Abstract:
The present invention is based upon the discovery that a MeOH extract of Anabaena affinis strain VS-1 showed strong cytotoxicity to L1210 murine leukemia cells, and from that extract was isolated the compounds having the following structures, Compounds 1 and 2. These two compounds are believed to be responsible for the cytotoxicity of the marine organism. This is the first report of the isolation and characterization of pyrrolo 3,2-d!pyrimidine derivatives as biosynthetic products. ##STR1##

Description:
STATEMENT OF GOVERNMENT SUPPORT 
     This invention was supported by funds from the National Institute of Allergy and Infectious Diseases, Grant No. AI 04769. 1  Thus the government of the United States of America has certain rights in this invention. 
    
    
     This is a continuation of application Ser. No. 08/039,984 filed on Mar. 30, 1993, now U.S. Pat. No. 5,569,757. 
    
    
     BACKGROUND OF THE INVENTION 
     Numerous nucleoside analogs have been synthesized and isolated from natural sources, and their biological activities have also been extensively investigated. While it would be almost impossible to isolate a new type of base per se since synthetic efforts have effectively provided examples of most variations on the base unit, 2  it would be important to isolate as a natural product a base unit which had previously been obtained only by chemical synthesis. 
     SUMMARY OF THE INVENTION 
     In the course of the screening of antiviral, antifungal, antibacterial, and cytotoxic compounds from cyanobacteria (i.e., blue-green algae), the present inventors discovered that a methanol (MeOH) extract of Anabaena affinis strain VS-1 showed strong cytotoxicity to L1210 murine leukemia cells, and following work up as described below, the assignment of the following structures was made for the two isolated compounds, designated herein as Compounds 1 and 2, which are believed to be responsible for cytotoxicity of the organism. Thus, the present invention is directed to the following isolated compounds, and to the use thereof in pharmaceutical compositions. ##STR2## 
     This is believed to be the first report of the isolation and characterization of pyrrolo 3,2-d!pyrimidine derivatives as biosynthetic products. 3   
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     A. affinis strain VS-1 was isolated from a cyanobacterial water-bloom collected from Star Lake, Norwich, Vt. 4  and cultivated in Z-8 mineral medium according to the conditions reported by Carmichael. 5  The lyophilized cells were extracted with MeOH--H 2  O (4:1), and the aqueous residue obtained after evaporation of the MeOH was passed through a CHP-20P column. The column was rinsed with H 2  O, and the active components were eluted with 15% EtOH-H 2  O and evaporated. Two active components, Compound 1 (0.25% of dried cell weight) and Compound 2 (0.028%), were isolated by bioassay-guided separation of the residue using HPLC with an ODS column. 6   
     Compound 1,  α! 28   D  +21.9° (C0.051, H 2  O), showed a molecular ion peak at m/z 429.1627 (C 17  H 24  N 4  O 9 , M+H, Δ-0.5 mDa) in the high-resolution (HR) FAB mass spectrum obtained with dithiothreitol/dithioerythritol (magic bullet) 7  as matrix. The  1  H NMR spectrum of Compound 1 contained two aromatic proton signals and 13 one-proton signals ascribable to a penrose and a hexose. 8  Six heteroatom-substituted aromatic  13  C signals were detected in the  13  C NMR spectrum of Compound 1, together with 11 signals due to the sugar units. 9  These data and the UV spectrum of Compound 1  δ max  (H 2  O) 287 (sh), 276, 268, and 229 nm; (0.01 n Hcl) 272 and 235 nm! suggested that Compound 1 is a nucleoside with two sugar units. 
