Cell-permeable esters of diazeniumdiolate-based nitric oxide prodrugs

Article abstract of DOI:10.1021/ol8020989

(Chemical Equation Presented) Although 0²-(2,4-dinitrophenyl) derivatives of diazeniumdiolate-based nitric oxide (NO) prodrugs bearing a free carboxylic acid group were activated by glutathione to release NO, these compounds were poor sources of intracellular NO and showed diminished antiproliferative activity against human leukemia HL-60 cells. The carboxylic acid esters of these prodrugs, however, were found to be superior sources of intracellular NO and potent inhibitors of HL-60 cell proliferation.

Full text of DOI:10.1021/ol8020989

ORGANIC
LETTERS
2008
Vol. 10, No. 22
5155-5158
Cell-Permeable Esters of
Diazeniumdiolate-Based Nitric Oxide
Prodrugs
Harinath Chakrapani,*,† Anna E. Maciag,‡ Michael L. Citro,‡ Larry K. Keefer,† and
Joseph E. Saavedra*,‡
Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, National Cancer
Institute at Frederick, Frederick, Maryland 21702, and Basic Research Program,
SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702
chakrah@ncifcrf.goV; saaVj@ncifcrf.goV
Received September 8, 2008
ABSTRACT
Although O2-(2,4-dinitrophenyl) derivatives of diazeniumdiolate-based nitric oxide (NO) prodrugs bearing a free carboxylic acid group were
activated by glutathione to release NO, these compounds were poor sources of intracellular NO and showed diminished antiproliferative
activity against human leukemia HL-60 cells. The carboxylic acid esters of these prodrugs, however, were found to be superior sources of
intracellular NO and potent inhibitors of HL-60 cell proliferation.
Nitric oxide (NO) donors of the diazeniumdiolate class are
routinely used as sources of nitric oxide for chemical and
biological applications.1 For example, the 1-[2-(carboxy-
lato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO)
anion dissociates in pH 7.4 phosphate buffer to form nitric
oxide with a half-life of 2 s (Scheme 1).2
† Laboratory of Comparative Carcinogenesis.
‡ SAIC-Frederick.
Scheme 1. Dissociation of PROLI/NO to Form NO
(1) (a) Scatena, R.; Bottoni, P.; Martorana, G. E.; Giardina, B. Expert
Opin. InVestig. Drugs 2005, 14, 835–846. (b) Keefer, L. K. Curr. Top. Med.
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(2) (a) Saavedra, J. E.; Southan, G. J.; Davies, K. M.; Lundell, A.;
Markou, C.; Hanson, S. R.; Adrie, C.; Hurford, W. E.; Zapol, W. M.; Keefer,
L. K. J. Med. Chem. 1996, 39, 4361–4365. (b) Chen, C.; Hanson, S. R.;
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H. C.; Bivalacqua, T. J.; Wang, R.; Kadowitz, P. J.; Keefer, L. K.; Saavedra,
J. E.; Hrabie, J. A.; Doherty, P. C.; Hellstrom, W. J. G. J. Urol. 1999, 161,
2013–2019. (d) Bivalacqua, T. J.; Champion, H. C.; De Witt, B. J.; Saavedra,
J. E.; Hrabie, J. A.; Keefer, L. K.; Kadowitz, P. J. J. CardioVasc. Pharmacol.
2001, 38, 120–129. (e) Waterhouse, D. J.; Saavedra, J. E.; Davies, K. M.;
Citro, M. L.; Xu, X.; Powell, D. A.; Grimes, G. J.; Potti, G. K.; Keefer,
L. K. J. Pharm. Sci. 2006, 95, 108–115.
Under aerobic conditions, one of the possible byproducts
of PROLI/NO decomposition is N-nitrosoproline (NPRO,
Scheme 2).2e
In contrast to most other N-nitrosamines,3 which are potent
carcinogens, NPRO showed no carcinogenic activity at a
10.1021/ol8020989 CCC: $40.75
Published on Web 10/29/2008
2008 American Chemical Society

