An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide prodrug

Article abstract of DOI:10.1016/j.tetlet.2009.05.089

An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide (NO) prodrug, in an overall yield of 26% in four steps from prolinol is reported; the previously published yield of this transformation was 1%. Using this revised strategy, the

Full text of DOI:10.1016/j.tetlet.2009.05.089

Tetrahedron Letters 50 (2009) 4545–4548
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An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric
oxide prodrug
a,
Sam Y. Hong a, Rahul S. Nandurdikar a, Larry K. Keefer a, Joseph E. Saavedra b, , Harinath Chakrapani
*
*
a Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702, USA
b Basic Science Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An improved synthesis of V-PROLI/NO, a cytochrome P450-activated nitric oxide (NO) prodrug, in an
overall yield of 26% in four steps from prolinol is reported; the previously published yield of this trans-
formation was 1%. Using this revised strategy, the sarcosine analogue (14) of V-PROLI/NO was prepared.
Finally, the methyl ester of V-PROLI/NO (15) was found to be an esterase-activated prodrug form of V-
PROLI/NO.
Received 13 May 2009
Revised 20 May 2009
Accepted 22 May 2009
Available online 28 May 2009
Published by Elsevier Ltd.
O2-(Vinyl) diazeniumdiolates are prodrugs that are designed to
be activated by cytochrome P450 to release nitric oxide (NO). O2-
Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO,
1), a member of this class of prodrugs, was shown to be a hepato-
protective agent against a variety of toxins in several animal mod-
els (Scheme 1).1–18
V-PROLI/NO was previously synthesized in four steps from pro-
linol in an overall yield of roughly 1% (Scheme 2).19 Diazeniumdi-
olation of prolinol provided 5 (73%), which was then subsequently
treated with 2-bromo-1-(trifluoromethanesulfonyloxy)ethane to
afford 6 in 48% yield (Scheme 2).19 Oxidation of 6 formed 7 in
19% yield; and dehydrohalogenation of 7 afforded V-PROLI/NO in
19% yield.19
Earlier, we reported that O2-vinyl [2-carboxylato)pyrrolidin-1-
yl]diazen-1-ium-1,2-diolate (V-PROLI/NO, 2), the
L
-proline
At the outset, our goal was to improve the overall yield of V-
PROLI/NO. Towards this aim, first, the diazeniumdiolation of pro-
linol was carried out numerous times and the yield of 5 varied from
73% to 82%. Next, the alkylation of 5 to form 6 was optimized and
the yield obtained was 78%.21 Recently, we reported the improved
synthesis of V-PYRRO/NO using an alkylation followed by a dehy-
drohalogenation of 8 by Verkade’s SuperBase (9) (Scheme 3).18,22
Under similar conditions, when 7 was treated with 9, we ob-
served complete disappearance of starting material but without
any trace of the desired product, V-PROLI/NO.18
analogue of V-PYRRO/NO, was metabolized by two isoforms of
cytochrome P450 (Scheme 1). The nitric oxide prodrug PROLI/NO
(4) may have a favorable toxicological profile as the expected prod-
ucts of decomposition are L-proline and NO, both naturally occur-
ring metabolites.19 Upon treatment of human liver HepG2 cells
with V-PROLI/NO, the formation of nitrite, a product of aerobic oxi-
dation of NO, in a time- and concentration-dependent manner, was
reported.20 Finally, V-PROLI/NO enhanced arsenite’s chemothera-
peutic efficacy in a HepG2 liver cell model.20
Instead, an inseparable mixture of presumably intra- and inter-
molecular substitution products resulted. This observation is con-
sistent with a previous study by Arumugam and Verkade who re-
ported that 1-bromo-propanoic acid failed to produce the
dehydrohalogenation product, acrylic acid, but instead formed
the nucleophilic substitution product, b-propiolactone, in a nearly
quantitative yield.22 Other bases such as DBU or proton sponge
failed to induce elimination of 7 and resulted in the recovery of
the starting material.
