Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem CellsIn Vitro

Article abstract of DOI:10.1021/acs.jmedchem.0c02194

A 3′-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. Thein vitrocytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Full text of DOI:10.1021/acs.jmedchem.0c02194

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Single Diastereomers of the Clinical Anticancer ProTide Agents
NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In
Vitro
Magdalena Slusarczyk,* Michaela Serpi, Essam Ghazaly, Benson M. Kariuki, Christopher McGuigan,
and Chris Pepper*
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ABSTRACT: A 3′-protected route toward the synthesis of the
diastereomers of clinically active ProTides, NUC-1031 and NUC-
3373, is described. The in vitro cytotoxic activities of the individual
diastereomers were found to be similar to their diastereomeric
mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have
preferential cytotoxic effects on leukemic stem cells (LSCs). These
effects were not diastereomer-specific and were not observed with
the parental nucleoside analogues gemcitabine and FUDR,
respectively. In addition, NUC-1031 preferentially targeted LSCs
in primary AML samples and cancer stem cells in the prostate
cancer cell line, LNCaP. Although the mechanism for this remains
incompletely resolved, NUC-1031-treated cells showed increased
levels of triphosphate in both LSC and bulk tumor fractions. As
ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be
caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.
studies of NUC-1031 are currently ongoing: a Phase III study
in metastatic pancreatic carcinoma (ACELARATE) and a
Phase III study in locally advanced or metastatic BTC
(NuTide:121). Results from PRO-001 revealed that NUC-
1031 has a favorable pharmacokinetic profile with a long
plasma half-life (8.3 h versus 1.5 h) and can generate
intracellular levels of the active anticancer metabolite dFdCTP
that are 217-times higher than those generated by gemcitabine.
Furthermore, NUC-1031 achieved high levels of disease
control (78%) in patients who had exhausted all other
treatment options, including those with tumors relapsed/
refractory to prior chemotherapy with gemcitabine.14 In June
2019, Acelarin, the NUC-1031_SP isomer enriched API (98%
SP-isomer: 2% RP-isomer), received orphan drug designation
from the FDA for the treatment of BTC.15 Currently, the SP
isomer of NUC-1031 is being investigated in all NUC-1031
clinical studies.
INTRODUCTION
■
The phosphoramidate (ProTide) approach1 applied to
antiviral and anticancer nucleoside analogues (NAs), and
more recently also to non-nucleoside compounds,2−8 con-
tinues to be of significant interest in the drug discovery field.
This approach is used to overcome the limitations of NAs that
are known to restrict their therapeutic potential. In the
oncology arena, the application of ProTide technology to
gemcitabine and floxuridine (FUDR) led to the discovery and
clinical development of two anticancer agents, NUC-10319 and
NUC-337310 (Figure 1). The first-in-class agent, NUC-1031,
was designed to overcome the cancer cell resistance
mechanisms that limit the clinical utility of gemcitabine
(dFdC). The three key resistance mechanisms involve down-
regulation of nucleoside transporter proteins (hENT1); down-
regulation of the key initial phosphorylating enzyme
deoxycytidine kinase (dCK) that is required to activate
dFdC to the 5′-monophosphate form dFdCMP; and up-
regulation of the catabolizing enzyme cytidine deaminase
(CDA).11−13 NUC-1031 has been assessed in four completed
clinical studies, including a Phase I study in patients with
advanced solid tumors (PRO-001), a Phase Ib study in patients
with recurrent ovarian cancer (PRO-002), a Phase Ib study in
patients with locally advanced or metastatic biliary tract cancer
(BTC) (ABC-08), and a Phase II study in patients with
platinum-resistant ovarian cancer (PRO-105). Two clinical
Received: December 19, 2020
Published: June 4, 2021
https://doi.org/10.1021/acs.jmedchem.0c02194
© 2021 American Chemical Society
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Figure 1. Phosphoramidate transformations of anticancer and FDA-approved antiviral nucleoside monophosph(on)ate analogues in the clinic and
ongoing clinical studies.
The second agent, NUC-3373, is a ProTide modification of
FUDR, the fluorinated pyrimidine nucleoside derivative of 5-
FU. These fluoropyrimidines are established clinical drugs used
in the treatment of solid tumors including gastrointestinal,
kidney, ovarian, and breast cancers.16 The key resistance
mechanisms that limit 5-FU and FUDR involve down-
regulation of the activating enzyme, thymidine kinase (TK),
required for the first phosphorylation step that transforms
FUDR to the active anticancer metabolite (FdUMP); up-
regulation of thymidylate synthase (TS), which is considered
to be the main target for 5-FU and FUDR; up-regulation of the
degradative enzyme thymidine phosphorylase (TP); and
decreased expression of nucleoside/nucleobase transport
proteins.17 In addition, more than 85% of administered 5-FU
is degraded by dihydropyrimidine dehydrogenase (DPD)
before it has an opportunity to enter cancer cells and exert
any therapeutic effect.18 In vitro data confirmed that NUC-
3373 exerts its cytotoxic activity independently of thymidine
kinase in TK-deficient tumor cell lines, and it is resistant to
degradative action by catabolic enzymes including TP and
DPD.19 In addition, NUC-3373 was found to generate 366-
fold higher intracellular levels of the active monophosphate
form, FdUMP, in the human colorectal cancer cell line HT29
compared with 5-FU and achieved significantly greater tumor
volume reduction than 5-FU in HT29 xenograft studies.20
NUC-3373, as a diastereomeric mixture, is currently being
assessed in two ongoing clinical studies. Interim pharmacoki-
netic data from the first-in-human Phase I study of NUC-3373
in patients with advanced solid tumors (NuTide:301)
demonstrated that NUC-3373 has a long plasma half-life
(9.7 h versus 8−14 min) and the ability to completely deplete
the pool of dTMP in patients PBMCs, compared to 5-FU. A
three-part Phase I study of NUC-3373 in combination with
standard agents used in colorectal cancer treatment (Nu-
Tide:302) is also ongoing and is designed to identify a
recommended Phase II dose (RP2D) and schedule for NUC-
3373 when combined with other agents.21 Recently, a third
anticancer agent, NUC-7738, the ProTide modification of 3′-
deoxyadenosine (3′-dA; also known as cordycepin), entered a
Phase I clinical study.22
NUC-1031 and NUC-3373 ProTides were obtained via a
coupling reaction between 3′-Boc-protected gemcitabine or
FUDR and phosphorylating agent (phosphorochloridate) in
the presence of either Grignard reagent (tert-butyl magnesium
chloride) or N-methylimidazole (NMI). In both cases, the
formation of a pair of diastereomers at the phosphate center
(1:1 ratio SP/RP) was achieved and, as such, NUC-1031 and
NUC-3373 were further biologically evaluated.9,10 Although
NMI favors the phosphorylation of the primary hydroxyl group
at the nucleoside’s 5′-position, the yield of the coupling
reaction is usually low. On the contrary, the Grignard reagent
is not selective, hence allowing the formation of a 5′-
phosphoramidate along with undesired 3′- and 3′,5′-
phosphoramidates. In order to prevent the formation of
these byproducts, a selective 3′-protection of the nucleosides is
required prior to the coupling reaction. This strategy involves
an additional deprotection step at the 3′-positions in the
obtained protected-phosphoramidates, as recently reviewed.23
Because the yield of coupling reaction in the presence of the
Grignard reagent is usually moderate to high, we adopted the
hydroxyl groups protection as a strategy to prepare NUC-3373
with higher yield. In the case of gemcitabine, 3′-OH protection
is more direct and selective due to the presence of fluorine
atoms at the 2′-position in the sugar moiety. For FUDR, which
lacks any substituents at the 2′-position, an initial protection of
both 3′- and 5′-OH and subsequent selective 5′-deprotection
was necessary. The 3′-OH protected FUDR was further used
for a coupling reaction with a phosphorochloridate in the
presence of the Grignard reagent to improve the overall yield
of NUC-3373 synthesis. A variety of hydroxyl protecting
groups can be used to prepare 3′-protected ProTides.23 A tert-
butyl silyl group (TBDMS) was selected at first and was found
to be the most optimal as a clear separation of two
diastereomers of the 3′-protected NUC-3373 mixture was
possible by normal-phase chromatography. Although a
regioselective strategy to obtain the desired 5′-ProTides was
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published,24 during the course of the present study, we decided
to proceed with our alternative methodology and to separate a
mixture of two diastereomers at the level of 3′-protected
ProTides. This alternative approach was of particular interest
in light of the notable difference in activity between SP and RP
isomers discovered for FDA-approved antiviral ProTide agents
sofosbuvir25 (18-fold increase in potency against HCV for the
SP isomer)26 and TAF27 (10-fold increase in potency against
HIV for the SP isomer)28 (Figure 1). In fact, chirality and chiral
separation of racemic compounds are considered important
topics in drug development. Most isomers of chiral drugs,
including diastereomers, differ in their pharmacology,
pharmacokinetics, metabolism, and biological activities.29
With this in mind, the current FDA recommendation is to
assess activity of each isomer of racemic therapeutic agents.30
Over the past decade, different diastereoselective methods for
the synthesis of P-chirogenic phosphoramidates have been
developed, including methods using a chiral auxiliary-bearing
phosphoramidating reagent,31 diastereomerically pure phos-
phoramidating agent with p-nitro-phenyl or 2,3,4,5,6-penta-
fluorophenyl as leaving aryloxy groups,32,33 or a method based
on the process for the diastereomeric enrichment due to
differences in solubility of SP and RP diastereomers in
alcohols.34
In our laboratory, we have focused on the development of
diastereoselective synthetic methods via a copper-catalyzed
reaction in the presence of different bases.35 Additionally,
methods for the separation of 3′-protected ProTides as a
diastereomeric mixture, in general being very difficult to isolate
as single diastereomers by standard chromatography (reverse-
phase chromatography or crystallization), were probed.
Herein, we report an alternative synthetic procedure to
obtain SP and RP isomers of the two clinical anticancer agents
NUC-3373 and NUC-1031. A selective TBDMS-protection
and deprotection manipulation of FUDR and gemcitabine was
followed by a coupling reaction to give 3′-protected
phosphoramidates which were separated to single diaster-
eomers. Further removal of 3′-TBDMS group in each
separated isomer yielded NUC-3373 and NUC-1031 as single
isomers. NUC-1031 diastereomers were obtained upon
column chromatography in crystalline form and the stereo-
chemistry at the phosphorus center was assigned via X-ray
crystallography. Moreover, for the first time, we disclose in
vitro biological data for single RP and SP isomers of NUC-1031
and NUC-3373 (stereochemistry to be determined) as well as
their ability to target cancer stem cells (CSC).