nitric oxide.5 For example, O2-(2,4-dinitrophenyl) diazeni-
umdiolates are reported to be activated by glutathione (GSH)
to form NO (Scheme 3).6 Glutathione is an essential
Scheme 2. Proposed Mechanism for the Formation of NPRO
from the Decomposition of PROLI/NO under Aerobic
Conditions
Scheme 3. Glutathione-Activated Nitric Oxide Prodrugs
range of dosages in numerous animal models.2e,4 For
example, N-nitrosodiethylamine was shown to induce tumors
in a rat model in different organs and sites (Table 1, entry
2).3b
component of the biochemical machinery, and its intracellular
distribution ranges from 0.1-10 mM.7
Earlier, we prepared the O2-(2,4-dinitrophenyl) derivative
of PROLI/NO, 2, from diazeniumdiolate salt 1 in two steps
(Scheme 4).8
Table 1. N-Nitrosamine-Induced Carcinogenesis in a Rat
Modela
% of treated animals
with tumors (organ
entry
N-nitroso
developing tumors)
Scheme 4. Synthesis of 2 from 1
1
2
3
4
5
6
proline (NPRO)b
diethylaminec
pyrrolidined
0
95 (esophagus), 65 (liver)
100 (liver)
100 (nasal), 67 (esophagus), 27 (liver)
piperidined
pipecolic acidd
0
0
isonipecotic acidd
a Administrated through drinking water; ref 3b. b Total dose was 100
mmol/animal. c Total dose was 1.0 mmol/animal. d Total dose was 3.9-4.6
mmol/animal.
However, even at 100-fold higher doses than those of
N-nitrosodiethylamine, NPRO showed no evidence of tumor
formation (Table 1, entry 1).3b Not just NPRO but other
N-nitrosamines with a carboxylic acid group were reported
to show no carcinogenic activity in a rodent model (Table
1, entries 5 and 6).3b Thus, the use of diazeniumdiolate anions
with a carboxylic acid functionality (such as PROLI/NO)
would be preferable in clinical settings due to the formation
of relatively innocuous byproducts.
Using a similar procedure, carboxylic acids 39 and 4 were
prepared (Figure 1).
O2-Derivatization of diazeniumdiolate anions using a
suitable protective group facilitates site-directed delivery of
(3) (a) Lijinsky, W. Chemistry and Biology of N-Nitroso Compounds;
Cambridge University Press: Cambridge, 1992. (b) Lijinsky, W. Cancer
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103–121. (g) Lijinsky, W.; Reuber, M. D.; Riggs, C. W. Cancer Res. 1981,
Figure 1. Compounds 3 and 4.
A chemiluminescence assay was used to study glutathione-
activated nitric oxide formation in aqueous buffer (Table 2).
Next, the intracellular NO release by these compounds was
determined using the nitric oxide-sensitive fluorophore,
4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate
(DAF-FM diacetate);10 briefly, human leukemia HL-60 cells
were preloaded with DAF-FM diacetate, followed by treat-
ment with DMSO solutions of 2, 3, and 4, and fluorescence
41, 4997–5003
.
(4) (a) Brunnemann, K. D.; Enzmann, H. G.; Perrone, C. E.; Iatropoulos,
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.
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Org. Lett., Vol. 10, No. 22, 2008

