Click reaction in conjunction with diazeniumdiolate chemistry: Developing high-load nitric oxide donors

Article abstract of DOI:10.1021/ol101645k

Figure Presented. The use of Cu(I)-catalyzed click reactions of alkyne-substituted diazeniumdiolate prodrugs with bis- and tetrakis-azido compounds is described. The click reaction for the bis-azide using CuSO₄/Na-ascorbate predominantly gave the expected bis-triazole. However, CuI/diisopropylethylamine predominantly gave uncommon triazolo-triazole products as a result of oxidative coupling. Neither set of click conditions showed evidence of compromising the integrity of the diazeniumdiolate groups. The chemistry developed has applications in the synthesis of polyvalent and dendritic nitric oxide donors.

Full text of DOI:10.1021/ol101645k

ORGANIC
LETTERS
2010
Vol. 12, No. 19
4256-4259
“Click” Reaction in Conjunction with
Diazeniumdiolate Chemistry: Developing
High-Load Nitric Oxide Donors
Oyebola A. Oladeinde,† Sam Y. Hong,† Ryan J. Holland,† Anna E. Maciag,‡
Larry K. Keefer,† Joseph E. Saavedra,*,‡ and Rahul S. Nandurdikar*,†
Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, National Cancer
Institute at Frederick, Frederick, Maryland 21702, and Basic Science Program,
SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702
saaVedjo@mail.nih.goV; nandurdikarr@mail.nih.goV
Received July 15, 2010
ABSTRACT
The use of Cu(I)-catalyzed “click” reactions of alkyne-substituted diazeniumdiolate prodrugs with bis- and tetrakis-azido compounds is described.
The “click” reaction for the bis-azide using CuSO4/Na-ascorbate predominantly gave the expected bis-triazole. However, CuI/diisopropylethylamine
predominantly gave uncommon triazolo-triazole products as a result of oxidative coupling. Neither set of “click” conditions showed evidence
of compromising the integrity of the diazeniumdiolate groups. The chemistry developed has applications in the synthesis of polyvalent and
dendritic nitric oxide donors.
Diazeniumdiolate prodrugs1 are efficient and reliable sources
of nitric oxide (NO), a potent bioregulatory agent.2,3 These
prodrugs on suitable hydrolysis or enzymatic activation
rapidly decompose to release NO at physiological pH.
Targeted NO-releasing prodrugs have potential applications
in treating cancer and other diseases. For example, JS-K (1,
Figure 1) is an anticancer lead compound activated by
gluthathione. JS-K (1) slowed tumor growth in several rodent
models of cancer including leukemia, prostate cancer,
multiple myeloma, and liver cancer.4 Another prodrug,
V-PYRRO/NO (2, Figure 1), is activated by cytochrome
P450 to release NO. It shows hepatoprotective properties
against a variety of toxins in several animal models.5
Glycosidase activated N-acetyl glucosaminylated diazeni-
umdiolate GlcNAc-DEA/NO (3) significantly decreased
Leishmania major parasite burden in infected macrophages.6
† Laboratory of Comparative Carcinogenesis.
‡ SAIC-Frederick.
(1) Hrabie, J. A.; Keefer, L. K. Chem. ReV. 2002, 102, 1135–1154.
(2) (a) Ignarro, L. J. Angew. Chem., Int. Ed. 1999, 38, 1882–1892. (b)
Furchgott, R. F. Angew. Chem., Int. Ed. 1999, 38, 1870–1880. (c) Moncada,
S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol. ReV. 1991, 43, 109–142
.
(3) (a) Miller, M. R.; Megson, I. L. Br. J. Pharmacol. 2007, 151, 305–
321. (b) Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk,
A. J. Chem. ReV. 2002, 102, 1091–1134. (c) Megson, I. L.; Webb, D. J.
(4) Maciag, A. E.; Saavedra, J. E.; Chakrapani, H. Anticancer Agents
Med. Chem. 2009, 9, 798–803.
Expert Opin. InVestig. Drugs 2002, 11, 587–601
.
10.1021/ol101645k  2010 American Chemical Society
Published on Web 09/02/2010

