Symmetrical Diamidates as a Class of Phosphate Prodrugs to Deliver the 5′-Monophosphate Forms of Anticancer Nucleoside Analogues

Article abstract of DOI:10.1002/cmdc.201800504

The application of phosphorodiamidate technology to pyrimidine and purine nucleosides with anticancer activity to potentially overcome the resistance mechanisms associated with parent nucleosides is reported. Sixteen symmetrical phosphorodiamidates were prepared from the natural amino acids l-alanine and glycine. All the compounds were evaluated for their cytotoxic activity against a wide panel of solid and leukaemic tumour cell lines. In addition, a carboxypeptidase Y assay was performed on a representative phosphorodiamidate in order to reveal the putative bioactivation pathway for the reported phosphorodiamidate-type prodrugs.

Full text of DOI:10.1002/cmdc.201800504

Accepted Article
Title: Symmetrical diamidates as a class of phosphate prodrugs to
deliver the 5'-monophosphate form of anticancer nucleoside
analogues
Authors: Magdalena Slusarczyk, Valentina Ferrari, Michaela Serpi,
Blanka Gonczy, Jan Balzarini, and Christopher McGuigan
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To be cited as: ChemMedChem 10.1002/cmdc.201800504
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ChemMedChem
Symmetrical diamidates as a class of phosphate prodrugs to
deliver the 5’-monophosphate form of anticancer nucleoside
analogues
Magdalena Slusarczyk,*1 Valentina Ferrari,1 Michaela Serpi,1 Blanka Gönczy,1 Jan
Balzarini,2 and Christopher McGuigan1
1School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, King Edward
VII Avenue, Cardiff CF10 3NB, U.K.
2Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research,
Herestraat 49, 3000 Leuven, Belgium
*Author for correspondence:
E-mail: SlusarczykM1@cardiff.ac.uk
Tel.: +44 02920874551
Dedicated to Professor Christopher McGuigan (1958-2016)
Abstract
We herein report on the application of phosphorodiamidate technology to both
pyrimidine and purine nucleosides with anticancer activity to potentially overcome
the resistance mechanisms associated with parent nucleosides. Sixteen symmetrical
phosphorodiamidates prepared from natural amino acids such as L-alanine and
glycine were synthesized. All the compounds were evaluated for their cytotoxic
activity in a wide panel of solid and leukemic tumour cell lines. In addition, a
carboxypeptidase Y assay was performed on a representative phosphorodiamidate in
order to reveal the putative bioactivation pathway for the reported
phosphorodiamidate-type prodrugs.
Introduction
Current chemotherapeutic treatment of cancer and viral infections is largely based on
the use of antimetabolites, in particular modified nucleoside analogues (NA)
mimicking natural purine or pyrimidine nucleosides.[1] In addition, there is also strong
evidence that NAs have some potential as antibacterial agents.[2,3] These molecules are
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10.1002/cmdc.201800504
ChemMedChem
often taken up by nucleoside transporters (NT) and then phosphorylated by specific
nucleo(s)(t)ide kinases to the corresponding 5’-mono, di-and triphosphate forms.
These metabolites interfere with de novo synthesis of DNA/RNA precursors leading
to inhibition of DNA/RNA synthesis, with subsequent suppression of tumour cell
growth or virus replication. However, NA activity is known to be limited by
mechanisms associated with (i) poor cellular uptake (i.e. by a down-regulation in the
expression of nucleoside transporters), (ii) poor conversion of the drug into an active
metabolite (i.e. by a down-regulation of nucleo(s)(t)ide kinases) or (iii) rapid
degradation into toxic byproducts (i.e. by up-regulation of deactivating enzymes such
as cytidine or cytidylate deaminases).[4,5] Many prodrug strategies aim to overcome
such resistance mechanisms associated with NAs to improve the NA’s effectiveness.
These include phosphate and phosphonate prodrug diesters pivaloyloxymethyl
(POM), isopropyloxycarbonyloxymethyl (POC), cyclosaligenyl (cycloSal), S-
acylthioalkyl (SATE), lipid and HepDirect and phosphoramidates in which an amino
acid ester promoiety is linked via P–N bond to a nucleoside aryl phosphate.[6-8] This
monophosphate prodrug approach, also called ProTide technology, is one of the most
successful prodrug strategies, with an established position in the nucleotide prodrug
area. This strategy, pioneered by Chris McGuigan’s group,[9] led to the discovery and
development of the clinically successful drugs sofosbuvir (Sovaldi)[10] and tenofovir
alafenamide (TAF)[11] by Gilead Sciences. The first drug is approved for the treatment
of chronic HCV infections, whereas the second to treat patients with either HIV or
chronic HBV infections (Figure 1). Other phosphoramidates are currently the subject
of ongoing clinical trials as antiviral agents. Stampidine is in Phase I for the treatment
of HIV infections[12] and GS-5734, more recently developed as a treatment for
filovirus infections,[13] was moved forward through a Phase I clinical trial[14] as a
consequence of the Ebola outbreak in West Africa in 2013-2016.[15] GS-9131 is an
orally bioavailable phosphonoamidate prodrug which has been shown to inhibit HIV-
1 reverse transcriptase (RT) in multiple HIV-1 clinical subtypes and has a unique
resistance profile toward N(t)RTI resistance mutations. GS-9131 is currently under
clinical evaluation.[16]
In the oncology arena, the first phosphoramidate to reach the clinic was
thymectacin (NB1011) which was evaluated in a Phase I trial for the treatment of
colon cancer.[17] Subsequently, our group designed and synthesised two
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10.1002/cmdc.201800504
ChemMedChem
phosphoramidate prodrugs named NUC-1031[18-20] and NUC-3373[21-23] (Figure 1).
The gemcitabine phosphoramidate NUC-1031 is currently being evaluated in several
clinical studies, including a Phase III study for patients with pancreatic cancer, a
Phase II study for patients with ovarian cancer and a Phase Ib study for patients with
biliary tract cancers. The 5’-fluoro-2’-deoxyuridine (FUdR) ProTide NUC-3373 is
currently being evaluated in a Phase I study for the treatment of a variety of solid
tumours including colorectal and breast cancers.
O
O
O
O
NH2
O
O
O
HN P O
O
O
NH
O
NH
H
N
N
N
N
N
O
P
O
N
O
HN P
O
O
O
O
O
N
O
F
O
HO
Sofosbuvir
Stampidine
Phase I, HIV
Tenofovir alafenamide (TAF)
In 2015 FDA-approved for HIV
In 2016 FDA-approved for HBV
Br
In 2013 FDA-approved for HCV
Br
NH2
NH2
O
O
O
O
O
O
O
N
O
N
N
N
F
O
O
HN P O
O
HN P O
O
O
NH
N
O
N
CN
OH
HN P
O
O
O
O
O
HO
HO
NUC-1031
F
HO
Thymectacin
Phase I/II, colorectal cancer
GS-5734
Phase I, Ebola
Phase I/II/III, solid tumours
NH
O
N
N
F
NH2
O
O
O
N
N
O
NH2
O
N
HN
O
O P
NH
N
HN
O
P
P
NH
O
O
N
N
O
O
O
N
NH
O
O
O
O
O
F
HO
GS-9191
PhaseI/II
Genital wart cause by HPV
O
GS-9131
Phase I/II, HIV
NUC-3373
Phase I, solid tumours
Figure 1. ProTides of antiviral and anticancer nucleoside monophosph(on)ate
analogues that have entered clinical studies.
Although less extensively investigated than the ProTide approach, a second
phosphoramidate strategy that has been reported by our group as a promising way for
the delivery of nucleoside monophosphates inside the cell, is the phosphorodiamidate
technology.[24-26] In this approach, two identical amino acid esters are introduced on
the monophosphate moiety through a P–N bond in order to mask the negative
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10.1002/cmdc.201800504
ChemMedChem
charges. Due to their symmetric structure, phosphoro(no)amidates, where no
phosphorus chirality arises, offer advantages over the aryloxy monoamidate
analogues.
Although stereoisomers have the same chemical structure they may exhibit
differences in their pharmacology, toxicology, pharmacokinetics and metabolism. For
example, the Sp diastereoisomer of TAF is 10-fold more potent against HIV than the
Rp diastereoisomer.[27] Likewise, in the HCV replicon assay, the Sp and Rp isomers of
sofosbuvir demonstrated an approximately 18-fold difference in activity against
HCV.[28] Though separation of the ProTide diastereoisomers by crystallization or by
chromatography can be achieved in some cases,[29,30] and different approaches to
obtain ProTides in a diastereoselective[31-34] and 5’-regioselective fashion[35] have
been recently reported, in general most of these methodologies are not very efficient
and are high-priced, time consuming and difficult to scale up. In this scenario the
diamidate approach, if as effective as the ProTides in terms of activity, could be
considered as a valid alternative, as demonstrated by 2’-C-methyl-6-O-
methylguanosine-based phosphorodiamidate agents and their nanomolar anti-HCV
activity in replicon assay.[24] Although the diamidate prodrug approach has been
validated both in vitro and in vivo, to our knowledge, only one phosphonodiamidate,
GS-9191 (Figure 1), has reached the clinic to date. GS-9191 is a nucleotide analogue
under clinical evaluation as a topical ointment in patients with external genital and
perianal warts caused by human papilloma virus infection.[36]
Based on our preliminary findings,[24-26] considering the potential advantage of
lack of chirality at the phosphorus atom for this class of prodrugs, and taking into
account the extraordinary results obtained with gemcitabine and FUdR ProTides, we
were interested to investigate the potential of the phosphorodiamidate approach when
applied to these two nucleosides. We also took the opportunity to expand our study to
other relevant anticancer NAs (1-7, Figure 2).
Herein, we report the synthesis, and biological activities of these target
compounds (8-23) against a wide panel of cancer cell lines. Moreover, in order to
support a putative bioactivation pathway for these prodrugs (in particular regarding
the first activation step), we also report on carboxypeptidase Y assays for the
gemcitabine phosphorodiamidate 8.
