Synthesis of 5-(6-hydroxy-7H-purine-8-ylthio)-2-(N-hydroxyformamido) pentanoic acid

Article abstract of DOI:10.3762/bjoc.6.93

We have developed a synthetic route for the preparation of a hybrid bisubstrate small molecule based on a nucleoside. A prototype compound was designed and docked into the catalytic domain of the AdSS enzyme bridging the region between the magnesium center of the protein to the nucleoside region. The synthesis involves coupling a brominated peptide fragment capable of complexing magnesium to a thiolated nucleoside to obtain the hybrid model compound.

Full text of DOI:10.3762/bjoc.6.93

Synthesis of 5-(6-hydroxy-7H-purine-8-ylthio)-
2-(N-hydroxyformamido)pentanoic acid
Yanmei Zhang, Greg Elliot, Adrian Saldanha, Igor Tsigelny, Dennis Carson
and Wolf Wrasidlo*
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2010, 6, 742–747.
Moores Cancer Center, University of California San Diego, La Jolla,
CA, 92093, USA
doi:10.3762/bjoc.6.93
Received: 08 June 2010
Email:
Accepted: 12 August 2010
Published: 01 September 2010
Yanmei Zhang - y2zhang@ucsd.edu; Wolf Wrasidlo* -
wwrasidlo@ucsd.edu
Associate Editor: N. Sewald
* Corresponding author
© 2010 Zhang et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
adenylosuccinate synthetase (AdSS); thiolated nucleoside
Abstract
We have developed a synthetic route for the preparation of a hybrid bisubstrate small molecule based on a nucleoside. A prototype
compound was designed and docked into the catalytic domain of the AdSS enzyme bridging the region between the magnesium
center of the protein to the nucleoside region. The synthesis involves coupling a brominated peptide fragment capable of
complexing magnesium to a thiolated nucleoside to obtain the hybrid model compound.
Introduction
During cell proliferation, adenine nucleotides for nucleic acid
synthesis are generated through a de novo biosynthetic route,
which requires the key enzyme adenylosuccinate synthetase
(AdSS). Adenine nucleotides are also recycled by a salvage
pathway which requires methylthioadenosine phosphorylase
(MTAP). In some cancers this salvage pathway is lost making
these cancers hypersensitive to cytotoxic effects of pharmaco-
logic AdSS inhibitors [1]. However, no direct binding inhibi-
tors of human AdSS are known. Recently, the crystal structure
of AdSS in the presence of their substrates IMP, GTP and
aspartic acid was published [2]. We have used this information
in computational models for the design of compounds capable
Figure 1: Structure of AdSS catalytic domain from Mg center (light
blue) to IMP loop (tan) with compound 13 docked.
of interfering with the catalytic function of this enzyme [3].
Figure 1 shows the orientation of our model compound within
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Beilstein J. Org. Chem. 2010, 6, 742–747.
the catalytic domain of AdSS. The synthesis of this hybrid treated with allyl bromide in the presence of powdered zinc in a
molecule is described below.
mixture of THF and aqueous ammonium chloride to produce
the allylglycine derivative 4 in excellent yield with high dia-
stereoselectivity [4]. Protection of the amino group as the
Results and Discussion
The synthetic pathway to the model compound 13 is outlined in N-trichloroethoxy-carbonyl derivative 5, followed by cleavage
Scheme 1. The reaction of 2-oxoacetic acid (1) with O-benzyl- of the chiral amide, esterification and reduction afforded the
hydroxylamine resulted in benzylhydroxylamino acetic acid 2 benzyl ester 8 (via intermediates 6 and 7) in good overall yield.
in near quantitative yield [4]. The latter compound was Treatment of 8 with triphenylphosphine in carbon tetrabromide
converted into the camphorsultam derivative 3, which was resulted in the bromide 9 [5,6], which was coupled to mercap-
Scheme 1: Synthesis of the model compound 13.
