Mitsunobu Alkylation of Cancerostatic 5-Fluorouridine with (2E)-10-Hydroxydec-2-enoic Acid, a Fatty Acid from Royal Jelly with Multiple Biological Activities

Article abstract of DOI:10.1002/cbdv.201500048

5-Fluorouridine (1) - a nucleoside antimetabolite with strong cancerostatic properties - was protected i) at the 2′- and 3′-OH groups with a heptan-4-ylidene residue and ii) at the 5′-OH group with a (4-methoxyphenyl)(diphenyl)methyl residue. This fully protected compound, 3, was submitted to a Mitsunobu reaction with the N-hydroxysuccinimide (NHS) ester, 5, of (2E)-10-hydroxydec-2-enoic acid (4) which gave nucleolipid 6. The latter was detritylated with Cl₂CHCOOH to yield the co-drug 7 as NHS ester.

Full text of DOI:10.1002/cbdv.201500048

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Mitsunobu Alkylation of Cancerostatic 5-Fluorouridine with (2E)-10-
Hydroxydec-2-enoic Acid, a Fatty Acid from Royal Jelly with Multiple
Biological Activities
by Vanessa Ottenhaus and Helmut Rosemeyer*
Organic Chemistry I – Bioorganic Chemistry, Institute of Chemistry of New Materials, University of
Osnabrück, Barbarastr. 7, DE-49069 Osnabrück (e-mail: Helmut.Rosemeyer@uos.de)
Dedicated to Prof. Dr. Zygmunt Kazimierczuk, Warsaw, on the occasion of his 70th birthday
5-Fluorouridine (1) – a nucleoside antimetabolite with strong cancerostatic properties – was
protected i) at the 2’- and 3’-OH groups with a heptan-4-ylidene residue and ii) at the 5’-OH group with
a (4-methoxyphenyl)(diphenyl)methyl residue. This fully protected compound, 3, was submitted to a
Mitsunobu reaction with the N-hydroxysuccinimide (NHS) ester, 5, of (2E)-10-hydroxydec-2-enoic acid
(4) which gave nucleolipid 6. The latter was detritylated with Cl2CHCOOH to yield the co-drug 7 as NHS
ester.
Introduction. – The transport of a ꢀsmall moleculeꢁ drug across a target cell
membrane can occur by four different mechanisms: i) by passive transport, ii) by
carrier-mediated passive transport, iii) by active transport, and iv) by corpuscular
absorption (phagocytosis or pinocytosis). If a pharmacologically active compound is
not taken up by an active transport mechanism, its internalization into a target cell
depends strongly on its lipophilicity. This is usually characterized by its partition
coefficient P between octan-1-ol and H2O; these data are often given as log10Pow
values [1].
According to ꢀLipinskiꢁs Rule of Fiveꢁ, the log10Pow value should be ꢀ5 to exhibit a
good oral bioavailability [2][3]. In a series of articles, we reported the synthesis of
hybrid molecules of both, canonical nucleosides, such as uridine, 5-methyluridine,
thymidine, and inosine, as well as of nucleoside antimetabolites, e.g., 5-fluorouridine
and 6-azauridine, with various kinds of lipids [4–11].
In this article, we describe the preparation of a conjugate between 5-fluorouridine
(1) [12–22] and (2E)-10-hydroxydec-2-enoic acid (HDEA; 4), a fatty acid from royal
jelly which is fed to the queen bee [23]. The fatty acid HDEA exhibits several
outstanding biomedical properties. For example, it increases the generation of neurons
and decreases that of astrocytes from neural stem/progenitor cells (NSCs) which have
self-renewal capacity and multipotent activity to differentiate into neurons, astrocytes,
and oligodendrocytes during development. This observation suggests that royal jelly
contains compounds that differently influence neuronal and/or glial lineages [24], and
that HDEA is one of such compounds of royal jelly that facilitates neurogenesis by
NSCs similar to docosahexaenoic acid.
ꢂ 2015 Verlag Helvetica Chimica Acta AG, Zürich

