N -phosphonocarbonylpyrrolidine derivatives of guanine: A new class of Bi-substrate inhibitors of human purine nucleoside phosphorylase

Article abstract of DOI:10.1021/jm201409u

A complete series of pyrrolidine nucleotides, (3R)- and (3S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acids and (3S,4R)-, (3R,4S)-, (3S,4S)-, and (3R,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N- ylcarbonylphosphonic acids, were synthesized and e

Full text of DOI:10.1021/jm201409u

Article
pubs.acs.org/jmc
N-Phosphonocarbonylpyrrolidine Derivatives of Guanine: A New
Class of Bi-Substrate Inhibitors of Human Purine Nucleoside
Phosphorylase
Dominik Rejman,*,† Natalya Panova,† Pavel Klener,*,‡ Bokang Maswabi,‡ Radek Pohl,†
and Ivan Rosenberg*,†
†Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i. Flemingovo nam
166 10 Prague 6, Czech Republic
́
. 2,
‡Institute of Pathological Physiology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Prague 2,
Czech Republic
S
* Supporting Information
ABSTRACT: A complete series of pyrrolidine nucleotides, (3R)-
and (3S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acids
and (3S,4R)-, (3R,4S)-, (3S,4S)-, and (3R,4R)-4-(guanin-9-yl)-3-hydroxy-
pyrrolidin-1-N-ylcarbonylphosphonic acids, were synthesized and
evaluated as potential inhibitors of purine nucleoside phosphorylase
(PNP) isolated from peripheral blood mononuclear cells (PBMCs)
and cell lines of myeloid and lymphoid origin. Two compounds,
(S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acid (2a)
and (3S,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonyl-
phosphonic acid (6a), were recognized as nanomolar competitive inhibitors of PNP isolated from cell lines with Ki values
within the ranges of 16−100 and 10−24 nM, respectively. The low MESGKi and PiKi values of both compounds for PNP isolated
from PBMCs suggest that these compounds could be bisubstrate inhibitors that occupy both the phosphate and nucleoside
binding sites of the enzyme.
PNP is widely expressed in human tissues, predominantly in
the cytosol and mitochondria of cells. The highest activity of
PNP was detected in both normal and malignant leukocytes,
including peripheral blood mononuclear cells (PBMCs),
neutrophil leukocytes (granulocytes), and lymphocytes. In-
creased levels of PNP in cancer cells and the relationship
between PNP activity and the degree of malignancy have been
reported.9
The first inhibitors of PNP were developed using structure-
based inhibitor design focused on iterative group alignment
established from the PNP crystal structure.10 Following a novel
approach based on the identification of the transition-state
structures stabilized by the target enzyme, the potent inhibitors
immucillin-H (IMH, 1-(9-deazahypoxanthin)-1,4-dideoxy-1,4-
imino-D-ribitol) and immucillin-G (IMG, 1-(9-deazaguanin)-
1,4-dideoxy-1,4-imino-D-ribitol) (Figure 1) of bovine, malarial,
Mycobacterium tuberculosis, and human PNPs were identi-
fied.11−14 IMH was reported to induce apoptosis and selectively
suppress the growth of human T-cell leukemia lines with an
IC50 of 5 nM.15 Furthermore, IMH has completed phase IIb
trials against cutaneous T-cell lymphoma (as BCX-1777 or
Forodesine).16,17 IMH was observed to selectively inhibit the in
vitro growth of malignant T-cell lines in the presence of dGuo
INTRODUCTION
■
In the purine salvage pathway, the enzyme purine nucleoside
phosphorylase (PNP, EC 2.4.2.1) catalyzes the reversible phos-
phorolytic cleavage of guanine and hypoxanthine nucleosides
in the presence of the second substrate, inorganic ortho-
phosphate (Pi), releasing the corresponding purine nucleobase
and sugar phosphate.1 In the absence of PNP, nucleoside
substrates, such as 2′-deoxyguanosine (dGuo), accumulate.2
The accumulation of dGuo has been observed in children with
an inherited PNP deficiency. These children exhibit severe
T-cell immunodeficiency but retain normal B-cell function.
Phosphorylation of dGuo (by 2′-deoxycytidine kinase, dCK,
EC 2.7.1.74) to 2′-deoxyguanosine triphosphate (dGTP) is
responsible for T-cell cytotoxicity. dGTP allosterically inhibits the
enzyme ribonucleoside-5′-diphosphate reductase (EC 1.17.4.1),
impairing DNA synthesis and initiating T-cell apoptosis.3
Human T-cells are unique in that they display a combination of
high dCK activity and relatively low nucleotidase activity, which
allows these cells to accumulate dGTP.4 PNP has been iden-
tified as a therapeutic target for the control of numerous T-cell
disorders, including organ transplant rejection, T-cell malig-
nancies, rheumatoid arthritis, psoriasis, and some other auto-
immune diseases.1,5−7 B-Cell chronic lymphocytic leukemia
(CLL) cells also exhibit high dCK activity. Recently, CLL cells
where shown to respond to PNP inhibition.8
Received: October 19, 2011
Published: January 23, 2012
© 2012 American Chemical Society
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Figure 1. Inhibitors of PNP.
without affecting non-T-cell tumor lines. Activated human peri-
pheral blood T-lymphocytes were also sensitive to inhibition by
IMH.15 Moreover, recent clinical and preclinical data showed
activity of Forodesine in B-cell acute lymphoblastic leukemia
(B-ALL), which supports the potential use of IMH for the
treatment of selected B-cell malignancies.8,18
More recently, the second generation of the immucillins, the
DADMe-immucillins (Figure 1), has been reported.19 In con-
trast to IMHs mimicking a substrate-like transition state, DADMe-
immucillin-H is more closely related to the product-like. Among
these new compounds, the most potent DADMe-immucillin-G
was eight times more active than IMH.
RESULTS AND DISCUSSION
■
Chemistry. Enantiomeric pyrrolidine derivatives of guanine
1a and 1b, prepared as previously reported,27 were treated with
phenyl diisopropylphosphonoformate28 in DMF at 90 °C
overnight.26 The diisopropyl ester groups were removed using
trimethylsilyl bromide in DMF, and the crude 2a and 2b were
purified by the preparative HPLC on C18 column. The
following conversion into sodium salts via Dowex 50 × 8 (Na+
form) and lyophilization from water afforded the final
enantiomeric phosphonic acids 2a and 2b (Scheme 1).
