V-PROU/NO, a prodrug of the nitric oxide donor, PROLI/NO

Article abstract of DOI:10.1021/ol701419a

The sensitivity to decomposition of the nitric oxide (NO) donor ion, 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), complicates direct electrophilic substitution to form useful prodrug derivatives. A modified general synthetic approach involving 1-[2-(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate ion (structure A, above) was used to prepare several PROLI/NO prodrugs including the previously Inaccessible O²vinyl derivative, V-PROLl/NO. Metabolism of V-PROLI/NO by liver microsomes enriched in human cytochrome P450 isoforms was demonstrated.

Full text of DOI:10.1021/ol701419a

ORGANIC
LETTERS
2007
Vol. 9, No. 17
3409-3412
V-PROLI/NO, a Prodrug of the Nitric
Oxide Donor, PROLI/NO
Harinath Chakrapani,† Brett M. Showalter,† Li Kong,† Larry K. Keefer,† and
Joseph E. Saavedra*,‡
Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, and Basic Research
Program, SAIC Frederick, National Cancer Institute at Frederick,
Frederick, Maryland 21702
saaVj@ncifcrf.goV
Received June 18, 2007
ABSTRACT
The sensitivity to decomposition of the nitric oxide (NO) donor ion, 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO),
complicates direct electrophilic substitution to form useful prodrug derivatives. modified general synthetic approach involving
A
1-[2-(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate ion (structure A, above) was used to prepare several PROLI/NO prodrugs including
the previously inaccessible O2-vinyl derivative, V-PROLI/NO. Metabolism of V-PROLI/NO by liver microsomes enriched in human cytochrome
P450 isoforms was demonstrated.
Secondary diazeniumdiolate ions (general structure at the
center of Scheme 1) are routinely used as reliable sources
a few seconds to several hours to suit the duration of the
experiment. Localization of therapeutic effects and site-
directed delivery of NO are highly desirable in clinical
settings and broadly, two strategies are employed (Scheme
1). An example of the first approach is incorporation of the
diazeniumdiolate into insoluble polymeric material, then
placing the NO donor in close proximity to the target tissue
(path a, Scheme 1).1c,2 When noninvasive access to certain
tissues or cell types is not possible, an alternate approach is
to protect the O2-position of the diazeniumdiolate (path b,
Scheme 1) with groups that can be selectively removed under
specific conditions.1b,d,3 Nitric oxide release from such
Scheme 1. Strategies for Site-Specific Delivery of NOa
a The solid sphere ) polymer, microparticle, etc.; R, R1 ) alkyl
groups; R2 ) protective group. Path a yields a material that
concentrates NO release at its surfaces; path b depicts conversion
of the starting ion into a neutral prodrug form that can circulate
freely in the blood stream but (path c) be metabolically converted
to the spontaneously NO-generating ionic form in the target organ.
(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. Chem. 2005, 5, 625-36. (c) Frost, M. C.; Reynolds, M. M.; Meyerhoff,
M. E. Biomaterials 2005, 26, 1685-1693. (d) Pavlos, C. M.; Xu, H.;
Toscano, J. P. Free Radical Biol. Med. 2004, 37, 745-752. (e) Thatcher,
G. R. Curr. Top. Med. Chem. 2005, 5, 597-601. (f) Hrabie, J. A.; Keefer,
L. K. Chem. ReV. 2002, 102, 1135-1154.
(2) (a) Stasko, N. A.; Schoenfisch, M. H. J. Am. Chem. Soc. 2006, 128,
8265-8271. (b) Smith, D. J.; Chakravarthy, D.; Pulfer, S.; Simmons, M.
L.; Hrabie, J. A.; Citro, M. L.; Saavedra, J. E.; Davies, K. M.; Hutsell, T.
C.; Mooradian, D. L.; Hanson, S. R.; Keefer, L. K. J. Med. Chem. 1996,
39, 1148-1156.
(3) Saavedra, J. E.; Dunams, T. M.; Flippen-Anderson, J. L.; Keefer, L.
K. J. Org. Chem. 1992, 57, 6134-6138.
of nitric oxide (NO) in chemical and biological studies.1 Such
NO donors have well-defined half-lives for NO release from
† Chemistry Section, Laboratory of Comparative Carcinogenesis.
‡ Basic Research Program, SAIC Frederick.
10.1021/ol701419a CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/20/2007