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10.1002/cmdc.201800504
ChemMedChem
NH2
N
NH2
N
O
N
N
N
F
NH
Cl
N
N
O
O
HO
HO
HO
O
O
O
F
F
OH OH
OH
OH
Gemcitabine (1)
FUdR (2)
8-Chloroadenosine (3)
O
S
N
NH2
N
N
N
N
N
NH
NH2
NH
N
N
N
X
N
F
HO
HO
HO
O
HO
O
O
HO
OH
OH OH
OH
Fludarabine (4)
X = H, Thioinosine (6)
X = NH2, Thioguanosine (7)
AraG (5)
Figure 2. Anticancer nucleosides considered for this study.
Results and discussion
Chemistry
The phosphorodiamidate prodrugs of gemcitabine (1), FUdR (2),
8-chloroadenosine (3), fludarabine (4), AraG (5), thioinosine (6-MP, 6), and
thioguanosine (6-TG, 7), were prepared according to reported procedure.[37] This
modified method, based on a Yoshikawa procedure to synthesize monophosphate
species,[38] was successfully applied previously to a number of antiviral and anticancer
nucleosides and led to identification of phosphorodiamidates, with L-alanine bearing
either benzyl or 2,2-dimethoxypropyl as the most promising compounds. [24-26] Thus,
in this work, we selected the above-mentioned amino acid esters as preferred entities
and synthesized in total sixteen phosphorodiamidate prodrugs of seven anticancer
NAs. Moreover, for gemcitabine and FUdR, cyclohexyl, pentyl and cyclopentyl esters
were also used, since these moieties afforded molecules with low submicromolar
anticancer activity when applied in the ProTide approach.[18,19] Additionally, for 8-
chloroadenosine, another natural amino acid such as glycine was also included. In the
first step of a general one-pot-two-stages strategy, unprotected nucleoside analogue
was treated with trimethylphosphate (TMP) and 1 equivalent of POCl3, leading to the
formation of the phosphorodichloridate intermediate as depicted in Scheme 1, the
formation of the latter intermediate (not isolated) was monitored by 31P NMR (δp = ~
7-8 ppm) and was accomplished within 4-12 hours. Following this, addition of the
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10.1002/cmdc.201800504
ChemMedChem
appropriate amino acid ester salt (5 equiv.) in the presence of DIPEA (10 equiv.) at –
78 °C afforded the desired phosphorodiamidates 8-23 with the yields between 3% and
49%.
O
R2
R3O
O
P O
O
P O
HN
Cl
HO
B
B
B
a
b
O
O
O
NH
R2
Cl
R1
R1
R1
R3O
OH
R
OH
R
OH
R
O
R = R1= F; B = a
8-23
1
2
3
4
5
6
7
phosphorodichloridate intermediate
R = R1= H; B = b
R2 = Amino acid side chain
R3 = Alkyl, cycloalkyl, aryl
R = R1= OH; B = c
R = H; R1= OH; B = d
R = H; R1= OH; B = e
R = OH; R1= H; B = f
R = OH; R1= H; B = g
NH2
NH2
O
N
NH2
N
B =
F
N
N
N
N
NH
N
Cl
N
N
F
N
N
O
O
a
c
b
d
O
S
N
S
N
N
N
N
N
N
N
NH
NH2
NH
NH2
NH
N
e
g
f
Scheme 1. General synthetic route to phosphorodiamidates 8-23. Reagents and
conditions: (a) (Me)3PO4 (1 mL), POCl3 (2 equiv.), 0-5 °C, 16 h, (b) anhydrous DCM,
amino acid ester salt (5 equiv.), DIPEA (10 equiv.), -78 °C (30 min.), then 0-5 °C,
72h.
Anticancer Activity
The anti-proliferative activities of nucleoside analogues and their related
phosphorodiamidates derived from gemcitabine (1; 8-10), FUdR (2; 11-14),
8-chloroadenosine (3; 15, 16), fludarabine (4, 17, 18), AraG (5; 19), 6-MP (6; 20, 21),
6-TG (7; 22, 23) are represented by their absolute EC50 (µM) and maximal inhibition
at the highest compound concentration (%) and are reported in Table 1 (solid tumour
cell lines) and Table 2 (hematologic cell lines). Gemcitabine was found to be the
most effective compound in Mia-Pa-Ca-2 cell cultures at an EC50 of 1.04 µM.
However, its prodrugs 8-10 were shown to be less active with EC50 ranging between
1.45-7.68 µM. In the same cell line, such loss of activity was also observed for the
diamidates of FUdR 2, with the prodrugs 11 and 13 showing EC50 of 3.54 µM and
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10.1002/cmdc.201800504
ChemMedChem
57.77 µM, respectively, versus an EC50 of 0.18 µM for the nucleoside 2. Conversely,
the diamidate of 8-ClA 16 showed a 2-fold increase in anticancer potency compared
with the parent compound 3 in the Mia-Pa-Ca-2 cell line. A similar trend and a 2-fold
boost in activity for prodrug over nucleoside analogue were noticed for 8-ClA 3 and
the phosphorodiamidate 16 in the MCF-7 cell line, which is generally (along with
HepG2) the least sensitive tumour cell line to our tested compounds. In a panel of
leukemia cell lines, while AraG 5 showed micromolar activity (EC50 of 1.35 µM for
CCRF-CEM and 1.79 µM for MOLT-4), its phosphorodiamidate 19 was found
ineffective (>198 µM) in the same cancer cell lines. This may be suggestive of a poor
cellular uptake and/or metabolic bioactivation and conversion of the
phosphorodiamidate 19 into the corresponding nucleoside monophosphate form. The
cytostatic activities of 6-MP 6 and 6-TG 7 were similar at submicromolar level (for 6:
EC50 of 0.64 µM in CCRF-CEM and 0.83 µM in MOLT-4; for 7: EC50 of 0.71 µM in
HL-60). The two L-Ala-OBn phosphorodiamidates 20 and 22 were ∼3-fold less active
than the parent 6 and 5-fold less active than the parent 7, respectively. A similar trend
was noted for the two L-Ala-Oneopentyl prodrugs 21 and 23, however with markedly
higher loss of activity in respect to the nucleosides 6 and 7 (55-fold in CCRF-CEM,
46-fold in MOLT-4 for 21; 54-fold in HL-60 for 23). This was also observed for
AraG phosphorodiamidate 19. Thus, as already mentioned above, the significant
reduction of potency of the prodrugs containing the neopentyl ester may indicate
either poor cellular uptake and/or metabolic activation and/or higher susceptibility of
L-Ala-Oneopentyl phosphorodiamidates as well as their metabolites to drug efflux
pumps.[22] Generally, the maximal inhibition observed at the highest compound
concentration (198 µM) for all tested compounds ranged between 79-100% except for
19, the diamidate of AraG (22% in CCRF-CEM and 25% in MOLT-4).
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a
Table 1. EC50 (µM) and Max Inhibition (%) (MI%) of selected phosphorodiamidates
for solid tumour cell lines.
Comp
NA
AA
Ester
(R3)
Ar
Cell lines
MCF-7
HepG2
HT29
Mia-Pa-Ca-2
EC50
MI%
EC50
MI%
EC50
MI%
EC50
MI%
Gem
1
1
1
>198
>198
>198
51
52
49
51.98
>198
50
39
55
2.23
>198
84.96
57
47
54
1.04[18]
1.45
80
74
75
8
9
Bn
L-Ala
L-Ala
CH2tB
178.12
7.68
u
10
NUC-1031
FUdR
11
1
1
2
2
cHex
Bn
>198
-
51
-
>198
-
53
-
70.54
2.89
0.20
8.58
56
-
4.48
0.44[18]
0.18
77
-
L-Ala
L-Ala
Ph
9.82
>198
78
52
94.16
>198
65
25
84
78
90
82
CH2tB
3.54
L-Ala
u
12
13
2
2
2
2
3
3
3
Pnt
cPnt
Bn
-
>198
-
-
48
-
-
>198
-
-
38
-
-
>198
-
-
-
-
74
-
L-Ala
L-Ala
L-Ala
L-Ala
47
-
57.77
14
-
-
NUC-3373
8-ClA
15
Bn
Naph
-
-
-
-
0.42
0.87
-
91
79
-
-
2.15
-
74
0.79
-
82
-
1.0
-
93
-
Bn
Bn
-
L-Ala
16
Gly
1.08
87
1.24
87
1.64
87
0.52
94
aEC50 Values (50% effective concentration on inhibition of cell viability), and MI(%)
(maximum inhibitory effect of the drug at the range of concentration considered). The
compounds were added to the cell in duplicate and tested in 9 serial concentrations
from 198 µM to 0.0199 µM.
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10.1002/cmdc.201800504
ChemMedChem
Table 2. aEC50 (µM) and Max Inhibition (%) (MI%) of selected phosphorodiamidates
for hematologic cell lines.
Comp NA
AA
Ester
(R3)
Cell lines
CCRF-CEM
MOLT-4
HEL92.1.7
HL-60
EC50
EC50
MI%
EC50
MI%
EC50
MI%
MI%
8-ClA
16
3
3
0.46
0.41
89
93
0.53
0.5
99
0.52
0.35
96
96
0.52
0.39
99
Bn
100
100
Gly
AraG
19
5
5
1.35
101
22
1.79
100
25
-
-
-
-
-
-
-
-
CH2tBu
>198
>198
L-Ala
6-MP
20
6
6
6
7
7
7
0.64
98
98
76
-
0.83
100
99
84
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Bn
CH2tBu
-
1.85
2.58
-
L-Ala
L-Ala
-
21
35.4
38.5
-
-
6-TG
22
-
-
-
-
-
-
0.71
3.70
38.6
95
92
79
Bn
-
-
L-Ala
L-Ala
23
CH2tBu
-
-
aEC50 Values (50% effective concentration on inhibition of cell viability) and MI(%)
(maximum inhibitory effect of the drug at the range of concentration considered). The
compounds were added to the cell in duplicate and tested in 9 serial concentrations
from 198 µM to 0.0199 µM.