Scheme 1. Synthesis of Enantiomeric Pyrrolidinyl
a
Nucleotide Analogues 2a and 2b
In addition to these nucleoside-based inhibitors, several acyclic
nucleotide analogues were found to inhibit human PNP (Figure 1)
in a nanomolar range. Acyclovir diphosphate (ACVDP)20−22
and Tenofovir-phosphate23 exhibited Ki values of 10 and 38
nM, respectively. However, neither ACVDP nor Tenofovir-
phosphate are considered as drug candidates for PNP inhibition
because of the instability of phosphate groups in vivo. High-
resolution differentiation X-ray data obtained from a crystal of
PNP with 9-(5′,5′-difluoro-5′-phosphonopentyl)guanine
(DFPP-G, Ki of 10.8 nM) confirmed DFPP-G as a multi-
substrate inhibitor. This crystal structure revealed that the
nucleoside and phosphonate parts of one molecule of DFPP-G
were bound simultaneously into the nucleoside and phosphate
binding sites of PNP, respectively.24 Recently, a variety of
deazaguanine analogues of DFPP-G have also been recognized
as potent PNP inhibitors.25
Our systematic search for nucleoside phosphonate-based
inhibitors of enzymes involved in nucleoside/nucleotide
catabolism has led to the synthesis of a series of pyrrolidine
nucleoside phosphonic acids. A recent study on the inhibition
of thymidine phosphorylase from rat T-cell lymphomas by
these type of compounds revealed their nanomolar inhibitory
effect, suggesting their possible role as bisubstrate inhibitors.26
On the basis of these findings, we synthesized the corres-
ponding guanine derivatives to examine their potential inhibitory
effect on PNP partially purified from human lymphoma and
leukemia cell lines of T- and B-cell origin as well as PNP from
the lymphocytes of healthy donors.
a
Reagents: PhO(O)CP(O)(OPr)2, DMF 90 °C; (i) Me3SiBr, DMF.
The synthesis of diastereoisomeric hydroxypyrrolidine deri-
vatives of guanine 5a−d was initiated from fully protected 3,4-
dihydroxypyrrolidines 3a−d29 (Scheme 2). The mesyl group of
these compounds was converted into an azido group by
treatment with sodium azide in DMF. The DMTr group of the
obtained azido derivatives was selectively removed with 1.5%
TFA in DCM.30 The N-Boc group was not cleaved under these
conditions. Subsequent hydrogenation over palladium catalyst
afforded good yields of amino compounds 4a−d, which were
transformed into guanine derivatives in several steps. The
reaction with 2,5-diamino-4,6-dichloropyrimidine followed by
cyclization to form the five-membered imidazole ring using
only 1.2 equiv of diethoxymethyl acetate in DMF, instead of
using diethoxymethyl acetate as a solvent, provided the desired
2-amino-6-chloropurine derivative.30 This modification elimi-
nated the formylation of the purine exocyclic 2-amino group
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a
Scheme 2. Synthesis of Diastereoisomeric Hydroxypyrrolidinyl Nucleotide Analogues 6a−d
a
Reagents: (i) NaN3, DMF, 110 °C; (ii) (i) 1.5% TFA/DCM; (ii) NaHCO3; (iii) H2, Pd/C, EtOH; (iv) 2,5-diamino-4,6-dichloropyrimidine, TEA,
nBuOH, 130 °C; (v) CH3COOCH(OCH2CH3)2, DMF, from rt to 110 °C; (vi) 1.5M aq HCl, 80 °C; (vii) PhO(O)CP(O)(OiPr)2, DMF, 90 °C;
(viii) Me3SiBr, MeCN.
that would otherwise render the isolation of the 2-amino-6-
chloropurine intermediate difficult.30 Final treatment of 6-
chloro-2-aminopurine intermediates with 1.5 M aq HCl
afforded guanine derivatives 5a−d in moderate yields.
The resulting diastereoisomeric hydroxypyrrolidine deriva-
tives of guanine 5a−d were treated with phenyl diisopropylphos-
phonoformate28 in DMF at 90 °C overnight.26 The diiso-
propyl ester groups were removed using trimethylsilyl bro-
mide in DMF, and the crude 6a−d were purified by the
preparative HPLC on C18 column. The following conversion
into sodium salts via Dowex 50 × 8 (Na+ form) and freeze-
drying from water afforded the final diastereomeric
phosphonic acids 6a−d.
molecular weight compounds and substantial part of balast
proteins, (b) performed the inhibition assays to select potent
inhibitors of PNP, and (c) performed inhibition study with
selected inhibitors and PNPs isolated from various human
lymphoma and leukemia cell lines of T- and B-cell origin to
check potential different sensitivity of the enzymes to inhibition
in comparison with PNP isolated from healthy donors.
PNP activity and inhibition assays were based on the phos-
phorolysis of 7-methyl-6-thioguanosine (MESG) to 7-methyl-6-
thioguanine catalyzed by PNP.32−34 This conversion resulted in
a significant spectrophotometric shift from 330 nm (MESG) to
360 nm (7-methyl-6-thioguanine). MSEG, a substrate of PNP,
was chosen with respect to the kinetics of cleavage, which are
well described by the Michaelis−Menten equation.32
Partial Purification of PNP. Partially purified PNPs
suitable for inhibition studies were obtained in two steps from a
105 × g supernatants of the cell homogenates including
ammonium sulfate precipitation, dialysis, and subsequent fast
anion-exchange chromatography on a HiTrap Q column (see
Supporting Information page S3). The purification protocol is
summarized in Table 1.
1H and 13C NMR spectra of each nucleoside phosphonate
(2a, 2b, and 6a−d) showed the presence of two compounds,
which were recognized as cis and trans rotamers around the
C6′−N1′ linkage. These rotamers arise as a consequence of the
conjugation of the carbonyl and pyrrolidine nitrogen electron
pairs, which partially prevents unrestricted free rotation around
the C6′−N1′ linkage (Figure 2).31
Determination of Km and Enzyme Activity. The Km and
V values for MESG and inorganic phosphate were determined
with PNP from different sources (Table 2). Using GraphPad
Prism 4,35 the measurement of the reaction rates of MSEG
cleavage at various concentrations (up to 200 μM) at a constant
potassium phosphate concentration (500 μM) provided the
appropriate MESGKm. Similarly, we obtained the PiKm values
from the reaction rate of the MESG cleavage (80 μM) and
various potassium phosphate concentrations (up to 2500 μM).
Simultaneously, V values were also obtained from the
appropriate plots. All Km and V values are averages of three
measurements.
Inhibition Assay. The initial inhibition experiments with
compounds 2a−b and 6a−d were carried out with PNP from
PBMCs at two inhibitor concentrations (10 and 1 μM). The
results summarized in Table 3 clearly show that only phos-
phonates 2a and 6a strongly inhibited the PNP even at an 80:1
Figure 2. Cis and trans rotamers of carbonylphosphonates 2a, 2b, and
6a−d.
Biochemistry. The main focus of this study was to assess
the inhibitory potential of novel N-phosphonopyrrolidine deri-
vatives of guanine toward PNP isolated from various cell lines.
To achieve this, we completed the following: (a) optimized two
step partial purification of PNP to remove a large excess of low
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Table 1. Partial Purification of PNP and Its Distribution in Cancer Cell Lines
protein concentration
PNP specific activity
(μmol·h−1 per mg of protein)
(μg/mL)
vol of 105 × g
supernatant
(mL)
purification factor
[fold]/ recovery
(%)
no. of cells
105 × g
supernatant
HiTrap Q
b
105 × g
supernatant
HiTrap Q
b
PNP source
× 106
column
column
a
PBMC
>1000
15
6000
250
2
27
14/4
JURKAT
K-562
300
<300
600
200
10
4
3
4000
nd
300
125
350
250
200
100
150
50
0.50
nd
11
10
24
35
54
23
11
26
60
24
21/41
nd/nd
8.6/36
16/42
14/49
14/28
56/33
87/22
36/37
10/73
DoHH2
3
3200
3000
2500
1300
8000
8000
2000
1500
2.40
2.32
3.50
1.59
0.25
0.30
1.62
1.09
MINO
3
MC-116
3
SU-DHL-1
RPMI 8226
OPM-2
412
235
235
300
nd
4
2.5
2.5
2.5
2.5
HBL2 (culture)
HBL2 (tumor)
50
125
c
e
e
CML
5000
5000
2000
2000
6
6.5
5
nd
450
400
250
150
nd
4
nd
nd
nd
nd
c
e
e
CML
nd
nd
8
d
e
e
AML
nd
nd
3.7
5.5
d
e
e
AML
5
nd
nd
a
b
c
Peripheral blood mononuclear cells were obtained as buffy coats from healthy donors. PNP activity was detected in only one 1 mL fraction. Two
d
e
different patients. One patient. Protein and PNP activity could not be measured due to the presence of a colored substance interfering with
spectrophotometric assay.