prodrugs will ideally not occur until the substituent at the
O2-position is cleaved in the target tissue to generate the
free ionic diazeniumdiolate (path c, Scheme 1). A specific
metabolic trigger resulting in selective removal of the O2-
protective group ensures site-directed delivery of therapeutic
NO.
Scheme 2. Synthesis of 2 and Attempted Synthesis of 6
of V-PYRRO/NO in good yield4a failed completely when
attempted with PROLI/NO (Scheme 2). The reaction of
PROLI/NO in the presence of 15-crown-5 with 2-bromo-1-
(trifluoromethanesulfonyloxy)ethane (5) also failed to pro-
duce the desired intermediate 6, necessitating a revised
strategy for the synthesis of 4 and other O2-derivatized
PROLI/NO prodrug forms.
Figure 1. PYRRO/NO, PROLI/NO, and their prodrug forms.
An agent designed for this latter approach is the O2-vinyl
derivative of PYRRO/NO (1), V-PYRRO/NO (2), which is
a NO prodrug that was shown in relevant animal models to
protect the liver in a variety of life-threatening situations,
including the ischemia-reperfusion injury associated with
organ transplantation and the toxicity of acetaminophen and
other agents (Figure 1).4
We postulated that, by diazeniumdiolating prolinol (7)
instead of proline (Scheme 3) to produce 8 as a starting
It would be desirable to modify its structure to improve
water solubility for formulation and site-directed delivery
while retaining the beneficial effects of rapid liver metabo-
lism to NO-releasing form. The diazeniumdiolate of L-
proline, PROLI/NO (3),5 is structurally similar to PYRRO/
NO, but has an additional carboxylic acid that may improve
solubility for its corresponding O2-protected prodrug forms
(Figure 1). Furthermore, this additional carboxyl functionality
serves as a synthetic handle for further derivatization by using
a peptide or polymer that potentially improves efficacy,
bioavailability, and/or site-directed delivery. Finally, the
products of decomposition of PROLI/NO are all naturally
occurring metabolites, suggesting a favorable toxicological
profile.5b Hence, V-PROLI/NO (4) may be an ideal candidate
for liver-specific delivery of therapeutic NO (Figure 2).
Unfortunately, the convenient synthetic strategy of O2-
bromoethylation/dehydrohalogenation used for the synthesis
Scheme 3. Proposed General Synthetic Route to V-PROLI/
NO (4) and other O2-Substituted PROLI/NO Derivatives
material, absence of the carboxyl group might allow substitu-
tion at the O2-position to occur without significant competing
decomposition. Subsequent oxidation of the primary alcohol
functionality might then be used to regenerate the carboxyl
group and produce the desired PROLI/NO derivative (Scheme
3).
As predicted, 4 could be prepared by this route. Treatment
of diazeniumdiolate 8 with 5 in the presence of 15-crown-5
afforded bromoethyl derivative 9 in 48% yield. Mild oxida-
tion with a modified Sharpless protocol (sodium periodate
and ruthenium trichloride)6 led to carboxylate 6 in 19% yield.
Dehydrobromination of 6 by treatment with sodium hydrox-
ide generated V-PROLI/NO in 19% yield (Scheme 4).
To examine the generality of this procedure, we reacted 8
with 2,4-dinitrofluorobenzene (FDNB). This produced 10a
in 54% yield (Table 1, entry 1), an outcome that contrasted
sharply with that seen when FDNB was reacted directly with
3. In the latter case, no combination of reaction conditions
(solvent, temperature, crown ether, or other additives) was
found that generated the desired product, 11a. Instead, the
major products isolated were N-nitrosoproline (12) and
N-(2,4-dinitrophenyl)proline (Scheme 5). Conversion of 10a
(4) (a) Saavedra, J. E.; Billiar, T. R.; Williams, D. L.; Kim, Y.-M.;
Watkins, S. C.; Keefer, L. K. J. Med. Chem. 1997, 40, 1947-1954. (b)
Liu, J.; Li, C.; Waalkes, M. P.; Clark, J.; Myers, P.; Saavedra, J. E.; Keefer,
L. K. Hepatology 2003, 37, 324-333. (c) Qu, W.; Liu, J.; Fuquay, R.;
Shimoda, R.; Sakurai, T.; Saavedra, J. E.; Keefer, L. K.; Waalkes, M. P.
Nitric Oxide Biol. Chem. 2005, 12, 114-120. (d) Inami, K.; Nims, R. W.;
Srinivasan, A.; Citro, M. L.; Saavedra, J. E.; Cederbaum, A. I.; Keefer, L.
K. Nitric Oxide Biol. Chem. 2006, 14, 309-315. (e) Ricciardi, R.; Foley,
D. P.; Quarfordt, S. H.; Saavedra, J. E.; Keefer, L. K.; Wheeler, S. M.;
Donohue, S. E.; Callery, M. P.; Meyers, W. C. Transplantation 2001, 71,
193-198.
(5) (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) 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. (c) Chen, C.;
Hanson, S. R.; Keefer, L. K.; Saavedra, J. E.; Davies, K. M.; Hutsell, T.
C.; Hughes, J. D.; Ku, D. N.; Lumsden, A. B. J. Surg. Res. 1997, 67, 26-
32. (d) Champion, 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. (e) 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.
(6) Prashad, M.; Lu, S.; Kim, H. Y.; Hu, B.; Repic, O.; Blacklock, T. J.
Synth. Commun. 1999, 29, 2937-2942.
3410
Org. Lett., Vol. 9, No. 17, 2007

