VOL.45
NO.
12
THE JOURNAL OF ANTIBIOTICS
1827
ISOLATION, PURIFICATION, SYNTHESIS, AND ANTIINVASIVE/ ANTIMETASTATIC ACTIVITY OF U-77863 AND U-77864
FROMStreptomyces
griseoluteus,
STRAIN WS6724
Don E. Harper* and Danny R. Welch*! Medicinal Chemistry Research, Upjohn Laboratories,
301 Henrietta St., Kalamazoo, Michigan 49001, U.S.A. n Jake Gittlen Cancer Research Institute, Department of Pathology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033, U.S.A. (Received
for publication
July 16, 1992)
In screening of actinomycetes for structures with differential solid tumor activity, Streptomyces griseoluteus, strain WS6724was found to produce U-77863 and U-77864. U-77863 exhibited antiinvasive activity in vitro in the membraneinvasion culture system (MICS) and a dose-dependent antimetastatic activity in vivo versus K1735-M2and B16-F10 murine melanomas. The isolation, purification, and synthesis of both structures and biological activity is reported.
In the course of screening actinomycetes for agents with differential solid tumor activity, U-77863 and U-77864 were isolated from Streptomyces griseoluteus, strain WS6724. Chromatography of extracts of the clear beer and mycelia yielded two pools ofeluate that were found to be active in vitro in the Wayne State University two-tumor soft agar assay.1} The
first
(U-77863)
(I,
pool
of
fractions,
6724A,
yielded
/ya7w-3-(2'-methylphenyl)-2-propene-l-carboxamide
Fig.
1)
and
second,
6724B,
yielded
the
3-hydroxy-3-(2'-methylphenyl)-propane-l-
carboxamide (U-77864) (II). U-77864 was found to be a novel structure while a structure corresponding to U-77863 was reported to have been synthesized.2) However, the analytical results reported in that paper did not match those obtained for U-77863. Synthesis was used to confirm both structures and to provide sufficient quantities of each for further biological studies. Wereport the results of in vitro two-tumor assays of these compounds,as well as preliminary results of in vivo testing for toxicity and antitumor activity. Because of their relatively low toxicity and their structural characteristics, U-77863 and U-77864 were tested in the membrane invasion culture system (MICS), an in vitro model for measuring the ability of tumor cells to invade through reconstituted basement membranematrices and these results are included here.3) Results
Isolation, In Vitro Anti-tumor Activity, and Identification Fermentations of the culture, WS-6724, were found to exhibit differential solid tumor activity in vitro (Table 1) and produced activity versus P388 leukemia in vivo (%T/C 155 at a 1 : 3 dilution of the whole beer, 1 ml injected ip, QID x 5, in BO1mice). This culture was isolated from a soil sample of unknown origin. Comparisons of the Ektachrome photographs and electron micrographs of this strain with those ofNRRLB-1315 and NRRL3412 were made. This strain exhibited an aerial mass color of white to sparse
grayish-pink,
was melanin-negative,
showed good growth at 28°C and no growth at 50°C, and gelatin
1828
THE JOURNAL OF ANTIBIOTICS
DEC.
1992
Table 1. WayneState University two-tumor soft agar assay. Zone diameter (/xm) Concen_.., . _ å o
1
Sample
*
*à"
tration
(mg/ml)
Dilution
faCt°r
6724-WB (1st fermentation) 6724-WB (2nd fermentation) 6724-CB 20 rc-BuOH ext/mycelia h-BuOH ext/6724-CB 6724A 10 6724B 10 U-77863 10 U-77864 10 Acrylamide 10 Cinnanamide 10 4-Methylcinnanamide III 10
Dose
T
,
Leukemias
(^g/dlsk) L1210
_,.,
;
P388
35 l x 0- 1 16 35 2 x 0 - 540 l x 330-370 10 1 x 500 460 10 l x 500 450 1/4 x 125 370 1/10 x 50 70 l x 500 50 l x 500 0 l x 500 1 30 1 x 500 1 0 1/2 x 250 0 1 x 500 0 -
Solid tumors
-
C38
PO3 -
320 > 950 780 950 900 480 450 90 0 0 -
PO1 0 0 0 -
VOL.
45 NO.
12
THE JOURNAL OF ANTIBIOTICS Fig.
2.