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Beilstein J. Org. Chem. 2010, 6, 742–747.
topurine 9’ in the presence of sodium hydride in DMF to yield lithium (2.5 M in hexane, 1.95 mL, 4.87 mmol) was added
10 [7,8] in excellent yield.
dropwise to a solution of sultam 2’ (1.0 g, 4.64 mmol) in dry
THF (20.0 mL) at −78 °C. The solution was stirred at −78 °C
Removal of the N-Troc group with zinc in acetic acid gave for 10 min, and then transferred, via a cannula, to the solution
intermediate 11 and subsequent N-formylation produced 12. of the mixed anhydride prepared above. The reaction mixture
Finally, debenzylation with boron tribromide afforded the was stirred at −78 °C for 5 min, and then at 0 °C for 1 h. Satu-
hybrid molecule 13 as a slightly tan powder [9-12]. All com- rated ammonium chloride solution (15.0 mL) was added and the
pounds were fully characterized and their structures confirmed mixture extracted three times with ethyl acetate. The combined
by 1H NMR and mass spectroscopy. The purity of the final pro- ethyl acetate extracts were washed successively with water and
duct was determined by HPLC and found to be 98%.
brine, dried with magnesium sulfate and concentrated under
reduced pressure. The residue was recrystallized from
Our model compound was docked into the catalytic domain of hexane–EtOAc to afford the title compound as colorless crys-
AdSS as shown in Figure 1. The compound exhibited a close fit tals (1.7 g, 98%). 1H NMR (300 MHz, CDCl3): δ 8.13 (s, 1H),
with interactions of the peptide part of the structure complexed 7.19–7.29 (m, 5H), 5.23 (s, 2H), 4.03 (t, J = 7.2 Hz, 1H),
to the Mg ion of the enzyme and the heterocyclic part located 3.34–3.47 (m, 2H), 1.96–2.08 (m, 2H), 1.79–1.90 (m, 3H),
near the IMP loop.
1.22–1.37 (m, 2H), 1.07 (s, 3H), 0.89 (s, 3H). [M + H]+: 377.4.
Experimental
Preparation of compound 2
Preparation of compound 4
Compound 2: Compound 1 (50% aqueous solution) (1.48 g,
0.02 mol) was added dropwise to the solution of O-benzylhy-
droxylamine (1.67 g, 0.014 mol) in 98.0 mL of water at room
temperature and the resulting mixture stirred for 4 h. The pH
was adjusted to 12 and washed with ethyl acetate. The pH of the
aqueous layer was adjusted to pH 1–2 with 2 N HCl and
extracted with ethyl acetate. The combined organic layers were
dried with sodium sulfate and concentrated in vacuum to give
compound 2 (1.57 g, 88%). 1H NMR (400 MHz, CDCl3): δ
7.54 (s, 1H), 7.34–7.41 (m, 5H), 5.32 (s, 2H). [M + H]+: 180.1.
Compound 4: To a vigorously stirred solution of compound 3
(1.76 g) in THF (9.0 mL) and saturated ammonium chloride
(9.0 mL) at 0 °C, was added allyl bromide (671.0 µL) followed
by zinc powder (0.62 g). The zinc dust was added portionwise
at such a rate that one portion was allowed to react before
adding the next. The mixture was stirred at 0 °C for 30 min,
then partitioned between ethyl acetate and water. The aqueous
layer was re-extracted with ethyl acetate. The combined ethyl
acetate extracts were washed with brine, dried with MgSO4 and
concentrated. The residue was recrystallized from
hexane–EtOAc to afford the desired product as colorless crys-
Preparation of compound 3
Compound 3: Pivaloyl chloride (0.80 mL, 6.49 mmol) was tals (1.0 g, 90%). 1H NMR (300 MHz, CDCl3): δ 7.26–7.37 (m,
added dropwise to a mixture of compound 2 (1.08 g, 6.04 5H), 5.69–5.78 (m, 1H), 5.02–5.10 (m, 2H), 4.65–4.75 (m, 2H),
mmol), Et3N (1.2 mL) and THF (30.0 mL) at −78 °C. The mix- 4.45–4.49 (m, 1H), 3.95–3.99 (m, 1H), 3.44–3.54 (m, 2H),
ture was stirred at −78 °C for 10 min then warmed to 0 °C, 2.27–2.40 (m, 2H), 2.04–2.08 (m, 2H), 1.86–1.92 (m, 3H),
stirred for 30 min. and then re-cooled to −78 °C. n-Butyl- 1.15(s, 3H), 0.94(s, 3H). [M + H]+: 419.5.
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Beilstein J. Org. Chem. 2010, 6, 742–747.
