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STUDIES IN CHEMISTRY OF O & N HETEROCYCLES
ABSTRACT .^.^
/ y ' / / '•*
: THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Boitor of ^l)tIo£opI)p w IN
CHEMISTRY
V
BY
MOHAMMAD ASAD
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2008
^ ^ S ^ ^
ABSTRACT
The work described in the thesis is based on the synthesis of heterocycHc
compounds from cheap and readily available starting materials. The compounds
chosen were 3-acetyl-4-hydroxycoumafin, 3-formyl-4-hydroxycoumarin, 6,7-
dimethyl-3-formyl-4-hydroxycoumarin, 5-chloro-3-methyl-1 -phenylpyrazole-4-
carboxaldehyde, 5-azido-3-methyl-l-phenylpyrazole-4-carboxaldehyde and 5-
amino -3-methyl-l -phenylpyrazole-4-carboxaldehyde.
The reaction of 3-acetyl-4-hydroxycoumarin was carried out with 3-
formylchromone. The reaction afforded heterochalcone 56. Further 56 was treated
with nitrogen bases such as hydrazine, phenylhydrazine and guanidine
hydrochloride to afford novel heterocycles 58a, 58b and 60c' respectively. The
structure of these compounds was inferred through spectral data.
He
Q
-Ha
Hb
Hd
HO / N N
58a-b 58a: R = H 58b: R = Ph
3-Formyl-4-hydroxycoumarin and 6,7-dimethyl-3-formyl-4-hydroxy
coumarin were readily converted to polyketomethylene compounds by treatment
with triacetic acid lactone under different reaction conditions. This led to the
formation of 68 and 69. The compounds 68 and 69 were transformed to pyrazole
derivatives 70a, 70b, 70c, 71a, 71b, 71c and isoxazole 70d by treatment with
nitrogen bases such as hydrazine hydrate, phenylhydrazine, hydrazine
benzothiazole and hydroxylammonium sulfate. The structure of these compounds
were established on the basis of spectroscopic studies and discussed at length in
the thesis.
R
O Hb O^ ,..0
68,69 H
\\ '
R'
Q o
0 \ ^ 0
CH^
O O N
70d
68: 69:
70a: 70b: 70c: 70d: 71a: 71b: 71c:
R' = R^ = H R ' = R^ = C H 3
R ' = R^ = H, R^=H R ' = R ^ = H , R^ = Ph R ' = R2 H, R' R' R' R'
R = H R^ = CH3, R = li
,3 R =CH., R .^
R'=R-=CH^, R - fo r
70a-c, 71a-c
In another reaction 3-formyl-4-hydroxycoumarin was treated with active
methylene compounds such as 5,5-dimethylcyclohexan-l,3-dione (dimedone) and
3-methyi-l-phenyl-5-pyrazolone to give 74 and 78 respectively. The structure of
these compounds was established through application of spectral data.
H,C \
. ^ ^ ^ ^ CH, t ' N 0 0 N Ph Ph
78
In another set of reaction 5-chloro and 5-azido-3-methyl-l-phenylpyrazole-
4-carboxaldehyde were treated with triacetic acid lactone and 4-hydroxycoumarin.
The reaction mixture afforded isomeric pyrones and benzopyrones viz. 94, 95 and
96, 97 respectively. The structure of these compounds was determined on the basis
of spectroscopic studies.
/ ^ ^
HiC
^^X 96 97
The reaction of 5-amino-3-methyl-l-phenylpyrazole-4-carboxaldehyde with
triacetic acid lactone was carried out to get the expected product 105 involving
translactonization type of rearrangement. The reaction however did not proceed as
visualized and afforded rather unexpected product 106.
0 0 Ph I H3C.
N. N N
H O
Ph
-CHi
105 106
The structure of the compound 106 was established through the application of
spectral data.
The compounds (68, 70a, 70b, 70c and 106) were investigated for anti
inflammatory, analgesic and antipyretic activities at the dose of 20 mg/kg
body weight in animal models. These compounds were found to possess
significant activities and their effect was compared with the standard drugs.
In anti-inflammatory effect, the compounds inhibited formalin induced
hind paw edema. The significant anti-inflammatory effect induced by the test
compounds 70a, 70b, 70c and 106 appeared at 1-2 hrs, progressively
increased, and reached 46.15, 88.46, 65.38 and 78,84% respectively at 5 hrs, while
the maximum anti-inflammatory effect of test compound 68 appeared at 1 hr (60%).
The anti-inflammatory effect induced by Diclofenac sodium (5 mg/kg) progressively
increased and reached a maximum 70.83% at 2 hrs as compared to control. These
compounds also significantly suppressed the formation of granuloma tissue in
cotton pellet induced chronic model of inflammation. The test compounds and
Diclofenac sodium significantly (P<0.05) reduced both wet weight as well as dry
weight in cotton pellet granuloma as compared to control. The effect of test
compound 70b in both reducing wet weight and dry weight of cotton pellet induced
granuloma was similar to that of Diclofenac sodium.
In analgesic test, the nociceptive response using hot plate test was
performed in rats. The test compounds produced significant analgesic activity
in hot plate test. The test compounds caused significant inhibition (P<0.05) of
the neurogenic (early phase) and inflammatory phases (late phase) of formalin
induced licking in rats. The Diclofenac sodium (5 mg/kg) also significantly inhibited
formalin induced licking in rats but only in late phase (15-30 minute). In contrast, the
reference antinociceptive drug Pentazocin (15 mg/kg) significantly reduced the
licking activity against both phases of formalin-induced nociception.
In the antipyretic tests, a model of pyrexia was used where baker's yeast
(135 mg/kg) was used to induce pyrexia in rats. The test compounds (68, 70a,
70b, 70c and 106) produced significant (P<0.05) antipyretic activity at 2 and 3
hrs. Among these compounds 70a and the standard drug Paracetamol (150
mg/kg) showed significant antipyretic activity throughout the observation
period up to 5 hrs. These test compounds and Paracetamol were also tested on
basal rectal temperature. The test compounds 68 and 70c were lowering of body
temperature at 2 hrs (0.12 and 0.5 °C respectively) following its administration. While
the maximum lowering of the rectal temperature noticed with the test compounds 70b
and 106 were 0.2 and 0.25 °C respectively at 1 hr and that of compound 70a and
Paracetamol were 0.1 and 0.05 °C at 1 and 3 hrs respectively.
The compounds (68, 69, 70a, 70b, 70c, 70d, 71a, 71b, 71c, 74, 78 and
106) were also screened for antibacterial activity against gram positive and
gram negative bacteria using Disc diffusion method. The compound 106
exhibited maximum antibacterial activity against both gram positive and gram
negative bacteria (zone diameter 22 mm against Staphylococcus aureus, 22 mm
against Escherichia coli, 15 mm against Pseudomonas aeruginosa, 18 mm against
Salmonella typhi and 25 mm against Klebsiella pneumoniae as compared to
antibiotic control Chloramphenicol (zone diameter 22, 23, Nil, 16, and 23 mm
respectively). Other compounds exhibited moderate activity against both gram
positive and gram negative bacteria. However, the compounds 71a and 71b
did not show any antibacterial activity.
STUDIES IN CHEMISTRY OF O & N HETEROCYCLES
/ > -THESIS /. : ^
SUBMITTED FOR THE AWARD OF THE DEGREE OF
. JBoitor of ^l)iIoiopI)p ^ t / / ;
IN
- C H E M I S T R Y ^ 1 1
^^^
W -j^;-^ '•D
-BY
/
MOHAMMAD ASAD
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
%-
2008
^x A»a<* f ,-^; ;
T6978
v_y
(Dr. Z£6a % Siddiqui M.Phil., Ph.D.
Reader
DEPARTMENT OF CHEMISTRY Aligarh Muslim University Aligarh-202 002 (India)
Ph. (Off) 0571-2703515 (Mob) 09412653054
E.mail: [email protected]
^Serdi^iedte
This is to certify that the thesis entitled "Studies in Chemistry of O & N
Heterocycles" submitted for the award of the degree of Doctor of Philosophy
(Ph.D.) in Chemistry to Aligarh Muslim University, Aligarh, India, is record of
bonafide research work carried out by Mr. Mohammad Asad under my guidance.
It is further certified that the thesis embodies the work of candidate himself and
has not been submitted for any degree either of this or any other university. The
present work is suitable for the submission for the above mentioned purpose.
hi CjJ.^ r. Zeba N. Siddiqui)
Supervisor
Res: C-23, Al-Hamd Apartment, Badar Bagh, Civil Lines, Aligarh-202 002
Primarily I would like to bow down my head in front of ALMIGHTY the most
beneficent the merciful for making this task reach its completion.
At the onset, I would like to express my thanks and gratitude to my supervisor
Dr. (Mrs.) Zeba N. Siddiqui. Her able guidance and meticulous supervision has
enabled me to overcome all hurdles in the light of her vast knowledge and her subject
expertise.
I would like to express my thanks to Prof. (Mrs.) Arunima Lai, Chairperson,
Department of Chemistry, AMU, Aligarh for providing all necessary research
facilities.
My overwhelming thanks go to my cousin brother Prof. Sartaj Tabassum and
Sister in law Dr. (Mrs.) Farrukh Arjmand, Department of Chemistry, AMU, Aligarh,
for their inspiration, scholarly advices and emotional support.
I am deeply indebted to Prof. Anil Kumar and Dr. Razi Ahmad, Department of
Pharmacology, Prof. Indu Shukla, Department of Microbiology, JNMC, AMU,
Aligarh, for providing facilities and carry out the biological studies.
My thanks to University Grants Commission, New Delhi, for their generous
financial assistance, and Regional Sophisticated Instrumentation Center, CDRI,
Lucknow and Punjab University, Chandigarh, for providing spectral facilities.
/ also acknowledge my colleagues Shagufta Praveen, Imrana Tabassum and
Mustafa for their practical support and candid discussion. I would like to express my
thanks to my friends Zishan Tabassum, Naved Azam and Mohd Azam for their
encouragement, cooperation, good company and healthy exchange of ideas. My
heartfelt thanks to all my relatives and my well-wishers especially my mamu Bahar-
Miyan Khan for his good wishes and prayers.
Last but not the least, I would fail in my duties if I do not acknowledge my
loving family and my parents for their unconditional support, yearning for my success
and accomplishments. To my sisters and brothers, I thank you for always praying for
my success and being a sweet support.
Aligarh ^ . p ^ ^ .
June, 2008 (Mohammad Asad)
Institutional Animal Ethics Committee (lAEC) Faculty of Medicine, Jawaharlal Nehru Medical College,
Aligarh Muslim University Aligarh (U.P.), India
Dated : 21st November, 2006
A meeting of the Institutional Animal pthics Committee was held on 21 •11-2006. The committee considered the research proposal entitled "Synthesis of pyrazole derivatives and screening them for their potential Medicinal use" submitted by Dr. Zeba N. Siddiqui, Reader Department of Chemistry, A.M.U., Aligarh for ethical clearance.
The committee did not find anything objectionable / unethical vis-a-vis animal subjects in the proposal. The proposal is, therefore, awarded ethical and biosafety clearance.
(Prof. Rabat Ali Khan) (Prof^^tbida Malik) Convener Department of Microbiology
Department of Pharmacology J. N. Medical College, J. N. Medical College, A. M.U., Aligarh
A. M.U., Aligarh
(Prof. Nafees Ahmad Farooqi) (Prof. Jamal Ahmad) Department of Anatomy Department of Medicine J. N. Medical College, J- N. Medical College,
A. M.U., Aligarh A. M.U., Aligarh
LIST OF PUBLICATIONS
> Synthesis and biological activity of heterocycles from chalcone.
Zeba N. Siddiqui, Mohammad Asad, Shagufta Praveen, Med Chem Res,
2007, DOI 10.1007/s00044-007-9067.
> New heterocyclic derivatives of 3-formyl-4-hydroxycoumarin.
Zeba N. Siddiqui, Mohammad Asad, Indian J. Chem, 2006, 45B, 2704-
2709.
CONTENTS
(Page Wo.
chapter 1
Introduction 1-3
Chapter 2
Theoreticat 4-22
Chapter 3
(Discussion 23-106
Chapter 4
(Jl) JLnti-infCammatory, anaCgesic 107-129
and antipyretic activities
((B) AntiB act eriaC activity 130-134
Chapter 5
'E^^perimentaC 135-162
(BiSCiograpfiy 163-171
Cfiapter 1
Introduction
1. Introduction
The work described in the thesis is based on the synthesis of heterocyclic
compounds from cheap and readily available starting materials. The compounds
selected were 3-acetyl-4-hydroxycoumarin, 3-formyl-4-hydroxycoumarin, 6,7-
dimethy 1-3-formyl-4-hydroxycoumarin, 5-chloro-3-methy 1-1 -phenylpyrazole-4-
carboxaldehyde, 5-azido-3-methyl-l-phenylpyrazole-4-carboxaldehyde and 5-
amino-3-methy 1-1 -phenylpyrazole-4-carboxaldehyde.
In the first an attempt was made to synthesize new heterochalcone
(2E)-1 -(4-hydroxy-1 -benzopyran-2-one-3-yl)-3-[ 1 ] (benzopyran-4-one-3-yl)-2-
propen-1-one 56 from 3-acetyl-4-hydroxycoumarin and 3-formylchromone.
Further heterochalcone 56 was converted to pyrazoline derivatives by carrying
out reaction in acetic acid with nitrogen bases such as hydrazine,
phenylhydrazine and guanidine hydrochloride. The reaction mixture, as
visualized, afforded 3-[4-hydroxy-[l]benzopyran-2-one-3-yr|-5-|5-(2-
hydroxylphenylpyrazol-4-yl]-pyrazolin 58a, l-phenyl-3-[4-hydroxy-[l]
benzopyran-2-one-3-yi]-5-[5-(2-hydroxyphenyl)-l-phenylpyrazol-4-yl]-
pyrazolin 58b and 3-amino-l-(4-hydroxy-1-benzopyran-2-one-3-yl]-3-( 1-
benzopyran-4-one-3-yl)-propen-l-one 60c' respectively.
In another series of reactions 3-formyl-4-hydroxycoumarin and
6,7-dimethyl-3-formyl-4-hydroxycoumarin were readily converted to poly
ketomethylenc compounds by treatment with triacetic acid lactone under
different reaction conditions. This led to the formation of 3-acetoacctylpyrano
[3.2-c] [IJ benzopyran-2,5-dione 68 and 8.9-dimelhyl-3-acetoacetylpyrano
[3,2-c] f 1] benzopyran-2,5-dione 69. The polykctomethylcne compounds 68
and 69 were transformed to pyrazole derivatives 3-(3-methylpyra/,ol-5-yl)-
pyrano |3 . 2-cJ 111 benzopyran-2,5-dione 70a, 3-(3-mctliyl-l-phenyl pyra/.olo-
yl)-pyrano [3, 2-cJ [IJ benzopyran-2, 5-dione 70b, 3-(3-methyl-l-
benzothiazolo pyrazol-5-yl)-pyrano [3,2-c] [1] benzopyran-2.5-dione
70c, 8,9-dimethyl-3-(3-methylpyrazol-5-yl)-pyrano [3,2-c] [1 | benzopy
ran-2,5-dione 71a, 8, 9-dimethyl-3-(3-methyl-l-phenylpyrazol-5-yi)-
pyrano[3,2-c][l]benzopyran-2,5-dione 71 b, 8,9-dimethyI-3-(3-methyI-I-
benzothiazoiopyrazoi-5-yi)-pyrano [3,2-c] [1] benzopyran-2,5-dione 71c
and isoxazolc 3-(3-methyl isoxazol-5-yl)-pyrano L3.2-c] [1] benzopyran-2,5-
dione 70d b}' treatment with nitrogen bases such as hydrazine hydrate,
phenylhydrazine, hydrazinobenzothiazole and hydroxylammonium sulfate. All
these compounds were synthesized easily and in one step. In another set of
reactions 3-formyl-4-hydroxycoumarin was treated with active methylene
compounds such as 5,5-dimethylcyclohexan-l.3-dione (dimedone) and 3-
methyl-I-phenyl-5-pyrazolone to give 7-(4-hydroxycoumarin-3-yl)-IO.IO-
dimethyl-8-oxo-8,9.10.l 1-tetrahydro pyrano [3,2-c| coumarin 74 and
methylidene-bis-4,4-(3-methyl-5-oxo-1 -phenylpyrazole) 78 respectively.
Ihe reaction of 5-chloro-3-methyl-l-phenylpyrazolc-4-carboxaldehyde
79 and 5-azido-3-mcthyl-l-phcnylpyrazole-4-carboxaldchyde 80 with cnol
lactones such as triacctic acid lactone and 4-hydroxycoumarin were carried out
under hydrolytic conditions, fhc reaction mixture afforded isomeric
benzopyrones namely. 4-(4-hydroxy-6-melhyl-2-oxo-2Il-pyran-2-onc-3-
yl)-3.7-dimelhyl-l-phenylpyrazol() 13,4:2,3|-41I-pyran() |3.2-b| pyran-5-
one 94, 4-(4-hydroxy-6-melhyl-2-oxo-2H-pyran-2-onc-3-yl)-3,7-
dimethyl-1-phenylpyrazolo [3,4:2.3]-4H-pyrano |3,2-c| pyran-5-onc 95.
4-(4-hydroxy-2-oxo-2H-l-benzopyran-2-one-3-yl)-3-mcthyl-l-phcnylpyr
azolo [3,4;2,3]-4H-pyrano [3,2-b]-l-benzopyran-5-one 96 and 4-(4-
hydroxy-2-oxo-2H-1 -benzopyran-2-one-3-yl)-3-methyl-1 -phenylpyrazolo
[3,4:2,3]-4H-pyrano [3,2-c]-l-benzopyran-5-one 97.
Lastly and in continuation of earlier work in the department,
efforts were directed towards the synthesis of pyranopyridines and
pyranocoumarins containing 1,3 dicarbonyl unit in the side involving
translactonization type of rearrangement''^ employing 3-formyl-4-
hydroxycoumarin, 2-amino-3-formylchromone and triacetic acid lactone.
The reaction when extended to 5-amino-3-melhyl-l-phenylpyrazole-4-
carboxaldehyde did not give the expected product. It instead, gave a
dimmer 3,6-dimethyl-l,8-diphenyl-diazocino [3,4-c:7,8-c'j bis pyrazole
106. The dimmer 106 was perhaps obtained through Friedlander
condensation reaction and is discussed in the thesis.
The structure of all compounds was established through
spectroscopic studies. Some of these compounds were evaluated for
their anti-inllammatory, analgesic, antipyretic and antibacterial
activities and arc discussed in chapter 4.
2. Theoretical
The work on the synthesis of new heterocyclic compounds of
pharmacological interest has made extensive use of 4-hydroxycoumarin
derivatives. The presence of OH group at 4-position gives nucleophilic
character at position 3 to 4-hydroxycoumarin. Another possibility is opening of
the lactone ring followed by loss of carbon dioxide. During the last thirty years
synthesis and the study of the biological activities of coumarin derivatives has
been the aim of many researchers. Also the structure activity relationship of
coumarins has revealed that the presence of substituents such as methyl,
formyl, acetyl, and amino groups at position 3 is an essential feature for their
pharmacological action. Based on the findings, we describe the work done by
different researcher for the synthesis of compounds featuring different
heterocyclic rings fused on to the coumarin moiety. Thus, in the context of
further work on 4-hydroxycoumarin a survey of the literature of thirty years
was done and some relevant examples are discussed below.
2.1. The reaction of 4-hydroxycouinarins with benzhydroxymoyi chloride.
The condensation reaction of 4-hydroxycoumarin la, 8-methyl-4-
hydroxycoumarin lb, and 8-chloro-4-hydroxy coumarin Ic with
benzhydroxymoyi chloride"^ 4 in the presence of EtsN gives 3-benzoyl-8-
chloro-4-hydroxycoumarin oximes 5a-c. The reaction involves nucleophilic
attack of double bond at position 3 of 4-hydroxycoumarin on benzonitrile oxide
generated in situ by base treatment of benzhydroxymoyi chloride (Scheme 1).
la-c O H ( B
C^H 6"5
5a-c
a: R = H b: R = CH3 c: R = C1
Scheme 1. Formation of oximes 5a-c by the reaction of 4-hydroxycoumarins la-c and benzhydroxymoyl chloride.
2.2. The reaction of 4-hydroxycoumarin with 1,4-Naphthoquinone.
One of the earliest example of 4-hydroxycoumarin 1 with 1,4-
naphthoquinone 6 involves Michael addition of 1 to the 1,4-naphthoquinone,
oxidation of the resulting quinol 7 by air to give 8 and by more unchanged 1,4-
naphthoquinone, a second Michael addition of 1 and another oxidation of the
product 9 to give 9a (Scheme 2) /
Scheme 2. Formation of quinone 9a by the reaction of 4-hydroxycoumarin 1 and 1,4-naphthoquinone 6, Formation of quinol 9 by the reduction of quinone 9a with sodium dithionite.
The acetylation of 9 forms tetra acetate derivative 10 whereas
methylation gives unexpected dimethoxycoumestan derivative 11.