Scheme 4. Successful Preparation of V-PROLI/NO (4) by the
Table 2. Reaction of PROLI/NO with Some Electrophiles
Procedure Outlined in Scheme 4
entry
electrophile
R
R1
compd
yield, %
1
2
3
Me2SO4
Et2SO4
ClSO2NMe2
Me
H
H
Me
Et
SO2NMe2
13
14
15
18
13
57
further expansion of PROLI/NO-based nitric oxide prodrugs.
Certain O2-2,4-dinitroaryl derivatives of diazeniumdiolates
are potent antitumor agents that are reported to operate
through activation by glutathione-S-transferase, which is
frequently overexpressed in tumor tissue.7 It will be of
interest to explore the anticancer activity of 11a and its
derivatives.
to 11a proceeded satisfactorily in 74% yield (Table 1, entry
3). Similarily, 8 was converted to 10b, which, after extensive
chromatography, was isolated in 10% yield (Table 1, entry
Table 1. Synthesis of Some O2-Substituted Derivatives of 3
entry
R
compd
yield, %
1
2
2,4-dinitrophenyla
10a
10b
54
10
2,3,4,6-tetra-O-acetyl-
â-D-(glucopyranosyl)b
2,4-dinitrophenylc
2,3,4,6-tetra-O-acetyl-
â-D-(glucopyranosyl)c
3
4
11a
11b
74
46
Figure 2. Metabolism of V-PROLI/NO by microsomes enriched
in human cytochrome P450 isoforms 2E1 (A) and 3A4 (B). No
significant decomposition of V-PROLI/NO was observed in the
presence of the NADPH-regenerating system alone during a 90-
min incubation, but the rate of metabolism increased progressively
as the enzyme was added at increasing levels (0.5, 1.0, and 2.0
mg/mL). All solutions contained NADPH-regenerating system (1.3
mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM magnesium
chloride, and 0.4 U/mL glucose-6-phosphate dehydrogenase) plus
100 µM V-PROLI/NO in 0.1 M phosphate buffer (pH 7.4). Samples
were incubated for 90 min at 37 °C.
a FDNB, 5% aq NaHCO3, t-BuOH. b 1-Bromo-2,3,4,6-tetraacetoxyglu-
cose, 5% aq NaHCO3, acetone. c NaIO4, RuCl3 (cat.), MeCN, EtOAc, H2O.
2); subsequent oxidation proceeded without affecting the
acetylated glucosyl group to afford 11b in 46% yield (Table
1, entry 4).
Scheme 5. Attempted Arylation of PROLI/NO
Nitric oxide release from the O2-glucosylated derivative
of PYRRO/NO upon treatment with â-D-glucosidase was
recently demonstrated.8 Here again, the presence of the
carboxyl group may lead to improved functional character-
istics relative to the V-PYRRO/NO-based materials.8
It might be noted that direct reaction of PROLI/NO dianion
with strong electrophiles did in some cases lead to isolable
O2-derivatives in reasonable yield. Thus, dimethyl sulfate
gave dimethylated product 13 in 18% yield, while diethyl
sulfate and N,N-dimethylsulfamoyl chloride led to O2-
substituted PROLI/NO derivatives in yields of 13% and 57%,
respectively (Table 2).
(7) (a) Shami, P. J.; Saavedra, J. E.; Bonifant, C. L.; Chu, J.; Udupi, 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) Saavedra, J. E.; Srinivasan, A.;
Buzard, G. S.; Davies, K. M.; Waterhouse, D. J.; Inami, K.; Wilde, T. C.;
Citro, M. L.; Cuellar, M.; Deschamps, J. R.; Parrish, D.; Shami, P. J.;
Findlay, V. J.; Townsend, D. M.; Tew, K. D.; Singh, S.; Jia, L.; Ji, X.;
Keefer, L. K. J. Med. Chem. 2006, 49, 1157-1164.
(8) (a) Wu, X.; Tang, X.; Xian, M.; Wang, P. G. Tetrahedron Lett. 2001,
42, 3779-3782. (b) Showalter, B. M.; Reynolds, M. M.; Valdez, C. A.;
Saavedra, J. E.; Davies, K. M.; Klose, J. R.; Chmurny, G. N.; Citro, M. L.;
Barchi, J. J., Jr.; Merz, S. I.; Meyerhoff, M. E.; Keefer, L. K. J. Am. Chem.
Soc. 2005, 127, 14188-14189.
The synthetic methodology described in this paper pro-
vides access to several derivatives of PROLI/NO, enabling
Org. Lett., Vol. 9, No. 17, 2007
3411

It seems possible that these PROLI/NO derivatives could
be therapeutically promising. With respect to V-PROLI/NO,
preliminary studies were conducted to determine whether it
can be metabolized by cytochrome P450 isoforms 2E1 and
3A4. Like V-PYRRO/NO, V-PROLI/NO was consumed in
the presence of an NADPH-generating system at rates that
increased with the concentration of enzyme (Figure 2). Future
work will focus on the pharmacological profiles of these
compounds and their analogues.
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
National Cancer Institute contract N01-CO-12400 to SAIC.
Supporting Information Available: Preparative proce-
dures, analytical data for new compounds, and details of the
metabolism study. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL701419A
3412
Org. Lett., Vol. 9, No. 17, 2007