The IC50 values of FUdR, 8-ClA and fludarabine and their respective
+
phosphorodiamidates versus mouse lymphocytic leukemia (L1210), human CD4
T-lymphocyte cells (CEM), and human cervical carcinoma (HeLa) cells are reported
in Table 3. FUdR (2) and its phosphorodiamidate prodrugs (11-14) showed activity in
a submicromolar range with IC50 of 0.0046-0.073 µM for FUdR and 0.01-0.40 µM for
the FUdR phosphorodiamidates against the wild-type cell lines. The L-alanine pentyl
prodrug 12 proved ~2-fold more active against HeLa cell cultures and ~2-fold less
active against L1210 and CEM cell cultures than FUdR. The loss of inhibitory
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10.1002/cmdc.201800504
ChemMedChem
activity for compound 14 versus FUdR ranged between 3-13-fold, and for compound
11 between ~3-15-fold.
The cytostatic activity of FUdR strongly depends on the thymidine kinase
(TK) enzyme responsible for the first phosphorylation step of FUdR to FdUMP.
Therefore the marked loss of activity for 2 was expected in the TK-deficient tumour
cell lines and was 10-fold against HeLa/TK-, 100-fold against CEM/TK- and 217-fold
against L1210/TK-. Although the potency of the four FUdR phosphorodiamidates was
also diminished in the TK-deficient tumour cell lines, the difference in fold- reduction
was less dramatic (11- to 51-fold instead of 217-fold for the parent FUdR).
However, for the CEM/TK- and HeLa/TK- cell lines the loss in activity found for the
FUdR phosphorodiamidates 11-14 was significantly higher than for FUdR. These data
clearly indicate that the phosphorodiamidates 11-14 require TK to eventually exert
biological activity, as they most likely release FUdR instead of the desired 5'-
monophosphate metabolite FdUMP. Strikingly, the cytostatic activity spectrum of L-
Ala-OBn-ONaph substituted FUdR prodrug, NUC-3373, that is not
a
phosphorodiamidate but contains the traditional ProTide structure, clearly differed
from the phosphorodiamidate prodrugs. NUC-3373 not only showed pronounced
cytostatic activity against the wild-type tumour cells (IC50: 0.011-0.068 µM), but it
remarkably retained considerable cytostatic potential against the TK-deficient tumour
cell lines, in particular for the L1210/TK- and CEM/TK- cell lines.
The drugs 3 and 4 along with their phosphorodiamidates 15, 16, 17 and 18 were also
investigated. Unlike for FUdR, TK activity is not a prerequisite for 8-Cl-adenosine
and fludarabine. 8-Cl-adenosine and fludarabine showed micromolar IC50 and both
were more active than their phosphorodiamidates. However, the difference in activity
was less significant in case of the 8-chloroadenosine series with the benzyl L-alanine
diamidate 15 being 2.6-3.4-fold less active than parent nucleoside 3. The benzyl
glycine diamidate 16 was found to be only ~1-5 to 2-fold less active than the parent
8-chloroadenosine.
Table 3. Cytostatic Activity (aIC50 in µM) reported for FUdR (2), 8-chloroadenosine
(3) and fludarabine (4), and phosphorodiamidate prodrugs (11-14, 15, 16, 17 and 18)
against tumour cell lines.
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10.1002/cmdc.201800504
ChemMedChem
Comp
NA
AA
Ester
(R3)
Ar
IC50 (µM)
L1210
L1210/
TK-
Ratio
CEM
CEM/
TK-
Ratio
HeLa
HeLa/
TK-
Ratio
TK+/TK-
TK+/TK-
TK+/TK-
FUdR
2
2
2
2
2
2
3
217
11
47
16
51
4
100
94
10
337
92
0.0046±
0.0027
1.0±0.3
0.76±0.06
0.47±0.0
2.5±0.1
0.013±0.008
1.3±0.1
15±8
0.073±0.032
0.73±
0.16
11
12
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
CH2tBu
Pnt
0.069±
0.006
0.16±
0.11
0.19±
0.03
64±6
3.4±1.1
68±9
150
233
223
4
0.01±
0.001
0.028±0.019
4.2±1.0
56±21
38±8
0.037±0.029
13
cPnt
Bn
170
67
0.16±
0.01
0.24±
0.02
0.4±
0.05
14
0.045±
0.013
2.3±0.6
0.17±
0.07
0.24±
0.05
16±8
NUC-3373
8-ClA
Bn
Naph
38[19]
0.011±
0.007
0.045±
0.027
0.068±0.035
1.1±0.6
0.31±
0.06
0.065±0.013
2.5±1.3
-
-
-
-
-
-
3.2±0.7
0.89±
0.5
15
3
3
4
4
4
L-Ala
Bn
Bn
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10±4
4.6±0.0
11±9
3.8±0.2
3.4±0.4
2.1±1.4
15±2
2.3±1.0
1.8±0.4
8.0±3.3
47±3
16
Gly
Fludarabine
17
18
L-Ala
L-Ala
CH2tBu
55±36
Bn
32±5
16±14
24±11
aIC50 or compound concentration required to inhibit tumour cell proliferation by 50%.
Data are the mean (±SD) of at least two to four independent experiments.
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10.1002/cmdc.201800504
ChemMedChem
Enzymatic Studies
The general putative mechanism of the bioactivation pathway for
phosphorodiamidates is depicted in Scheme 2 and is similar to the metabolic route
postulated for the phosphoramidates[39,40] involving four (a-d) consecutive steps: a)
esterase or carboxypeptidase-mediated hydrolysis of one of the two esters; b)
spontaneous intracellular displacement of the second amino acid through an
intramolecular attack of the carboxylate anion on the phosphorus and further
formation of a mixed five-membered ring anhydride; c) spontaneous hydrolysis of the
anhydride intermediate; and finally d) P–N bond cleavage mediated by a
phosphoramidase-type enzyme.[41,42]
O
O
R2
O
R2
O
O
R3O
H
R2
O
N
HN
HN
P
O
P
O
P
O
B
B
B
a
b
O
O
O
O
NH
R2
NH
R2
O
R1
R1
R1
R3O
O
OH
R
OH
R
OH
R
O
O
R2
O
B = purine, pyrimidine
R, R1 = H, OH, F
R2 = Amino acid side chain
R3 = Alkyl, cycloalkyl, aryl
O
O
P
O
HN
O
O P
O
O
c
d
B
B
O
O
O
R1
R1
OH
R
OH
R
Scheme 2. General putative activation pathway of phosphorodiamidate prodrugs: a)
carboxypeptidase-mediated ester hydrolysis; b) spontaneous intracellular
displacement; c) spontaneous hydrolysis; d) enzyme-mediated P–N bond cleavage.
Here, we report the carboxypeptidase Y assay performed on the gemcitabine
phosphorodiamidate 8 in the presence of d6-acetone and TRIZMA buffer (pH = 7.6),
showing ester hydrolysis of one of the amino acid ester moieties during the
bioactivation process. Upon the addition of carboxypeptidase Y, within the first
10 minutes of incubation, phosphorodiamidate 8 (δp = 14.14 ppm, Figure 2) was
converted to the intermediate 8-A lacking both ester moieties (peak at 14.82 ppm).
Finally, the formation of the metabolite 8-C lacking one of the amino acid (thought to
be mediated by spontaneous cyclisation and hydrolysis) (8-B) was confirmed by the
presence of the single peak at δp 7.04 ppm. This is analogous to results reported in our
previous study.[24] No direct 31P-NMR evidence was obtained for the formation of the
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10.1002/cmdc.201800504
ChemMedChem
cyclic compound 8-B, consistent with it being a short-lived metabolite. Another
possibility could be that the metabolite 8-C is formed spontaneously from the
metabolite 8-A, without a cyclisation step being involved. Compound 8 was fully
processed to the aminoacyl metabolite 8-C within approximately 3 h, with an
estimated half-life of 6 minutes.
NH2
N
O
NH2
N
O
O
O
O
O
O
O
NH
P
N
O
NH
P
N
O
O
O
(a)
O
O
NH
O
NH
F
F
CPY
F
F
OH
OH
8-A
O
8
(b)
spont.
NH2
NH2
O
O
NH2
N
N
N
H
N
NH
O
O
O
P
N
O
N
O
N
O
(c)
spont. hydrolysis
(d)
P
O
P
O
O
O
O
O
O
O
O
O
Hint
O
F
F
F
F
F
F
OH
8-B
OH
8-C
OH
8-D
Scheme 3. Putative mechanism of bioactivation for phosphorodiamidate 8.
The same activation pathway with a similar estimated half-life was obtained with
inactive compound 18, the phosphorodiamidate of AraG (data not shown). These
results indicate that discrimination between active and inactive compounds might be
ascribed to the last step in the activation pathway, catalysed by Hint, a
phosphoramidase type enzyme.[41-43]
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10.1002/cmdc.201800504
ChemMedChem
tE=E185Emin
tE=E157Emin
tE=E115Emin
tE=E73Emin
tE=E59Emin
tE=E45Emin
tE=E38Emin
tE=E24Emin
8)A
tE=E17Emin
tE=E10Emin
8)C
8
blankEtE=E0
16
15
14
13
12
11
10
9
8
7
6
5
4
3
ppm
Figure 3. Carboxypeptidase Y-mediated cleavage of compound 8 as monitored by 31P NMR.
Conclusion
In conclusion, we report on the application of the phosphorodiamidate
approach leading to sixteen prodrugs derived from amino acids and seven anticancer
nucleoside analogues. The biological in vitro screening of the phosphorodiamidates
against a wide panel of tumour cell lines revealed a potential of this approach in case
of FUdR and 8-ClA, since some of their corresponding phosphorodiamidate prodrugs
showed similar or improved inhibitory activities when compared with the parent
nucleosides. The carboxypeptidase Y assay supported the postulated bioactivation
pathway as reported for one gemcitabine phosphorodiamidate 8, for which a similar
activity versus its parent compound was observed for the Mia-Pa-Ca-2 tumour cell
line. The lack of activity found in the other families of compounds may indicate either
poor bioactivation of the phosphorodiamidate motif with a subsequent poor release of
the free 5’-monophosphate form (last step) or susceptibility of inactive
phosphorodiamidates and their corresponding metabolites to drug efflux pump
mechanisms. Overall, the wide range of in vitro data presented for the selected
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10.1002/cmdc.201800504
ChemMedChem
phosphorodiamidates may demonstrate that the biological properties and activities of
nucleoside analogues and their prodrugs are unpredictable due to the complexity of
the nature of distinct cell systems and differences in levels and activity of enzymes
involved in the uptake and conversion of the prodrugs to the eventual intracellular
active metabolite.
