Doyle et al.
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704 1
Antitumor Agents f r o m the Bohemic Acid Complex
Antitumor Agents from the Bohemic Acid Complex, 4. Structures of Rudolphomycin, Mimimycin, Collinemycin, and Alcindoromycin Terrence W. Doyle,* Donald E. Nettleton, Robert E. Grulich, David M. Balitz, David L. Johnson, and Albert L. Vulcano Contribution from Bristol Laboratories, P.O. Box 657, Syracuse, New York 13201. Received December 21, 1978
Abstract: The structures of four new anthracyclines (rudolphomycin (1 1), collinemycin (12), mimimycin (13), alcindoromycin (14)) have been determined by a combination of chemical degradations and spectral interpretation. The use of 13CN M R spectroscopy has been extensive and assignments to the 13C N M R spectra of these compounds have been made.
The marked clinical effectiveness of the antitumor antibiotics adriamycin and daunomycin (1 and 2) (Figure 1) has led to an intensive search for new members of this interesting class of compounds.* This search has resulted in the isolation and characterization of a number of new agents: carminomycin3 (3), cinerubins A and B,4 aclacinomycins A and B,5 the rhodirubins,6 b a ~ m y c i n snogalamycin,* ,~ and marcellomycin and musettamycing from our own laboratories. Recent work from these laboratories has shown that some of the t-pyrromycinone-based anthracyclines as well as aclacinomycin A (10) (based on 1-deoxypyrromycinone) possess a mode of action which distinguishes them from the adriamycin class of anthracyclines.I0 In the preceding paper’’ of this series we have described the isolation of several new members of this unique class of agents. It is the purpose of this paper to give the structure determination of these compounds as well as to provide further details of the structure elucidations of musettamycin and m a r c e l l ~ m y c i nThe . ~ structures are illustrated i n Table I. Structures of Musettamycin (7) and Marcellomycin (8) The structural assignments to musettamycin and marcellomycin which were made earlier were based on a number of lines of evidence as outlined below. Both compounds were reddish-orange solids having elemental formulas C36H45N014 and C42H55N017, respectively. Total acid hydrolysis of either 7 or 8 led to the isolation of 6-pyrromycinone (4) in addition to trace amounts of 17-pyrromycinone (21) (Scheme I) which possessed identical properties with those reported by Brockmann and Lenk for compounds 4 and 21.12 Mild alcoholysis of 7 or 8 gave t-pyrromycin ( 6 ) ,the N M R spectrum of which was identical with that reported for the partial hydrolysis product of cinerubin A (9).4 Thus the aglycone as well as the first sugar for both these products was established. From the nonanthracycline portion of the methanolysis experiments the methyl glycoside of 2-deoxy-~-fucose(22) as a mixture of a and p anomers was obtained with the a anomer predominating. The [ H N M R spectrum of musettamycin exhibited three signals in the anomeric region at 6 5.50,5.24, and 5.00 as broad singlets which have been assigned to the C-1’, C-7, and C-1” protons, respectively, by comparing the line positions for a series of related compounds (Table 11). The small coupling constants indicated that the protons in question were equatorial rather than axial. Thus the structure of musettamycin was Scheme I
P
21
I
0002-7863/79/1501-7041$01.00/0
