Facile rearrangement of N4-(α-aminoacyl)cytidines to N-(4-cytidinyl)amino acid amides

Article abstract of DOI:10.1016/j.tetlet.2008.02.015

Under near neutral and mildly basic conditions, primary N⁴-(α-aminoacyl)cytidines (4a-g) undergo a facile rearrangement to form N-(4-cytidinyl)amino acid amides (5a-g). Secondary aminoacyl derivatives rearrange with other competing pathways. Tertiary aminoacyl derivatives do not rearrange.

Full text of DOI:10.1016/j.tetlet.2008.02.015

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 2052–2055
Facile rearrangement of N4-(a-aminoacyl)cytidines
to N-(4-cytidinyl)amino acid amides
Deyi Zhang *, David M. Bender, Frantz Victor, Jeffrey A. Peterson, Robert D. Boyer,
Gregory A. Stephenson, Adam Azman, James R. McCarthy *
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
Received 7 January 2008; accepted 4 February 2008
Available online 8 February 2008
Abstract
Under near neutral and mildly basic conditions, primary N4-(a-aminoacyl)cytidines (4a–g) undergo a facile rearrangement to form
N-(4-cytidinyl)amino acid amides (5a–g). Secondary aminoacyl derivatives rearrange with other competing pathways. Tertiary amino-
acyl derivatives do not rearrange.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Rearrangement; N4-(a-Aminoacyl)cytidines; N-(4-Cytidinyl)amino acid amides; Nucleoside
Nucleosides and their analogues are an important class
of anticancer and antiviral agents. However, poor biophar-
maceutical properties and an unfavorable therapeutic index
often limit their use as therapeutic agents.1 Recently, pro-
drug strategies have been utilized to improve the pharma-
cological properties of nucleoside analogues and to
reduce toxic side effects of the parent molecules.2 Amidon
and co-workers have prepared amino acid ester prodrugs
of various nucleoside analogues.3 Wipf reported the syn-
thesis of N4-peptidyl-ara-C derivatives as chemoreversible
prodrugs of ara-C.4 In the 1960 s, Chheda’s group reported
that N6-glycyladenine rapidly underwent a Dimroth rear-
rangement to N-(6-purinyl)glycine in aqueous solution at
near neutral pH.5 They further demonstrated that 9-
alkyl-N6-(aminoacyl)adenine derivatives can undergo a
similar reaction to produce N-[6-(9-alkylpurinyl)]glycine.6
In contrast, Stella and co-workers7 reported in 1994 that
aminoacyl amide prodrugs of prazosin undergo an intra-
molecular rearrangement involving the a-amine of the
amino acid forming a spiro intermediate that opens to
the a-alkyl amides. No citations of this report have
appeared in the literature. However, two publications8
and three patents9 on aminoacyl amides of cytosine nucleo-
sides isolated as HCl or TFA salts have appeared.
Amidon3d recently published the synthesis of water sol-
uble amino acid ester prodrugs of gemcitabine (GemzarÒ)
(1), an effective anticancer drug widely used for pancreatic
and non-small cell lung cancers.10 Due to our interest in
potentially more stable water soluble prodrugs of gemcita-
bine, a number of aminoacyl amides of gemcitabine were
selected to synthesize. The results reported by Stella and
co-workers7 were of interest and in contrast to the reports
on the aminoacyl amides of cytosine.8,9 Therefore, we felt it
important to utilize X-ray data to confirm structures. The
synthesis of aminoacyl-gemcitabines 4a–g is outlined in
Scheme 1. Thus, the amino acid amides 3a–g were prepared
through a PyBOP/HOAt mediated selective acylation at
the N4-position of 1 using Boc-protected amino acids.
Removal of the Boc-protecting group with TFA/CH2Cl2
(1:1) afforded the TFA salts of the corresponding N4-(a-
aminoacyl)gemcitabines (4a–g). The L-proline derivative
4h was prepared in a similar fashion. To synthesize N4-
(N,N-dimethylphenylalanyl)gemcitabine 8 (Scheme 2), the
*
Corresponding authors. Tel.: +1 317 433 1591 (D.Z.); tel.: +1 317 433
1345; fax: +1 317 433 0552 (J.R.M.).
E-mail addresses: zhang_deyi@lilly.com (D. Zhang), jmccarthy@lilly.
com (J. R. McCarthy).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.02.015