Preparation of compound 5
Preparation of compound 7
Compound 5: To a mixture of compound 4 (1.9 g, 4.54 mmol), Compound 7: To a solution of the acid 6 (491.0 mg, 1.24
DMAP (20.0 mg) and dichloromethane (16.0 mL) at 0 °C, was mmol) in anhydrous THF (20.0 mL) at 0 °C, was added 1-(3-
added pyridine (0.43 g, 5.44 mmol) followed by the dropwise dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
addition of trichloroethoxy chloroformate (1.17 g, 5.52 mmol). (EDC, 475.0 mg, 2.48 mmol) followed by DMAP (302.0 mg,
The mixture was stirred at 0 °C for 2 h, then warmed to room 2.48 mmol). After stirring at 0 °C for 30 min, benzyl alcohol
temperature and stirred at r.t. overnight. The reaction mixture (402.0 mg, 3.72 mmol) was added. The reaction mixture was
was diluted with DCM and quenched with saturated ammoni- stirred at 0 °C for 1 h, then warmed to room temperature and
um chloride. The organic layer was washed successively with stirred for 18 h. The mixture was diluted with ethyl acetate, and
water and brine, dried with sodium sulfate and concentrated. washed successively with water and brine, dried (MgSO4) and
The residue was purified by chromatography with concentrated. The residue was purified by column chromatog-
DCM–MeOH (120:1) as eluent to yield the title compound 5 as raphy with hexane–EtOAc (4:1) as eluent to give the desired
a white foam (2.1 g, 77%). 1H NMR (400 MHz, CDCl3): δ product as a colorless oil (240.0 mg, 40%). 1H NMR (300 MHz,
7.40–7.43 (m, 2H), 7.26–7.30 (m, 3H), 5.71–5.79 (m, 1H), CDCl3): δ 7.32–7.47 (m, 10H), 5.76–5.89 (m, 1H), 4.73–5.40
4.67–5.31 (m, 7H), 3.83–3.89 (m, 1H), 3.39–3.46 (m, 2H), (m, 9H), 2.70–2.86 (m, 2H). [M + H]+: 487.7.
2.67–2.86 (m, 2H), 2.01–2.07 (m, 2H), 1.79–1.90 (m, 3H),
1.24–1.39 (m, 2H), 1.08 (s, 3H), 0.91 (s, 3H). [M + H]+: 594.9. Preparation of compound 8
Preparation of compound 6
Compound 8: A solution of 9-BBN (0.5 M in THF, 4.84 mL,
2.4 mmol) was added slowly to a solution of compound 7
(587.0 mg, 1.2 mmol) in anhydrous THF (4.0 mL) at 0 °C. The
Compound 6: A 1 N lithium hydroxide solution (5.25 mL, 5.25 reaction mixture was warmed to room temperature. After stir-
mmol) was added dropwise to a solution of compound 5 (2.1 g, ring at this temperature for 23 h, the reaction mixture was
3.54 mmol) in THF (22.0 mL) at 0 °C. The mixture was stirred cooled to 0 °C. 3 M NaOAc (3.43 mL) and 30% H2O2 (1.71
at 0 °C for 1 h, then at room temperature for 5 h. The reaction mL) were added successively. The mixture was heated and
mixture was diluted with ethyl acetate (25.0 mL) and water stirred at 50 °C for 4 h. After cooling to room temperature, the
(25.0 mL). The aqueous phase was extracted with ethyl acetate aqueous layer was extracted with ethyl acetate. The ethyl
(2 × 25 mL) to remove camphor sultam, then acidified with 2 N acetate extracts were washed in turn with water and brine, dried
HCl (until pH 2). The cloudy aqueous solution was then with sodium sulfate and concentrated. The residue was purified
extracted with ethyl acetate (3 × 25 mL). The combined ethyl by silica gel column chromatography. The column was eluted
acetate solution was dried with sodium sulfate and concen- first with hexane–EtOAc (10:1) and then with
trated. The residue was purified by column chromatography hexane–EtOAc–Et3N (80:40:1) to give the desired product as a
with DCM–MeOH–AcOH (120:20:1) as eluent to give the colorless oil (483.0 mg, 79%). 1H NMR (300 MHz, CDCl3): δ
desired product 6 as a colorless oil (1.0 g, 71%). 1H NMR (400 7.22–7.34 (m, 10H), 4.91–5.19 (m, 4H), 4.62–4.76 (m, 3H),
MHz, CDCl3): δ 7.34–7.42 (m, 5H), 5.77–5.86 (m, 1H), 3.54–3.62 (m, 2H), 1.91–2.15 (m, 2H), 1.36–1.70 (m, 2H). [M
4.76–5.30 (m, 6H), 2.76–2.82 (m, 2H). [M + H]+: 397.6. + H]+: 505.7.
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Beilstein J. Org. Chem. 2010, 6, 742–747.