measurements after 40 min provided estimates of levels of
intracellular NO (Table 2).
nonionizable species, an ester should be able to cross the
cell membrane.12 Subsequent intracellular ester hydrolysis
and glutathione activation should generate the spontaneously
nitric oxide-forming diazeniumdiolate anion, which upon
decomposition would generate a secondary amine linked to
a carboxylic acid such as L-proline (Figure 2).
Table 2. Nitric Oxide Release, Fluorescence Measurements, and
in Vitro Anti-Proliferative Activity
nitric oxide fluorescencec (
relative
IC50
compd yield (%)a
S. D.d (a.u.)
fluorescence (%) (µM)e
DMSO
0
7.7 ( 0.25
9.5 ( 0.13
7.9 ( 0.09
7.4 ( 0.18
17.0 ( 0.49
28.9 ( 1.26
17.0 ( 0.49
24.3 ( 0.85
26.7 ( 0.85
24.9 ( 0.70
100
124
103
96
221
375
221
315
346
324
-
2
100
87b
96
>20f
9.6g
>20
5.4
2.7
6.0
3.4
4.8
1.4
3
4
5
98
6
100b
100
100
100
99
7
8
9
10
a Nitric oxide yields from the decomposition of the compound (50-100
µL of a 0.1 mM DMSO solution) in the presence of GSH (3.6 mM) in 0.1
M phosphate buffer (3.5 mL) containing 50 µM diethylenetriamine
pentaaceticacid(DTPA)atpH7.4and37°Casmeasuredbychemiluminescence.
b Calculated based on 4 moles of NO per mole of compound. c Mean
intracellular NO release measured using the NO-sensitive DAF-FM diacetate
dye in HL-60 cells measured in arbitrary units (a.u.). d Standard deviations
of fluorescence measurements (three independent experiments). e The 50%
inhibitory concentrations are reported for activity against proliferation of
HL-60 cells. f Ref 11. g Ref 9.
Figure 2. Design of cell-permeable nitric oxide prodrugs.
Accordingly, 5, the methyl ester of 2, was prepared by
treating the carboxylic acid with diazomethane (Scheme 5).
While they were excellent sources of nitric oxide in the
presence of glutathione in aqueous phosphate buffer, the
carboxylic acids 2, 3,9 and 4 did not form significantly higher
levels of intracellular NO than the DMSO control (Table
2). These intracellular NO release observations are consistent
with their diminished ability to inhibit in vitro proliferation
of human leukemia HL-60 cells (Table 2).9,11
Scheme 5. Synthesis of 5 by Methylation of 2
To improve cell permeability, it was envisaged that a free
carboxylic acid group be masked as an ester. As a neutral,
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Using a similar procedure, compounds 6 and 7 were prepared
from 3 and 4, respectively (Figure 3). Next, diazeniumdi-
olation of the requisite secondary amines, followed by
arylation, produced esters of isonipecotic acid, 8 and 9, and
nipecotic acid, 10 (Figure 3).
Glutathione-activated nitric oxide release for these esters
was determined. Quantitative nitric oxide yields from a
majority of the prodrug esters were observed (Table 2). Then,
these esters were tested for their ability to deliver nitric oxide
intracellularly using the DAF-FM diacetate assay. All the
esters were found to release much higher levels of intracel-
lular NO than their carboxylic acid counterparts (Table 2).
Under these conditions, the prodrug 6, which released 4
moles of NO in the presence of GSH, produced the most
intracellular nitric oxide. A plot of the relative levels of
intracellular NO shows that esters 8, 9, and 10 formed nearly
3-fold higher levels of NO than 2 (Figure 4).
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V.; Malaviya, S.; Carr, B. I.; Kar, S.; Wang, M.; Jia, L.; Ji, X.; Keefer,
L. K. J. Med. Chem. 2006, 49, 4356–4366. (b) Chakrapani, H.; Wilde, T. C.;
Citro, M. L.; Goodblatt, M. M.; Keefer, L. K.; Saavedra, J. E. Bioorg. Med.
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Finally, the ability of these compounds to inhibit prolifera-
tion of human leukemia HL-60 cells was determined.
2003, 4, 461–485
.
Org. Lett., Vol. 10, No. 22, 2008
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Figure 4. Relative levels of intracellular nitric oxide formation upon
treatment of HL-60 cells with compounds 2-10 (5 µM DMSO
solutions) and DMSO (control) as determined by DAF-FM diacetate
fluorescence study.
permeability of O2-(2,4-dinitrophenyl) diazeniumdiolates to
release nitric oxide intracellularly appears to be a crucial
determinant of inhibitory potential.
Figure 3. Compounds 6, 7, 8, 9, and 10.
Acknowledgment. This research was supported in part
by the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research, as well as by
National Cancer Institute contract N01-CO-12400 to SAIC-
Frederick.
Esterification of the carboxylic acids 2-4 significantly
improved in vitro antiproliferative activity of the resulting
esters 5-7 against HL-60 human leukemia cells (Table 2).
For example, the PROLI/NO ester analogues 5 and 6 were
superior inhibitors of HL-60 cell proliferation relative to their
carboxylic acid counterparts 2 and 3 (Table 2). All the other
esters (7-10) showed excellent antiproliferative activity that
was consistent with elevated levels of intracellular nitric
oxide. While other mechanisms might be operational, cell
Supporting Information Available: Preparative and cell
culture procedures, analytical data, and NMR spectra for new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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