Synthesis and antitumor activities of phosmidosine A and its N- acetylated derivative

Article abstract of DOI:10.1016/S0040-4039(00)00926-6

A new antitumor active phosmidosine A, which was isolated from Streptomyces sp., was successfully synthesized by a series of reactions involving construction of the N-acyl phosphoramidate linkage which was achieved by the reaction of the 5'-O-phosphoramid

Full text of DOI:10.1016/S0040-4039(00)00926-6

Tetrahedron Letters 41 (2000) 5881±5885
Synthesis and antitumor activities of phosmidosine A
and its N-acetylated derivative
Tomohisa Moriguchi,a Norio Asai,a Takeshi Wada,a Kohji Seio,a Takuma Sasakib
and Mitsuo Sekinea,*
aFaculty of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan
bCancer Research Institute, Kanazawa University, Takara-machi, Kanazawa 920-8640, Japan
Received 23 March 2000; revised 25 March 2000; accepted 2 June 2000
Abstract
A new antitumor active phosmidosine A, which was isolated from Streptomyces sp., was successfully
synthesized by a series of reactions involving construction of the N-acyl phosphoramidate linkage which
was achieved by the reaction of the 50-O-phosphoramidite derivative with a prolinamide derivative in the
presence of 5-(3,5-dinitrophenyl)-1H-tetrazole. The growth inhibitory e€ect of phosmidosine A and its
N-acetyl analog on the various human cell lines was examined. These results showed that both compounds
have a signi®cant growth inhibitory activity and that the 6-amino group is not required for the growth
inhibitory activity of phosmidosine A. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: phosmidosine A; 8-oxoadenosine; l-proline; N-acyl phosphoramidate linkage; antitumor activity.
Phosmidosine (1) was found as a new type of antifugal antibiotic isolated from a culture ®ltrate
of Streptomyces durhameusis1 by Uramoto and Isono et al. Later, it was found by mass
spectrometry and NMR spectroscopy that phosmidosine was a novel nucleotide-type antibiotic,
having an N-acyl phosphoramidate linkage which connects a nucleoside analog, 8-oxoadenosine,
with an l-proline residue.2 Recently, Osada et al. have reported that phosmidosine suppresses
S-phase entry and arrests cell cycle progression at the G1 phase in human lung ®broblast WI-38
cells.3 Moreover, phosmidosine A (2) and B were also isolated as compounds having the
morphological reversion activity from the fermentation broth of Streptomyces sp. strain RK-16,
which is a producer strain of phosmidosine.4 It was shown that phosmidosine A (2) displays
inhibitory activity against the cell cycle progression and the morphological reversion activity on
srcts-NRK cells in a manner similar to that described in the case of phosmidosine (1).4
* Corresponding author. Fax: +81 45 924 5772; e-mail: msekine@bio.titech.ac.jp
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00926-6

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It was reported that the N-acyl phosphoramidate linkage of phosmidosine is extremely
unstable under weakly basic conditions,1 therefore the base±labile protecting groups were not
used for the synthesis of phosmidosine. However, the demethylated N-acyl phosphoramidate
linkage has proven to be stable under both basic and acid conditions.5a In this paper, we report the
synthesis and p53 phenotype-independent cytotoxicity of phosmidosine A, i.e. a demethylated
derivative of phosmidosine. The key step for the synthesis of this natural product is the construction
of the N-acyl phosphoramidate linkage. We have recently reported an e€ective method for the
synthesis of a new type of aminoacyl-adenylate analog, having an N-acyl phosphoramidate
linkage.5 In this approach, the N-acyl phosphoramidate linkage was formed by condensation of
adenosine 50-phosphoramidite derivatives with amino acid amide derivatives in the presence of
5-(3,5-dinitrophenyl)-1H-tetrazole as an activating reagent of the phosphoramidite derivatives.
To synthesize phosmidosine A, we prepared the 50-O-phosphoramidaite derivative 9 of
8-oxoadenosine as shown in Scheme 1. 8-Bromoadenosine (3) was hydrolyzed and the resulting
8-oxoadenosine derivative was acetylated by treatment with acetic acid and acetic anhydride in
the presence of sodium acetate at 120ꢀC.6 The peracetylated derivative 4 was treated in situ with
aqueous ammonia to give the N-acetylated 8-oxoadenosine derivative 5 in 83% yield, from 3. The
selective silylation of 5 with TBDMSCl in pyridine gave the 50-O-TBDMS derivative 6 in 94%
yield. Treatment of 6 with benzoic anhydride in the presence of DMAP gave the 20,30-di-O-benzoyl
Scheme 1. (i) AcONa, Ac2O, AcOH, 120ꢀC, 3 h; (ii) conc. NH3±pyridine (9:1, v/v), rt, 6 h; (iii) TBDMSCl (1.2 equiv.),
.
pyridine, rt, 12 h; (iv) Bz2O (2.4 equiv.), DMAP (0.1 equiv.), pyridine, rt, 4 h; (v) TBAF H2O (1.3 equiv.), AcOH (1.3
equiv.), THF, rt, 12 h; (vi) TSEOP(N(i-Pr)2)2 (1.5 equiv.), diisopropylammonium tetrazolide (0.5 equiv.), CH2Cl2, rt,
1.5 h

