Synthesis of peptide-diazeniumdiolate conjugates: Towards enzyme activated antitumor agents

Article abstract of DOI:10.1016/S0040-4039(01)00263-5

The development of NO donors with site-specific and time-controlled properties is of great interest. We have designed a novel prodrug class as possible agents against metastatic prostate cancer by coupling a diazeniumdiolate to the terminal carboxyl groups of amino acids or peptides, such as Ser-Ser-Tyr-Tyr, Ser-Ser-Phe-Tyr, and Gly-Ile-Ser-Ser-Phe-Tyr. These prodrugs can be activated by α-chymotrypsin or prostate specific antigen and are potentially potent compounds for prostatic cancer.

Full text of DOI:10.1016/S0040-4039(01)00263-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2625–2629
Synthesis of peptide-diazeniumdiolate conjugates: towards
enzyme activated antitumor agents
Xiaoping Tang,a Ming Xian,a Mohit Trikha,b Kenneth V. Honnb and Peng George Wanga,*
aDepartment of Chemistry, Wayne State University, Detroit, MI 48202, USA
bDepartment of Radiation Oncology and Pathology, Wayne State University, Detroit, MI 48202, USA
Received 22 January 2001; accepted 18 February 2001
Abstract—The development of NO donors with site-specific and time-controlled properties is of great interest. We have designed
a novel prodrug class as possible agents against metastatic prostate cancer by coupling a diazeniumdiolate to the terminal carboxyl
groups of amino acids or peptides, such as Ser-Ser-Tyr-Tyr, Ser-Ser-Phe-Tyr, and Gly-Ile-Ser-Ser-Phe-Tyr. These prodrugs can be
activated by a-chymotrypsin or prostate specific antigen and are potentially potent compounds for prostatic cancer. © 2001
Published by Elsevier Science Ltd.
There is currently no effective therapy for men with
metastatic prostate cancer with relapse after androgen
ablation.1 We recognized the need to develop new
strategies for the delivery of cytotoxic agents specifi-
cally to sites of metastatic prostate cancer while avoid-
ing systemic toxicity. One such approach would be to
develop prodrugs that are inactive when given systemi-
cally but become activated when processed proteolyti-
cally within prostate cancer metastases by a prostate
specific antigen (PSA).2–4 Recently reported conjugates
of peptide with doxorubicin2,3 or mustard agents4 were
designed according to this strategy. PSA is a serine
protease with chymotrypsin-like substrate specificity;5 it
is synthesized and secreted in large quantities by normal
and malignant prostatic epithelial cells.6 The elevated
levels of PSA in serum have been correlated with the
benign and metastatic disease, making PSA a useful
marker for the growth of prostate cancer.7 However,
PSA in blood serum is inactive due to the presence of
high concentrations of natural inhibitory proteins, such
as a1-antichymotrypsin and a2-macroglobulin.8
Nitric oxide (NO) has been identified as a major media-
tor in many physiological processes.9–11 In addition to
its well-documented vasoactive,12,13 and neurotransmit-
ter properties,14,15 NO produced by macrophages,
Kupffer cells, natural killer T-cells, and endothelial cells
participate in tumoricidal activity against many types of
tumors.16,17 It may interact with oxygen-derived radi-
cals to generate molecules that can nitrosate proteins,
modify their functions and mediate damage to DNA.18
Although NO can be endogeneously produced from
Scheme 1. PSA activated peptide-diazeniumdiolate prodrug targeting prostate cancer.
* Corresponding author. Tel.: 313-993-6759; fax: 313-577-2554; e-mail: pwang@chem.wayne.edu
0040-4039/01/$ - see front matter © 2001 Published by Elsevier Science Ltd.
PII: S0040-4039(01)00263-5

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X. Tang et al. / Tetrahedron Letters 42 (2001) 2625–2629
prostate cancer cell, where it will be specifically
hydrolysed by PSA and release toxic NO radicals to
destroy the cancer cells (Scheme 1). The work described
herein chooses a diazeniumdiolate, PYRRO/NO, as the
specific NO donor. Although a number of high-affinity
substrates for PSA have been identified, peptides con-
taining amino acid sequence Ser-Ser-(Tyr/Phe)-Tyr
were initially selected as the PSA substrate based on
synthetic consideration.29 These units will be linked
together via an ‘acetal’ linkage, which was previously
used in our study of cupferron prodrugs.30
Scheme 2. General synthetic scheme for PSA-activated pro-
drugs.
