Synthesis and Biological Activity of 5-Fluorotubercidin

Article abstract of DOI:10.1081/NCN-120027825

The electrophilic fluorination of 4-chloropyrrolo[2,3-d]pyrimidine (1) was studied culminating a 59% conversion of compound 1 to 4-chloro-5-fluoropyrrolo[2,3-d]-pyrimidine (2) using Selectfluor. This transformation proceeded via the 4-chloro-5,6-dihydro-5

Full text of DOI:10.1081/NCN-120027825

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Synthesis and Biological Activity of
5‐Fluorotubercidin
Xiaojing Wang a , Punit P. Seth a , Ray Ranken a , Eric E. Swayze a & Michael T. Migawa a
a Ibis Therapeutics Division , Isis Pharmaceuticals, Inc. , 2292 Faraday Avenue, Carlsbad,
California, 92008, USA
Published online: 01 Jun 2007.
To cite this article: Xiaojing Wang , Punit P. Seth , Ray Ranken , Eric E. Swayze & Michael T. Migawa (2004) Synthesis and
Biological Activity of 5‐Fluorotubercidin , Nucleosides, Nucleotides and Nucleic Acids, 23:1-2, 161-170, DOI: 10.1081/
NCN-120027825
To link to this article: http://dx.doi.org/10.1081/NCN-120027825
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 1 & 2, pp. 161–170, 2004
Synthesis and Biological Activity of 5-Fluorotubercidiny
Xiaojing Wang, Punit P. Seth, Ray Ranken, Eric E. Swayze,
*
and Michael T. Migawa
Ibis Therapeutics, A Division of Isis Pharmaceuticals, Inc.,
Carlsbad, California, USA
ABSTRACT
The electrophilic fluorination of 4-chloropyrrolo[2,3-d]pyrimidine (1) was studied
culminating a 59% conversion of compound 1 to 4-chloro-5-fluoropyrrolo[2,3-d]-
pyrimidine (2) using Selectfluor. This transformation proceeded via the 4-chloro-5,6-
dihydro-5-fluoro-6-hydroxypyrrolo[2,3-d]pyrimidine (3) in a 9:1 trans:cis ratio. The
trans isomer of compound 3 was studied by 1H NMR and 19F NMR, and the 5-H
tautomer (4) was observed as another intermediate. A modified Vorbruggen procedure
of compound 2 and tetra-O-acetylribose gave 4-chloro-5-fluoro-7-(2,3,5,-tri-O-
benzoyl-b-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (6) in a 65% yield. Treatment
of compound 6 with ammonia (l) in dioxane gave 5-fluorotubercidin (7). No
antibacterial activity was observed. An MTT assay (Promega) against Huh-7 liver
cells, normal mouse spleen cells stimulated with Con A (a T-cell mitogen), and
normal mouse spleen stimulated with LPS (a B-cell mitogen) showed no significant
toxicity. Increased activity of 7 over tubercidin was observed against L-1210 cells and
toxicity in fibroblast cells was reduced.
Key Words: 5-fluorotubercidin; MTT assay; LPS.
yIn honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
*
Correspondence: Michael T. Migawa, Ibis Therapeutics, A Division of Isis Pharmaceuticals, Inc.,
2292 Faraday Ave., Carlsbad, CA 92008, USA; E-mail: mmigawa@isisph.com.
161
DOI: 10.1081/NCN-120027825
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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INTRODUCTION
Pyrrolo[2,3-d]pyrimidine nucleosides have received considerable interest as
biologically active molecules.[1,2] In particular, extensive synthetic and biological
studies[3] have been carried out on the naturally occurring tubercidin and its 5-sub-
stituted derivatives, toyocamycin and sangivamycin (Figure 1). Tubercidin is closely
related in structure to adenosine and is rapidly converted to its 5’-monophosphate by
adenosine kinase and subsequently to the higher phosphates.[4] Not surprisingly then, a
vast array of biological activities have been reported for tubercidin and its derivatives
which include; in vitro cytotoxicity in mammalian cell strains,[5] significant in vivo
antitumor activity,[6,7] inhibition of RNA and DNA virus replication[8–11] and the
inhibition of the growth of a variety of microorganisms.[12] Despite the reported
antitumor activity of tubercidin, its toxicity has precluded its use in the clinic.[13]
Subsequently, considerable effort has been expended in the search for derivatives with
a greater selectivity in their biological response. For example, the related halogenated
analogs such as 5-iodotubercidin, 5-bromotubercidin and 5-chlorotubercidin have been
prepared (Figure 1), and were found to have cytotoxic effects and antiviral
activities.[13–17] However, the 5-fluoro analog of tubercidin, or any pyrrolo[2,3-d]-
pyrimidine, has never been reported in the literature, perhaps due to the synthetic
difficulties associated with direct electrophilic fluorinations. The present study
describes the one-pot electrophilic fluorination of a pyrrolo[2,3-d]pyrimidine culmi-
nating in the synthesis of 5-fluorotubercidin and its subsequent evaluation in a variety
of cell lines for antiproliferative activity.
RESULTS AND DISCUSSION
In general 5-halogenated pyrrolo[2,3-d]pyrimidines nucleosides can be prepared by
a direct halogenation of an appropriately protected tubercidin. For example, direct
bromination of tubercidin with NBS in DMF gives the 5-bromotubercidin, while the
use of NBS in a KOAc-buffered medium gives 6-bromotubercidin.[18] In a similar
fashion, the 5-iodo and 5-chloro analogs can also be prepared. However, we found that
the direct fluorination of pyrrolo[2,3-d]pyrimidine nucleosides resulted in no reaction or
cleavage of the glycosidic bond, as determined with a variety of electrophilic
fluorinating conditions (vida infra).
Figure 1. Several 5-substituted pyrrolo[2,3-d]pyrimidines.