Table 2. Km and V Values for MESG and Inorganic
Phosphate Determined for the Phosphorolysis Catalysed by
Partially Purified PNPs
are competitive inhibitors of the PNPs studied with respect to
MESG. In the case of PNPs from cancer cell lines, the Ki values
of compound 2a varied from 16 to 100 nM, whereas lower
values (10−24 nM) were obtained for the hydroxy derivative
6a. The low MESGKi and PiKi values of 2a and 6a for PNP from
PBMCs suggest that these compounds could be bisubstrate
inhibitors, occupying both the phosphate and nucleoside
binding sites of the enzyme.
MESGKm
(μM)
PiKm
(μM)
MESGV
(nmol/min)
PiV
PNP source
(nmol/min)
a
PBMC
80 20
60 10
50 10
2.5 0.1
1.05 0.05
1.4 0.06
b
hrPNP
100 20
1.4 0.09
The inhibitory activity of 2a, in contrast to 6a, varied widely
between 16 and 100 nM (Table 4). Because the plausibility of
these data is given by the arrangement of kinetic experiments
(both 6a and 2a were measured simultaneously with the same
batch of the enzyme), one can speculate that the obtained data
for 2a resulted from different affinity of 2a toward PNP iso-
forms,37 the ratio and type of which could vary in dependence
on the type of cancer cell lines. On the other hand, it seems that
the inhibitor 6a providing a relatively narow window of inhi-
bitory activity lost a selectivity due to the presence of hydroxy
group allowing the formation of additional hydrogen bond(s)
and thus a more tight binding to the enzyme catalytic site
(regardless of the PNP isoforms).
Time-Dependent Inhibition. To quantify the possible
time- and concentration-dependent inactivation of PNP during
preincubation with the most potent inhibitor 6a, we measured
the remaining enzyme activity using MESG as a substrate at
various inhibitor concentrations (0−250 nM) during 10 and
20 min periods of preincubation. The results demonstrated an
increase in inhibition activity at each inhibitor concentration
(Figure 3). In contrast, there was no difference between 10 and
20 min of preincubation. We observed that the IC50 values
decreased from approximately 40 nM (no preincubation) to 10 nM
within 10 or 20 min of preincubation. This time-dependent
inhibition could be explained by a two-step mechanism where
binding involves the rapid formation of an enzyme−inhibitor
collision complex followed by slow conformational changes that
DoHH2
MINO
40
5
nd
nd
nd
1.6 0.04
1.8 0.4
1.5 0.2
nd
nd
nd
120 20
MC-116
70 10
a
b
From healthy donors. Human recombinant PNP.
ratio of substrate to inhibitor. Therefore, these compounds
were assayed with hrPNP and PNP from PBMCs and cell
lines derived from different human tumors to obtain Ki values
with respect to MESG with the aim to find possible differ-
ences in the inhibitory potency. The results are summarized in
Table 4. The initial reaction rates of the cleavage of MESG
(at 40 and 80 μM) in the absence and presence of inhibitors
2a and 6a (up to 500 nM) and the constant inorganic phos-
phate concentration (500 μM) were measured. The appro-
priate MESGKi values were calculated from Dixon plots using
SigmaPlot software (see Experimental Section and the Supporting
Information for plots).36
In the case of PNP from PBMCs, Ki values for 2a and 6a
with respect to the inorganic phosphate were also determined.
The measurements of the initial rates of MESG (80 μM)
cleavage at three inorganic phosphate concentrations (250, 500,
and 1000 μM) in the absence and presence of inhibitors (up to
Pi
500 nM) provided Ki values of 50 and 20 nM for 2a and 6a,
respectively. All Ki values in Table 4 represent the average
values of three independent measurements. The patterns of
Dixon plots (Supporting Informationo) suggest that 2a and 6a
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Table 3. Inhibition of PNP Isolated from PBMCs of Healthy
Donors by Enantiomeric and Diastereomeric Guanine-Based
Pyrrolidine Nucleoside Phosphonic Acids
CONCLUSION
■
A series of enantiomeric and diastereomeric N-phosphonocar-
bonylpyrrolidine (2a,b) and N-phosphonocarbonyl-3-hydrox-
ypyrrolidine (6a−d) derivatives of guanine, respectively, were
synthesized and assayed with native human PNPs partially
purified from PBMCs of healthy donors and various cancer cell
lines. From the series of potential inhibitors, compounds 2a and
6a were recognized as potent, potential bisubstrate inhibitors of
PNP from PBMCs with nanomolar Ki values. The patterns of
the Dixon plots obtained suggested that these compounds are
competitive inhibitors of PNPs from various sources with
respect to the MESG substrate. The Ki values of compound 2a
for PNPs from cancer cell lines varied from 16 to 100 nM,
whereas lower values (10−24 nM) were obtained for the
hydroxy derivative 6a, suggesting a slightly higher selectivity of
2a toward several PNPs. The low MESGKi and PiKi values for 2a
and 6a may suggest that these compounds are bisubstrate
inhibitors of PNP from PBMCs. Regarding the stereochemistry
of inhibitors 2a and 6a, these compounds undergo the cis ↔
trans isomerism around the N−C(O) linkage and therefore
exist as cis and trans conformers as confirmed by 1H and 13C
NMR spectroscopy (Figure 2). One can conclude that only
one of these conformers is capable of binding efficiently to
the PNP phosphorus binding site. The availability of X-ray
coordinates of the enzyme with 6a and 2a would not only be
an instructive tool for determining the conformational and
binding properties of these nucleotide analogues but also a
starting point for the rational design of potent bisubstrate
inhibitors of human PNP based on pyrrolidine nucleotide
analogues. Such a study is underway and the results will be
published in due course.
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise stated, all solvents used were
anhydrous. Pyrrolidine derivatives of guanine (1a, 1b) and mesyl
derivatives (3a−d) were synthesized according to previously described
procedures.27,28 TLC was performed on TLC plates precoated with
silica gel (silica gel/TLC-cards, UV 254, Merck). Compounds were
detected using UV light (254 nm), heating (for the detection of
dimethoxytrityl group; orange color), spraying with 1% ethanolic
solution of ninhydrine to visualize amines, or by spraying with a 1%
solution of 4-(4-nitrobenzyl)pyridine in ethanol, followed by heating
and treating with gaseous ammonia (for the detection of alkylating
agents, such as mesyl derivatives; blue color). The purity of the final
compounds was greater than 95%. The purity of the prepared
compounds was determined by LC-MS using a Waters AutoPur-
ification System with 2545 quarternary gradient module and 3100
single quadrupole mass detector using Luna C18 column (100 mm ×
4.6 mm, 3 μm, Phenomenex) at a flow rate of 1 mL/min. The
following mobile phase was used, where A, B, and C represent 50 mM
NH4HCO3 and 50 mM NH4HCO3 in 50% aq CH3CN and CH3CN,
respectively: A → B over 10 min, B → C over 10 min, and C for 5 min.