10
11
However, when the reaction is extended to 5,5-dimethyicyclohexan-l,3-
dione, the reaction occurs in the same manner except that in the final step,
cyclization of the disubstituted quinol 12 takes place in preference to oxidation
(Scheme 3).
>\^bo^
Scheme 3. Formation of 3,3,9,9-tetramethyl-3,4,9,10-tetrahydrobenzo [ 1,2-b:4,5-b'] bis (benzofuran-1,7(2H,8H)-dione 13 by the reaction of 1,4-benzoquinone and dimedone.
The reaction of 1,4-benzoquinone with 4-hydroxycoumarin in aqueous
acetic acid is similar to that reported earlier^ except that the major product is
colourless quinol 14. Oxidation of 14 with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone gives the quinone 15 which was identical with that obtained
from the aqueous acetone reaction.
14
An extension to such type of reaction of 4-hydroxycoumarin la-g is
117 treatment with a-chlorobenzylphenyl ketone 16 in anhydrous potassium
carbonate to give 4-(l,2-diphenyl-2-oxoethyloxy) coumarins 17a-g which on
stirring with PPA (P2O5 and H3PO4) undergoes cyclization to furnish the
cyclized compound 18a-g (Scheme 4).
18a-g l,18(a-g) : R, R2 R3 R4
a: H H H H b: CH3 H H H c: H CH3 H H d: H H CH3 H e: H H NO2 H f: R, = R2 = benzo H H g: H H R3 = R4=benzo
Scheme 4. Formation of coumarins 18a-g by the reaction of 4-hydroxy coumarins la-g and a-chlorobenzylphenyl ketone 16.
In a similar type of reaction 3-(2'-cyclopentenyl)-4-hydroxy [1] benzopyran
20 is obtained by refluxing 4-hydroxycoumarin 1 and 3-chlorocyclopentene 19 in
acetone potassium carbonate. Compound 20 is finally converted to its acetate
derivative 21 by carrying out reaction with Ac20/NaOAc. The acetate 21 was then
converted to chromone derivative 22 by conducting the reaction with pyridine
hydrobromide (PyHBrs) in dichloromethane. The same compound 22 is also
obtained when compound 20 is treated with PyHBrs in CH2CI2. The formation of
chromone 22 is indicated by Infrared (IR) spectroscopy showing absorption band
at 1650 cm"' and a double doublet for proton per/ to carbonyl group at 5 8.30 in its
nuclear magnetic resonance (NMR) spectrum. However, the isomerisation of
chromone 22 into coumarin 23 takes place when the compound 22 is heated with
50% of H2SO4 (Scheme 5).
^ ^ o
Acetone / K2CO3
-HCI
50% H2SO4 Br
H
/ ^ o o^ "o
Scheme 5. Formation of chromone 22 and coumarin 23 by the reaction of 4-hydroxycoumarin 1 and 3-chlorocyclopentene 19.
i n
2.3. The reaction of 4-hydroxycoumarin with l-aryloxy-4-ehlorobut-2-
ynes.
Another interesting reaction of 4-hydroxycoumarin is formation of ether
4-(4-aryloxybut-2-ynyloxy) [1] benzopyran-2-ones 25(a-f). The reaction takes
place between l-aryloxy-4-chlorobut-2-ynes 24 and 4-hydroxycoumarin 1 in
refluxing acetone in the presence of anhydrous potassium carbonate
(Scheme 6).^
? ^ .
MejCO-KjCOj
^ O A r
OAr 25a-f
24
a, Ar=2-methylphenyl
b, Ar=4-methylphenyl
c, Ar=3-methylphenyl
d, Ar = 2,4-dimethylphenyl
e, Ar = 3,5-dimethylphenyl
f, AT = 4-tert-butylphenyl
Scheme 6. Formation of ether 25a-f by the reaction of 4-hydroxycoumarin 1 and l-aryloxy-4-chlorobut-2-ynes 24.
When ether 25a is heated in chlorobenzene, two products are obtained.
One of the product has been identified as 4-(2-cresoxymethyl) pyrano [3, 2-c]
[1] benzopyran-5-(2H)-one 26a and the other product as 27. Ether 25b when
refluxed in chlorobenzene also furnishes two products. One of the product is
identified as 4-(4-cresoxymethyl) pyrano [3,2-c] [1] benzopyran-5(2H)-one
26b, and the other product as 28 (Scheme 7).
11
OAr
CfiHcCl / A -6"5' 15h
25
OAr
26a-b
26a : Ar = 2-methylphenyl 26b : Ar = 4-methylphenyl
Scheme 7. Formation of pyranopyran 26a-b by the reaction of ether 25a-b and chlorobenzene.
The fonnation of 27 from 25 may be explained by an initial [3,3]
sigmatropic rearrangement to give allene moiety 29 followed by isomerisation
to butadiene 30 by a [1,3] H^ shift"' and acid catalysed cyclisation (enol may
also act as an acid) of 30 to 27. The product 26 is also formed from
intermediate 29 via enolisation followed by [1,5] H* shift and electrocyclic ring
closure. ' Further the compound 27 is formed rapidly in preference to
product 26a. The product ratio, however, is not changed even when the
reaction is carried out in purified chlorobenzene or in the presence of toluene 4-
sulfonic acid (Scheme 8).
12
? ^ . .OAr
^ / ^ OAr O ^ O
29
27 -*
^ / ^ OAr "O O
i = [3,3] sigmatropic rearangement ii = isomerization by 1,3 H® shift
26(a-f)
^ - ^ . OAr ^O O
Scheme 8. Formation of coumarin 27 and 26(a-f) from ether 25.
2.4. The reaction of 3-formyl-4-hydroxycoumarin with amines.
3-Formyl-4-hydroxycoumarin 31a-d is an interesting starting material
for the synthesis of a variety of heterocycles. One such example is treatment of
31 with ethanolic solution of hydrazinehydrate, phenylhydrazine and
hydroxylamine hydrochloride in acetic acid to give corresponding IH, 4H-4-
oxo-benzopyrano [4,3-c] pyrazoles 32a-d, 4H-4-oxo-l-phenylbenzopyrano
[4,3-c] pyrazoles 32a-h and 4H-4-oxo benzopyran [3,4-c] isoxazoles 33a-d
(Scheme 9).
13
RNHNH, CH3COOH
EtOH
O^^O
32a-h 33a-d
32a, 33a
32b, 33b
32c, 33c
32d, 33d
32e
32f
32g
32h
RI — R2 ~ R3 = H, R — H
Ri = CH3, R2 — R3 = H, R = H
R2 ~ CH3 Ri = R3 = H, R = H
R3 — CH3 R] = R2 = H, R — H
Ri = R2=R3=H, R = Ph
R, = CH3,R2=R3=H,R = Ph
R2=CH3,Ri = R3=H,R = Ph
R3=CH3,Ri = R2=H,R = Ph
Scheme 9. Formation of pyrazoles 32a-h and isoxazoles 33a-d by the reaction of 3-formyl-4-hydroxycoumarin 31 a-d with hydrazines and hydroxylamine hydrochloride.
Another example'" in this category is the reaction of 3-formyl-4-
hydroxycoumarin 31 a-d with malonic acid in the presence of zinc chloride to
give 2H, 5H-2,5-dioxo-3-pyrano [3,2-c] benzopyranoic acids 34a-d which on
treatment with methanol in the presence of thionylchloride furnishes methyl
2H, 5H-2,5-dioxopyrano [3,2-c] benzopyran-3-oates 35a-d. The compounds
35a-d was further converted into its acid hydrazides 36a-d, upon treatment
with hydrazine (Scheme 10).
14
+ HjC / COOH ZnCU
CHO 'COOH
H2NNH2
C—NHNH2
36a-d O O
SOCUin ^ MeOH
COOH
COOCH3
35a-d O
36a : R,,R2, R s ^ H 36b : Ri = CH3, R2R3 = H 36c : R,,R3 = H, R2 = CH3. 36d : Ri,R2 = H, R3 = CH3
Scheme 10. Formation of hydrazides 36a-d by the reaction of 3-formyl-4-hydroxy coumarin 31a-d with malonic acid, thionylchloride and hydrazine.
K. Rad-Moghadam and M. Moheseni have reported'' the reaction of 3-
formyl-4-hydroxycoumarin 31 with an in situ generated 1:1 adduct of
triphenyiphosphine and dialkyl acetylenedicarboxylate to form the dialkyl-2H,
5H-pyrano [3,2-c] [l]benzopyran-5-one-2,3-dicarboxylates (pyranocoumarins),
38a-c. The reaction may be rationalized on the basis of well-known Chemistry
of trivalent phosphorus nucleophiles. Addition of triphenyiphosphine to
acetylenic ester results in a zwitterionic 1:1 adducts which upon abstraction of
a proton from 31 and concomitant addition of thus formed 3-formylcoumarin-
15
4-hydroxylate anion produces the key intermediate phosphorane 37. The
reaction uUimately entails with an intramolecular Wittig reaction of the
postulated phosphorane 37 (Scheme 11).
COOR CHO HC
, IK®
O O COOR
31
COOR
Intramolecular Wittig reaction
COORcoOR
38a : R = Me 38b : R = Et 38c : R = t-Bu
PPh,
38a-c
Scheme 11. Formation of coumarins 38a-c by the reaction of 3-formyl-4-hydroxy coumarin 31 with triphenylphosphine and dialkyl acetylenedicarboxylate.
2.5. The reaction of 3-formyl-4-hydroxycoumarin with phosphorus
hydrazides.
3-Formyl-4-hydroxycoumarin 31 reacts with phosphorus hydrazine
derivatives NH2-NR2-P(S)(ORi)2 to give phosphorus hydrazide of coumarin
40a-c/41a-c.''* The same product 40a-c/41a-c is also obtained when chromone-
3-carboxylic ester 39 is treated with phosphorus hydrazines. The reaction
16
involved conversion of chromone-3-carboxylic ester to 3-formyl-4-
hydroxycoumarin under basic conditions followed by reaction with phosphorus
hydrazine to give 40a-c which tautomerized to 41a-c (Scheme 12: Route A).
However, formyl group of 31 also directly reacts with phosphorus hydrazine to
give 40a-c which then tautomerizes to 41a-c (Scheme 12: Route B). Both the
tautomers are present in solution form as well as solid form and are
inseparable.
OH /OH
OCH,
^ ) O- H
Toutomerization O OH O OH
O ^ ^ O
41a-c 40a-c R=1|J-P(S)(0R,)2
R2
3! Rj ~ C2H,5, R2 ~ CH3 b: R, = CH3 R2 = CHj
Scheme 12: Route A. Synthetic route to the phosphorus hydrazides of coumarin 41a-c and benzopyran-2,4-dione 40a-c.
17
NH,R OH /NHR
41a-c
R = ]j4-P(S)(OR,)2
R2 &'. Rj = C2H5, R2 — CH3 b:R, =CH3 R2 = CH3 c: R, = C2H;, R2 = H
Scheme 12: Route B. Synthetic route to the phosphorus hydrazides of coumarin 40a-c and benzopyran-2,4-dione 41a-c.
2.6. The reaction of 3-acetyl-4-hydroxycouinariii with hydroxylamine.
3-Acetyl-4-hydroxycoumarin 42 is another interesting starting material for
the synthesis of heterocyclic compounds of pharmacological value. Thus, in 1955
Klosa'^ reported that 43 affords crystalline oximes on treatment with excess of
hydroxylamine hydrochloride and potassium acetate in refluxing ethanol.
However, Desai and coworkers'^ suggested that the oxime of Klosa was a
18
cyclodehydrated product 44. They also said that the oxime 43 is obtained only at
room temperature and did not undergo Beckmann rearrangement upon treatment
with SOCI2 or PCI5 and instead gives cyclodehydrated product 44 (Scheme 13).
Some authors'^ also have reported the formation of isoxazole 44 at room
temperature under basic conditions. In order to clarify this discrepancy the reaction
of 3-acetyl-4-hydroxycoumarin 42 with hydroxylamine hydrochloride was
reinvestigated by Chantegrel et al and found that the reaction of 42 with
hydroxy lamine under Klosa's reaction conditions affords a mixture of 45 and 46.
The formation of 45 involves the oxime 43 as intermediate and a nucleophilic
attack at the C-2 lactone carbonyl by the hydroxyimino group with ring opening
(Scheme 14). The Compound 46 is obtained by interaction of 45 with an excess of
hydroxylamine (Scheme 15).
NHzOH/EtOH
Room temperature
Scheme 13. Formation of isoxazole 44 by the reaction of 3-acetyl-4-hydroxycoumarin 42 and hydroxylamine.
19
Scheme 14. Formation of isoxazole 45 by the reaction of 3-acetyI-4-hydroxycoumarin 42 and hydroxylamine under the Klosa's reaction conditions.
\ NH^OH. HCl TM *'
/ (-H2O) ^^^::^oHo^?
O-H N—Q
-=Hl vVt
zHl +H
^---^QH^C^ ^N-OH
N—Q N—
0 46
-0
CH3
Scheme 15. Formation of isoxazole 46 by the reaction of 45 and excess of hydroxylamine.
20
2.7. The reaction of 3-acetyI-4-hydroxycoumarm with thiourea and
arylsulfonamides.
In another reaction, 3-acetyl-4-hydroxycoumarin 42 is used for the
synthesis of some antibacterial compounds by carrying out reaction with thiourea
and benzenesulfonamides 19
In this context, 3-acetyl-4-hydroxycoumarin 42 is treated with phenyl
trimethyl ammonium bromide to afford 3-bromoacetyl-4-hydroxycoumarin 47
which is then treated with thiourea to give 48 in the form of a bromide salt. The
fmal compound sulfonamide 49a-d is obtained by treating 48 with arenesulfonyl
chlorides (Scheme 16).
OH o OH O
(I)
42
^HSOr{Q^^ J OH N-
NH,HBr
49a-d 48
a : R = CH3 b : R = C1 c : R = OCH3 d : R = NH2
(I) Phenyl trimethyl ammonium tribromide, THF, 25 °C, 15 min.
(II) (NH2)2CS, ethanol, reflux 30 min.
(III) Ar SO2CI, pyridine, 25 °C, 12 hrs.
Scheme 16. Formation of sulfonamide 49a-d from 3-acetyl-4-hydroxycoumarin 42.
21
2.8. The reaction of 3-acetyl-4-hydroxycoumarin with phenylhydrazine.
Recently authors have shown the conversion of 3-acetyl-4-
hydroxycoumarin 42 into its hydrazone derivatives by performing the reaction
under microwave irradiation using Zn [L-proline]2 complex as catalyst^^ in
order to obtain the high yield of the product as compared to the one obtained
under the conventional heating procedure. The reaction involved nucleophilic
addition of hydrazine on acetyl carbon followed by cyclodehydration to form
the product 50a-c (Scheme 17) 21
o^ ^o
MW/120°C Zn[L -Proline]2
R-NHNH2 -H^
RNHNH,
:^Z R"
,N N
50(a-c)
50a : R = Ph 50b : R = ClPh 50c: R =N02Ph
Scheme 17. Formation of pyrazoles 50a-c by the reaction of 3-acetyl-4-hydroxy coumarin 42 and phenylhydrazines under the microwave irradiation.
22
3. Discussion
3-Acetyl-4-hydroxycoumarin 42 has served as starting material for the
synthesis of many novel heterocycles. Thus, pyrazole derivatives are obtained
when the reaction of 3-acetyl-4-hydroxycoumarin is carried out with
phenylhydrazine. ' The hydrazone 51 has been earlier shown to undergo
nucleophilic ring opening followed by cyclodehydration to give 52 on heating
in acetic acid (Scheme 18) 22
O CH3
Scheme 18. Formation of pyrazole 52 by the reaction of 3-acetyl-4-hydroxycoumarin 42 and phenylhydrazine.
23
It is also shown that on heating the hydrazone 51 with alcoholic HCl, it
is converted to 53.
However, when the hydrazone 51 is heated with another mole of
phenylhydrazine the isomeric compound 54 is obtained (Scheme 19).
o^ ^o
HN-NHPh N-^NHPh
Scheme 19. Formation of isomeric pyrazole 54 by the reaction of hydrazone 51 and phenylhydrazine.
3.1. Synthesis of chalcones
These above mentioned reaction involves nucleophilic addition on
carbonyl carbons of pyrone ring as well as of acetyl group. Thus, it seemed
interesting to utilize acetyl group of 3-acetyl-4-hydroxycoumarin for the
synthesis of new heterocycles such as heterochalcones.
24
,23 Chalcones are important precursors of flavonoids and are generally
synthesized from acetophenones and aromatic aldehydes under basic
conditions.' '* Thus, it was thought worthwhile to synthesize new
heterochalcones employing 3-acetyl-4-hydroxycoumarin 42 and 3-
formylchromone 55, which are heterocycles of pharmaceutical value, under
very mild basic conditions. The reaction mixture, as expected, afforded
heterochalcone 56 exclusively as shown in Scheme 20.
^\(?=^ Pyridine (few drops)
Scheme 20. Formation of chalcone 56 by the reaction of 3-acetyl-4-hydroxycoumarin 42 and 3-fonnylchromone 55.
25
The compound 56 gave positive lest with ferric chloride due to phenolic
4 -OH group. The compound was characterized on the basis of spectroscopic
data. The IR spectrum (Fig. 1) of 56 showed sharp bands for chromone and
coumarin carbonyl groups at 1650 and 1734 cm' respeetivel}. fhe broad band
at 3318 cm"' was due to OH group. Another sharp band at 1610 cm' was
assigned to carbon-carbon double bond. The 'H N M R spectrum (Fig, 2) of 56
showed trans olefmic protons Ha and Hb as ortho coupled doublets at 6 9.10
(J = 15.6 Hz) and 8.18 (J = 15.9 Hz) respectively. The appearance of doublet
for Ha proton in very low field was due anisotropic effect exerted by two
carbonyl groups (C-4 and C-1). The presence of chromone moiety was inferred
through a sharp singlet integrating for one proton (C-2) at 5 8.56 and a doublet
(J = 7.8 Hz) integrating for one proton (C-5) at 8 8.30. The remaining three
protons of chromone unit and four protons of coumarin moiety appeared as
multiplet in the region 8 7.45-7.93. The mass spectrum (Fig. 3) of 56 showed
M^ at m/z 360 as base peak. The other important peaks were obtained as shown
in Scheme 21(a-b).
26
OH ^O O m/z 360
56
OH 90
O ^ ^ O H ^ , ^ / 0 ^
OH O
0 0 O m/z 360
OH O
®
OH O
H
f-CH3
OH O m/z 204
O m/z 157
Scheme 21a. Mass fragmentation of chalcone 56.
27
0 0 O m/z 360
56
RDA Cleavage
m/z 120
+ •
+
-CO
c=o
m/z 92
m/z 240
(CH=C=0)
-X5^ + •
O O m/z 199
Scheme 21b. Mass fragmentation of chalcone 56.
28
( ;
ei_
(gvoo.vvtiztr);
(996*01-'/»•/*/)
-(-996«)k-'«:9«8-)—i g
(698i'9-'8?'00lO
UJ a. CO
o
M
lO
T -
0 >-eo
I II X
^ i ; ;
!Se«Mi- ' S00S91) --fz60se-'»i>£/:i)
i ; j ;
t ^ •
;
i :
j .'
Lt-' I • i 1
;'
;'
;'
• — t —
I — -
;
> 1 ,' '
y • • •
< > . • • 1
; ^ .'
: : :
— • • •
o o N
,_ o o n
o
£
o £ 3 C «
5
II >•
00 o>
II X
o
J
Fig. 1: IR spectrum of 56
29
• -S - -=,? = £ - = - • : ! ; ,
681 0-
~V
i eascc !
00P9 E
-OJ
OSS' ; - ,
;=;; rGC 5JC
5t6 lei
fSt
8/y
flQ U'b
gf,';
bt^l
uc
i -; ! •
^-5 -
£ - '
a •••
P —'
e-; B ' b -_
1 •.,t i=
• ; ~
• • /
'1
+ u-fir:
;9r..
;
I
1
561-re
-LD
-CD
E
Fig. 2: 'll NMR spcclmm of 56
30
I S
Q
03
C3 - m
o j
OJ
in G)
(J3
CS
s .-\J
> 0 2
1
ru r \ j
fN ,
• • ." iS V en 4-> ; * t 10
O I oc cr a f -H
_l c
+ 11 1.
a; • D
O >. c o
1—1 r - i L. fO
ai r — J
*-> m -r^l '
^ O — •
CJ"
0)
o _J
+-•
u
en m CQ ' y
C>1 U~)
CSJ
o
s
s
E
t"
i n
^ e OJ 3 r\ j
.. > *J O a Z
a LT) ir.
w
Q n U)
cr a 21 O i -
fV a en 1
X •
• J
u ai u
r::
( i >
(V
n
(U
r) X
1 -
fa
:U
t J
Ul
c — (. -
('•) 3 '
_ r
ro N IV tS)
—. <o (") .NJ
C
(S) U l
CJO CO
V cv. r fx) 4-
IM
I
1
CI j 1
ra s
133 •Ji
, X
s
CD CO
—^
-
cs
s
3 - 3
—
CS
1
CD T"
m fS CSC l i '
3 S
- CD
m
- c i
S
C2
c:i L i 2 Lfi Q: i;i o
Fig. 3: Mass spectrum of 56
31
The condensation of a, P-unsaturated carbonyl compounds with
hydrazines usually results in the formation of pyrazolines. "^ Due to presence of
a, (3 unsaturated carbonyl unit in compound 56 we also thought that reaction of
56 with hydrazines would occur in usual manner to give 57.
