Synthesis of a protected keto-lysidine analogue via improved preparation of arabino-isocytosine nucleosides

Article abstract of DOI:10.1021/acs.orglett.9b00086

Anhydrouridines react with aliphatic amines to give N-alkyl isocytosines, but reported procedures often demand very long reaction times and can be low yielding, with narrow scope. A modified procedure for such reactions has been developed, using microwave

Full text of DOI:10.1021/acs.orglett.9b00086

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of a Protected keto-Lysidine Analogue via Improved
Preparation of Arabino-isoCytosine Nucleosides
§
‡
∥
‡
Joseph B. Sweeney,*,† Paul A. Bethel, Duncan M. Gill, Agata M. Ochocinska, Anthony E. J. Walsh,
́
and Scarlett M. Walton‡
†Department of Chemistry, Lancaster University, Lancaster LA1 4YB, U.K.
‡Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, West
Yorkshire HD1 3DH, U.K.
§AstraZeneca Pharmaceutical Development, Global Chemical Development S46/4, Etherow, Silk Road Business Park,
Macclesfield SK10 2NA, U.K.
∥School of Chemistry, Food and Pharmacy, University of Reading, PO Box 224, Whiteknights, Reading RG6 6AP, U.K.
S
* Supporting Information
ABSTRACT: Anhydrouridines react with aliphatic amines to give N-alkyl isocytosines, but reported procedures often demand
very long reaction times and can be low yielding, with narrow scope. A modified procedure for such reactions has been
developed, using microwave irradiation, significantly reducing reaction time and allowing facile access to a diverse range of novel
nucleosides on the gram scale. The method has been used to prepare a precursor to a novel analogue of lysidine, a naturally
t
occurring iminonucleoside found in RNA.
motifs, compounds rarely reported in the literature, we sought a
synthetic entry to arabino-configured isocytosines 4.
oncoded (“unnatural”) nucleoside analogues are priv-
Nileged chemical motifs, possessing a diverse range of bio-
logical and clinical properties. Most nucleoside drug candidates
contain modified heterocyclic components; however, modifica-
tion of the native ribose core can also confer significant bio-
logical activity, and 2′-modified nucleosides are found at the
heart of marketed antisense oligonucleotide1 medications,
including mipomersen (Kynamro, used to treat homozygous
familial hypercholesterolemia) and nusinersen (Spinraza, for
spinal muscular atrophy). 2′-Modified mononucleosides also
exhibit biological potency, and arabino-configured nucleosides
Cytarabine (ara-C 1, acute myeloid and lymphocytic leukemias,
and lymphomas; Figure 1), Clevudine (2, hepatitis B), and
Fludarabine (3, chronic lymphocytic leukemia, non-Hodgkin
lymphoma) are used in the clinic, leading to great interest in
methods for synthesis of this class of compound.2,3 In addition
to therapeutic significance, modified nucleosides are also of
great utility as structural probes and as chemical start points for
functional synthetic polynucleotides. Arabino-configured anti-
sense systems have been prepared and shown to possess inter-
esting properties,4 and aminated arabino-isocytosines 4 are also
biologically active nucleoside analogues, possessing anticancer
activity,5 and as precursors to polynucleotides able to form
duplexes with isoG-containing sequences.6 As part of a research
program directed toward synthesis and testing of novel catalytic
polynucleotides possessing both modified base and ribose
In addition, for similar purposes, we also sought access to
t
arabino-configured analogues of the naturally occurring RNA
nucleoside Lysidine 5, as potential inhibitors of lysidine
synthetase.7
We envisaged entry to arabino-isocytosines, including the
previously unreported lysidine analogue 6, by means of ring
opening of 2,3′-anhydrouridines 7 by aliphatic amines (Figure 2):
thus, reaction of a suitably protected lysine 8 would directly give
the protected lysidine analogue. However, we found existing
methods capricious and low yielding and so sought an alternative
entry to these noncoded nucleoside analogues; we report here a
modified procedure which ameliorates these problems, delivering
the target nucleosides on the gram scale and representing a
significant advance over the existing methodology.