Experimental Section
MTS Cell Viability Assay. The solid and hematologic tumour cell lines were seeded
at a range of density 0.5 to 100 x 103 cells/well in the wells of a 96-well plate the day
before compound incubation, then incubated for 72 hours with the different
concentrations of the test compound. Then 50 µL of MTS was added and the 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.
Cell cultures and Cytostatic Activity Assays. Murine leukemia L1210, human
lymphocyte CEM, human cervix carcinoma HeLa and their corresponding
TK-deficient congeners L1210/TK-, CEM/TK- and HeLa/TK- were seeded at –50 to
75 x 103 cells/200 µl in the wells of a 96-well microtiter plate that contained a variety
of drug concentrations. After 48 hours (L1210) or 72-96 hours (CEM; HeLa) the cell
number was determined by counting with a Coulter Counter. The IC50 (50%
inhibitory concentration) was determined as the drug concentration required to inhibit
tumor cell proliferation by 50%.
Carboxypeptidase Y (EC 3.4.16.1) Assay. The experiment was carried out by
dissolving diamidate 8 (5.0 mg) in acetone-d6 (0.15 mL) followed by addition of
31
0.30 mL of Trizma buffer (pH 7.6). After recording the control P 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
analysis (at 25 °C). The spectra were recorded every 7 minutes for over 14 hours. 31P
NMR recorded data were processed and analyzed with the Bruker Topspin 2.1
program.
Chemistry. General Procedures. Solvents and Reagents. The following anhydrous
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10.1002/cmdc.201800504
ChemMedChem
solvents were purchased from Sigma-Aldrich: dichloromethane (CH2Cl2),
trimethylphosphate ((Me)3PO4), triethylphosphate ((Et)3PO4), diethyl ether (Et2O),
tetrahydrofuran (THF), dimethylformamide (DMF), and any other reagents used.
Amino acid esters commercially available were purchased from Novabiochem. All
reagents commercially available were used without further purification.
Thin Layer Chromatography (TLC). Precoated aluminium-backed plates (60 F254,
0.2 mm thickness, Merck) were visualized under both short and long wave ultraviolet
light (254 and 366 nm) or by burning using the following TLC indicators: (i)
molybdate ammonium cerium sulfate; (ii) potassium permanganate solution.
Preparative TLC plates (20 cm × 20 cm, 500-2000 µm) were purchased from Merck.
Flash Column Chromatography. Flash column chromatography was carried out
using silica gel supplied by Fisher (60A, 35-70 µm). Glass columns were slurry
packed using the appropriate eluent with the sample being loaded as a concentrated
solution in the same eluent or pre-adsorbed onto silica gel. Fractions containing the
product were identified by TLC, and pooled and the solvent was removed in vacuo.
High Performance Liquid Chromatography (HPLC). The purity of the final
compounds was verified to be >95% by HPLC analysis using either I)
ThermoSCIENTIFIC, SPECTRA SYSTEM P4000, detector SPECTRA SYSTEM
UV2000, Varian Pursuit XRs 5 C18, 150 x 4.6 mm (as an analytic column) or II)
Varian Prostar (LC Workstation-Varian Prostar 335 LC detector), Thermo
SCIENTIFIC Hypersil Gold C18, 5 µm, 150 x 4.6 mm (as an analytic column). For
the method of elution see the experimental part. The purification of one final
compound was achieved on Pursuit XRs 5 C18, 150 x 21.2 mm (as the semi-
preparative column), Varian Prostar (LC Workstation-Varian Prostar 335 LC
detector).
1
13
Nuclear Magnetic Resonance (NMR). H NMR (500 MHz), C NMR (125 MHz),
19
31P NMR (202 MHz) and F NMR (470 MHz) were recorded on a Bruker Avance
500 MHz spectrometer at 25 ºC. Chemical shifts (δ) are quoted in parts per million
(ppm) relative to internal CH3OH-d4 (δ 3.34 1H-NMR, δ 49.86 13C-NMR) and CHCl3-
1
13
31
d4 (δ 7.26 H NMR, δ 77.36 C NMR) or external 85 % H3PO4 (δ 0.00 P NMR).
Coupling constants (J) are measured in Hertz. The following abbreviations are used in
the assignment of NMR signals: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), bs (broad singlet), dd (doublet of doublet), dt (doublet of triplet), app
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10.1002/cmdc.201800504
ChemMedChem
1
13
(apparent). The assignment of the signals in H NMR and C NMR was done based
on the analysis of coupling constants and additional two-dimensional experiments
(COSY, HSQC, HMBC, PENDANT).
Mass spectromethry (MS). Low resolution mass spectra were performed on Bruker
Daltonics microTof-LC, (atmospheric pressure ionization, electron spray mass
spectroscopy) in either positive or negative mode.
Standard procedure A: synthesis of phosphorodiamidates. To a solution of
nucleoside (1 eq) in trimethyl phosphate (1 mL per 1 mmol of nucleoside, unless
otherwise stated), phosphoryl chloride (1-2 eq) was added dropwise at - 5 °C and the
reaction mixture was left stirring for 4 - 12 hours. The formation of the intermediate
31
was monitored by P-NMR. A suspension of the appropriate amino acid ester (5 eq)
in dichloromethane (4 mL per 1 mmol of amino acid ester) was added followed by
diisopropyl ethyl amine (10 eq) at - 78 °C. After stirring at room temperature for
20 hours, water (10 mL per mmol of nucleoside) was added and the layers were
separated. The aqueous phase was extracted with CH2Cl2 (10 mL per mmol of
nucleoside) and the organic phase washed with brine (10 mL per mmol of
nucleoside). The combined organic layers were dried over Na2SO4 and concentrated.
The residue was purified unless otherwise stated either by column chromatography or
Biotage Isolera One to give the title compound as a white solid.
2’-Deoxy-2’,2’-difluoro-D-cytidine-5’-O-bis(benzoxy-L-alaninyl)] phosphate (8):
Prepared according to the standard procedure A from gemcitabine (0.25 g, 0.95
mmol), POCl3 (0.29 g, 0.17 mL, 1.89 mmol), followed by addition of L-Ala benzyl
ester tosylate salt (1.67 g, 4.75 mmol), DIPEA (1.23 g, 1.65 mL, 9.50 mmol). Column
purification followed by preparative TLC purification gave the product 8, as a white
solid (0.079 g, 12%). 31P NMR (202 MHz, CD3OD): δ = 13.87; 19F NMR (470 MHz,
1
CD3OD): δ = – 118.14 (d, J = 239 Hz), –119.70 (d, broad signal, J = 239 Hz); H
NMR (500 MHz, CD3OD): δ = 7.67 (d, J = 7.53 Hz, 1H, H-6), 7.38 – 7.31 (m, 10H,
H-Ar), 6.27 (t, J = 8.05 Hz, 1H, H-1’), 5.97 (d, J = 7.51 Hz, 1H, H-5), 5.18 – 5.10 (m,
4H, 2 x CH2Ph), 4.31 – 4.17 (m, 3H, H-5’, H-3’, H-5’), 4.02 – 3.95 (m, H-4’, 3H, 2 x
NHCHCH3), 1.38 (d, J = 7.14 Hz, 3H, NHCHCH3), 1.36 (d, J = 7.14 Hz, 3H,
13
3
NHCHCH3); C NMR (125 MHz, CD3OD): δ = 175.43 (d, JC-P = 5.40 Hz, C=O,
ester), 167.68 (C-2), 157.81 (C-4), 142.63 (C-6), 137.29, 137.28 (C-Ar), 129.85,
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10.1002/cmdc.201800504
ChemMedChem
1
129.66, 129.65, 129.40, 129.39, 129.32, 127.02 (CH-Ar), 123.69 (apparent t, JC-F
=
2
258 Hz, CF2), 96.87 (C-5), 86.02 (apparent t, broad signal, JC-F = 27.0 Hz, C-1’),
3
2
80.46 (apparent t, JC-F = 8.20 Hz, C-4’), 71.28 (apparent t, JC-F = 23.44 Hz, C-3’),
2
2
68.00, 67.99 (CH2Ph), 64.52 (d, JC-P = 4.78 Hz, C-5’), 51.15 (d, JC-P = 5.41 Hz,
NHCHCH3), 20.78, 20.67 (2 x d, 3JC-P = 5.53 Hz, NHCHCH3); MS [ES+] m/z: found
666.22 [M + H+], 688.21 [M + Na+], C29H34F2N5O9P required 665.58 [M]. HRMS
[ESI] m/z: calculated for C29H34F2N5O9P: 665.2062; found 666.2058 [M + H]+.
Reverse-phase HPLC eluting with H2O/CH3OH from 100/0 to 0/100 in 35 min), F = 1
mL/min, λ = 254, tR = 24.15 min.
