mm. Solvent d6-
DMSO. Observed chemical shifts (ppm): 170.8, 170.7, 161.8(d),
161.8(d), 143.2(d), 141.5(d), 130.1, 128.7, 126.0(d), 125.9(d),
120.9, 119.3, 118.2, 118.1, 117.7, 116.5, 108.8(d), 108.6(d),
98.9(d), 98.3(d), 84.7, 78.6, 76.5, 73.1, 67.5, 58.3.
SolubilitY: Soluble in DMSO, DMF, THF, MeOH, acetone, EtOAc.
Thin LaYer ChromatoqraPhy (Rf values): Normal phase (silica
gel 60); EtoAc: 0.40. EtoAc-MeOH (9:1 v/v): 0.63. Reversed phase
(C18); 0.lM NH4OAc-MeOH-CH3CN (2:4:4: v/v): 0.60.
Compound of Formula VIII
Description: Bright yellow amorphous solid
Molecular Formula: C27H21F2N37
Molecular Weiqht: 537.481
Mass sPectrum: Kratos MS 25 Mass Spectrometer. FABMS 538
(M + H)+, 361 (M-176, loss of 4-0-methylglucose).
Ultraviolet sPectrum: Hewlett Packard 8452A Diode Array
Spectrophotometer. Concentration 1.2 mg/100 ml MeOH. Neutral Amax
nm (E 1%/lcm): 398(103), 316(1050), 280(387), 256(454), 228(780).
Infrared Spectrum: Perkin-Elmer 1800 FTIR Spectrometer. KBr
Pellet (cm-l): 3324, 2938, 1747, 1703, 1623, 1580, 1491, 1471,
1452, 1412, 1384, 1330, 1233, 1172, 1140, 1116, 1087, 1054, 963,
918, 828, 764, 649, 635, 618, 498.
360 MHz lH-NMR: Bruker Model AM-3000 Spectrometer. Duel
carbon-proton probe, 5mm. Solvent d6-DMSO. Observed chemical
-16-
2~37783
shifts (ppm): 11.77 (s,lH), 11.18(s,lH), 9.12(dd,lH), 9.05(dd,lH),
7.88(dd,1H), 7.41(dd,1H), 7.23(m,2H), 6.25(d,1H), 6.24(s,1H),
5.36(dd,lH), 5.31(d,lH), 5.04(d,lH), 3.97(m,2H), 3.60-3.95(m,4H).
90 MHz 13C-NMR: Bruker Model AM-3000 Spectrometer. Proton
decoupled spectrum. Duel carbon-proton probe, 5mm. Solvent d6-
DMSO. Observed chemical shifts (ppm): 170.8, 170.7, 161.8(d),
161.7(d), 143.1(d), 141.5(d), 130.1, 128.7, 126.0(d), 125.9(d),
120.9, 119.4, 118.2, 118.1, 117.7, 116.5, 108.8~d), 108.6(d),
98.7(d), 98.3(d), 84.4, 77.2, 77.2, 76.2, 73.2, 59.9, 58.5 ppm.
SolubilitY: Soluble in DMSO, DMF, THF, acetone, EtOAc, MeOH.
Thin Layer Chromatoqraphy (Rf values): Normal phase (silica
gel 60); EtoAc: 0.72. EtoAc-MeOH (9:1 v/v): 0.89. Reversed phase
(C18); 0.lM NH4OAc-MeOH-CH3CN (2:4:4: v/v): 0.59.
8iological Properties:
Representative compounds of the present invention were tested
against the transplanted mouse leukemia P388 to determine in vivo
antitumor activity (Tables 1-3). CDF1 mice were implanted
intraperitioneally ("ip") with 106 P388 leukemia cells obtained
from DBA/2 donor mice bearing this transplantable murine leukemia.
The CDF1 leukemic mice were treated ip with either saline (control
mice) or doses of the compound of Formulas IV, V and VIII once at
Day 1 post-tumor inoculation. These animals were observed daily
and their deaths recorded. Average body weight changes (from the
day of leukemia implant to the day of last treatment) were
determined for all groups as a means of reflecting drug toxicity.
