Title:
Process for Novel 2-(3, 5-dimethyl-1H-pyrazol-1-y1)-1-arylethanones as DNA
photocleaving Agents.
Field of invention:
The present invention relates to a regioselective synthesis of some novel 2-(3,5-dimethyl-IHpyrazol-
1 -yl)- 1 -arylethanones under mild conditions, and evaluation of their DNA
photocleavage activities.
Background of Invention:
Pyrazole derivatives have attracted much attention in recent period owing to their diverse
biological activities as scaffolds in drug discovery and agricultural field. They are
extensively used as antifungal, antiviral, anti tubercular, anticonvulsant and antiinflammatory
agents. Moreover, they act as inhibitors against DNA gyrase and topoisomerase
IV at their respective ATP binding sites, GABA receptor antagonists and also found to
I
exhibit important chemotherapeutics properties like anticancer, antiproliferative and
antineoplastic. Some pyrazole derivatives having methyl group at position-3 and 5 were
found to act as potent antibacterial, antioxidant and anticancer agents. Literature survey
revealed that some phenacyl and napthacyl substituted azoles exhibit a wide range of
I
biological activities. N-Phenacylpyrazoles act as key precursors for the discovery of
regulators for apoptosis and autophagy in A549 lung cancer cells, N-(4-substituted
phenacy1)imidazoles and N-(4-substituted phenacy1)-2-methylimidazole act as potent
neuronal nitric oxide synthase inhibitors used in the treatment of cerebral ischemia and
I neurodegenerative diseases.
!
I Need of Invention:
Great biological potential and importance of 3,5-dimethylpyrazole, phenacylazoles and
napthacylazoles prompted us to continue our ongoing research work on the synthesis of novel
pyrazole derivatives as potential biological active agents. Therefore, we planned to synthesize
series of compounds having phenacyl or napthacyl moiety at position-1 and methyl group at
1
position-3 and 5 of the pyrazole nucleus. Some synthetic approaches for a few Nphenacylpyrazole
derivatives have already been reported in literature but suffer from certain
drawbacks such as use of hazardous organic solvents, extremely high temperature conditions,
prolonged reaction time, low yields and formation of side products. Therefore, an efficient,
mild and environmentally benign method for the synthesis of N-phenacyl I
napthacylpyrazoles is still highly desirable.
5960/DELNP/2010 concerns a process for the regioselective synthesis of 1-alkyl-3-haloalkylpyrazole-
4-carboxylic acid derivatives by cyclization of 2,3-disubstituted acrylic acid
derivatives, and to the hydrazones formed as intermediates in the process.
6348/DELNP/2010 relates to a process for the regioselective synthesis of 1-alkyl-3-haloalkylpyrazole-
4-carboxylic acid derivatives by cyclization of 2,3-disubstituted acrylic acid
derivatives with hydrazines in the presence of carbonyl compounds.
2673/DEL/2011 relates to an improved acid catalysed one pot synthesis of 2- styryl
quinoline.
None of the prior art indicate above either alone or in combination with one another disclose
what the present invention has disclosed.
Summary of the invention:
The present invention discloses the regioselective synthetic approach to novel 2-(3,5-
dimethyl-l H-pyrazol- 1 -yl)- 1 -arylethanones under facile and extremely mild solvent free
conditions. Among a series of compounds, 1- (4-bromopheny1)-2-(3,Sdimethyl-1H -pyrazol-
1 -yl)ethanone and 2-(3,5-dimethyl- 1 H-pyrazol- 1 -yl)- 1 -(4-nitropheny1)ethanone were found
to be highly active which completely degraded the both forms of DNA (SC and OC), even at
a very low 1 pg concentration only under irradiation of UV light. Compounds can be used as
such or be the basis of some novel efficient bioactive agents in future.
Detailed description of the invention:
Chemistry:
In continuation of our work related to the developments of greener approaches, we report a
highly regioselective and one pot method for synthesis of some 2-(3,5-dimethyl-IH-pyrazol-
1-y1)-1-arylethanones using sodium carbonate as a mild base under solvent free conditions.
The compounds were evaluated for their DNA photocleavage study with an aim to explore
their biological potential. Development of green synthetic protocols constitutes a major
challenge to the modern organic chemists. Recently, solvent free organic synthesis has gained
much importance due to high selectivity, clean reaction profile and mild reaction conditions.
In view of these observations, we developed a greener protocol to achieve regioselective
synthesis of 2-(3,5-dimethyl-1 H-pyrazol- 1 -yl)- 1 -arylethanone derivatives 4a-k using one pot
grind stone methodology (Scheme- 1).