2’-Deoxy-2’,2’-difluoro-D-cytidine-5’-O-bis(2,2-dimethylpropoxy-L-alaninyl)]-
phosphate (9): Prepared according to the standard procedure A from gemcitabine
(0.25 g, 0.95 mmol), POCl3 (0.29 g, 0.17 mL, 1.89 mmol), and (Et)3PO (1.0 mL),
followed by addition of L-Ala neopentyl ester tosylate salt (1.57 g, 4.75 mmol),
DIPEA (1.23 g, 1.65 mL, 9.50 mmol). Column purification followed by preparative
TLC purification gave the compound 9 as a white solid (0.041 g, 7%); 31P NMR (202
MHz, CD3OD): δ = 13.93; 19F NMR (470 MHz, CD3OD): δ = –118.1 (d, J = 245 Hz),
1
–119.6 (d, J = 245 Hz); H NMR (500 MHz, CD3OD): δ = 7.70 (d, J = 7.58 Hz, 1H,
H-6), 6.29 (t, J = 8.20 Hz, 1H, H-1’), 6.01 (d, J = 7.58 Hz, 1H, H-5), 4.37 – 4.33 (m,
1H, H-5’), 4.30 – 4.23 (m, 2H, H-5’, H-3’), 4.08 – 4.06 (m, 1H, H-4’), 4.03 – 3.96 (m,
2H, 2 x NHCHCH3), 3.90, 3.93 (AB system, JAB = 10.55 Hz, 2H, CH2C(CH3)3), 3.79,
3.73 (AB system, JAB = 11.0 Hz, 2H, CH2C(CH3)3), 1.45 (d, J = 7.08 Hz, 3H,
NHCHCH3), 1.43 (d, J = 7.08 Hz, 3H, NHCHCH3), 0.97 (s, 18H, 2 x CH2C(CH3)3);
13C NMR (125 MHz, CD3OD): δ = 175.71 (d, 3JC-P = 3.44 Hz, C=O, ester), 175.67 (d,
3JC-P = 3.44 Hz, C=O, ester), 167.70 (C-2), 157.79 (C-4), 142.68 (C-6), 123.65
(apparent t, 1JC-F = 258 Hz, CF2), 96.84 (C-5), 86.04 (apparent t, 2JC-F = 26 Hz, C-1’),
80.48 (apparent t, 3JC-F = 8.51 Hz, C-4’), 75.46, 75.43 (CH2C(CH3)3), 71.30 (t, 2JC-F
=
2
2
23.0 Hz, C-3’), 64.57 (d, JC-P = 4.73 Hz, C-5’), 51.17 (d, JC-P = 7.78 Hz,
NHCHCH3), 32.35 (CH2C(CH3)3), 32.33 (CH2C(CH3)3), 26.7 (CH2C(CH3)3), 21.08
3
3
(d, JC-P = 6.10 Hz, NHCHCH3), 20.96 (d, JC-P = 6.10 Hz, NHCHCH3); MS [ES+]
m/z: found 626.28 [M + H+], 648 [M + Na+], C25H42F2N5O9P required 625.60 [M].
Reverse-phase HPLC eluting with H2O/CH3CN from 100/0 to 0/100 in 35 min), F = 1
mL/min, λ = 254, tR = 17.27 min.
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10.1002/cmdc.201800504
ChemMedChem
2’-Deoxy-2’,2’-difluoro-D-cytidine-5’-O-bis(cyclohexoxy-L-alaninyl)]-phosphate
(10): Prepared according to the standard procedure A from gemcitabine (0.25 g, 0.95
mmol), POCl3 (0.29 g, 0.17 mL, 1.89 mmol), followed by addition of L-Ala
cyclohexyl ester HCl salt (0.99 g, 4.75 mmol), DIPEA (1.23 g, 1.65 mL, 9.50 mmol).
Column purification followed by preparative TLC purification gave the product 10, as
a white solid (0.029 g, 5%); 31P NMR (202 MHz, CD3OD): δ = 13.94; 19F NMR (470
MHz, CD3OD): δ = –118.30 (d, J = 241 Hz), –119.78 (d, broad signal, J = 245 Hz);
1H NMR (500 MHz, CD3OD): δ = 7.70 (d, J = 7.15 Hz, 1H, H-6), 6.29 (t, J = 7.97
Hz, 1H, H-1’), 6.01 (d, J = 7.51 Hz, 1H, H-5), 4.79 – 4.73 (m, 2H, 2 x OCH-
cyclohexyl), 4.37 – 4.33 (m, 1H, H-5’), 4.32 – 4.22 (m, 2H, H-3’, H-5’), 4.08 – 4.06
(m, 1H, H-4’), 3.93 – 3.89 (m, 2H, 2 x NHCHCH3), 1.87 – 1.84 (m, 4H, 2 x CH2-
cyclohexyl), 1.77 – 1.74 (m, 4H, 2 x CH2-cyclohexyl), 1.58 – 1.56 (m, 2H, CH2-
13
cyclohexyl), 1.48 – 1.33 (m, 16H, 5 x CH2-cyclohexyl, 2 x NHCHCH3); C NMR
3
(125 MHz, CD3OD): δ = 175.64 (d, JC-P = 5.37 Hz, C=O, ester), 167.69 (C-2),
1
157.79 (C-4), 142.66 (C-6), 123.66 (apparent t, JC-F = 259 Hz, CF2), 96.85 (C-5),
2
3
86.00 (apparent t, broad signal, JC-F = 30 Hz, C-1’), 80.48 (apparent t, JC-F = 8.50
Hz, C-4’), 74.66, 74.56 (2 x CH-cyclohexyl), 71.29 (apparent t, 2JC-F = 25.0 Hz, C-3’),
64.54 (d, 2JC-P = 4.23 Hz, C-5’), 51.13 (d, 2JC-P = 13.0 Hz, NHCHCH3), 32.55, 32.53,
32.48, 32.34, 26.58, 26.44, 24.79, 24.69 (CH2-cyclohexyl), 21.15 (d, 3JC-P = 5.52 Hz,
NHCHCH3), 20.93 (d, 3JC-P = 5.52 Hz, NHCHCH3); MS [ES+] m/z: found 650.28 [M
+ H+], 672.26 [M + Na+], C27H42F2N5O9P required 649.62 [M]. Reverse-phase HPLC
eluting with H2O/CH3CN from 100/0 to 0/100 in 35 min, F = 1 mL/min, λ = 254, tR =
17.72 min.
5-Fluoro-2′-deoxyuridine-5′-O-bis(2,2-dimethylpropoxy-L-alaninyl) phosphate
(11): Prepared according to the standard procedure A from FUdR (0.25 g, 1.01
mmol), POCl3 (0.18 mL, 2.03 mmol), the tosylate salt of 2,2-dimethylpropoxy-L-
alanine (1.68 g, 5.07 mmol) and DIPEA (1.77 mL, 10.15 mmol). After work-up and
silica gel column chromatography phosphorodiamidate 11 was obtained as an off-
31
19
white solid (0.020 g, 3%). P NMR (202 MHz, CD3OD): δ = 13.94; F NMR (470
MHz, CD3OD): δ = –167.40; 1H NMR (500 MHz, CD3OD): δ = 7.86 (d, J = 6.2 Hz,
1H, H-6), 6.35 (t, J = 5.2 Hz, 1H, H-1′), 4.48 – 4.45 (m, 1H, H-3′), 4.26 – 4.20 (m,
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10.1002/cmdc.201800504
ChemMedChem
2H, H-5′), 4.18 – 4.15 (m, 1H, H-4′), 4.08 – 4.00 (m, 2H, 2 x NHCHCH3), 3.86 –
3.78 (m, 4H, 2 x CH2C(CH3)3), 2.38 – 2.35 (m, 1H, H-2′), 2.33 – 2.29 (m, 1H, H-2′),
1.48 – 1.43 (m, 6H, 2 x NHCHCH3), 1.00 (s, 18H, 2 x CH2C(CH3)3); 13C NMR (125
MHz, CD3OD): δC 175.6 (d, 3JC–P = 3.7 Hz, C=O, ester), 159.8 (d, 2JC–F = 25.0 Hz, C-
1
2
4), 151.0 (C-2), 141.7 (d, JC–F = 232.5 Hz, C-5), 125.9 (d, JC–F = 33.75 Hz, C-6),
3
86.8 (C-1′), 86.6 (d, JC – P = 8.75 Hz, C-4′), 75.4, 75.3 (CH2C(CH3)3), 71.9 (C-3′),
2
2
66.4 (d, JC–P = 5.0 Hz, C-5′), 51.2 (d, JC–P = 7.5 Hz, CHCH3), 40.7 (C-2′), 32.4
3
(CH2C(CH3)3), 26.8 (CH2C(CH3)3), 20.7, (2 x d, JC–P = 5.0 Hz, NHCHCH3). MS
[ES+] m/z: found 631.64 [M + Na+], C25H42FN4O10P required 608.59 [M]. Reverse
HPLC, eluting with H2O/CH3OH from 100/0 to 0/100 in 30 min, F = 1 mL/min, λ =
254, tR = 22.59.
5-Fluoro-2′-deoxyuridine-5′-O-bis(pentoxy-L-alaninyl) phosphate (12): Prepared
according to the standard procedure A from FUdR (0.35 g, 1.42 mmol), POCl3 (0.26
mL, 2.84 mmol), the tosylate salt of pentoxy-L-alanine (1.39 g, 7.10 mmol) and
DIPEA (2.47 mL, 14.20 mmol).
After work-up and silica gel column
chromatography phosphorodiamidate 12 was obtained as an off-white solid (0.050 g,
6%). 31P NMR (202 MHz, CD3OD): δ = 13.91; 19F NMR (470 MHz, CD3OD): δ = –
1
167.35; H NMR (500 MHz, CD3OD): δ = 7.90 (d, J = 6.2 Hz, 1H, H-6), 6.30 (t, J =
5.5 Hz, 1H, H-1′), 4.49 – 4.45 (m, 1H, H-3′), 4.20 – 4.15 (m, 7H, 2 x OCH2, 2 x H-5′,
H-4′), 3.90 – 3.85 (m, 2H, 2 x NHCHCH3), 2.38 – 2.35 (m, 1H, H-2′), 2.29 – 2.20
(m, 1H, H-2′), 1.72 – 1.60 (m, 4H, 2 x CH2), 1.43 – 1.30 (m, 14H, 4 x CH2, 2 x
13
NHCHCH3), 1.00 – 0.96 (m, 6H, 2 x CH2CH3); C NMR (125 MHz, CD3OD): δ =
3
2
175.70, 175.67 (2d, JC–P = 3.7 Hz, C=O, ester), 160.10 (d, JC–F = 26.0 Hz, C-4),
1
2
151.30 (C-2), 141.78 (d, JC–F = 235.0 Hz, C-5), 125.85 (d, JC–F = 35.0 Hz, C-6),
3
86.90 (C-1′), 86.78 (d, JC–P = 7.5 Hz, C-4′), 71.95 (C-3′), 66.48, 66.47 (OCH2),
2
2
66.38 (d, JC–P = 5.0 Hz, C-5′), 51.15, 50.64 (2d, JC–P = 8.5 Hz, NHCHCH3), 40.79
3
(C-2′), 29.43, 29.20 (CH2), 23.61, 23.40 (CH2), 20.95, 20.26 (2 x d, JC–P = 6.25 Hz,
NHCHCH3). MS [ES+] m/z: found 631.59 [M + Na+], C25H42FN4O10P required
608.59 [M]. Reverse HPLC, eluting with H2O/CH3CN from 100/0 to 0/100 in 30 min,
F = 1 mL/min, λ = 254, tR = 15.88.