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Due to this difficulty, we determined to first prepare the 5-fluoropyrrolo[2,3-
d]pyrimidine heterocycle followed by coupling to an appropriately substituted ribose.
We chose 4-chloropyrrolo[2,3-d]pyrimidine (1) as our starting material, as we believed
that a 4-amino group would interfere with the electrophilic fluorination process.
Generally electrophilic fluorination of heterocycles is carried out using the highly
reactive fluorine gas or acetyl hypofluorites.[19] We, however, had hoped that a more
mild fluorination could be carried out directly on the electron-rich 5-position of
pyrrolo[2,3-d]pyrimidine 1 using commercially available reagents, although such a
reaction was unprecedented. A variety of conditions and electrophilic fluorinating
reagents[20,21] were tested (NFSI, Selectfluor[22] in acetonitrile, DMF and dichloromethane
at various temperatures), Selectfluor/acetonitrile proved to be the most promising.
Treatment of compound 1 with Selectfluor in wet acetonitrile for 4 h at room
temperature, gave compound 3 as a (9:1) mixture of diastereoisomers as the main
products (Scheme 1). Alternatively, a similar reaction in scrupulously dried acetonitrile
in the presence of activated molecular sieves gave product 2; however, the reaction was
very sluggish and we never achieved greater than 33% conversion. Additionally, we
found it laborious to achieve the anhydrous conditions required to consistently prevent
the formation of compound 3. We then attempted to affect a one-pot hydro-
fluorination–elimination by adding TFA or AcOH, as both of these acids had been
successfully used in pyrrolo[2,3-d]pyrimidine 5,6-dehydrations.[23] However, there was
no reaction with either acid at room temperature. Increasing the reaction temperature to
70–80°C for 30 min in the presence of TFA resulted in a mixture of starting material
Scheme 1. Electrophilic fluorination of 5-chloropyrrolo[2,3-d]pyrimidine.

