Synthesis and biological evaluation of a novel phenyl substituted sydnone series as potential antitumor agents

Article abstract of DOI:10.1016/S0960-894X(03)00487-6

A series of compounds containing an N-(4′-substituted-3′- nitrophenyl)sydnone moiety with potential antitumor activity was prepared based on active analogues. The rationale behind the design of these compounds is presented along with the 4-step synthetic route to the derivatives in the 4′-position of the phenyl sydnone framework. Out of the six novel compounds, the 4′-fluoro derivative has an improved activity against all three cell lines as compared to the earlier leads.

Full text of DOI:10.1016/S0960-894X(03)00487-6

Bioorganic & Medicinal Chemistry Letters 13(2003) 2899–2901
Synthesis and Biological Evaluation of a Novel Phenyl
Substituted Sydnone Series as Potential Antitumor Agents
Christopher S. Dunkley and Charles J. Thoman*
Department of Chemistry and Biochemistry, University of the Sciences in Philadelphia, 600 S. 43rd St., Philadelphia, PA 19104, USA
Received 25 February 2003; revised 8 May 2003; accepted 9 May 2003
Abstract—A series of compounds containing an N-(40-substituted-30-nitrophenyl)sydnone moiety with potential antitumor activity
was prepared based on active analogues. The rationale behind the design of these compounds is presented along with the 4-step
synthetic route to the derivatives in the 40-position of the phenyl sydnone framework. Out of the six novel compounds, the 40-fluoro
derivative has an improved activity against all three cell lines as compared to the earlier leads.
# 2003Elsevier Ltd. All rights reserved.
Since their discovery mesoionic compounds have shown
a variety of biological activities including antitumor.1ꢀ4
It is thought that the ionic resonance structures of the
heterocyclic ring promote significant interactions with
biological molecules. In 1992 a series of 40-substituted-
30-nitrophenylsydnones were synthesized and evaluated
by Grynberg et al.5 for anticancer activity and it was
found that the 40-chloro and the 40-pyrrolidino com-
pounds significantly enhanced the survival of Sarcoma
180 (S180), Ehrlich carcinoma (Ehrlich) and Fibrous
histiocytoma (B10MCII) tumor bearing mice (Fig. 1). It
was also found that the larger hetero rings; p-piperidino
and p-morpholino, were less potent.
since the six-membered analogue (40-piperidino) was
inactive, we prepared the 40-azetidino compound to see
if ring size plays a role in activity.
It should be noted that no mechanism has been defini-
tively determined for the activity of these compounds,
but we may speculate about one. Fliedner6 found that
the acidity of (3-sydnonyl)acetic acid (pKa=1.70) was
almost identical to that of nitroacetic acid (pKa=1.68),
which means that the 3-sydnonyl moiety is as strong an
electron withdrawing group as the nitro. In nucleophilic
aromatic substitution, activation of a leaving group
(often chloro) is promoted by nitro groups in the ortho
and para positions. We propose that the facile substitu-
tion of the nucleophiles for the chloro in these studies
occurs because the o-nitro and p-(3-sydnonyl) groups
are acting like o,p-dinitro groups in normal nucleophilic
aromatic substitutions. This may also explain the mode
of activity of the active sydnone compounds against
cancerous cells; nucleophilic groups on DNA or some
other biological compound might be attacking the syd-
none compound in the same way, becoming attached to
it and somehow deactivated by it.
Herein we report the design, synthesis, and biological
evaluation of six para-substituted analogues of Gryn-
berg’s molecules. These were tested for anti-carcino-
genic activity against Sarcoma 180 (S180), Ehrlich
carcinoma (Ehrlich) and fibrous histiocytoma
(B10MCH) tumor bearing mice. Since the 40-Cl deriva-
tive was the most active of Grynberg’s compounds, the
chlorine was replaced with a fluorine to ascertain whe-
ther a stronger inductive effect or the substituent’s size
were important in determining activity. The 40-pyrroli-
dino-derivative was also active against two of the
strains, leading us to synthesize the two benzo deriva-
tives of it (the indolino and the isoindolino-). In addi-
tion, we made the open-ring analogue of the
40-pyrrolidino moiety, the diethylamino- group. Finally,
Figure 1. Structures of N-(40-chloro and 40-pyrrolidino-30-nitro-
phenyl)sydnones.
*Corresponding author. E-mail: c.thoman@usip.edu
0960-894X/03/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00487-6