     Collisionally induced tandem FABMS (FABMS/CID/MS) of Compound 1 showed three major fragment ion peaks at m/z 177, 163, and 147 (Scheme I), together with a strong fragment ion peak at m/z 267 generated by the loss of the hexose unit, but a prominent peak due to (base+H 2 ) +   was not detected, suggesting a C-nucleoside. 10  This was confirmed by the chemical shift of the anomeric center (δ H , 4.87; δC, 78.1), which were observed at relatively high fields in the  1  H and  13  C NMR spectra of Compound 1. ##STR3## 
     Subtraction of the sum of the two sugar units from the molecular formula of Compound 1 gave C 6  H 5  N 4  (133 Da) as the base unit. One-bond  1  H- 13  C coupling constants of  13  C signals at δ 128.0 ( 1  J C ,H =190 Hz) and 149.9 ( 1  H C ,H =207 Hz) were characteristic for carbons attached to, respectively, one and two nitrogen atoms. 11  The  1  H signal (δ H  7.72) for the hydrogen attached to the former carbon (δ C  128.0) showed long-range coupling (J=1.0 Hz) to an anomeric proton (δ H  4.89, H-1&#39;). These data suggested that the base unit is either pyrrolo 2,3-d!pyrimidine (i.e., 9-deazaadenine). The  13  C signals due to the aromatic carbons of Compound 1 resemble those reported for 9-deazaadenine derivatives 12  rather than 7-deazaadenine derivatives, 13  although no pyrrolo 3,2-d!pyrimidine derivative has been reported from natural sources. 3  UV spectra of Compound 1, especially the shifts of absorption maxima in acidic solution, were also more like those of 9-deazaadenine 14  than those of tubercidin (7-deazaadenosine). 15  Accordingly, the base unit in Compound 1 is most likely 9-deazaadenine. 
     The  13  C signals assigned to the hexose unit of Compound 1 closely resembled those of methyl α-D-glucopyranoside, 16  suggesting that Compound 1 is the α-D-glucopyranoside of 9-deazaadenosine. 5&#39;-α-D-Glucopyranosides of tubercidin and toyocamycin (Compounds 3 and 4, respectively, Scheme II) have been isolated from cyanobacteria. 17  1 H and  13  C NMR data for the sugar units of Compound 1 were very similar to those for Compounds 3 and 4, except for the signals due to the C-1&#39; position. Moreover, enzymatic deglycosidation of Compound 1 with α-D-glucosidase gave D-glucose and Compound 2, which was isolated as the minor component (11% of Compound 1) from the same cyanobacterium. ##STR4## 
     Compound 2,  α! 28   D  -28.4° (C 0.016 H 2  O), showed a molecular ion peak at m/z 267.1090 (C 11  H 15  N 4  O 4 , M+H, Δ+0.3 Mda) by HRFABMS. FABMS/CID/MS of Compound 2 gave the same fragment ion peaks at m/z 177, 163 and 147 observed for Compound 1 (Scheme I). The  1  H NMR spectrum of Compound 2 showed the signals ascribable to a ribose unit and two aromatic proton signals. 18   
     From the results above, the structure of Compound 2 can be assigned as 9-deazaadenosine, which has been synthesized by Lim and Klein as a cytotoxic C-nucleoside isostere of adenosine. 19  The direct comparison of Compound 2 with a synthetic sample of 9-deazaadenosine 20  by HPLC, TLC, and UV spectra confirmed that Compound 2 was identical to synthetic 9-deazaadenosine. 6  1 H NMR data for natural Compound 2 hydrochloride were also identical with those for synthetic Compound 2 hydrochloride. 21   
     Consequently, the structure of Compound 1 was assigned as the 9-deazaadenosine 5&#39;-α-D-glucopyranoside, as shown in Scheme I. Compounds 1 and 2 are pyrrolo 3,2-d!pyrimidine derivatives which have not been reported previously as biosynthetic products, 3  i.e., from natural sources. Their biosynthesis will be of considerable interest. 