As shown in Scheme 2, the sodium salt of PYRRO/NO
(1) was first O-alkylated with chloromethyl methyl-
sulfide, and the resulting compound (2) was subse-
quently treated with sulfuryl chloride. Such in situ
generated chloride (3) can be coupled to the C-terminal
carboxylic acids of the peptides in the presence of
cesium carbonate to give the desired prodrugs. Three
single amino acids (Ala, Phe, and Tyr) were chosen to
test the feasibility of the coupling strategy. Then three
peptides (Ser-Ser-Tyr-Tyr, Ser-Ser-Phe-Tyr, and Gly-
Ile-Ser-Ser-Phe-Tyr) were successfully coupled to the
diazeniumdiolate moiety. All of the peptide-diazonem-
diolate conjugates (4–9) were obtained in very good
yield (Table 1). The peptides were prepared by standard
solid phase peptide synthesis techniques using Fmoc
strategy.31
L
-arginine, mediated by nitric oxide synthase
(NOS),19,20 exogenous sources of NO have been widely
used as therapeutic agents.21–25 Diazeniumdiolates
(NONOates) are compounds containing the [N(O)NO]−
structural unit, this class of compound is known as an
excellent source for a controlled release of NO both in
vitro and in vivo.26,27 Keefer’s group has recently intro-
duced a new class of diazeniumdiolate prodrugs which
are acetoxymethylated at the anionic oxygen of the
parent NONOates.28 These esterase-sensitive com-
pounds reveal significant antileukemic activity in vitro.
We are interested in the design of PSA substrate-nitric
oxide donor conjugates as potential enzyme activated
antitumor agents. This class of conjugate prodrugs, if
properly designed, will be inactive until they reach the
Enzymetic assays were performed to study the hydroly-
sis of prodrugs by the a-chymotrypsin (EC 3.4.21.1)
Table 1. The synthetic result of peptide-diazonemdiolate prodrugs (4–9)

X. Tang et al. / Tetrahedron Letters 42 (2001) 2625–2629
2627
Experimental
All reagents were purchased from commercial suppliers
and were used without further purification. Melting
points were measured on an Electrothermal IA 9100
1
digital apparatus without correction. H and 13C NMR
spectra were recorded on a Varian Gemini-300, a Mer-
cury-400, or a Varian Unity-500 spectrometer. MS
(ESI) and HRMS (EI) were performed at the Depart-
ment of Chemistry, Wayne Sate University; HRMS
(DCI or FAB) were performed at the Department of
Chemistry, University of California, Riverside. Silica
gel F254 plates (Merck) and silica gel 60 (70–230 mesh,
Merck) were used in analytical thin-layer chromatogra-
phy (TLC) and flash column chromatography,
respectively.
Figure 1. Enzymatic release of NO from prodrugs 5 (2), 6
(ꢀ), 8 (ꢁ) and 9 (ꢂ) in the presence of PSA (substrate: 25
mM, PSA: 20 mg/mL).
Sodium 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (1):
Prepared by Keefer’s method.28 1H NMR (D2O, 400
MHz): l 2.99 (m, 4H), 1.68 (m, 4H); 13C NMR (D2O,
100 MHz): l 51.58, 22.60.
and purified PSA (Cortex Biochem. Inc., San Leandro,
CA) at room temperature. Each prodrug was first
dissolved in 1.0 mL of anhydrous methanol, a portion
of the solution was then diluted with 10 mL of 0.1 M
Tris–Cl buffer (pH 7.0) followed by the addition of an
aqueous solution of pure enzyme. The generation of
NO from the hydrolysis could be quantitatively mea-
sured with an Electrochemical ISO-NO Mark II Iso-
lated Nitric Oxide Meter manufactured by World
Precision Instruments, Inc. (Saraota, Florida). All the
prodrugs were essentially stable at neutral pH, and no
detectable amount of NO was released even after sev-
eral hours. When the prodrug was treated either with
pure PSA (Fig. 1) or a-chymotrypsin (Fig. 2), the NO
release was significantly accelerated and a substantial
amount of NO was detected. The difference in the NO
release rate between the prodrugs may reflect different
binding affinity to the enzyme.
