An efficient synthesis of gougerotin and related analogues using solid- and solution-phase methodology

Article abstract of DOI:10.1021/ol0507322

(Chemical Equation Presented) The first solid-phase synthesis of the natural product gougerotin has been accomplished. The synthetic route is versatile and allows for diversification at position C-4 of the heterocycle, C-6′ of the sugar ring, and both residues of the peptidic moiety at N-4′ in a parallel fashion.

Full text of DOI:10.1021/ol0507322

ORGANIC
LETTERS
2005
Vol. 7, No. 16
3429-3432
An Efficient Synthesis of Gougerotin
and Related Analogues Using Solid- and
Solution-Phase Methodology
Michael T. Migawa,* Lisa M. Risen, Richard H. Griffey, and Eric E. Swayze
Ibis Therapeutics, a DiVision of Isis Pharmaceuticals, Inc., 1896 Rutherford AVenue,
Carlsbad, California 52008
mmigawa@isisph.com
Received April 5, 2005 (Revised Manuscript Received June 22, 2005)
ABSTRACT
The first solid-phase synthesis of the natural product gougerotin has been accomplished. The synthetic route is versatile and allows for
diversification at position C-4 of the heterocycle, C-6 of the sugar ring, and both residues of the peptidic moiety at N-4 in a parallel fashion.
′
′
Natural products that target RNA include the macrolides
(e.g., erythromycin, clarythromycin, and azithromycin)1 and
the aminoglycosides (e.g., tobramycin and gentamicin).2-4
Each of these has a remarkable ability to efficiently inhibit
protein synthesis, resulting in excellent antibacterial activity,
and has been the subject of many reports. However, the
hexopyranosyl cytosines, a large class of RNA-binding
natural products, have been less well explored. Members of
this class are structurally related, containing a cytidine base
attached to a six-membered ring sugar/s and often a modified
peptidic moiety. Compounds in this class include bagoug-
eramine A, blasticidin S, and gougerotin (Figure 1).5-7
Experiments have suggested a common binding site for each
Figure 1. Several hexopyranosyl cytosines.
(1) Zhanel, G. G.; Dueck, M.; Hoban, D. J.; Vercaigne, L. M.; Embil, J.
M.; Gin, A. S.; Karlowsky, J. A. Drugs 2001, 61, 443.
(2) Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C.-H. J. Am. Chem. Soc.
1998, 120, 1965.
(3) Haddad, J.; Kotra, L. P.; Llano-Sotelo, B.; Kim, C.; Azucena, E. F.,
Jr.; Liu, M.; Vakulenko, S. B.; Chow, C. S.; Mobashery, S. J. Am. Chem.
Soc. 2002, 124, 3229.
compound which is located near the peptidal transferase
region of the large subunit of rRNA.8,9 Common points of
contact of these compounds with the structured rRNA have
been previously noted.10
(4) Sucheck, S. J.; Wong, A. L.; Koeller, K. M.; Boehr, D. D.; Draker,
K.-a.; Sears, P.; Wright, G. D.; Wong, C.-H. J. Am. Chem. Soc. 2000, 122,
5230.
(5) Takahashi, A.; Ikeda, D.; Naganawa, H.; Okami, Y.; Umezawa, H.
J. Antibiot. 1986, 39, 1041.
(7) Lichtenthaler, F. W.; Morino, T.; Winterfeldt, W. Nucleic Acids Res.,
Spec. Publ. 1975, 1, S33.
(8) Vazquez, D.; Barbacid, M.; Carrasco, L. Haematol. Bluttransfus.
1974, 14, 327.
(6) Tsukada, K. Seitai no Kagaku 1984, 35, 557.
(9) Barbacid, M.; Vazquez, D. Eur. J. Biochem. 1974, 44, 445.
10.1021/ol0507322 CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/13/2005