Preparative RP HPLC was performed on an LC5000 liquid
chromatograph (INGOS-PIKRON, CR) using a Luna C18 (2)
column (250 mm × 21.2 mm, 5 μm) at a flow rate of 10 mL/min.
A gradient elution of methanol in pH 7.5 0.1 M TEAB (A, 0.1 M
TEAB; B, 0.1 M TEAB in 50% aq methanol; C, methanol) was used.
All final compounds were lyophilized from water. The optical rotation
values were measured on an AUTOPOL IV (Rudolph Research
Analytical, USA) polarimeter for the sodium D line at 20 °C. Mass
spectra were collected on an LTQ Orbitrap XL (Thermo Fisher
Scientific) using ESI ionization. The phosphorus content in the
compounds was determined using a simultaneous energy-dispersive X-
ray fluorescence spectrometer SPECTRO iQ II. NMR spectra were
collected in DMSO-d6 or D2O on a Bruker AVANCE 400 (1H at 400.0
MHz, 13C at 100.6 MHz, and 31P at 162.0 MHz) and/or Bruker
a
Measured at 80 μM of MSEG and 500 μM of inorganic phosphate.
Table 4. Km and Ki Values of Partially Purified PNP from
PBMCs and Cancer Cell Lines
a
b
c
PiKi values. Human recombinant enzyme. Two different patients.
d
One patient.
lead to a more stable enzyme−inhibitor complex, as observed
for immucillin H13 or DFPP-DG.25
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Figure 3. The effects of 0, 10, and 20 min preincubation of hrPNP with inhibitor 6a on the remaining PNP activity measured by the phosphorolysis
of MESG ((a) remaining activity measured after 2 min; (b) remaining activity measured after 5 min).
AVANCE 500 (1H at 500.0 MHz, 13C at 125.7 MHz, and 31P at 202.3
washed with water, and the product was eluted with 3% aq ammonia.
The final product was obtained by preparative RP-HPLC using a linear
gradient of methanol in water.
(S)-3-(Guanin-9-yl)pyrrolidin-N-ylcarbonylphosphonic Acid (2a).
Compound 2a was prepared from the pyrrolidine derivative 1a (60 mg,
0.27 mmol) in a 46% yield (45.9 mg, 0.123 mmol) as a white
amorphous solid using general method A. 1H NMR (500.0 MHz, D2O,
25 °C): 2.28−2.56 (m, 4H, H-4′-cis+trans), 3.55−3.70 (m, 2H, H-5′-cis),
3.83 (ddd, 1H, Jgem = 13.4, J2′b,3′ = 5.5, JHP = 1.6, H-2′b-trans), 3.99
(ddd, 1H, Jgem = 13.4, J2′a,3′ = 7.2, JHP = 1.8, H-2′a-trans), 4.08 (dt, 1H,
MHz) NMR spectrometers. Chemical shifts (in ppm, δ scale) were
1
referenced to the residual DMSO-d6 signal (2.5 ppm for H and 39.7
1
ppm for 13C) or to the 1,4-dioxane signal (3.75 ppm for H and 69.3
ppm for 13C) as an internal standard in D2O. 31P NMR spectra were
referenced to H3PO4 (0 ppm) as an external standard. Coupling
constants (J) are given in Hz. The complete assignment of 1H and 13
C
signals was performed by analysis of the correlated homonuclear
H,H−COSY and heteronuclear H,C-HSQC and H,C-HMBC spectra.
The relative configuration of compounds was checked using DPFGSE-
NOE and 2D-ROESY techniques. The numbering for signal
assignment is shown in Figure 2.
Jgem = 12.3, J5′b,4′ = 6.7, H-5′b-trans), 4.11 (dt, 1H, Jgem = 12.3, J5′a,4′
=
7.7, H-5′a-trans), 4.37 (d, 2H, J2′,3′ = 5.4, H-2′-cis), 5.01 (m, 1H, H-3′-cis),
5.03 (m, 1H, H-3′-trans), 7.85 and 7.86 (2 × s, 2 × 1H, H-8-cis+trans).
13C NMR (125.7 MHz, D2O, 25 °C): 31.64 (CH2-4′-cis), 34.13 (CH2-
4′-trans), 46.34 (d, JC,P = 4.2, CH2-5′-cis), 48.54 (CH2-5′-trans), 52.79
(d, JC,P = 4.7, CH2-2′-trans), 54.38 (CH2-2′-cis), 54.80 (CH-3′-trans),
56.76 (CH-3′-cis), 118.93 (C-5-cis+trans), 140.15, 140.25 (CH-8-cis+trans),
154.15, 154.19 (C-4-cis+C-4-trans), 156.74, 156.79 (C-2-cis+trans),
162.14, 162.17 (C-6-cis+trans), 181.08, 181.18 (d, JC,P = 189.4, C
O-cis+trans). 31P{1H} NMR (162.0 MHz, D2O, 25 °C): −1.65. IR
νmax(KBr) 3125 (m, br), 2960 (m, sh), 1694 (vs), 1652 (m, br), 1601
(s, sh), 1581 (s), 1536 (m), 1179 (m), 1085 (m, br), 574 (m, br) cm−1.
HR-ESI C10H12O5N6P [M − H]− calcd 327.0608, found 327.0612.
[α]20= −15.8 (c 0.253, H2O). Elem. Anal.: P, found 7.18% (MW 431.4).
(R)-3-(Guanin-9-yl)pyrrolidin-N-ylcarbonylphosphonic Acid (2b).
Compound 2b was prepared from pyrrolidine derivative 1b (0.41 g,
1.86 mmol) in a 33% yield (228.4 mg, 0.614 mmol) as a white
General Method A: Phosphonocarbonylation and Preparation of
Phosphonic Acids 2a, 2b, and 6a−d. Phenyl diisopropylphospho-
noformate (2 mmol) was added to a DMF solution (10 mL) of
pyrrolidine derivative of nucleobase (1a, 1b, and 5a−d; 1 mmol)
previously (dried by coevaporation with DMF) at 90 °C. The course
of the reaction was followed by LC-MS or TLC (10% EtOH/CHCl3).
After 2 h, the solvent was removed in vacuo, and the respective
diisopropyl esters of compounds 2a, 2b, and 6a−d were recovered by
silica gel chromatography using a linear gradient of ethanol in
chloroform (0 → 10%). The obtained diesters were coevaporated with
DMF and dissolved in the same solvent (10 mL/mmol). Then
bromotrimethylsilane (1.32 mL/mmol) was added, and the reaction
mixture was stirred at 70 °C. The reaction was monitored by LC-MS.