5883
derivative 7 in 88% yield, which was further converted by treatment with TBAF to the
50-hydroxyl derivative 8 in 93% yield. It is known that phosmidosine of the O,O-dialkyl
phosphoramidate type is extremely unstable even under weak basic conditions.1 Therefore, the
2-(trimethylsilyl)ethyl (TSE) group7 was used as a phosphate-protecting group that can be
removed by treatment with TBAF under neutral conditions. The 50-OH derivative 8 was allowed
to react with 2-(trimethylsilyl)ethyl N,N,N0,N0-tetraisopropylphosphorodiamidite in the presence
of diisopropylammonium tetrazolide to give the 50-O-phosphoramidite derivative 9 in 81% yield.
As the protecting group of the amino group of prolinamide, the base-labile 4,40,400tris-
(benzoyloxy)trityl (TBTr) group8,9 was employed. We previously found that the solubility of
amino acid amide derivatives is important for the smooth P N bond formation between
phosphoramidite derivatives and amino acid amide derivatives.5 Moreover, this base-labile
protecting group can be removed together with other acyl protecting groups at the same time.
0
5
Thus, N-TBTr prolinamide (10) was allowed to react with the 5 -O-phosphoramidite derivative
9 in the presence of 5-(3,5-dinitrophenyl)-1H-tetrazole. This reaction proceeded to give the con-
densation product, which was treated in situ with t-BuOOH to give the phosphoramidate
.
derivative 11. Further subsequent treatment of 11 with TBAF H2O and AcOH a€orded the
desired N-acyl phosphoramidate derivative 12 in 43% yield. It was found that the N-acetyl group
remained after treatment of 12 with aqueous ammonia, and the N-acetylated derivative 13 was
obtained in 72% yield. Additional treatment of 13 with 1 M NaOH at 70ꢀC for 30 min gave the
desired product, phosmidosine A (2), which could be isolated in 53% yield by C-18 reversed-
phase chromatography (Scheme 2). The structure of 2 was con®rmed by NMR spectroscopy and
MALDI-TOF mass spectroscopy.10
Scheme 2. (i) 10 (0.67 equiv.), 5-(3,5-dinitrophenyl)-1H-tetrazole (2 equiv.), CH3CN, rt, 10 min; (ii) t-BuOOH (3.3
.
equiv.), rt, 5 min; (iii) TBAF H2O (3 equiv.), AcOH (3 equiv.), THF, rt, 18 h; (iv) conc. NH3±dioxane (1:1, v/v), rt, 8 h;
(v) 1 M NaOH, 70ꢀC, 30 min
The growth inhibitory activities of phosmidosine A (2) and the N-acetylated derivative (13)
against various human cancer cells were tested in the MTT assay.11 These results are summarized
in Table 1, which also shows the data of the inhibitory activity of CDDP as a positive control.

5884
Table 1
The growth inhibitory activities of phosmidosine A (2) and 13 against various human cancer cellsa
Phosmidosine A (2) showed signi®cant growth inhibitory activities against the various human
cancer cell lines in the range of 8.8±190 mM. Furthermore, the di€erence in antitumor activity
between phosmidosine A and its N-acetylated derivative 13 was little observed in this assay.
These results would be helpful for the design of phosmidosine analogs as antitumor agents. The
antitumor activity of phosmidosine A is not in¯uenced by the status of p53 phenotype in tumor
cells, while CDDP showed signi®cant selective inhibitory activity against the p53 wild type cell
lines. Further studies are required to elucidate the mechanism of the p53-independent growth
inhibitory action of phosmidosine analogs.
The synthesis of phosmidosine is now under investigation, and the structure±activity relationship
between the phosmidosine and the antitumor activity is also being studied.
Acknowledgements
This work was supported by a Grant from `Research for the Future' Program of the Japan
Society for the Promotion of Science (JSPS-RFTF97I00301) and a Grant-in-Aid for Scienti®c
Research from the Ministry of Education, Science, Sports and Culture, Japan.

5885
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10. Compound 2: H NMR (D2O) 1.89±1.98 (3H, m, 400-Ha, Hb, 300-Ha), 2.39 (1H, m, 300-Hb), 3.36 (2H, m, 500-Ha,
1
Hb), 4.07±4.18 (3H, m, 40-H, 50-Ha, Hb), 4.36 (1H, m, a-H), 4.53 (1H, dd, J=5.3 Hz, J=4.6 Hz, 20-H), 5.11 (1H,
dd, J=5.3 Hz, J=5.3 Hz, 30-H), 5.85 (1H, d, J=4.9 Hz, 10-H), 8.09 (1H, s, 2-H); 13C NMR (D2O) ꢀ 26.20 (400-C),
32.09 (300-C), 49.00 (500-C), 63.02 (d, J=13.4 Hz, a-C), 67.98 (d, J=4.9 Hz, 50-C), 72.38, 72.92 (20-C, 30-C), 84.84
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