R = H R = Ph
However, the reaction did not take place as visualized to afford 57. Due
to the presence of chromone unit in compound 56, the 4-pyrone ring suffered
ring cleavage ' at C-2 by the nucleophilic hydrazines (hydrazine and
phenylhydrazine) to form pyrazole moiety along with the formation of
pyrazoline moiety with a, P-unsaturated carbonyl unit (Scheme 22).
32
NH-NH-R
OH ^NH-NH-R
R-NH
^ ^ ^ O H2NNHR '^A (-H2O)
HO / J — N
^ 58a-b
58a: R = H 58b: R = Ph
Q =
Scheme 22. Formation of bipyrazoles 58a-b by the reaction of chalcone 56 and hydrazines.
The reaction of 56 with hydrazine hydrate afforded 58a and
phenylhydrazine gave 58b. The ring opening of chromone moiety by
hydrazines was also confirmed by positive ferric chloride test (thin layer
chromatography plate developed in chloroform-methanol mixture in the ratio
3:1 and sprayed with ferric chloride when a black colored spot was observed)
and absence of diagnostic signal for C-2' proton in the nmr spectra of both the
compounds.
33
Both the compounds (58a and 58b) were identified on the basis of
spectroscopic data. The IR spectrum (Fig. 4) of 58a disphiyed broad bands at
3618, 3456, 3396, and 3269 cm"' due to the presence of two OH and two NM
groups. A sharp and strong absorption band at 1684 cm' indicated a carbonyl
group in the compound. Since absorption band for chromone carbonyl groups
generally appear in the region 1620-1650 cm"', ^ the band at 1684 was assigned
to coumarin rather than chromone carbonyl group. The presence of pyrazoline
unit in 58a was clearly established by two double doublets at 5 3.66, 3.72 (Ha),
4.04. 4.09 (Hb) and a multiplet at 6 5.51 (He). The singlet (0 ,0 exchangeable)
at 8 6.36 was assigned to NH proton of pyrazoline unit. The aromatic region of
the spectrum showed eight protons of coumarin and phenolic units in the form
of multiplet at 8 6.91-7.58. A sharp singlet integrating for one proton was
assigned to Hd proton of pyrazole unit. Two other broad singlets (D2O
exchangeable) at 5 10.6 and 12.8 were assigned to two OH groups (phenolic
OH group and 4-OH group of coumarin unit) (Fig. 5). Further confirmation of
the structure was provided by mass spectrum (Fig. 6) which showed M' at 388.
The other prominent peaks were obtained as shown in Scheme 23(a-c).
/ N N H m/zl35
Scheme 23a. Mass fragmentation of bipyrazole 58a.
35
HO / N N H
m/z 186
ni/zl59
Scheme 23b. Mass fragmentation of bipyrazole 58a.
36
o^^o.
H m/z388 58a
H m/z295
m/z387
H*
m/z359
Scheme 23c. Mass fragmentation of bipyrazole 58a
- 7
a. (A 00 o o « i r IP cs
«o
CO
II > •
•A O at o es II X
• • • • • f -
-;
- i r -
™ir_
(8<ioo9zseofr)
(9Z00-9'98 8 « )
(8Z!00'9'WBOfi)
(Uo/8e' sf sze)
. ( iW9* '.E8046 ) . . . . . (oSisVKiedi)
(SS89Z 09KU)
(;s6sie'9S'eEzi.)
i e8^^"^' e i p w i ) (:z6si-€'?8qpci.)
- ^~-—HszEzr/fiosi)
(E96Ce'990tit)
(e96«e'89l-9Bl)
( o H c f s z z r t t )
1 i
Kg
a o o S2
E 3 c »
1
o o o
(E99S7^'W69Ze)j
S6Zi 2E' Biseee) - : (•9E9VZV ' »S«9t^ )
«B9^-z-V9sm9€) - m»9£^
•o
cd « II
>-o rvi a> o C4 II X
o iA
§ O o
Fig. 4; IR spectrum of 58a
38
r o
M
tt.
r = os.- as-c oj
X z m D .1 ^ o> " — ; 0 1 0 0 0 0 0 t^ 10 in in O o • O " lO r^ (U (\1 • ID Q
o o o
" «
>? ^
- 1 -
3 O X
B99 0
90f ee.
- " g e t I
~^9to e
_ 000 i
V
f7ff 0
/
O t/l — 2 1 O
^ o
-OJ
-UD
-CO
L o
n n
Fig. 5: n NMR spectrum of 58a
39
in
en nj
(S
1 o
1 — «
t-T i_ 4-> u ll) o
U l
1-1
i n
_• ^ •
r j iM
> < ) y. i t i f i
Q en St
r (T U
_ l (1
3
tr • (1
(n LL
I o
O' a (M
— 1!
;.
c n
~'
• f j
( ] OJ t
^ LJ
L i i j
r
— 1
r n
ro E
n
<r
(11
(; X t—
1-
^ r':
f \ .
* r iX: f ;
u:
c — h
rvi
—
U) ' • " ^
'^
* - •
r
(\) (M W N
m (T) CO
• ^
CD
C)
( n
0
i n r^
tt)
cn
ai O l I It) i -
N
\ fc
(S f
IS LD
in
G) m
(3 OJ
S
(n
OD
:E
- ^
Z
=
~
r<j
Q
i M
G) - c n
CD
- CD
I D
( S - i n
'XI
IN. ' cn
j i
— r e
LD - ' '^•. — ( s
— iD CTl ' - ^ un —
"~ JJ
- rr
(X
in
S
-^ g-
(s:i
t \
OJ ^
(.n (V (\i
^ —
~ --
IT.'
r i j
n - a ;
tJ G
CD r ~
^1 —
111 rn C C c 1 LL 3 •.n 0-. rn c^
Fig. 6: Mass spectrum of 58a
40
When the reaction of heterochalcone 56 was carried out with guanidine
59 in relluxing AcOIl, the reaction mixture turned reddish brown after
completion of the reaction, fhe reaction mixture was then poured into cold
water to afford a dirty white solid. The pure compound, however, was obtained
as white powder after repeated crystalHzation.
O NH2 O
60c'
As a result of above mentioned reaction the compound 60a or 60b
should be formed in the normal course of the reaction. The compound, melting
point 156-158 ''C, showed M' at m/z 377 in its mass spectrum (Fig. 7).
Therefore, if the nucleophilic guanidine would have reacted with (x. P
unsaturated unit, 60a should be the possible structure for the compound. If
pyrone ring of chromonc unit suffers ring cleavage, then structure 60b could be
assigned to the compound. Hut both the structures were ruled out on the basis
41
of M ' . The III spectrum (Fig. 8) showed strong and slightly notched absorption
bands at 1686 cm' . There was also present a sharp and strong bands at 1620
cm' . These absorption bands could be easily assigned to a eoumarin, a ketone
carbonyl and a chromone carbonyl group respectively. In addition to this a
broad band was also present in the IR spectrum at 3437 cm"' which indicated
the presence of OH/NH group/s in the compound. The 'll NMR spectrum
(Fig. 9) of the compound showed presence ofpyrazoline unit due to two double
doublets each integrating for one proton at 5 3.31 (Ha), 3.63 (Hb) and a
multiplet also integrating for one proton at 5 4.88 (He).
The intact chromone ring was inferred by the presence of a sharp singlet
integrating for one proton at 6 8.45 for C-2 proton. The proton per/ to carbonyl
group (C-5) was present in the form of double doublets at 8 8.27. There was
also present a double doublet integrating for one proton at 5 7.99 and may be
due to C-5 proton of eoumarin unit. Usually four benzenoid protons of
eoumarin appear as multiplet,^^ but some times splitting of C-5 proton of
eoumarin moiety also take place.' Rest of three protons of eoumarin and
chromone units were present as multiplet in the region 6 7.36-7.76. Combining
these spectroscopic features one arrives at structure 60c/60c' for the compound.
Probable mechanism for the formation of 60c/60c' is depicted in Scheme 24.
42
H 56
.0> H2N-rc—NH2
59
HO .NH H2N—C
We NH2
NHn 60c/60c'
O NH
Ac-^0-^C=NH
t I
O NH ®
OAc H2N
^ N H 2
Scheme 24. Formation of 3-amino-l-(4-hydroxy-l-benzopyran-2-one-3-yl]-3-(l-benzopyran-4-one-3-yl)-propen-l-one 60c/60c' by the reaction of chalcone 56 and guanidine hydrochloride.
Coumarins are also phenolic in nature and therefore give positive test
with ferric chloride but compound 60c did not give any color when TLC plate
was sprayed with ferric chloride solution. It may be due to predominant
existence of compound in the tautomeric form 60c'. The IR spectrum also
showed only one broad absorption band and that may be givens to NH2 group
rather than OH group. The structure 60c' was well accorded with M*, which
was at m/z 377 in its mass spectrum. The other important peaks were obtained
as shown in Scheme 25.
43
QCl> + •
-CO
n I 0 H
m/zl71
W°l 0
m/z 199
Scheme 25. Mass fragmentation of 60c'.
44
f > CO •
<N \ n r (O r g
Ok (O
V-
f " * ; • c
o o
^ SS:
'1- O
;; o » ". ?? » o
: 4
I fc c U J o « o >n O i o c. m o >« o "Ji o m o i/> o o
5. ^ SDuepunciv a*!lE|Sb
Fig. 7: Mass spectrum of 60c'
45
S ;
3 ! 0> I ; CO
I to I n
(W6l€Z'W»S)
: (>k?n.'«fiSPA : (i»fez u • oz t so t ) •
(6e8fZl'96'99£l)
" ~ r ^ —<«6«^=;*j-
(^Z.'9t
•3
••h
(j(:i8»8i
I
zisz)
esot)
ZEW)
999U
n
I
o o
o o o
o o 10 o o r
o
Fig, 8: Mass spectrum of 60c'
46
m r-, a .t= ry 5
I..
["•'
Fig. 9: H NMR spectrum of 60c'
47
3.2. The reaction of 3-forinyI-4-hydroxycouniarin (31) with active
methylene compounds.
3-Formyl-4-hydroxycoumarin 31 is as acidic as acetic acid. The formyl
group in 31 is labile under strongly acidic or basic conditions. Therefore, the
condensation reactions of 31 with active methylene compounds such as 4-
hydroxy-6-methyl-2-oxo-2H-pyran-2-one (triacetic acid lactone), 5,5-dimethyl
cyclohexan-l,3-ciione (dimedone) and 3-methyl-I-phenyl-5-pyrazolone was
carried out under mild conditions.
The reaction of 3-formyl-4-hydroxycoumarin (31) and 6,7-dimcthyl-4-
hydroxycoumarin (67) with 4-hydroxy-6-methyl-2-oxo-2H-pyran-2-one
(Triacetic acid lactone) (64).
o-Hydroxybenzaldehyde (salicylaldehyde) 61 reacts with 4-hydroxy
coumarin 1 to give product 62.' In a later study 62 was found to be untenable
and structure 62 was revised to 63 29
OTH
Rearrangement
48
In a series of papers, Spanish workers have shown that structures of type
62 are unstable and consistently rearrange to type 63 through intramolecular
translactonization.^^ Thus, when salicylaldehyde 61 was treated with triacetic
acid lactone 64, the intermediate 65 could not be obtained due to its conversion
to 66 involving translactonization (Scheme 26).
/ < : > ^ Q H Q J P > \ / C H 3
o o
Scheme 26. Formation of 3-acetoacetylcoumarin 66 by the reaction of salicylaldehyde 61 and triacetic acid lactone 64.
Similar type of rearrangement was observed when the reaction was
extended to 3-formyl-4-hydroxycoumarin 31. Thus, when 31 and 6,7-dimethyl-
3-formyl-4-hydroxycoumarin 67 were treated with triacetic acid lactone 64 in
alcohol and methanol respectively, the rearranged products 68 and 69 were
obtained quantitatively (Scheme 27).
49
OTH OH fOii O
0 - ^ 0 0 ^ 0 ' -CH3
31:R' = R^=H 67:R' = R^ = CH3
R O ^ O O ^ O - -CH3 O^ /CH,
Intramolecular translactonization
68:R' = R^ = H 69:R' = R^ = CH3
Scheme 27. Formation of 3-acetoacetylpyrano [3,2-c] [1] benzopyran-2,5-dione 68,69 by the reaction of 3-formyl-4-hydroxycoumarins 31,67 and triacetic acid lactone 64.
The structure of compounds 68, 69, obtained as a result of trans
lactonization, was established on the basis of spectroscopic data. Thus, the IR
spectrum (Fig. 10) of 69 showed a broad band at 3377 cm"' and a strong and
sharp band at 1720 cm'' along with another sharp & slightly weak absorption
band at 1756 cm''. These bands were assigned to OH, coumarin and enol
lactone carbonyl groups respectively. The ' H N M R (Fig. 11) of 69 exhibited
50
the presence of three methyl singlets at 6 2.27. 2.38 and 2.42. Two more
singlets each integrating for one proton at 6 6.88 and 8.91 were assigned to
Ha and Hb protons respectively. Two aromatic protons (C-5 and C-8)
appeared as singlets at 8 7.84 and 7.26. Further confirmation for structure 69
was provided by mass spectroscopy showing M at m/z 326 in its mass
spectrum (M^+1 peak at m/z 327 in Fig. 12). The other peaks were obtained
as shown in Scheme 28.
51
iWz 92
( i ) -CH^C \ / \ / ^ ^ I
v^P (ii) RDA Ov
m/z 120 O m/z 213
Scheme 28. Mass fragmentation of 8,9-dimethyl-3-acetoacetylpyrano [3,2-c] [1] benzopyran-2,5-dione 69.
52
< z o z
(DJ91.-8!'19 265) '.
<o i o
(O CO
II >-!>» 00 o!
II
o o o
: ' ^ = r r " ~ — r (i69£'6Z ' 96i9Sei)
c I T - - irr-^rteeee^^ez' gi-*??!)
. jn:!7^^-=--<TMpeK • tt^wcz)
(6990 9Z • Z6 9i£C) - ;
o o a> eo o O
to o in o o
CO
o 1 O
1 •
T -
E in hm
V J2
E 3 C ID
> n ^
o o o fO
i
1 <0 ;' i
g ^ 1 C»j <o , eo { II
>- • 1 ^ ; CO 1
«> O)
" CM II
X
r-
J. -*• 1-
•
^•i t -
r'
< > ; ^fr-' . 4 . { ::• i
Fig. 10: IR spectrum of 69
53
I . •—
Cl Z :•} —
( - U.1 I ' l o f^ • • fj> l O i . ) .. u~) O
a; !_'
f \ j
I (VJ U l
n i r i
"'
X o •T:
T
o.
<ii
o r-l o I D - y •ct .-• i n I P
? o o C--
' T
« ^ o c. IJ-l
•r:
o
o ..-> ' " • '
i; 1:,
( J
<I>
s s III iri
C '-1 o o o o .M O
r^ ;:
s :' U l 1
... : o
•y
1^ T .J ~
3 o c i :::
-. ^ u: ^' L'l o '-i^ y:* j : — '
geooo 0-
SOKqs
fsaee G^f8^ tBoze
Bocge
mm JfCiE /^s'ljs'
0 I
'. I
1
c'-
c-e-
--, -^" _ \ - - —
: / " —-.
.'— ^^''
-%
%
\
^ ,
Z'ii?.L\ f-
90a9c i -
f ^'^.'t i9-,a I
G 0 i I 5 9 - ._J OOOU 1
uidU
Fig. I I : 'll NMR spectrum of 69
54
3
n
G) OJ (M
en m OJ
c\j -OJ
en
IS
I D
V CD 1 n
en >%
en
.•vj I a OJ
CD rv j
CD
OJ
C Q CE tn tr a I o
OJ r
^ m a E CD a 3 U V u OJ * j CO OJ U OJ I HI III CE Q. i x Z
13 'D O CD LD Z
CD
Li .
OJ • o O
c o
(S
D U
cs en
CD
en in
J S
a ^ a-, rr 0) o j a . CD c - • • - — fK LD
_ l — V n
LD
n
D
C C 13 t-' ^ O O c C3
I— J) i-< r\S
e C-. 3
CD
(S
s n
cs OJ
IS J —
o
z S G S3 C i 3 C
i n (S
QJ C • C!. - T >~ E in
I— — Q
E CS M 3 • \ V CS
(S (S
00
o i n
(S in
0) i-" a . I - 0 - 3
en Qi DQ o
n , -4- •^
cs
IS LC
IS i n
IN. CS
en CD OJ ,
cr IV o-' ,
E l
(S OJ
CS
s
IS en OJ
iS CD O j
(3
(S
Fig. 12: Mass spectrum of 69
55
Due to presence of 1,3-dicarbonyl chain, it was thought to obtain
pyrazoles, isoxazole from 68, 69 and hydrazines, hydroxylamine. Thus, the
reaction of 68, 69 with hydrazinehydrate, phenylhydrazine,
hydrazinobenzothiazoie and hydroxyiammonium sulfate afforded pyrazoles
70a-c, 71a-c and isoxazole 70d (Scheme 29).
R -NH-NH-AcOH/Reflux
(-H2O)
CH3
NH2OH.H2SO4 HCl/HiO/AcOH
Reflux
70d
68: R ' = R^ = H 69: R ' = R^ = CH3
70a:R' = R = H,R^ = H 70b:R' = R = H,R^ = Ph 70c: R R!=H,R^=
71a:R' = R = CH3,R^ = H 70d:R' = R2 = H 71a:R' = R = CI _ 71b: R ' = R = CH3, R = Ph
71c:R' = R2 = CH3,R3=(QCg^
70a-c, 71a-c
Thus, the IR speclrum (Fig. 13) of 71a showed a broad band al 3215
cm"' which can be assigned to NH group. (Wo sharp bands at 1746 and 1721
cm"' were assigned to enol lactone and coumarin carbonyl groups respectively.
Two more sharp bands at 1630 and 1559 were assigned to >C=N and >C =C<
groups respectively. The 'H N M R spectrum (Fig. 14) of 71a exhibited three
methyl singlets at 5 2.31, 2.38 and 2.40. The aromatic region of the spectrum
clearly showed four singlets each integrating for one proton at 6 6.71, 7.39.
7.79 and 8.42. The up field singlet at 6 6.7 was assigned to Ha proton of
pyrazole moiety. The low field singlet at 5 8.42 was assigned to Hb proton
(peri to carbonyl group) and two singlets at 7.39 and 7.79 were due to two
aromatic protons (C-7, C-10). Further confirmation for structure 71a was
provided by its mass spectrum, which exhibited M^ at m/z 322 (M^+1 peak at
m/z 323 in the Fig. 15). The peak at m/z 307 was due to loss of methyl group
from molecular ion peak. The other important peaks were obtained as shown in
Scheme 30a-b.
57
H,C
O m/z 238
3 2
m/z 154 (Base peak)
Scheme 30a. Mass fragmentation of pyrazole 71a.
58
H.C
N N m/z 289
Scheme 30b. Mass fragmentation of pyrazole 71a.
59
(9W6f •
(98Z6>'
•tafSSE: -StttSiW)
6SEZ)
91.6Z)
( Z9»'9' 86 /.soe) 8UE)
(e/.89S'2fe't'I.Z€
CO
n >• r». ca o> 0>
n X
(eiz i>|zsow) ( e u t > 1 ioi88)
(9626>' SSWU)
(9828>'()rSKl>-
(zzeze'jsszQH) ( 8W9Z ; IJ'SStrl ) -
-4eK9-2!9f6SSl) —
—< ew9'z i SS0C91)
o
o
Zt@6V)
;69'iQ9)
o o o N
o o o
S2 o JQ
E
00 a>
<o H >-oo o>
H X
(B O o
CM
f
Fig. 13: IR spectrum of 71a
60
;ilil s s s i: £ o o 9 9 p :l
r,si J.ill i J ^ -"• 5. ff ? p g 5 s I ^ g g I o >- o o
r(:6c;c t — 6-;9 • 0
Sl9'rb seeoE 1880£
fftM-ri^SZf
I-2 -
5 -
r-
^ • - ^
-'" >
o
_ l i 7 E
I J i i O :
pccu/. g- ~rO I
: 9 o • I
0 1 0 I
9 0 0 I P 9 - OOl''
ii'do
Fig. 14: 'll NMR spectrum of 71a
SI
§
^ V
CD
a X a-cr I—1 - J a
z: a; • in cr a X
n £ B DC n i- u . r-i
•^ CD r\ i o rvj OJ Ld a rv
in
CE
r ai
(D
CE
OJ • n c r c o
(S
cs
3
CD I . U3 (O '-• (77 00 0) 'X (X U) C - . . - in rv C7>
_ j - ^ IV r^
l.O
I S en ru
is> OJ
— (S
m m ^ t\] ivj — ( T J , -'
(S - rvj
rvj
en -S — (S <^ -.