O2 -Methylthiomethyl 1-(pyrrolidin-1-yl)diazen-1-ium-
1,2-diolate (2): A slurry of compound 1 (3.20 g, 20.9
mmol) and anhydrous sodium carbonate (2.22 g, 20.9
mmol) in 40 mL of dry THF was cooled with an
ice-water bath, to which 2.28 mL (27.2 mmol) of
chloromethyl methylsulfide was slowly added via a
syringe, followed by 15 mL of DMF. After 5 min, the
bath was removed and a catalytic amount of KI (200
mg) was added. The reaction mixture was stirred under
nitrogen overnight. Then it was diluted with 200 mL of
ether and washed with cold water. The separated
aqueous layer was again extracted with ether. The
combined ether solution was washed with brine, dried
over anhydrous sodium sulfate, and concentrated under
vacuum. The residue was chromatographed on silica
gel. Elution with 1:4 ethyl acetate/hexane provided 1.80
g (45%) of 2 as a colorless oil, which solidified when
kept inside a refrigerator. 1H NMR (CDCl3, 300 MHz):
l 5.09 (s, 2H), 3.44 (m, 4H), 2.15 (s, 3H), 1.83 (m, 4H);
13C NMR (CDCl3, 75 MHz): l 78.06, 50.94, 23.02,
15.61; UV umax (MeOH, o): 255 nm (10.0 mM−1 cm−1);
MS (ESI) 214 (M+Na+), 405 (2M+Na+); HRMS (EI)
calcd for C6H13N3O2S (M+) 191.0728, found 191.0727.
In summary, we have developed an efficient synthetic
strategy to synthesize peptide-diazeniumdilote conju-
gates. Such prodrugs can be activated by cellular
proteases, such as a-chymotrypsin or prostate-specific
antigen, to release nitric oxide. Further systemic cyto-
toxic studies against PSA secreting tumor cell lines will
be reported in due course.
O2-Chloromethyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-dio-
late (3): A solution of compound 2 (91 mg, 0.48 mmol)
in 10 mL of dry CH2Cl2 was cooled with an ice-water
bath, to which 0.48 mL of sulfuryl chloride (1.0 M in
CH2Cl2) was added dropwise. After 10 min, the ice-
water bath was removed, and the mixture was stirred
for another 30 min. Evaporation under vacuum gave 3
as a yellow oil, which was immediately used in the
following reactions without further purification. 1H
NMR (CDCl3, 400 MHz): l 5.80 (s, 2H), 3.60 (t, 4H),
1.95 (m, 4H); 13C NMR (CDCl3, 100 MHz): l 79.86,
50.86, 23.32.
General procedure for the synthesis of peptide-diazenium-
diolate prodrugs: To a DMF solution of freshly pre-
pared compound 3 was added amino acid or peptide
(one equivalent) in one portion, followed by the addi-
Figure 2. Enzymatic release of NO from prodrugs 5 (2), 6
(ꢀ), 7 (×), 8 (ꢁ) and 9 (ꢂ) in the presence of a-chy-
motrypsin (substrate: 100 mM, a-chymotrypsin: 50 mg/mL).

2628
X. Tang et al. / Tetrahedron Letters 42 (2001) 2625–2629
a
O2-(N -Acetyl
L
-serine-
L-serine-L-phenylalanine-L-tyro-
tion of cesium carbonate (one equivalent). The mixture
was stirred at room temperature until the disappear-
ance of starting material as monitored by TLC. Then
the mixture was diluted with water and extracted with
CH2Cl2. The separated organic phase was combined,
washed with brine, dried over anhydrous sodium sul-
fate, and concentrated under vacuum to give the crude
product, which was further purified by flash column
chromatography.
sine)methyl
1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate
1
(8): Prepared from the peptide Ac-SSFY-OH (41%). H
NMR (CD3OD, 500 MHz): l 7.26–7.16 (m, 5H), 7.00
(m, 2H), 6.69 (m, 2H), 5.78 (d, 1H, J=7.5 Hz), 5.69 (d,
1H, J=7.0 Hz), 4.60 (m, 2H), 4.42 (t, 1H, J=6.0 Hz),
4.36 (t, 1H, J=5.5 Hz), 3.80–3.67 (m, 4H), 3.50 (m,
4H), 3.12–2.83 (m, 4H), 2.01 (s, 3H), 1.92 (m, 4H);
partial 13C NMR (CD3OD, 125 MHz): l 130.21,
129.18, 129.11, 128.27, 127.05, 126.57, 115.12, 87.60,
61.82, 61.42, 55.90, 55.60, 54.97, 54.42, 50.50, 37.38,
36.24, 22.65, 21.34; UV umax (MeOH, o): 254 nm (5.0
mM−1 cm−1); MS (ESI) found 710 (M+Na+).
