Detour of prenostodione synthesis towards pyrazolones for antibacterial activity

Article abstract of DOI:10.1016/j.bmcl.2013.03.123

Our attempts to prepare indolyl acid (3), enroute to prenostodione (2), from phenyl-hydrazine following a reported procedure of Fischer-Indole synthesis rather lead to ethyl 2-(5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-3-yl)acetate as a major product, which underwent facile condensation with aldehydes to provide the pyrazolones. Intrigued by the opportunity for the diversity oriented synthesis of substituted pyrazolones, we have developed a facile one pot approach for pyrazolones and screened for antibacterial activity and, herein, results are reported.

Full text of DOI:10.1016/j.bmcl.2013.03.123

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3235–3238
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Detour of prenostodione synthesis towards pyrazolones
for antibacterial activity
a
a
a
a
a
Sivappa Rasapalli a, , Yingjun Fan , Menglong Yu , Christiaan Rees , John T. Harris , James A. Golen ,
⇑
Jerry P. Jasinski b, Arnold L. Rheingold c, Steven M. Kwasny d, Timothy J. Opperman d
a Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA
b Department of Chemistry, Keene State College, 229 Main Street, Keene, NH 03435-2001, USA
c Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0358, USA
d Microbiotix Inc., One Innovation Drive, Worcester, MA 01605, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Our attempts to prepare indolyl acid (3), enroute to prenostodione (2), from phenyl-hydrazine following
a reported procedure of Fischer-Indole synthesis rather lead to ethyl 2-(5-oxo-1-phenyl-2,5-dihydro-1H-
pyrazol-3-yl)acetate as a major product, which underwent facile condensation with aldehydes to provide
the pyrazolones. Intrigued by the opportunity for the diversity oriented synthesis of substituted pyrazo-
lones, we have developed a facile one pot approach for pyrazolones and screened for antibacterial activity
and, herein, results are reported.
Received 27 February 2013
Revised 26 March 2013
Accepted 27 March 2013
Available online 6 April 2013
Keywords:
Scytonemin
Published by Elsevier Ltd.
Prenostodione
Pyrazolone
Fischer-Indole synthesis
Anti-bacterial activity
Pyrazolones are pharmacophores of numerous compounds
(Fig. 1) that display activities such as analgesic and antipyretic
(propylphenazone, phenazone, metamizole etc.),1 anti-cancer
(TELIN),2 anti-ischemic (edaravone),3 and anti-anxiolytic.4 Pyrazo-
lones are gaining importance especially in drug discovery pro-
grams towards cerebral ischaemia5 and cardiovascular diseases.6
Due to its diverse pharmacological properties, the chemistry of
pyrazolones is gaining attention, and there have been numerous
novel methodologies reported recently.7 We describe herein our
research findings in this area.
Admittingly, our foray in to pyrazolones was rather a detour of
our efforts towards synthesis of prenostodione (2),8 a natural prod-
uct found in various cyanobacterial species and a precursor of scy-
tonemin (1).9 The cyanobacteria, formed on the bottoms of lakes,
ponds, and oceans, when matured are filled with air, and rise to
the surface to form bacterial mats where they encounter harmful
doses of ultraviolet radiation, triggering the production the bacte-
rial sunscreen, scytonemin. Scytonemin’s highly conjugated unique
dimeric indole-phenolic molecular structure protects the cyano-
bacterial cells from exposure to UV radiation.10 Scytonemin is an
obvious candidate for applications in the cosmetics industry, par-
ticularly in the realm of UVR absorbing sunscreens as it absorbs
in the electromagnetic spectrum of 325–425 nm akin to melanin,
the human pigment responsible for absorbing UVR for protection
of altering the stratified squamous cells in epithelium tissue.
Scytonemin also exhibits anti-proliferative and anti-inflammatory
bioactivities and is potent at micromolar concentrations. Scytone-
min (1) is the only small molecule natural product known to target
the polo-like kinase in the G2/M phase of mitosis of cancer cells
while remaining non-toxic to surrounding healthy tissue.11 It is
also known to target synovial tissue inflammation, and play role
in reducing arthritis symptoms.12 Due to the above properties, scy-
tonemin (1) has attracted considerable interest among synthetic
chemists. Our initial plan was to mimic this biosynthesis of scy-
tonemin, and we had set out to access prenostodione (2) via indolyl
O
N
O
O
O
S
O-
N
Na+
N
F
F
N
O
N
N
N
F
ARONIS023059
Metamizole sodium
O
propyphenazone
O
O
Cl
Cl
N
N
N
N
Cl
N
N
Cl
TELIN
Edaravone
Phenazone
⇑
Corresponding author.
E-mail address: srasapalli@umassd.edu (S. Rasapalli).
Figure 1. Biologically important pyrazolones.
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.03.123