The incidence of mice alive in each group on Day 5 post-tumor
implant was record~-, as an additional means of assessing drug
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2~37783
toxicity. No therapeutic result was considered as meaningful if
more than 1 mouse per treatment group had died by Day 5. Each
treatment group consisted of 4 to 6 mice; control groups contained
10 mice. The number of mice, if any, surviving to Day 30 (the 1st
day of the experiment) was also recorded.
Therapeutic efficacy was evaluated by determining the median
survival time ("MST") of mice treated with the compound of Formula
IV, V and VIII and comparing it to the MST of parallel control
mice. This comparison was made by dividing the MST of the former
by the latter and multiplying by 100 to derive a parameter called
the percent T/C value. A percent T/C of 2125% was considered to
represent a meaningful increase in lifespan and, hence, an active
result.
As shown in Table 1, the compound of Formula IV is active
against P388 leukemia at dose levels ranging from 10 to 90 mg/kg.
The best effect was achieved at a dosage of 30 mg/kg and consisted
of a percent T/C of 175%. Toxicity was not observed even at the
highest dose (90 mg/kg) tested.
As shown in Table 2, the compound of Formula V is active
against P388 leukemia at dose levels ranging from 0.8 to 102.4
mg/kg. The best effect was achieved at a dosage of 102.4 mg/kg and
consisted of a percent T/C of 178%. Toxicity was not observed even
at the highest dose (102.4 mg/kg) tested.
As shown in Table 3, the compound of Formula VIII is active
against P388 leukemia at dose levels ranging from 0.8 to 102.4
mg/kg. The best effect was achieved at a dosage of 51.2 mg/kg and
consisted of a percent T/C of 206%. Toxicity was not observed at
the highest dose (102.4 mg/kg) tested.
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20377~3
Table 1. Effect of Compound of Formula IV on P388 Leukemiaa
- (Day 1 Treatment)
Median Average No. of Mice
Dose, ip. Survival % Weight Alive on
(mg/kg/inj) Times (Days) T/C Chanqe (q~ Day 5 Day 30
90 16.5 165 -0.9 4/4 o/4
30 17.5 175 0.6 4/4 0/4
10 14.5 145 0.7 4/4 0/4
Control 10.0 100 10/10 0/10
a Mice were implanted with 106 P388 leukemia cells, and treatments
with the compound of Formula IV were begun 1 day later. Control
mice were given saline injections.
Table 2. Effect of Compound of Formula V on P388 Leukemiaa
(Day 1 Treatment)
Median Average No. of Mice
Dose, ip. Survival % Weight Alive on
(mg/kg/inj) Times (Days) T/C Chanqe (q) Day 5 Day 30
102.4 16.0 178 -0.1 6/6 0/6
51.2 15.0 167 -0.2 6/6 0/6
25.6 14.5 161 -0.2 6/6 0/6
12.8 15.0 167 0.8 6/6 0/6
6.4 15.0 167 -1.1 6/6 0/6
3.2 14.0 156 0.0 6/6 0/6
1.6 14.0 156 -0.7 6/6 0/6
0.8 13.0 144 0.1 6/6 0/6
Control 9.0 100 1.3 10/10 0/10
a Mice were implanted with 106 P388 leukemia cells and treatments
with the compound of Formula V were begun 1 day later. Control
mice were given saline injections.
--19--
2037~83
Table 3. Effect of Compound of Formula VIII on P388 Leukemiaa
_ (Day 1 Treatment)
Median Average No. of Mice
Dose, ip. Survival % Weight Alive on
(mq/kq/inj) Times (Days) T/C Chanqe (q) Day 5 DaY 30
102.4 14.5 161 0.2 6/6 0/6
51.2 18.5 206 0.3 6/6 0/6
25.6 14.0 156 0.1 6/6 0/6
12.8 14.5 161 -0.1 6/6 0/6
6.4 16.0 178 0.1 6/6 0/6
3.2 16.5 183 -0.3 6/6 0/6
1.6 14.0 156 -0.1 6/6 0/6
0.8 14.0 156 -0.4 6/6 0/6
Control 9.0 100 1.3 10/10 0/10
a Mice were implanted with lo6 P388 leukemia cells and treatments
with the compound of Formula VIII were begun 1 day later. Control
mice were given saline injections.