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Moreover, HDEA has been reported to show immune-modulatory properties [25],
antitumor activity [26], collagen production-promoting activity [27], as well as
antibiotic activity [28], a finding which is not really surprising, since royal jelly would
otherwise be an excellent substrate for bacterial growth. Very recently, Herdewijn and
co-workers used a- and b-OH as well as a-amino fatty acids for the synthesis of
nucleolipids with different nucleobases and different sugar moieties and investigated
these new co-drugs for their anti-HIV activities [29–31].
Results and Discussion. – In an earlier study, we have shown that a Mitsunobu
alkylation of a pyrimidine nucleoside occurs only then swimmingly and without
formation of many side products, if all glyconic OH groups are protected (Scheme)
[7][8].
Therefore, 5-fluorouridine (1) was first reacted with heptan-4-one to the O-2’,3’-
heptylidene derivative, 2, which was subsequently protected at the 5’-OH group with a
(4-methoxyphenyl)(diphenyl)methyl (MMTr) residue to give 3.
In the following, HDEA (4) was reacted with N-hydroxysuccinimide (NHS) to give
NHS ester 5. Compound 5 was then submitted to a Mitsunobu reaction with 3 to give
the highly lipophilic nucleolipid 6. The latter was detritylated with 4% Cl2CHCOOH
solution in CH2Cl2 (10 min, r.t.). Upon this reaction, several by-products were formed
so that 7 could be isolated only in moderate yield (11%). The latter compound
represents a novel co-drug combining two molecules with completely different
pharmacological activities. Further reactions of 7 with a fluorescent label at the
NHS ester moiety, as well as phosphitylation at 5’-position, will be published else-
where.
The Figure displays the stepwise increase of lipophilicity of the compounds during
the synthesis of 7 – expressed in terms of log10Pow values.
1
All novel substances were characterized by H- and 13C-NMR spectroscopy, using
DEPT-135 spectra for the assignment of 13C resonances, as well as by ESI-MS.
Conclusions. – Only a fully protected nucleoside, such as 3, can be regioselectively
N-alkylated at the base with an w-OH fatty acid NHS ester under Mitsunobu condi-
tions.
The authors gratefully acknowledge the financial support of the BBraun AG (Melsungen,
Germany), as well as the Bundesministerium für Wirtschaft (FKZ KF 2369401 S B9 and FKZ
2369501 S B9). We thank Prof. Dr. Uwe Beginn (Organic Materials Chemistry, University of Osnabrück)
for excellent laboratory facilities. Moreover, we thank Mrs. Marianne Gather-Steckhan for NMR spectra
and Dr. Stefan Walter for ESI-MS.
Experimental Part
General. All chemicals were purchased from SigmaÀAldrich (Deisenhofen, Germany) or TCI –
Europe (Zwijndrecht, Belgium). Solvents were of laboratory grade and were distilled before use.
Compounds 2 and 3 were prepared according to [9]; they were identical in all respects to authentic
samples. Thin-layer chromatography (TLC): aluminum sheets, silica gel 60 F254 (SiO2; 0.2 mm; Merck,
1
Germany). Column chromatography (CC): SiO2 60 (2Â20 cm). H- and 13C-NMR spectra: AMX-500
(Bruker, Rheinstetten, Germany; 500.14, and 125.76 MHz, resp.); in (D6)DMSO; d in ppm rel. to Me4Si
as internal standard, J in Hz. ESI-MS: Bruker Daltonics Esquire HCT instrument (Bruker Daltonics,

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1309
Scheme
a) Heptan-4-one, (EtO)3CH, 4m HCl in 1,4-dioxane, DMF. b) MMTrCl, pyridine. c) NHS, DCC, DMF.
d) Benzene, DIAD, Ph3P. e) 4% Cl2CHCOOH in CH2Cl2.