In order to synthesize 2-(3,5-dimethyl- 1 H-pyrazol- 1 -yl)- 1 -phenylethanone 4a, acetylacetone
1 (1 mole eq.) was treated with hydrazine hydrate 2 (1.1 mole eq.) at 0 OC and the resultant
reaction mixture was ground with phenacyl bromide 3a (0.9 mole eq.) in the presence of
sodium carbonate (0.5 mole eq.) under solvent free conditions. The desired product 4a was
formed regioselectively within 10-15 minutes in excellent yield with high purity. The
structure of 4a was established on the basis combined use of IR, 'H NMR, I3c NMR
spectroscopy and mass spectrometry.
The 'H NMR spectrum of 4a displayed two sharp singlets due to six protons at 6 2.17 and
2.24 which affirm the presence of two methyl groups at positions- 3' and 5' of the pyrazole
nucleus, respectively. The CH2 protons resonated downfield as a singlet at 6 5.46 due to its
attachment with carbonyl carbon and nitrogen at position-1' of the pyrazole ring. Another
sharp singlet corresponding to one proton at 6 5.91 was observed due to 4'-H pyrazole ring,
besides a multiplet centered around 6 7.26-7.98 due to five aromatic protons. Further
evidence in support of an exclusive formation of 4a came by recording its I3c NMR spectrum
which showed the three characteristic signals of pyrazole system at 8 148.29, 105.91, and
140.73 due to C-3', C-4', and C-5', respectively. The formation of pyrazole was further
supported on the basis of its mass spectrum which showed a molecular ion peak at m/z =
214.1 (M') in conformity with molecular formula CI3Hl4N2o0f 4a.
ArCOCH2Br
+ NH2NH2H20
3a - k 2 d I 2 1
N-N
Na2C03
1 2
grind, 0 - 100 OC
For compounds 3,4 Ar =
Scheme-1: A highly regioselective one pot solvent free synthesis of 2-(3,5-
dimethyl- 1H-pyrazol-1-yl)-1-arylethanones 4a-k
In order to generalize the protocol, the reaction was carried out with different acyl bromides
3b-k under similar conditions and reaction progress was monitored through thin layer
chromatography (TLC) using petroleum ether and ethyl acetate as an eluent in ratio (5.7:l).
This method successfully resulted into the regioselective formation of 2-(3,Sdimethyl-1Hpyrazol-
1 -yl)- 1 -arylethanones 4b-k in excellent yields.
IR spectra of compounds 4a-k showed the characteristics absorption bands at 168 1 - 1699,
1584-1 628, 1552-1 554 cm-1 due to C=O, C=N and C=C stretching vibrations, respectively
DNA photocleavage activity:
The development of DNA cleaving agents is very important in the field of chemical and
medical sciences. In the area of drug design, considerable efforts have been made by the
chemists and biochemists to identify and characterize new biologically active moieties which
are capable of mediating scissions in nucleic acids strands. Especially, designing of the
compound based on their ability to cleave DNA under photo irradiation is of great importance
4
not only from the hndamental biological point of view but also in terms of photodynamic
therapeutic approach to develop potent antitumor agents. The photo irradiation technique has
a very high potential to strike at specific targeted sites. Photocleavage efficiency of the drug
is basically related to the tendency of relaxation of the super coiled (SC) DNA into open
circular (OC) DNA or more specifically degradation of both the SC and OC form of DNA.
To see the potential of 4a-k, agarose gel electrophoresis was used for the analysis of DNA
photocleavage activity. The cleavage potential of the test compounds were assessed by
comparing the bands appeared in control and test compounds in absence and presence of UVirradiation.
It has been observed that in the absence of UV-irradiation, the test compounds 4ak
showed no DNA degradation, because the intensity of both forms were found to be same in
comparison to control. However, under UV-irradiation 60 pg of compounds 4c, 4d, 4f, 4h,
and 4k degraded both the forms of DNA. To see the effects at low concentrations, experiment
was carried at 5 yg, 2.5 yg and 1 yg of concentration of the candidates that showed DNA
degradation at 15 pg and 30 pg (Fig. I). It was found that compound 4c cleaved the DNA
completely at all the three concentrations, while 4d showed complete cleavage at 5 pg and
2.5 pg and partial cleavage was found at 1 pg concentration. However, no significant effect
was observed in case of 4h at all the three tested concentrations.