Preparation of compound 9
Preparation of compound 11
Compound 9: To a mixture of compound 8 (0.48 g, 1 equiv),
CBr4 (0.48 g, 1.5 equiv) and DCM (9.0 mL) at 0 °C, was added
a solution of PPh3 (0.38 g, 1.5 equiv) and DCM (4.0 mL). The
resulting mixture was stirred at room temperature until the
starting materials disappeared, the solvent was removed in
vacuum and the residue was purified by silica gel column chro- Compound 11: Compound 10 (90.0 mg) was dissolved in
matography to give the product (0.22 g, 39%). 1H NMR (400 acetic acid (1.0 mL) and treated with zinc dust (160.0 mg). The
MHz, CDCl3): δ 7.23–7.29 (m, 10H), 5.04–5.16 (m, 2H), mixture was stirred at room temperature for 5 h, diluted with
4.88–5.01 (m, 2H), 4.58–4.77 (m, 3H), 3.26–3.31 (m, 2H), ethyl acetate, and filtered through a Celite pad. The ethyl
1.86–2.12 (m, 4H). [M + H]+: 568.6.
acetate solution was washed successively with saturated potas-
sium carbonate, water and brine, dried with sodium sulfate and
concentrated. The residue was purified by column chromatog-
raphy with DCM–MeOH (95:5) as eluent to give the crude pro-
duct. The crude product was used in the next step without
further purification (30.0 mg, 45%). [M + H]+: 480.5.
Preparation of compound 10
Preparation of compound 12
Compound 12: To a solution of compound 11 (26.0 mg) in
formic acid (96%, 800.0 µL), was added acetic anhydride (15.0
µL). The mixture was stirred at 20 °C for 1 h. After pouring into
iced-water, the aqueous layer was extracted with ethyl acetate.
The combined ethyl acetate phase was washed successively
with potassium carbonate aqueous solution and brine, dried
over sodium sulfate, concentrated, and the residue purified with
preparative TLC with DCM–MeOH (97:3) as eluent (devel-
oped 5 times) to give the desired product 12 as a syrup (10.0
mg, 36%). 1H NMR (400 MHz, MeOD): δ 8.12 (s, 1H), 7.83 (s,
1H), 7.20–7.48 (m, 10H), 5.09 (s, 2H), 4.49 (s, 2H), 2.06–2.07
(m, 2H), 1.68–1.77 (m, 2H), 1.12–1.27 (m, 2H). [M + H]+:
507.5.
Compound 10: To a solution of compound 9’ (50.0 mg, 1
equiv) in DMF (1.0 mL) at 0 °C, was added sodium hydride
(11.9 mg, 1.1 equiv). After stirring at 0 °C for 2 h, a solution of
compound 9 (169.0 mg, 1 equiv) in DMF (0.3 mL) was added
within 1 min. The resulting mixture was stirred for 25 min, then
quenched with ammonium chloride The mixture was extracted
with ethyl acetate, dried, concentrated and purified by column
chromatography on silica gel to yield compound 10 (90.0 mg,
46%). 1H NMR (400 MHz, CDCl3): δ 13.30 (brs, 1H), 12.20 (s,
1H), 7.91 (s, 1H), 7.27–7.36 (m, 10H), 5.09–5.17 (m, 2H),
4.80–4.96 (m, 5H), 3.20–3.28 (m, 2H), 2.01–2.19 (m, 2H),
1.75–1.88 (m, 2H). [M + H]+: 655.9.
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Beilstein J. Org. Chem. 2010, 6, 742–747.
9. Iyengar, R.; Schildknegt, K.; Morton, M.; Aube, J. J. Org. Chem. 2005,
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Preparation of compound 13
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License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Compound 13: To a mixture of compound 12 (30.0 mg) and
DCM (10.0 mL) at −70 oC, was added a solution of BBr3 (130.0
mg) in DCM (1.0 mL). The mixture was allowed to warm to
room temperature, stirred for one additional hour and then
poured into iced-water. The aqueous layer was washed with
dichloromethane and adjusted to pH 3–4. The remaining
aqueous solution was concentrated in vacuum, and the residue
was washed with THF. The THF layer was concentrated and
purified by preparative HPLC to give the product (5.0 mg,
25%). 1H NMR (400 MHz, CDCl3): δ 12.80 (brs, 1H), 12.21 (s,
1H), 10.06 (s, 0.5H), 9.81 (brs, 0.5H), 8.36 (s, 0.5H), 8.02 (s,
0.5H), 7.93 (s, 1H), 4.74–4.78 (m, 0.5H), 4.49–4.52 (m, 0.5H),
3.22–3.27 (m, 2H), 1.88–1.95 (m, 2H), 1.62–1.70 (m, 2H). [M
+ H]+: 328.3.
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.6.93
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