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Wang et al.
and both products 2 and 3. Prolonged heating resulted in decomposition. In contrast,
the dehydration was successful using AcOH at elevated temperature, as treatment of 1
with Selectfluor in the presence of AcOH at 70–80°C affected smooth conversion to
the desired compound 2. Furthermore, this process was amenable to scale-up, as
compound 2 was obtained in a 59% yield on a 5 gram reaction.
It is of interest to note that after about 30 min at 70°C, most of starting material 1
was consumed and one LC/MS peak (retention time 2.6 min) corresponding to the mass
of compound 2 was observed. After 2 h, an additional peak (retention time 2.7 min)
was formed having a mass spectrum identical to that of the peak at 2.6 min. After
running the reaction overnight, only the peak at 2.7 min remained. We had speculated
that the 5-H tautomer 4 (Scheme 2) might be the peak observed at 2.6 min, and is
formed as an intermediate between compounds 2 and 3. This tautomer is unprecedented
in the pyrrolo[2,3-d]pyrimidine ring system; however, an analogous tautomer has been
reported for indoles.[24]
In order to more fully explore our hypothesis, the major diastereomer (9:1 ratio) of
compound 3 was isolated by silica gel chromatography. The structure was determined
to have the hydroxy and fluoro groups in a trans orientation (3 trans) since the coupling
constant (close to 0 Hz) of the 5,6-hydrogens for the major isomer is less than the one
for minor isomer (5.8 Hz). The dehydration of compound 3 trans was investigated by
1H NMR in presence of AcOD–d3 in Acetonitrile–d3 at 70°C (Figure 2) and by 19F
NMR (not shown). It can be clearly observed that after heating for 2.5 h at 70°C,
tautomer 4 is present as a significant product (together with small amount of tautomer 2
and compound 3 trans). After further heating, compound 2 was observed as the major
product. This confirms our hypothesis that 4 is an intermediate between compound 3
trans and 2; however, we cannot conclusively rule out that some portion of 3 trans is
converted directly to 2. Also we do not know the fate of 3 cis; however, it seems that a
direct 3 cis to 2 conversion is more likely, as the H-5 proton and hydroxy group are
anti-periplanar.
Scheme 2. Mechanism of electrophilic fluorination.

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165
Figure 2. 1H NMR spectra of dehydration. A) 25°C, 5 min; B) 70°C, 2.5 h; C) 70°C, 14 h.
With 4-chloro-5-fluoropyrrolo[2,3-d]pyrimidine (2) in hand we set out to prepare
5-fluorotubercidin (7). Our first attempts using sodium salt glycosylation[25,26] were
unsuccessful. This is potentially due to the electron withdrawing effects of the
5-fluorine substitution, which are likely to stabilize the pyrrolo[2,3-d]pyrimidine anion,
and thereby reduce its nucleophilicity. Subsequently we used a modified Vorbruggen
procedure[27,28] and achieved a successful conversion to the 4-chloro-5-fluoro
pyrrolo[2,3-d]pyrimidine nucleoside 6 in a 65% yield. Treatment of compound 6 with
ammonia (l) in dioxane gave the target compound 7 (Scheme 3).
5-Fluorotubercidin, along with 5-iodotubercidin and tubercidin, were tested for
their ability to inhibit bacterial trancription/translation as well as antibacterial activity
versus E. coli and S. aureus. No antibacterial activity was observed at concentrations
of up to 100 uM. Additionally, evaluations of cellular cytotoxicity were carried out in
an MTT assay (Promega) against Huh-7 liver cells, normal mouse spleen cells
Scheme 3. Synthesis of 5-fluorotubercidin.