Scheme 1. General Synthetic Strategy Toward 3′-TBDMS-
a
Protected Nucleosides 5 and 6
a
Reagents and conditions: (a) imidazole (5.0 equiv), TBDMSCl (2.2
equiv), DMAP (0.3 equiv), DMF, 0 °C to rt, 1 h; (b) CSA (1.0
equiv), MeOH, rt, 10 min or TCA (28.0 equiv), THF, H2O, rt, 2 h.
in the presence of their secondary equivalents.36 Thus, first
FUDR (1) was treated with TBDMS, imidazole, and DMAP at
elevated temperature37 to give the 5′,3′-TBDMS protected
FUDR derivative (3) in 98% yield. Next, the FUDR
intermediate 3 was subjected to different deprotection
conditions to selectively remove the 5′-TBDMS moiety. The
primary silyloxy groups are known to be cleaved under acidic
conditions more efficiently in comparison with their secondary
counterparts. Therefore, we investigated a variety of acidic
conditions starting with a TFA/H2O/THF (1:1:4) mixture,
reported by Zhu et al. as the most suitable for selective 5′-
desilylation of the multisilylated nucleoside analogues.36
However, when compound 3 was treated under the above-
mentioned conditions, 3′-TBDMS-FUDR derivative (5) was
obtained in low yield (20%). Due to poor selectivity of
trifluoroacetic acid toward 5′-desilylation, different acids such
as p-toluenesulfonic acid (p-TsOH) or camphorsulfonic acid
(CSA) were further tested as summarized in Table 1. No
selectivity was observed when desilylation was performed with
0.6 equiv of p-TsOH in methanol at room temperature. After 1
h of reaction, a mixture of 3′- and 5′-monosilylated products in
almost 1:1 ratio were detected along with a significant amount
of starting material 3 (entry 1, Table 1). Decreasing the
reaction temperature to −15 °C considerably improved the
selectivity, and the desired product 5 was recovered in 45%
yield after 4 h (entry 2, Table 1). Replacing MeOH with DCM
drastically slowed down the reaction and significantly
decreased the 5′-desilylation selectivity (data not reported).
1
The yields were estimated by H and 19F NMR of the crude
reaction mixture. Given the unsatisfactory result obtained with
p-TsOH, we turned our attention to a new strategy with 10-
camphorsulfonic acid (CSA), which has been reported in a few
cases to be able to selectively deprotect primary silyl ether in
multisilylated nucleosides to give free primary alcohol.37,38
Thus, when the reaction was carried out in a 1:1 mixture of
DCM/MeOH at low temperature (0−5 °C), slow depro-
tection was detected; however, a remarkable selectivity was
observed. The desired compound 5 was obtained in 38% crude
RESULTS AND DISCUSSION
■
Chemistry. The selective protection and deprotection of
hydroxyl groups in nucleoside analogues subjected to ProTide
technology and the coupling reaction represents a solid
strategy to improve the overall yield of phosphoramidate
synthesis. This alternative approach may be considered as
advantageous over the general method in which nonprotected
nucleoside starting materials are used, as only the desired 5′-
phosphorylated ProTides are formed. The alternative
procedure for the preparation of clinical anticancer agents
NUC-3373 and NUC-1031 from 3′-protected FUDR and
gemcitabine, respectively, is depicted in general in Scheme 1.
In our approach to improve the efficiency of the route to
obtain the desired 5′-ProTides, we selected the tert-
butyldimethyl silyl group reported for its ability to be
selectively cleaved from the primary TBDMS hydroxyl groups
1
yield as estimated by H and 19F NMR. By increasing the
temperature to 25 °C, the selectivity toward 5′-desilylation was
retained and the reaction was completed within 2.5 h,
providing compound 5 with 37% yield (data not shown). A
major improvement was obtained when only MeOH was
employed as a solvent. In this case the reaction was
accomplished much faster, and after 1 h the desired compound
1
5 was formed in 60% crude yield based on H and 19F NMR
(entry 5, Table 1). Performing the reaction under the same
conditions but at room temperature increased the rate of the
deprotection. However, after 45 min, a significant amount of
FUDR was formed, suggesting that meticulous monitoring is
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Table 1. Selective 5′-Desilylation of 3′,5′-TBDMS-FUDR (3) and 3′,5′-TBDMS-Gemcitabine (4): Conditions Optimization
a b
,
entry
nucleoside analogue
acid/equiv
solvent
time/temp
yield (%)
1
2
3
4
5
6
7
8
9
FUDR
FUDR
FUDR
FUDR
FUDR
FUDR
FUDR
FUDR
p-TsOH/0.6
p-TsOH/1.0
p-TsOH/1.0
CSA/0.6
CSA/0.6
CSA/0.5
CSA/0.6
CSA/1.0
CSA/1.0
MeOH
MeOH
DCM
MeOH
MeOH
MeOH
MeOH
MeOH
AcCN
1 h/rt
a
4 h/−15 °C
6 h/15 °C−rt
30 min/0 °C
1 h/0 °C
25 min/rt
45 min/rt
10 min/rt
1 h/rt
45
a
56
a
60
b
50
b
60
FUDR
10
11
12
FUDR
gemcitabine
gemcitabine
CSA/1.0
CSA/1.0
TCA/28.0
acetone
MeOH
THF/H2O
10 min/rt
10 min/rt
30 min/rt
b
82
a
b
1
5′-Desilylated product, calculated yield by H and 19F NMR. Isolated yield.
required (entry 7, Table 1). The best result was obtained when
performing the reaction in MeOH with 1 equiv of CSA at
room temperature in 10 min. Under these conditions the
desired product 5 was recovered after work up in 60% yield
(entry 8, Table 1). Additional attempts to improve the yield of
5′-TBDMS removal by changing solvents were unsuccessful
(entries 9 and 10, Table 1).
Scheme 2. General Synthetic Strategy Toward Separated
Diastereomers of NUC-3373 and NUC-1031 (9a,9b and
10a,10b)
a
Next, the same conditions for protection and selective 5′-
deprotection were applied to gemcitabine (2) to form the key
intermediate 6. However, instead of 3′-TBDMS-protected
gemcitabine 6, formation of its 5′-TBDMS-protected analogue
was observed along with the starting bis-silylated compound 4.
While an attempt to selectively remove a silyl group from the
5′-position with oxalic acid (up to 3.0 equiv) was found
unsuccessful (data not reported), the use of trichloroacetic acid
(28 equiv) in a mixture of THF/H2O (3.5:1) gave the key
intermediate 6 with 82% yield (entry 12, Table 1).
The key intermediate 5 was further subjected to the
standard coupling conditions with L-Ala-OBn-ONaph phos-
phorochloridate10 in the presence of the Grignard reagent
(Scheme 2). 3′-TBDMS-protected compound 7 obtained as a
mixture of diastereomers was purified using normal-phase
chromatography to give single isomers: fast eluting (7_isomer
A, 7a or 7b) and slow eluting (7_isomer B, 7a or 7b). A final
deprotection of the 3′-silyl group in isomers 7a and 7b was
performed with a 4:1:1 mixture of THF/TFA/H2O at 0 °C
and yielded single diastereomers of NUC-3373, herein
reported as NUC-3373_isoA (9a or 9b) (obtained from
7_isoA) and NUC-3373_isoB (9a or 9b) (obtained from
7_isoB) as stereochemistry at the phosphorus atom remained
undetermined at the time of preparation of this manuscript.
Attempts to crystallize NUC-3373_isoA (9a or 9b) and NUC-
3373_isoB (9a or 9b) as well as their 3′-protected precursors
(7_isomer A and 7_isomer B) from various solvents including
MeOH, 2-propanol, CHCl3 and a combination of CHCl3/
Et2O (1:1), and MeOH/CHCl3 (3:1), CHCl3/H2O (1:1)
were unsuccessful. In addition, the vapor diffusion method,
using 2-propanol/hexane, MeOH/H2O and MeOH/petroleum
ether as the binary solvent system, did not lead to formation of
crystalline form. In parallel, a coupling reaction between L-Ala-
OBn-OPh phosphorochloridate9 and 6 gave 3′-TBDMS-
protected NUC-1031 analogue 8, further isolated as single
isomers 8a and 8b via column chromatography. Removal of
the 3′-TBDMS-group in separated diastereomers 8a and 8b
under acidic conditions provided isomers of NUC-1031 10a
and 10b (Scheme 2), which after normal-phase chromatog-
a
Reagents and conditions: (a) (ArO)(XOC(O)CHCH3NH)P(O)Cl
(2.0 equiv), tert-BuMgCl, THF, rt, 16 h; (b) separation of
diastereomers; and (c) THF/TFA/DCM (4:1:1), 0 °C for 2 h,
then rt for 12 h.
raphy were obtained as a crystalline solid. The molecular
structure and stereochemistry at the phosphate center was
elucidated by single-crystal X-ray crystallography for the two
isomers of NUC-1031 and determined to be SP and RP for
diastereomers 10a and 10b, respectively (Figure 2).
In Vitro Cytotoxic Activity. The single isomers of NUC-
1031 and NUC-3373 were biologically evaluated for cytotoxic
activity in a panel of solid tumor cell lines (Mia-Pa-Ca-2, Bx-
PC-3, LNCaP, HT-29, HCT-116, MDA-MB-231, and SW-
620), the acute myeloid leukemia (AML) cell line, KG1a, and
primary AML blasts. The activity of the ProTides was
compared with the corresponding diastereomeric mixtures
NUC-3373 and NUC-1031 as well as with the parent
nucleosides FUDR (1) and gemcitabine (2), respectively
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Figure 2. X-ray crystal structures of 10a (NUC-1031_SP) and 10b (NUC-1031_RP).