D. Zhang et al. / Tetrahedron Letters 49 (2008) 2052–2055
2053
R1 R2
H
N
NH2
NHBoc
O
a
O
O
+
N
F
N
N
F
N
O
HO
HO
NHBoc
R1 R2
2a-g
HO
O
O
F
F
HO
HO
3a-g
1
a: R1 = R2 = H
e: R1 = Bn, R2 = H
f: R1 = R2 = Me
g: R1,R2 = CH2CH2
b: R1 = Me, R2 = H
c: R1 = i-Pr, R2 = H
d: R1 = t-Bu, R2 = H
O
R1 R2
H
H
N
N
NH2
NH2 TFA
.
O
b
O
c
R2 R1
N
N
F
N
F
N
O
HO
HO
O
O
F
HO
F
HO
5a-g
4a-g
H
N
N
N
H
.TFA
O
O
c
N
F
N
N
N
O
NH2
HO
HO
O
F
O
O
F
F
HO
HO
4h
5h
Scheme 1. Reagents: (a) PyBOP, HOAt, Et3N, DMF; (b) TFA, CH2Cl2 (1:1); (c) saturated aqueous NaHCO3, MeOH (1:1, v/v).
NH2
NH2
O
O
a
b
N
F
N
N
N
BocO
BocO
HO
F
O
O
F
F
HO
1
6
Ph
N
Ph
N
H
N
H
N
O
O
c
N
F
N
O
N
N
O
HO
BocO
BocO
TFA
F
O
O
F
HO
F
7
8
Scheme 2. Reagents: (a) (i) dioxane, 1 M aqueous KOH (1:1, v/v), Boc2O (5 equiv); (ii) dioxane, Boc2O (5 equiv), then 1 M aqueous KOH (1:1 to
dioxane); (b) PyBOP, HOAt, Et3N, DMF; (c) TFA, CH2Cl2 (1:1, v/v).
30 and 50-hydroxy groups of gemcitabine were selectively
protected to obtain compound 6. Coupling of 6 with
N,N-dimethylphenylalanine under PyBOP/HOAt condi-
tions afforded amide 7. Removal of the protecting group
provided 8 as the TFA salt.
Interestingly, compound 4d with the bulky t-butyl group
was 100% degraded at pH 8 at 40 °C in 4 h to a water insol-
uble product. While some amide hydrolysis to gemcitabine
occurred under acidic conditions (pH 1–2), the observed
degradation product at pH 8 was inconsistent with simple
hydrolysis to gemicitabine. Comparison of the UV spectra
of this product and 4d indicated a change in electron den-
sity in the cytosine moiety (Fig. 1). Treatment of 4d with a
mixture of saturated aqueous NaHCO3 and MeOH
(Scheme 1) provided a single new compound (5d) with
the same molecular weight as 4d that exhibited a batho-
chromic shift in the UV spectrum (see Fig. 1).
1
The structure of 5d was assigned based on both 1-D H
and 2-D homonuclear 1H and heteronuclear 1H–13C NMR
experiments.11 The significant long-range correlations
observed in the HMBC experiment are also shown in
Figure. 2. A single crystal X-ray on the HCl salt of 5d
confirmed the structure (Fig. 3).12 Similar studies using
aqueous NaHCO3–MeOH were performed on additional
N4-(aminoacyl)gemcitabines and the results are summa-
rized in Table 1. Compounds 4a–h were found to undergo
rearrangement to 5a–5h. The amide bond on the glycyl
analog 4a was very labile. Products arising from both