A 6
Fig.7. DNA photocleavage activity of compounds 4c-d and 4h at 30 pg -1 pg
concentration in the presence of UV irradiation, 7A: Lane-1: DNA + 4c (30
pg), Lane 2: DNA+ 4f (30pg), Lane- 3: DNA + 4d (30pg), Lane-4: DNA +
4h (30pg), Lane-5: DNA + 4c (15~g)L, ane-6: DNA + 4f (15 pg), Lane-7:
DNA + 4d (lspg), Lane-8: DNA+ 4h (ISpg), Lane-9: Untreated control, 78:
5
Lane-1: Untreated control, Lane-2: DNA + 4c (5pg), Lane-3: DNA+ 4d
(5pg), Lane-4: DNA + 4h (5pg), Lane-5: DNA + 4c (2.5pg), Lane-6: DNA
+ 4d (2.5pg), Lane-7: DNA + 4h (2.5pg), Lane-8: DNA + 4c (lpg), Lane-9:
DNA+ 4d (lpg), Lane-10: DNA+ 4h (lpg)
Outcome of the Invention:
In conclusion a highly regioselective and one pot practical synthetic protocol has been
developed to achieve seven unknown and four known 2-(3,Sdimethyl- 1 H-pyrazol- 1 -yl)- 1 -
arylethanones. The structures of all compounds were established on the basis of rigorous
analysis of IR, NMR ('H and I3c) and mass spectral data. DNA photocleavage study showed
that the compound 4c and 4d having p-bromo and p-nitro substitution on phenyl ring,
respectively resulted in the complete DNA degradation even at lower concentration lpg,
whereas the compound 4f and 4h having p-fluoro and o-p-fluoro substituted phenyl ring,
respectively showed complete DNA degradation at 30 pg concentration. Therefore, the
compounds 4c and 4d may be used as DNA photocleaving agent in future.
Highlights of the Invention:
*3 Simple, Efficient, Economical and Fast reaction rate
*:* Regioselectivity under mild reaction conditions
*:* Does not need microwave irradiation etc..
Q Catalyst used is very cheap
*:* Environmentally benign practical appraoch
*3 Explored two novel and highly effective DNA photocleaving agents
Experimental:
Chemistry:
Melting points were determined by open capillary method and are uncorrected. The IR
spectra of the compounds were recorded on FT Infra-Red Spectrometer Model RZX (Perkin
6
Elmer) using KBr pellets. The 'H and I3c NMR spectra were recorded on Bruker Advance I1
400 NMR Spectrometer at 400 MHz and lOOMHz, respectively; chemical shifts are
expressed on &scale downfield from TMS as an internal standard. Mass spectra were
recorded on Waters Micromass Q-Tof Micro Mass spectrometer equipped with electrospray
ionization (ESI) and atmospheric pressure chemical ionization (APcI) sources having mass
range of 4000 amu in quadruple and 20000 arnu in ToF.
Substituted acyl bromides 3a-k were synthesized according to the literature procedure.
General procedure for synthesis of 2-(3,5-dimethyl- 1 H-pyrazol- 1 -yl)- 1 -arylethanones.
In dry mortar, acetylacetone (0.01 mole) was charged and cooled to 0 OC, and to it was added
hydrazine hydrate (0.01 1 mole) dropwise. The resultant mixture was further ground with
sodium carbonate (0.005 mol) and differently substituted acylbromides (0.009 mole) at 100
"C for an appropriate period of time as shown in Table-1. The progress of reaction was
monitored by thin layer chromatography. After completion of reaction, the reaction mixture
was washed well with ice cold water to remove the sodium carbonate, dried, and melting
point of the was noted.
Characterization data of 4c and 4d.
1 -(4-Bromopheny1)-2-0, 5-dimethyl- 1 H-pyrazol- 1 -yl)ethanone 4c. IR (v,,, cm-' ) : 1690
(CO str.), 1584 (C = N str.), 1552 (C = C str.); 'H NMR (400 MHz; CDC13, 6 ~2).0 6 (s, 3H,
3'-CH3), 2.14 (3H, S, 5'-CH3), 5.31 (s, 2H, 2-CH2), 5.82 (s, IH, 4'-H), 7.55 (d, 2H, 3", 5"-H,
3~H+1 = 7.56 Hz), 7.75 (d, 2H, 2", 6"-H, 3~H.H = 7.56 Hz) ; MS: m/z 292.0 (M') and 294
(~'+2) in the ratio showing typical bromine isotopic profile (1:l); Anal. Calcd for
CI3Hl3BrN20N: , 9.59%. Found: 9.49%.