Table
2.
Sample
1829
Modified Dose (mg/disk)
L1210 (units)
C38 (units)
50 50 50
10 70 45
0 > 120 25
U-77863 U-77864 Flavone acetic acid 1 unit= Table
two-tumor assay.
150/mi. 3. LI210 tube dilution
Sample U-77863 U-77864
ID50
fag/ml) > 100 > 100
assay. ID90
(/zg/ml) > 100 > 100
liquefaction. From these studies, it was concluded Acrylamide 58 > 100 Cinnanamide 52 > 100 that this culture is a strain identified as Streptomycet 4-Methylcinnamide 1 00 > 1 00 Flavone acetic acid >5 >5 griseoluteus, strain WS6724. Extraction of the clear beer at basic pH (8.0] and the mycelia with «-butyl alcohol after filtration in the presence of filter aid and subsequent evaporation of the extract recovered the majority of the activity present in the whole and clear beers of this fermentation (Table 1). Chromatography of the combined concentrated extracts on silica gel with a mobile phase of toluene - methyl isobutyl ketone (MIBK) - methyl alcohol yielded only two pools of fractions which upon evaporation showed activity in the two-tumor assay. The first, 6724A, showed essentially equal activity against leukemia and solid tumor cells while the second, 6724B, showed differential activity toward solid tumor cells. The structures
of the compounds isolated
from pools 6724A and 6724B (U-77863 and U-77864,
respectively) were determined by chemical, spectroscopic, optical rotation, and X-ray diffraction analyses. U-77863 was determined to be of the trans configuration by *H NMRspectroscopy due to the coupling constant (16 Hz) for the vinylic hydrogens.4) As mentioned previously, a structure corresponding to that of U-77863 was reported, but the only data for the previously synthesized structure that was identified as 2'-methyl-3-phenylpropene-l-carboxamide
was a melting point of 178°C.3) Their reference to a reported
melting point of 179°C for this structure,5* was not supported by our inspection of that article. Wehave found the corrected melting point of 2'-methyl-3-phenylpropene-l-carboxamide to be 151 ~ 153°C. An optical rotation, [a]£5 = 0°, suggested that U-77864 consisted of a racemic mixture of the two stereoisomers generated by the chiral ^-carbon. The X-ray diffraction spectra (Fig. 2) of U-77864 recrystallized from methyl alcohol
confirmed that structure.
After recrystallization from MIBK,neither of the compounds isolated from the pools, 6724Aand 6724B (U-77863 and U-77864, respectively) showed significant activity in vitro in the Wayne State University two-tumor assay, but did produce activity in a modified two-tumor assay (Table 2)6) that was comparable to that shown for the evaporated pools, 6724A and 6724B. Both structures showed insignificant activity in the L1210 tube dilution assay (Table 3).7) The supernatants from these recrystallizations did not produce any other active structures. Wehypothesize that these compoundsare more solubilized in the in vitro cells system of the modified two-tumor assay than in the WayneState University two-tumor soft agar assay. Synthesis
of U-77863
and U-77864
A modified Perkin reaction involving o-tolualdehyde, acetamide, and potassium acetate produced
1830
THE JOURNAL OF ANTIBIOTICS Fig.
Scheme
1.
Scheme
2.
DEC.
1992
3.
Scheme 3.
U-77863 in 27% yield (Scheme 1, Fig. 3).8) The synthetic route to U-77864 (Scheme 2) involved the condensation of o-toluoyl chloride with ethyl acetoacetate in isopropyl alcohol -water (1 : 1) yielding III in 50% yield.9) The jS-ketoester was reduced with sodium borohydride to IV in 70% yield.10) This /Miydroxy ester was then converted to U-77864 in 27% yield with excess NH4OHin pyridine.11} A sample of the synthesized
U-77864
/7-toluenesulfonic
was then converted
to U-77863
in 64%
yield
by refluxing
for 66 hours
with
acid in toluene (Scheme 3).12) Dehydration did not occur under milder conditions.
VOL.
45
NO.
12
THE JOURNAL OF ANTIBIOTICS
1831
Spectroscopic (mass, NMR,IR and UV) and elemental analyses verified that the synthetic compounds were identical to those isolated from the beers. In Vivo Antitumor Activity Both structures were non-toxic (lethality) in mice at single doses
NO.