Table 2. Cytotoxicity of NUC-3373 and NUC-1031 as a Diastereomeric Mixture and Single Isomers RP and SP Reported as
a
LC50 (μM) Values
LC50(μM) SEM in different cell lines
a
a
a
cmpd
Mia-Pa-Ca-2
Bx-PC-3
LNCaP
HT-29
HCT-116
SW-620
MDA-MB-231
KG1a
5-FU
FUDR
5.5 0.8
0.8 0.3
1.7 0.6
2.1 0.6
1.5 0.4
3.7 0.7
1.6 0.4
3.1 0.8
4.3 1.0
2.6 0.9
7.98
1.55
1.84
2.37
1.46
12.6 1.1
2.4 0.7
2.8 0.9
3.6 0.6
2.5 0.6
1.4 0.5
1.18 0.4
0.053 0.02
0.052 0.01
0.055 0.02
NUC-3373
NUC-3373_IsoA (9a or 9b)
NUC-3373_IsoB (9a or 9b)
gemcitabine
NUC-1031
NUC-1031_SP(10a)
NUC-1031_RP(10b)
0.01
0.1
0.03
0.19
0.74
2.29
2.44
1.29
2.8 0.5
3.1 0.6
2.6 0.7
3.4 0.5
0.7 0.2
1.3 0.5
1.5 0.3
1.0 0.4
1.8 0.3
0.28 0.07
0.31 0.1
0.25 0.1
a
LC50 values (the concentration of drug required to induce 50% cell death) were calculated using an MTS assay performed in duplicate and tested
in 9 serial concentrations from 198 μM to 0.0199 μM (data expressed as mean LC50 values) or an Annexin V/7-AAD assay performed in triplicate
(data expressed as mean LC50 values SEM).
(Table 2). In the solid tumor cell lines, the LC50 values for
both NUC-3373 isomers were comparable to those of NUC-
3373 and FUDR and ranged between 0.61−2.37 μM and
0.31−1.46 μM in HT-29 and SW-620 for NUC-3373_IsoA
(9a or 9b) and NUC-3373_IsoB (9a or 9b), respectively. In
comparison to 5-FU, a significant increase in potency was
observed and ranged between 13 and 25-fold and 3−5-fold for
NUC-3373_IsoA (9a or 9b) and NUC-3373_IsoB (9a or 9b),
respectively, in HT-29 and SW-620 cell lines. The cytotoxic
activity of the gemcitabine ProTide NUC-1031 and its isomers
were similar, except for the NUC-1031_SP isomer (10a) in the
Mia-Pa-Ca-2 cell line, which was found to be 6-fold more
potent than its RP analogue 10b (0.03 μM vs 0.19 μM) and 3-
fold more potent than its diastereomeric mixture (0.03 μM vs
0.10 μM). In the HT-29 cancer cell line, the submicromolar
LC50 values for both SP and RP NUC-1031 isomers and NUC-
1031 ranged between 0.34 and 0.74 μM, similar to the
gemcitabine LC50 value of 0.21 μM. NUC-1031 and both
isomers were equipotent in the Bx-PC-3 tumor cell line. When
considering the KG1a cell line, NUC-1031 was approximately
6-fold more potent than gemcitabine, again, with no significant
difference between the diastereomers (Figure 3A−C). We
went on to confirm that NUC-1031 was significantly more
potent than gemcitabine in primary AML blasts (Figure
3G,H). Similarly, NUC-3373 was approximately 23-fold more
potent than FUDR with no significant difference between the
diastereomers (Figure 3D−F).
3373_IsoA (9a or 9b) was found to be slightly more stable
than NUC-3373_IsoB (9a or 9b) and its diastereomeric
mixture NUC-3373, with a half-life of 53 min. In the case of
the gemcitabine family, NUC-1031_SP (10a) was found to
have a shorter half-life of 37 min in human hepatocytes
compared with NUC-1031_RP (10b), which had a half-life
>120 min. This intriguing discrepancy in half-life of both
NUC-1031 isomers in human hepatocytes could potentially be
explained by different carboxylesterases (CESs) substrate
specificity also toward isomers. Mammalian carboxylesterases
(CES1 and CES2), the crucial enzymes involved in the
metabolism of endogenous esters and ester or amide-
containing xenobiotics, may exhibit different hydrolysis rate
between the R- and S-isomers. Thus, their reactivity might be
affected by the conformational orientation into the active
pocket of CESs.39
Biological Activation: Carboxypeptidase Y Assay. The
mechanism of activation of NUC-1031 and NUC-3373 in their
diastereomeric forms was previously reported9,10 and was
confirmed to follow the commonly accepted putative route
(Scheme 3). The activation pathway involves (i) carbox-
ypeptidase-mediated hydrolysis of the ProTide ester moiety to
form the intermediate A, (ii) displacement of the aryl moiety
and spontaneous cyclization of A to form metabolite B, and
(iii) spontaneous hydrolysis of cyclic anhydride B to the
intermediate C. The latter metabolite would then be converted
to the monophosphate D via P−N bond cleavage catalyzed by
a phosphoramidase-type enzyme. In this work, we carried out
an enzymatic assay for the two separated isomers NUC-
3373_isoA (9a or 9b) and NUC-3373_isoB (9a or 9b) and for
the gemcitabine ProTide NUC-1031_SP (10a) and NUC-
1031_RP (10b) in the presence of carboxypeptidase Y
The metabolic stability of NUC-3373, NUC-1031, and the
separated diastereomers (9a, 9b and 10a,10b) was investigated
by incubation with human hepatocytes. The half-lives and
percentage of compounds remaining after 1 h were measured
and are outlined in Table 3. In human hepatocytes, NUC-
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Figure 3. Overlaid sigmoidal dose−response curves. (A) Comparison of gemcitabine and NUC-1031_mix (diasteromeric mixture) and (B) the
separated diastereomers, NUC-1031_SP (10a) and NUC-1031_RP (10b). (C) Comparison of the LD50 values for gemcitabine, NUC-1031_mix,
NUC-1031_SP (10a), NUC-1031_RP (10b). (D) Comparison of FUDR and NUC-3373_mix (diasteromeric mixture) and (E) the separated
diastereomers NUC-3373_isoA (9a or 9b) and NUC-3373_isoB (9a or 9b). (F) Comparison of the LD50 values for 5-FU, FUDR, NUC-
3373_mix, NUC-3373_isoA (9a or 9b), and NUC-3373_isoB (9a or 9b). All assays carried out using KG1a cells are presented as mean ( SEM)
of five independent experiments. (G,H) Relative sensitivity of primary AML blasts to gemcitabine and NUC-1031_mix (n = 4).
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similar cell surface markers (CD34+/CD38−) to normal
hematopoietic stem cells implying that a similar hierarchical
organization may occur in tumors. Subsequently, CSCs have
been found in a wide range of solid tumors including breast,
lung, colon, prostate, ovarian, skin, and pancreas.42 CSCs are
also linked with the development of resistance to therapy and
recurrence of cancer, as reported for human colon cancer cell
resistance to 5-FU in vitro43 and for human pancreatic
adenocarcinoma resistance to gemcitabine.44
Table 3. Metabolic Stability of ProTides Incubated with
Human Hepatocytes
intrinsic clearance (cryopreserved human hepatocytes)
half-life
(min)
% compound remaining after
1 h
cmpd
NUC-3373
NUC-3373_IsoA (9a or
9b)
34
53
30
40
NUC-3373_IsoB (9a or 9b)
NUC-1031
NUC-1031_SP (10a)
NUC-1031_RP (10b)
47
76
37
40
58
33
97
Preferential Targeting of Cancer Stem Cells by
ProTides. Given the growing interest in the treatment of
cancer by targeting tumor stem cells, we assessed the relative
abilities of NUC-1031_mix and NUC-3373_mix, their single
diastereomers, and the parental nucleoside analogues to
preferentially kill LSCs (as defined by the phenotype
CD34+CD38−) in the acute myeloid leukemia cell line,
KG1a, and in primary AML blasts.45 Figure 5A,B shows that
gemcitabine did not significantly alter the percentage of LSCs
in the KG1a cell cultures at concentrations ≤2.5 × 10−6 M. In
contrast, the NUC-1031 diastereomers significantly reduced
the LSC fraction at concentrations ≥2.5 × 10−7 M; both
diastereomers had a similar effect (Figure 5B). Importantly, we
were also able to demonstrate that NUC-1031_mix preferen-
tially depleted LSCs in primary AML blasts (Figure 5C).
Furthermore, consistent with the results generated with
gemcitabine, neither 5-FU nor FUDR showed any preferential
toxic effect on LSCs when compared to the bulk tumor at
concentrations ≤5 × 10−6 M (Figure 5D). In contrast, both
NUC-3373 diastereomers preferentially depleted the LSC
compartment at concentrations ≥7 × 10−8 M (Figure 5D). In
order to establish whether this phenomenon was specific for
KG1a cells and AML blasts, the prostate cancer cell line,
LNCaP, was employed as an alternative cancer stem cell
model. In this case, the cancer stem cells were identified by a
CD44+CD133+ phenotype; this population of cells accounted
for just 1.4% of the cell line. Although the LC50 values for
gemcitabine and NUC-1031 were not significantly different (P
= 0.24), NUC-1031 showed a preferential effect on cancer
stem cell population when compared to gemcitabine at
concentrations ≥2.5 × 10−6 M (Figure 5E).
>120
according to the standard procedure.40 Within 10 min of
incubation (Figure 4), NUC-3373_isoA (9a or 9b) was fully
processed to metabolite A, as indicated by the lack of the single
peak at δ 4.04 ppm ascribed to the NUC-3373_isoA (9a or
9b) and appearance of a single peak at δ 5.16 ppm (metabolite
A). During this time, a single peak at δ 6.80 ppm
corresponding to achiral metabolite C was also detected.
Slightly slower processing to metabolites A and C was
observed for the isomer NUC-3373_isoB (9a or 9b) within
the first 10 min of incubation, during which 28% of NUC-
3383_isoB remained unprocessed by the enzyme (Supporting
Information, Figure S1a).
In the case of the gemcitabine ProTide NUC-1031, only the
SP isomer (10a) was processed to its metabolites A (δ 4.51
ppm, 68%) and C (δ 6.80 ppm, 18%) within the first 10 min of
incubation with carboxypeptidase Y. NUC-1031_RP (10b) was
converted to metabolite C (without metabolite A detection)
within approximately 120 min (Supporting Information, Figure
S1b,c).
CANCER STEM CELL TARGETING
■
The putative existence of cancer stem cells (CSCs) has been
suggested in many human cancers, including leukemias and
solid tumors. The cancer stem cell hypothesis postulates that
only a small subpopulation of tumor cells are responsible for
tumor formation, maintenance of the bulk of the tumor, as well
as its invasion and metastasis. In 1994, Lapidot and co-workers
demonstrated that only a small percentage of acute myeloid
leukemia cells had the capability to initiate leukemia in mice.41
These leukemic stem cells (LSCs) were shown to express
Intracellular Triphosphate Accumulation and ABC
Transporter Expression. LSC and bulk tumor fractions (5 ×
106) were isolated by high-speed cell sorting and were
Scheme 3. General Metabolic Activation Pathway for NUC-3373 and NUC-1031 ProTides
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Figure 4. (A) 31P NMR spectra of NUC-3373_isoA (9a or 9b) over time after enzymatic incubation with carboxypeptidase Y. (B) Chart
representation of percentage of NUC-3373_isoA (9a or 9b) and metabolites A and C over time after enzymatic incubation with carboxypeptidase
Y. Conditions 202 MHz, 25 °C, d6-acetone, 0.05 M Trizma buffer, pH 7.6.
subsequently cultured for 1 h with either 1 μM gemcitabine or
1 μM NUC-1031_mix. The level of intracellular triphosphate
was assessed using mass spectrometry and quantified by
running defined concentrations of nucleoside through the mass
spectrometer in order to construct a standard curve (Figure
6A). In both fractions, NUC-1031 resulted in higher
intracellular accumulation of the triphosphate when compared
to the parental gemcitabine nucleoside (Figure 6B).