2-(3,5-Dimethyl-1 H - p y r a z o l - 1 - y 1 ) - 1 - ( 4 - n i n e 4d. IR (v,,,,, cm-I): 1699 (CO
str.), 1602 (C = N str.), 1553 (C = C str.); 'H NMR (400 MHz; CDC13, 6 ~1).7 4 (s, 6H, 3', St-
CH3), 5.26 (s, 2H, 2-CH2), 5.49 (s, 1 H, 4'-H ), 7.76 (d, 2H, 2", 6"-H, 3~H.H = 8.48 Hz ), 7.86
(d, 2H, 3", 5"-H, 3~H.H = 8.36 Hz); MS: rn/z 259.1 (M'); Anal. Calcd for C13H13N303N: ,
16.2 1 %. Found: 16.15%.
DNA Photocleavage activity:
7
Treatment of DNA with rhe samples
The stock solutions for all tested compounds were prepared by dissolving 0.01 g of
compound in 0.5 ml of DMSO. All synthesized compounds 4a-k (60 pg - 1 pg) in DMSO
were added separately to volume of 10 pl containing plasmid DNA in TE (Tris 10 mM.
EDTA 0.01 mM, pH 8.0) buffer. The same volume of DMSO that was used to make the
solution of the tested compounds was added into the control (C). The reaction volumes were
held in caps of polyethylene microcentrifuge tubes, which were irradiated directly on the
surface of a trans-illuminator (8000 mW/cm) at 360 nm for 30 minutes at room temperature.
Irradiated samples were further incubated at 37 "C for 1 hour.
Agarose gel electrophoresis.
Following the treatment of DNA samples, electrophoresis of the samples was done according
to the procedure mentioned below.
To a 2 ml 50X tris-acetate EDTA buffer (TAE) (40 mM Tris, 20 mM acetic acid, 1 mM
EDTA, pH: 8.0). added 98 ml of autoclaved water to make it 1X TAE buffer. To the resultant
mixture agarose (0.8 g) was dissolved by boiling. When the gel attained 55 "C, 10 mglml of
ethidium bromide (ETBR) was added and then it was poured into gel cassette fitted with a
comb. The gel was then allowed to solidify. The comb was carefully removed and the gel was
placed over electrophoresis chamber flooded with tris-acetate EDTA buffer (40 mM Tris, 20
mM acetic acid. 1 mM EDTA, pH: 8.0). The treated DNA sample mixed with 6X loading dye
(0.25% bromophenol blue and 30% glycerol) was carefully loaded into the wells along with
untreated plasmid DNA, which acts as a control. and electrophoresis was carried out at
5VIcm for 2.0 h and the bands were observed under UV transilluminator.

' We claim:
---A- % *
l~flp.t0 7 44!74 ~JR
5- -
JAN 2014
I 1. A Process for regioselective synthesis of 2-(3,5-dimethyl-I H-pyrazol- 1 -yl)- 1 -
arylethanones of the formula (I) under solvent free conditions
(1)
in which
Ar is selected from the group consisting of
methyl groups at position 3 and 5
comprising of the reaction sequences (A), (B),(C) and (D)
(A) Charging of acetyl acetone(0.01 mole) in dry mortar and then cooling to O'C
(B) Adding hydrazine hydrate (0.01 1 mole) dropwise.
(C) Grounding of resultant mixture with sodium carbonate (0.005 mol) and differently
substituted acylbromides (0.009 mole) at 100 'C
I
a
WE$* 9 7 N 2014
(D) Washing of reaction mixture with ice cold water after completion of reaction to
I remove sodium carbonate and drying. I
2. The process as claimed in claim 1, wherein bicarbonates or carbonates of alkali and I
I alkaline earth metals are used under solvent free conditions. I
I 3. The process as claimed in claim 1, wherein the compounds 4c and 4d are used at
lower concentration of 1 pg under irradiation of UV light.
4. The process as claimed in claim 1, wherein the compounds 4f and 4h are used at
concentration of 30pg under irradiation of UV light.
5. The process as claimed in claim 1, wherein sodium carbonate are used upto
temperature of 100°C.
6. The process as claimed in claim 1, wherein the compounds 4c, 4d, 4f and 4h are used
I
I as DNA photocleaving agents.
Dated this January 1 7, 20 14
(Satya PAUTHORIZED
AGENT FOR THE APPLICANT
PATENT AGENT REGISTRATION NO. INIPA-389