gests that conformationally constrained 1,3-bis-azides 4
and 5 preferably form bis-triazole derivatives 6 and 7,
respectively, over less hindered 8 and 9 (Figure 2), even
Figure 1. Structures of NO-releasing prodrugs JS-K (1), V-PYRRO/
NO (2), and GlcNAc-DEA/NO (3).
The 2,4-dinitrophenyl, vinyl, and GlcNAc protecting groups
are well studied for their trigger mechanisms to release NO
from diazeniumdiolates.
Figure 2. Preferential bis-triazole formation by hindered bis-azides
4 and 5 (ref 11).
Our laboratory is involved in NO-drug development
mainly aimed at increasing the potency of these prodrugs7
and their effective site-directed delivery of NO. Some
strategies we employ to achieve these ends are increasing
the payload of NO per mole of prodrug (high-load NO-
donors) and conjugating our NO-prodrugs with other bio-
logically significant molecules. However, many of these
prodrugs decompose under certain reaction conditions, thus
limiting the scope of suitable reagents/reaction conditions.
The Cu(I)-catalyzed azide-alkyne cycloaddition (“click”)
reaction, independently discovered by the Sharpless8 and
Meldal9 groups, involves mild reaction conditions. They are
popular in medicinal chemistry because of their ease of
execution, functional group tolerance, and potential to
produce a library of compounds.10 Therefore the “click”
reaction was an attractive method to achieve the above-
mentioned properties in NO-drug development.
if a 10-fold excess of bis-azide over alkyne was used. In
the case of 4 and 5, formation of the first triazole ring
catalyzes the subsequent cycloaddition to give the required
bis-triazole predominantly.11
We envisioned that addition of benzylidene protection to 2,2-
di(azidomethyl)propane-1,3-diol 4 would further add to con-
formational strain in the system and hence lead to preferential
bis-triazole formation. Furthermore, suitably substituted ben-
zylidenes have potential applications in synthesis of dendritic
azides. Thus, the diol 4 was transformed into bis-azide 10
and tetrakis-azide 11 (Figure 3) (details in the Supporting
Information).
The “click” reaction between a suitably functionalized
diazeniumdiolate prodrug having a terminal alkyne group
and a polyazide can lead to multivalency and increased
payload of NO. We planned to investigate the reaction of
a bis-azide with the alkyne group attached to the diaz-
eniumdiolate prodrug. Finn and co-workers reported
mechanistic studies and reactivities of bis-azides in ligand-
free Cu(I)-catalyzed “click” reactions.11 The report sug-
Figure 3. Structures of bis-azide 10 and tetrakis-azide 11 synthe-
sized for the “click” reaction.
The diazeniumdiolate prodrugs 12-15 with terminal
alkynes are shown in Figure 4 (details of their synthesis and
(5) (a) Hong, S. Y.; Saavedra, J. E.; Keefer, L. K.; Chakrapani, H.
Tetrahedron Lett. 2009, 50, 2069–2071. (b) Liu, J.; Saavedra, J. E.; Lu, T.;
Song, J.-G.; Clark, J.; Waalkes, M. P.; Keefer, L. K. J. Pharmacol. Exp.
Ther. 2002, 300, 18–25.
(6) Valdez, C. A.; Saavedra, J. E.; Showalter, B. M.; Davies, K. M.;
Wilde, T. C.; Citro, M. L.; Barchi, J. J., Jr.; Deschamps, J. R.; Parrish, D.;
El-Gayar, S.; Schleicher, U.; Bogdan, C.; Keefer, L. K. J. Med. Chem. 2008,
51, 3961–3970.
(7) Nandurdikar, R. S.; Maciag, A. E.; Hong, S. H.; Chakrapani, H.;
Citro, M. L.; Saavedra, J. E.; Keefer, L. K. Org. Lett. 2010, 12, 56–59.
(8) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. 2001,
113, 2056–2075. (b) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2001, 40, 2004–2021.
(9) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057–3064.
(10) For selected reviews, see: (a) Meldal, M.; Tornoe, C. W. Chem.
ReV. 2008, 108, 2952–3015. (b) Moorhouse, A. D.; Moses, J. E. ChemMed-
Chem 2008, 3, 715–723. (c) Lutz, J. Angew. Chem., Int. Ed. 2007, 46, 1018–
1025. (d) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org.
Chem. 2006, 2006, 51–68. (e) Kolb, H. C.; Sharpless, K. B. Drug DiscoVery
Today 2003, 8, 1128–1137.
Figure 4. Diazeniumdiolate prodrugs 12-15 with terminal alkyne
groups synthesized for use in the “click” reaction.
Org. Lett., Vol. 12, No. 19, 2010
4257