Furthermore, the LSC fraction retained high levels of the
triphosphate following treatment with NUC-1031_mix, which
may contribute to the preferential depletion of these cells when
exposed to ProTide. In contrast, LSCs treated with
gemcitabine showed approximately a 30% reduction in
intracellular triphosphate when compared with bulk tumor
cells. Based on the intracellular triphosphate data, we next
evaluated the expression of ABC transporters in LSC and bulk
tumor fractions both at the level of protein and mRNA
transcript. Figure 6C shows that LSCs show a trend toward
higher expression of ABCB1 and significantly higher expression
of ABCG2 in LSCs versus bulk tumor cells. Furthermore, at
the level of transcript, ABCB1, ABCG2, ABCC4, and ABCC5
all showed significantly increased expression in LSCs versus
bulk tumor cells (Figure 6D). A number of these transporters
have been previously implicated in gemcitabine resistance.46,47
obtained in crystalline form and their molecular structure and
stereochemistry at the phosphate center was assigned via X-ray
crystallography. The cytotoxic activities of RP and SP isomers of
NUC-1031 were similar and there was no significant difference
in the in vitro potency when compared to NUC-1031 as a
diastereomeric mixture. Similarly, the in vitro potency of NUC-
3373 and its single isomers NUC-3373_isoA and NUC-
3373_isoB was comparable in the cancer cell lines tested.
Attempts to crystallize two diastereomers of NUC-3373 were
unsuccessful at the time of preparation of this manuscript. Both
NUC-1031_mix and NUC-3373_mix and their separated
diastereomers were shown to preferentially target leukemic
cancer stem cells, whereas the parent nucleosides gemcitabine
and FUDR did not. Mechanistically, increased intracellular
accumulation of triphosphate may contribute to the prefer-
ential killing of CSCs by the ProTide in contrast to the
parental analogue, but other factors may also be involved.
Given that one of the gemcitabine resistance mechanisms has
been associated with ABC transporter expression,46,47 we
investigated their expression in LSC and bulk tumor cells.
LSCs showed significantly increased expression of ABCB1,
ABCG2, ABCC4, and ABCC5 at the level of transcript and
increased expression of ABCG2 at the level of protein. We
postulate that the increased intracellular triphosphate in
ProTide-treated cells is caused, at least in part, by the
differential bioavailability of the ProTide versus the parental
nucleoside analogue. As LSCs preferentially express ABC
transporters, the increased intracellular accumulation of
triphosphate in these cells, following treatment with ProTide,
results in increased cell death of these LSCs. Importantly, the
same effect was observed in cancer stem cells from a prostate
cancer cell line (LNCaP). As such, ProTides may prove to be
an important addition to the therapeutic repertoire, particularly
in the context of eradication of cancer stem cells.
CONCLUSION
■
The biological potencies and activities of single enantiomers or
diastereomers of chiral compounds such as phosphoramidates
(ProTides), with their stereocenter at the phosphorus atom,
may differ markedly. In light of the FDA recommendation to
assess activity of each isomer of racemic or diastereomeric
mixtures, it became even more important to develop either
stereoselective synthetic strategies or separation methods to
obtain the desired isomers. Herein, we described an alternative
synthesis of the two clinical anticancer agents NUC-1031 and
NUC-3373 via a 3′,5′-hydroxyl group protection and 5′-
deprotection strategy, which allowed separation of the single SP
and RP diastereomers. Two diastereomers of NUC-1031 were
EXPERIMENTAL SECTION
■
Materials and Methods. Cell Lines. KG1a, HT-29, HCT-116,
LNCaP, and MDA-MB-231 cell lines were all purchased from DSMZ
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Figure 5. Differential cytotoxic effects of nucleosides and ProTides on cancer stem cells. (A) Comparison of the viable fraction of KG1a cells
exposed to gemcitabine and NUC-1031_mix for 48 h. (B) Both diastereomers of NUC-1031 caused a dose-dependent, statistically significant,
reduction in the LSC (CD34+/CD38−) fraction of KG1a cells. In contrast, gemcitabine had no significant effect on the LSC compartment at
concentrations ≤2.5 × 10−6 M. (C) The diastereomeric mixture of NUC-1031 induced a dose-dependent decrease in the LSC fraction of primary
AML blasts. (D) Both diastereomers of NUC-3373 caused a dose-dependent, statistically significant reduction in the LSC (CD34+/CD38−)
fraction of KG1a cells. In contrast, 5-FU and FUDR had no significant effect on the LSC compartment at concentrations ≤5 × 10−6 M. *P < 0.05,
**P < 0.01. (E) A similar effect was observed in the prostate cancer cell line, LNCaP. NUC-1031 preferentially depleted the cancer stem cells
(CD44+/CD133+) at concentrations ≥2.5 × 10−6 M when compared with gemcitabine.
(Germany). All lines were initially expanded and then frozen down in
aliquots (107 cells) at low passage number. Cell lines were never
expanded beyond 8−10 passages and were routinely confirmed as
mycoplasma-free to ensure the integrity of each cell line.
MTS Cell Viability Assay. The assay was contracted and carried-out
by WuXi AppTec (Shanghai) Co., Ltd. The tumor cell lines Mia-
PaCa-2, Bx-PC-3, HT-29, and SW620 were seeded at cell densities of
0.5 to 100 × 103 cells/well in a 96-well plate the day before drug
incubation. Then the plates were incubated for 72 h with the different
concentrations of compound to be tested. After the incubation period,
50 μL of MTS was added and the tumor cells were incubated for 4 h
at 37 °C. The data were read and collected by a Spectra Max 340
absorbance microplate reader. The compounds were tested in
duplicate with 9 serial concentrations (3.16-fold titrations with 198
μM as the highest concentration), and the data were analyzed by XL-
fit software.
Annexin V/7-AAD Cell Viability Assay. Cell lines were grown in
T175 flasks until confluent. Cells were then aliquoted (105 cells/100
μL) into 96-well plates and were incubated at 37 °C in a humidified
5% CO2 atmosphere for 72 h in the presence of tested compounds at
the concentrations that were experimentally determined for each
compound. In addition, control cultures were set up to which no drug
was added. Cells were subsequently harvested by centrifugation and
were analyzed by flow cytometry using the Annexin V/7-AAD assay.
All experiments were performed in triplicate. LC50 values (the
concentration of compound required to kill 50% of the cells in
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Figure 6. Measurement of intracellular triphosphate ABC transporters in LSC and bulk tumor cell fractions from KG1a cells. (A) Defined
concentrations of nucleoside were run through the mass spectrometer in order to construct a standard curve (B) Purified LSCs and bulk tumor
cells were treated with 1 μM of gemcitabine or NUC-1031_mix for 1 h prior to cell lysis and mass spectrometry. Both LSC and bulk tumor
fractions showed increased intracellular triphosphate following treatment with NUC-1031_mix. (C) Protein expression of ABCB1 and ABCG2 on
purified bulk tumor (CD34+/CD38+) and LSCs (CD34+/CD38−). Cell sorted LSCs showed significantly higher levels of ABCG2 when compared
with bulk tumor cells. (D) Relative gene transcription of ABC transporter genes in purified bulk tumor cells (solid bars) and LSCs (hatched bars).
LSCs showed significantly increased relative transcription of ABCB1, ABCG2, and ABCC5 when compared with bulk tumor cells. All data are the
mean ( SEM) of five independent experiments, performed in triplicate.
culture) were calculated by nonlinear regression modeling using
GraphPad Prism software and are shown as mean SEM for each
replicate data set.
scans every 7 min unless otherwise stated. 31P NMR recorded data
were processed and analyzed with the Bruker Topspin 2.1 program.
Cell Culture Conditions. The acute myeloid leukemia (AML)
KG1a cell line and the prostate cancer cell line, LNCaP, were
maintained in RPMI medium (Invitrogen, Paisley, U.K.) supple-
mented with 100 units/mL penicillin, 100 μg/mL streptomycin, and
20% fetal calf serum. The colorectal cancer cell lines, HT-29 and
HCT-116, and the breast cancer cell line, MDA-MB-231, were
maintained in Dulbecco’s modified essential medium with high
glucose supplemented with 100 units/mL penicillin, 100 μg/mL
streptomycin, and 10% fetal calf serum.
Measurement of In Vitro Apoptosis. Cultured cells were harvested
by centrifugation and resuspended in 195 μL of calcium-rich buffer.
Subsequently, 5 μL of Annexin V was added to the cell suspension
and cells were incubated in the dark for 10 min prior to washing. Cells
were finally resuspended in 190 μL of calcium-rich buffer together
with 10 μL of propidium iodide. Apoptosis was assessed by dual-color
immunofluorescent flow cytometry48 as described previously.
Subsequently LC50 values (the doses required to kill 50% of the
cells in culture) were calculated for each nucleoside analogue and
ProTide.
Metabolic Stability (Cryopreserved Hepatocytes, Human) Assay.
The assay was contracted and performed by Cerep (Seattle, WA, USA
Laboratories, 15318 N. E. 95th Street Redmond, WA, 98052, U.S.)
according to the published procedure (Cerep ref. 1432). Pooled
cryopreserved hepatocytes were thawed, washed, and resuspended in
Krebs−Heinslet buffer (pH 7.3). The reaction was initiated by adding
the test compound (1 μM final concentration) into cell suspension
and incubated in a final volume of 100 μL on a flat-bottom 96-well
plate for 0 and 60 min, respectively, at 37 °C/5% CO2. The reaction
was stopped by adding 100 μL of acetonitrile into the incubation
mixture. Samples were then mixed gently and briefly on a plate shaker,
transferred completely to a 0.8 mL V-bottom 96-well plate, and
centrifuged at 2550g for 15 min at room temperature. Each
supernatant (150 μL) was transferred to a clean cluster tube, followed
by HPLC-MS/MS analysis on a Thermo Electron triple quadrupole
system.