The present invention includes within its scope pharmaceutical
compositions which comprise an effective or tumor-inhibiting amount
of a rebeccamycin analog of the present invention, or a
pharmaceutically acceptable acid addition salt thereof, in
combination with an inert pharmaceutically acceptable carrier or
diluent.
According to another aspect of the invention, a method is
provided for therapeutically treating an animal (preferably
mammalian) host effected by a malignant tumor which comprises
administering to such host an effective tumor-inhibiting dose of
the antibiotic of the present invention or a pharmaceutically
acceptable acid addition salt thereof.
Examples of suitable compositions include solid compositions
-20-
2~3~733
for oral administration such as tablets, capsules, pills, powders
and granules, liquid compositions for oral administration such as
solutions, suspensions, syrups and elixirs and preparations for
parenteral administration such as sterile solutions, suspensions or
emulsions. They may also be manufactured in the form of sterile
solid compositions which can be dissolved in sterile water,
physiological saline or some other sterile injectable medium
immediately before use.
It will be appreciated that the actual preferred dosages of
the rebeccamycin analogs of the present invention will vary
according to the particular compound being used, the particular
composition formulated, the mode of application and the particular
situs, host and disease being treated. Many factors that modify
the action of the drug will be taken into account by those skilled
in the art, e.g. age, body weight, sex, diet, time of
administration, rate of excretion, condition of the host, drug
combinations, reaction sensitivities and severity of the disease.
Administration can be carried out continuously or periodically
within the maximum tolerated dose. Optimal application rates for
a given set of conditions can be readily ascertained by those
skilled in the art using conventional dosage determination tests.
The present invention is illustrated by the following examples
which are not intended to be construed as limiting the scope of the
invention.
Exam~le 1. Preparation of cryopreservative culture of
Saccharothrix aerocoloniqenes strain C38,383-RK2 (ATCC 39243)
Saccharothrix aerocolonigenes strain C38,383-RK2 was
2037~3
maintained as a cryopreservative culture stored at -80C in a Revco
ultralow temperature freezer. To prepare a cryopreservative
culture, strain C38,383-RK2 was transferred in test tubes on slants
of yeast-malt extract agar supplemented with CaC03 which consisted
of
dextrose 4.0g
yeast extract4.0g
malt extract lOg
CaC03 1.5g
agar 15g
deionized waterq.s. to 1 liter
The agar slant was incubated at 28C for 7-10 days.
Vegetative culture was prepared by transferring the surface growth
from the slant culture to a 500 ml Erlenmeyer flask containing 100
ml of a sterile vegetative medium consisting of
Cerelose (Corn Products) 30g
Pharmamedia (Traders Oil Mill Co.) lOg
Nutrisoy (Archer Daniels Midland Co.) lOg
CaC03 3g
deionized water q.s. to 1 liter
This vegetative culture was incubated at 28C for 48 hours on
a rotary shaker set at 250 revtmin. The vegetative culture was
mixed with equal volume of cryoprotective solution consisting of
Sucrose lOOg
glycerol 200g
deionized water q.s. to 1 liter
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2037783
Four ml portions of this mixture were transferred to
sterile cryogenic tube (5 ml capacity, Corning) and were frozen
in a dry ice-acetone bath. The frozen vegetative cultures were
then stored at -80C in a Revco ultralow temperature freezer.
Example 2. Preparationofvegetative culture ofSaccharothrix
aerocolonigenes strain C38,383-RK2 (ATCC 39243)
Vegetative culture was prepared by transferring 4 ml
of the cryopreservative culture to a 500 ml Erlenmeyer flask
containing 100 ml of a sterile vegetative medium having the same
composition as the vegetative medium described in Example 1. The
vegetative culture was incubated at 28C for 48 hours on a rotary
shaker set at 250 rev/min.