assigned as shown below for 7 (Figure 2). The methanolysis of marcellomycin (8) gave only 22 in addition to pyrromycin. An examination of the proton spectrum of 8 indicated the presence of an additional anomeric proton ( a anomer) as well as other signals attributable to a third sugar in the molecule. Examination of the I3C N M R spectrum of 8 in comparison with that of 7 confirmed the existence of a third sugar and indicated that it was probably a second 2-deoxyfucosyl residue. At this juncture there was some question as to the point of attachment of the 2-deoxy-~-fucoseresidues. The ‘H N M R spectra of both 7 and 8 exhibited three exchangeable signals downfield for the phenolic protons a t C - I , C-4, and C-6, thus ruling out attachment to these centers. It did not appear likely that the sugar residues could be attached to the tertiary C-9 hydroxyl, thus leaving only the C-4’ carbon as the point of attachment. A comparison of the I3C N M R spectra of pyrromycin, musettamycin, and marcellomycin confirmed that these residues were linked through the C-4’ carbon (Figure 3). Note the shift of the C-4’ carbon on formation of the glycosidic linkage to the 2-deoxy-~-fucosein musettamycin (7). Similarly, a large shift for the C-4” carbon was noted on going to marcellomycin from musettamycin. Details of the assignments are given in a later section on the I3C N M R spectra of a number of these compounds. The conformation shown for the D ring of the aglycone portion is based on the narrow coupling for the proton a t C-7 as well as an examination of molecular models which indicates this to be the conformation in which the peri interactions between the sugar residue at C-7 and the C-6 hydroxyl and the C- I O carbomethoxy and C-1 1 proton are minimized. Structure of Rudolphomycin (11) The third anthracycline present in major amounts in the bohemic acid complex is rudolphomycin (11). It was isolated as an orange-red solid which analyzed for C42H52N2O16a 3/2H20.13High-resolution field desorption mass spectrometry confirmed this elemental formula exhibiting ions at m/e 841 ( M I ) , 586, and 428 corresponding to the molecular ion and cleavages at the C- 1’’ and C-I’ anomeric carbons, respectively. The ultraviolet and visible spectra of rudolphomycin were superimposable on that of marcellomycin with the exception of a new absorption band a t 280 nm which did not shift on addition of either dilute base or dilute acid (Figure 4). The IR spectrum of 11 showed carbonyl bands at 1735 and 1600 cm-I and was similar in this respect to both 7 and 8 with the exception that the 1600-cm-’ band was relatively more intense than in either 7 or 8. As in the case of marcellomycin extensive hydrolysis of rudolphomycin gave t-pyrromycinone while partial methanolysis yielded pyrromycin, thus establishing the structure of the ag-
+
0 1979 American Chemical Society
7042
Journal of the American Chemical Society
/
101.23
/ November 7 , 1979
Table 1. Structures
14
4 5 6
R2
R3
OH H OH
C02CH3 C02CH3 C02CH3 C02CH3 C02CH3 C02CH3 C02CH3 C02CH3 H H C02CH3
H H H H H H H H C02CH3 C02CH3 H
OH
7 8 9
OH OH H
IO
OH OH OH
11 12 13 14
OH
R4 OH a-Rh a-Rh a-Rh-4’-a-DF L~-R~-~’-~-DF-~’’-~Y-DF a-Rh-4’-a-DF-4’’-aY-Cin a-Rh-4’-a-DF-4’’-a-Cin a-Rh-4/-a-DF-4”-a-R a-Rh-4/-a-DF ~-R~-~’-~-DF-~’’-cY-DF
t-pyrromycinone aklavin pyrrom ycin musettamycin marcellomycin cinerubin A aclacinomycin A rudolphomycin collinem ycin mimimycin CX-NDMR~-~’-CX-DF-~’’-CI-DF alcindorom ycin
cHFoH cHFoH cHF-oH HO
15, 16,
R’
NR1R2
R1 = R 2 = H RI = R 2 = C H 3 17, R’ = H; R 2 = CH3
daunosamine rhodosamine N-demethylrhodosamine
(D) (Rh) (NDMRh)
2-deoxy-~-fucose
(DF)
(Cin)
rednose
(R)
19
O =H:
OH
cinerulose
”>
20
IR
PI
F1
?E
W
E
R’O
0
E
2
0
1
P N,CH3
O ’ “ 1J
cn3
-
&,
5
2
R1-Ez-CBJ
cB3 E2
-
m cB20H
2 p - x R 2 - 9 Figure 1. Structures of adriamycin ( l ) , daunomycin (2), and carminomycin (3).