5-Fluoro-2′-deoxyuridine-5′-O-bis(cyclopentoxy-L-alaninyl) phosphate (13):
Prepared according to the standard procedure A from FUdR (0.35 g, 1.42 mmol),
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10.1002/cmdc.201800504
ChemMedChem
POCl3 (0.26 mL, 2.84 mmol), the tosylate salt of cyclopentoxy-L-alanine (2.34 g, 7.10
mmol) and DIPEA (2.47 mL, 14.20 mmol). After work-up and silica gel column
chromatography phosphorodiamidate 13 was obtained as an off-white solid (0.075 g,
31
19
9%). P NMR (202 MHz, CD3OD): δ = 13.99; F NMR (470 MHz, CD3OD): δ = –
167.47; 1H NMR (500 MHz, CD3OD): δ = 8.00 (d, J = 6.5 Hz, 1H, H-6), 6.26 (t, J =
5.5 Hz, 1H, H-1′), 5.24 – 5.15 (m, 2H, OCH), 4.48 – 4.45 (m, 1H, H-3′), 4.32 – 4.29
(m, 2H, H-5′), 4.15 – 4.08 (m, 1H, H-4′), 3.95 – 3.86 (m, 2H, 2 x NHCHCH3), 2.38 –
2.35 (m, 1H, H-2′), 2.29 – 2.24 (m, 1H, H-2′), 2.00 – 1.89 (m, 4H, 2 x CH2), 1.72 –
1.59 (m, 10H, 5 x CH2), 1.59 – 1.52 (m, 2H, CH2), 1.48 – 1.30 (m, 6H, 2 x
13
3
NHCHCH3); C NMR (125 MHz, CD3OD): δ = 175.45, 175.41 (2d, JC–P = 2.5 Hz,
2
1
C=O, ester), 159.76 (d, JC F = 25.0 Hz, C-4), 151.06 (C-2), 142.0 (d, JC–F = 232.5
Hz, C-5), 125.95 (d, 2JC–F = 34.0 Hz, C-6), 86.86 (C-1′), 86.78 (d, 3JC–P = 7.5 Hz, C-
4′), 79.50 (OCH), 71.95 (C-3′), 66.42 (d, 2JC–P = 5.0 Hz, C-5′), 51.20, 50.80 (2d, 2JC–P
= 8.5 Hz, NHCHCH3), 40.75 (C-2′), 33.84, 33.70, 33.52, 33.49 (CH2), 24.70, 24.69
(CH2), 20.94, 20.85 (2d, 3JC–P = 6.25 Hz, CHCH3). MS [ES+] m/z: found 627.222 [M
+ Na+], C25H38FN4O10P, required 604.56 [M]. HRMS [ESI] m/z: calculated for
C25H38FN4O10P: 604.2310; found 605.2314 [M + H]+. Reverse HPLC, eluting with
H2O/CH3CN from 100/0 to 0/100 in 30 min, F = 1 mL/min, λ = 254, tR = 13.39.
5-Fluoro-2′-deoxyuridine-5′-O-bis(benzoxy-L-alaninyl) phosphate (14): Prepared
according to the standard procedure A from FUdR (0.25 g, 1.01 mmol), POCl3 (0.18
mL, 2.03 mmol), the tosylate salt of benzoxy-L-alanine (1.78 g, 5.07 mmol) and
DIPEA (1.76 mL, 10.15 mmol). After work-up and silica gel column chromatography
31
phosphorodiamidate 14 was obtained as an off-white solid (0.030 g, 5%). P NMR
(202 MHz, CD3OD): δ =13.82; 19F NMR (470 MHz, CD3OD): δ = –167.51; 1H NMR
(500 MHz, CD3OD): δ = 7.90 (d, J = 6.5 Hz, 1H, H-6), 7.38 – 7.31 (m, 10 H, Ar-H),
6.25 – 6.22 (m, 1H, H-1′), 5.22 – 5.10 (m, 4H, 2 x CH2Ph), 4.39 – 4.36 (m, 1H, H-3′),
4.16 – 4.07 (m, 2H, H-5′), 4.01 – 3.95 (m, 3H, H-4′, 2 x NHCHCH3), 2.31 – 2.26 (m,
1H, H-2′), 2.20 – 2.14 (m, 1H, H-2′), 1.42 – 1.34 (m, 6H, 2 x NHCHCH3); 13C NMR
(125 MHz, CD3OD): δC = 175.3 (C=O, ester), 159.3 (C-4), 150.5 (C-2), 141.5 (d, 1JC–
= 238.0 Hz, C-5), 137.3, 137.1 (2 x C ipso-Ph), 129.7, 129.6, 129.5, 129.4, 129.3,
F
2
129.2, 128.0 (CH-Ar), 125.8 (d, JC–F = 33.75 Hz, C-6), 122.5, 116.3 (CH-Ar), 86.8
(C-1′), 86.7 (d, 3JC–P = 8.7 Hz, C-4′), 71.3 (C-3′), 68.0, 67.9 (CH2Ph), 66.4 (d, 2JC–P
=
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10.1002/cmdc.201800504
ChemMedChem
3
5.0 Hz, C-5′), 51.2, 51.1 (NHCHCH3), 40.7 (C-2′), 20.7, 20.6 (2 x d, JC–P = 6.2 Hz,
NHCHCH3). MS [ES+] m/z: found 671.20 [M + Na+], C29H34FN4O10P required
648.57 [M]. Reverse HPLC, eluting with H2O/CH3CN from 100/0 to 0/100 in 30 min,
F = 1 mL/min, λ = 254, tR = 13.88.
8-Chloroadenosine
5’-O-bis(benzoxy-L-alaninyl)phosphate
(15):
Prepared
according to the general procedure A, using 8-chloroadenosine (0.25 g, 0.83 mmol),
in (CH3O)3PO (5 mL), POCl3 (77 µL, 0.83 mmol), L-alanine-O-benzyl ester tosylate
salt (1.46 g, 4.15 mmol) in CH2Cl2 (5 mL) and DIPEA (1.45 mL, 8.3 mmol). The
crude was purified by column chromatography (gradient elution of 4-7%
CH3OH/CH2Cl2) to give the title compound 15 as a white solid (0.24 g, 42%).31P
NMR (202 MHz, CD3OD): δ = 13.54; 1H NMR (500 MHz, CD3OD): δ = 8.21 (s, 1H,
H-2), 7.36 – 7.26 (m, 10H, H-Ph), 6.02 (d, J = 4.8 Hz, 1H, H-1’), 5.30 (t, J = 5.5 Hz,
1H, H2’), 5.14 – 5.02 (m, 4H, 2 x CH2Ph), 4.62 (dd, J = 7.5, 5.0 Hz, 1H, H-3’), 4.31 –
4.24 (m, 1H, H-5’), 4.18 – 4.11 (m, 2H, H-5’, H-4’), 3.95-3.82 (m, 2H, NHCHCH3),
1.28 (d, J = 7.2 Hz, 3H, CHCH3), 1.23 (d, J = 7.2 Hz, 3H, CHCH3);13C NMR (125
3
3
MHz, CD3OD): δ = 175.34 (d, JC-P= 5.0 Hz, C=O), 175.30 (d, JC-P= 5.0 Hz, C=O),
156.35 (C-6), 154.22 (C-2), 151.71 (C-4), 139.83 (C-Ar), 137.32 (C-Ar), 137.27 (C-
Ar), 129.58 (CH-Ar), 129.56 (CH-Ar), 129.32 (CH-Ar), 129.28 (CH-Ar), 119.55 (C-
3
5), 91.28 (C-1’), 84.50 (d, JC-P= 7.3 Hz, C-4’), 72.65 (C-2’), 71.47 (C-3’), 67.91
2
(CH2Ph), 67.86 (CH2Ph), 66.17 (d, JC-P= 4.4 Hz, C-5’), 51.01 (NHCHCH3), 50.98
3
3
(NHCHCH3), 20.81 (d, JC-P= 6.0 Hz, NHCHCH3), 20.53 (d, JC-P= 6.0 Hz,
NHCHCH3). MS [ES+] m/z: found 704.1 [M + H+], 726.1 [M + Na+], C30H35ClN7O9P
required 704.07 [M]. HRMS [ESI+] m/z: calculated for C30H35ClN7O9P: 703.1922;
found 704.0612 [M + H]+. Reverse-phase HPLC eluting with H2O/CH3CN from
90/10 to 0/100 in 35 minutes, F = 1 mL/min, λ = 254, tR 24.98 min.
8-Chloroadenosine 5’-O-bis(benzoxy-glycinyl) phosphate (16): Prepared according
to the general procedure A, using 8-chloroadenosine (0.10 g, 0.33 mmol), in
(CH3O)3PO (2.5 mL), POCl3 (31 µL, 0.33 mmol), glycine-O-benzyl ester
hydrochloride salt (0.33 g, 1.65 mmol), in CH2Cl2 (5 mL) and DIPEA (0.57 mL, 3.3
mmol). The crude was purified by column chromatography (gradient elution of 4-7%
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10.1002/cmdc.201800504
ChemMedChem
CH3OH in CH2Cl2) to give the title compound as a white solid (0.06 g, 27%). 31P
NMR (202 MHz, CD3OD): δ = 15.96; 1H NMR (500 MHz, CD3OD): δ = 8.23 (s, 1H,
H2), 7.35 – 7.26 (m, 10H, H-Ph), 6.03 (d, J = 5.1 Hz, 1H, H-1’), 5.31 (dd, J = 5.5, 5.1
Hz, 1H, H-2’), 5.11 – 5.08 (m, 4H, 2 x CH2Ph), 4.59 (dd, 5.5 Hz, 5.1 Hz, 1H, H-3’),
4.34 – 4.28 (m, 1H, H-5’), 4.27 – 4.16 (m, 2H, H-5’, H-4’), 3.70 – 3.64 (m, 4H, 2 x
NHCH2); 13C NMR (125 MHz, CD3OD): δ = 172.91 (d, 3J = 4.7 Hz, C=O), 172.89
C-P
3
(d, JC-P= 4.7 Hz, C=O), 156.24 (C6), 154.08 (C-2), 151.69 (C-4), 139.88 (C-Ar),
137.22 (C-Ar), 129.57 (CH-Ar), 129.56 (CH-Ar), 129.33 (CH-Ar), 129.31 (CH-Ar),
3
119.56 (C-5), 91.16 (C-1’), 84.70 (d, JC-P= 7.2 Hz, C-4’), 72.62 (C-2’), 71.58 (C-3’),
67.86 (CH2Ph), 67.84 (CH2Ph), 66.17 (d, 2J = 4.6 Hz, C-5’), 43.56 (NHCH2), 43.47
C-P
(NHCH2). MS [ES+] m/z: found 704.1 [M + H+], 726.1 [M + Na+], C28H31ClN7O9P
required 676.01 (M). Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to
0/100 in 35 minutes, F = 1 mL/min, λ = 254, tR 23.23 min.