Carboxypeptidase Y (EC 3.4.16.1) Assay. The experiment was
carried out by dissolving ProTides (3.5 mg) in acetone-d6 (0.15 mL)
followed by addition of 0.30 mL of Trizma buffer (pH 7.6). After
recording the control 31P NMR at 25 °C, a previously defrosted
carboxypeptidase Y (0.1 mg dissolved in 0.15 mL of Trizma) was
added to the sample, which was then immediately submitted to the
31P NMR experiments (at 25 °C). The spectra were recorded with 64
Immunophenotypic Identification of the Cancer Stem Cells.
KG1a cells or LNCaP cells were cultured for 72 h in the presence of a
wide range of concentrations of each nucleoside analogue and their
respective ProTides. Cells were then harvested, and KG1a cells were
labeled with anti-CD34 (FITC) and anti-CD38 (PE) and LNCaP
cells were labeled with anti-CD44 (APC) and anti-CD133 (Alexa
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488). The subpopulations expressing a cancer stem cell (CSC)
phenotype were subsequently identified and were expressed as a
percentage of all viable cells left in the culture. The percentages of
stem cells remaining were then plotted on a dose−response graph,
and the proportion of cancer stem cells was compared across the
various concentrations of ProTides and their respective parental
nucleoside in order to determine if they preferentially target cancer
stem cells.
Cell Sorting. KG1a cells were cultured until confluent in order to
generate 108 cells. Subsequently, 5 × 106 of CD34+/CD38− LSCs and
CD34+/CD38+ bulk tumor cells were purified by high-speed cell
sorting using a FACS Melody cell sorter (Becton Dickenson) and
were placed back into culture prior to the addition of 1 μM
gemcitabine or the ProTide NUC-1031_mix.
were solved and refined using SHELX (see Table S2, Supporting
Information).49,50 Geometric restraints were applied during refine-
ment of orientationally disordered components. Non-hydrogen atoms
were refined with anisotropic displacement parameters. Hydrogen
atoms were inserted in idealized positions, and a riding model was
used.
General Methods. All solvents and reagents were used as obtained
from commercial sources unless otherwise indicated. All reactions
were performed under a nitrogen atmosphere. The 1H and 13C NMR
spectra were recorded on a Varian spectrometer operating at 500
1
MHz for H and 125 MHz for 13C. Deuterated chloroform was used
as the solvent for NMR experiments, unless otherwise stated. 1H
chemical shifts values (δ) are referenced to the residual nondeuterated
components of the NMR solvents (δ = 7.26 ppm for CHCl3, etc.).
The 13C chemical shifts (δ) are referenced to CDCl3 (central peak, δ
= 77.0 ppm). Fluorine chemical shifts are referenced to CFCl3. Mass
spectra were measured in positive mode electrospray ionization (ESI).
Thin layer chromatography (TLC) was performed on silica gel 60
F254 plastic sheets. Column chromatography was performed using
silica gel (35−75 mesh). Purity of prepared compounds was
determined by high-pressure liquid chromatography-ultraviolet
(HPLC-UV) analysis (Thermo HPLC connected with UV detector).
The purity of all final compounds was determined to be >95% by
reversed phase-high-pressure liquid chromatography (RP-HPLC)
using the eluents water (eluent A), acetonitrile (eluent B), and the
following conditions: Varian Pursuit XS, 4.6 mm × 150 mm, 5.0 μm,
1.0 mL/min, gradient 30 min 10% → 100% eluent B in eluent A
(method 1). The purity of the intermediates was >90%, unless
otherwise stated.
Mass Spectrometry Assessment of Intracellular Nucleoside/
ProTide. Defined concentrations of nucleoside were run through the
mass spectrometer in order to construct a standard curve (Figure 6A).
This was used to quantify the levels of nucleoside/ProTide
triphosphate in each of the samples provided. KG1a cells (5 ×
106), cultured in DMEM + 10% FCS, were exposed to 1 μM of
gemcitabine or NUC-1031_mix for 1 h prior to harvesting by
centrifugation. Cells were then FACS sorted into LSC (CD34+/
CD38-) and bulk tumor cell fractions (CD34+/CD38+). Purified
cells were then pelleted prior to lysis and mass spectrometry analysis
for intracellular triphosphate content of gemcitabine or NUC-1031.
Immunopheotypic Expression of ABCB1 and ABCG2. KG1a cells
were harvested from culture and labeled with anti-CD34 (FITC) and
anti-CD38 (PE) to identify the LSC and bulk tumor fractions. In
addition, they were labeled with anti-ABCB1 (Alexa 647) and anti-
ABCG2 (APC). Subsequently, the LSC and bulk tumor fractions were
gated and the expression levels of the two ABC transporters were
quantified using mean fluorescence intensity values.
Transcription of ABC Transporter Genes. A volume of 1 mL of
TRIzol was added to each pellet of 2 × 105 harvested bulk tumor or
LSC cells. A volume of 200 μL of chloroform was added and
centrifuged at 8000g for 15 min at 4 °C. The aqueous layer was gently
removed and added to an equal volume of 70% ethanol. A volume of
700 μL of the sample was then added to an RNeasy spin column, and
the RNeasy Mini Kit protocol was followed. The elution step used 50
μL of RNase-free water, and RNA quantification was assessed using a
Nanodrop:1000 spectrophotometer. Duplicate 1 μL samples were
evaluated, and the RNA concentration, A260/280 ratio, and A260/
A230 ratios were obtained. Aliquots of 0.5 μg of RNA were then
added to 0.5 mL microfuge tubes containing 10 μL of master mix and
made up to 20 μL with sterile RNase-free water. Samples were added
to the thermal cycler and run as per the kit’s protocol. Once the run
was complete, 20 μL of newly converted cDNA was diluted 5:1 with
sterile DNA and RNase-free water to give a working concentration of
2.5 ng/μL. Samples were stored at −20 °C. The TaqMan Fast
Advanced Master Mix kit protocol was followed. Samples were run in
triplicate, including a minus RT sample and a minus cDNA sample.
Samples were run on a 96-well plate with a running volume 10 μL per
well. This was composed of 2.5 μL of thawed cDNA, 0.5 μL of
forward and reverse primers (ABC transporter gene or GAPDH as a
housekeeping control; see Table S1, Supporting Information), 5 μL of
TaqMan master mix, and 2 μL of RNase-free water. Samples were run
on a ViiA7 real-time PCR system using the TaqMan FAST program.
Results were analyzed using the Thermo Fisher Cloud software using
the Relative Quantification qPCR application.
General Procedure 1 for the Preparation of 3′,5′-TBDMS
Protected FUDR (3) and of 3′,5′-TBDMS Protected Gemcitabine
(4). TBDMSCl (2.2 equiv) was added to a stirred solution of
nucleoside (1.0 equiv), imidazole (5.0 equiv), and DMAP (0.3 equiv)
in DMF (10 mL) at 0 °C, and the mixture was allowed to warm to 25
°C and stirred for 1 h. The reaction was quenched with saturated
aqueous solution of ammonium chloride, followed by extraction with
EtOAc (3 × 5 mL). The combined organic layers were dried over
Na2SO4, and the solvent was removed under vacuum to give a crude
product as an oil used for the next step without further purification.
2′-Deoxy-5-fluoro-3′,5′-bis(O-tert-butyldimethylsilyl-uridine (3)
was obtained from FUDR (2.0 g, 8.10 mmol), imidazole (40.62
mmol, 2.76 g), DMAP (2.43 mmol, 0.297 g), and TBDMSCl (17.87
1
mmol, 2.70 g) in DMF (15 mL) as an oil. Yield, 83% (3.20 g). H
NMR (500 MHz, CD3OD): δH 8.02 (d, J = 6.5 Hz, 1H, H-6), 6.30 (t,
J = 7.50, 1H, H-1′), 4.40 (q, J = 3.5 Hz, 1H, H-3′), 3.97−3.86 (m, 2H,
H-4′, H-5′), 3.74 (dd, 1H, J = 12.0, 3.5 Hz, H-5′), 2.40−2.31 (m, 1H,
H-2′), 2.10−2.00 (m, 1H, H-2′), 0.93, 0.92 (2s, 18H, C(CH3)3), 0.12,
0.11 (2s, 12H, Si(CH3)2). 13C NMR (125 MHz, CDCl3): δC 157.23
(d,2JC−F = 26.0 Hz, C-4), 149.17 (C-2), 141.54 (d,1JC−F = 235.0 Hz,
C-5), 124.12 (d,2JC−F = 33.7 Hz, C-6), 87.98 (C-4′), 85.46 (C-1′),
71.38 (C-3′), 62.58 (C-5′), 41.73 (C-2′), 25.65 (C(CH3)3), 18.37,
18.06 (C(CH3)3), −4.67, −4.93 (Si(CH3)2).
2′-Deoxy-2′,2′-difluoro-3′,5′-bis(O-tert-butyldimethylsilyl-D-cyti-
dine (4) was obtained from gemcitabine (2.0 g, 7.59 mmol),
imidazole (2.58 g, 37.95 mmol), DMAP (0.278 g, 2.27 mmol), and
TBDMSCl (2.52 g, 16.69 mmol) in DMF (15 mL) as an oil. Yield
82% (3.05 g). 1H NMR (500 MHz, CD3OD): δH 7.74 (d, J = 8.0 Hz,
1H, H-6), 6.27 (dd, JH−F = 9.50, 5.50 Hz, 1H, H-1′), 5.77 (d, J = 7.5
Hz, 1H, H-5), 4.38−4.28 (m, 1H, H-3′), 4.02 (dd, 1H, J = 11.5, 3.5
Hz, H-5′a), 3.92−3.88 (m, 1H, H-4′), 3.80 (dd, 1H, J = 2.0, 11.5 Hz,
H-5′b), 0.93, 0.92 (2s, 18H, C(CH3)3), 0.12, 0.11 (2s, 12H,
Si(CH3)2).
2′-Deoxy-5-fluoro-3′-(O-tert-butyldimethylsilyl-uridine (5). CSA
(6.74 mmol, 1.56 g) was added to 2′-deoxy-5-fluoro-3′,5′-bis(O-tert-
butyldimethylsilyl-uridine (3) (6.74 mmol, 3.20 g) dissolved in
MeOH (15 mL), at 0 °C and the reaction mixture was stirred under
an argon atmosphere for 10 min. After the reaction was completed,
the solvent was removed under reduced pressure to yield the crude
residue that was purified by silica gel column chromatography with a
gradient of MeOH (1% to 10%) in DCM as an eluent to afford 5 as a
Statistical Analysis. The data obtained in these experiments were
evaluated using one-way ANOVA. All data was confirmed as Gaussian
or a Gaussian approximation using the omnibus K2 test. LC50 values
were calculated from the nonlinear regression and line of best-fit
analysis of the sigmoidal dose−response curves. Values are shown as
mean SEM from each replicate data set. All statistical analyses were
performed using Graphpad Prism 6.0 software (GraphPad Software
Inc., San Diego, CA).