Cytochrome
P450
R
R
N
N
N
N
pH 7.4
NO
O
N
O
O
N
O
R = H, PYRRO/NO, 3
R = COO−, PROLI/NO, 4
1
R = H, V-PYRRO/NO,
R = COOH, V-PROLI/NO, 2
Thus, a revised strategy to synthesize V-PROLI/NO was neces-
sary. Instead of oxidizing 6 to 7 and then attempting to dehydro-
halogenate 7 to form V-PROLI/NO, a reversal of the order of the
aforementioned reactions was envisaged (Scheme 4). Dehydro-
halogenation of 6 is expected to produce 10 and subsequent oxida-
tion of 10 should produce V-PROLI/NO.
Scheme 1. The nitric oxide prodrugs V-PYRRO/NO (1) and V-PROLI/NO (2) and their
corresponding devinylated metabolites, the spontaneously NO-releasing anions
PYRRO/NO (3) and V-PROLI/NO (4).
* Corresponding authors. Tel.: +1 301 846 1602 (J.E.S.); tel.: +1 301 846 1601; fax:
+1 301 846 5946 (H.C.).
E-mail addresses: saavj@ncifcrf.gov (J.E. Saavedra), chakrah@ncifcrf.gov (H.
Chakrapani).
Indeed this strategy to synthesize V-PROLI/NO was successful
(Scheme 5). The alcohol 10 was isolated in 83% yield from the
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2009.05.089

4546
S. Y. Hong et al. / Tetrahedron Letters 50 (2009) 4545–4548
NaIO4
O
Br
RuCl3
N
N
OTf
N
N
NO (3 atm)
N
N
NaOH
N
H
OH
OH
OH
Na
V-PROLI/NO, 2
OH
NaOMe,
MeOH
O
N
O
MeCN,
THF,
THF, H2O
O
N
O
O
N
O
EtOAc, H2O
15-crown-5
48%
Br
Br
19%
73%
19%
Prolinol
5
6
7
Scheme 2. Reported synthesis of V-PROLI/NO (2).19
P
O
N
N
N
N
N
Diazomethane
Ether
O Me
N
V-PROLI/NO, 2
N
O
TfO
Br
9
N
N
O
1
V-PYRRO/NO,
3
PYRRO/NO,
70%
49%
15
81%
Br
O
N
O
Scheme 7. Synthesis of 15.
8
Scheme 3. Improved synthesis of V-PYRRO/NO (1).18
The methyl ester 15 was found to be a prodrug form of V-PROLI/
NO. In pH 7.4 buffer at 37 °C, gradual disappearance of 15
(Fig. 1A) over several days (Fig. 1B) with concomitant formation
of V-PROLI/NO (Fig. 1C) was observed.30 A time course of this ester
hydrolysis to form V-PROLI/NO is shown in Figure 2. Finally, treat-
ment of 15 with esterase formed V-PROLI/NO in a nearly quantita-
tive yield (by HPLC).31 Taken together, these results indicate that
15 is a prodrug for V-PROLI/NO.
N
N
OH
2
6
V-PROLI/NO,
O
N
O
10
Scheme 4. Revised retrosynthetic analysis of V-PROLI/NO (2).
Jones
9
N
N
oxidation
50%
OH
6
V-PROLI/NO, 2
83%
O
N
O
10
Scheme 5. Improved synthesis of V-PROLI/NO from 6.
treatment of 6 with 9.23 Next, oxidation of 10 was carried out using
the Jones oxidation method to produce V-PROLI/NO in a yield of
50%; this step was also convenient as no chromatography was re-
quired during isolation of the product.24 Thus, starting from prolin-
ol, the overall yield of V-PROLI/NO was 26% in four steps.