a
O2-(N -Acetyl
L-alanine)methyl 1-(pyrrolidin-1-yl)dia-
zen-1-ium-1,2-diolate (4): Prepared from Na-acetyl ala-
1
nine and collected as a colorless oil (60%). H NMR
(CDCl3, 400 MHz): 6.45 (d, 1H, J=6.4 Hz), 5.78 (d,
1H, J=6.4 Hz), 5.67 (d, 1H, d=6.4 Hz), 4.55 (m, 1H),
3.52 (m, 4H), 1.95 (s, 3H), 1.91 (m, 4H), 1.34 (d, 3H,
J=6.8 Hz); 13C NMR (CDCl3, 100 MHz): l 171.79,
169.78, 87.63, 50.48, 47.82, 28.81, 17.81; UV umax
(MeOH, o): 253 nm (6.5 mM−1 cm−1); MS (ESI) found
297 (M+Na+), 313 (M+K+).
a
O2-(N -Acetyl
L
-glycine-
L
-isoleucine-
L
-serine-
L
-serine-
L
-
phenylalanine-L-tyrosine)methyl 1-(pyrrolidin-1-yl)diaz-
en-1-ium-1,2-diolate (9): Prepared from the peptide Ac-
1
GISSFY-OH (45%). H NMR (CD3OD, 500 MHz): l
7.23–7.17 (m, 5H), 7.00 (m, 2H), 6.67 (m, 2H), 5.78 (d,
1H, J=7.5 Hz), 5.69 (d, 1H, J=7.0 Hz), 4.60 (m, 2H),
4.38 (t, 1H, J=6.0 Hz), 4.22 (m, 1H), 3.72–3.60 (m,
4H), 3.50 (m, 4H), 3.12–2.85 (m, 4H), 2.00 (s, 3H), 1.92
(m, 4H), 1.17 (m, 3H), 0.90 (m, 6H); partial 13C NMR
(CD3OD, 125 MHz): l 174.00, 173.65, 173.21, 171.91,
171.43, 157.42, 138.39, 131.37, 130.31, 129.45, 128.25,
127.73, 116.30, 88.78, 62.95, 59.54, 56.69, 56.00, 55.63,
51.69, 43.67, 38.59, 38.04, 37.40, 25.93, 23.83, 22.41,
16.99, 11.60; UV umax (MeOH, o): 252 nm (7.1 mM−1
cm−1); MS (ESI) found 880 (M+Na+).
a
O2-(N -Acetyl
L-phenylalanine)methyl 1-(pyrrolidin-1-
yl)diazen-1-ium-1,2-diolate (5): Prepared from Na-acetyl
phenylalanine and collected as colorless needles (60%).
Mp 91–92°C. 1H NMR (CDCl3, 500 MHz): l 7.30–7.70
(m, 3H), 7.08 (d, 2H, J=8.0 Hz), 5.96 (d, 1H, J=7.5
Hz), 5.87 (d, 1H, J=7.0 Hz), 5.70 (d, 1H, d=7.0 Hz),
4.92 (m, 1H), 3.56 (m, 4H), 3.16 (dd, 1H, J=14.0, 6.0
Hz), 3.11 (dd, 1H, J=14.0, 5.5 Hz), 1.96 (m, 7H); 13C
NMR (CDCl3, 125 MHz): l 170.41, 169.62, 135.39,
129.26, 128.56, 127.11, 97.90, 52.84, 50.58, 37.33, 23.00,
22.93; UV umax (MeOH, o): 252 nm (8.4 mM−1 cm−1);
MS (ESI) found 373 (M+Na+), 723 (2M+Na+); HRMS
(DCI) calcd for C16H23N4O5 (M+H+) 351.1668, found
351.1655.
Acknowledgements
This work was supported by grants from the National
Institutes of Health (GM 54074).
a
O2-(N -Acetyl
L-tyrosine)methyl 1-(pyrrolidin-1-yl)dia-
zen-1-ium-1,2-diolate (6): Prepared from Na-acetyl
tyrosine and collected as a yellow solid (54%). Mp
97–102°C; H NMR (CD3OD, 500 MHz): l 6.99 (d,
References
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Hz), 4.62 (m, 1H), 3.53 (m, 4H), 3.03 (dd, 1H, J=14.5,
6.0 Hz), 2.87 (dd, 1H, J=14.5, 6.0 Hz), 1.96 (m, 4H),
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