Crystal Structure Determination. Single-crystal XRD data were
collected on an Agilent SuperNova Dual Atlas diffractometer with a
mirror monochromator using Cu (λ = 1.5418 Å) . Crystal structures
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white solid. Yield, 60% (1.45 g). 1H NMR (500 MHz, DMSO-d6): δH
8.22 (d, JH−F = 8.0 Hz, 1H, H-6), 6.25 (td, J = 7.5, 2.0 Hz 1H, H-1′),
5.20 (t, J = 3.0 Hz, 1H, 5′-OH), 4.53 (q, J = 3.5 Hz, 1H, H-3′), 3.92
(qr, J = 3.5 Hz, 1H, H-4′), 3.80 (dd, J = 12.0, 3.5 Hz, 1H, H-5′), 3.73
(dd, J = 12.0, 3.5 Hz, 1H, H-5′), 2.26−2.21 (m, 2H, H-2′), 0.94 (s,
9H, C(CH3)3), 0.15 (s, 6H, Si(CH3)2). 13C NMR (125 MHz,
CD3OD): δC 159.46 (d,2JC−F = 26.2 Hz, C-4), 150.83 (C-2), 141.87
(d,1JC−F = 231.0 Hz, C-5), 126.24 (d,2JC−F = 35.0 Hz, C-6), 89.58 (C-
4′), 86.83 (C-1′), 73.46 (C-3′), 62.38 (C-5′), 41.99 (C-2′), 26.41
(C(CH3)3), 18.84 (C(CH3)3), −4.65 (d,1JC−Si = 18.7 Hz, (Si(CH3)2).
MS (MSI+): m/z calcd for C15H25FN2O5Si: 360.45 [M]+; found
383.149 [M + Na]+.
2′-Deoxy-2′,2′-difluoro-3′-(O-tert-butyldimethylsilyl)-D-cytidine
(6). A solution of trichloroacetic acid (113.88 mmol, 11.42 mmol) in
8.7 mL of water was added to a solution of compound 4 (2.0 g, 4.06
mmol) in THF (38 mL) at 0 °C. The reaction mixture was stirred for
2 h at rt, and then the reaction solution was neutralized with solid
NaHCO3 until evolution of gas was finished. Next, 100 mL of water
was added to the reaction mixture, followed by extraction with EtOAc
(3 × 50 mL). The volatiles were concentrated under reduced pressure
to afford the title compound 6 as a white solid. Yield, 82% (1.25 g).
1H NMR (500 MHz, DMSO-d6): δH 7.54 (d, J = 7.5 Hz, 1H, H-6),
7.55, 7.53 (2 × bs, 2H, NH2), 6.03 (t, JH−F = 8.0 Hz, 1H, H-1′), 5.68
(d, J = 7.5 Hz, 1H, H-5), 5.15 (bs, 1H, OH-5′), 4.25−4.18 (m, 1H, H-
3′), 3.71−3.64 (m, 2H, H-4′, H-5′a), 3.46 (dd, J = 12.5, 3.5 Hz, 1H,
H-5′b), 0.77 (s, 9H, C(CH3)3), 0.00, −0.02 (2s, 6H, Si(CH3)2). 13C
NMR (125 MHz, CD3OD): δC 165.99 (C−NH2), 156.16 (CO
base), 140.43 (CH−base), 122.61 (t,1JC−F = 256 Hz, CF2), 95.16
(CH−base), 84.35 (t,2JC−F = 31.7 Hz, C-1′), 80.67 (t,3JC−F = 4.0 Hz,
C-4′), 68.39 (t,2JC−F = 22.8 Hz, C-3′), 60.35 (C-5′), 25.09, 24.92
(C(CH3)3), 17.92 (C(CH3)3), −6.65 (d,1JC−Si = 12.9 Hz, Si(CH3)2).
General Procedure 2 for a Preparation of 3′-TBDMS-Protected
ProTides. tert-BuMgCl (1.0 M solution in THF, 1.2 equiv) was added
to a solution of 3′-O-(tert-butyldimethylsilyl)-protected nucleoside
(1.0 equiv) in dry THF (10 mL), in one portion followed by addition
of an appropriate phosphorochloridate (2.0 equiv) dissolved in
anhydrous THF (3 mL). The reaction mixture was stirred for 16 h
and then evaporated under reduced pressure to give a crude residue
that was purified either by silica gel column chromatography, eluting
with a gradient of MeOH (0−2%) in DCM or with a gradient of
MeOH (3−6%) in a mixture of EtOAc (15%) and DCM as an eluent,
followed by TLC preparative purification unless stated otherwise to
afford 3′-TBDMS-protected ProTides as single diastereoisomers.
2′-Deoxy-5-fluoro-3′-O-tert-butyl-dimethylsilyl-D-uridine-5′-O-
[naphthyl-(benzyloxy-L-alaninyl)] phosphate (7) was prepared
according to general procedure 2 from 2′-deoxy-5-fluoro-3′-(O-tert-
butyldimethylsilyl-uridine (5) (2.77 mmol, 1.0 g) and L-Ala-OBn-
ONaph phosphorochloridate10 (5.55 mmol, 2.24 g), (3.33 mmol, 3.33
mL) in THF (15 mL). The crude mixture was purified by silica
column chromatography with a slow gradient of MeOH (0−2%) in
DCM as an eluent to give the fast eluting isomer A (7a,SP) or (7b,RP)
and the slow eluting isomer B (7a, SP) or (7b,RP).
CD3OD): δH 8.17−8.14 (m, 1H, H-Ar), 7.88−7.86 (m, 1H, H-Ar),
7.70 (d, JH−F = 6.5 Hz, 1H, H-6), 7.60 (d, J = 8.5 Hz, 1H, H-Ar),
7.54−7.50 (m, 3H, H-Ar), 7.37 (t, J = 8.0 Hz, 1H, H-Ar), 7.33−7.27
(m, 5H, H-Ar), 6.06 (t, J = 7.5 Hz 1H, H-1′), 5.06 (apparent s, 2H,
OCH2Ph), 4.32−4.27 (m, 2H, H-3′, H-5′), 4.23−4.19 (m, 1H, H-5′),
4.15−4.11 (m, 1H, CHCH3), 3.99−3.97 (m, 1H, H-4′), 1.98 (ddd, J
= 13.5, 5.5, 2.5 Hz, 1H, H-2′), 1.59−1.54 (m, 1H, H-2′), 1.38 (d, J =
7.0 Hz, 3H, CHCH3), 0.87 (s, 9H, C(CH3)3), 0.04 (d,2JSi−H = 5.5 Hz,
6H, Si(CH3)2). 13C NMR (125 MHz, CD3OD): δC 173.08 (d,3JC−P
=
4.8 Hz, CO, ester), 157.95 (d,2JC−F = 26.2 Hz, C-4), 149.12 (C-2),
146.41 (d,2JC−P = 7.8 Hz, OC-Naph), 140.32 (d,1JC−F = 232.0 Hz, C-
5), 135.74, 134.90 (C-Ar), 128.19, 127.96, 127.89, 127.60, 126.51
(CH−Ar), 126.47, 126.42 (C-Ar), 126.20, 125.17, 124.84 (CH−Ar),
124.28 (d,2JC−F = 34.1 Hz, C-6), 121.20 (CH-Ar), 115.17 (d,3JC−P
=
3.2 Hz, CH−Ar), 85.77 (d,3JC−P = 7.7 Hz, C-4′), 85.55 (C-1′), 72.10
(C-3′), 66.63 (OCH2Ph), 66.11 (d,2JC−P = 5.5 Hz, C-5′), 50.46
(CHCH3), 39.80 (C-2′), 24.81 (C(CH3)3), 18.99 (d,3JC−P = 6.9 Hz,
CHCH3), 17.34 (C(CH3)3), −6.06 (d,1JC−Si = 14.2 Hz, Si(CH3)2).
MS (MSI+): m/z calcd for C35H43FN3O9PSi: 727.78 [M]+; found
750.25 [M + Na]+.
2′-Deoxy-2′,2′-difluoro-3′-O-(tert-butyl-dimethylsilyl)-D-cytidine-
5′-O-[phenyl(benzyloxy-L-alaninyl)] phosphate (8) was obtained
from 3′-O-(tert-butyldimethylsilyl)-gemcitabine 6 (2.66 mmol, 1.0
g) in dry THF (14 mL), tert-BuMgCl (2.90 mmol, 2.9 mL), and L-
Ala-OBn-OPh phosphorochloridate9 (3.97 mmol, 1.40 g) dissolved in
anhydrous THF (5 mL). The reaction mixture was stirred for 16 h
and then evaporated in vacuum to give a crude residue that was
purified by column chromatography on silica gel, eluting with a
gradient of DCM/EtOAc/MeOH (82%/15%/3%) to (79%/15%/
6%) to afford 8b as the fast eluting fraction and 8a as the slow eluting
fraction.
2′-Deoxy-2′,2′-difluoro-3′-tert-butylsilyl-D-cytidine-5′-O-[phenyl-
(benzyloxy-L-alaninyl)] phosphate (8a) was the slow eluting fraction
obtained as a white solid. Yield, 30% (0.55 g). 31P NMR (202 MHz,
CD3OD): δP 3.56. 1H NMR (500 MHz, CD3OD): δH 7.40 (d, J = 7.5
Hz, 1H, H-6), 7.22−7.16 (m, 7H, Ar-H), 7.09−7.04 (m, 3H, Ar-H),
6.07 (t, JH−F = 8.5 Hz 1H, H-1′), 5.71 (d, J = 7.5 Hz, 1H, H-5), 5.00,
4.96 (AB q, JAB = 12.0, 9.0 Hz, 2H, OCH2Ph), 4.31−4.27 (m, 1H, H-
3′), 4.22−4.16 (m, 1H, H-4′), 4.13−4.08 (m, 1H, H-5′), 3.89−3.83
(m, 2H, H-5′, CHCH3), 1.23 (dd, J = 7.0, 1.5 Hz, 3H, CHCH3), 0.78
(s, 9H, C(CH3)3), 0.0 (s, 6H, Si(CH3)2). 13C NMR (125 MHz,
CD3OD): δC 173.07 (d,3JC−P = 5.3 Hz, CO, ester), 166.26 (C-
NH2), 156.28 (CO base), 150.68 (d,2JC−P = 3.3 Hz, C-Ar), 141.11
(CH-base), 135.79 (C-Ar), 129.50, 128.22, 127.98, 127.88, 124.94
(CH-Ar), 122.83 (d,1JC−F = 258 Hz, CF2), 120.02, 119.98 (CH-Ar),
95.28 (CH-base), 79.39 (broad signal, C-1′), 79.35 (C-4′), 71.05
(apparent t,2JC−F = 26.1 Hz, C-3′), 66.60 (OCH2Ph), 64.02 (d,2JC−P
4.9 Hz, C-5′), 50.29 (CHCH3), 24.63 (C(CH3)3), 19.02 (d,3JC−P
=
=
6.8 Hz, CHCH3), 17.47 (C(CH3)3), −6.20 (d,1JC−Si = 29.1 Hz,
Si(CH3)2). MS (ESI+) m/z calcd for C31H41F2N4O8PSi: 694.73
+
[M+]; found 717.45 [M + Na] .