CD
m. CD
i n CD
- a ru
en
§
(S
ID
c "> I-' in O o c rvj
M t n >-H —
at
o 2:
s
E in
_: CS
I- GJ
o -- ••
C2.1 - Q. i n Qi m
cv r~
S IS
CD
CS (S
f v
(S ID
S I
Q
OJ
S
IS
(2 CD
00 0 1
s
CD 00
IS i n
IS
T
o
(S CXJ
s -
(S
(X I T
I S
00 OJ
X "
IN (S
03 CM ^
' lAl
-
_~
~^
r
_ i :
Q oo
OJ
" •
f j 1
Fig. 15: Mass speclrum of 71 a
62
3.3. The reaction of 3-formyI-4-hydroxycoumarin (31) with 5,5-dimethyl
cycIohcxan-l,3-dione (72)
Another reaction of 3-formyl-4-hydroxycoumarin 31 which seemed
interesting, was carried out with active methylene compound, namely. 5.5-
dimethylcyclohexan-l,3-dione (dimedone) 72 in alcohol. The reaction did not
give 73 as expected from the reaction. Instead, it afforded 74 as a sole product.
Since formyl group of 3-formyl-4-hydroxycoumarin is very labile under strong
conditions, it seemed that when the reaction was heated for more than an hr
deformylation of 3-formyl-4-hydroxycoumarin had taken place to form 4-
hydroxycoumarin which was then added to the double bond of 75 followed by
removal of OH group from the molecule to form the dimer 74 (Scheme 31).
63
C I " H ^
iiCk^^^^^xs^O OH
74
10^
Scheme 31. Formation of 7-(4-Hydroxycouma^in-3-yl)-10,10-dimethyl-8-oxo-8,9,10,l l-tetrahydropyrano [3,2-c] coumarin 74 by the reaction of 3-formyl-4-hydroxycoumarin 31 and dimedone 72.
The formation of high molecular weight dimeric structure 74 was also
indicated by mass spectroscopy. Thus, mass spectrum (Fig. 16) of the
compound 74 showed M* at m/z 456. The base peak at m/z 295 was obtained
64
as a result of loss of 4-hydroxycoumarin fragment from molecular ion peak
(Scheme 32).
Scheme 32. Mass fragmentation of 74.
65
Ihe IR spcclrum (Fig. 17) exhibited slightly broad and strong
absorption band at 1724 cm"' along with another notched peak at 1698 cm" .
The band at 1724 cm"' indicated presence of more than one coumarin carbonyl
group in the compound. The other band at 1698 was easily assigned to a ketone
group. The ' H N M R spectrum (Fig. 18) exhibited two methyl singlets at 5 1.11
and 1.18. Two more singlets each integrating for two protons at 5 2.36 and 2.38
were assigned to two methylene (CH2) protons. Another singlet integrating for
one proton was assigned to methine proton (H-7). Two double doublets each
integrating for one proton at 6 8.02 (.1 = 7.8 Hz, 1.2 Hz) and 7.92 (.1 = 7.8 Hz,
1.2 Hz) were assigned to H-1 and H-5' of coumarin units. The remaining six
aromatic protons were present as a multiple! in the region 5 7.17-7.61. A broad
singlet (D2O exchangeable) at 5 10.55 was due to OH proton. The presence of
OH group of coumarin moiety was also confirmed by characteristic ferric
chloride test. Combining this spectroscopic featuring one arrives at structure 74
for the compound.
66
LD in
S nj I en
(S en
0) X *j or •o IX CD (J
a:
o
C I
cn 03 rvj
en rx - -
(X rvj
IS - S
IS - CD
r LD
+ a:
2 c o
IS cs
u
w ^ L D ID lU \r \r r^ C - • •
- — CO LP _l ^ i;) tn I LT
L _ •• ••
^ o
* " •
fc 3 I .
I J
u OJ a
U l
i/i
'/) rd
>_
m r x i n 3 L " H l i l vr
^ 4-»
(/; '21 M
a:
CK •
•<T
1 (]' L.
11
— O t
' u I) v_
a
- M
(U 1)
^ —
c o
e i_ o ^
(U Q.
>> (-i-3
\--t-J U lU
H i
c -£
(\' — e
r 1—(
rg rs 1") en OJ
N \ e
i n
fx
n j CO
lU OT
^ tti
\-M
S
^-^ 3 a
*-i
CD n n.i i n n j O)
1
CS ( S
ID CVJ < ^ s .
—
i \ ) - fXJ
r\ j
to
in in
S
0^ CO ,
s
cs :- CD
(S ID
CS
CS
IS in
CD
OJ CD
nj CO
i"0
CO -
en
rvj nj 1^1,
in C7)
en CD -
CS
in
CS IS in
CS CO
(S
IS r- IS
in u? -
(S IXI
IS in
Cil
ro .0 o C CL I— LL 3
"TD oT z: cn ct: CD o
Fig. 16: Mass spectrum of 74
67
I
« •o
s R
>• u> ; " i •l i ! M CO II X
i I
\
-.IZKVK'iyoaez) -;(Z898W'iies«)
._ o
8
E
E e
I I
CO
._ o ^ o
e 8 o
i lO
II
Fig. 17; IR spectrum of 74
r'
68
; o in ^ :) 1
'? „ ^ X
-"= °g 1^ ' i£J r - X
uu J •- O _ e VI o ^ _ -J - i a 3 o o i/j • - - ?. ?
x g >~- C3 l a I u.' uj " I* •< a o o — G
. - ?v cu a LL u. 1 a
• I [I,i9 8
t - o
£ 1 9 2 - -
108 e -
j e g o I vV8SE 0 l e g ; o
-CM
S^E-
. - #
/ - / /
:l 5£0 8-
0000 1
•ID
-CD
O
OJ
mad a.
Fig. 18: 'H N M R spectrum of 74
69
3.4. The reaction of 3-formyl-4-hydroxycoumarin (31) with 3-methyl-l-
phenyl-5-pyrazolone (76).
The reaction of 3-formyl-4-hydroxycoumarin 31 with 3-methyl-l-
phenyl-5-pyrazolone 76 was carried out in the hope of getting 77. The reaction,
however, did not give the expected product and instead, afforded 78
(Scheme 33).
H-,a H,C PTS :i
Nv ^A, Ethanol N^ A O H ^ N O Reflux ^ N 0-^H "
Ph 76
Ph
OH o H L> H •
H,C
I Ph
Q]^ I Ph
4HC H3C^^ . ^ ^ ^CH,
I I Ph Ph
I I Ph „„ Ph
78
Scheme 33. Formation of bis pyrazole 78 by the reaction of 3-formyl-4-hydroxy coumarin 31 and 3-methyl-l-phenyI-5-pyrazolone 76.
70
I he IR spectrum (Fig. 19) olthe compound 78 did not show ain band
for coumarin carbonyl group. Besides, there was no characteristic signal for 11-
5 proton of coumarin in its 'll NMR spectrum (Fig. 20). It exhibited a singlet
integrating lor six protons at 6 2.32, which was due to presence of two methyl
groups in the compound. Another singlet integrating for one proton at 6 7.18
was assigned to methylene proton. Two multiplets each integrating for two and
four protons in the region 6 7.24-7.28 and 7.40-7.45 were assigned to 11-4, H-4'
and 3, 5, 3', 5' protons. Two downfield singlets each integrating for two protons
at 5 7.88, 7.91, however, were assigned to 2, 6 and 2', 6' protons. It seemed that
only aldehydic group of 31 reacted with 76 to give dimeric product 78 which
was supported by its synthesis from 76 and moist triethylorthoformate
containing a catalytic amount ofp-toluenesulfonic acid (PTS) (Scheme 34).
71
OEt 1
H—C OEt " HCr=OEt
•OEt ®
OEt I
H — C \
-^ EtO
OEt OEt
^®0-Et I H
H—OEt/
XH, H—C.
I Ph
-EtOH
HO' ^N I Ph
^ N ^ O O - ^ N ^ I I Ph Ph
I Ph
H
H3C.
Ph
-CH, H.C.
^ N OHO ^ N
r ^ ^ ^ ^ 'CH.
2' 6f
4' 4
^ N OHO ^ N 1 1 Ph Ph
78
Scheme 34. Formation of bis pyrazole 78 by the reaction of 3-formyl-4-hydroxy coumarin 31 and triethylorthofomiate.
72
t809lZl-'9S>OV)-
(pwcu- 'wf is)-
E
« E 3 C
S i
•o o
CO
Fig. 19: IR spectrum of 78
73
. . - - mi W !8 . : S 8 3 [IrT!
Ilii t\t « - Z C !S^!£IS * — o 19 X UJ lU •stui
S. a
- . I s ,
J —
eec a-
88] £
^ ^ S B O O
— ^ 6Ee •
=c;^soto
0
0 I 0
055a 0
s " f ^
uiOO
KOS 0
^^D
- 0 3
r\j
e a Q
Fig. 20: 'H N M R spectrum of 78
74
3.5. Reaction of 5-chIoro-3-niethyl-l-phenylpyrazole-4-carboxaldehydc (79)
and 5-azido-3-niethyl-l-phenylpyrazoIe-4-carboxaldehyde (80) with enol
lactones.
5-Chloro and 5-azido derivatives have been employed as versatile
intermediates for the preparation of biologically active compounds.'''^' In the
heterocyclic area chloroformyl and azidoformylpyrazoles of type 79 and 80 are
interesting starting materials for two reasons. Firstly CI and N3 groups are easily
substituted by nucleophiles and secondly the CHO group is ideally suited for
carrying out functionalization. Since 79 and 80 are easily synthesized in the
laboratoryZ"*^^ a number of polyheterocyclic compounds are synthesized
conveniently as shown in Scheme 35"^ and 36.''
H,C
N \ N I Ph
79
r I Ph
80
,R H3C Allyl isothiourea / Base^
"CI
,CHO
'N,
75
H,C
H,C
R 81a: CHO,
81c: C=NMe-0',
81b:CH = N0H
81d:CH = N-NHTs
COzEt
81e: (2-Phenyltetrazol-5-yi), 81f:C^N.NPh
81g:CH = NMe-CHC02Et. 81h: CH = NMe-NCOCHjPh
81i: C=N-0-
Scheme 35. Formation of isoxazoles 82,83 oxathiazocine 84, pyrazole-2-carboxylate 86 and dipyrazoles 85,87 by the reaction of allylsulfanyl-4-formylpyrazole 81a-i with NH2OH, CH3NHOH, tosylhydrazide, ethyl iV-methylglycinate and A^-methyl-A^-phenylacetylhydrazide.
76
HjC
N.
^^» AMrf^^'>^
,CHO Ar'-NH;
H3C.
1 Ph
80
RT, Ethanol 6 hrs
N
^ ^ N - A r ' Ph^P
\ N N3 I Ph 88
CH2CH2, RT, 15 hrs
H.C. / ^ N - A r '
H,C.
N
Ar^-NCO r ^ ^ ^ N
.Ar'
\ N N=PPh3 I Ph 89
RT,CH2CH2,4hrs N ^ ^ > ^ ^ ^ > ^ ^ _ ^ ^ 2
H,C
CS RT, CH2CH2, 6 hrs
^ N - A r '
N N=C=S
I Ph
91
Ph 90
H,C. r ^ ^ ^ l - ^
N N I Ph
92
N
Scheme 36. Formation of pyrimidine 92 from azidoformylpyrazole 80.
Due to reactive aldehydic group in 79 various fiinctionalization (inter and
intra molecular) have been carried out using active methylene compovmds
(Scheme 37).^^
77
H.C
> N.
^ ^ ^ V / ^ 2 H3C
I Ph
^ 0 - ^ 0 N. r °
H3C.
Ethyl glycinate hydrochloride in bioling pyridine
'N I Ph
^OH N ^ ^O
CA\ 6"5
H3C
hippuric acid / NaOAc / AC2O
H,C. CH2(CN)2
Piperidine ^ PI Knoevenagel condesation ^ N ^
Ph
T l
/ Ethanol/Piperidine
CH2(CN)2/NH40Ac at 120°C without solvent
XN
PhCHjCN / PTC
N.
,CN H3C
~ N ^ CI I Ph 93
COOC2H5 N
CN H3C
CONH, N CI
I Ph
CN
C6H5
Scheme 37. Formation of pyrazole derivatives from chloroformylpyrazole 79.
The addition of nitrogen nucleophiles into a, P unsaturated system in 93
led to the removal of ethylcyanoacetate moiety with the formation of schiffs base
93a (Scheme 38).
78
NHR XN
H^NR
HO 93 S)
— Z—C^r^ / C N XH
I COOC2H5
-(CHfC CN COOC2H5
NR
-*- Z—C 93a
Z = 5-Chloropyrazol-4-yl R = OH,NHPh
Scheme 38. Formation of schiffs base 93a from 93.
Further 93 is treated with other nitrogen nucleophiles to give cyclized products as
shown in Scheme 39 38
^ N ^ O
H,C N^ ^ - ^ N -
CH.
Ph i
^COCHj
H3C.
J 2
N.
•^ ^ 7 p-anisidine j |
COOC2H5 ^
^--^^.^^^^^^OCH,
~N^ CI I Ph 93
CH3NH2/ BoilingMeOH
N N 1 Ph
^ ' ^ x ^
H3C
( J /piperidine
N. "N CI
Ph
CN
CCXDCHj
HaC
^ 0 - ^ 0 + >
^ - N - ^ 0 I Ph
Scheme 39. Reaction of a-cyano-p-(5-chlDro-3-methyl-l-phenylpyrazol-4-yl) acrylate 93 with amines and active methylene compounds.
79
In the light of above mentioned reactions, the reaction of 79 and 80 was
carried out with enol lactones such as triacetic acid lactone and 4-hydroxy
coumarin. The reaction of chloropyrazole carboxaldehyde 79 and azidopyrazole
carboxaldehyde 80 with triacetic acid lactone 64 afforded 94 and 95 whereas 96
and 97 were obtained with 4-hydroxycoumarin 1. Perhaps the reactions occurred
as shown in Scheme 40.
iN O O ^ CHj I 9 8 Ph
94
96
80
79: X = CI 80: X = N3 64, 94, 95: Z =CH=C-CH3 '? 1,96,97:Z = 0 Ph 95,97
N a
0 C-4 Carbonyl is involved in
Z cyclization
Scheme 40. Formation of pyrazolopyrones 94,95,96,97 by the reaction of chioro and azidoformylpyrazoles 79,80 with 4-hydroxycoumarin 1 and triacetic acid lactone 64.
81
The isomeric pyranones 94, 95 exhibiting M^ al 418 were distinguished on
the basis oflR and 'H NMR spectroscopy. Thus, the IR of 94 (Fig. 21) exhibited a
broad band at 3442 cm"' ibr OH group, a sharp and very strong absorption band at
1726 cm"' for lactone carbonyl groups and a sharp, slightly less strong band at
1642 cm"' for chromone carbonyl group. On the other hand the IR spectrum of 95
(Fig. 22) exhibited slightly broad and strong absorption band for lactone carbonyl
group at 1703 cm"' in addition to a sharp band 1621 cm" for carbon-carbon double
bond and a broad band at 3378 cm"' for OH group. The ' H NMR spectrum of 94
(Fig. 23) showed singlet integrating for six protons at 5 2.05 and another singlet
integrating for three protons at 5 2.29. These singlets may be assigned to two
methyl groups of lactone moiety and one methyl group of pyrazole moiety. Two
more singlets integrating for one and two protons at 5 4.53 and 6.01 were assigned
to methine protons and C-5 protons of two lactone moieties. The aromatic protons
of phenyl group attached to nitrogen of pyrazole moiety were present as multiplet
in the region 5 7.068-7.62 excluding the singlet at 5 7.28 which was due the
solvent CDCI3. The ' H NMR of 95 (Fig. 24) exhibited the methyl singlet of
lactone nucleus at 6 2.15 and another methyl singlet at 5 2.31 of pyrazole nucleus.
Besides these three singlets each integrating for one proton at 5 5.13, 5.93 and
6.20 were assigned to methine proton and C-5 protons of two lactone moieties. In
the aromatic region protons of phenyl group of pyrazole moiety were seen as
multiplet situated in the region 7.26-7.71. A broad singlet (D2O exchangeable) at
10.12 was due to OH proton.
82
The isomeric pyrones 94,96 and 95,97 were probably formed by involving
intramolecular cyclization involving C-2 carbonyl as well as C-4 carbonyl groups
of 97a. The formation of isomeric pyranones was justified by drawing analogy
from the reaction of a phenol 98 with a 3-oxoester 99 to give a coumarin 100, a
chromone 101 or a mixture of both (Scheme 41) 39
98
EtO
+ o=c
Me
99
CH2
H2S04 ^ ^ o
O, Me OH V
/CH2
P,0 2^5
EtO—C
II o
Scheme 41. Formation of coumarin 100, chromone 101 by the reaction of phenol 98 and 3-oxoester 99.
>40 41 The formation of coumarin 100 and chromone 101 involved Simonis
-42,43 condensation ' and was further explored to show that product may be one or two
isomeric benzopyranones.'*'*''*^ Further confirmation for 94 and 95 was done by
mass spectroscopy as shown in Scheme 42 and 43.
83
CH,
O
O
H3C ^ o ^ ^Q /U^A../ " ni/z418 Ph
-CH,
m/zl37
H3C ^O ^O
m/z245(100)
CH,
O
H O ' ^ ^ . ^ ^ O
O O"
m/z403
I Ph
r N
CH, -CO
CHi
HO
O
O
-CH,
,y N O O N
I m/z375 ^^
Scheme 42. Mass fragmentation of pyrazolopyrone 94.
84
H, °^-^oOv^°^-N/^"3
N N Ph m/z418^
9§
H,
H 3 C ^ / 0 \ ^ 0
HX^O^O V OH
H
0 < > ^ C H 3
Ph /
N N
m/z292(100)
' ^ \ ^ 0 | ^ ^ V - C H 3
CH pK / N N
m/zl55 O
m/z 137
Scheme 43. Mass fragmentation of pyrazolopyrone 95.
The identification of coumarin products 96 and 97 was again done with the
help of IR and ' H N M R spectroscopy as both 96 and 97 were having same
molecular weight, ie. mass spectrum depicted M* for both 96 and 97 at m/z 490.
Obviously, both benzopyrans were obtained as a resuh of cyclization involving C-
2 and C-4 carbonyl groups as explained earlier. The IR spectra (Fig. 25 and 26) of
both the compoimds 96 and 97 exhibited broad bands for OH groups at 3451 and
3078 cm' respectively. The carbonyl region of the spectrum showed a pronounced
and strongly absorbed band for coumarin carbonyl groups at 1729 cm' of 96
whereas the compound 97 exhibited two strong absorption bands at 1731 and
85
1670 cm''. The former value in 97 was assigned to coumarin carbonyl group and
the later value to chromone carbonyl group. The ' H N M R spectrum (Fig. 27) of 96
exhibited a methyl singlet at 5 2.62. The value is. however, slightly high and may
be due solvent DMSO in which the spectrum was recorded. Another singlet
integrating for one proton at 5 4.75 was assigned to methine proton. The aromatic
region exhibited a doublet (J = 7.9 Hz) integrating for two proton and was
assigned to C-5 proton of two coumarin units. Usually aromatic protons of 4-
hydroxycoumarin appear as muliplet.''^ But we have observed the splitting of C-5
protons of coumarin unit in a compound associated with another project. The rest
of eleven protons (six protons of coumarin units + five protons of phenyl group of
pyrazole moiety) appeared as a multiplet in the region 5 7.25-7.79. The ' H NMR
spectrum of 97 (Fig. 28) indicated singlet for methyl group at its normal value
ie. 6 2.12 as the spectrum was recorded in CDCI3. Another singlet integrating for
one proton at 5 5.36 was assigned to methine proton. Two doublets each
integrating for one proton at 5 7.97 and 8.04 were assigned to C-5 protons of
chromone and coumarin units. The remaining eleven protons were present in the
form of multiplet in the region 5 7.30-7.83. It is' difficult to say which doublet
corresponds to C-5 protons of chromone or coumarin unit but it appears that the
value at 8.04 may be assigned to C-5 protons of chromone unit as the value for
this proton has been reported at 5 8.21.'''' The mass spectrum of compound 96
exhibited M ' at m/z 490 as base peak (Fig. 29). The appearance of peak at m//
317 was shown in Scheme 44.
86
' ° - ^ 0 0 ^ / 0 .
N N Ph" m/z490
96
.0 0
o -OH-
O
o .0
N N
CHi
Ph"
m/z317
Scheme 42. Mass fragmentation of pyrazolopyrone 96.
The mass spectrum (Fig. 30) of 97 exhibited M^ at m/z 490. The other
peaks were obtained as shown in Scheme 45.
87
O O N 97 m/z 490 I ^
Fn
-
OH
O H
.CH,
^^A, O O N m/z 329
RDA Cleavage
Ph
H
. ^ ^ ' ^C
m/z 120
.0
H*
O O N m/z 489
RDA Cleavage
m/z 120 O N
m/z 369 I Ph
4HC
.CH,
+
:N
O N m/z 209 I
Ph
-Ph
/CH3
.^^
O N z208
CH3 ^ y " 2«« Ph
:N-Ph
o m/z 90
O m/z 167
O N m/z 193 Ph
Scheme 42. Mass fragmentation of pyrazolopyrone 97.