characterization in Supporting Information). These alkynes
represent a sample of O2-protected diazeniumdiolates with
various modes of activation. Compound 14 is reported and
has shown antiproliferative activity comparable to JS-K (1)
against HL-60 and U-937 leukemia cell lines.12
reaction of alkynes 12-15 with 10 using CuI and diisopro-
pylethylamine (DIPEA) as base predominantly gave the
cycloaddition/oxidative coupling products (Table 2). Thus,
The “click” reaction of the alkynes 12-15 with bis-azide
10 was performed using CuSO4/Na-ascorbate in THF/water
(Table 1). The reaction proceeded quickly (15-45 min).
Table 2. 1,3-Dipolar Cycloaddition Using CuI/DIPEA for
Bis-azide 10a
Table 1. 1,3-Dipolar Cycloaddition Using CuSO4/Na-Ascorbate
for Bis-azide 10a
azide alkyne % yield (bis-triazole) % yield (triazolo-triazole)
10
10
10
10
12
13
14
15
trace (16)
28 (18)
20 (20)
19 (22)
74 (17)
44 (19)
72 (21)
56 (23)
a Conditions: CuI (2 equiv)/DIPEA (2 equiv), isolated product yields.
azide alkyne % yield (bis-triazole) % yield (triazolo-triazole)
10
10
10
10
12
13
14
15
60 (16)
67 (18)
75 (20)
63 (22)
12 (17)
14 (19)
10 (21)
trace (23)
steric congestion can be an additional factor for such
oxidative dimerization reaction in Cu(I)-catalyzed cycload-
ditions.
a Conditions: CuSO4·5H2O (40 mol %)/Na-ascorbate (80 mol %), THF/
H2O (3:1), isolated product yields.
In the case of the tetrakis-azide 11, an additional partially
coupled product (triazolo-triazole bis-triazole adduct) along
with the expected tetrakis-triazole and bis(triazolo-triazole)
may occur. To minimize the formation of these byproducts
and avoid difficulties of their chromatographic separations,
the 1,3-dipolar cycloadditions of tetrakis-azide 11 with
alkynes 12 and 13 were carried out in the absence of oxygen
using CuSO4/Na-ascorbate (Table 3). Under nitrogen atmo-
However, we observed formation of two products. The major
product in each case was the expected bis-triazole. The minor
product was 5,5′-triazolo-triazole. No formation of monot-
riazole product was observed.
Formation of such triazolo-triazoles is reported in the
literature, but often as undesired and uncharacterized impuri-
ties.13 They are a result of the oxidative coupling reaction
of copper species after the triazole formation. To the best of
our knowledge, there has only been one detailed report
showing base dependence for the formation of triazolo-
triazoles in an intermolecular cycloaddition reaction.14 In the
cycloaddition reaction of azides 10 and 11, the benzylidene
protection may bring the two newly formed triazole rings
fairly close to enhance the formation of triazolo-triazole
by oxidative coupling. These 5,5′-triazolo-triazoles remain
under-explored heterocycles. Therefore, we attempted to
synthesize triazolo-triazoles as the predominant product
formed in the case of bis-azide 10, as they may be good
additions to heterocyclic linkers in medicinal chemistry. The
Table 3. 1,3-Dipolar Cycloaddition Using CuSO4/Na-Ascorbate
for Tetrakis-azide 11
azide
alkyne
% yield (tetrakis-triazole)
11
11
12
13
54 (24)
46 (25)
a Conditions: under N2 reaction, CuSO4·5H2O (80 mol %)/Na-ascorbate
(11) Rodionov, V. O.; Fokin, V. V.; Finn, M. G. Angew. Chem., Int.
Ed. 2005, 44, 2210–2215.
(160 mol %), THF/H2O (3:1), isolated product yields.
(12) Nandurdikar, R. S.; Maciag, A. E.; Citro, M. L.; Shami, P. J.;
Keefer, L. K.; Saavedra, J. E.; Chakrapani, H. Bioorg. Med. Chem. Lett.
2009, 19, 2760–2762.
(13) Vsevolod, V. R.; Luke, G. G.; Valery, V. F.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
sphere, the cycloaddition reactions of 12 and 13 gave the
desired tetrakis-triazoles 24 and 25, respectively, in accept-
able yields. Efforts are underway to make tetrakis-triazoles
(14) Angell, Y.; Burgess, K. Angew. Chem., Int. Ed. 2007, 46, 3649–
3651.
4258
Org. Lett., Vol. 12, No. 19, 2010