Subsequently, the reaction mixture was concentrated in vacuo, the
residue was dissolved in a mixture of 2 M TEAB (1 mL) and ethanol
(5 mL), and the solution was concentrated in vacuo.
The preparative RP HPLC afforded desired products 2a, 2b, and
6a−d as triethylammonium salts, which were converted into the
sodium salts using Dowex 50 (Na+ form). The triethylammonium salts
of phosphonic acids were subjected to HR ESI measurements, whereas
the sodium salts was checked, after lyophilization from water, for
phoshorus content, which was used for the calculation of molecular
weights.
General Method B: Nucleosidation, Preparation of Compounds
5a−d. A mixture of the appropriate amino derivative (4a−d) (1 mmol),
2,5-diamino-4,6-dichloropyrimidine (0.27 g, 1.5 mmol), and triethyl-
amine (0.56 mL, 4 mmol) in n-butanol (10 mL) was stirred in a
pressure vessel at 130 °C for two days. The course of the reaction was
monitored by LC-MS or TLC (10% EtOH/CHCl3). The chloropyr-
imidine intermediate, obtained by chromatography on silica gel using a
linear gradient of ethanol in chloroform, was treated with diethoxy-
methyl acetate (1.2 equiv) in DMF (10 mL/mmol) at rt for 2 h and then
at 110 °C for 2 d. The reaction mixture was concentrated in vacuo, and
the resulting residue was treated with 3 M HCl (10 mL/mmol) at 70 °C
overnight. The solution was diluted with water (20 mL/mmol) and
applied onto a Dowex 50 column in H+ (30 mL/mmol). The resin was
1
amorphous solid using general method A. H NMR, 13C NMR, and
31P{1H} NMR spectra are identical to 2a. HR-ESI C10H12O5N6P [M − H]−
calcd 327.0608, found 327.0607. [α]20 = +19.4 (c 0.237, H2O). Elem.
Anal.: P, found 7.66% (MW 404.4).
(3S,4R)-4-Amino-1-N-tert-butyloxycarbonyl-3-hydroxypyrrolidine
(4a). A mixture of the mesyl derivative 3a (19.8 g, 34 mmol) and
sodium azide (11 g, 170 mmol) in DMF (340 mL) was stirred at
100 °C overnight. The solvent was removed in vacuo. The azido deri-
vative, obtained by silica gel chromatography using a linear gradient of
ethyl acetate in toluene (0 → 100%), was treated with 1.5% TFA in
DCM (350 mL) for 30 min (TLC in 10% EtOH/CHCl3). Solid
NaHCO3 (20 g) and methanol (150 mL) were then added, and the
suspension was stirred until a neutral pH value was reached. The
suspension was filtered, and the filtrate was concentrated in vacuo. The
azido derivative obtained by silica gel chromatography using a linear
gradient of ethanol in chloroform was dissolved in methanol and
subjected to hydrogenation in the presence of Pd/C catalyst (1.5 g)
overnight. After filtration over Celite and evaporation of the solvent,
compound 4a was obtained in a 55% yield (3.78 g, 18.7 mmol) as a
colorless, viscous oil. NMR of trifluoroacetate salt: 1H NMR (500.0 MHz,
1617
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Journal of Medicinal Chemistry
Article
DMSO-d6, 80 °C): 1.41 (s, 9H, (CH3)3C)), 3.25 (dd, 1H, Jgem = 10.7,
J5b,4 = 7.0, H-5b), 3.27 (dd, 1H, Jgem = 11.6, J2b,3 = 3.1, H-2b), 3.44 (dd,
1H, Jgem = 11.6, J2a,3 = 5.0, H-2a), 3.57 (dd, 1H, Jgem = 10.7, J5a,4 = 7.4,
H-5a), 3.62 (ddd, 1H, J4,5 = 7.4, 7.0, J4,3 = 4.7, H-4), 4.28 (ddd, 1H,
J3,2 = 5.0, 3.1, J3,4 = 4.7, H-3). 13C NMR (125.7 MHz, DMSO-d6, 80
°C): 28.06 ((CH3)3C), 46.96 (CH2-5), 51.50 (CH-4), 51.86 (CH2-2),
67.80 (CH-3), 78.66 (C(CH3)3), 117.25 (q, JC,F = 299.7, CF3COO),
153.41 (CO), 158.33 (q, JC,F = 31.1, CF3COO). HR-ESI
C9H18O3N2Na [M + Na]+ calcd 225.1210, found 225.1209.
(3S,4S)-4-Amino-1-N-tert-butyloxycarbonyl-3-hydroxypyrrolidine
(4b). Compound 4b was prepared from mesyl derivative 3b (15.5 g,
26.55 mmol) following the same procedure used to obtain the amino
derivative 4a in a 64% yield (3.44 g, 19.38 mmol). Compound 4b was
obtained as a colorless, viscous oil that solidified to an amorphous
solid upon standing in the refrigerator. 1H NMR (500.0 MHz, DMSO-
(3R,4S)-4-(Guanin-9-yl)-3-hydroxypyrrolidine (5c). Compound 5c
was prepared according to general method B from 4c (0.95 g, 4.32 mmol)
in a 14% overall yield (142.2 mg, 0.61 mmol) as an amorphous solid. 1H
NMR and 13C NMR spectra are identical to 5a. HR-ESI C9H13O2N6
[M + H]+ calcd 237.10945, found 237.10940. [α]20= −64.7 (c 0.306,
DMSO).
(3R,4R)-4-(Guanin-9-yl)-3-hydroxypyrrolidine (5d). Compound 5d
was prepared in a 21% overall yield (142 mg, 0.6 mmol) as an amor-
phous solid according to general method B from 4d (0.52 g, 2.57
1
mmol). H NMR and 13C NMR spectra are identical to 5b. HR-ESI
C9H13O2N6 [M + H]+ calcd 237.10945, found 237.10941. [α]20=
−31.1 (c 0.228, H2O).
(3S,4R)-4-(Guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphos-
phonic Acid (6a). Compound 6a was prepared from pyrrolidine
derivative 5a (58 mg, 0.25 mmol) in a 15% yield (14.5 mg, 0.037
mmol), using general method A, as a white amorphous solid. 1H NMR
(400.0 MHz, D2O, 25 °C): 3.60 (dt, 1H, Jgem = 13.6, J2′b,3′ = JH,P = 2.1,
H-2′b-cis), 3.84 (ddd, 1H, Jgem = 13.6, J2′a,3′ = 5.2, JH,P = 1.8, H-2′a-cis),
3.97 (ddd, 1H, Jgem = 12.8, J5′b,4′ = 8.4, JH,P = 1.9, H-5′b-trans), 4.09
(ddd, 1H, Jgem = 12.8, J5′a,4′ = 8.2, JH,P = 1.7, H-5′a-trans), 4.17 (d, 2H,
J2′,3′ = 3.9, H-2′-trans), 4.31 (dd, 1H, Jgem = 11.5, J5′b,4′ = 9.5, H-5′b-cis),
4.60−4.69 (m, 3H, H-3′-cis+trans, H-5′a-cis), 5.02 (ddd, 1H, J4′,5′ = 9.5,
7.7, J4′,3′ = 4.3, H-4′-cis), 5.07 (ddd, 1H, J4′,5′ = 8.4, 8.2, J4′,3′ = 4.3, H-4′-
trans), 7.96, 7.97 (2 × s, 2 × 1H, H-8-cis+trans). 13C NMR (125.7
MHz, D2O, 25 °C): 49.00 (d, JC,P = 4, CH2-5′-trans), 50.34 (CH2-5′-
cis), 54.15 (d, JC,P = 4, CH2-2′-cis), 55.75 (CH2-2′-trans), 57.18 (CH-4′-
trans), 58.69 (CH-4′-cis), 70.53 (CH-3′-cis), 72.54 (CH-3′-trans),
118.44, 118.47 (C-5-cis+trans), 141.40, 141.47 (CH-8-cis+trans),
154.78 (C-4-cis+trans), 154.89 (C-2-cis+trans), 156.56 (C-6-cis+trans),
181.54 (d, JC,P = 189, CO-cis+trans). 31P{1H} NMR (162.0 MHz,
D2O, 25 °C): −1.63, −1.75. HR-ESI C10H12O6N6P [M − H]− calcd
343.0561, found 343.0565. [α]20= +47.4 (c 0.177, H2O). Elem. Anal.:
P, found 7.64% (MW 405.4).