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C. S. Dunkley, C. J. Thoman / Bioorg. Med. Chem. Lett. 13 (2003) 2899–2901
The first step, a condensation, involves neutralizing an
aqueous solution of chloroacetic acid with an equimolar
equivalent of NaOH and adding this solution over a
period of 4 h to an aqueous solution of either 40-chloro-
30-nitroaniline (a) or 40-fluoro-30-nitroaniline (b) while
under refux (Scheme 1). Heating is continued for 48 h
and the clear liquor is then vacuum filtered while hot, to
remove any decomposition products, and refrigerated
overnight. The resulting crystals are vacuum filtered and
rinsed with ether to obtain compounds N-(40-X-30-
nitrophenyl)glycine 1a (X=Cl) or 1b (X=F). A second
crop of crystals is obtained by evaporating the filtrate
and boiling the resulting solid in a 20% (v/v) ammonia
solution which dissolves the product. Once the ammo-
nia solution is cooled to room temperature, the starting
material is removed by filtration and the filtrate is acid-
ified with concentrated HCl and rinsed with water to
afford a second crop of 1a or 1b.
resulting crystals are filtered, rinsed with cold hexane
and recrystallized from water.
Sydnones 4b–d are all prepared following a slightly dif-
ferent protocol than that of 4a. The three nucleophiles;
azetidine hydrochloride, indoline and isoindoline, are
each treated with NaH in dimethoxyethane followed by
the slow addition of 3a. Each reaction has slightly dif-
ferent conditions; however, the progress of each reac-
tion is monitored using thin layer chromotography.
Both the indoline and isoindoline are extracted with
ethyl acetate which precipitates out the products 4c and
4d. These products are rinsed with cold hexanes and
recrystallized from ethanol. The azetidine sydnone 4b
does not precipitate out during extraction; therefore,
after concentrating the solution under reduced pressure,
column chromotography is used to further purify the
product.
The resulting 4-substituted-3-nitrophenylglycines 1a and
1b are then mechanically stirred at 0 ꢁC while an aque-
ous solution of sodium nitrite is slowly added over a 30-
min period. After an additional 10 min, the solid is
vacuum filtered, washed with water and allowed to dry
overnight to yield 2a and 2b quantitatively. The nitro-
sated compounds, 2a and 2b, are then treated with ace-
tic anhydride and stirred at room temperature for 12 h
to yield N-(40-X-30-nitrophenyl) sydnones 3a (X=Cl)
and 3b (X=F). The 40-chloro-30-nitrophenylsydnone 3a
precipitates upon formation and is recrystallized from
ethanol, but 40-fluoro-30-nitrophenylsydnone 3b must be
purified via column chromotography from a red paste.
Compounds 3a–b and 4a–d were prescreened at the
National Cancer Institute for antitumor activity. This
primary assay consists of a 3-cell line panel; MCF7
(Breast), NCI-H460 (Lung) and SF-268 (CNS). The
protocol consists of inoculating and incubating each cell
line on a microtiter plate. Test agents are then added at
a single concentration and the culture is incubated for
48 h. End-point determinations are then made with
alamar blue. Results for each test agent are reported as
the percent of growth of the treated cells when com-
pared to the untreated cells. Compounds that reduce the
growth of any one of the cell lines to 32% or less are
passed on for evaluation in the full panel of 60 cell lines
over a 5-log dose range. Although both 40-halogen sub-
stituted sydnones passed the primary assay by inhibiting
the growth of at least one cell line to less than 32%, the
novel 40-fluorosydnone derivative 3b proved to be both
more active and more versatile against each cell line
(Table 1). The four nitrogen containing analogues 4a–d
proved to be neither active nor versatile against any cell
line in the primary assay. In second stage testing com-
pounds 3a and 3b were evaluated against a full panel of
Subsequently, sydnones 4a–d are obtained by readily
replacing the 40-chloro group of 3a with the corres-
ponding nucleophiles.7 This ease of replacement is due
to the 10-sydnonyl and 30-nitro electron-withdrawing
groups which drastically increase the rate of the
nucleophilic aromatic substitution. Thus, the N-(40-di-
ethylamino-30-nitrophenyl)sydnone 4a is obtained by
stirring 3a with diethylamine neat at 40 ꢁC for 12 h. The
Scheme 1. Synthetic route to sydnone derivatives 4a–d. (i) ClCH2COOH (1 equiv), NaOH (1 equiv), reflux, 48 h; (ii) NaNO2 (1.1 equiv), 0 ꢁC, 1 h;
(iii) Ac2O neat, 40 ꢁC, 12 h; (iv) 4a diethylamine, 40 ꢁC, 12 h; 4b azetidine hydrochloride (1 equiv), NaH (1 equiv), 60 ꢁC, 24 h; 4c indoline (1 equiv),
NaH (1 equiv), rt, 18 h; 4d isoindoline (1 equiv), NaH (1 equiv), 50 ꢁC, 50 min.