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Figure. log10Pow Values of 1–7
Leipzig, Germany); ionization with 2% aq. HCOOH soln.; in m/z. log10Pow Values were calculated using
the http://eadmet.com/de/physprop.php site with ePhysChem that contains ALOGPS v.3.0.
1-{[(2E)-10-Hydroxydec-2-enoyl]oxy}pyrrolidine-2,5-dione (5). HDEA (4; 745 mg, 4 mmol) and
NHS (461 mg, 4 mmol) were dissolved in DMF and cooled to 58. Then, N,N’-dicyclohexylcarbodiimide
(DCC; 824 mg, 4 mmol) was added, and the mixture was stirred for 24 h at ambient temp. Precipitated
1,3-dicyclohexylurea was filtered off, and the filtrate was evaporated to dryness and subsequently dried in
vacuo. The residue was suspended in CH2Cl2, residual 1,3-dicyclohexylurea was filtered off, and the
i
filtrate was evaporated to dryness. Product 5 was crystallized from PrOH. Yield: 590 mg (52%). Rf
1
3
3
3
(CH2Cl2/MeOH 98 :2) 0.36. H-NMR: 7.25 (dt, J(8,7)¼15.8, J(8,9)¼7.0, HÀC(8)); 6.19 (d, J(7,8)¼
15.8, HÀC(7)); 4.27 (t, 3J(HO,15)¼2.5, OH); 3.39–3.36 (m, CH2(15)); 2.82 (s, CH2(3,4)); 2.31 (Yq,
3J(9,8)¼7.0, 3J(9,10)¼3.3, CH2(9)); 1.48–1.40 (m, CH2(13,14)); 1.29 (br. s, CH2(10, 11, 12)). 13C-NMR:
170.19 (C(2,5)); 161.34 (C(6)); 156.57 (C(7)); 115.11 (C(8)); 60.60 (C(15)); 32.39 (C(9)); 31.97 (C(14));
28.51, 26.90, 25.37, 25.35 (4 CH2). ESI-MS: 283.59 ([MþH]þ , C14H21NO5þ ; calc. 283.14), 567.18
([2MþH]þ ).
5-Fluoro-5’-O-[(4-methoxyphenyl)(diphenyl)methyl]-3-{(8E)-10-oxo-10-[(2,5-dioxopyrrolidin-1-
yl)oxy]dec-8-en-1-yl}-2’,3’-O-(1-propylbutylidene)uridine (6). Ph3P (262 mg, 1 mmol) was added to a
clear soln. of 3 (750 mg, 1.2 mmol) and 5 (280 mg, 1 mmol) in THF (7 ml) under stirring at ambient temp.
Then, the mixture was warmed to 458 for 2 min. Subsequently, diisopropylazodicarboxylate (DIAD;
200 mg, 1 mmol) was added, and the mixture was stirred overnight at r.t. After evaporation of the
i
solvent, the residue was dissolved in PrOH, and the soln. was cooled to À208, whereupon a slightly
1
yellowish solid precipitated. Yield: 890 mg (97%). Rf (CH2Cl2/MeOH 98 :2) 0.76. H-NMR: 8.17 (d,
3J(6,F)¼5.0, HÀC(6)); 7.66–7.54 (m, HÀC(8’’’’)); 7.41–7.15 (m, 4 HÀC(3’’’), 4 HÀC(4’’’), 2 HÀC(5’’’),
3
2 HÀC(7’’’)); 6.86–6.82 (m, 2 HÀC(8’’’)); 6.19 (d, J(9’’’’,8’’’’)¼15.0, HÀC(9’’’’)); 5.81 (br. s, HÀC(1’));
5.06–4.97 (m, HÀC(2’)); 4.77–4.63 (m, HÀC(3’)); 4.26–4.17 (m, HÀC(4’)); 3.72 (s, Me(10’’’)); 3.69–3.61
(m, CH2(1’’’’)); 3.13–3.04 (m, CH2(5’)); 2.82 (br. s, CH2(12’’’’,13’’’’)); 2.37–2.24 (m, CH2(7’’’’)); 1.70–1.59
(m, CH2(2’’)endo); 1.53–1.41 (m, CH2(2’’)exo); 1.39–1.11 (m, 2 CH2(3’’), CH2(2’’’’–6’’’’)); 0.91 (t,
3J(4’’endo,3’’endo)¼7.5, Me(4’’)endo)); 0.84 (t, 3J(4’’exo,3’’exo)¼7.5, Me(4’’)exo)). 13C-NMR: 170.17
(C(11’’’’,14’’’’)); 161.34 (C(10’’’’)); 158.13 (C(9’’’)); 156.49 (C(8’’’’)); 156.30 (d, 2J(F,4)¼26.4, C(4));
148.67 (C(2)); 144.02, 138.36 (2 C(2’’’)); 142.74 (d, 1J(F,5)¼303.0, C(5)); 134.72 (C(6’’’)); 131.41, 131.33,
129.83, 128.68, 128.58, 127.87, 127.77, 127.68, 126.81, 126.75 (10 C (MMTr)); 126.24 (d, 2J(F,6)¼34.0,