88
(eiZSME'ZCZiS) i
(Zi01.l-C'0i889) :
(?8S«0EStK8) :
.(.9wj);pE'.K/ZB)..'... (ZSZ4 K • W BWU -•
(osvSez'gzftcti) :
-(SSlKK'ZSOWl) -J
-—=• .-^.--(WJoez' ijit£>) -p
" "-Z^~ = "— ; liEBesz-Ms-aai*;) J.- ^
; i - - -
§ o
J Ul
z o 2
(0E»9-8Z'lt>l«eZ)
( W 6 6 i Z ' S e 9 J «
•a-
o - = GO <o c<i "> . II
>• ii 09 |l
(et)Es'zz'£^»KK
o (D
O
E u
"i" ID
E 3 C
o o
Iv. o CO <o rJ H> H
> • N. W a>
M II X
Fig. 21: IR spectrum of 94
89
(««8-»'»»C9)^
•Xizus'.u^sit— :(izz6s;8si.M):
: (?s«t'8ieo6;)
;(«s»>r9*»sot;) -
• (wic- 'eo-QGit) — : (wict'zc-wU)—
(wwt'ssiso)—
' : (8witjp.»-wtl.)—
i!ZirsBsk6^yif»Ki.:r-
10
e e
1
>
I
1 ( M S ^ l ' M ' l ^ ) -
. O
r "> M
1 o o
e o o rt N T-
-1 H >-
n X
Fig. 22: IR spectrum of 95
90
u) 'O r- [iJ
a; c, -z. M W 2 (J M X .1. O P < X tt U Z u: a.
< 3 a o z ti I ct; X u: u lu
I ft) (U H (H 1^ ^' Q iJ (3 W O -3 1- .T: Q C 3 fO
J n ti^ O y) 03 CQ O J W to 3: to .J O ^'
0000- L =•
cfs r - 'JtV
ID
960
ooi
.-1 .-I Sfih'OI — I (J
m T; T. ;;
9t-<i
Fig. 23: 'H N M R spectrum of 94
= I mu'um ^ O O G O
' u< u ^ O O f\j "» — tn "1 o
3) o in
li r i nr 1 o « a i
: — O Cr
bti Ii?!iiei..iis..«., •ri
1000 0-as io 0
8£^B0
sees I
II Eei9-
^808 g
"8189 S
90C! 2 96Et a 5I tE '2 '^
E976 a
9E6I' £
g i E i g
l ege '5
9S02 9
l E 9 g i
ooBav
letE z
60Q> i 9E9^ i; aoi I.
Z" V
SSiCO
CQ/i' 0
nrassf 0 ^ " S E 9 > 0
OtS8 0 6iE0 1 0000 I
Fig. 24: 'H N M R spectrum of 95
WLE
92
zid^i te&^i^zsi)
(fiteo' 65-688)-
" tl8iasfe';s£;eo9i
;|ji9a;z-J;iB;6za!
ui S < z O z
(/SEEo'zgSiez)-
CO
§ (M
II
>-00
0 CM II
: ^
i;;
; . ;
;
;i
0 (0
] "> (ijsceo'srwoe)
• 5
; V
> (iiTW'o'ei'wt'C)
-IS?"
.=•?
0 0 0 0 0 U> • * M CM T -
o o o
o o
o o o
E
12 o j i E 3 e
>
u> II >-o>
00
A o N II . X
1
Fig. 25: IR spectrum of 96
93
o o
o o
E
12
E 3 C
>
s ; : I CO 1 II > lO on
Fig. 26: IR spectrum of 97
94
I X
E Ui
o ^ I—;
^ rt: w w u y: z OS > CO . <
3:
(]>
n M
4-1
U u. Q < <
C O (/J
>, J j
CO
0)
-1 _C C VJ
Z> (0
a-.a H ra n
• n C c ra
(X, CJ
W) )-l
y i
(U
h ( 0
•0
u
,. t : a t
a
u
r-o
' . • I
1
.-( ^
S
o OJ
r -
r O 2 2
^^^f'
i a) o r\i e r-i n r : i j - . . 1-. o m
' 0 C - r-H l i . O
o C CM
! - •
(-1
t r
< i
* J 1 O l O C; ^ O CC f ^ l r o { j ^ .
(t> CO a ^ i T
a M 10 O
CO EC
E
C a O DC p: a:
1 <U <!} H CO ' ^ •»-' S (JO O 1-1
r s i <D - 1 2 a : p
'X>
U )
:z u;
>
y c^ CD 1 « i
cr
n: 1
S O M t / l S
M 01 i r : (/)
o% 1 ^
o\ r-n -^ f n Ct o vo T-< OJ
- r -
o •
t o [ c ]
a: u
< r ( N
'-
M C O
s o w
5
u •V
,-o
X
' i J d -<D
•Xf (T( O
w
r s j
Ul
T /
O
""•
^ o
II II 11 II
II II II
•i-t
!z
r;
1) II
11
tt
rr n
=>
•if
-1
o ; n
f t
,
0 1
c - j O
C
, •<
1-)
rr. c:?
o
. 'M 7^
—( o
• - I -
o J U^
t.)
J-> 11
fc: T l
»-< T l
v l
H
-1"!
Q
LU
' r'
( Y ,
'*; r
f^T
*~*
i . , 1 -
r -
i T . - N ' • —
=:
L u
[ : :
:v P ( Q i O . E - ' t O Z Q t J l t . ^ r t O i ^ O a f - O ? fj. fvl U) S: CO 1-1 iJ tL,
Fig. 27: 'H N M R spectrum of 96
95
1 ill'
iii I i l l ! !;i?iil9i!*siPsssatt^
;cioo U--
I' iZb 0
SEED I c:i'90 I
0955 I
ecEt' ;
t555 (
1 -TV '" J! /
_ . - • ' ' 2£L
n':': t
\i'sF" 0"
Fig. 28: 'll NMR spectrum of 97
96
o o
o in
o - o
(O
o - in in
o c\i en
LU CO CM
O O
in
o -in
I f>5
o o o
o o o
CD CO
o m
> <
•<i-
K-
m 00
CO
O N -CD >
o - i n
CO
^-h o o CO
o o
< a:
o CO
o in (M
2, o o
2 ^.< ; 5 gS !
F: U ,
O U cfl <
tr
CO
«? CD
r^ o
o ^-"
O OT <f LU I." LU . . h- 1-
o - o
C\]
o in
o - o
6 o C7> 00 e' r i r : 1
O ^T riTT o o
CO o CM
"a"'' o i n
aouepunqv sAiieiay
Fig. 29: Mass spectrum of 96
97
(S IS
2 : I
(S
I a-o
( £
a 01 a:
. IS
IS)
ED X
-o o
u
cs ~ en fM i n ao D
V -^ CO — 00 i n
0,J
J - LO
CX)
en
in
LL.
3 U C — 1— 01 ' - u )
: - Q no
CD
I S - r\ i
G)
1 m-
S
(S
IX)
I S
OS • ra
' 3 CO
1 - LD
(\i T ^ O 10 r
t; IV o. 3 a. V 21 CU ^ eg n j u 1"^ I 0) I d fX a r . i -
IJl
nj <0 U. tij
IS) I S U O 01
. V i n -<a g ^ oc
C3 K)
ts "1
K)
- i
eg
L3 :J1 Z •-• i n 3' C1 O
Fig. 30: Mass spectrum of 97
98
3.6. The reaction of 5-aniino-3-inethyI-l-phenylpyrazole-4-carboxaldehyde
(102) with triacetic lactone (64).
Ahluwalia et. al. synthesized 5-amino-3-methyl-l-phenylpyrazole-4-
carboxaldehyde 102 by reduction of 5-azido-3-methyl-l-phenylpyrazole-4-
carboxaldehyde 80 with H2S in dry methanol.'' When we tried to synthesize 102
by above method, no reaction occurred and work up of the reaction mixture gave
only starting material. A survey of Uterature showed the reduction of azide to
amine by lithium aluminum hydride'* ' ^ or by catalytic reduction.'' "^^ '' However,
F. Rolla has reported the conversion of azide to amine by using sodium
borohydride in the presence of water in toluene. ^ We successfully converted azide
80 to amine 102 by the method reported by F. Rolla.
The reaction of 2-amino-3-formylchromone 103 reacts with triacetic acid
lactone 64 to afford 104. The formation of 104 involved translactonization type of
rearrangement (Scheme 46). The reaction of amine 102 with triacetic acid lactone
was attempted to get the expected product 105 through same type of
rearrangement (Scheme 47) as we had earlier observed it in the formation of 104.
99
O T H
0 \ / N H 2 .NHjO^^O^^CHj
+
103 W6 O 64
CH3
Rearrangement
- H^, + H^
Scheme 46. Formation of 3-acetoacetyl-5-oxo-5H-[l]benzopyrano [3,2-e]pyridin-2-one 104 by the reaction of 2-amino3-fonnylchromone 103 and triacetic acid lactone 64.
HiCv % :
H
— IT
Ph ^ H
C?" H CH
N NH2O O CH3
Ph
102
H3C. Intramolecular
transiactonizatioit type rearrangement
Nv. / \ ^ ^^>~^ / " ^ ^ N - ^ N H 2 d ^ C ^ ' ^ ^ C H 3
Ph
O O
H,C
N N O I H Ph
105 Scheme 47. Formation of 3-acetoacetyl pyrazolopyridone 105 by the reaction of
aminoformylpyrazole 102 and triacetic acid lactone 64.
100
However, the reaction did not proceed as thought. It did not give the
expected product 105 because the compound did not give the characteristic ferric
chloride test. The IR (Fig. 31) of the compound did not show any absorption band
for carbonyl group. It exhibited only bands for C=C groups at 1594 and C=N at
1550 cm''. The 'H N M R spectrum (Fig. 32) of the compound showed a sharp
singlet integrating for six protons at 5 2.48. This could be singlet of methyl groups.
Besides this, there was another singlet integrating for two protons in the down
field region i.e. at 5 8.19. The aromatic region also depicted multiplets integrating
for ten protons in the region 5 7.34-7.65. Combining these spectral features one
arrives at structure 106 for the compound.
H,C
N I Ph
H
I
I H
106
XH,
-N N I Ph
The compound 106 was probably obtained as a result of Friedlander
condensation reaction"" between two molecules of 5-amino-3-methyl-l-
phenylpyrazole-4-carboxaldehyde 102 (Scheme 48).
10!
H,C
N N-. , Ph H OHV
102
H3C
-2H2O
Ph
N
N N ^ I Ph 106
^CHi
Scheme 48. Formation of diazocinopyrazole 106 from aminoformylpyrazolel02.
Further confirmation for 106 was provided by mass spectrum (Fig. 33)
which showed M^ at m/z 366 ( M ^ l peak at m/z 367) . T h e other important peaks
were obtained as shown in Scheme 49a-b.
HiC -NPh- . li
N
Ph 106 ni^z366
N I Ph m/z 275
-CH3*
HiC-^ /^N. H,Cv
N-
•N
m/z 169
•CH3
. -NPh- " ] [ N^O
Vr^ Ph m/z 260
N- 0 -CN N-
m/z 154
=Nv
m/z 128
-CN
=Nv
m/z 102
Scheme 49a. Mass fragmentation of diazocinopyrazole 106.
102
CH.
Ph m/z 366
106
Ph
H,C
Ph*
' = N ^ / N .
• ^xPh
=N^ / N .
Ph m/z 289
"N I N
m/z 182
HjC.
N. + HC^N +
N I Ph
m/z 156
Ph
N=C' CH3
H m/z 183 (100)
CH3, - HCN HCN
.Nv r H,C
N=C' CH3
H
I m/z 106 m/z 141
I Ph
m/z 156
Scheme 49b. Mass fragmentation of diazocinopyrazole 106.
103
S < z o
CO H
! >-
II
X
(zeoEK- • nas
(sz!esic-'66 £?9 > •
^ • i ; - = . „ -:— (Bzte- te-' S9'
'-; ^ ,...; ,'. - • 1 ^ " " ' " • ( JEOC'eZ- • Z88SOI
Z96)
^ • = * 8 « t ^ Z ' ' E8SZZI
2 ^ = — ( z c o E i e e - • IC8I
""^.r"-- tBS8,!'6S- • 00
l^t
1
:r_-i£SSeB2-' 61 81-22
2s=fe089? OS-' 8S'6ei)e
I i
o o o
o
o CO
£ a.
E 3
c >
I S !
II
II X
o CO
3 o CM
Fig. 31: IR spectrum of 106
104
='" 3 2 r :? 3 •*: / I m
O O *I3 O O o o • o o o o o o
: - - lO O O ;D O O •
,1 / Q. ^- : 5 a -
< .3C o Q t - r i >
aooc 0--
1609 I - • r osoe•0
6e«7 £ r ; / 5 P l
as9P / -J
869C
geoQ
ost'g
1551
• . \
4 t
1r J
coac'i
3!3t' 0
Fig. 32: ' H N M R spectrum of 106
105
i n
CD
U 3 CD
(S
(S (S n j 1
>-m 2 :
— m
u •!->
rd
D
(S ^-1 n
rvi g >-3 cr (- r • J —
u m OJ L J Q . r v
LO
n M T) fU
s : • ^ »n
i -> •
* I Q: cc ( J 1—(
?J cc
r ir Q oc
E I
g ^ C3 O j
1
OQ i ;
1
OJ . .
— 0 . QJ G ^ • 'U -J
'S\ Z
+ m (1 i i
(U T )
a
r n
1—(
• H
c; a i i -
^ u
4-»
Ql
— t ^ - 1
(—1 l -10 01 t
— 1
l l i.. '"* r 0
10 f -
D
7-
(U t J . >v
I—
-1 t^
4->
U 'U VI
U l
<—. (T)
-r ^
r <D
<}
C
— P
i n 0 0
(si
1 -
IX
,— (L
> 1
* - J
u
CD IS cn (S -
• n ts • G 3 r ^
^ 0
*-> 4-» UD
C r l-H CD
i n
cn
(S
S 01 (S C
• 10 V 1-cn — N
\
E •^ 7i
• • U -• - J
a 3 EC 0
CD
n
03 r>j
Q
n
3
ts tv j
IS)
OJ
CS 53
CD
00
S
G)
(S
f\l
IS
G)
CJl
• r s - o)
m
- CO
0 1 00 nj
tS3 'JD
\ E
S) ID
3 in
C3
CS) (S
IS cn OJ
IS CD OJ
U3
CO r -CHrn CS (^ CD ~
e
(S
Fig. 33: Mass spectrum of 106
106
Cfiapter 4
(yi) Anti-ittflamniatoty, analgesic and
antipyretic activities
(<B) JbitiSacteriaC activity
A. Anti-inflammatory, analgesic and antipyretic activities.
1. Introduction
Coumarins, reported to possess multiple biological activities^ are used
in the treatment of vitiligo, psoriasis and other dermal diseases. The
physiological properties of natural and synthetic [1] benzopyran-2[H]-ones
have been reviewed by various workers.^^ In recent times [1] benzopyran-2[H]-
ones have been extensively used as laser materials,''*^ photosensitizers.^'
brightner, as intermediates for dyes, pesticides and pharmaceuticals as well
as in perfume formulations "*' ^ and in enzymology as biological probes.^^
Coumarins show activities such as antifungal,^^ anticoagulant, *^ antibacterial, *^
antipyretic,^^ analgesic,^^ and anti-inflammatory.''*'" '
Pyrazole derivatives have been reported to show a broad spectrum of
biological activities such as antimicrobial,^^ fungicidal,'^ anti-inflammatory7''
analgesic,'^ antipyretic,'^ and peptide deformylase inhibitor.'^ Due to
bioactivity associated with pyrazoles, researchers and chemist are very much
interested in pyrazole chemistry.''"'^
Drugs having anti-inflammatory, analgesic and antipyretic properties are
one of the most widely used drugs for various medical and surgical conditions
to the patients. A large number of drugs having the above effects exist with
potent activity but adverse drug reaction also. So, safe drug is required in order
to avoid adverse drug reaction. Keeping this in view, the present study has been
undertaken to investigate the anti-inflammatory, analgesic and antipyretic
107
activities of the synthetic heterocycHc compounds in experimental animal
models.
2. Materials and methods
2.1. Chemicals and test compounds
Following heterocyclic compounds were tested for anti-inflammatory,
analgesic and antipyretic activities on animal models in the Department of
Pharmacology, Jawahar Lai Nehru Medical College, A.M.U Aligarh.
1. 3-Acetoacetyl pyrano [3,2-c] [1] benzopyran 2,5-dione 68.
The compound 68 was prepared from intramolecular translactonization of 3-
formyl-4-hydroxycoumarin and triacetic acid lactone. The resulting compound
68 which possessed a 1,3-diketone unit in its structure were converted to
pyrazoles by treatment with hydrazine, phenylhydrazine and
hydrazinobenzothiazole to afford.
2. 3-(3-Methyl pyrazol-5-yl)-pyrano [3,2-c] [1] benzopyran 2,5-dione 70a
3. 3-(3- Methyl-1-phenyl pyrazol-5-yl)- pyrano [3,2-c] benzopyran-2,5-
dione 70b
4. 3-(3-Methyl-l-benzothiazolopyrazol-5-yl)-pyrano [3,2-c] [1] benzo
pyran-2,5-dione 70c.
5. 3.6-Dimethyl-l,8-diphenyl-diazocino [3,4-c:7,8-c'] bispyrazole 106
The compound 106 was obtained from the reaction of 5-amino-3-methyl-l-
phenylpyrazole-4-carboxaldehyde and triacetic acid lactone.
The test compounds were dissolved in 2.5% DMSO (Dimethyl sulph
oxide) prior to administration in different concentration so that animal received
108
equal volume each time (5 ml/kg). Dose selection of the test compounds were
based on preliminary trial carried out in our laboratory over a dose range 5
mg/kg to 40 mg/kg in geometric increasing order and maximal effect was
found at the dose of 20 mg/kg.
Drugs used:
• Formalin (Merck, India)
• Diclofenac sodium (Novartis, India)
• Baker's yeast (Britannia food products, India)
• Paracetamol (IPCA, India)
• Pentazocin (Ranbaxy, India)
2.2. Experimental Animals
For anti-inflammatory and analgesic activities adult male Wistar Albino
rats (weight 100-150 gm) and for antipyretic activity young male Wistar
Albino rats 28-30 days of age (weight 90-100 gm) were used. They were
obtained from Laboratory Animal Breeding and Research Center Jamia
Hamdard University, New Delhi. The animals were given a week time to get
acclimatized with laboratory conditions
The animals were housed in polypropylene cage (4 per cage) with
sterilized paper cuttings as bedding material under laboratory conditions with
control environment of temperature 22 ± 3 °C, humidity (60% ± 10%) and 12
hrs light/dark cycle. They were given free access to food with standard rodent
pellet diet (from Lipton, India) and drinking water. The animals were
transferred to the experimental room 2 hrs before the experiment. All
109
experiments were taken between 10:00 to 16:00 hrs, when the rectal
temperature reported to be stable.^''
The study protocol was approved by the institutional ethical committee.
2.3. Experimental Protocol
The following experimental models were used for test compounds.
a. Anti-inflammatory activity
a. i. Acute anti-inflammatory model (Paw edema induced by Formalin).
Method describe by Northover and Subramanian in which 0.05 ml of
3.5% formalin in normal saline was injected in the subcutaneous tissue of the
planter surface of right hind paw to produce sub maximal degree of swelling.
The paw volume was measured at 0, 0.5, 1, 2, 3, 4 and 5 hrs after injection of
formalin.
The volume (in milliliters) of the inflamed paw was measured by standard
volumetric technique, using a calibrated plethysmometer. The paw was
immersed up to the tibiotarsic articulation (marked with ink) in a cylinder filled
with mercury. The increased level, consequent on the increase of the mercury
meniscus, was measured from the increase of dyed ethanol in a glass tube
connected to the surface of the mercury so that variation of the mercury level
corresponded to increase in the dyed ethanol in a calibrated glass tube. The
increase in volume of the paw was calculated by subtracting the initial volume
from the volume obtained after formalin administration and expressed as paw
volume increase over time (ml h S.E.M). The effect (percent of negative
control) for each rat and each group was obtained as follows.
(Vt - Vo) control ~ (Vt -Vo) treated Percentage of inhibition = X 100
(Vt ~ Vo) control
(Vo = is average volume of right paw before injection of formalin i.e. at 0 hr
and Vt = is average volume of right paw after injection of formalin)
Experimental design and drug treatment:
Rats were divided into three groups of six rats each.
Group I: Received 2.5% DMSO 1 hr prior to formalin and served as control.
Group II: Received test compounds (20 mg/kg, orally) 1 hr before injection of
formalin.
Group III: Received Diclofenac sodium (5 mg/kg, orally) 1 hr before injection
of formalin.
a. a. Chronic anti-inflammatory activity (Cotton pellets-induced granuloma
test).
This method was used, under light ether anesthesia, sterile cotton
pellets (10 ± 1 mg) were implanted subcutaneously in the groin regions of the
rats. The test compounds. Diclofenac sodium and control vehicle 2.5% DMSO
were administered once daily orally for seven consecutive days from the day of
cotton pellet implantation. The animals were anesthetized on the eighth day and
cotton pellets were removed surgically and made free from fat and extraneous
tissues. The wet weights of granuloma were estimated and than pellet were
dried overnight at 60 "C in hot-air oven to constant weight. The weight of the
cotton pellet before implantation was subtracted from the weight of the dried,
dissected pellet. The mean weight was calculated for the pellets from a group
of rats, and compared with the mean for a group of controls.