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Wang et al.
stimulated with Con A (a T-cell mitogen), normal mouse spleen stimulated with LPS
(a B-cell mitogen), L-1210 lymphoblastoid cells and fibroblast cells. We found that all
compounds were non toxic (IC50 > 200 uM) for Huh-7 liver epithelial cells, normal
mouse spleen cells stimulated with Con A (a T-cell mitogen), and normal mouse
spleen stimulated with LPS (a B-cell mitogen). However, activity was observed
against L-1210 cells (> 10 mM for 5-iodotubercidin, 2–3 mM for tubercidin and 1 mM
for 5-fluorotubercidin). Interestingly, toxicity in fibroblast cells was observed at
concentrations above 12 mM for tubercidin, while no toxicity was observed for
5-fluorotubercidin in concentration of up to 200 mM. This indicates that 5-fluoro-
tubercidin may be less cytotoxic than its parent tubercidin, which could translate to a
greater therapeutic index for antitumor indications if the antitumor activity of
tubercidin is maintained. Further studies to more rigorously assess the antitumor
potential of 5-fluortubercidin are ongoing.
EXPERIMENTAL
General. NMR spectra were recorded on a 300 M Hz Bruker. Silica gel 60 from
EM Science was used for purification. Compound 1 was purchased from Toronto
Research Chemicals. Selectfluor was purchased from Aldrich.
Coupled bacterial transcription/translation assay. The DNA template, pBest
TM
Luc (Promega), is a plasmid containing a reporter gene for firefly luciferase fused to
a strong tac promoter and ribosome binding site. Messenger RNA from 1 mg pBestLuc
was transcribed and translated in E. coli S30 bacterial extract in the presence or
absence of test compound. Compounds were tested in a black 96 well microtiter plate
with an assay volume of 35 mL. Each test well contained: 5 mL test compound, 13 mL
S30 premix (Promega), 4 mL 10 ꢀ complete amino acid mix (1 mM each), 5 mL E. coli
TM
S30 extract and 8 mL of 0.125 mg/mL pBest Luc . The transcription/translation reaction
was incubated for 35 minutes at 37°C followed by detection of functional luciferase
TM
with the additon of 30 mL LucLite (Packard). Light output was quantitated on a
Packard TopCount.
Minimum inhibitory concentrations (MICs). The assays are carried out in
150 mL volume in duplicate in 96-well clear flat-bottom plates. The bacterial suspension
from an overnight culture growth in appropriate medium is added to a solution of test
compound in 2.5% DMSO in water. Final bacterial inoculum is approximately 102–103
CFU/well. The percentage growth of the bacteria in test wells relative to that observed
for a control wells containing no compound is determined by measuring absorbance at
595 nm (A595) after 20–24 h at 37°C. The MIC is determined as a range of
concentration where complete inhibition of growth is observed at the higher
concentration and bacterial cells are viable at the lower concentration. Both ampicillin
and tetracycline are used as antibiotic positive controls in each screening assay for E.
coli (ATCC 25922) and S. aureus (ATCC13709).
MTT assays. MTT proliferation assays were purchased as kits from Promega and
were run according to the manufacturers protocol.