This improved strategy was applied to the synthesis of the sar-
cosine analogue of V-PROLI/NO (Scheme 6). A reported method
was used to prepare the diazeniumdiolate salt 11.25 Next, 11 was
alkylated to afford 12 in 71% yield.26 Dehydrohalogenation of 12
produced 13 in 55% yield;27 and finally, the alcohol 13 was oxi-
dized to form 14 in 57% yield.28 The overall yield of 14 from the
commercially available 2-(methylamino)ethanol was 16% (four
steps).
Figure 1. HPLC traces of: (A) 15 in pH 7.4 phosphate buffered saline (PBS) at
time = 0; (B) 15 and its hydrolysis product in pH 7.4 PBS at time = 14 days, the
hydrolysis product was identified as V-PROLI/NO; (C) V-PROLI/NO in pH 7.4 PBS.
Next, the methyl ester of V-PROLI/NO was prepared in 70% yield
by reacting V-PROLI/NO with diazomethane in ether (Scheme 7).29
Me
O
Br
Me
O
Me
O
O
N
N
Me
O
OTf
CrO3
N
N
NO (3 atm)
9
N
N
N
N
Me
OH
OH
OH
N
H
NaOMe,
MeOH
H2SO4
THF,
OH
N
O
MeCN
N
O
N
O
15-crown-5
71%
N
O
OH
Na
73%
57%
55%
Br
11
13
12
14
Scheme 6. Synthesis of 14 in 4 steps from N-methyl-N-(2-hydroxyethyl)amine.

S. Y. Hong et al. / Tetrahedron Letters 50 (2009) 4545–4548
4547
bromo-1-(trifluoromethanesulfonyloxy)ethane (6.18 g, 24.0 mmol, 1.1 equiv)
was added and the mixture was stirred overnight at room temperature. The
solution was washed with 10% NaOH and the organic layer was separated,
dried (Na2SO4) and filtered through
a plug of MgSO4. The filtrate was
concentrated under reduced pressure and the resulting oil was then purified
by silica gel flash chromatography using a mixture of 12?75% EtOAc/hexanes
to afford 6 (4.40 g, 16.4 mmol, 75%) as an oil. The spectral features of this
material were consistent with the previously published values (Ref. 19).
22. Arumugam, S.; Verkade, J. G. J. Org. Chem. 1997, 62, 4827–4828.
23. O2-Vinyl 1-[2-(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (10): To a
solution of 6 (1.70 g, 6.3 mmol) in anhydrous acetonitrile (64 mL), 9 (2.74 g,
2.27 mmol) was added and the resulting mixture was stirred overnight at room
temperature. The solvent was removed and the resulting oil was purified using
silica gel flash chromatography with a mixture of 12?75% EtOAc/hexanes to
afford 10 (0.99 g, 5.27 mmol, 83%) as an oil: UV (EtOH) kmax (e) 267 nm
(8.84 mM-1 cmÀ1); 1H NMR (400 MHz, CDCl3) d 6.80 (dd, J = 14.1, 6.7 Hz, 1H),
4.84 (dd, J = 14.1, 2.5 Hz, 1H), 4.39 (dd, J = 6.7, 2.5 Hz, 1H), 4.24–4.09 (m, 1H),
3.89–3.48 (m, 4H), 2.66 (t, J = 5.9 Hz, 1H), 2.25–1.80 (m, 4H); 13C NMR
(100 MHz, CDCl3) d 148.5, 91.9, 65.1, 64.0, 52.8, 26.8, 23.1. Anal. Calcd for
C7H13N3O3Á0.1 EtOAc: C, 45.35; H, 7.10; N, 21.44. Found: C, 45.35, H, 6.93; N,
21.08.