2054
D. Zhang et al. / Tetrahedron Letters 49 (2008) 2052–2055
hydrolysis to gemcitabine and rearrangement to 5a were
observed. Increasing the steric bulk at the a-position
(4b–g) improved the hydrolytic stability of the aminoacyl
derivatives and afforded the rearrangement product in
good yields (64–81%) with the exception of a,a-dimethyl
glycyl amide (4f) that provided 5f in 52% yield.
It is interesting to note that the proline derivative 4h
underwent rearrangement similar to the open-chain ana-
logs providing 5h, albeit in 38% yield. The N,N-dimethyl
derivative 8, when subjected to the same reaction condi-
tions was found to not undergo rearrangement but instead
degrades to several products including gemcitabine. These
results indicate the involvement of the amino group in
the N4-(a-aminoacyl)-gemcitabine degradation process.
Compounds 4a–4g that contain a free primary a-amino
group primarily followed the rearrangement pathway.
Under the reaction conditions, variation in the pKa values
of the amino acids was not a major factor as demonstrated
by the conversion of 4g–5g.
Fig. 1. Overlay of UV spectra of 4d and 5d.
However, steric effects do contribute to the change in
degradation pathways. Increasing the steric bulk of the a-
substituent facilitated the rearrangement while increasing
the bulkiness at the amino group (4h) slowed the rearrange-
ment process and instead, favored the hydrolytic pathway.
A mechanism for this rearrangement similar to the one
proposed by Stella and co-workers7 for the aminoacyl
prazosin analogs is in Scheme 3. The reaction involves a
nucleophilc attack of the free a-amino group in 4 at the
4-position of the cytidine ring, resulting in the displacement
of the amide nitrogen.
In summary, N4-(a-aminoacyl)gemcitabines are not
stable in solution and undergo rapid pH dependent degra-
dation. Under near neutral and basic conditions, primary
N4-(a-aminoacyl)-gemcitabines rearrange to novel N-(4-
gemcitabinyl)amino acid amides that are no longer of inter-
est as soluble prodrugs of gemcitabine. For secondary
gemcitabine derivatives rearrangement is accompanied by
amide hydrolysis. At low pH or when a tertiary amino acid
moiety is present, the hydrolytic pathway predominates.
This novel rearrangement process is germane to reports8,9
of aminoacylamide cytosine nucleosides and would limit
their in vivo utility. Equally important is the potential
applicability of this rearrangement to other aminoacyl
nucleosides.
Fig. 2. ORTEP drawing of 5d.
H
O
5
8
6
1
N
N
9
7
NH2
4
10
5'
11
O
1'
4'
3'
H
N 3
HO
2
12
F
13
O
2'
HO
F
Fig. 3. Structure of 5d with HMBC correlations.
Table 1
Results from rearrangement reactions
R2 R1
Product
Amino acid moiety
Rearrangement yield (%)
O
RHN
5a
5b
5c
5d
5e
5f
5g
5h
8
L-Glycyl
L-Alanyl
L-Valyl
L-t-Leucyl
L-Phenylalanyl
a,a-Dimethylglycyl
a-Aminocyclopropylcarboxyl
L-Prolyl
51
75
64
78
81
52
69
38
R
N
O
NH
NH2
O
O
N
N
R2 R1
N
N
F
HO
HO
F
O
O
F
HO
F
HO
5
4
N,N-Dimethyl-L-phenylalanyl
N/A
Scheme 3. Proposed mechanism for the rearrangement reaction.

D. Zhang et al. / Tetrahedron Letters 49 (2008) 2052–2055
2055
7. Pochopin, N. L.; Charman, W. N.; Stella, V. J. Int. J. Pharm. 1994,
105, 169.
Acknowledgements
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11. Compound 5d: 1H NMR (600, DMSO-d6) d 0.95 (s, 9, t-Bu), 3.61 (m,
1, H50a), 3.76 (m, 1, H50b), 3.78 (m, 1, H40), 4.14 (m, 1, H30), 4.60 (d,
1, J = 9.6 Hz, H8), 5.19 (t, 1, J = 5.5 Hz, 50-OH), 6.12 (t, 1,
J = 9.6 Hz, H10), 6.16 (d, 1, J = 7.6 Hz, H5), 6.22 (d, 1, J = 6.6 Hz,
30-OH), 7.08 (s, 2, 9-NH2), 7.62 (s, 1, 9-NH2), 7.65 (d, 1, J = 7.6 Hz,
H6), 7.85 (d, 1, J = 9.6 Hz, H7).
12. Crystallographic data (excluding structure factors) for 5d have been
deposited with the Cambridge Crystallographic Data Centre as
supplementary publication number CCDC 622780. Copies of the data
can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (fax: +44 (0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
The authors would like to thank Patric J. Hahn and
Robert Barbuch for helpful discussions on this work.
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