3236
S. Rasapalli et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3235–3238
HO
HO
O
O
O
OH
O
O
OR
HN
N
NH
H
N
OR
H
O
OH
HO
Scytonemin (1)
Indole acid(3)
Prenostodione (2)
Figure 2. Biomimetic retrosynthetic relation of scytonemin and prenostodione.
acid (3) as shown in our the retrosynthetic analysis (Fig. 2). Stano-
vinik’s et al., have reported the synthesis of indolyl acid (3) from
phenyl hydrazine (5) and diethyl-1,3-acetonecarboxalate (4)13 by
modification of the Mill’s reaction conditions.14 Protonation of
nitrogen of hydrazone (6) under strong acidic conditions was pro-
posed to have prevented the further nucleophilic reactions with
the ester carbonyl to form the pyrazolone (7) and rather facilitated
the formation of indolyl acid 3 via Fisher-Indole cyclization path.
However, this reaction failed in our hands despite of large number
of attempts and modifications to the procedure and the major
product isolated was pyrazolone (7) or its tautomer 8 in our trials.
It appears that the reaction of phenylhydrazine and diethyl-1,3-
acetonecarboxalate was extremely temperature dependent and
seems to be a difficult prep to reproduce, at least in our hands.15
To form the desired indolyl acid 3, [3,3]-sigmatropic rearrange-
ment would need to occur from the intermediate hydrazone (6)
at À10 °C. We reasoned that the 3,3 sigmatropic rearrangement
is not favorable at such a low temperature which normally occurs
at higher temperatures in conventional Fischer-Indole synthesis,
especially in the absence of special conditions. Instead, the car-
bonyl of the initial hydrazone seems to have been protonated in
strong acidic medium initiating the intramolecular nucleophilic at-
tack by b-nitrogen of hydrazone forming a pyrazolone (7) that tau-
tomerizes to a hydroxyl pyrazole (8) that is inturn etherified. The
structures of the tautomeric compounds were confirmed through
single crystal X-ray structures.16 A brief literature search revealed
that this pyrazolone core was surprisingly under explored for anti-
bacterial activity17 though it offers a versatile scaffold for medici-
nal chemistry purposes due to its various intrinsic diversity
points.18 As part of our ongoing quest for new anti-biofilm and
anti-bacterial inhibitors,19 we thought of exploring the hitherto
unexplored pyrazolones further for antibacterial activity. There-
fore, we sought to shorten the synthesis (see Scheme 1).
aldehydes. Thus after investigation of different conditions, we were
able to find the conditions for one pot synthesis of pyrazolones in
decent yields by carrying out the pyrazolone formation and aldol
condensation in one pot in acetic acid as a solvent.20 The yields
and the structural details of the pyrazolones have been depicted
in Scheme 2. Although one pot approach seems to give somewhat
inferior yields compared to the two step procedure, the operational
simplicity and the use of milder conditions make the new synthesis
desirable for library generation. We have generated a small collec-
tion of pyrazolones and the details of the antibacterial and antibio-
film activities of some of the intermediates have been listed in
Table 1.
Antibacterial activity assays to measure the inhibition of plank-
tonic growth for each compound were carried out as described by
Clinical and Laboratory Standards Institute.21 The antibiofilm activ-
ity assays were performed according to the literature protocol.22
The antibacterial assay results of the pyrazolones were not partic-
ularly encouraging with inhibition ranging from modest to none.
However, of all the derivatives screened for various medically rel-
evant strains, the compounds Y4 and Y10 showed significant anti-
biofilm activity towards Staphylococcus aureus strains. Compound
Y4 also inhibited biofilm formation of Staphylococcus epidermidis.
We are currently evaluating the efficacy of pyrazolones for other
activities, and exploring the ways to further functionalize the scaf-
fold by condensation of the carbonyl compounds with the active
methylene group, by 1,4 additions to the benzylidine carbon, and
through cycloadditions. We are also exploring the alternative ways
to access modified indole acid (3) compound to complete the syn-
thesis of prenostodione, nostodione and will report the progress in
due course.
Conclusion
We reasoned that there was no need for concentrated sulfuric
acid, low temperatures and a separate step for condensation with
In summary, enroute to the synthesis of prenostodione and scy-
tonemin through Fischer-Indole synthesis, we developed a facile
OR
O
O
O
OH
O
HO
X
NH
N
OR
COOR
COOR
RO
RO
H
NH2
3
H
N
O
O
O
2
N
H
5
OH
N
O
HO
6
N
N
2 drops acetic acid
ether,
N
H
N
hydrazone intermediate
-10 oC, 0.5 h
+
OR
OR
O
O
Added dropwise
8
0
oC, 1 h
7
to Con. H2SO4
4
Scheme 1. Synthesis of pyrazolone as a major component in the attempted Fischer-Indole synthesis of acetoacetate.