lycone and the first sugar in rudolphomycin. This also proved that the 280-nm U V chromophore as well as the 1600-cm-’ IR band was located in the remaining sugar residues. The ’H N M R spectrum of rudolphomycin exhibited signals in the anomeric region of the spectrum which integrated for five protons. Three of the resonances appeared at 6 5.53,5.28, and 5.10 as broad singlets and could be assigned to the C-l’, C-7, and C-I” protons (Table 11). These resonances were almost superimposable on those of musettamycin and marcellomycin. In addition, two sharp singlets integrating for one proton each were observed a t 6 5.26 and 5.32. The methyl region of the spectrum integrated for 12 protons, indicating that rudolphomycin contained 4 methyl groups. One of the methyl resonances was shifted downfield from the rest to 6 1.38 and appeared as a doublet. When the N M R spectrum of 11 was run
OH 7
R - E
8,
R -a-2-deoaytuconl
Figure 2. Structures of musettamycin (7) and.marcellomycin (8)
in deuterated pyridine the protons on the oxygenated carbons were shifted apart, permitting a first-order analysis of the signals. In addition to those signals assignable to the aglycone and rhodosamine there were two methyl groups present, one of which was coupled to a proton appearing at 6 5.52 which was not coupled to any other protons. Furthermore, this methyl group appeared farthest downfield of the methyl signals. This suggested the following fragment in which the low-field position of the methyl group was accommodated.
I n view of the structures of marcellomycin and musettamycin we suspected that the second sugar residue in rudolphomycin might also be 2-deoxy-~-fucose.The ‘H N M R
Doyle et al.
I
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/ Antitumor Agents from the Bohemic Acid Complex
1 0 0
I
90
I 7 0
80
6 ‘0
I 40
5’ 0
3‘0
I
2 0
1’0
PPM
Figure 3. I3C N M R line positions for the aliphatic portions of (a) pyrromycin (6), (b) musettamycin (7), (c) marcellomycin (8), (d) rudolphomycin (11). Lines given relative to internal MedSi.
0. 9
Rudolphoqcin
O.,B
- - .- . ... M r c e l l w y c i n
0. 7
Wavelength (ma)
Figure 4. Comparative U V and visible spectra of marcellomycin and rudolphomycin. Table 11. I H N M R Chemical Shifts of Selected Protons
compd 4 6
7 8 9 11 12 13 14
29
C- I’H
C-7H
5.54 5.50 5.52 5.53 5.53 5.50 5.50 5.49
5.38 5.32 5.24 5.32 5.28 5.28 5.27 5.28 5.29
C-1”H
5.00 5.05 5.05 5.10 5.02 5.00 4.96 4.75
spectrum of 11 (both in CDCl3 and C5D5N) supported this view, as did a comparison of the 13C N M R spectrum of Tudolphomycin (1 1) with those of 7 and 8. The 13C NMR spec-
C-1””
C-3””
4.90 5.05 5.26
5.32
5.00 4.96 5.28
5.37
C- 1OH 4.12 4.12 4.10 4.12 4.12 4.14 4.00 4.00 4.14
~
COXHI ~~~
3.72 3.70 3.68 3.69 3.70 3.12 3.88 3.88 3.73
trum of 11 was similar to that of musettamycin (7) with the exception that the C-4” carbon was shifted to 83.1 ppm (vs. 84.6 observed for marcellomycin). While 11 gave signals for
7044
1 101:23 1 Nocember 7, I979
Journal of the American Chemical Society
r: OCH-
6''
J
F"3
P
25
21
ZQ
1M)o
tr
c-
"V
mx
280
33c
mr
-
Cm-l
I
28
1598 cm-'
3.36 s
3.15
C-1H C-2H
4 . 8 0 br
4. 79
1.60-2.10 m
1.6-2. I
C-3H
4.0
4 . 0 4 ddd
4
C-LH
1.66
3.94 be
I
C-5H
3 . 9 3 dq
3.98 d
3. 92 dq
CdHl
1.10 d
1.30
1.28
OHe 287 m
m
OH
C-2'"
1 5 7 . 8 ~
164.7 ppm
C-3"'
95.1 ppm
C-4"'
194.1 ppm
98.2 ppm 196.2 pp"
Figure 5. Structure of rednosyl residue 25 in rudolphomycin
the third anomeric carbon (97.1 ppm) and the C-5"' and C-6"' carbons (at 70.7 and 15.7 ppm, respectively), no other aliphatic-type carbons could be seen. An examination of the downfield portion of the spectrum showed signals a t 95.1, 157.8, and 194.1 ppm. The signal a t 194.1 ppm could only be for the carbonyl carbon of a n a$-unsaturated ketone which would account for the three missing carbons of the hexose. This joined with the earlier derived fragment gave the partial structure shown in 23.