2-Fluoroadenine-9-β-D-arabinofuranoside-5′-O-bis(2,2-dimethylpropoxy-L-
alaninyl) phosphate (17): Prepared according to the standard procedure A from
fludarabine (0.25 g, 0.88 mmol), POCl3 (0.16 mL, 1.75 mmol), the tosylate salt of
2,2-dimethylpropoxy-L-alanine (1.45 g, 4.38 mmol) and DIPEA (1.52 mL, 8.76
mmol). After work-up and silica gel column chromatography phosphorodiamidate 17
31
was obtained as an off-white solid (0.052 g, 9%). P NMR (202 MHz, CD3OD): δ =
13.84; 19F NMR (470 MHz, CD3OD): δ = –53.28; 1H NMR (500 MHz, CD3OD): δ =
8.25 (s, 1H, H-8), 7.40 – 7.20 (m, 10 H, Ar-H), 6.32 (d, J = 4.3 Hz, 1H, H-1′), 4.46 –
4.25 (m, 4H, H-2ʹ, H-3ʹ, 2 x H-5ʹ), 4.20 – 4.15 (m, 1H, H-4ʹ), 3.98 – 3.94 (m, 2H, 2 x
NHCHCH3), 3.87, 3.84 (AB system, JAB = 10.5 Hz, 2H, CH2C(CH3)3), 3.74, 3.72
(AB system, JAB = 10.5 Hz, 2H, CH2C(CH3)3), 1.44 – 1.39 (m, 6H, 2 x NHCHCH3),
13
0.99 (s, 18H, CH2C(CH3)3); C NMR (125 MHz, CD3OD): δ = 175.74, 175.66 (2d,
3JC–P = 5.0 Hz, C=O, ester), 160.70 (d, 1JC–F = 207.5 Hz, C-2), 158.90 (d, 3JC–F = 20.0
Hz, C-6), 152.26 (d, 3JC–F = 18.8 Hz, C-4), 142.69 (C-8), 117.84 (d, 4JC–F = 3.75 Hz,
4
C-5), 86.07 (C-1′), 84.06 (d, JC–P = 7.5 Hz, C-3ʹ), 77.31, 77.15 (C-2ʹ, C-4ʹ), 75.42
2
2
(CH2C(CH3)3), 66.35 (d, JC–P = 5.0 Hz, C-5′), 51.2 (d, JC–P = 8.75 Hz, CHCH3),
3
32.34, 32.32 (CH2C(CH3)3), 26.82 (CH2C(CH3)3), 21.15 (d, JC–P = 6.30 Hz,
3
NHCHCH3), 21.02 (d, JC–P = 6.30 Hz, NHCHCH3). [ES+] m/z: found 670.61 [M +
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10.1002/cmdc.201800504
ChemMedChem
Na+], C26H43FN7O9P required 647.63 [M]. HRMS [ESI+] m/z: calculated for
C26H43FN7O9P: 647.2844 found 647.2839 [M + H]+. Reverse HPLC, eluting with
H2O/CH3OH from 100/0 to 0/100 in 30 min, F = 1 mL/min, λ = 254, tR = 25.52.
2-Fluoroadenine-9-β-D-arabinofuranoside-5′-O-bis(benzoxy-L-alaninyl)
phosphate (18): Prepared according to the standard procedure A from fludarabine
(0.25 g, 0.88 mmol), POCl3 (0.16 mL, 1.75 mmol), the tosylate salt of benzoxy-L-
alanine (1.50 g, 4.38 mmol) and DIPEA (1.52 mL, 8.76 mmol). After work-up and
silica gel column chromatography phosphorodiamidate 18 was obtained as an off-
31
19
white solid (0.016g, 3%). P NMR (202 MHz, CD3OD): δ = 13.76; F NMR (470
MHz, CD3OD): δ = –53.30; 1H NMR (500 MHz, CD3OD): δ = 8.25 (s, 1H, H-8), 7.40
– 7.20 (m, 10 H, Ar-H), 6.32 (d, J = 4.4 Hz, 1H, H-1′), 5.22 – 5.15 (m, 4H, 2 x
CH2Ph), 4.40 – 4.36 (m, 1H, H-2ʹ), 4.32 – 4.24 (m, 3H, 2 x H-5ʹ, H-4ʹ), 4.19 – 4.00
(m, 1H, H-3′), 3.98 – 3.94 (m, 2H, NHCHCH3), 1.45 – 1.38 (m, 6H, 2 x NHCHCH3);
3
13C NMR (125 MHz, CD3OD): δ = 175.56, 175.48 (2d, JC–P = 5.0 Hz, C=O, ester),
160.60 (d, 1JC–F = 207.5 Hz, C-2), 158.85 (d, 3JC–F = 20.0 Hz, C-6), 152.18 (d, 3JC–F
=
20.0 Hz, C-4), 142.69 (C-8), 137.26, 137.21 (2 x C ipso-Ph), 129.86, 129.63, 129.61,
129.38, 129.33, 129.32, 129.29, 129.25 (CH-Ar), 117.86 (C-5), 86.02 (C-1′), 83.96
4
(d, JC–P = 7.5 Hz, C-3ʹ), 77.25 (C-4ʹ), 77.15 (C-2ʹ), 67.98, 67.85 (CH2Ph), 66.30 (d,
2JC–P = 5.0 Hz, C-5′), 51.2 (d, 2JC–P = 8.75 Hz, NHCHCH3), 20.85 (d, 3JC–P = 6.30 Hz,
3
NHCHCH3), 20.72 (d, JC–P = 6.30 Hz, NHCHCH3). [ES+] m/z: found 710.22 [M +
Na+] C30H35FN7O9P required 687.61 [M]. Reverse HPLC, eluting with H2O/CH3OH
from 100/0 to 0/100 in 30 min, F = 1 mL/min, λ = 254, tR = 23.08.
Guanine-9-β-D-arabinofuranoside-5’-O-bis(2,2-dimethoxypropoxy-L-alaninyl)
phosphate (19): Prepared according to general procedure A from AraG (0.40 g, 1.41
mmol), POCl3 (0.216 g, 0.131 mL), the tosylate salt of 2,2-dimethoxypropoxy-L-
alanine (2.33 g, 7.05 mmol) and DIPEA (2.45 mL, 14.1 mmol). After the reaction was
completed the solvent was removed under vacuum and the crude material was
purified by column chromatography on silica gel followed by preparative HPLC
(reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 20 minutes, F
= 20 ml/min, l = 254 nm, tR = 10.72 min) to afford phosphorodiamidate 19 as a white
31
1
solid (0.050 g, 6%). P NMR (202 MHz, CD3OD): δ = 13.88; H NMR (500 MHz,
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10.1002/cmdc.201800504
ChemMedChem
CD3OD): δH 7.90 (s, 1H, H-8), 6.37 (d, J = 4.6 Hz, 1H, H-1ʹ), 4.33 – 4.27 (m, 4H, 2 x
H-5ʹ, H-4ʹ, H-2ʹ), 4.07 – 4.05 (m, 2H, H-3ʹ), 3.99 – 3.95 (m, 2H, 2 x NHCHCH3),
3.88, 3.78 (AB system JAB = 10.50 Hz, 2H, CH2C(CH3)3), 3.87, 3.77 (AB system, JAB
= 10.50 Hz, 2H, CH2C(CH3)3), 1.42 (d, J = 7.5 Hz, 3H, NHCHCH3), 1.40 (d, J = 7.5
Hz, 3H, NHCHCH3), 0.98 (s, 18H, CH2C(CH3)3); 13C NMR (125 MHz, CD3OD): δ =
175.70, 175.65 (C=O, ester), 159.35 (C-6), 155.36 (C-2), 152.99, (C-4), 139.61 (C-8),
117.09 (C-5), 85.82 (C-1ʹ), 84.00 (d 3JC-P = 7.36 Hz, C-4ʹ), 77.59 (C-2ʹ), 77.25 (C-3ʹ),
2
75.39, 75.37 (CH2C(CH3)3, 66.49 (d, JC-P = 5.26 Hz, C-5ʹ), 51.14 (CHCH3), 32.31
(CH2C(CH3)3), 26.66 (CH2C(CH3)3), 21.09 (d, 3JC-P = 5.97 Hz, NHCHCH3), 20.95 (d,
3JC-P = 6.44 Hz, NHCHCH3); MS [ES+] m/z: found 646 [M + H+], 669 [M + Na+],
C26H44N7O10P required: 645.28 (M). HRMS [ESI+] m/z: calculated for
C26H44N7O10P: 645.2887; found 646.2881 [M + H]+. HPLC Reverse-phase HPLC
eluting with H2O/CH3CN from 90/10 to 0/100 in 30 minutes, F = 1ml/min, l = 254
nm, tR = 14.93 min.