Figure 2. Time course of ester hydrolysis of 15 (A) forming V-PROLI/NO (B) in pH
7.4 Hank’s Balanced Salt Solution (HBSS) at 37 °C during 15 days.
24. Oxidation of 10: To 10 (100 mg, 0.53 mmol) in acetone (5 mL) at rt, Jones
reagent (40 g CrO3 + 32 mL 98% sulfuric acid + 75 mL H2O) was added dropwise
until TLC showed a complete disappearance of starting material. The solution
was then quenched with 2-propanol (1 mL) and concentrated. The ensuing
residue was taken up in water (5 mL) and washed with DCM (4 Â 4 mL). The
organic fractions were collected and concentrated. The pale yellow oil was
dissolved in a NaOH solution (1.5 M, 3 mL) and washed with DCM (3 Â 2 mL).
The aqueous phase was then acidified with HCl (6 M) until pH 2.0 was reached.
The aqueous phase was washed with DCM (5 Â 4 mL). The organic fractions
were collected and concentrated under reduced pressure affording 2 (54 mg,
0.267 mmol, 50% yield) as a waxy brown solid. The spectral features of this
solid matched with previously reported values (Ref. 19). Note: to address any
possible chromium impurities, additional purification by silica gel flash
column chromatography (9:1 CH2Cl2/ethyl acetate), was conducted; the yield
of 2 in such a case was 21%.
Carboxylic acid esters are reported to have improved bioavail-
ability in comparison with their carboxylic acid counterparts.32 Re-
cently, we reported that ester derivatives of PROLI/NO were
superior cell penetrators and inhibitors of proliferation of HL-60
leukemia cells than their corresponding free carboxylic acid coun-
terparts.33 Studies on the cell permeability and the efficacy of 14
and 15 as hepatoprotective agents will be reported in due course.
Acknowledgments
This research was supported in part by the Intramural Research
Programme of the NIH, National Cancer Institute, Center for Cancer
Research, as well as National Cancer Institute contract NO1-CO-
2008-00001 to SAIC.
25. Nguyen, J.-T.; Velazquez, C. A.; Knaus, E. E. Bioorg. Med. Chem. 2005, 13, 1725–
1738.
26. O2-(2-Bromoethyl)
1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-
diolate (12): To a slurry of 11 (100 mg, 0.64 mmol) and 15-crown-5 (100 L)
l
in THF (7 mL), 2-bromo-1-(trifluoromethanesulfonyloxy)ethane (213 mg,
0.83 mmol, 1.3 equiv) was added and the resulting reaction mixture was
stirred overnight at room temperature. The solution was washed with 10%
NaOH and the organic layer was separated, dried (Na2SO4) and filtered through
a plug of MgSO4. The filtrate was concentrated under reduced pressure and the
resulting oil was then purified by silica gel flash chromatography using a
mixture of 12?75% EtOAc/hexanes to afford 12 (110 mg, 0.45 mmol, 71%) as
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173–180.
an oil: UV (MeCN) kmax (e
) 261 nm (7.00 mMÀ1 cmÀ1); 1H NMR (400 MHz,
CDCl3) d 4.50 (t, J = 8.0 Hz, 2H), 3.79–3.74 (m, 2H), 3.58 (t, J = 8.0 Hz, 2H), 3.45–
3.42 (m, 2H), 3.05 (s, 3H), 2.14 (t, J = 5.8 Hz, 1H);13C NMR (100 MHz, CDCl3) d
72.7, 59.6, 57.1, 42.0, 27.8. Anal. Calcd for C5H12BrN3O3Á0.1 EtOAc: C, 25.85; H,
5.14; N, 16.75; Br, 31.85. Found: C, 25.97; H, 5.14; N, 16.48; Br, 31.76.