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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C(6)); 116.56 (C(1’’)); 115.11 (C(9’’’’)); 113.10, 113.05 (2 C(8’’’)); 93.83 (C(4’)); 86.54 (C(1’)); 85.86
(C(1’’’)); 83.75 (C(2’)); 80.95 (C(3’)); 64.32 (C(5’)); 54.88 (C(10’’’)); 40.97 (C(1’’’’)); 38.64 (C(2’’)endo);
38.51 (C(2’’)exo); 31.95 (C(7’’’’)); 28.34 (C(5’’’’)); 28.25 (C(6’’’’)); 26.88 (C(4’’’’)); 26.52 (C(2’’’’)); 26.03
(C(3’’’’)); 25.32 (C(12’’’’,13’’’’)); 16.87 (C(3’’)endo); 16.20 (C(3’’)exo); 14.01 (2 C(4’’)). ESI-MS: 936.3 ([Mþ
Na]þ , C51H62FN3O 1þ1 ; calc. 912.05).
5-Fluoro-3-{(8E)-10-oxo-10-[(2,5-dioxopyrrolidin-1-yl)oxy]dec-8-en-1-yl}-2’,3’-O-(1-propylbutyli-
dene)uridine (7). Compound 6 (400 mg, 0.44 mmol) was dissolved in CH2Cl2 (9 ml). After addition of
4% Cl2CHCOOH soln. in CH2Cl2 (6 l), the mixture was stirred at r.t. for 15 min and then neutralized by
addition of 5% aq. NaHCO3 soln. The org. layer was separated, dried (Na2SO4), filtered, and evaporated
to dryness. CC (SiO2 60; CH2Cl2/MeOH 98 :2) gave 7 (50 mg, 11%) after evaporation of the solvent and
2
drying in vacuo. Rf (CH2Cl2/MeOH 98 :2) 0.24. 1H-NMR: 8.22 (d, J(F,6)¼7.0, HÀC(6)); 7.27–7.24 (m,
HÀC(8’’’’)); 6.19 (d, 3J(9’’’’,8’’’’)¼16.0, HÀC(9’’’’)); 5.88 (s, HÀC(1’)); 4.91–4.90 (m, HÀC(2’)); 4.77–4.77
(m, HÀC(3’)); 4.15–4.14 (m, HÀC(4’)); 3.79–3.74 (m, CH2(1’’’’)); 3.66–3.51 (m, CH2(5’)); 2.83 (s,
CH2(12’’’’,13’’’’)); 2.33–2.27 (m, CH2(7’’’’)); 1.68–1.64 (m, CH2(2’’)endo); 1.55–1.35 (m, CH2(2’’)exo
,
3
CH2(2’’’’,6’’’’)); 1.35–1.18 (m, 2 CH2(3’’), CH2(3’’’’–5’’’’)); 0.92 (t, J(4’’endo,3’’endo)¼7.0, Me(4’’)endo); 0.87
(t, J(4’’exo,3’’exo)¼7.0, Me(4’’)exo)). 13C-NMR: 169.49 (C(11’’’’,14’’’’)); 161.63 (C(10’’’’)); 156.30 (C(8’’’’));
3
155.29 (C(4)); 149.64 (C(2)); 142.60 (d, 1J(F,5)¼325.0, C(5)); 124.93 (C(6)); 118.50 (C(1’’)); 115.63
(C(9’’’’)); 96.33 (C(4’)); 87.26 (C(1’)); 84.62 (C(2’)); 80.66 (C(3’)); 63.08 (C(5’)); 42.17 (C(1’’’’)); 39.52
(C(2’’)endo); 39.48 (C(2’’)exo); 33.03 (C(7’’’’)); 29.18 (C(5’’’’)); 29.07 (C(6’’’’); 28.93 (C(4’’’’)); 27.70 (C(2’’’’));
27.53 (C(3’’’’)); 26.84 (C(12’’’’)); 25.85 (C(13’’’’)); 17.70 (C(3’’)endo); 17.10 (C(3’’)exo); 14.51 (C(4’’)endo);
14.47 (C(4’’)exo). ESI-MS: 624.26 ([MþH]þ , C30H43FN3O1þ0 ; calc. 624.29).
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Received February 10, 2015