12
THE JOURNAL OF ANTIBIOTICS
1827
ISOLATION, PURIFICATION, SYNTHESIS, AND ANTIINVASIVE/ ANTIMETASTATIC ACTIVITY OF U-77863 AND U-77864
FROMStreptomyces
griseoluteus,
STRAIN WS6724
Don E. Harper* and Danny R. Welch*! Medicinal Chemistry Research, Upjohn Laboratories,
301 Henrietta St., Kalamazoo, Michigan 49001, U.S.A. n Jake Gittlen Cancer Research Institute, Department of Pathology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033, U.S.A. (Received
for publication
July 16, 1992)
In screening of actinomycetes for structures with differential solid tumor activity, Streptomyces griseoluteus, strain WS6724was found to produce U-77863 and U-77864. U-77863 exhibited antiinvasive activity in vitro in the membraneinvasion culture system (MICS) and a dose-dependent antimetastatic activity in vivo versus K1735-M2and B16-F10 murine melanomas. The isolation, purification, and synthesis of both structures and biological activity is reported.
In the course of screening actinomycetes for agents with differential solid tumor activity, U-77863 and U-77864 were isolated from Streptomyces griseoluteus, strain WS6724. Chromatography of extracts of the clear beer and mycelia yielded two pools ofeluate that were found to be active in vitro in the Wayne State University two-tumor soft agar assay.1} The
first
(U-77863)
(I,
pool
of
fractions,
6724A,
yielded
/ya7w-3-(2'-methylphenyl)-2-propene-l-carboxamide
Fig.
1)
and
second,
6724B,
yielded
the
3-hydroxy-3-(2'-methylphenyl)-propane-l-
carboxamide (U-77864) (II). U-77864 was found to be a novel structure while a structure corresponding to U-77863 was reported to have been synthesized.2) However, the analytical results reported in that paper did not match those obtained for U-77863. Synthesis was used to confirm both structures and to provide sufficient quantities of each for further biological studies. Wereport the results of in vitro two-tumor assays of these compounds,as well as preliminary results of in vivo testing for toxicity and antitumor activity. Because of their relatively low toxicity and their structural characteristics, U-77863 and U-77864 were tested in the membrane invasion culture system (MICS), an in vitro model for measuring the ability of tumor cells to invade through reconstituted basement membranematrices and these results are included here.3) Results
Isolation, In Vitro Anti-tumor Activity, and Identification Fermentations of the culture, WS-6724, were found to exhibit differential solid tumor activity in vitro (Table 1) and produced activity versus P388 leukemia in vivo (%T/C 155 at a 1 : 3 dilution of the whole beer, 1 ml injected ip, QID x 5, in BO1mice). This culture was isolated from a soil sample of unknown origin. Comparisons of the Ektachrome photographs and electron micrographs of this strain with those ofNRRLB-1315 and NRRL3412 were made. This strain exhibited an aerial mass color of white to sparse
grayish-pink,
was melanin-negative,
showed good growth at 28°C and no growth at 50°C, and gelatin
1828
THE JOURNAL OF ANTIBIOTICS
DEC.
1992
Table 1. WayneState University two-tumor soft agar assay. Zone diameter (/xm) Concen_.., . _ å o
1
Sample
*
*à"
tration
(mg/ml)
Dilution
faCt°r
6724-WB (1st fermentation) 6724-WB (2nd fermentation) 6724-CB 20 rc-BuOH ext/mycelia h-BuOH ext/6724-CB 6724A 10 6724B 10 U-77863 10 U-77864 10 Acrylamide 10 Cinnanamide 10 4-Methylcinnanamide III 10
Dose
T
,
Leukemias
(^g/dlsk) L1210
_,.,
;
P388
35 l x 0- 1 16 35 2 x 0 - 540 l x 330-370 10 1 x 500 460 10 l x 500 450 1/4 x 125 370 1/10 x 50 70 l x 500 50 l x 500 0 l x 500 1 30 1 x 500 1 0 1/2 x 250 0 1 x 500 0 -
Solid tumors
-
C38
PO3 -
320 > 950 780 950 900 480 450 90 0 0 -
PO1 0 0 0 -
VOL.
45 NO.
12
THE JOURNAL OF ANTIBIOTICS Fig.
2.
Table
2.