d6, 80 °C): 1.41 (s, 9H, (CH3)3C)), 2.95 (dd, 1H, Jgem = 10.7, J5b,4
=
3.5, H-5b), 3.06 (dd, 1H, Jgem = 11.2, J2b,3 = 3.1, H-2b), 3.12 (dt, 1H,
J4,5 = 5.9, 3.5, J4,3 = 3.5, H-4), 3.42 (dd, 1H, Jgem = 10.7, J5a,4 = 5.9,
H-5a), 3.47 (dd, 1H, Jgem = 11.2, J2a,3 = 5.1, H-2a), 3.75 (ddd, 1H, J3,2
=
5.1, 3.1, J3,4 = 3.5, H-3), 4.74 (bs, 1H, OH). 13C NMR (125.7 MHz,
DMSO-d6, 80 °C): 28.10 ((CH3)3C), 51.55 (CH2-2), 51.70 (CH2-5),
56.90 (CH-4), 75.30 (CH-3), 77.80 (C(CH3)3), 153.79 (CO). IR νmax
(KBr) 2981 (s), 2942 (m), 2887 (m), 1687 (vs), 1678 (vs, sh), 1478
(m), 1455 (m), 1415 (s), 1394 (s), 1368 (s), 1251 (m), 1162 (s),
1078 (m) cm−1. HR-ESI C9H18O3N2Na [M + Na]+ calcd 225.1210,
found 225.1209.
(3R,4S)-4-Amino-1-N-tert-butyloxycarbonyl-3-hydroxypyrrolidine
(4c). Compound 4c was prepared from mesyl derivative 3c (13.9 g,
23.81 mmol) in an 81% yield (3.92 g, 19.38 mmol) following the same
procedure used to obtain the amino derivative 4a. Compound 4c was
obtained as colorless, viscous oil that solidified to an amorphous solid
on standing in refrigerator. 1H NMR and 13C NMR spectra are
identical to 4a. HR-ESI C9H18O3N2Na [M + Na]+ calcd 225.1210,
found 225.1209.
(3S,4S)-4-(Guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphos-
phonic Acid (6b). Compound 6b was prepared from pyrrolidine
derivative 5b (100 mg, 0.42 mmol) in an 18% yield (30 mg, 0.077
mmol), using general method A, as a white amorphous solid. 1H NMR
(500.0 MHz, D2O, 25 °C): 3.50 (ddd, 1H, Jgem = 13.3, J2′b,3′ = 4.2,
JH,P = 1.1, H-2′b-cis), 3.82 (ddd, 1H, Jgem = 13.3, J2′a,3′ = 6.2, JH,P = 1.2,
H-2′a-cis), 3.92 (m, 2H, H-2′b,5′b-trans), 4.12 (m, 1H, H-5′a-trans),
(3R,4R)-4-Amino-1-N-Boc-3-hydroxypyrrolidine (4d). Compound
4d was prepared from mesyl derivative 3d (31.22 g, 53.49 mmol) in a
48% yield (5.24 g, 25.91 mmol) following the same procedure as used
to obtain the amino derivative 4a. Compound 4d was obtained as
colorless, viscous oil that solidified to an amorphous solid upon
standing in the refrigerator. 1H NMR and 13C NMR spectra are
identical to 4b. HR-ESI C9H18O3N2Na [M + Na]+ calcd 225.1210,
found 225.1209.
4.36 (dd, 1H, Jgem = 12.5, J2′a,3′ = 5.9, H-2′-trans), 4.46 (dd, 1H, Jgem
=
12.9, J5′b,4′ = 5.4, H-5′b-cis), 4.55 (dd, 1H, Jgem = 12.9, J5′a,4′ = 6.9, H-5′a-
cis), 4.69 (m, 2H, H-3′-cis+trans), 4.85 (m, 2H, H-4′-cis+trans), 7.85,
7.86 (2 × s, 2 × 1H, H-8-cis+trans). 13C NMR (125.7 MHz, D2O, 25 °C):
(3S,4R)-4-(Guanin-9-yl)-3-hydroxypyrrolidine (5a). Compound 5a
was prepared in an overall yield (134 mg, 0.57 mmol) of 15% as an
amorphous solid according to general method B from compound 4a
1
50.13 (d, JC,P = 4.6, CH2-5′-trans), 51.72 (CH2-5′-cis), 52.90 (d, JC,P
=
(0.83 g, 3.77 mmol). H NMR (400.0 MHz, DMSO-d6, 25 °C): 2.79
4.1, CH2-2′-cis), 54.73 (CH2-2′-trans), 60.66 (CH-4′-trans), 62.52
(CH-4′-cis), 73.97 (CH-3′-cis), 75.69 (CH-3′-trans), 118.89, 118.91
(C-5-cis+trans), 140.15, 140.40 (CH-8-cis+trans), 154.53, 154.58 (C-4-
cis+trans), 156.43 (C-6-cis+trans), 161.71, 161.74 (C-2-cis+trans),
181.07, 181.20 (d, JC,P = 190.0, CO-cis+trans). 31P{1H} NMR (202.3
MHz, D2O, 25 °C): −1.84, −1.82. IR νmax(KBr) 3414 (m, vbr), 3126
(m, vbr), 1696 (vs), 1587 (s, br), 1537 (m), 1184 (m), 1085 (m)
cm−1. HR-ESI C10H12O6N6P [M − H]− calcd 343.05614, found
343.05606. [α]20= −25.5 (c 0.247, H2O). Elem. Anal.: P, found 7.61%
(MW 407.0).