C. S. Dunkley, C. J. Thoman / Bioorg. Med. Chem. Lett. 13 (2003) 2899–2901
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Table 1. In vitro antitumor activity of sydnones 3a–b and 4a–d against
3-cell lines. Results for each test agent are reported as the percent of
growth of the treated cells when compared to the untreated cells
1H and 13C NMR and elemental analysis. Sydnone 3b: mp
110–113 ꢁC. H NMR (acetone-d6) d 7.96 (dd, J=6.3, J=2.7,
1
1H), 8.59 (ddd, J=3.8, J=2.7, J=9.1, 1H), 8.80 (dd, J=10.4,
J=9.1, 1H); 13C NMR (acetone-d6) d 95.8 (s), 121.2 (d,
J=23.5, 1C), 121.5 (d, J=1.62, 1C), 130.1 (d, J=9.7, 1C),
131.7, 138.2 (d, J=2.10, 1C), 157.8 (d, J=268.6, 1C), 169.3.
calcd for C8H4N3O4F (225.14): C, 42.68: H, 1.79: N, 18.67:
O, 28.43: F, 8.44. Found: C, 42.74: H, 2.13: N, 17.68: F, 8.74.
Compd
MCF7 (%)
NCI-H460 (%)
SF-268 (%)
3a
3b
4a
4b
4c
4d
44
0
85
75
82
75
91
0
92
95
87
76
9
0
95
95
96
95
Sydnone 4a: mp 118–120 ꢁC. H NMR (chloroform-d1): d 1.23
1
(tr, J=7.3, 6H), 3.35 (q, J=7.3, 4H), 7.26 (d, J=9.3, 1H), 7.72
(dd, J=9.2, J=2.7, 1H), 8.14 (d, J=2.7, 1H); 13C NMR
(chloroform-d1): d 12.9, 46.6, 93.4, 120.5, 121.4, 124.0, 125.2,
139.3, 147.0, 169.1. calcd for C12H14N4O4 (278.27): C, 51.8: H,
5.07: N, 20.13: O, 23.00. Found: C, 1.79: H, 5.07: N, 20.14.
60 tumor cell lines at a minimum of five concentrations
at 10-fold dilutions and both compounds proved either
inactive or caused cell death, thereby terminating fur-
ther testing.
Sydnone 4b: mp 139–140 ꢁC. H NMR (chloroform-d1): d 2.5
1
(quin, J=1.9, 2C), 4.18 (tr, J=1.9, 4H), 6.68 (s, 1H), 6.75 (d,
J=2.4, 1H), 7.72 (dd, J=2.4, J=0.7, 1H), 8.20 (d, J=0.7,
1H); 13C NMR (chloroform-d1): d 16.4, 54.5, 93.3, 116.0,
120.3, 122.7, 125.7, 134.0, 146.8, 169.1. calcd for C11H10N4O4
(262.22): C, 50.38: H, 3.84: N, 21.37. Found: C, 50.47: H, 3.93:
N, 21.4. Sydnone 4c: mp 224–225 ꢁC. H NMR (DMSO-d6): d
1
References and Notes
3.18 (tr, J=7.9, 2H), 4.02 (tr, J=7.9, 2H), 6.64 (d, J=8.0,
1H), 6.94 (tr, J=6.8, 1H) 7.10 (tr, J=6.8, 1H), 7.31 (d, J=8.0,
1H), 7.88 (d, J=9.0, 1H), 7.89 (S, 1H), 8.16 (dd, J=9.1,
J=2.5, 1H) 8.61 (d, J=2.5, 1H); 13C NMR (DMSO-d6): d
28.5, 53.7, 95.1, 99.2, 120.2, 122.0, 122.9, 125.7, 126.5, 126.6,
126.7, 132.5, 138.8, 139.8, 144.9, 168.2. calcd for C16H12N4O4
(324.3): C, 59.26: H, 3.73: N, 17.28. Found: C, 59.04: H, 3.87:
1. Kier, L. B.; Roche, E. B. J. Pharm. Sci. 1967, 56, 149.
2. Shinzato, T. O.; Grynberg, N. F.; Gnomes, R. M.; Eche-
varria, A.; Miller, J. Med. Sci. Res. 1989, 17, 865.
3. Satyanarayna, K.; Rao, M. J. Pharm. Sci. 1995, 84, 263.
4. Kavali, Jyoti R.; Badami, Bharati V. Farmaco 2000, 55,
406.
5. Grynberg, Noema; Gomes, Rosa; Shinzato, Toshiko;
Echevarria, Aurea; Miller, Joseph. Anticancer Res. 1992, 12,
1025.
N, 17.17. Sydnone 4d: mp 211–212 ꢁC. H NMR (DMSO-d6):
1
d 4.73(s, 4H), 7.38 (m, 5H), 7.78 (s, 1H), 8.06 (dd, J=9.2,
J=2.4, 1H), 8.41 (d, J=2.4, 1H); 13C NMR (DMSO-d6): d
56.3, 95.2, 119.1, 120.7, 123.1, 123.3, 126.2, 128.4, 136.5, 136.6,
143.5, 169.3. calcd for C16H12N4O4 (324.3): C, 59.26: H, 3.73:
N, 17.28. Found: C, 59.06: H, 3.84: N, 17.12.
6. Fliedner, L. Doctoral Thesis, University of Mass.
(Amherst), 1964, 21.
7. Compounds 3b, and 4a–b were characterized by IR, MS,