2′-Deoxy-2′,2′-difluoro-3′-tert-butylsilyl-D-cytidine-5′-O-[phenyl-
(benzyloxy-L-alaninyl)] phosphate (8b), the fast eluting fraction, was
obtained as a white solid. Yield, 25% (0.45 g). 31P NMR (202 MHz,
CD3OD): δP 3.71. 1H NMR (500 MHz, CD3OD): δH 7.45 (d, J = 7.5
Hz, 1H, H-6), 7.24−7.18 (m, 7H, Ar-H), 7.09−7.07 (m, 3H, Ar-H),
6.12 (t, JH−F = 8.5 Hz 1H, H-1′), 5.78 (d, J = 7.5 Hz, 1H, H-5), 5.04,
5.01 (AB q, JAB = 12.5, 3.0 Hz, 2H, OCH2Ph), 4.39−4.36 (m, 1H, H-
3′), 4.23−4.13 (m, 2H, H-5′, H-4′), 3.93−3.87 (m, 2H, H-5′,
CHCH3), 1.24 (dd, J = 7.0, 1.5 Hz, 3H, CHCH3), 0.80 (s, 9H,
C(CH3)3), 0.0 (s, 6H, Si(CH3)2). 13C NMR (125 MHz, CD3OD): δC
173.35 (d,3JC−P = 4.2 Hz, CO, ester), 166.26 (C-NH2), 156.26
(CO base), 150.63 (d,2JC−P = 2.4 Hz, C-Ar), 140.89 (CH-base),
135.82 (C-Ar), 129.47, 128.21, 127.98, 127.92, 125.57 (CH−Ar),
123.90 (d,1JC−F = 266 Hz, CF2), 120.11, 120.07 (CH-Ar), 95.38 (CH-
base), 79.24 (broad signal, C-1′), 75.23 (broad signal, C-4′), 70.94
(broad signal, C-3′), 66.59 (OCH2Ph), 63.59 (d,2JC−P = 5.0 Hz, C-5′),
50.47 (CHCH3), 24.62 (C(CH3)3), 18.82 (d,3JC−P = 7.7 Hz,
CHCH3), 17.47 (C(CH3)3), −6.21 (d,1JC−Si = 28.1 Hz, Si(CH3)2).
2′-Deoxy-5-fluoro-3′-O-(tert-butyl-dimethylsilyl)-D-uridine-5′-O-
[naphthyl-(benzyloxy-L-alaninyl)] phosphate (7_Isomer A) was the
fast eluting fraction obtained as a white solid. Yield, 9% (0.19 g) 31P
NMR (202 MHz, CD3OD): δP 4.64. 1H NMR (500 MHz, CD3OD):
δH 8.15 (d, J = 8.0 Hz, 1H, Ar-H), 7.89 (d, J = 8.0 Hz, 1H, H-Ar),
7.73 (d, JH−F = 8.0 Hz, 1H, H-6), 7.65 (d, J = 6.5 Hz, 1H, Ar-H),
7.54−7.50 (m, 3H, Ar-H), 7.43. (t, J = 8.0 Hz, 1H, Ar-H), 7.34−7.28
(m, 5H, Ar-H), 6.08 (t, J = 6.5 Hz 1H, H-1′), 5.06 (ABq, JA‑B = 12.0
Hz, 2H, OCH2Ph), 4.30−4.28 (m, 3H, 2 × H-5′, H-3′), 4.15−4.11
(m, 1H, CHCH3), 3.97−3.95 (m, 1H, H-4′), 1.96 (ddd, J = 13.5, 5.5,
2.5 Hz, 1H, H-2′), 1.55−1.50 (m, 1H, H-2′), 1.38 (d, J = 7.0 Hz, 3H,
CHCH3), 0.89 (s, 9H, C(CH3)3), 0.01 (d,2JSi−H = 5.5 Hz, 6H,
Si(CH3)2). MS (MSI+): m/z calcd for C35H43FN3O9PSi: 727.78
[M]+; found 750.25 [M + Na]+.
2′-Deoxy-5-fluoro-3′-O-(tert-butyl-dimethylsilyl)-D-uridine-5′-O-
[naphthyl-(benzyloxy-L-alaninyl)] phosphate (7_Isomer B) was the
slow eluting fraction obtained as a white solid. Yield, 12% (0.25 g).
31P NMR (202 MHz, CD3OD): δP 4.28. 1H NMR (500 MHz,
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MS (ESI+) m/z calcd for C31H41F2N4O8PSi: 694.73 [M+]; found
717.45 [M + Na] .
1.38 (d, J = 7.2 Hz, 3H, CHCH3). 13C NMR (125 MHz, CD3OD): δC
174.61 (d,3JC−P = 5.0 Hz, CO, ester), 167.64 (C-NH2), 157.74
(CO base), 152.10 (d,2JC−P = 7.0 Hz, C-Ar), 142.41 (CH-base),
137.22 (C-Ar), 130.91, 129.64, 129.40, 129.33, 126.33 (CH-Ar),
124.51 (d,1JC−F = 257 Hz, CF2), 121.47, 121.44 (CH−Ar), 96.67
(CH-base), 86.08 (broad signal, C-1′), 80.32 (C-4′), 71.27 (apparent
t,2JC−F = 23.7 Hz, C-3′), 68.04 (OCH2Ph), 65.74 (d,2JC−P = 5.30 Hz,
C-5′), 51.67 (CHCH3), 20.44 (d,3JC−P = 6.25 Hz, CHCH3). (ESI+)
m/z, found: (M + Na+) 603.14. C25H27F2N4O8NaP required: (M+)
580.47. Reverse HPLC, eluting with H2O/MeOH from 100/0 to 0/
100 in 35 min, tR = 18.32 min.
+
General Procedure 3 for the 3′-Desilylation of 3′-TBDMS-
Protected ProTides 7a, 7b and 8a, 8b. The separated diaster-
eoisomer of 3′-TBDMS-protected ProTide (7a, 7b, 8a, and 8b) was
treated with a mixture of THF/TFA/DCM (4:1:1) at 0 °C. The
resulting reaction mixture was stirred at 0 °C for 2 h and then at room
temperature for 12 h. After the reaction was completed, the solvents
were evaporated, and the residue was purified on silica gel with a
gradient of MeOH (2−8%) in DCM as an eluent.
2′-Deoxy-5-fluoro-D-uridine-5′-O-[naphthyl-(benzyloxy-L-alanin-
yl)] phosphate (9a or 9b, NUC-3373_IsoA) was obtained from the
fast eluting 7_isomer A (7a or 7b) (0.19 g, 0.26 mmol) as a white
solid. Yield, 75% (0.12 g). 31P NMR (202 MHz, CD3OD): δP 4.60.
2′-Deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzyloxy-L-ala-
ninyl)] phosphate (10b, NUC-1031_RP) was obtained from 8b (0.45
g, 0.65 mmol) as a white, crystalline solid. Yield, 78% (0.29 g). 31P
NMR (202 MHz, CD3OD): δP 3.83. 19F NMR (470 MHz, CD3OD):
δF −118.3 (d, J = 241 Hz, F), −120.38 (broad d, J = 241 Hz, F). 1H
NMR (500 MHz, CD3OD): δH 7.56 (d, J = 7.5 Hz, 1H, H-6), 7.38−
7.31 (m, 7H, Ar-H), 7.23−7.19 (m, 3H, Ar-H), 6.26 (t, J = 7.5 Hz,
1H, H-1′), 5.88 (d, J = 7.5 Hz, 1H, H-5), 5.20 (s, 2H, OCH2Ph),
4.49−4.46 (m, 1H, H-5′), 4.38−4.34 (m, 1H, H-5′), 4.23−4.17 (m,
1H, H-3′), 4.07−4.01 (m, 2H, H-4′, CHCH3), 1.38 (d, J = 7.2 Hz,
1
19F NMR (470 MHz, CD3OD): δF − 167.05. H NMR (500 MHz,
CD3OD): δH 8.14 (dd, J = 8.5, 2.0 Hz, 1H, Ar-H), 7.87 (d, J = 7.0 Hz,
1H, H-Ar), 7.72 (apparent s, 1H, Ar-H), 7.69 (d, JH−F = 6.5 Hz, 1H,
H-6), 7.54−7.49 (m, 3H, Ar-H), 7.42 (t, J = 8.0 Hz, 1H, Ar-H), 7.34−
7.28 (m, 5H, Ar-H), 6.14 (t, J = 6.0 Hz 1H, H-1′), 5.10 (ABq, JA‑B
=
12.5 Hz, 2H, OCH2Ph), 4.35−4.33 (m, 2H, H-3′, H-5′), 4.30−4.28
(m, 1H, H-5′), 4.14−4.08 (m, 1H, CHCH3), 4.07−4.05 (m, 1H, H-
4’), 2.11 (ddd, J = 14.0, 6.0, 3.0 Hz, 1H, H-2’), 1.73−1.68 (m, 1H, H-
2’), 1.33 (dd, J = 7.5, 1.5 Hz, 3H, CHCH3). 13C NMR (125 MHz,
CD3OD): δC 174.92 (d,3JC−P = 3.9 Hz, CO, ester), 159.27 (d,2JC−F
= 25.9 Hz, C-4), 150.52 (C-2), 147.90 (d,2JC−P = 7.3 Hz, OC-Naph),
141.73 (d,1JC−F = 232.3 Hz, C-5), 137.19, 136.29 (C-Ar), 129.59,
129.36, 128.91, 127.90 (CH-Ar), 127.85, 127.80 (C-Ar), 127.60 (CH-
Ar), 126.50 (d,3JC−P = 1.4 Hz, CH-Naph), 126.18 (CH-Ar), 125.53
(d,2JC−F = 34.0 Hz, C-6), 122.64 (CH-Ar), 116.30 (d,3JC−P = 3.12 Hz,
CH-Ar), 86.93 (C-1′), 86.88 (d,3JC−P = 8.0 Hz, C-4′), 72.18 (C-3′),
68.10 (OCH2Ph), 67.86 (d,2JC−P = 5.2 Hz, C-5′), 51.96 (CHCH3),
40.84 (C-2′), 20.24 (d,3JC−P = 7.5 Hz, CHCH3). MS (MSI+): m/z
calcd for C29H29FN3O9P: 613.53 [M]+; found 636.15 [M + Na]+.