Example 3. Preparation of the compound of Formula IV
Three ml of the vegetative culture of Example 2 was
inoculated into 500 ml Erlenmeyer flasks each containing 100 ml
of production medium consisting of
Staclipse* J-UB starch (A.E. Staley) lOg
KH2PO4 2g
MgSO4 lg
L-threonine 2.5g
CaCO3 2g
deionized water q.s. to 1 liter
The production culture was incubated at 28C on a
rotary shaker set at 250 rev/min. After 48 hours of
fermentation, DL-5-fluorotryptophan was added to the culture at
a final concentration
* a trade-m~
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of 1 mg/ml. The culture was allowed to incubate at 28C and shaken
at 250 revtmin for additional 4 days. Production of the compound
of Formula IV was monitored by HPLC. Optimal production of the
compound of Formula IV at a concentration of 23-32 mg/ml was
generally obtained at 6 days of fermentation (i.e. 4 days after the
addition of DL-5-fluorotryptophan).
Example 4. Preparation of the compound of Formula V and VIII.
Three ml of vegetative culture of Example 2 was inoculated
into 500 ml Erlenmeyer flasks each containing 100 ml of production
medium having the same composition as described in Example 3.
The production culture was incubated at 28C on a rotary shaker set
at 250 rev/min. After 48 hours of fermentation, DL-6-
fluorotryptophan was added to the culture at a final concentration
of 1 mg/ml. The culture was allowed to incubate at 28C and shaken
at 250 rev/min for additional 4 days. Production of the compounds
of Formula V and VIII were monitored by HPLC. Optimal production
of the compound of Formula V and VIII were generally obtained at 6
days of fermentation (i.e. 4 days after the addition of DL-6-
fluorotryptophan) at a concentration of 36-58 ~g/ml and 31-42 ~g/ml
respectively.
Example 5. Isolation and purification of the compounds of Formulas
IV, V and VIII.
a) General Method
Solvents were not redistilled before use. Methanol, acetone,
ethyl acetate, isopropyl ether, chloroform,tetrahydrofuran, ethyl
ether and hexanes were ACS reagent grade. Water for HPLC refers to
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in-house deionized water from a Barnstead Nanopure* II system.
Tetrahydrofuran, methanol and acetonitrile for HPLC use were B & J
Brand HPLC grade solvents. Ammonium acetate was Fisher ~PLC grade.
Normal phase thin layer chromatograph (tlc) was carried out on
Silica gel 60, F-254 plates (EM Reagents, Cat. #5765, 5 x 10 cm,
by 0.25 mm thick). Reversed phase tlc was accomplished with
Whatman MKC18 plates (Cat. #4803-110, 0.2 mm thick). Plates were
developed in Whatman cylindrical jars with caps and 10 ml of
eluant. Rebeccamycin analogs were visible as yellow zones in
normal lighting or as yellow fluorescing zones with 254 nm or 366
nm ultraviolet light.
To whole broths was added Dicalite (speed plus) filter aid.
After brief stirring the broths were filtered on large Buchner
funnels or on a Tolhurst Centerslung Centrifugal Filter Unit (Model
lB15, Ametek, Inc.). Filtrates were discarded. Mycelial mats were
stirred in THF or THF-acetone mixtures for one hour, filtered, and
the Dicalite further rinsed with acetone until it no longer
fluoresced yellow under W light. The combined filtrates were
concentrated under reduced pressure to yield crude extracts.
A vacuum liquid chromatography (VLC) apparatus consists of a
Buchner funnel (Kontes, Art. #K-954100) containing a sealed-in
sintered glass disc (M porosity), a side hose connection for vacuum
and a lower 24/40 joint for attachment of receiving flasks.
Initially, the least polar eluting solvents pulled through under
vacuum to form tightly packed 5 cm adsorbent bed heights. Samples
were preadsorbed onto adsorbent and applied to funnels as slurries,
or applied in a solution of the least polar eluting solvent. Step
gradients were carried out where predetermined volumes of
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increasingly polar eluant constituted the fractions. The funnel
was sucked dry after each volume of eluant. Fractions were
concentrated and combined on the basis of tlc analysis.