(dd, 1H, Jgem = 11.9, J2′b,3′ = 2.9, H-2′b), 3.10 (dd, 1H, Jgem = 11.0, J5′b,4′
8.9, H-5′b), 3.15−3.22 (m, 2H, H-2′a,5′a), 4.14 (td, 1H, J3′,4′ = J3′,2′a
=
=
5.6, J3′,2′b = 2.9, H-3′), 4.56 (td, 1H, J4′,5′ = 8.9, J4′,3′ = 5.6, H-4′), 5.12
(bs, 1H, OH), 6.43 (bs, 2H, NH2), 7.69 (s, 1H, H-8). 13C NMR
(100.6 MHz, DMSO-d6, 25 °C): 48.94 (CH2-5′), 54.19 (CH2-2′),
56.74 (CH-4′), 69.61 (CH-3′), 116.17 (C-5), 137.34 (CH-8), 151.65
(C-4), 153.54 (C-2), 157.06 (C-6). IR νmax(KBr) 3373 (m, vbr), 3125
(m, vbr), 1694 (vs), 1680 (vs), 1639 (s, sh), 1612 (m), 1584 (m, sh),
1568 (m, sh), 1538 (m), 1477 (m), 1433 (m), 1420 (m, sh), 1384
(m), 1365 (m) cm−1. HR-ESI C9H11O2N6 [M − H]− calcd 235.09490,
found 235.09491. [α]20= +69.2 (c 0.416, DMSO).
(3R,4S)-4-(Guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphos-
phonic Acid (6c). Compound 6c was prepared from pyrrolidine
derivative 5c (40 mg, 0.17 mmol) in a 28% yield (18 mg, 0.05 mmol)
(3S,4S)-4-(Guanin-9-yl)-3-hydroxypyrrolidine (5b). Compound 5b
was prepared from the amino derivative 4b (0.8 g, 3.96 mmol) in a
25% overall yield (237 mg, 1 mmol) as an amorphous solid according
as a white amorphous solid using general method A. 1H NMR and 13
C
NMR are identical to 6a HR-ESI C10H12O6N6P [M − H]− calcd
343.05614, found 343.05617. [α]20= −46.5 (c 0.256, H2O). Elem.
Anal.: P, found 7.69% (MW 402.8).
1
to general method B. H NMR (400.0 MHz, DMSO-d6, 25 °C): 2.72
(dd, 1H, Jgem = 11.6, J2′b,3′ = 4.4, H-2′b), 2.96 (dd, 1H, Jgem = 11.8,
J5′b,4′ = 4.9, H-5′b), 3.24 (dd, 1H, Jgem = 11.6, J2′a,3′ = 6.1, H-2′a), 3.31
(dd, 1H, Jgem = 11.8, J5′a,4′ = 7.3, H-5′a), 4.32 (ddd, 1H, J3′,2′ = 6.1, 4.4,
J3′,4′ = 3.5, H-3′), 4.52 (ddd, 1H, J4′,5′ = 7.3, 4.9, J4′,3′ = 3.5, H-4′), 5.84
(bs, 2H, NH2), 7.75 (s, 1H, H-8). 13C NMR (100.6 MHz, DMSO-d6,
25 °C): 50.93 (CH2-5′), 53.92 (CH2-2′), 62.55 (CH-4′), 76.57 (CH-3′),
114.01 (C-5), 135.24 (CH-8), 152.75 (C-4), 154.96 (C-6), 159.46 (C-2).
HR-ESI C9H13O2N6 [M + H]+ calcd 237.10945, found 237.10942.
[α]20= +29.1 (c 0.326, H2O).
(3R,4R)- 4-(Guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonyl-
phosphonic Acid (6d). Compound 6d was prepared from pyrrolidine
derivative 5d (150 mg, 0.635 mmol) in an 11% yield (25.7 mg, 0.07
mmol) using general method A, as a white amorphous hygroscopic
1
solid. H NMR and 13C NMR spectra are identical to 6b. HR-ESI
C10H12O6N6P [M − H]− calcd 343.05614, found 343.05624. [α]20= +21.7
(c 0.240, H2O). Elem. Anal.: P, found 6.77% (MW 457.515).
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Journal of Medicinal Chemistry
Article
Biochemistry. The instruments used to perform enzyme
purification were the following: (a) a Beckman Coulter Allegra X-22R
centrifuge, (b) a Beckman Coulter Optima L-100 XP ultracentrifuge,
and (c) a Cell ultrasound desintegrator Soniprep 150 (Sanyo). Spectro-
photometric enzyme assays were performed on an Infinite F500 Tecan
reader. Protease inhibitor cocktail was obtained from Sigma, and 7-
methyl-6-thioguanosine (MESG) was purchased from Berry &
Associates. HiTrap Q ion exchange columns (1 mL volume) were
obtained from GE Healthcare Life Sciences.
Infinite F500 Tecan reader. Initial steady-state rates were cal-
culated from a linear part of the time-dependent absorbance
changes over a wide range of MESG concentrations at a constant
concentration of inorganic phosphate. The data obtained for
partially purified PNP from various sources fit the Michaelis−
Menten equation. The GraphPad Prism433 software was used to
calculate a Km and Vmax
.
Inhibition Study. Compounds 2a, 2b, and 6a−d were tested for
their inhibition activity against PNP with respect to MESG and
inorganic phosphate substrates. The reaction mixture (200 μL) for
each assay contained 50 mM Tris buffer (pH 7.5), MESG (40 or 80 μM),
potassium phosphate (500 or 250 μM), and the inhibitor (0−500 nM).
Phosphorolysis was initiated by the addition of the appropriate
amount of enzyme sample (2−5 uL; approximately 3 μg) and
monitored for 5−7 min at 360 nm. The type of inhibition and Ki
values were determined from Dixon plots (1/v versus [I]) using
SigmaPlot software. Ki values were calculated from three independent
measurements.
Time-Dependent Inhibition. Human recombinant enzyme (0.002
units, 2 μL of stock solution in 20 mM Tris buffer in 50% aqueous
glycerol, pH 7.5) was preincubated with various concentrations of
inhibitor 6a (0, 0.1, 10, 25, 50, 100, and 250 nM) in 50 mM Tris buffer
(pH 7.5, 445 μL) at 20 °C for 0, 10, and 20 min. Then 50 mM
potassium dihydrogenphosphate (5 μL) followed by 110 μM MSEG
(100 μL) in Tris buffer were added to final concentrations of 500 and
20 μM, respectively, (total volume 550 μL).
Normal peripheral blood mononuclear cells (PBMCs) were isolated
from the buffy coats of healthy donors who signed informed consent
forms. Primary chronic myeloid leukemia (CML) and acute myeloid
leukemia (AML) cells were obtained from patients who underwent
cytapheresis therapy, all of whom signed an informed consent form.
The cells were provided by the Institute of Hematology and Blood
Transfusion in Prague. Cell lines and cell line xenografts were provided
by the Institute of Pathological Physiology, First Faculty of Medicine,
Charles University, in Prague. The following cell lines were used in the
study: K562 (erythroleukemia progressed from chronic myeloid leukemia),
MC-116 (B-cell lymphoma), JURKAT (acute T-cell lymphoblastic
leukemia), DoHH2 (diffuse large B-cell lymphoma progressed from
follicular lymphoma), MINO (mantle cell lymphoma), HBL2 (mantle
cell lymphoma), SU-DHL-1 (anaplastic large T-cell lymphoma),
OPM-2 (multiple myeloma), and RPMI-8226 (multiple myeloma).