of diazeniumdiolates with 2,4-dinitrophenyl and GlcNAc
protecting groups.
the oxidatively coupled triazole adducts, leading to unex-
plored heterocyclic linkers for biological applications. These
results underline the need to further investigate the role of
steric congestion for the formation of such Cu-catalyzed
oxidative coupling products. A proper substitution in the
phenyl ring of the synthesized substrates along with a proper
coupling reaction may allow the synthesis of well-defined
dendritic NO-donors. These dendritic NO-donors can have
potential applications in NO-releasing materials. The chem-
sitry established has potential applications in synthesizing
hybrids of biologically important molecules and NO-donors.
Efforts are underway for the synthesis of such NO-donor
hybrids using the “click” reaction.
The hydrolysis or enzymatic activation to release NO for
these types of prodrugs is well established in the litera-
ture.4-7,12 However, the NO release profile for the bis-
triazole-, triazolo-triazole-, and tetrakis-triazole-linked pro-
drugs may have differences in their kinetics. In addition, the
steric effect on enzymatic activation, their intracellular NO-
release, biological evaluation, toxicity, and physiological
clearance of the linker15,16 need to be investigated. Efforts
are underway in these directions. If required, the phenyl ring
of benzylidene protection in these compounds can be suitably
substituted to improve their cell permeability and/or solubil-
ity.
Thus, the Cu-mediated “click” reaction was utilized to
tether two and four molecules of NO-donor prodrugs
together, which would serve to increase the payload of NO.
The reaction conditions did not compromise the integrity of
the diazeniumdiolate functional group, which is sensitive to
certain other reaction conditions. The reactions showed
formation of unusual oxidative coupling products, which
were well characterized. Use of CuI/DIPEA in acetonitrile
to carry out the dipolar cycloaddition preferentially formed
Acknowledgment. This project has been funded with
Federal funds from the National Cancer Institute, National
Institutes of Health, under contract HHSN261200800001E
and by the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research. We thank Dr.
Sergey Tarasov and Ms. Marzena A. Dyba of the Biophysics
Resource in the Structural Biophysics Laboratory, NCI-
Frederick, for assistance with the high-resolution mass
spectrometry studies.
Supporting Information Available: Experimental details
and full spectroscopic data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(15) The triazole linkers are considered inert to metabolic transformation.
However, the toxicological profile or physiological clearance of the bis-
triazole or triazolo-triazole linkers is not reported. The synthesis of such
compounds makes them available for such studies.
(16) For a review on five-membered heterocyclic compound biotrans-
formation, see: Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh-Bakht, S. C.;
Obach, R. S.; O’Donnell, J. P. Chem. Res. Toxicol. 2002, 15, 269–299
.
OL101645K
Org. Lett., Vol. 12, No. 19, 2010
4259