Apparatus for size exclusion chromatography consisted of the
following: A Glenco column (2.5 I.D. x 100 cm) equipped with
solvent resistant teflon and plates; Fluid Metering, Inc. FMI lab
pump (Model RP-G150); Glenco glass reservoir (500 ml); Isco Model
328 fraction collector. Columns were slurry packed with Sephadex*
LH-20 (Pharmacia) preswollen in the eluting solvent. Solvent was
delivered in a downward manner through the column at a rate
controlled by the lab pump.
The following components were used to construct a semi-
preparative HPLC system: Waters Associates Model 590 Solvent
Delivery System pump; Knauer model 87 Variable Wavelength Detector.
Waters Associates model SR-204 Strip Chart Recorder; Whatman
Partisil 10 ODS-3 column (10 mm x 50 cm); 316 stainless steel
tubing (0.23 mm i.d.).
b.) Isolation and purification of the comPound of Formula IV
Whole broth (5 liters) was filtered with Dicalite and the
mycelial mat extracted by stirring in THF (2 liters) for 1 hour.
After further filtering, and an additional THF rinse (1 liter), the
combined filtrates were concentrated under reduced pressure to
yield 4.5 g crude extract. The extract was adsorbed onto 6.5g
Lichroprep Si 60 Silica gel (EM Science, Art. 9336, 15-25 microns)
and applied to a 60 ml VLC funnel containing an additional 24.5 g
silica gel. A hexane-ethyl acetate step gradient was carried out
(200 ml volumes), followed by a 200 ml volume THF wash. The THF
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.~
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wash (156 mg) was dissolved in 4 ml THF and applied to a column
containing 160 g Sephadex LH-20 equilibrated with THF (bed height
90 cm, bed volume 430 ml). Flow rate 3.75 ml/min. As could be
determined visually, a major yellow band (62 mg) eluted in the
first one-fourth of the second bed volume. Final purification was
accomplished by reversed phase (C18) HPLC chromatography with a
flow rate of 4 ml/min (0.1 M NH40Ac-THF (60-40)). Detection was
at 320 nm. Eluting at 39 minutes was the major analog (23 mg)
designated as the compound of the Formula IV.
c.) Isolation and purification of the compounds of Formula V and
VIII
Whole broth (2 liters) was filtered using Dicalite filter aid.
The mycelial mat, after stirring in THF (2 liters) for 45 minutes,
was filtered, and rinsed with an additional volume of THF (1.5
liters). The filtrate was concentrated under reduced pressure to
yield 3.39 g crude extract. The crude extract was triturated with
five 50 ml volumes of THF, which were combined and concentrated to
yield 0.74 g of THF soluble residue. This mass contained the bulk
of the yellow fluorescing materials. The THF soluble material was
adsorbed onto 2 g Silica Gel H (Merck, 10-40 microns) and applied
to a 30 ml VLC funnel containing an additional 11 g Silica Gel H.
A hexane-ethyl acetate step gradient was carried out using 100 ml
volumes of eluant. The two major rebeccamycin analogs were
separated cleanly in this manner. The less polar yellow zone (141
mg) eluted with hexane-ethyl acetate (1:1) and the more polar
analog (105 mg) with hexane-ethyl acetate (1:3).
The more polar analog (105 mg) was dissolved in 2 ml THF and
~ f ~
applied to a column containing 160 g Sephadex LH-20 (bed heigth 90
cm, bed volume 430 ml) preswollen in THF. The flow rate was 4
ml/min. The main yellow band eluted at 1.25 bed volumes as could
be determined visually to yield the compound of Formula V (77 mg).
The less polar analog from VLC (141 mg) was dissolved in 2 ml
MeOH and applied to a column containing 110 g Sephadex LH-20
preswollen in MeOH (bed heigth 80cm, bed volume 400 ml). The flow
rate 3.5 ml/min. The main yellow zone eluted at the end of the
fourth bed volume to yield the compound of formula VIII (70 mg).
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