     The IC 50  s of Compounds 1 and 2 vs L1210 murine leukemia cells were 0.01 and 0.002 μg/mL, respectively. These compounds also showed lethal toxicity to the aquatic invertebrate Ceriodaphnia dubia; the LC 50  s for acute (48 hr) and chronic (7 day) toxicities were, respectively, 0.5 and 0.3 μg/mL for Compound 1 and 0.3 and 0.1 μg/mL for Compound 2. Thus, these compounds are expected to be useful as pharmaceutical agents in mammals, including humans. 
     FOOTNOTES &amp; REFERENCES 
       1  Grant No. AI 04769 to K. L. Rinehart and a subcontract from the same grant to W. W. Carmichael. 
       2  Chemistry of Nucleoside and Nucleotides; Townsend, L. B., Ed.; Plenum: New York 1991; Vol. 2. 
       3  Otter, B. A.; Patil, S. A.; Klein, R. S.; Ealick, S. E., J. Am. Chem. Soc., 1992, 114, 668-671. 
       4  Gilbert, J. J., Ecology, 1990, 71, 1727-1740; culture strain sample received from Dr. Gilbert of Dartmouth College. 
       5  Carmichael, W. W., in Fundamental Research in Homogeneous Catalysis; Shilow, V., Ed.; Gordon and Breach: New York, 1986; Vol. 3, pp 1249-1262. 
       6  HPLC retention times (min. Nucleosil 7 C 18 , 10 mm×250 mm, 2 mL/min) for Compounds 1 and 2, respectively; MeOH-0.5% AcOH (1:9), 11.1 and 13.1; MeOH-0.05% TFA (1:9) 17.8 and 21.3; MeCN-0.1% NH 4  OAc (.1:20), 15.4 and 20.2 TLC (R 1  value, silica gel, 0.25-mm thick) and Compounds 1 and 2, respectively: CHCl 1  --MeOH--H 2  O (26:15:3) 0.18 and 0.63; EtOAc-2-OrOH--H 2  O (4:3:2), 0.24 and 0.50; 1-BuOH--AcOH--H 2  O (4:1:1), 0.20 and 0.40. 
       7  Witten, J. L.; Schaffer, M. H.; O&#39;Shea, M.; Cook. J. S.; Hemling. M. E.; Rinehart, K. L., Jr., Biochem. Biophys. Res. Commun., 1984, 124, 350-358. 
       8  1 H NMR data (500 MHz, 26° C.) for Compound 1 in DMSO-d 6  (2.40 ppm); δ8.05 (s, H-2), 7.72 (d, J=1.0 Hz, H-6), 4.87 (d, J=5.0 Hz, H-1&#39;), 4.74 (d, J=3.5 Hz, H-1 &#34;), 4.25 (dd, J=5.0, 5.0 Hz, H-2&#39;), 4.136 (dd, J=5.0, 4.5 H-5&#39;), 3.59 (dd, J=11.5, 2.0 Hz, H-6&#34;), 3.52 (dd, J=11.0, 3.0 Hz, H-5&#39;), J=9.0, 5.5, 2.0 Hz, H-5&#34;), 3.23 (dd, J=9.5, 3.5 Hz, H-2&#34;), 3.08 (dd, J=9.0, 9.0 Hz, H-4&#34;), all signals for 1 H; assigned by  1  H-- 1  H COSY and single-frequency decoupling experiments. 
       9  13 C NMR data (125 MHz, 26° C.) for Compound 1 in DMSO-d 6  (39.5 ppm); δ150.8 (s), 149.9 (d,  1  J C ,H =207 Hz, C-2), 144.4 (s), 128.0 (d,  1  J C ,H =190 Hz, C-6), 114.2 (s), 114.1 (s), 98.7 (d, C-1&#34;), 81.8 (d, C-4&#39;), 78.1 (d, C-1&#39;), 74.9 (d, C-2&#39;), 73.4 (d, C-3&#34;), 72.8 (d, C-5&#34;), 72.3 (d, C-2&#34;), 70.9 (d, C-3&#39;), 70.1 (d, C-4&#34;), 66.9 (t, C-5═), 61.0 (t, C-6&#34;); assigned by  1  H- 13  C COSY experiment. 