Dry pellets were obtained either from in vitro cultures of the
aforementioned cell lines or ex vivo from murine xenografts. The
xenografts were derived using the following procedure: 106 cells
were subcutaneously injected into the right flank of 6−8 week old
immunodeficient mice NOD-SCID; tumors were allowed to grow
subcutaneously until they reached 2−3 cm in any diameter; the
mice were sacrificed; tumors were excised and passed through
45 μM nylon mesh; and the cells were counted and frozen in the
form of dry pellets. Human recombinant purine nucleoside phos-
phorylase was kindly provided by Dr. Larissa Balakireva (NovoCIB,
Lyon, France).
Partial Purification of Purine Nucleoside Phosphorylase (PNP).
All steps were carried out at 4 °C. A suspension of human PBMCs
from healthy donors was centrifuged at 4000g, the sedimented cells
were resuspended in buffer A (pH 7.5 20 mM TRIS-citric acid, 135
mM sodium chloride, 2.5 mM EDTA, 5.5 mM glucose, 100 mL) and
washed twice with the same buffer. The washed cells were stored at
−80 °C. Cells (109) were suspended in 50 mM Tris-HCl buffer pH 7.5
(15 mL) containing a protease inhibitor cocktail (0.1 mL), disrupted
by freezing and thawing, and the suspension was subsequently soni-
cated three times for 10 s. The crude lysate was centrifuged at 104g for
30 min to remove cell debris and then at 105g for 1 h. The resulting
supernatant (15 mL) was brought to 60% saturation by adding solid
ammonium sulfate (5.5 g). After 1 h, the precipitate was collected by
centrifugation at 104g for 40 min and dissolved in pH 7.5 50 mM Tris-
HCl buffer (10 mL), and the solution was dialyzed against the same
buffer (2 L) overnight. The desalted solution was loaded on a HiTrap
Q column equilibrated with 50 mM Tris-HCl buffer (pH 7.5; 20 mL).
The column was eluted with a linear gradient (0−1 M) of NaCl in the
same buffer at a flow rate of 1 mL/min. Fractions (1 mL, 20 fractions
totally) were collected and analyzed for PNP activity. PNP activity was
detected only in one fraction (at approximately 200 mM NaCl), which
was used immediately for assays. Protein concentration was
determined by the method of Bradford38 using bovine serum albumin
as a standard.
After 2 and 5 min of incubation at 25 °C, aliquots (100 μL) of each
mixture were removed and quenched with 10 μL of 20% HCl. The
extent of MESG phosphorolysis was subsequently analyzed using
HPLC (Luna C18 3 μm, 100 mm × 4.6 mm).
The enzyme activity is expressed as the remaining activity related to
assays without inhibitor. See Figure 3 and Figure S13 in Supporting
Information for these results.
ASSOCIATED CONTENT
* Supporting Information
■
S
Graphical data used to determine the Ki and Km values. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For I.R.: phone, +420 220 183 381; E-mail, rosenberg@uochb.
cas.cz. For D.R.: phone, +420 220 183 371; E-mail, rejman@
uochb.cas.cz. For P.K.: phone, +420 608 177 782; E-mail,
pklener@yahoo.com.
ACKNOWLEDGMENTS
■
Support by grants 2B06065, MSM 0021620806, MSM
0021620808, SVV-2010-254260507, and Research Centre
LC06077 (Ministry of Education, CR) and Research Centre
KAN200520801 (Academy of Sciences, CR) under research project
Z40550506 is gratefully acknowledged. The authors are indebted to
Larissa Balakireva, PhD, President, Femme en Or 2011 - Femme
d′Innovation, NovoCIB, France for provision of sample of the
human recombinant purine nucleoside phosphorylase.
PNPs from cancer cell lines and blood cells of patients suffering
from chronic myeloid leukemia (CML) and acute myeloid leukemia
(AML) were purified using the same method described above (Table 1).
Determination of PNP Activity. To determine PNP activity, we
employed a spectrophotometric method described by Webb et al34
that uses 7-N-methyl-6-thiopurineriboside (MESG) as a substrate. The
incubation mixture (200 μL) contained 50 mM Tris-HCl buffer (pH 7.5),
500 μM potassium phosphate, 20−100 μM MSEG, and a partially
purified PNP. Enzyme phosphorolytic activity was calculated from
changes in absorbance at 360 nm using ε360 nm = 11000 M−1 cm−1.
Assays were performed in 96-well plates and monitored using the
ABBREVIATIONS USED
■
ACVDP, acyclovir diphosphate; AML, acute myeloid leukemia;
B-ALL, B-cell acute lymphoblastic leukemia; B-CLL, B-cell
chronic lymphocytic leukemia; CML, chronic myeloid
leukemia; dCK, deoxycytidine kinase; DFPP-G, 9-(5′,5′-
difluoro-5′-phosphonopentyl)guanine; dGuo, 2′-deoxyguano-
sine; DoHH2, diffuse large B-cell lymphoma progressed from
follicular lymphoma; HBL2 , mantle cell lymphoma (culture =
in vitro cultured cells, tumor = ex vivo obtained cells grown in
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(16) Bantia, S.; Ananth, S. L.; Parker, C. D.; Horn, L. L.; Upshaw, R.
Mechanism of inhibition of T-acute lymphoblastic leukemia cells by
PNP inhibitor BCX-1777. Int. Immunopharmacol. 2003, 3, 879−887.
(17) BioCryst Pharmaceuticals, Inc.; BioCryst Pharmaceuticals, Inc.:
Durham, NC, 2012; www.biocryst.com (Accessed September 2011).
(18) Alonso, R.; Lopez-Guerra, M.; Upshaw, R.; Bantia, S.; Smal, C.;
Bontemps, F.; Manz, C.; Mehrling, T.; Villamor, N.; Campo, E.;
Montserrat, E.; Colomer, D. Forodesine has high antitumor activity in
chronic lymphocytic leukemia and activates p53-independent
mitochondrial apoptosis by induction of p73 and BIM. Blood 2009,
114, 1563−1575.
(19) Evans, G. B.; Furneaux, R. H.; Lewandowicz, A; Schramm, V. L.;
Tyler, P. C. Synthesis of second-generation transition state analogues
of human purine nucleoside phosphorylase. J. Med. Chem. 2003, 46,
5271−5276 and references therein.
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metabolites on purine nucleoside phosphorylase. J. Biol. Chem. 1993,
259, 4085−4069.
(21) Gilbertsen, R. B.; Sircar, J. C. Enzyme Cascades: Purine
metabolism and immunosuppression. In Comprehensive Medicinal
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form of subcutaneous tumors in immunodeficient mice);
hrPNP, human recombinant purine nucleoside phosphorylase;
IMG, immucillin-G; IMH, immucillin-H; JURKAT, T-cell
leukemia; K-562, erythroleukemia progressed from chronic
myeloid leukemia; kcat, catalytic constant; Ki, inhibition
constant; PiKi, inhibition constant related to inorganic
phosphate; Km, Michaelis constant; MESGKi, inhibition constant
related to MESG; MESGKm, Michaelis constant for MESG; PiKm,
Michaelis constant for Pi; MC-116, B-cell lymphoma; MESG, 7-
methyl-6-thioguanosine; MINO, mantle cell lymphoma; OPM-
2, multiple myeloma; PBMCs, peripheral blood mononuclear
cells; Pi, inorganic phosphate; PNP, purine nucleoside
phosphorylase; RPMI 8226, multiple myeloma; SU-DHL-1,
anaplastic large cell lymphoma; V, reaction velocity
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