-
C-1'H c-3 '
C-5'H
-
C-6'H3 OCHl
5.57
1.5-2.0
4.0 3. 76
8
5.41
5.44 s
4.86
4.65 q
4.85
1.45
1.45 3.80
6 . 3 6 br
"2
Figure 6.
ddd
3.34
'HN M R spectra of 2-deoxy-~-fucoseand disaccharides from
rudolphomycin. Scheme I1
23
Addition of an anomeric carbon and the extra nitrogen atom revealed by the elemental analysis and field desorption mass spectrum suggested the structure 24. reaction. A sample of 11 was dissolved in methanol-methylene chloride and methanolic hydrogen chloride added (Scheme 11). The reaction was followed by monitoring the disappearance ", of rudolphomycin (11) using high-pressure liquid chroma24 tography." As the amount of 11 decreased the levels of pyrromycin increased as well as two new peaks (in -7:3 ratio) Placement of the amino function a t C-2"' was fully consiswhich eluted a t greater retention volumes. When all of the tent with the physical data and so structure 25 is proposed for rudolphomycin disappeared the reaction mixture was worked the third carbohydrate residue in rudolphomycin. Upon up to yield pyrromycin and two slower moving components. searching the literature we found that this probably represents the first example of a 2-amino-2,3,6-trideoxyhex-2-enopyra- These were purified by semipreparative high-pressure liquid chromatography to yield 29a and 29b, respectively, the a and nos-4-ulose, although the synthesis of a closely related compound was reported by Meyer zu R e ~ k e n d 0 r f . In I ~ view of the P anomers of the disaccharide derived from the terminal sugars of rudolphomycin. If the methanolysis reaction was allowed origin of this sugar we propose the trivial name rednose for to proceed for 24 h (rather than -1 h), compounds 29a and 29b 25. While there are no known carbohydrates containing the disappeared only to be replaced by compound 30 (a mixture of a and anomers of the disaccharide derived from methaP-enamino ketone function in the literature, there are a number nolysis of the 2"'-amino function in 29). The structures of 29 of P-enaminones which may serve as models (Figure 5). The and 30 follow from their spectra as outlined below. The great IR absorption of the carbonyl function in 26 has been reported stability of the anomeric linkage of the rednosyl residue with as 1598 cm-I compared to that of rednose a t -1600 cm-I.I5 the 4 oxygen of the 2-deoxy-~-fucoseexplains the failure to As a model for the UV absorption of rednose 25 one may use observe 22 on methanolysis of 11. the P-enaminone 27 the UV maximum of which has been reAs in the parent compound 11, the disaccharide 29 exhibited ported to occur at 287 nm3.16The failure of 25 to exhibit a shift a maximum in the UV a t 280 nm. In contrast, the maximum on addition of acid is probably due to the fact that P-enamiwas shifted to 250 nm in compound 30, which would be exnones only exhibit a shift when the UV is run in 0.1 N acid pected on replacement of an amino function by an alkoxy solution.17Finally the 13C N M R of compound 28 has been function in an a,P-unsaturated ketone.I9 Compound 29 was reported.I8 Comparison of the carbon resonances of 28 with silylated using TriSil and the mass spectrum of the resulting those assigned to 25 shows a good correspondence, the small silyl ether measured. A molecular ion at m/e 359 was observed shift differences seen being ascribable to the substitution of the and the fragmentation pattern was consistent with the proposed nitrogen atom in 28. structure. The IH N M R was measured in both CDC13 and We next attempted to confirm the proposed structure by examining the nonanthracycline products resulting from the CSD~N and using spin decoupling techniques it was possible methanolysis of rudolphomycin. Attempts to detect the methyl to assign the resonances for the 2-deoxy-L-fucose protons. Comparison of the spectrum of 29 with that of 2-deoxy-~glycosides of 2-deoxy-~-fucosein the methanolysis mixture failed completely and led to a more careful examination of the fucose a-methyl glycoside gave almost superimposable spectra
Doyle et al. / Antitumor Agents from the Bohemic Acid Complex Table 111.