27. O2-Vinyl 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate (13):
To a solution of 12 (2.20 g, 9.1 mmol) in anhydrous acetonitrile (90 mL), 9
(3.93 g, 18.18 mmol) was added and the mixture was stirred overnight at room
temperature. The solvent was removed under reduced pressure and the
resulting oil was purified by silica gel flash chromatography with a mixture of
12?75% EtOAc/hexanes to afford 13 (0.80 g, 4.98 mmol, 55%) as an oil: UV
(EtOH) kmax (e
) 264 nm (8.23 mMÀ1 cmÀ1); 1H NMR (400 MHz, CDCl3) d 6.83
(dd, J = 14.1, 6.7 Hz, 1H), 4.87 (dd, J = 14.1, 2.5 Hz, 1H), 4.42 (dd, J = 6.7, 2.5 Hz,
1H), 3.81 (dt, J = 5.6, 4.8 Hz, 2H), 3.53 (t, J = 4.8 Hz, 2H), 3.14 (s, 3H), 2.13 (t,
J = 5.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 148.4, 92.4, 59.7, 56.7, 41.6. Anal.
Calcd for C5H11N3O3: C, 37.26; H, 6.88; N, 26.07. Found: C, 37.34, H, 6.99; N,
25.91.
8. Liu, J.; Li, C.; Waalkes, M. P.; Clark, J.; Myers, P.; Saavedra, J. E.; Keefer, L. K.
Hepatology 2003, 37, 324–333.
9. Gong, P.; Cederbaum, A. I.; Nieto, N. Mol. Pharmacol. 2004, 65, 130–138.
10. Liu, J.; Qu, W.; Saavedra, J. E.; Waalkes, M. P. J. Pharmacol. Exp. Ther. 2004, 310,
18–24.
11. Balogh, G. T.; Dalmadi, B.; Bielik, A.; Keserü, G. M. Comb. Chem. High Throughput
Screening 2005, 8, 347–352.
12. Liu, J.; He, Y.-Y.; Chignell, C. F.; Clark, J.; Myers, P.; Saavedra, J. E.; Waalkes, M. P.
Biochem. Pharmacol. 2005, 70, 144–151.
13. Liu, J.; Waalkes, M. P. Toxicology 2005, 208, 289–297.
14. 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.
15. 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.
16. Qu, W.; Liu, J.; Fuquay, R.; Saavedra, J. E.; Keefer, L. K.; Waalkes, M. P. Cancer
Lett. 2007, 256, 238–245.
28. O2-Vinyl 1-[N-(carboxymethyl)-N-methylamino]diazen-1-ium-1,2-diolate (14):
To a vigorously stirred solution of 13 (800 mg, 4.96 mmol) at 0 °C in acetone
(50 mL), Jones reagent (40 g CrO3 + 32 mL 98% sulfuric acid + 75 mL H2O) was
added dropwise; TLC showed a complete disappearance of starting material.
The reaction was quenched with 2-propanol (1 mL). The organic solvent was
removed using a rotary evaporator and the aqueous residue was extracted with
DCM. The crude product was purified by dissolving in 1.5 M NaOH and
washing with DCM. The aqueous layer was acidified using 6 M HCl until the pH
reached 3. The product was collected after DCM extraction followed by
removal of the organic layer under reduced pressure affording 14 (499 mg,
2.85 mmol, 57%) as an oil: UV (EtOAc) kmax (e
) 261 nm (9.2 mMÀ1 cmÀ1); 1H
NMR (400 MHz, CDCl3) d 8.43 (s, 1H), 6.79 (dd, J = 14.0, 6.7 Hz, 1H), 4.82 (dd,
J = 14.0, 2.4 Hz, 1H), 4.39 (dd, J = 6.7, 2.4 Hz, 1H), 4.30 (s, 2H), 3.32 (s, 3H). 13C
NMR (100 MHz, CDCl3) d 148.6, 91.9, 53.0, 40.6. Anal. Calcd for C5H9N3O4Á0.1
EtOAc: C, 35.17; H, 5.48; N, 22.60. Found: C, 35.39, H, 5.48; N, 22.60.
17. Edwards, C.; Feng, H.-Q.; Reynolds, C.; Mao, L.; Rockey, D. C. Am. J. Physiol.
Gastrointest. Liver Physiol. 2008, 294, G1311–G1317.
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50, 2069–2071.