S. Rasapalli et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3235–3238
3237
H
CHO
R
N
COOR
NH2
ROOC
N
N
H
6
O
N
O
O
O
NH4OAc, AcOH
10
N
R
O
RO
OR
NH2
5
NH4OAc, AcOH
CHO
N
H
Y1-10
[Yield over 2 steps]
6
(65%)
R
(40-55%)
[Yield of one step synethesis]
O
O
O
O
O
O
O
O
O
O
N
O
N
O
H
H
N
N
H
H
N
N
N
H
N
N
H
N
N
N
O
O
O
O
O
O
O
O
O
O
N
O
Y6
Y1
Y2
Y4
Y5
Y3
Cl
H
O
OH
O
O
O
O
O
O
O
O
N
H
N
N
N
N
N
N
H
H
H
O
N
Br
O
O
O
O
S
N
O
Y8
Y7
Y9
Y10
Scheme 2. Synthesis of pyrazolones in one pot three components and two step approaches.
Table 1
Antibacterial and antibiofilm activity studies of the pyrazolones
Organism strain 120802
Percent inhibitiona of overall growth or biofilm formation by 50
Staphylococcus epidermidis Staphylococcus aureus
GTC 18972 1457 ATCC 35556 MSSA
Growth Biofilm Biofilm Growth Biofilm Biofilm
l
M of following compoundsb
Pseudomonas aeruginosa
MRSA 1094 PA01
Growth Biofilm Biofilm
15.174
Growth
Growth
Growth
Y1
Y2
Y3
Y4
Y5
Y6
Y7
Y8
Y9
Y10
21.968
30.201
33.780
32.348
À2.371
25.953
27.695
33.780
15.564
2.596
4.080
15.021
15.751
23.045
À7.590
13.864
9.1862
14.292
À1.755
32.350
À1.834
3.695
10.632
6.0844
47.67
9.8639 À15.359
1.2414 À18.355
2.0267 À17.471
À2.456 34.016
À7.756 À0.723
À3.339 31.239
10.350 88.071
À19.679 62.024
À0.402
À5.327
1.491
21.951
À19.346
À5.327
57.819
9.955
2.808
7.952
20.947
38.003
À1.253
14.129
À5.607
À5.965
10.998
9.684
À7.0063
12.764
20.421
54.509
12.129
3.7488
10.788
19.789
86.697
8.0224
À15.605
À27.336
2.884
À0.864
À5.147
À10.794
À14.849
21.468
0.9137 À126.00
À38.520
6.4864
20.392
11.472
À166.74
35.856
À23.791
37.654
87.936
13.920
9.7021 À11.391
2.4461 À79.795
0.3554
1.531
À3.0547
À6.085
8.690
À5.7912 14.991
10.819
À3.784
9.212
À4.4127
À31.113
À22.853
20.536 À5.555
À0.983
À5.547
À61.898
À0.1692
54.976 82.624
50.814
À6.961 65.846
46.471
53.468
⁄
The compounds that showed decent activity have been shown indicated in bold.
Percent inhibition of overall growth and biofilm formation was calculated as follows: (OD600 + compound/OD600 untreated control) Â 100. Negative values are indicative
a
of overall growth or biofilm formation that was higher in the samples treated with compound than in the untreated control. Abbreviations: OD600, optical density at 600 nm.
b
Overall growth, which refers to bacterial growth in the liquid phase of the culture medium and to growth of bacteria attached to the surface of the assay well, is measured
as the OD600 of the assay well. Biofilm growth refers to bacteria surface attached bacteria only. To measure biofilm growth, nonadherent bacteria are removed by repeated
washing and adherent bacteria are fixed, stained with crystal violet, and quantified by measuring OD600 as described [Ref. 22].
one pot multi component approach for pyrazolones (Y1–Y10) in
decent yields. We have developed a small library of pyrazolones
varying different substituents on aromatic core and screened to-
wards various medically relevant bacterial screens with the hope
to tap the diversity of pyrazolones for lead development. However,
the activities are modest and not very encouraging for anti-bacterial
activity. It is pertinent to mention that, though the antibacterial
activity of the pyrazolones was not at useful levels, the decent
anti-biofilm activities of Y4 and Y1023 suggest that non-microbici-
dal compounds can be developed for external applications and
efforts are underway to generate a bigger library and further
screening, and also to explore the potential of the same for differ-
ent activities.
Acknowledgments
S.R. thanks Microbiotix Inc. and UMass Dartmouth for financial
support and JPJ acknowledges the NSF-MRI program (Grant No.
CHE1039027) for funds to purchase the X-ray diffractometer.
Supplementary data
Supplementary data (experimental details of synthesis and the
spectral & X-ray structural data of the compounds) associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2013.03.123.