Sample
1829
Modified Dose (mg/disk)
L1210 (units)
C38 (units)
50 50 50
10 70 45
0 > 120 25
U-77863 U-77864 Flavone acetic acid 1 unit= Table
two-tumor assay.
150/mi. 3. LI210 tube dilution
Sample U-77863 U-77864
ID50
fag/ml) > 100 > 100
assay. ID90
(/zg/ml) > 100 > 100
liquefaction. From these studies, it was concluded Acrylamide 58 > 100 Cinnanamide 52 > 100 that this culture is a strain identified as Streptomycet 4-Methylcinnamide 1 00 > 1 00 Flavone acetic acid >5 >5 griseoluteus, strain WS6724. Extraction of the clear beer at basic pH (8.0] and the mycelia with «-butyl alcohol after filtration in the presence of filter aid and subsequent evaporation of the extract recovered the majority of the activity present in the whole and clear beers of this fermentation (Table 1). Chromatography of the combined concentrated extracts on silica gel with a mobile phase of toluene - methyl isobutyl ketone (MIBK) - methyl alcohol yielded only two pools of fractions which upon evaporation showed activity in the two-tumor assay. The first, 6724A, showed essentially equal activity against leukemia and solid tumor cells while the second, 6724B, showed differential activity toward solid tumor cells. The structures
of the compounds isolated
from pools 6724A and 6724B (U-77863 and U-77864,
respectively) were determined by chemical, spectroscopic, optical rotation, and X-ray diffraction analyses. U-77863 was determined to be of the trans configuration by *H NMRspectroscopy due to the coupling constant (16 Hz) for the vinylic hydrogens.4) As mentioned previously, a structure corresponding to that of U-77863 was reported, but the only data for the previously synthesized structure that was identified as 2'-methyl-3-phenylpropene-l-carboxamide
was a melting point of 178°C.3) Their reference to a reported
melting point of 179°C for this structure,5* was not supported by our inspection of that article. Wehave found the corrected melting point of 2'-methyl-3-phenylpropene-l-carboxamide to be 151 ~ 153°C. An optical rotation, [a]£5 = 0°, suggested that U-77864 consisted of a racemic mixture of the two stereoisomers generated by the chiral ^-carbon. The X-ray diffraction spectra (Fig. 2) of U-77864 recrystallized from methyl alcohol
confirmed that structure.
After recrystallization from MIBK,neither of the compounds isolated from the pools, 6724Aand 6724B (U-77863 and U-77864, respectively) showed significant activity in vitro in the Wayne State University two-tumor assay, but did produce activity in a modified two-tumor assay (Table 2)6) that was comparable to that shown for the evaporated pools, 6724A and 6724B. Both structures showed insignificant activity in the L1210 tube dilution assay (Table 3).7) The supernatants from these recrystallizations did not produce any other active structures. Wehypothesize that these compoundsare more solubilized in the in vitro cells system of the modified two-tumor assay than in the WayneState University two-tumor soft agar assay. Synthesis
of U-77863
and U-77864
A modified Perkin reaction involving o-tolualdehyde, acetamide, and potassium acetate produced
1830
THE JOURNAL OF ANTIBIOTICS Fig.
Scheme
1.
Scheme
2.
DEC.
1992
3.
Scheme 3.
U-77863 in 27% yield (Scheme 1, Fig. 3).8) The synthetic route to U-77864 (Scheme 2) involved the condensation of o-toluoyl chloride with ethyl acetoacetate in isopropyl alcohol -water (1 : 1) yielding III in 50% yield.9) The jS-ketoester was reduced with sodium borohydride to IV in 70% yield.10) This /Miydroxy ester was then converted to U-77864 in 27% yield with excess NH4OHin pyridine.11} A sample of the synthesized
U-77864
/7-toluenesulfonic
was then converted
to U-77863
in 64%
yield
by refluxing
for 66 hours
with
acid in toluene (Scheme 3).12) Dehydration did not occur under milder conditions.
VOL.
45
NO.
12
THE JOURNAL OF ANTIBIOTICS
1831
Spectroscopic (mass, NMR,IR and UV) and elemental analyses verified that the synthetic compounds were identical to those isolated from the beers. In Vivo Antitumor Activity Both structures were non-toxic (lethality) in mice at single doses