The ring opening of anhydrouridine by ammonia was first
reported by Todd et al.;8 the method was later refined9 and
broadened in scope, to include higher amines.10 In general, the
ring opening of anhydrouridine with alkylamines to give ara-
isocytosines is a slow process,11 with reactions typically taking
several days to reach completion (e.g., ring opening of anhydro-
uridine by cyclohexylamine takes 32 days to reach completion10).
Moreover, the isolation of aminated products is inefficient, with
Received: January 8, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b00086
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. continued
reaction
time/h product yield/%
7
R
conditions
7c12
Me THF 10 equiv of amine,
1
4ca
4bb
4cb
4cb
4cb
4db
65
μW, 80 °C
7b
7c
nBu THF 10 equiv of amine,
1
mixture
μW, 120 °C
nBu THF 10 equiv of amine,
0.5
0.5
0.5
1
55
0
μW, 80 °C
7c
nBu THF 10 equiv of amine,
μW, 120 °C
7c
nBu THF 10 equiv of amine,
0
ZnCl2, μW, 120 °C
7d13
nBu THF 10 equiv of amine,
68
μW, 80 °C
a
b
Ref 10. Yield of corresponding triacetate.
Table 2. Microwave-Mediated Anhydrouridine Amination:
Yield Comparison
Figure 1. arabino-Nucleosides: privileged chemical motifs
reaction
time/h
7
R
conditions
product yield/%
a
7a
7d
nBu
nBu
DMF, 7 equiv of amine, rt10
36
4ab
4db
71
THF 10 equiv of amine,
1
68
μW, 80 °C
a
7a
7d
allyl
allyl
DMF, 7 equiv of amine, rt10
48
1
4ac
4dc
77
THF 10 equiv of amine,
μW, 80 °C
66
a
7a
7d
benzyl DMF, 7 equiv of amine, rt10
benzyl THF 10 equiv of amine,
180
1
4ad
4dd
90
56
Figure 2. Arabino-ketolysidine synthesis: strategy.
μW, 80 °C
a
7a
7d
cHex
cHex
DMF, 7 equiv of amine, rt10
384
4ae
4de
72
Table 1. Nucleoside Amination by Ring Opening of
Anhydrouridine: Traditional vs Microwave Conditions
THF 10 equiv of amine,
μW, 80 °C
1
51
a
Yield of corresponding triacetate.
postreaction derivatization of the unprotected product neces-
sary to allow reasonable yields to be achieved.8 Prior protection
of the 5′-hydroxyl group directly delivers tractable products but
does not noticeably improve the rate of reaction. The use of
microwave irradiation to accelerate reactions, in particular those
performed in polar solvents, is a contemporary paradigm, and we
envisaged that this would be a valuable method to accelerate the
ring opening of anhydronucleosides by amines. After a screen of
reaction conditions, we were, therefore, gratified to observe that
n
anhydrouridines 7b−d reacted with methylamine and butyl-
amine in THF at 80 °C at 300 W to give the aminated products,
arabino-isocytosines 4ba, 4ca, 4cb, and 4db, in good yields, after
only 1 h (Table 1). The reaction with methylamine had not been
reported in the literature, presumably due to the volatility of the
amine component.
Although the ring-opened products proved stable to chro-
matography, showing little tendency to return to the anhydro
reaction
7
R
conditions
time/h product yield/%
7a
Me DMF, 7 equiv of amine,
rt
nBu DMF, 7 equiv amine, rt
N/A
4aa
N/A
a
10
a
b
7a
7b12
72
1
4ab
4ba
71
Me THF 10 equiv of amine,
89
μW, 80 °C
B
DOI: 10.1021/acs.orglett.9b00086
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Thus, the microwave-mediated procedure reduced the
reaction time by 97%; when compared to the reported proce-
dure10 using other amines, the increase in the rate of reaction
was even more striking (Table 2). The use of THF rather than a
high-boiling polar solvent (such as DMF10) also represents a
significant simplification in the process.
The process can be applied to a range of aliphatic amines
(Figure 3), delivering arabino-2-(alkyl)amino isocytosines in
good yields. The simplicity of the method enables gram-scale
reactions to be carried out in short order, and the ability to
isolate pure products directly from the reactions enables rapid
and convenient access to novel nucleosides.