6-Thioinosine-5′-O-bis(benzoxy-L-alaninyl) phosphate (20): Prepared according to
the standard procedure A from 6-thioinosine (0.15 g, 0.53 mmol), POCl3 (0.05 mL,
0.53 mmol), the tosylate salt of benzoxy-L-alanine (0.88 g, 2.50 mmol) and DIPEA
(0.91 mL, 5.01 mmol). After work-up and silica gel column chromatography
31
phosphorodiamidate 20 was obtained as an off-white solid (0.014 g, 4%). P NMR
(202 MHz, CD3OD): δ = 13.71; 1H NMR (500 MHz, CD3OD): δ = 8.40 (s, 1H, H-2),
8.15 (s, 1H, H-8), 7.35 – 7.29 (m, 10 H, Ar-H), 6.01 (d, J = 4.5 Hz, 1H, H-1′), 5.15,
5.13, 5.10, 5.07 (2 x AB system, JAB = 12.5, 4.5 Hz, 4H, 2 x CH2Ph), 4.70 (t, J = 4.5
Hz, 1H, H-2’), 4.40 (t, J = 4.5 Hz, 1H, H-3ʹ), 4.25 – 4.14 (m, 3H, 2 x H-5ʹ, H-4ʹ), 3.94
13
– 3.91 (m, 2H, 2 x NHCHCH3), 1.32, 1.29 (2 x d, J = 7.0 Hz, 2 x NHCHCH3); C
3
NMR (125 MHz, CD3OD): δ = 178.27 (C=S), 175.44, 175.38 (2d, JC–P = 5.0 Hz,
C=O, ester), 145.03 (C-2), 142.90 (C-4), 142.80 (C-8), 137.31, 137.27 (2 x C ipso-
Ph), 137.05 (C-5), 129.58, 129.57, 129.31, 129.30, 127.17 (CH-Ar), 90.42 (C-1′),
3
84.74, 84.70 (d, JC–P = 8.2 Hz, C-4ʹ), 75.53 (C-2ʹ), 71.57 (C-3’), 67.99, 67.96
(CH2Ph), 66.22, 66.18 (C-5′), 51.14, 51.10 (d, 2JC–P = 6.25 Hz, NHCHCH3), 20.85 (d,
3
3JC–P = 6.25 Hz, NHCHCH3), 20.62 (d, JC–P = 6.30 Hz, NHCHCH3). [ES+] m/z:
found 709.71 [M + Na+] C30H35N6O9PS required 686.67 [M].
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10.1002/cmdc.201800504
ChemMedChem
6-Thioinosine-5′-O-bis(2,2-dimethoxypropoxy-L-alaninyl)phosphate
(21):
Prepared according to the standard procedure A from 6-thioinosine (0.15 g, 0.53
mmol), POCl3 (0.05 mL, 0.53 mmol), the tosylate salt of 2,2-dimethoxypropoxy-L-
alanine (0.87 g, 2.50 mmol) and DIPEA (0.91 mL, 5.01 mmol). After work-up and
silica gel column chromatography phosphorodiamidate 21 was obtained as an off-
31
1
white solid (0.017 g, 5%). P NMR (202 MHz, CD3OD): δ = 13.84; H NMR (500
MHz, CD3OD): δ = 8.43 (s, 1H, H-2), 8.19 (s, 1H, H-8), 6.04 (d, J = 5.0 Hz, 1H, H-
1′), 4.72 (t, J = 5.0 Hz, 1H, H-2’), 4.43 (t, J = 5.0 Hz, 1H, H-3ʹ), 4.30 – 4.21 (m, 3H, 2
x H-5ʹ, H-4ʹ), 3.97 – 3.93 (m, 2H, 2 x NHCHCH3), 3.88, 3.86, 3.76, 3.74 (2 x AB
system, JAB = 10.5, 4.5 Hz, 4H, 2 x CH2C(CH3)3), 1.39, 1.37 (2 x d, J = 7.0 Hz, 2 x
NHCHCH3), 0.95 (s, 18H, 2 x CH2C(CH3)3); 13C NMR (125 MHz, CD3OD): δ =
178.44 (C=S), 175.07, 175.02 (2d, 3JC–P = 6.25 Hz, C=O, ester), 146.47 (C-2), 145.03
3
(C-4), 142.76 (C-8), 137.19 (C-5), 90.44 (C-1′), 84.82, 84.76 (d, JC–P = 7.8 Hz, C-
2
4ʹ), 79.47 (CH2C(CH3)3), 74.89 (C-2ʹ), 71.58 (C-3’), 66.27, 66.23 (d, JC–P = 5.0 Hz,
2
C-5′), 51.19, 51.09 (d, JC–P = 6.0 Hz, NHCHCH3), 32.53, 32.50, 32.46, 32.44
3
(CH2C(CH3)3), 30.73, 26.44, 24.67 (CH2C(CH3)3), 21.13 (d, JC–P = 5.6 Hz,
3
NHCHCH3), 20.89 (d, JC–P = 5.60 Hz, NHCHCH3). [ES+] m/z: found 669.83 [M +
Na+] C26H43N6O9PS required 646.69 [M].
6-Thioguanosine-5′-O-bis(benzoxy-L-alaninyl) phosphate (22): Prepared according
to the standard procedure A from 6-thioguanosine (0.15 g, 0.50 mmol), POCl3 (0.05
mL, 0.53 mmol), the tosylate salt of benzoxy-L-alanine (0.88 g, 2.50 mmol) and
DIPEA (0.91 mL, 5.01 mmol). After work-up and silica gel column chromatography
31
phosphorodiamidate 22 was obtained as an off-white solid (0.010 g, 3%). P NMR
(202 MHz, CD3OD): δ = 13.70; 1H NMR (500 MHz, CD3OD): δ = 8.15 (s, 1H, H-8),
7.35 – 7.30 (m, 10 H, Ar-H), 6.01 (d, J = 5.5 Hz, 1H, H-1′), 5.16 –5.06 (m, 4H, 2 x
CH2Ph), 4.68 (t, J = 5.5 Hz, 1H, H-2’), 4.48 (t, J = 5.5 Hz, 1H, H-3ʹ), 4.24 – 4.20 (m,
1H, 1 x H-5ʹ), 4.16 – 4.14 (m, 2H, 1 x H-5ʹ, H-4ʹ), 3.97 – 3.91 (m, 2H, 2 x
13
NHCHCH3), 1.32, 1.29 (2 x d, J = 7.0 Hz, 2 x NHCHCH3); C NMR (125 MHz,
CD3OD): δ = 177.12 (C=S), 175.44, 175.38 (2d, 3JC–P = 6.25 Hz, C=O, ester), 163.19
(C-2), 161.34 (C-4), 151.47 (C-8), 140.49 (C-5), 137.25, 137.20 (2 x C ipso-Ph),
3
129.61, 129.37, 129.34, 129.32, 127.17 (CH-Ar), 90.02 (C-1′), 84.49, 84.41 (d, JC–P
= 8.2 Hz, C-4ʹ), 74.63 (C-2ʹ), 71.69 (C-3’), 68.07, 68.02 (CH2Ph), 66.38, 66.32 (d,
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10.1002/cmdc.201800504
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2
3
2JC–P = 7.5 Hz, C-5′), 51.14, 51.08 (d, JC–P = 3.8 Hz, NHCHCH3), 20.85 (d, JC–P
=
5.8 Hz, NHCHCH3), 20.62 (d, 3JC–P = 5.80 Hz, NHCHCH3). [ES+] m/z: found 724.75
[M + Na+] C30H36N7O9PS required 701.68 [M].
6-Thioguanosine-5′-O-bis(2,2-dimethoxypropoxy-L-alaninyl)phosphate
(23):
Prepared according to the standard procedure A from 6-thiguanosine (0.15 g, 0.50
mmol), POCl3 (0.05 mL, 0.50 mmol), the tosylate salt of 2,2-dimethoxypropoxy-L-
alanine (0.83 g, 2.50 mmol) and DIPEA (0.91 mL, 5.01 mmol). After work-up and
silica gel column chromatography phosphorodiamidate 23 was obtained as an off-
31
1
white solid (0.016 g, 5%). P NMR (202 MHz, CD3OD): δ = 13.83; H NMR (500
MHz, CD3OD): δ = 8.05 (s, 1H, H-8), 5.87 (d, J = 5.0 Hz, 1H, H-1′), 4.71 (t, J = 5.0
Hz, 1H, H-2’), 4.41 (t, J = 5.0 Hz, 1H, H-3ʹ), 4.30 – 4.19 (m, 3H, 2 x H-5ʹ, H-4ʹ), 4.00
– 3.95 (m, 2H, 2 x NHCHCH3), 3.88, 3.86, 3.76, 3.74 (2 x AB system, JAB = 10.5, 4.5
Hz, 4H, 2 x CH2C(CH3)3), 1.38, 1.35 (2 x d, J = 7.0 Hz, 2 x NHCHCH3), 0.94 (s,
18H, 2 x CH2C(CH3)3); 13C NMR (125 MHz, CD3OD): δ = 177.1 (C=S), 175.66,
3
175.58 (2d, JC–P = 5.8 Hz, C=O, ester), 164.19 (C-2), 148.82 (C-4), 140.71 (C-8),
3
130.28 (C-5), 90.11 (C-1′), 84.70, 84.64 (d, JC–P = 7.5 Hz, C-4ʹ), 75.44
2
(CH2C(CH3)3), 75.41 (C-2ʹ), 74.71 (C-3’), 66.56, 66.50 (d, JC–P = 7.70 Hz, C-5′),
2
51.17, 51.09 (d, JC–P = 10.0 Hz, NHCHCH3), 32.38, 32.35 (CH2C(CH3)3), 26.79
3
3
(CH2C(CH3)3), 21.12, 21.10 (d, JC–P = 5.6 Hz, NHCHCH3), 20.89 (d, JC–P = 5.60
Hz, NHCHCH3). [ES+] m/z: found 684.78 [M + Na+] C26H44N7O9PS required 661.70
[M].
Acknowledgements
We thank NuCana plc for financial support. Part of the cytostatic evaluations were
performed by Mrs. Lizette van Berckelaer at the Rega Institute for Medical Research,
KU Leuven (GOA 15/19 TBA).
Conflict of interest
The authors (M. Serpi and M. Slusarczyk) receive research funding from NuCana plc,
whose two agents NUC-1031 and NUC-3373 are cited here.
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10.1002/cmdc.201800504
ChemMedChem
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