Increment in the dry weight of the pellets was taken as measure of granuloma
formation.
The difference in wet and dry weights of granuloma from control group to that
of treated group indicated the anti-inflammatory activity.
Experimental design and drug treatment:
Rats were divided into three groups of six rats each.
Group I: Received 2.5% DMSO orally daily for seven days and served as
control.
Group II: Received test compounds (20 mg/kg) orally daily for seven days.
Group III: Received Diclofenac sodium (5 mg/kg) orally daily for seven days.
b. Analgesic activity
Adult rats weighing 100-150 gm were divided into three groups (n = 6)
and analgesic activity was tested by (i) Hot-plate method (ii) Formalin test.
Group I: Received 2.5% DMSO orally 30 min before experiment.
Group II: Test compound 20 mg/kg were administered orally 30 min before
experiment.
Group III: Pentazocin (15 mg/kg) was given intraperitoneal 15 min prior to
experiment.
f^gSlS 12
Experimental design and drug treatment:
b. i. Hot-plate method
The method was described by Eddy and Leimbach. Rats were
screened by placing them on the hot plate (Eddy's hot plate from Techno India)
maintained at 55 ± 1 °C and reaction time in seconds for hind paw licking or
jumping was recorded. Only rats, which reacted within 5 to 10 seconds, were
used in the study. Those animals in which the reaction time is increased to at
least twice the mean reaction time for control animals or control reading plus
eleven seconds (control+11 seconds) were taken as showing significant
analgesia. Pentazocin was used as standard drug.
b. a. Formalin test
Thirty minutes after administration of the test compounds or Diclofenac
sodium and 15 minutes after Pentazocin intraperitonealy, 20 ^1 of 2.5%
formalin in saline was injected subcutaneously to a hind paw of the rat. The rat
was observed for 30 min after the injection of formalin, and the amount of time
spent licking the injected hind paw was recorded and the data were expressed
as total licking time in the early phase (0-5 min) and the late phase (15-30 min)
after formalin injection. The early phase represents neurogenic pain while the
latter phase is of inflammatory pain.
c. Antipyretic activity
c.i. Baker's yeast induced pyrexia.
I'cvcr was induced by intraperitoneal injection of baker's yeast 135
mg/kg, which induced a sustained increase in rectal temperature for 5 hrs.
Paracetamol and other novel antipyretics, reverted baker's yeast-induced
fever."' The test compounds and Paracetamol (standard drug) were
administered 1 hr after injecting yeast when there was an average increase in
temperature of about 1 °C.
Rats were divided into four groups (n = 6). The animals were set in
their cages individually throughout the experiment. Rectal temperature was
measured with a lubricated thermister probe inserted 3 cm deep into the
rectum. The probe was linked to telethermometer (range 31-41 "C with O.l °C
precision) for 5 hrs, Rectal temperature was measured every 15 min for each 5
hrs and recorded manually at specified intervals.
To minimize the stress response of the animals to the lightly restrained
condition, we made a careful handling and performed two sets of acclimatizing
training in the cage for 2 days before starting the experiments.
Experimental design and drug treatment:
Group 1: (Control) only yeast was injected and continuously temperature
was monitored and recorded at specified interval for 5 hrs.
Group II: Received 2.5% DMSO orally 1 hr after administering yeast.
Group III: Test compounds (20 mg/kg) was administered orally 1 hr after
administering yeast.
Group IV: Paracetamol (150 mg/kg) was given orally 1 hr after administering
yeast.
114
c. /'/. Basal rectal temperature.
Test compounds and Paracetamol were given orally and rectal
temperature was measured every 15 min for each 5 hrs and recorded manually
at specified intervals.
Group I: Received 2.5% DMSO given orally.
Group II: Test compounds (20 mg/kg) was administered orally.
Group III: Paracetamol (150 mg/kg) was given orally.
d. Toxicity study
The acute oral toxicity was carried out as per the guidelines set by the
organization for the economic co-operation and development (OECD) received
from the committee for the purpose of control and supervision of experimental
animals (CPCSEA).
Experimental design and drug treatment:
or
In toxicity study two rats (one from either sex) were dosed at
predetermined [250, 500 and 1000 mg/kg dissolved in fixed amount (1.5 ml) of
DMSO] and administered by stomach feeding cannula. They were observed
continuously for the first 2 hrs for toxic symptoms and up to 24 hrs for
mortality. If there was no mortality or if no more than one rat of either sex
died at the highest level tested (1000 mg/kg) with the total of 10 rats (5/sex)
dosed at 1000 mg/kg and monitored for 7 days period LD50 was considered to
more than 1000 mg/kg.
115
Statistical analysis
Experimental data were expressed as mean ± S.E.M. Student's t-test was
applied for expressing the significance and P value <0.05 was considered as
significant.
3. Results
a.i. Acute inflammation model (formalin induced paw edema)
The results of the anti-inflammatory effect of the test compounds on formalin
induced edema in rat's right hind paws were presented in Table 1& 2. There
was a gradual increase in edema paw volume of rats in the control (Formalin
treated). However, in the test groups, the compounds showed a significant
reduction in the edema paw volume. As indicated in Table 2. the significant
anti-inflammatory effect induced by test compounds 70a, 70b, 70c and 106
appeared at 1-2 hrs and progressively increased and reached a maximum 46.15,
88.46, 65.38, 78.84% respecfively at 5 hrs, while the maximum anti
inflammatory effect of test compound 68 appeared at 1 hr (60%). The anti
inflammatory effect induced by Diclofenac sodium progressively increased and
reached a maximum (70.83%) at 2 hrs. It was maintained up to 5 hrs. The anti
inflammatory effect of 70b was more potent as compared to others.
a. a. Cotton pellet induced granuloma (Chronic model)
In the chronic model (cotton pellet induced granuloma) the Icsl
compounds and Diclofenac sodium significantly reduced both wet as vvcll as
dry weights in cotton pellet granuloma (Table 3). I'he efiect of test compound
116
70b in botli reducing wet weight and dry weight of cotton pellet induced
granuloma was similar to that of Diclofenac sodium.
b. i. Hot plate reaction time in Rats
The results of hot-plate test indicated a significant increase in reaction
time at 2 hrs (2.5 fold), 3 hrs (3.0 fold) and 4 hrs maximum effect up to cut-off
time) with the test compounds, whereas reference drug Pentazocin, a centrally
acting analgesic drug, markedly increased pain latency at 1 hrs (2.5 fold) and
achieving maximum effect (up to cut-off time) at 2 and 3 hrs (Table 4).
b. a. Formalin test
As shown in Table 5 the pretreatment with test compounds caused a
significant inhibition of the neurogenic (early phase) and inflammatory phases
(late phase) of formalin induced licking in rats.
The standard drug, Diclofenac sodium (5 mg/kg) also significantly
inhibited formalin induced licking in rats but only in late phase (15-30 minute)
In contrast, the reference antinociceptive drug Pentazocin (15 mg/kg)
significantly reduced the licking activity against both phases of formalin-
induced nociception.
c.i. Effect of test compounds and Paracetamol on Yeast-induced pyrexia
The experimental rats showed a mean increase of about 1 °C in rectal
temperature 1 hr after backer's yeast injection (135 mg/kg, i.p). The test
compounds (68, 70a, 70b, 70c and 106) produced significant (P<Q.05)
antipyretic activity at 2 and 3 hrs. Among these test compounds, 70b and the
117
reference drug Paracetamol (150 mg/kg) showed significant antipyretic activity
throughout the observation period up to 5 hrs (Fig 34).
c. a. Effect of test compounds and Paracetamol on basal rectal temperature.
The result showed by the test compounds and paracetamol on normal
body temperature in rats was presented in Fig. 35. The test compounds 68 and
70c were lowering of body temperature at 2 hrs (0.12 and 0.5 °C respectively)
following its administration. While the maximum lowering of the rectal
temperature noticed with the test compounds 70b and 106 were 0.2 and 0.25 °C
respectively at 1 hr and that of compound 70a standard drug Paracetamol were
0.1 and 0.05 °C at 1 and 3 hrs respectively.
d. Acute toxicity study evaluation
In acute toxicity study the test compounds did not show any toxicity and
mortality up to maximum dose of 1000 mg/kg body weight in rats. No gross
change in behavior was observed at this dose. Weight of rats had a normal
variation after 7 days of observations.
4. Discussion
Various coumarin and pyrazole-related derivatives were recognized as
inhibitors of lipoxygenase and cycloxygenase pathways of arachidonate
metabolism and also of nculrophile-dependcnl super oxide anion generation."'
Several natural or synthetic coumarins and pyra/oles with various hydroxy! and
118
other substituents were found to inhibit lipid peroxidation, to scavenge
hydroxyl radicals and superoxide anion^" and lo influence processes involving
free radical-mediated injury, as can some plant phenolics and flavonoids.
The results of this study indicate the synthetic new heterocyclic
derivatives of coumarin and then conversion to pyrazoles by addition of
different groups possess acute and chronic anti-inflammatory, antipyretic and
analgesic activities on animal's models.
It is well known that inhibition of edema induced by formalin in rats is one
of the most suitable test procedures to screen antiarthritic and antiinflammatory
agents, as it closely resembles human arthritis.^' Arthritis induced by formalin
is a model used for the evaluation of an agent with probable antiproliferative
• • 92
activity.
The formalin-induced inflammation in the rats foot may be conveniently
divided into two parts, the first involving 5-hydroxytryptamine as mediator and
the second some mediator which is unrelated to 5-hydroxytryptamine. The
portion of the total response, which is due to the release of 5-
hydroxytryptamine, can be prevented by either depleting the skin of 5-
hydroxytryptamine or by giving the rats an antagonist of 5-hydroxytryptamine.
It also seems probable that the portion of the total response which is due to the
second mediator" can be prevented by treatment with certain analgesic-
antipyretic drugs (acetylsalicylic acid) and other substances like the
hydroxybenzoates. the pyrazolones, the flavone and flavanone glycosides are
inactive against 5-hydroxytryptamine-induccd inflammation but they produce
Iheir action against a formalin-induced inflammation by inactivating the second
factor.' ' Another possibility is that an anti-inflammatory agent might operate
by releasing or activating some endogenous factor, which is anti-inflammatory.
It is well known that the salicylates, for example, release both adrenal cortical
and adrenal medullary hormones*^ and although the adrenal cortical hormones
are inactive against formalin-induced inflammation in the rats foot, the adrenal
medullary are active. "' ^
The test compound 68 exhibited significant anti-inflammatory
activity with maximum effect at 3 hrs. However, the compounds 70b and 106
exhibited markedly improved anti-inflammatory activity and was as good as
Diclofenac sodium. The compounds 70a and 70c also exhibited significant
anti-inflammatory activity but to a lesser extent.
As the test compounds significantly inhibited this model of inflammation,
it can be thought to possess antiproliferative and antiarthritic activities similar
to Diclofenac and salicylates, the cyclooxygenase inhibitors.
The cotton pellet method is widely used to evaluate the transudative and
proliferative components of the chronic inflammation. Inflammation and
granuloma develops during the period of several days. The Inflammation
involves proliferation of macrophages, neutrophils and fibroblasts, which are
basic sources of granuloma formation. The wet weight of the cotton pellets
correlates with the transuda; the dry weight of the pellets correlates with the
amount of the granulomatous tissue."^ '' Hence, the decrease in the weight of
granuloma indicates the ability of the test compounds in reducing the synthesis
120
of proteins, collagen and infiltration of macrophages. Administration of test
compounds (20 mg/kg) and Diclofenac sodium (5 mg/kg) appear to be
effective in inhibiting both the wet weight and dry weight of cotton pellet
(Table 3). The test compounds 70b and 106 (20 mg/kg) appear to be equally
effective to that of Diclofenac sodium (5 mg/kg) in inhibiting both the wet
weight and the dry weight of cotton pellets.
Thermic painful stimuli (hot-plate test) are known to be selective to
ng
centrally, but not peripherally, acting analgesic drugs. The test compounds
produced a significant inhibitory effect on the nociceptive response at 2, 3 and
4 hrs though less potent than that of the Pentazocin, a centrally acting analgesic
drug, which significantly increased the reaction time in hot-plate test at 1,2, 3,
and 4 hrs.
The formalin test is another pain model, which assesses the way an
animal responds to moderate, continuous pain generated by injured tissue.' ''
Centrally acting drugs such as morphine inhibited both of the early and late
phases equally while peripherally acting drugs such as Aspirin only inhibited
the second phase.'""""
In the present study the test compounds significantly inhibited both the
neurogenic pain (early phase) and inflammatory phase (later phase) except 70a
that has no significant role in inhibiting neurogenic pain. Pentazocin
significantly reduced the licking activity in both phases while Diclofenac
decreased the licking activity only in the late phase.
121
one of the possible mechanisms that contribute to the central antinociceptive, as
well as antipyretic activities of the test compounds seen in the present study.
The involvement of the opioid system in the antinociceptive activity could also
be suggested, based on the claim by Chan et al.'°^ and Hosseinzadeh and
Younesi'^* that centrally acting drugs like opioids affect both phases of the
formalin and hot plate tests, respectively.
Based on the results it can be concluded that the new heterocyclic
derivatives possess significant role in inhibition of both acute and chronic
phases of inflammation.
Additions of different functional groups have varying effects.
Significant increase in anti-inflammatory effect of compound 68 was observed
after addition of phenylhydrazine. The synthetic new coumarin and pyrazole-
related derivatives have potent analgesic and antipyretic activity.
123
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Table 2: Percentage Inhibition in formalin induced paw edema by test compounds
68, 70a, 70b, 70c, 106 and Diclofenac sodium.
Test % Inhibition
Compounds Dose/kg 0.5 hr 1 hr 2 hr 3 hr 4 hr 5 hr
DMSO 5ml . . . . . .
68 20 mg/ 40 60 45.83 38.70 22.22 25
0.067 mmole
70a 20 mg/ 00 40 41.66 38.70 38.88 46.15
0.068 mmole
70b 20 mg/ 40 53.33 62.50 70.96 77.77 88.46
0.054 mmole
70c 20 mg/ 00 46.66 58.33 45.16 50 65.38
0.046 mmole
106 20 mg/ 40 60 62.50 67.74 75 78.84
0.054 mmole
Diclofenac 5 mg/ 40 66.66 70.83 67.74 66.66 65.38 Sodium 0.017 mmole
125
Table 3: Effects of test compounds 68, 70a, 70b, 70c, 106 and Diclofenac sodium on
cotton pellet induced granuloma.
Weight of cotton pellets
Test
Compounds Dose/kg Wet weight % inhibition Dry weight % inhibition
DMSO 5ml 183.7±11.5 79.3 ±3.5
68 20 mg/ 100 ±9.5*
0.067 mmole
45.56 39.6 ±2.6* 50.06
70a 20 mg/ 99.5 ± 8.2*
0.068 mmole
45.83 42.0 ±2.3* 47.03
70b 20 mg/ 79.0 ±4.5*
0.054 mmole
56.99 30.35 ±1.6* 61.72
70c 20 mg/ 115.0 ±10.2* 37.39
0.046 mmole
44.0 ±2.9* 44.51
106 20 mg/ 81.5 ±4.0*
0.054 mmole
55.63 30.4 ±2.1* 61.66
Diclofenac 5 mg/ 84.5 ± 6.3*
Sodium 0.017 mmole
54.00 30.0 ±1.8*
The results given are mean ± S.E.M; number of animals used (n = 6). *P value of < 0.05 was considered as significant in comparison to control.
62.17
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Table 5: Anti-nociceptive activity of test compounds 68, 70a, 70b, 70c, 106,
Pentazocin and Diclofenac sodium on formalin induced pain.
Test Total time spent in Paw licking time (s)
compounds Dose/ kg
Phase (0-5) % inhibition Phase (15-60) % inhibition
DMSO 5ml 62.2 ±5.2 - 146.4±12.3
68 20 mg/ 40.1 ±2.2* 35.53 88.3 ± 7.0* 39.68
0.067 mmole
70a 20 mg/ 46.2 ±1.4 25.72 96.2 ±4.4* 34.28
0.068 mmole
70b 20 mg/ 34.3 ±2.0* 44.85 52.4 ±6.2* 64.20
0.054 mmole
70c 20 mg/ 38.7 ±6.3* 37.78 96.2 ±8.4* 34.28
0.046 mmole
106 20 mg/ 35.2 ±2.0* 43.40 56.5 ± 6.2* 61.36
0.054 mmole
Pentazocin 15 mg/ 18.2 ±3.2* 70.79 40.6 ±8.7* 72.26
0.052 mmole
Diclofenac 5 mg/ 54.3 ±2.4 12.70 64.2 ±5.3* 56.14 Sodium 0.017 mmole
The results are mean ± S.E.M from 6 animals *P<0.05, when compared to vehicle control (DMSO).
128
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-1 ' 1 2 3
Time in hrs
Fig. 34: The effect of test compounds 68, 70a, 70b, 70c, 106 and Paracetamol on yeast induced pyrexia in rats.
0.2
0 . 1 -
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- • - DMSO - • - 6 8 A 70a • - 7 0 b • 70c
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Fig. 35: The effect of test compounds 68, 70a, 70b, 70c, 106 and paracetamol on basal rectal temperature in rats.
129 t)S^ ^i^
B. Antibacterial activity
Introduction
Infectious diseases continue to be one of the most dreaded diseases
leading to a large number of premature deaths in the entire world. The
enhancement of multiple drug resistant bacteria threatens the world's
population. Hence, in the present scenario of antibiotic therapy, there is a
continuing quest of new antimicrobial' drugs.
In this chapter we have discussed antibacterial activity of compounds
68, 69, 70a, 70b, 70c, 70d, 71a, 71b, 71c, 74, 78 and 106 synthesized in our
laboratory.
1. Materials and Method
(i Microorganisms Used:
The test organisms used included Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853,
Streptococcus pneumoniae. Shigella dysenteriae. Salmonella typhi and
Klebsiella pneumoniae.
(ii) Culture Media and Inoculum :
The microbial cultures were diluted in nutrient broth to obtain a cell
suspension of 10 CFU/ml.
130
(iii) Antimicrobial assays:
The Disc diffusion method'"' with little modification was used to
determine the antibacterial activity of some compounds. Briell}' 0.1 ml of
diluted inoculum (10' CFU/ml) of test bacteria was spread on nutrient agar
plates. Sterile paper disc impregnated with 70 |j,g of compound in DMSO and a
disc without compound was used as a negative control. The plates were
incubated for 18 hrs at 37 °C for test bacteria. The antibacterial activity was
evaluated by measuring the zone of inhibition around the disc of tested
compound. Antibiotic Chloramphenicol (30^g/disc) was used as positive
control.
Except 71a, 71b which didn't show any antibacterial activity. All
compounds exhibited antibacterial activity towards various gram positive and
gram negative bacteria. Interestingly the compound 68 showed moderate
activity against gram positive bacteria Staphylococcus aureus and compound
70c showed antibacterial activity (zone diameter 18 mm) against gram negative
bacteria Pseudomonas aeruginosa for which the antibacterial drug
Chloramphenicol did not show any activity. All other compounds showed
pronounced to moderate broad spectrum antibacterial activity (both gram
positive and gram negative bacteria). Thus, it was clearly observed from Tabic
6 and 7 that the compounds 70a, 70b showed good activity against gram
positive bacteria. Staphylococcus aureus and no activity against Streptococcus
pneumoniae. Interestingly, both the compounds exhibited some activity against
gram negative bacteria, Pseudomonas aeruginosa (zone diameter 11 and 21
[31
mm respectively) for which the antibacterial drug Chloramphenicol didn't show
activity. The compound 70a also showed good activity against gram negative
bacteria Shigella dysenteriae and klebsiella pneumoniae whereas compound
70b showed pronounced activity against gram negative bacteria, Escherichia
coli, Salmonella typhi and Klebsiella pneumoniae as compared to
Chloramphenicol. Further compounds 69 and 71c exhibited moderate activity
against gram negative bacteria, Escherichia coli. Shigella dysenteriae and no
activity against Pseudomonas aeruginosa, Salmonella typhi, Klebsiella
pneumoniae as compared to antibacterial drug Chloramphenicol.
The compounds 70d, 74 and 78 were not active against both gram positive
bacteria {Staphylococcus aureus and Streptococcus pneumoniae) and gram
negative bacteria Klebsiella pneumoniae. However, compound 70d showed
moderate activity against Salmonella typhi and Shigella dysenteriae and no
activity against Escherichia coli and Pseudomonas aeruginosa. The compound
74 again did not show any activity against Salmonella typhi. Both 74 and 78
were moderate active against Escherichia coli and Shigella dysenteriae. Further
74 did not show any activity Salmonella typhi whereas 78 was not active
against Pseudomonas aeruginosa. Among all these compounds the most potent
compound was 106 which showed maximum activity against both gram
positive as well as gram negative bacteria (comparable to Chloramphenicol).
However the compound was not active against two bacteria only viz.
Streptococcus pneumoniae (gram positive) and Shigella dysenteriae (gram
negative).