Armed with a new and efficient entry into 2-(alkyl)amino
isocytosine nucleosides, we turned our attention to our original
target molecule, ketocytosine analogue 6, and we were delighted
to observe that reaction of lysine derivative 8 with anhydrour-
idine 7d delivered the key product 9 (a protected precursor to 6)
in excellent yield (Scheme 1).
In summary, we have reported an improved method for
amination of 2,2′-anhydrouridine, enabling a practical and high
yielding entry to a range of 2-aminated nucleosides. We are
currently engaged in the use of this technology to deliver novel
analogues of bioactive nucleosides.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.9b00086.
Experimental details and associated spectral data (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: j.sweeney1@lancaster.ac.uk.
ORCID
Joseph B. Sweeney: 0000-0001-7593-9598
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank EPSRC, AstraZeneca, and the Universities of
Huddersfield and Reading for funding (studentships to A. E. J.
W., S. M. W., and A. M. O., respectively). J. B. S. thanks the Royal
Society for provision of an Industry Fellowship.
Figure 3. Amination of 5′-O-TBDPS-2,2′-anhydrouridine: scope.
Scheme 1. Synthesis of Novel Ketolysidine Derivative 9
REFERENCES
■
(1) Khvorova, A.; Watts, J. K. Nat. Biotechnol. 2017, 35, 238−248.
(2) For recent synthetic activity in the area, see: Dostie, S.; Prevost,
́
M.; Guindon, Y. J. Org. Chem. 2012, 77, 7176−7186.
(3) Ewald, B.; Sampath, D.; Plunkett, W. Oncogene 2008, 27, 6522−
6537.
(4) (a) Resmini, M.; Pfleiderer, W. Helv. Chim. Acta 1993, 76, 158.
(b) Damha, M. J.; Wilds, C. J.; Noronha, A.; Brukner, I.; Borkow, G.;
Arion, D.; Parniak, M. A. J. Am. Chem. Soc. 1998, 120, 12976−12977.
(c) Li, F.; Sarkhel, S.; Wilds, C. J.; Wawrzak, Z.; Prakash, T. P.;
Manoharan, M.; Egli, M. Biochemistry 2006, 45, 4141−4152.
̌
́
̌
́
̌
́
(5) Krizmanic, I.; Visnjevac, A.; Luic, M.; Glavas-Obrovac, L.; Zinic,
̌
́
M.; Zinic, B. Tetrahedron 2003, 59, 4047−4057.
(6) Ozaki, H.; Nakajima, K.; Tatsui, K.; Izumi, C.; Kuwahara, M.;
Sawai, H. Bioorg. Med. Chem. Lett. 2003, 13, 2441−2443.
(7) (a) Suzuki, T.; Miyauchi, K. FEBS Lett. 2010, 584, 272−277.
(b) Shapiro, A. B.; Plant, H.; Walsh, J.; Sylvester, M.; Hu, J.; Gao, N.;
starting material, the products could more conveniently be
isolated by precipitation from the crude reaction medium.
C
DOI: 10.1021/acs.orglett.9b00086
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Livchak, S.; Tentarelli, S.; Thresher, J. J. Biomol. Screening 2014, 19,
1137−1146.
(8) Brown, D. M.; Parihar, D. B.; Todd, A. R.; Varadarjan, S. J. Chem.
Soc. 1958, 0, 3028−3035.
(9) Doerr, I. L.; Fox, J. J. J. Org. Chem. 1967, 32, 1462−1471.
́
(10) Delia, T. J.; Beranek, J. J. Carbohydr., Nucleosides Nucleotides
1977, 4, 349−362.
(11) See, for instance: Ozaki, H.; Nakajima, K.; Tatsui, K.; Izumi, C.;
Kuwahara, M.; Sawai, H. Bioorg. Med. Chem. Lett. 2003, 13, 2441−
2443.
(12) Ferreri, C.; Costantino, C.; Romeo, R.; Chatgilialoglu, C.
Tetrahedron Lett. 1999, 40, 1197−1200.
(13) Sebesta, D. P.; Rourke, S. S. O.; Martinez, R. L.; Piekcn, W. A.;
McGee, D. P. C. Tetrahedron 1996, 52, 14385−14402.
D
DOI: 10.1021/acs.orglett.9b00086
Org. Lett. XXXX, XXX, XXX−XXX