       10  Crow, F. W.; Tower, K. B.; Gross, M. L.; McCloskey, J. A.; Bergstrom, D. E., Anal. Biochem., 1984, 139, 243-262. 
       11  Breitmaier, E.; Voelter, W., Carbon-13 NMR Spectroscopy; VCH: Weinheim, Germany, 1987; p. 289. 
       12  Buchanan, J. G.; Craven, D. A.; Wightman, R. H.; Harnden, M. R., J. Chem. Soc., Perkin Trans., 1, 1991, 195-202. 
       13  Chenon, M. T.; Pugmire, R. J.; Grant, D. M.; Panzica, R. P.; Townsend, L. B., J. Amer. Chem. Soc., 1975, 97, 4627-4636. 
       14  Imai, K., Chem. Pharm. Bull., 1964, 12, 1030-1042. 
       15  Anzai, K.; Nakamura, G.; Suzukim S., J. Antibiot., Ser. A, 1957, 10, 201-204. 
       16  Bock, K., Pedersen, C., in Advances in Carbohydrate Chemistry and Biochemistry; Tipson, R. S.; Horton, D., Eds.; Academic: New York, 1983; Vol. 41, pp 27-66. 
       17  Stewart, J. B., Bornemann, V.; Chen, J. L.; Moore, R. E.; Caplan, F. R.; Karuso, H.; Larsen, L. K.; Patterson, G. M. L., J. Antibiot., 1988, 41, 1048-1056. The assignments of  1  H and  13  C signals for the C-3&#34; and 4&#34; positions must be interchanged. 
       18  1 H NMR data (500 MHz, 18° C.) for Compound 2 in DMSO-d 6  (2.49 ppm); δ11.95 (1 H, br s, NH), 8.17 (1 H, s, H-2), 7.72 (2 H, br s, NH 2 ), 7.58 (1 H, s, H-6), 4.85 (1 H, d, J=3.1 Hz, OH), 4.77 (1 H, d, J=7.4 Hz, H-1&#39;), 4.22 (1 H, dd, J=7.4, 5.1 Hz, H-2&#39;), 4.14 (1 H, d, J=3.9 Hz, OH), 4.00 (1 H, dd, J=5.1, 2.8 Hz, H-3&#39;), 3.86 (1 H, ddd, J=3.1, 3.1, 2.8 Hz, H-4&#39;), 3.60 (1 h, dd, J=12.0, 3.1 Hz, H-5&#39;), 3.51 (1 H, dd J=12.0, 3.1 Hz, H-5&#39;); assigned by single-frequency decoupling experiments. 
       19  Lim, M. I.; Klein, R. S., Tetrahedron Lett., 1981, 22, 25-28. 
       20  The synthetic sample of 9-deazaadenosine hydrochloride was provided by Dr. Robert S. Klein, Montefiore Medical Center. 
       21   1  H NMR data (500 MHz, 18° C.) for Compound 2 hydrochloride in DMSO-d 6  (2.49 ppm): δ12.80 (1 H, s, NH) 9.03 and 8.99 (each 1 H, s, NH 2 ), 8.50 (1 H, s, H-2), 7.86 (1 H, d, J=1.0 Hz, H-6), 4.86 (1 H, d, J=7.0 Hz, H-1&#39;), 3.97 (1 H, dd, J=7.0, 5.1 Hz H-3&#39;), 3.94 (1 H, dd, J=5.1, 3.1 Hz, H-2&#39;). 3.87 (1 H, dt, J=3.2, 3.1 Hz, H-4&#39;), 3.62 (2 H, d, J=3.2 Hz, H 2  -5&#39;). 
     The present invention has been described in detail, including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and/or improvements on this invention and still be within the scope and spirit of this invention as set forth in the following claims.