132
Table 6: Antibacterial activity of compounds 68, 69, 70a, 70b, and 70c.
Organism Test organisms Inhibition zone size in mm
68 69 70a (70^g (70^g (70^g /disc) /disc) /disc)
70b 70c (70^g (70^g /disc) /disc)
Antibiotic control*
Gram +ve Staphylococcus 9.0
Bacteria aureus
12 18 22
Streptococcus
pneumoniae
21
Gram -ve
Bacteria
Escherichia coli
Pseudomonas
aeruginosa
10
11
22
21 18
23
Salmonella
typhi
19 16
Shigella
dysenteriae
10 12 23
Klebsiella
pneumoniae
13 21 23
•Antibiotic control: Chloramphenicol (30|ig/disc). - No activity detected.
133
Table 7: Antibacterial activity of test compounds 70d, 71c, 74, 78 and 106.
Organism
Gram +ve
Bacteria
Gram -ve
Bacteria
Test organisms
Staphylococcus
aureus
Streptococcus
pneumoniae
Escherichia coli
Pseudomonas
aeruginosa
Salmonella
typhi
Shigella
dysenteriae
Klebsiella
pneumoniae
70d (70 ig /disc)
_
-
-
7.0
10
-
Inhibition zone size in mm
71c 74 78 (70^g (70ng (70^g /disc) /disc) /disc)
_ _ _
-
11 8.0 10
7.0
8.0
8.0 8.0 8.0
-
106 (70^g /disc)
22
-
22
15
18
-
25
Antibiotic control*
22
21
23
16
23
23
* Antibiotic control: Chloramphenicol (SO^g/disc). No activity detected.
134
Experimental
General
The melting points were taken in open capillaries and are
uncorrected. The Infrared spectra were recorded on Interspec 2020
spectrometer using KBr. Ultra-voilet spectra in 95% methanol, DMSO
were measured on USB 2000 Ocean-Optical spectrophotometer and
wave lengths, X ax were expressed in nm. The 300 MHz ' H N M R and
high resolution FAB mass spectra were provided by Sophisticated
Analytical Instrument Facility (SAIF), CDRI, Lucknow. The ' H NMR
spectra were recorded using DMSO and deuterated chloroform as the
solvent, in which the chemical shifts were reported in 5 values relative
to TMS as internal standard. m-Nitrobenzyl alcohol was used as the
matrix for recording the FAB mass spectra. Peaks at m/z 136, 137,
154, 289, 307 which appear in mass spectrum were due to matrix.
G.C.M.S were recorded on a Thermofinnigan LCQ Advantage max ion
trap mass spectrometer. The purity of the compounds was checked by
TLC on glass plates coated with silica Gel G (Merck Germany) using
benzene - ethyl acetate (4:1), chloroform-methanol (9:1) as mobile
phase and visualized by iodine vapour and alcoholic ferric chloride.
All the solvents and chemicals used were AR grade. 4-
Hydroxycoumarin, dehydroacetic acid, triethylorthoformate, 3-methyl-l-
phenyl-5-pyrazolone, acetophenone, benzaldehyde, N,N-dimethyl
^1':
formamide, Guanidine hydrochloride and phosphorous oxychloride
were obtained from E. Merck (Germany). Dimedone and
hydroxylammonium sulfate were obtained from CDH (India).
3-Formyl-4-hydroxycoumarin,"'^ 3-acetyl-4-hydroxycoumarin,
3-formylchromone,"^ triacetic acid lactone,"^ hydrazinobenzothiazole,
5-chloro-3-methyl-1 -phenyl-pyrazole-4-carboxaldehyde,^'' 5-azido-4-
formyl-3-methyl-l-phenyl pyrazole were synthesized by reported
method.
The reaction of 3-acetyl-4-hydroxycouinarin (42) with 3-formyIchroinone
(55).
Formation of (2E)-l-(4-hydroxy-l-benzopyran-2-one-3-yl)-3-[l] (benzo
pyran-4-one-3-yl)-2-propen-l-one (56).
To a well stirred solution of 3-acetyl-4-hydroxycoumarin 42 (1.0
gm, 4.9 mmole) in ethanol (30 ml) containing pyridine (0.5 ml) was
added 3-formylchromone 55 (0.85 gm, 4.9 mmole). The reaction mixture
was refluxed on water bath for 12 hrs, cooled at room temperature and
poured into ice-cold water (200 ml). The light yellow solid (2E)-l-(4-
hydroxy-1 -benzopyran-2-one-3-yl)-3-[ 1 ] (benzopyran-4-one-3-yl)-2-propen-1 -
one 56, as obtained, was filtered, washed with water, alcohol, dried and
recrystallized from chloroform as shining needles; yield 65%; m.p.260-
262 "C.
Spectral Data:
UV (MeOH) X max : 210.07, 270.29, 307.09, 347.24 nm.
136
IR (KBr) v„ax 3318, 1734, 1650 and 1610 cm"
' H N M R ( 3 0 0 M H Z , CDCI3) 5 7.45-7.93 (m, 7H, Ar-H), 8.18
(d, IH, J=15.9 Hz, Hb), 8.30 (d,
IH, J=7.8 Hz, H-5' ), 8.56 (s, IH,
H-2'), 9.10 (d, IH, J=15.6 Hz, Ha).
MS (% rel. Int.)
Analyzed for C21 H12 06
m/z 361 (M*+l, 100), 342 (30),
240 (5), 204 (90), 199 (35), 171
(20), 156 (20), 120 (40), 107 (15),
92(10).
Calculated C - 70, H = 3.33%;
Found C = 7.25, H = 3.53%.
The Reaction of (2E)-l-(4-hydroxy-l-benzopyran-2-one-3-yl)-3-[l]
(benzopyran-4-one-3-yl)-2-propen-l-one (56) with hydrazine
hydrate.
Formation of 3-[4-hydroxy-[l] benzopyran-2-one-3-yl]-5-[5-(2-
hydroxyphenylpyrazoI-4-yI]-pyrazolin (58a).
(2E)-1 -(4-hydroxy- l-benzopyran-2-one-3-yl)-3-[ 1 ] (benzopyran-
4-one-3-yl)-2-propen-l-one 56 (1.0 gm, 2.7 mmole) was dissolved in
alcohol (25 ml) and hydrazine hydrate (1.6 ml, 5.4 mmole) added to it.
The reaction mixture was refluxed on water bath for 30 min. On cooling
at room temperature, a light green solid 3-[4-hydroxy-[l] benzopyran-2-
I I T
one-3-yl]-5-[5-(2-hydroxyphenylpyrazol-4-yl]-pyrazolin 58a was
obtained. It was filtered, washed with ethanol-water, dried and
recrystallized from DMF; yield 79%; m.p.l62 "C.
Spectral Data:
UV (MeOH) I n,a,x 206.72, 248.54, 343.89, 348.91,
439.24 nm.
IR (KBr) V max
3618, 3456, 3396, 3269, 1684,
1608 and 1578 cm 1
' H N M R (300 MHz, DMSO-dfi) 5 3.72 (dd, IH, J - 17.7 Hz, 8.1
Hz, Hb), 4.09 (dd, IH, J = 17.1
Hz, 8.7 Hz, Ha), 5.15 (m, IH, He),
6.37 (br s, IH, exchangeable,
NH), 7.81 (s, IH, Hd), 6.91-7.58
(m, 8H, Ar-H), 10.60 (br s, IH,
exchangeable, OH), 12.80 (br s,
IH, exchangeable, OH).
M S (% rel int) m/z 389 (M^+1, 100), 388 (50), 387
(40), 386(70), 370(15), 342(5),
295 (7), 268 (8), 240 (40), 229
(50), 227(10), 161 (20), 159(5),
134(25), 136(25), 118 (20), 107
(10).
Analyzed for C2I H,6 N4 04 Calculated C = 64.94, H = 4.12,
N = 14.43%; Found C - 65.14,
H = 4.35,N = 14.63%.
The reaction of (2E)-l-(4-hydroxy-l-benzopyran-2-one-3-yl)-3-(l]
(benzopyran-4-one-3-yl)-2-propen-l-one (56) with phenylhydrazine.
Formation of l-phenyl-3-(4-hydroxy-(lJbenzopyran-2-one-3-yI]-5-(5-
(2-hydroxyphenyl)-l-phenylpyrazoI-4-yI]-pyrazolin (58b).
(2E)-1 -(4-hydroxy-1 -benzopyran-2-one-3-y l)-3-[ 1 ] (benzopyran-
4-one-3-yl)-2-propen-l-one 56 (1.0 gm, 2.7 mmole), phenylhydrazine
(0.55 ml, 5.4 mmole) were taken in methanol (25 ml) and 5 drop of
acetic acid were added to it. The reaction mixture was refluxed on water
bath for 45 min. It was cooled at room temperature and poured into ice-
cold water (200 ml), filtered, washed with ethanol-water mixture, and
dried. The yellow solid l-phenyl-3-[4-hydroxy-[l] benzopyran-2-one-3-
yl]-5-[5-(2-hydroxyphenyl)-1 -phenylpyrazol-4-yl]-pyrazolin 58b as
obtained was recrystallized from chloroform-benzene; yield 53%;
m.p.l85-190°C.
Spectral Data:
UV (DMSO) X niax : 283.15, 365.55 nm.
IR(KBr)Vn,ax •• 3614, 3452, 1710, 1610 and 1553
cm"'.
' H N M R ( 3 0 0 M H Z , DMS0-d6) : 5 3.74 (m, IH, Ha), 4.17 (m, HI,
Hb), 5.2l(m, IH, He), 6.75-7.62
(m, 17H, Ar-H), 7.45 (s, IH, Hd),
8.04 (d, IH, J = 7.2Hz, H5");
MS (% rel int)
Analyzed for C33 H24 N4 O4
m/z 540 (M^, 90), 539 (50), 522
(5), 496 (5), 463 (6), 447 (10), 420
(5), 379 (5).
Calculated C = 73.33, H = 4.44,
N = 10.37%; Found C = 73.54,
H = 4 .75 ,N= 10.55%.
The reaction of (2E)-l-(4-hydroxy-l-benzopyran-2-one-3-yI)-3-(lJ
(benzopyran-4-one-3-yI)-2-propen-l-one (56) with guanidine hydro
chloride (59).
Formation of 3-amino-l-(4-hydroxy-l-benzopyran-2-one-3-yl]-3-(l-
benzopyran-4-one-3-yl)-propen-l-one (60c').
(2E)-1 -(4-hydroxy-1 -benzopyran-2-one-3-y l)-3-[ 1 ] (benzopyran-
4-one-3-yl)-2-propen-l-one 56 (1.0 gm, 2.7 mmole), was dissolved in
acetic acid (20 ml) and guanidine hydrochloride (0.51 gm, 5.4 mmole)
and catalytic amount of sodium acetate (0.44 gm, 5.4 mmole) added to
it. The reaction mixture was refluxed on an oil bath for 10 hrs, cooled
and allowed to stand at room temperature to afford white solid 60c'. It
was filtered, washed with water and dried. The filtrate was poured into
ice-cold water (50 ml) to get more 60c'. The two solids were combined
and recrystallized from chloform-methanol mixture to afforded
3-amino-1 -(4-hydroxy-1 -benzopyran-2-one-3-yl]-3-( 1 -benzopyran-4-one-
3-yl)-propen-l-one 60c'; yield 66.2%; m.p.156-158 °C.
Spectral Data:
UV (MeOH) X 234 JS, 302.89 nm.
IR (KBr) Vr 3437, 1686 and 1620 cm"'.
'H NMR (300 MHz, CDCI3) 5 3.31 (dd, IH, Ha), 3.63 (dd, IH,
Hb), 4.88 (m, IH, He), 8.45 (s,
IH, H-2'), 8.27 (dd, IH, H-5'),
7.99 (dd, IH, H-5'), 7.36-7.76 (m,
3H, Ar-H).
MS (% rel int)
Analyzed for C21 H15 N 06
m/z 377 (M^, 100), 333 (10), 257
(5), 216 (5), 213 (5), 199 (5),
171(5), 161 (10), 117(5), 120(5),
92 (5).
Calculated C = 66.84, H = 3.97,
N = 3.71%; Found C = 67.10,
H = 4.22, N = 3.85%.
141
Synthesis of 6,7-dimethyl-4-hydroxy-2-oxo-2H-l-benzopyran-3-carboxal
dehyde (67).
Triethylorthoformate (150 ml) was moist by adding water (20 ml)
and shaking in a separating funnel. The aqueous layer was removed and
organic layer was transferred to a 250 ml of round bottom flask. To this
moist triethylorthoformate was added a catalytic amount of p-toluene
sulfonic acid (100 mg) and heated on water bath for 0.5 hr. Now 6,7-
dimethyl-4-hydroxy-2-oxo-2H-l-benzopyran (5.0 gm) was added in
portion with simultaneous thorough shaking of the content of the flask.
After complete addition was made, the reaction mixture was refluxed for
0.5 hr (completion of the reaction was checked by TLC).The reaction
mixture was concentrated under reduced pressure and extracted with
diethyl ether (40 ml). The ethereal layer was washed with water, dried
over anhydrous sodium sulfate and concentrated. The residue, after
evaporation of the ether was crystallized from the chloroform-petrol to
give 6,7-dimethyl-4-hydroxy-2-oxo-2H-1 -benzopyran-3-carboxaldehyde
67; yield 60%; m.p.MO^'C.
The compound was identified on the basis of m.p., mixed m.p. and co-
TLC from an authentic sample.
142
The reaction of 4-hydroxy-2-oxo-2H-l-benzopyran-3-carboxaldchyde (31)
with triacetic acid lactone (64).
Formation of 3-acetoacetyIpyrano [3,2-c] [1] benzopyran-2,5-dione (68).
4-Hydroxy-2-oxo-2H-l-benzopyran-3-carboxaldehyde 31 (1.0 gm,
5.3 mmole) and triacetic acid lactone 64 (0.65 gm, 5.1 mmole) were taken in
ethanol (20 ml) and refluxed on boiling a water bath for 0.5 hr. On cooling
yellow solid of 3-acetoacetylpyrano [3,2-c] [1] benzopyran-2,5-dione 68 was
obtained. It was filtered, washed with ethanol and purified by recrystallization
from chloroform; yield 72%; m.p. 242-243 °C.
Spectral Data:
UV (MeOH) X niax : 230, 270, 385, and 410 nm.
IR (KBr) v ax : 3500, 1760, 1730, 1640 and 1550 cm '.
'H N M R (300 MHz, DMSO-dfi) : 5 2.30 (s, 3H, CH3), 7.11 (s, IH, H^),
7.23-8.21 (m, 4H, Ar-H), 8.52 (s,
lH,Hb), 10.5(brs, IH,).
MS (% rel. int.) : m/z 298 (M^ 47), 283 (10), 270 (5),
256 (10), 255 (10), 242 (20), 241
(100), 213 (10), 185 (25), 120 (35)
and 92 (30).
Analyzed for C,6 HioOfi : Calculated C = 64.42, H = 3.35%.
Found C = 64.53, H = 3.55%.
143
The reaction of 6,7-diniethyl-4-hydroxy-2-oxo-2H-l-benzopyran-3-
carboxaldehyde (67) with triacetic acid lactone (64).
Formation of 8,9-dimethyl-3-acetoacetylpyrano |3,2-c] [1] bcnzo
pyran-2,5-dione (69).
A mixture of 6,7-dimethyl-4-hydroxy-2-oxo-2H-l-benzopyran-3-
carboxaldehyde 67 (1.0 gm, 4.5 mmole) and triacetic acid lactone 64
(0.57 gm, 4.5 mmole) was taken in methanol (30 ml) and stirred at 60 "C
for 3 hrs. A greenish yellow solid 8,9-dimethyl-3-acetoacetylpyrano
[3,2-c] [1] benzopyran-2,5-dione 69 deposited. The crystals were
filtered, washed with methanol and dried; yield 66.2%; m.p.240 °C.
Spectral Data:
UV (MeOH) X ax : 236.57, 288.52, 340.47 nm.
IR (KBr) Vmax 3377, 1756 and 1720 cm' .
'H N M R (300 MHz, CDCI3) 5 2.27 (s, 3H, CH3), 2.38 (s, 3H, CH3),
2.42 (s, 3H, CH3), 6.88 (s, IH, Ha),
7.26 (s, IH, Ar-H), 7.84 (s, IH, Ar-
H), 8.91 (s, lH,Hb).
MS (rel. int.) : m/z 327 (M" + 50), 326 (M^ 20), 311
(10), 298 (5), 283 (10), 270 (35), 269
(30), 241(20), 213 (10), 197 (15), 148
(80), 128(15), 120(20).
Analyzed for C,8 H,4 06 : Calculated C = 66.25, H = 4.29%;
Found C = 66.40, H = 4.35%.
144
The reaction of 3-acetoacetylpyrano |3, Z-cJ [IJ benzopyran-2,5-dionc (68)
with hydrazine hydrate.
Formation of 3-(3-methyI pyrazol-5-yI)-pyrano (3, 2-c) (IJ benzopyran-2,5-
dione (70a).
3-Acetoacetylpyrano [3, 2-c] [1] benzopyran-2,5-dione 68 (1.0 gm, 3.3
mmole) was dissolved in acetic acid (20 ml) and hydrazine hydrate (1 ml, 20.2
mmole) added to it. The reaction mixture was refluxed for 1 hr on an oil bath.
On cooling light yellow solid 3-(3-methyl pyrazol-5-yl)-pyrano [3,2-c] [1]
benzopyran-2,5-dione 70a was obtained. It was filtered, washed with water and
dried. The filtrate was poured into crushed ice-water (50 ml) when more 70a
was collected by filtration. The two solids were combined and purified by
recrystallization from chloroform-benzene mixture as yellow shining needles;
yield 70%; m.p. 135-140 °C.
Spectral Data:
UV (MeOH) X 258.58, 233.49, 211.74 nm.
IR (KBr) V, 3250, 1767, 1711, 1628 and 1557 cm"
'H N M R (300 MHz, CDCI3) 2.35 (s, 3H, CH3), 6.75 (s, IH, Ha),
7.16-8.11 (m, 4H, Ar-H), 7.71 (s, IH,
Hb), 8.61 (brs, NH).
145
MS (rel. int.)
Analyzed for Ci6 Hio N2 O4
: m/z 294 (M^ 20), 293 (20), 279 (5)
253 (5), 161 (20), 153 (100), 137 (50),
133(40), 120 (20) and 104(10).
: Calculated C = 65.30, H = 3.40,
N = 9.52%; Found C = 65.55, H =
3.65, N = 9.72%.
The reaction of 3-acetoacetyIpyrano [3, 2-c] [1] benzopyran-2,5-dione (68)
with phenylhydrazine.
Formation of 3-(3-methyl-l-phenyI pyrazoI-5-yI)-pyrano [3, 2-c] [1] benzo
pyran-2, 5-dione (70b).
3-Acetoacetylpyrano [3,2-c] [1] benzopyran 2,5-dione 68 (1.0 gm, 3.3
mmole) was taken in acetic acid (20 ml) and phenylhydrazine (1 ml, 10.2
mmole) added to it. The reaction mixture was refluxed for 1 hr on an oil bath.
On cooling at room temperature a yellow solid was obtained. It was filtered,
washed with water and dried. The filtrate was poured into crushed ice-water
(50 ml) when more 3-(3-methyl-l-phenyl pyrazol-5-yl)-pyrano [3, 2-c] [1]
benzopyran-2, 5-dione 70b was obtained. The two solids were combined and
recrystallized from chloroform; yield 65%; m.p.225-230 °C.
Spectral Data:
UV (MeOH) X
IR(KBr)v„ax
208.40,373.64,367.31 nm.
1760, 1726, 1637 and 1565 cm"
146
' H N M R (300 MHz, CDC13)
MS (% rel. int.)
Analyzed for C22 Hu N2 O4
5 2.39 (s, 311, CH3), 6.59 (s. III, Ha),
7.35-8.04 (m, 9H, Ar-H), 7.81
(s, lH,Hb);
m/z 370 (M^ 100), 369 (20), 329 (5),
213 (5), 185 (10), 181 (5), 161 (30),
157 (30), 153 (70), 137 (50), 133 (40),
120(30), 104 (30) and 92 (35).
Calculated C = 71.35, H = 3.78,
N = 7.56%. Found C = 71.55, H =
3.90, N = 7.70%.
The reaction of 3-acetoacetyIpyrano [3,2-c] [1] benzopyran-2,5-dione (68)
with hydrazinobenzothiazole.
Formation of 3-(3-methyl-l-benzothiazolo pyrazol-5-yI)-pyrano
(3,2-c] [1] benzopyran-2,5-dione (70c).
3-Acetoacetyl pyrano [3,2-c] [1] benzopyran 2,5-dione 68 (1.0 gm, 3.3
mmole) was dissolved in alcohol (25 ml) containing PTS in catalytic
amount and hydrazinobenzothiazole (0.6 gm, 3.3 mmole) added to it.
The reaction mixture was refluxed on boiling water bath for 16-18 hrs. It
was cooled at room temperature to afford 3-(3-methyl-l-benzothiazolo
pyrazol-5-yl)-pyrano [3,2-c] [1] benzopyran-2,5-dione 70c as light green
crystalline solid. It was filtered, washed with alcohol and dried; yield
72%; m.p.270-75 "C.