7045
I3C N M R Chemical Shifts“
carbon no.
I 2 3 4 6 II 5 I2 4a Sa I la 6a I Oa I2a 7 8 9 IO 13 14 15 16 I’ 2’ 3‘ 4’ 5’ 6’ N(CH3)2 I” 2’’ 3” 4“ 5” 6“ 1 ”’ 2“‘ 3Iff 4”’ 5”’ 6”‘
39h
5c
6‘
7‘
1s
Sd
13c
119.5 137.7 125.0 162. I 156.6 156.7 190. I 185.9 115.8 110.9 I 1 1.0 134.7 132.9 139.3 61.2 35.0 71.5 51.8 32.7 7.1 171.3 52.6
120.0 137.1 124.6 162.3 161.9 120.8 192.4 181.0 115.6 114.4 131.2 132.7 142.5 133.3 71.0 33.7 71.7 57.1 32.1 6.7 171.0 52.4 101.3 28.8 59.5 65.9 66.4 17.0 41.9
157.9 129.6 130. I 158.5 162.4 120.5 190.6 185.6 112.5 114.9 131.7 132.9 142.8 112.6 71.1 34.2 71.8 57.4 32.3 6.7 171.2 52.3 101.5 28.8 60.1 66.4 66.9 17.0 42.0
157.4 129.5 129.9 158.1 161.9 120.0 189.8 184.9 I 1 1.7 114.0 131.1 132.2 142.1 111.9 71.2 34.0 71.4 56.9 32.5 6.6 171.0 52.4 101.3 29.2 61.5 73.6 68.2 17.8 42.7 98.9 32.0 65.5 70.5 66.4 16.6
157.7 129.9 130.0 158.2 162.2 120.5 190.4 185.7 112.5 114.3 132.3 132.2 143.5 112.4 71.4 36.8 72.0 56.4 33.1 7.1 171.4 52.2 100.6 29.3 61.8 74.1 68.4 18.0 43.3 99.4 32.9 65.6 70.3 66.6 17.1
158.7 130.5 130.8 159.3 163.2 121.0 191.6 186.7 113.1 115.5 132.5 133.5 143.5 113.3 71.3 34.9 71.7 57.9 33.5 7.1 172.0 52.9 102.4 29.7 62.2 74.6 69.0 18.2 43.5 100.0 34.4 66.2 84.6 67.5 17.4 101.6 32.8 66. I 71.9 67.9 17.1
157.6 129.9 129.5 158.2 161.4 120.8 190.2 185.5 112.4 114.1 132.3 131.1 142.6 112.2 70.9 33.3 71.9 55.8 29.6 8.0 171.2 52.3 100.6 29.1 61.3 73.9 69.1 17.9 43.0 100.5 34.2 65.5 83.7 67.1 17.0 99.2 32.9 65.4 70.9 66.7 16.8
14‘
157. I 129.5 131.8 157.6 161.8 119.5 189.3 184.6 1 1 1.8 114.0 131.2 129.4 141.9 1 1 1.6 70.5 33.9 70.8 56.8 32.6 6.6 170.5 52.2 101.4 31.5 54.4 76.8 67.7 17.4 33.0 100.1 33.8 64.9 81.0 67.2 17.1 100.1 31.9 64.8 70.8 67.1 16.9
11‘
loc
158.5 130.1 129.6 159.0 162.3 120.4 190.6 185.6 112.5 114.8 132.8 131.6 142.6 I 12.4 70.7 34.3 72.6 51.3 32.3 6.7 171.2 52.4 101.6 29.3 61.7 74.3 68.5 17.9 43.2 99.4 34.1 65.5 83.1 66.8 17.4 97.1 158.5 95.1 194.0 71.7 15.7
120.0 137. I 124.6 162.3 161.9 120.7 192.4 181.0 1 1 5.6 114.4 131.2 132.6 142.4 133.3 70.5 33.7 71.6 57.1 32. I 6.7 171.1 52.4 101.5 29.2 61.4 73.9 68.3 17.8 43.2 100.0 33.7 65.3 82.9 66.6 16.9 99.2 27.6 33.5 209.7 71.7 14.8
29