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S. Rasapalli et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3235–3238
M.; Bernardo, V.; Bechi, B.; Castagnolo, D. Tetrahedron Lett. 2009, 50, 6572; (c)
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(0.54 g, 5 mmol), dimethyl 3-oxopentanedioate (0.87 g, 5 mmol) and aldehyde
(0.5 mmol) in acetic acid (8 ml) and NH4OAc (20% mmol, 0.08 g) was stirred for
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to afford the product as a red colored compound.
Data for Y4: Yield: 51%; mp: 138–140 °C. 1H NMR (DMSO-d6, 75 MHz): d (ppm)
3.69 (s, 3H), 3.98 (s, 2H), 7.21 (t, J = 7.4 Hz, 1H), 7.43 (t, J = 7.8 Hz, 2H), 7.65 (d,
J = 8.6 Hz, 2H), 7.86 (d, J = 8.1 Hz 1H), 7.93 (s, 1H), 8.59 (d, J = 8.6 Hz, 3H). 13C
NMR (CDCl3, 75 MHz): 34.41, 118.97, 119.52, 125.54, 126.87, 129.04, 129.23,
130.94, 131.42, 135.37, 138.18, 139.93, 146.64, 147.23, 161.81, 169.34. HRMS:
Calcd for C19H16ClN2O: 355.0849; Found: 355.0844. IR (neat): mmax: 1728, 1691,
1489, 1340, 852, 756, 669 cmÀ1
.
15. Through personal communication and through the foot notes of the
manuscript of Stanovnik et al. Ref.3
16. The structures have been deposited to the Cambridge Crystallographic Data
Centre. CCDC# 922934 (7), 922933 (8), 922932 (Y4) 922931 (Y9). Please see the
Supplementary data for the structural data.
Data for Y10: Yield: 47%; mp: 175–180 °C. 1H NMR (CDCl3, 300 Hz): d (ppm) 3.72
(s, 3H), 3.99 (s, 2H), 7.20 (t, J = 7.4 Hz, 1H), 7.42 (m, 5H), 7.74–8.02 (m, 2H), 8.44
(m, 1H). 13C NMR (CDCl3): 34.44, 36.37, 118.93, 119.34, 125.49, 127.45, 130.56,
133.29, 133.36, 133.56, 133.94, 144.49, 145.88. HRMS: Calcd for C19H16BrN2O3:
399.0344; Found: 399.0338.; IR (neat): m .
max: 1741, 1694, 1317, 850, 753 cmÀ1
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