147
Spectral Data:
UV (MeOH) X n,ax 208.40, 226.80, 248.54, 288.69,
300.40 nm.
IR (KBr) v„,ax
'H N M R (300 MHz, CDCI3)
MS (% rel. int.)
Analyzed for C23 H,3 N3 O4 S
1740, 1643, 1557, and 1442 cm'.
5 2.40 (s, 3H, CH3), 6.50 (s, IH, Ha),
8.01 (s, IH, Hb), 7.32-8.01 (m, 8H,
Ar- H).
m/z 427 (M\ 100), 399 (10), 383
(20), 371 (10), 214 (5), 170 (5), 126
(5).
Calculated C - 64.63, H = 3.04,
N = 9.83%; Found C = 64.72, H =
3.15, N = 9.95%.
The reaction of 3-acetoacetyIpyrano [3,2-cl [1] benzopyran-2,5-dione (68)
with hydroxylammonium sulfate.
Formation of 3-(3-niethyl isoxazol-5-yI)-pyrano [3,2-c] [1] benzopyran-2,5-
dione (70d).
Hydroxylammonium sulfate (0.54 gm, 3.3 mmole) was dissolved in 15
ml of water and 10 drops of dil HCl added to it. This solution was added to a
solution of 3-acetoacetylpyrano [3,2-c] [1] benzopyran-2,5-dione 68 (1.0 gm,
3.3 mmole) in acetic acid (12 ml). The reaction mixture was refluxed on an oil
bath for 1 hr. The Pale yellow crystalline solid 3-(3-methyl isoxazol-5-yl)-
148
pyrano [3,2-c] [1] benzopyran-2,5-dione 70d as obtained on cooling was
filtered, washed with cold water and dried; yield 70%; m.p. 230 °C.
Spectral Data:
UV(MeOH)X„a,
IR (KBr) V max
'H N M R (300 MHz, CDCI3)
MS (% rel. int.)
206.72, 233.49, 283.67, 375.68 nm.
1765, 1725, 1628, 1603, 1566 and
1555 cm'
5 2.40 (s, 3H, CH3), 6.98 (s, IH, Ha),
7.44-8.14 (m, 4H, Ar- H), 8.67 (s, IH,
Hb);
m/z 295 {M\ 50), 254 (20), 213 (10),
185 (10), 157 (100), 134 (75), 120
(25), 106 (40) and 76 (30).
Analyzed for C|6 H9 N Of Calculated C = 65.08, H = 3.05,
N = 4.74%; Found C = 65.15, H =
3.10, N = 4.78%.
The reaction of 8,9-diinethyl-3-acetoacetylpyrano [3,2-c] [1]
benzopyran-2, 5-dione (69) with hydrazine hydrate.
Formation of 8,9-dimethyI-3-(3-niethylpyrazoI-5-yI)-pyrano [3,2-c]
[1] benzopyran-2,5-dione (71a).
8,9-Dimethyl-3-acetoacetylpyrano [3,2-c] [1] benzopyran-2, 5-
dione 69 (1.0 gm, 3.0 mmole) was dissolved in acetic acid (20 ml) and
hydrazine hydrate (1 ml, 20.2 mmole) added to it. The reaction mixture was
149
refluxed for 1 hr on an oil bath. On cooling light yellow solid 8,9-diinethyl-3-
(3-methylpyrazol-5-yl)-pyrano [3,2-c] [1] benzopyran-2,5-dione 71a was
obtained. It was filtered, washed with water and dried. The filtrate was poured
into crushed ice water (50 ml) when more 71a was collected by filtration. The
two solids were combined and purified by recrystallization ft-om chloroform-
benzene mixture as yellow shining needles; yield 65%; m.p. 315-320 °C.
Spectral Data:
UV (MeOH) X „,a>
IR (KBr) V max
' H N M R (300 MHz, DMSO-dfi)
MS (% rel. int.)
Analyzed for Cig H H N2 O4
264.40,280.45, 310.60 nm.
3215, 1746, 1721, 1630 and 1559 cm''.
5 2.31 (s, 3H, CH3), 2.38 (s, 3H,
CH3), 2.40 (s, 3H, CH3), 6.71 (s,
IH, Ha), 8.42 (s, IH, Hb), 7.39 (s, IH,
H-7),7.79(s, 1H,H-10).
m/z 322 (M^ 85), 307 (80), 289 (65),
279 (20), 242 (5), 238 (10), 198 (20),
154(100).
Calculated C = 67.08, H = 4.34,
N = 8.69%; Found C = 67.25, H =
4.55, N = 8.85%.
150
The reaction of 8,9-dimethyl-3-acetoacetylpyrano 13,2-c] |1)
benzopyran-2, 5-dione (69) with phenylhydrazinc.
Formation of 8, 9-dimethyi-3-(3-methyl-l-phenylpyrazoI-5-yl)-
pyrano [3,2-c] [l]benzopyran-2,5-dione (71b).
8,9-Dimethyl-3-acetoacetylpyrano [3,2-c] [1] benzopyran-2, 5-
dione 69 (1.0 gm, 3.0 mmole) was taken in acetic acid (20 ml) and
phenylhydrazinc (1 ml, 10.2 mmole) added to it. The reaction mixture was
refluxed on an oil bath for 1 hr. On cooling at room temperature a yellow solid
was obtained. It was filtered, washed with water and dried. The filtrate was
poured into crushed ice-water (50 ml) when more 8, 9-dimethyl-3-(3-methyl-
l-phenylpyrazol-5-yl)-pyrano [3,2-c] [1] benzopyran-2,5-dione 71b was
obtained. The two solids were combined and purified by recrystallization from
chloroform as yellow crystals; yield 70%; m.p. 245-250 °C.
Spectral Data:
UV (MeOH) X
IR (KBr)
'H NMR (300 MHz, CDCI3)
MS (% rel. int.)
210.07, 235.16, 287.02, 370.66 nm.
1748, 1721, 1635 and 1559 cm"'.
6 2.34 (s, 3H, CH3), 2.39 (s, 6H,
2CH3), 6.57(s, IH, Ha), 7.16- 7.81
(m, 8H, Ar-H+H-6).
m/z 398 (M\ 100), 397 (20), 370
(10), 357 (5), 241 (5), 213 (5), 209
(5), 189 (5), 185 (5), 181 (5), 165
151
Analyzed for C24 Hig N2 O4
(10), 161 (5), 148(35), 132(10). 120
(10).
Calculated C = 72.36, H = 4.52,
N = 7.03%; Found C = 72.50, H =
4.74, N = 7.18%.
The reaction of 8,9-dimethyl-3-acetoacetyIpyrano [3,2-c] [1]
benzopyran-2, 5-dione (69) and hydrazinobenzothiazole.
Formation of 8,9-dimethyI-3-(3-methyI-l-benzothiazoIopyrazol-5-
yl)-pyrano [3,2-c] [1] benzopyran-2,5-dione (71c).
8,9-Dimethyl-3-acetoacetylpyrano [3,2-c] [1] benzopyran-2,5-
dione 69 (1.0 gm, 3.0 mmole) was dissolved in alcohol (25 ml) containing
p-toluene sulfonic acid (30 mg) and hydrazinobenzothiazole (0.53 gm,
3.0 mmole) added to it. The reaction mixture was refluxed on boiling
water bath for 16-18 hrs. It was cooled at room temperature to afford
8,9-dimethyl-3-(3-methyl-l-benzothiazolopyrazol-5-yl)-pyrano [3,2-c]
[1] benzopyran-2,5-dione 71c as light green crystalline solid. It was
filtered, washed with alcohol and dried; yield 66%; m.p. 285-290 °C.
Spectral Data:
UV (MeOH) X max
IR (KBr)
210.07, 288.69, 365.64 nm.
1768, 1742, 1684 and 1640 cm"
152
'H N M R (300 MHz, CDCI3) 2.30 (s, 9H, 3CH3), 6.48 (s. IH, Ha),
6.97-8.17 (m, 7H, Ar-H), 8.81 (s, IH.
Hb).
MS (% rel. int.) m/z 455 (M\ 100), 427 (10), 411 (5),
399 (10), 321 (2), 308 (5), 153 (70),
167(10), 123(3).
Analyzed for C25 H,? N3 O4 S Calculated C = 65.93, H = 3.73,
N = 9.23%; Found C = 66.12, H
3.98, N = 9.35%.
The reaction of 4-hydroxy-2-oxo-2H-l-benzopyran-3-carboxaldehyde (31)
with 5,5-dimethyIcyclohexan-l,3-dione (72).
Formation of 7-(4-hydroxycoumarin-3-yI)-10,10-diniethyl-8-oxo-8, 9,
10, 11-tetrahydro pyrano [3,2-c] coumarin (74).
A mixture of 4-hydroxy-2-oxo-2H-l-benzopyran-3-carboxaldehyde
31 (1.0 gm, 5.3 mmole) and 5,5-dimethylcyclohexan-l,3-dione (dimedone) 72
(0.72 gm, 5.1 mmole) was refluxed in ethanol (20 ml) for 1 hr. The cream
coloured solid 7-(4-Hydroxycoumarin-3-yl)-10,10-dimethyl-8-oxo-8,9,10,
11-tetrahydropyrano [3,2-c] coumarin 74 obtained on cooling, was filtered,
washed with ethanol and purified by recrystallization from chloroform; yield
60%; m.p. 240-245 °C.
153
Spectral Data:
UV(MeOH)X,
IR(KBr)v,„ax
211.74, 258.58, 302.07 nm.
1724, 1612, 1369, 1304, 1199, 1037
and 754 cm -1
'HNMR(300MHz,CDCl3) 5 1.11 (s, 3H, CH3), 1.18 (s, 3H,
CH3), 2.36 (s, 2H), 2.38 (s, 2H), 5.113
(s, IH), 7.17-7.94 (m, 6H, Ar-H), 8.04
(dd, IH, H-5', J = 7.8, 1.2 Hz), 7.92
(dd, IH, H-1, J = 7.8, 1.2 Hz), 10.55
(brs, IH);
M S (% rel. int.)
Analyzed for C27 H20 O7
m/z 456 (M^ 40), 335 (20), 307 (5),
295 (100), 239 (25), 121 (4) and 107
(10).
Calculated C = 71.05, H = 4.38%;
Found C = 71.23, H = 4.55%.
The reaction of 4-hydroxy-2-oxo-2H-l-benzopyran-3-carboxaldehyde
(31) with 3-methyI-l-phenyl-5-pyrazolone (76).
Formation of methylidene-bis-4,4-(3-niethyl-5-oxo-l-phenylpyrazole) (78).
4-Hydroxy-2-oxo-2H-l-benzopyran-3-carboxaldehyde 31 (1 gm, 5.3
mmole) was dissolved in ethanol (15 ml) and 3-methyl-l-phenyl-5-pyrazolone
154
76 (0.9 gm, 5.2 mmole) added to it. The reaction mixture was relluxed for 10-12
115 hrs. Yellow solid methylidene-bis-4, 4-(3-methyl-5-oxo-l-phenylpyrazole) 78
as obtained on cooling was filtered, washed with ethanol and recrystallized from
chloroform or benzene; yield 61.5%; m.p. 182-185 °C.
Spectral Data:
UV (MeOH) X 208.40, 332.18 nm.
' H N M R ( 3 0 0 M H Z , CDCI3)
MS (% rel. int.)
IR(KBr)v,^ax 3350, 1627, 1592, 1550, 1498 and
1328 cm-'.
: 5 2.32 (s, 6H, CH3), 7.26-7.92 (m,
IIH, Ar-H+ methylene proton).
: m/z 358 (M^ 100), 357 (50), 340 (5),
281 (6), 117 (4), 104 (4), 90 (20) and
77 (35).
: Calculated C = 70.39, H = 5.02,
N = 15.64%; Found C = 70.15,
H = 5.32, N = 15.25%.
To refluxing moist triethylorthoformate (40 ml) containing a catalytic
amount of p-toluene sulfonic acid was added 3-methyl-l-phenyl-5-pyrazolone
76 (0.8 gm) in portions during 0.5 hr. The additions were so regulated that no
solid remained before further addition was made. After complete addition the
refluxing was continued for another 15 min., when yellow crystals of
Analyzed for C21 Hig N4 O2
155
' H N M R (400 MHz, CDCI3)
MS (% rel. int.)
5 2.05 (s, 6H, 2CH3), 2.29 (s, 3H,
CH3), 4.53 (s, IH, CH), 6.01 (s,
2H, H-5), 7.068-7.62 (m, 5H, Ar-
H), 10.49 (s, IH, OH).
m/z 418 ( M \ 50), 403 (5), 375 (5),
245 (100), 262 (5), 156 (5), 137
(5).
Analyzed for C23 Hig N2 Oe Calculated C = 66.02, H = 4.30,
N = 6.69%; Found C = 66.23, H =
4.55, N = 6.40%.
The reaction of 5-azido-3-methyI-l-phenylpyrazole-4-carboxaI
dehyde (80) with triacetic acid lactone (64).
Formation of 4-(4-hydroxy-6-methyl-2-oxo-2H-pyran-2-one-3-yl)-
3,7-dimethyI-l-phenylpyrazolo [3,4:2,3]-4H-pyrano [3,2-c] pyran-5-
one (95).
5-Azido-3-methyl-l-phenylpyrazole-4-carboxaldehyde 80 (1.0 gm,
4.4 mmole) was dissolved in methanol (20ml) and triacetic acid lactone
64 (0.53 gm, 4.4 mmoles) added to it. The resultant mixture was refluxed
on water bath for 6 hrs. It was concentrated and allowed to stand at room
temperature when cream coloured solid crystallized out from the reaction
mixture. It was filtered, washed with ethanol, dried and recrystallized
from chloroform-benzene to afford 4-(4-hydroxy-6-methyl-2-oxo-2H-
157
pyran-2-one-3-yl)-3,7-dimethyl-l-phenylpyrazolo [3,4:2,3]-4H-pyrano
[3,2-c] pyran-5-one 95; yield 65.3%; m.p.230-32 °C.
Spectral Data:
UV (MeOH) X max
IR (KBr) v^ax
' H N M R (500 MHz, CDCI3)
MS (% rel. int.)
207.91, 265.23 nm.
3378, 1703 and 1621 cm'
5 2.15 (s, 6H, 2CH3), 2.31 (s, 3H,
CH3), 5.13 (s, IH, CH), 5.93 (s,
IH, H-5'), 6.20 (s, IH, H-5), 7.26 -
7.71 (m, 5H, Ar-H), 10.12 (br s, IH,
OH, D2O exchangeable),
m/z 418 (M^ 20), 292 (100), 137
(50), 155(20).
Analyzed for CzsHigNz 06 Calculated C = 66.02, H = 4.30,
N = 6.69%; Found C = 66.23, H =
4.55, N = 6.40%.
Reaction of 5-chloro-3-methyI-l-phenyIpyrazoIe-4-carboxaldehyde
(79) with 4-hydroxycoumarin (1).
Formation of 4-(4-hydroxy-2-oxo-2H-l-benzopyran-2-one-3-yl)-3-
methyl-l-phenylpyrazolo [3,4:2,3]-4H-pyrano (3,2-b]-l-benzopyran-
5-one (96).
To a solution of 5-chloro-3-methyl-l-phenylpyrazole-4-
carboxaldehyde 79 (1.0 gm, 4.5 mmole) and 4-hydroxycoumarin .1 (0.74
gm, 4.5 mmole) in alcohol (20 ml) was added anhydrous sodium acetate
158
(0.37 gm, 4.4 mmole). The reaction mixture was refluxed on water bath
for 18 hrs. It was then cooled at room temperature and poured into ice
cold water. The white solid which precipitate out was filtered,
washed with cold water, dried and recrystallized from DMF to
give 4-(4-hydroxy-2-oxo-2H-l-benzopyran-2-one-3-yl)-3-methyl-l-
phenylpyrazolo [3,4:2,3]-4H-pyrano [3,2-b]-l-benzopyran-5-one 96 as
white crystals; yield 74.3%; m.p. 270 °C.
Spectral Data:
UV(MeOH)^„3x 210.07, 246.87, 255.23, 308.76 nm.
IR (KBr) V,,, 3451 and 1729 cm 1
'H NMR (400 MHz, DMSO-de)
MS (% rel. int.)
5 2.62 (s, 3H, CH3), 4.75 (s, IH,
CH), 7.25-7.79 (m, IIH, Ar-H),
8.20 (d, 2H, J = 7.9 Hz).
m/z491 (M^+1, 100), 317(90).
Analyzed for C29 H,8 N2 Og : Calculated C = 71.02, H = 3.67,
N = 5.71%; Found C = 71.35, H =
3.95, N = 5.40%.
159
Reaction of 5-azido-3-inethyl-l-phenyipyrazole-4-carboxaldehydc
(80) with 4-hydroxycoumarin (1).
Formation of 4-(4-hydroxy-2-oxo-2H-l-benzopyran-2-one-3-yl)-3-
methyI-1-phenylpyrazolo [3,4:2,3]-4H-pyrano [3,2-cl-l-benzopyran-
5-one (97).
A mixture of 5-azido-3-methyl-l-phenylpyrazole-4-
carboxaldehyde 80 (1.0 gm, 4.5 mmole) and 4-hydroxycoumarin 1 (0.71
gm, 4.4 mmole) in methanol (20 ml) was refluxed on water bath for 2
hrs and allowed to cool at room temperature. The solid which
crystallized out from the reaction mixture, was filtered, washed with
ethanol, dried and recrystallized from chloroform to give 4-(4-hydroxy-
2-OXO-2H-1 -benzopyran-2-one-3-yl)-3-methyl-1 -pheny Ipyrazolo
[3,4:2,3]-4H-pyrano [3,2-c]-l-benzopyran-5-one 92 as white solid; yield
70%; m.p. 290-292 °C.
Spectral Data:
UV (MeOH) >. max : 211.74, 246.87, 255.23, 325.49 nm.
IR(KBr)v^a, : 3078, 1731 and 1670 cm"'.
'H NMR (500 MHz, CDCI3) : 6 2.12 (s, 3H, CH3), 5.36 (s, IH, CH),
7.30-7.83 (m, IIH, Ar-H), 7.97 (d, IH,
H-5), 8.04 (d, IH, H-5).
MS (% rel. int.) : m/z 490 (M^ 50), 489 (10), 369 (5),
329 (55), 209 (5), 208 (5), 193 (5),
167(5), 120(10), 90(15).
160
Analyzed for C29H,8N2 06 : Calculated C = 71.02, H - 3.67,
N = 5.71%; Found C = 71.35, H =
3.95, N = 5.40%.
Synthesis of 5-ainino-3-methyl-l-phenyIpyrazole-4-carboxaldehyde (102).
To a stirred solution of sodium borohydride (0.6 gm) in water (3 ml)
were added 5-azido-3-methyl-l-phenylpyrazole-4-carboxaldehyde 80 (1.0 gm,
4.5 mmole) and toluene (1 ml). The reaction mixture was stirred for 4-5 hrs at
room temperature. A reddish brown solid was obtained after completion of the
reaction. The solid was filtered, washed thoroughly with water and adsorbed on
a column of silica gel. Elution of the column by a mixture of benzene-ethyl
acetate (99:1 v/v) afforded 5-amino-3-methyl-l-phenyl pyrazole-4-
carboxaldehyde 102 as white solid; yield 60%; m.p. 110-112 °C (lit."* m.p. 92-
93 °C).
The reaction of 5-amino-3-methyl-l-phenylpyrazole-4-carboxal
dehyde (102) with triacetic acid lactone (64).
Formation of 3,6-diniethyl-l,8-diphenyl-diazocino (3,4-c:7,8-c'] bis
pyrazole (106).
5-Amino-3-methyl-l-phenylpyrazole-4-carboxaldehyde 102 (1.0
gm, 5.0 mmole) and triacetic acid lactone 64 (0.6 gm, 5.0 mmoles) were
taken in pyridine (20 ml) and catalytic amount of piperidine (0.5 ml)
added to it. The reaction mixture was stirred at room temperature for 48-
50 hrs. The reaction mixture was acidified with HCI and extracted
with chloroform. The reaction mixture was concentrated and
161
chromatographed over silica gel. Elution of the column with benzene-
ethylacetate (90:10 v/v) afforded a white crystalline solid labeled as 3,6-
dimethyl-l,8-diphenyl-diazocino [3,4-c:7,8-c'] bis pyrazole 106; yield
40.6%; m.p. 98 °C.
Spectral Data:
UV(MeOH)).,ax
IR(KBr)v„,ax
' H N M R (300 MHz, CDCI3)
MS (% rel. int.)
Analyzed for C22 Hig Ng
206.12, 263.44 nm.
1594 and 1550 cm"'.
5 2.48 (s, 6H, 2CH3), 8.19 (s, 2H),
7.34-7.65 (m, lOH, Ar-H).
m/z 366 (M^ 15), 336 (10), 289 (15),
275 (10), 260 (15), 183 (75), 182 (50),
169 (15), 156 (10), 154 (50), 141 (15),
128(20), 106(25), 102(10).
Calculated C = 72.13, H = 4.91,
N = 22.95%; Found C = 72.30,
H = 5.18, N = 22.55%.
162
(BiBGograpfiy
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