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167
4-Chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine (2). 4-Chloro-7H-pyrrolopyri-
midine 1 (5 g, 32.7 mmol) and Selectfluor (17.35 g, 49 mmol) were placed in a r.b.
flask, followed by the addition of dry acetonitrile (250 mL) and AcOH (50 mL). The
solution was then heated at 70°C for 14 h under N2. After cooling to rt, the solvent was
removed in vacuo and co-evaporated with toluene (50 mL ꢀ 2). The solid was
dissolved in a mixture of DCM:EtOAc (1:1) and filtered through a pad of silica gel
which was thoroughly washed. The combined washings were evaporated. And the
crude product was then subjected to column chromatography with DCM:EtOAc (4:1) to
1
give 3.3 g (59% yield) of 2 as a white solid. H NMR (DMSO–d6) d 7.73 (s, 1H), 8.64
(s, 1H), 12.5 (br s, 1H); 13C NMR (DMSO–d6) d 105.3 (d, J = 14.3 Hz), 111.0 (d,
J = 25.5 Hz), 139.6 (d, J = 244.5 Hz), 146.7 (d, J = 1.5 Hz), 148.5 (d, J = 3.8 Hz),
151.0; 19F NMR (DMSO–d6) d ꢁ 170.7 (d, J = 1.6 Hz): MS calcd for C6H3ClFN3
(M + H): 172.0, observed: 172.0.
Trans-4-Chloro-5-fluoro-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-6-ol (3
Trans). 4-Chloro-7H-pyrrolopyrimidine 1 (10 mg, 0.065 mmol) and Selectfluor
(115 mg, 0.33 mmol) were placed in a r.b. flask, followed by the addition of
acetonitrile (1 mL) and two drops of water. The mixture was then stirred at rt for 4 h.
After evaporation, the crude product was purified by column chromatography using
1
DCM:MeOH (98:2) to give 5 mg of a brownish solid. H NMR (CD3CN) d 8.19 (d,
1H, J = 1.9 Hz), 5.51 (d, 1H, J = 53.9 Hz), 5.19 (d, 1H, J = 21.9 Hz); 19F NMR
(CD3CN) d ꢁ 177.5 (ddd, J = 1.9, 21.6, 53.6 Hz): MS calcd for C6H5ClFN3O
(M + H): 190.0, observed: 190.1.
4-Chloro-5-fluoro-7-(2,3,5,-tri-O-benzoyl- -D-ribofuranosyl)pyrrolo[2,3-d]py-
rimidine (6). N,O-Bis(trimethylsilyl)acetamide (BSA, 0.16 mL, 0.64 mmol) was
added to a stirred suspension of 4-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine (0.1 g,
0.58 mmol) in dry acetonitrile (4 mL). After stirring at rt for 10 min, 1-O-acetyl-
2,3,5,-tri-O-benzoyl-b-D-ribofuranose (0.322 g, 0.64 mmol) was added, followed by the
addition of trimethylsily trifluoromethanesulfonate (0.115 mL, 0.64 mmol). The
reaction was stirred at rt for 15 min after which it was transferred to a preheated oil
bath at 80°C. After stirring for 1 h at 80°C, the reaction was cooled to rt and diluted
with EtOAc (25 mL). The organic phase was then sequentially washed with sat.
NaHCO3 and brine, dried (MgSO4) and concentrated to provide the crude nucleoside.
Purification by column chromatography (SiO2, 10–25% EtOAc in hexanes) provided
1
protected nucleoside (6) as a white foam (232 mg, 65%). H NMR (CDCl3) d: 8.63 (s,
1H), 8.11 (d, 2H, J = 7.1 Hz), 8.01 (d, 2H, J = 7.1 Hz), 7.91 (d, 2H, J = 7.1 Hz),
7.65–7.33 (m, 9H), 7.17 (d, 1H, J = 2.6 Hz), 6.69 (dd, 1H J = 6.2, 1.1 Hz), 6.09 (m,
2H), 4.87 (dd, 1H, J = 12.1, 3.0 Hz), 4.78 (dd, 1H, J = 7.2, 3.7 Hz), 4.67 (dd, 1H,
J = 12.1, 3.7 Hz). 13C NMR (CDCl3) d: 166.1, 165.4, 165.1, 151.9, 151.1, 147.1,
143.7–140.3 (C–F-coupling), 133.8, 133.7, 133.6, 129.8, 129.8, 129.7, 129.3, 128.7,
128.7, 128.6, 128.5, 128.4, 109.5–109.1 (C–C–F-coupling), 108.3–108.1 (C–C–F-
coupling), 86.3, 80.6, 73.9, 71.4, 63.6. MS (M + H): 616.1, observed: 616.1.
5-Fluorotuberidicin (7). 4-Chloro-5-fluoro-7-(2,3,5,-tri-O-benzoyl-b-D-ribofura-
nosyl)pyrrolo[2,3-d]pyrimidine 6 (230 mg, 0.42 mmol) was dissolved in dioxane
(3 mL) and liquid ammonia (8–10 mL). The reaction was sealed in a steel bomb and

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Wang et al.
heated in an oil bath at 75°C for 14 h. The reaction was then cooled and the solvent
evaporated to provide the crude nucleoside. Purification by column chromatography
(SiO2, 5–15% MeOH in CHCl3) provided 5-fluorotubericidin (7) as a white solid
1
(77 mg, 73%). Mp 221–222°C; H NMR (DMSO–d6) d: 8.06 (s, 1H), 7.34 (d, 1H,
J = 2 Hz), 6.99 (s, br, 2H), 6.06 (dd, 1H, J = 6.1, 1.7 Hz), 5.28 (d, 1H, J = 6.4 Hz),
5.10 (m, 2H), 4.31 (m, 1H), 4.05 (m, 1H), 3.85 (m, 1H), 3.7–3.14 (m, 2H). 13C NMR
(DMSO–d6) d: 156.3, 152.6, 146.7, 144.7–141.4 (C–F coupling), 105.0–104.6 (C–
C–F coupling), 93.1–92.9 (C–C–F coupling), 86.4, 84.9, 73.7, 70.4, 61.5. 19F NMR
(DMSO–d6) d: ꢁ 167.73. MS (M + H)+: 285.1, observed: 284.9.
ACKNOWLEDGMENTS
Dr. Swayze and Dr. Migawa thank Prof. Leroy B. Townsend for his mentorship
and guidance, and are grateful for the lessons in life and science that were learned in
his laboratory. We also thank Lisa Risen for the T/T and MIC experiments.
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In Book of Abstracts, 214th ACS National Meeting, Las Vegas, NV, September
7–11, 1997; MEDI-250.
Received August 22, 2003
Accepted October 6, 2003

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