Scheme 1. Gougerotin Synthesis
The most frequently studied hexopyranosyl cytosine,
appeared to be compatible with current solid-phase meth-
odology, which would overcome the previous obstacle to
advanced structure-activity relationship studies; i.e., the
solution phase synthesis is too complex to be practical for
the preparation of a large number of analogues for biological
studies. A successful adaptation of the synthesis of goug-
erotin to solid-phase would allow us unprecedented access
into a vast array of analogues and we could rapidly evaluate
the hexopyranosyl cytosine class for biological activity.
gougerotin, has been obtained either by isolation from the
fermentation broth of Streptomyces gougeroti11 or through
total synthesis.12,13 No semisynthetic analogues have been
reported in the literature, and no more than 30 analogues
based on the total synthesis have been reported.7,14-16
Additionally, compounds with improved activity relative to
the parent compound have not been identified, and literature
reports of additional synthetic work on gougerotin have
remained absent since the 1970s. As part of our program to
discover novel classes of compounds that bind RNA, we
were intrigued by the fact that a synthesis of gougerotin
Therefore, we initiated studies toward a solid-phase total
synthesis of gougerotin that would not only be amenable to
high-throughput analogue production but would allow us to
access previously unexplored positions (e.g., N-4, C-5).
(10) Menzel, H. M.; Lichtenthaler, F. W. Nucleic Acids Res., Spec. Publ.
1975, 1, S155.
(11) Clark, J. M., Jr. Antibiotica 1967, 1, 278.
(12) Fox, J. J.; Watanabe, K. A. Pure Appl. Chem. 1971, 28, 475.
(13) Lichtenthaler, F. W.; Morino, T.; Winterfeldt, W.; Sanemitsu, Y.
Tetrahedron Lett. 1975, 3527.
(14) Coutsogeorgopoulos, C.; Bloch, A.; Watanabe, K. A.; Fox, J. J. J.
Med. Chem. 1975, 18, 771.
(15) Lichtenthaler, F. W.; Morino, T.; Menzel, H. M. Tetrahedron Lett.
1975, 665.
In light of the previous difficulties in obtaining analogues
synthetically, our initial efforts were directed toward the total
synthesis of gougerotin with a focus on key steps initially
developed by Fox and Watanabe.12,16-24 Using literature
methods (Scheme 1), we were able to prepare the starting
glycosyl donor 2 from commercially available galactoside
1 on a multigram basis. This compound was then coupled
to give 3 and deacetylated under the conditions developed
(16) Watanabe, K. A.; Falco, E. A.; Fox, J. J. J. Am. Chem. Soc. 1972,
94, 3272.
3430
Org. Lett., Vol. 7, No. 16, 2005