Reverse HPLC eluting with (H2O/AcCN from 100/0 to 0/100) in 30
min, tR 16.61 min.
3H, CHCH3). 13C NMR (125 MHz, CD3OD): δC 174.65 (d,3JC−P
=
5.0 Hz, CO, ester), 167.65 (C-NH2), 157.75 (CO base), 152.10
(d,2JC−P = 7.0 Hz, C-Ar), 142.28 (CH-base), 137.50 (C-Ar), 130.86,
129.63, 129.40, 129.32, 126.31 (CH-Ar), 123.73 (d,1JC−F = 252 Hz,
CF2), 121.44, 121.40 (CH-Ar), 96.67 (CH-base), 85.90 (broad signal,
C-1′), 80.27 (C-4′), 71.02 (apparent t,2JC−F = 23.7 Hz, C-3′), 68.04
(OCH2Ph), 65.52 (d,2JC−P = 5.30 Hz, C-5′), 51.85 (CHCH3), 20.23
(d,3JC−P = 7.5 Hz, CHCH3). (ESI+) m/z, found: (M + Na+) 603.14.
C25H27F2N4O8NaP required: (M+) 580.47. Reverse HPLC, eluting
with H2O/MeOH from 100/0 to 0/100 in 35 min, tR = 19.15 min.
General Method for Crystallization of 7_isoB from the Column
Purified Amorphous 7_isoB Using the Modified Procedure.26 The
chromatographed fraction containing the compound 7_isoB (50 mg,
97%) was dissolved in 5 mL of 2-propanol and diluted with hexane
until cloudy. The solution was seeded and stirred at room
temperature for 5 h. The resulting solid was filtered, washed with
hexane (2 × 2 mL), and dried under a high vacuum. The obtained
solid was not suitable for single crystal X-ray analysis. Multiple
attempts to crystallize 7_isoA, 7_isoB, 9_isoA, and 9_isoB by slow
evaporation of MeOH, 2-propanol, CHCl3, and a combination of
CHCl3/Et2O (1/1, v/v), and MeOH/CHCl3 (3/1, v/v), CHCl3/
H2O (1/1, v/v) also failed to give crystals of sufficient quality for
single crystal X-ray analysis.
2′-Deoxy-5-fluoro-D-uridine-5′-O-[naphthyl-(benzyloxy-L-alanin-
yl)] phosphate (9a or 9b, NUC-3373_IsoB) was obtained from the
slow eluting 7_isomer B (7a or 7b) (0.24 g, 0.36 mmol) (0.19 g, 0.26
mmol) as a white solid. Yield, 75% (0.15 g). 31P NMR (202 MHz,
1
CD3OD): δP 4.26. 19F NMR (470 MHz, CD3OD): δF −167.31. H
NMR (500 MHz, CD3OD): δH 8.17−8.14 (m, 1H, Ar-H), 7.90−7.87
(m, 1H, Ar-H), 7.72 (apparent s, 1H, Ar-H), 7.69 (d, JH−F = 6.0 Hz,
1H, H-6), 7.54−7.48 (m, 3H, Ar-H), 7.38 (t, J = 8.0 Hz, 1H, Ar-H),
7.34−7.28 (m, 5H, Ar-H), 6.10 (t, J = 6.0 Hz 1H, H-1′), 5.12 (s, 2H,
OCH2Ph), 4.36−4.25 (m, 3H, H-3′, 2 × H-5′), 4.14−4.08 (m, 1H,
CHCH3), 4.05 (apparent q, J = 2.5 Hz, 1H, H-4′), 2.14 (ddd, J = 14.0,
6.0, 3.0 Hz, 1H, H-2′), 1.75−1.69 (m, 1H, H-2′), 1.36 (dd, J = 7.0, 0.5
Hz, 3H, CHCH3). 13C NMR (125 MHz, CD3OD): δC 174.57
(d,3JC−P = 4.5 Hz, CO, ester), 159.38 (d,2JC−F = 26.0 Hz, C-4),
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02194.
■
sı
150.48 (C-2), 147.80 (d,2JC−P = 7.3 Hz, OC-Naph), 141.67 (d,1JC−F
=
232.0 Hz, C-5), 137.15, 136.27 (C-Ar), 129.63, 129.40, 129.36,
128.97, 127.89 (CH-Ar), 127.86, 127.81 (C-Ar), 127.59 (CH-Ar),
126.56 (d,3JC−P = 1.4 Hz, CH-Naph), 126.22 (CH-Ar), 125.62
(d,2JC−F = 34.1 Hz, C-6), 122.63 (CH-Ar), 116.56 (d,3JC−P = 3.0 Hz,
CH-Ar), 86.98 (C-1′), 86.68 (d,3JC−P = 7.6 Hz, C-4′), 72.02 (C-3′),
68.08 (OCH2Ph), 67.85 (d,2JC−P = 5.5 Hz, C-5′), 51.84 (CHCH3),
40.90 (C-2′), 20.44 (d,3JC−P = 6.7 Hz, CHCH3). MS (MSI+): m/z
calcd for C29H29FN3O9P: 613.53 [M]+; found 636.15 [M + Na]+.
Reverse HPLC eluting with (H2O/AcCN from 100/0 to 0/100) in 30
min, tR 16.03 min.
Compound molecular formula strings (CSV)
Carboxypeptidase Y assay for NUC-3373_isoB, 10a, and
10b (Figures S1a−c); list of primers used for RT-PCR
(Table S1); compound purity analyses; and crystal data
and structure refinement for the compounds 10a and
10b (Table S2) (PDF)
Accession Codes
CCDC 2049153 and 2049154 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/structures.
2′-Deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzyloxy-L-ala-
ninyl)] phosphate (10a, NUC-1031_SP) obtained from 8a (0.55 g,
0.79 mmol) as a white, crystalline solid. Yield, 76% (0.35 g). 31P NMR
(202 MHz, CD3OD): δP 3.66. 19F NMR (470 MHz, CD3OD): δF
1
−118.0 (d, J = 241 Hz, F), −120.24 (broad d, J = 241 Hz, F). H
AUTHOR INFORMATION
Corresponding Authors
■
NMR (500 MHz, CD3OD): δH 7.58 (d, J = 7.5 Hz, 1H, H-6), 7.38−
7.32 (m, 7H, Ar-H), 7.26−7.20 (m, 3H, Ar-H), 6.24 (t, J = 7.5 Hz,
1H, H-1′), 5.84 (d, J = 7.5 Hz, 1H, H-5), 5.20 (AB q, JAB = 12.0, 7.2
Hz, 2H, OCH2Ph), 4.46−4.43 (m, 1H, H-5′), 4.36−4.31 (m, 1H, H-
5′), 4.25−4.19 (m, 1H, H-3′), 4.07−4.00 (m, 2H, H-4′, CHCH3),
Magdalena Slusarczyk − Cardiff School of Pharmacy and
Pharmaceutical Sciences, Cardiff University, Cardiff CF10
3NB, U.K.; orcid.org/0000-0002-4707-7190;
8191
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Parkinson’s disease associated PTEN-induced putative kinase 1
(PINK1) independent of mitochondrial depolarization. J. Med.
Chem. 2017, 60, 3518−3524.
Phone: +44 (0)2920874551; Email: SlusarczykM1@
cf.ac.uk
Chris Pepper − Brighton and Sussex Medical School,
University of Sussex, Brighton BN1 9PX, U.K.; Phone: +44
(0)1273 678644; Email: C.Pepper@bsms.ac.uk
(8) Davey, M. S.; Malde, R.; Mykura, R. C.; Baker, A. T.; Taher, T.
E.; Le Duff, C. S.; Willcox, B. E.; Mehellou, Y. Synthesis and biological
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aryloxy triester phosphoramidate prodrugs as activators of Vγ9/Vδ2
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G.; Blagden, S.; Thompson, E.; Ghazaly, E.; McGuigan, C.
Application of protide technology to gemcitabine: a successful
approach to overcome the key cancer resistance mechanisms leads
to a new agent (NUC-1031) in clinical development. J. Med. Chem.
2014, 57, 1531−1542.
Authors
Michaela Serpi − Cardiff University, School of Chemistry,
Cardiff CF10 3AT, U.K.
Essam Ghazaly − Centre for Haemato-Oncology, Barts Cancer
Institute, Queen Mary University of London, London EC1M
6BQ, U.K.
Benson M. Kariuki − Cardiff University, School of Chemistry,
Cardiff CF10 3AT, U.K.; orcid.org/0000-0002-8658-
3897
Christopher McGuigan − Cardiff School of Pharmacy and
Pharmaceutical Sciences, Cardiff University, Cardiff CF10
3NB, U.K.
(10) McGuigan, C.; Murziani, P.; Slusarczyk, M.; Gonczy, B.; Vande
Voorde, J.; Liekens, S.; Balzarini, J. Phosphoramidate protides of the
anticancer agent FUDR successfully deliver the preformed bioactive
monophosphate in cells and confer advantage over the parent
nucleoside. J. Med. Chem. 2011, 54, 7247−7258.
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transporters are required for gemcitabine influx and manifestation of
toxicity in cancer cell lines. Cancer Res. 1998, 58, 4349−4357.
(b) Gourdeau, H.; Clarke, M. L.; Ouellet, F.; Mowles, D.; Selner, M.;
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02194
Notes
The authors declare no competing financial interest.
̀
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ACKNOWLEDGMENTS
■
The authors would like to thank NuCana plc for funding part
of the research work described in this manuscript and Andrew
William Gibbs for technical assistance.
ABBREVIATIONS USED
■
BTC, biliary tract cancer; CDA, cytidine deaminase; CSA,
camphorsulfonic acid; dCK, deoxycytidine kinase; DPD,
dihydropyrimidine dehydrogenase; hENT1, human equilibra-
tive nucleoside transporter 1; TP, thymidine phosphorylase;
TS, thymidylate synthase
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