19. Chakrapani, H.; Showalter, B. M.; Kong, L.; Keefer, L. K.; Saavedra, J. E. Org. Lett.
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20. Qu, W.; Liu, J.; Dill, A. L.; Saavedra, J. E.; Keefer, L. K.; Waalkes, M. P. Cancer Sci.
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29. O2-vinyl [2Àcarboxylato)pyrrolidinÀ1Àyl]diazen-1-ium-1,2-diolate methyl ester
(15): To a solution of 2 (100 mg, 0.57 mmol) in methanol (4 mL), a solution of
diazomethane in ether (1.5 mL) was added. The ensuing reaction mixture was
stirred at room temperature for 30 min and then concentrated under reduced
pressure. The crude product was purified by silica gel flash column
chromatography (9:1 CH2Cl2/ethyl acetate) to afford 15 (76 mg, 0.40 mmol,
21. The reaction was carried out in the same way as reported earlier (Ref. 19) but
was initiated at room temperature and not on ice. Detailed procedure: To a
71%) as a yellow oil: UV (EtOH) kmax (e
) 266 nm (8.70 mMÀ1 cmÀ1); 1H NMR
slurry of 5 (4.00 g, 21.8 mmol) and 15-crown-5 (100 lL) in THF (44 mL), 2-

4548
S. Y. Hong et al. / Tetrahedron Letters 50 (2009) 4545–4548
(400 MHz, CDCl3) d 6.74 (dd, J = 14.1, 6.7 Hz, 1H), 4.78 (dd, J = 14.1, 2.4 Hz,
solutions were carried out once per day. The data plotted were
of three independent experiments.
a mean
1H), 4.60 (dd, J = 8.5, 3.8 Hz, 1H), 4.34 (dd, J = 6.7, 2.4 Hz, 1H), 3.96–3.87 (m,
1H), 3.76 (s, 3H), 3.76–3.68 (m, 1H), 2.40–2.26 (m, 1H), 2.19–2.00 (m, 3H). 13C
NMR (100 MHz, CDCl3) d 171.6, 148.6, 91.4, 61.8, 52.4, 50.7, 27.9, 22.2. Anal.
Calcd for C8H13N3O4: C, 44.65; H, 6.09; N, 19.54. Found: C, 44.74; H, 6.10; N,
19.43.
31. In Hank’s Balanced Salt Solution (HBSS, 3 mL), 15 (30
lL, 10 mM DMSO stock
solution) was added. Porcine liver esterase (5 L, 14 units) was then added and
l
the resulting solution was incubated overnight at 37 °C. HPLC analysis was
carried out according to Ref. 30.
30. Stock solutions (10 mM) of the compounds in DMSO were prepared and
32. (a) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Jarvinen, T.;
Savolainen, J. Nat. Rev. Drug Disc. 2008, 7, 255–270; (b) Liderer, B. M.;
Borchardt, R. T. J. Pharm. Sci. 2006, 95, 1177–1195; (c) Potter, P. M.; Wadkins, R.
M. Curr. Med. Chem. 2006, 13, 1045–1054; (d) Beaumont, K.; Webster, R.;
Gardner, I.; Dack, I. Curr. Drug Metabol. 2003, 4, 461–485.
diluted in
concentration of 100
1
Â
pH 7.4 phosphate buffered saline (PBS) to
a
final
PBS)
lM
(10 stock solution in DMSO + 990 lL
l
L
and incubated at 37 °C in the dark. An Agilent 1100 series HPLC fitted
with
C-18 reverse-phase column (Phenomenex Luna 250 Â 4.60 mm)
operating at 262 nm and run isocratically with acetonitrile:water (75%,
v/v) was used to analyze the reaction course. Injections (20 L) of these
a
33. Chakrapani, H.; Maciag, A. E.; Citro, M. L.; Keefer, L. K.; Saavedra, J. E. Org. Lett.
2008, 10, 5155–5158.
l