by Fox and Watanabe. A one-step oxidation using TEMPO/
bis(acetoxy)iodobenzene (BIAB)25 was then carried out in
CH3CN (aq) to give the carboxylic acid 5, in good yield.
Protection of the N-4 exocyclic amino group was ac-
complished to give the Teoc-protected26 precursor 6. Our
precursor was then coupled with HATU to ArgoGel Rink
resin to give the resin-bound precursor 8a. Reduction of the
azido group with Sn(II) chloride liberated the 4′-amino group
to give amine 11a. A subsequent HATU coupling with OtBu-
Fmoc-D-serine followed by Fmoc removal with 10% piper-
idine/DMF gave amine 14a. A second HATU-mediated
coupling with Boc-sarcosine gave the fully protected resin-
bound gougerotin. Compound 14a was treated sequentially
with 0.4 M NaOH/MeOH (1:5) to remove the benzoates and
Teoc carbamate,27 and then TFA to give gougerotin (17a)
as its TFA salt. The structure of gougerotin was determined
deprotected with TFA to give the desired product (LCMS
[M + H] ) 679.2 m/z, >95% purity). The remainder of the
sequence was then affected (coupling both fragments and
deprotection) to give the N-substituted gougerotin precursor
19b. A similar sequence was carried out on the unfunction-
alized uracil derivative 9a and 9b to give the uracil
derivatives of gougerotin, 18a and 18b, respectively. A
similar sequence was carried out on compound 6 using the
piperazine-resin to give compound 17b. In all cases, purities
of 85 to >95% were achieved after cleavage from the resin,
as determined by LC/MS, and these compounds could be
assesed for biological activity without the need for any further
purification.
A small test library of 12 compounds was prepared and
screened for biological activity (Table 1) by appropriate
1
by 1- and 2-D, H NMR, and 13C NMR, and LCMS was
used to determine purity (>90%).
Table 1. Functionalized Gougerotin Analogues
Next, we coupled uracil to our glycosyl donor 2 under
modified Vorbruggen conditions28 using 3 equiv of TMS-
OTf under refluxing conditions to give an 80% yield of
compound 4. In contrast, the SnCl4 conditions gave only a
small amount of product. After deacetylation and oxidation
using the conditions developed for the protected cytidine,
we were able to obtain our precursor 7. A HATU-mediated
coupling then gave our resin-bound uracil derivative 9a.
MICb
Nucleophilic displacement of an appropriately installed
leaving group on N-4 was then envisaged as a route to those
analogues. Treatment of uracil 9a with a preformed tri-
azolating reagent29 (i.e., 1,2,4-triazole, POCl3, and Et3N in
CH3CN) for 4 h followed by nucleophilic aromatic substitu-
tion with BnNH2 failed to give any desired product. This
was, however, believed to be due to either the primary amide
or homogeneous nature of the reagent, as this reaction had
never been reported on the solid phase. Therefore, compound
7 was coupled to ArgoGel-MBCHO10 resin, functionalized
as a piperazine, to give compound 9b. Subjecting this
compound with the preformed triazolating reagent followed
by treatment with benzylamine in ethanol gave the putative
intermediate 10b. A small amount of this resin was then
T/Ta
IC50
series (µM)
MICb C. albi-
E. coli
(µM)
cans
(µM)
compd
R1
R2
17ac NH2
18a OH
20a NH2
21a OH
17b NH2
18b OH
19b NHBn
20b NH2
21b OH
CH2OH
CH2OH
CH2CH2NH2
CH2CH2NH2
CH2OH
CH2OH
CH2OH
CH2CH2NH2
CH2CH2NH2
A
A
A
A
B
B
B
B
B
B
B
B
0.5
>100
0.2
>100
0.4
>100
0.5
>200 50-100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1-3
0.3 50-100
>100
>100
>100
22b NHCH2Bn CH2CH2NH2
23b NHCH2Bn CH2OH
24b NHBn
0.5 25-50
2.3
0.4 50-100
NTd
>100
NTd
CH2CH2NH2
6-12
a T/T ) inhibition of the transcription/translation sequence in procaryotic
(17) Fox, J. J.; Kuwada, Y.; Watanabe, K. A. Tetrahedron Lett. 1968,
6029.
system. b Minimum inhibitory concentration. c Gougerotin. d Not tested.
(18) Kotick, M. P.; Klein, R. S.; Watanabe, K. A.; Fox, J. J. Carbohydr.
Res. 1969, 11, 369.
(19) Watanabe, K. A.; Kotick, M. P.; Fox, J. J. Chem. Pharm. Bull.
(Tokyo) 1969, 17, 416.
(20) Watanabe, K. A.; Kotick, M. P.; Fox, J. J. J. Org. Chem. 1970, 35,
231.
(21) Watanabe, K. A.; Fox, J. J. Chem. Pharm. Bull. 1973, 21, 2213.
(22) Chiu, T. M. K.; Warnock, D. H.; Watanabe, K. A.; Fox, J. J. J.
Heterocycl. Chem. 1973, 10, 607.
(23) Watanabe, K. A.; Falco, E. A.; Fox, J. J. J. Org. Chem. 1972, 37,
1198.
substitutions of the procedure outlined in Scheme 1 (i.e.,
D-serine to D-diaminobutyric acid, benzylamine to pheneth-
ylamine). The results were very encouraging. Small changes
in structure were found to have a dramatic impact on the
activity of inhibition of peptide bond formation (i.e., inhibi-
tion of transcription/translation, T/T) and against pathogenic
E. coli and C. albicans. The presence of a nitrogen at
position-4 was absolutely required for activity. Additionally,
substitutions at the amide portion of the sugar (series B),
the presence of an alkylaryl group on N-4, and the D-
diaminobutyric acid substitution at the first peptidic moiety
all had positive effects on activity. For example, compound
22b showed an approximate 4-fold increase over the parent
against pathogenic E. coli (the MIC for gougerotin is >200
(24) Chiu, T. M. K.; Watanabe, K. A.; Fox, J. J. Carbohydr. Res. 1974,
32, 211.
(25) Epp, J. B.; Widlanski, T. S. J. Org. Chem. 1999, 64, 293.
(26) Shute, R. E.; Rich, D. H. Synthesis 1987, 346.
(27) Treatment of a small sample of compound 6 with 0.4M NaOH/
MeOH (1:5) rapidly gives compound 5, indicating that the Teoc comes off
in the presence of base. Bjoerkman, S.; Chattopadhyaya, J. Chem. Scr. 1982,
20, 201.
(28) Vorbrueggen, H. Acc. Chem. Res. 1995, 28, 509.
(29) Krawczyk, S.; Migawa, M. T.; Drach, J. C.; Townsend, L. B.
Nucleosides, Nucleotides Nucleic Acids 2000, 19, 39.
Org. Lett., Vol. 7, No. 16, 2005
3431

µM). Most interestingly, compound 20b showed <3 µM
MIC against C. albicans, which demonstrates a potential new
class of antifungal therapeutic.
analogues this methodology makes available. We are cur-
rently seeking to prepare several hundred analogues and
further expand our methodology.
In conclusion, we have developed a solid-phase synthesis
of the natural product gougerotin and prepared a small test
library demonstrating this methodology. Positions amenable
to substitution include N-4, the 6′-amide, and the first and
second peptide residues and should be amenable to substitu-
tions at C-5 and C-6. The purity of our analogues allows us
to assess our compounds for biological activity without the
need for chromatography or other purification. Most encour-
aging though, is the new and improved biological activities
discovered using only a very small subset of the potential
Acknowledgment.
We acknowledge USAMRID
DAMD717-02-2-0023 for financial support. The U.S. Army
Medical Research Acquisition Activity, 820 Chandler Street,
Fort Detrick, MD 21702-5014, is the awarding and admin-
istering office.
Supporting Information Available: Experimental pro-
cedures and spectral data for all compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL0507322
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Org. Lett., Vol. 7, No. 16, 2005