Benzylidine indane-1,3-diones: As novel urease inhibitors; synthesis, in vitro, and in silico studies

Article abstract of DOI:10.1016/j.bioorg.2018.09.030

Current study deals with the evaluation of indane-1,3-dione based compounds as new class of urease inhibitors. For that purpose, benzylidine indane-1,3-diones (1–30) were synthesized and fully characterized by different spectroscopic techniques including

Full text of DOI:10.1016/j.bioorg.2018.09.030

Accepted Manuscript
Benzylidine indane-1,3-diones: As Novel Urease Inhibitors; Synthesis, In vitro,
and In silico Studies
Bilquees Bano, Kanwal, Khalid Mohammed Khan, Farida Begum, Arif Lodhi,
Uzma Salar, Ruqaiya Khalil, Zaheer-Ul-Haq, Shahnaz Perveen
PII:
S0045-2068(18)30501-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.030
YBIOO 2527
Reference:
Bioorganic Chemistry
To appear in:
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Accepted Date:
20 May 2018
18 September 2018
Please cite this article as: B. Bano, Kanwal, K. Mohammed Khan, F. Begum, A. Lodhi, U. Salar, R. Khalil, Zaheer-
Ul-Haq, S. Perveen, Benzylidine indane-1,3-diones: As Novel Urease Inhibitors; Synthesis, In vitro, and In silico
Studies, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.09.030
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Benzylidine indane-1,3-diones: As Novel Urease Inhibitors; Synthesis, In vitro, and In silico
Studies
Bilquees Bano,a Kanwal,a Khalid Mohammed Khan,a,e Farida Begum,b Arif Lodhi,b Uzma
Salar,a Ruqaiya Khalil,c Zaheer-Ul-Haq,c Shahnaz Perveend
aH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University, Mardan, Pakistan
cPanjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
dPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280,
Pakistan
eDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 31441,Dammam Saudi Arabia
Abstract: Current study deals with the evaluation of indane-1,3-dione based compounds as new
class of urease inhibitors. For that purpose, benzylidine indane-1,3-diones (1-30) were
synthesized and fully characterized by different spectroscopic techniques including EI-MS,
HREI-MS, 1H-, and 13C-NMR. All synthetic molecules 1-30 were evaluated for urease inhibitory
activity and showed good to moderate inhibitory potential within the range of (IC50 = 11.60 ±
0.3- 257.05 ± 0.7 µM) as compared to the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5
µM). Compound 1 (IC50 = 11.60 ± 0.3 µM) was found to be most potent inhibitor amongst all
derivatives. The key binding interactions of most active compounds within the enzyme pocket
were evaluated through in silico studies.
Keywords: Synthesis; indane-1,3-dione; urease inhibitory activity; structure-activity
relationship; molecular docking study
Introduction
Urease (EC 3.5.1.5) is a nickel containing enzyme that is involved in the hydrolysis of urea to
ammonia and CO2 or carbamate which further decomposes into another molecule of ammonia
[1]. Among other binuclear metallohydrolases of the super family, ureases are unique due to
having Ni (II) ions in the active site. Ureases are extensively found in nature and biosynthesized
*Corresponding Authors: khalid.khan@iccs.edu, drkhalidhej@gmail.com; Tel.: +922134824910, Fax:
+922134819018
1

by several organisms, including bacteria, plants, algae, fungi, invertebrates, and are found in soil
as a soil enzyme [2,3]. This enzyme is also involved in nitrogen metabolism and germination
process of plants [4]. High amount of ammonia released during the fertilization of urea cause a
significant increase in pH thus increasing the alkalinity of soil which leads to the damage of the
plants by depriving them from their essential nutrients [5,6]. Urease plays a vital role in many
pathogenic processes in humans as well as in animals. It showed a major role in urinary catheter
incrustation, peptic ulceration, pyelonephritis, kidney stone, hepatic encephalopathy, urolithiasis,
and arthritis [7-9]. Therefore, strategies based on urease inhibition are now considered as the first
line of treatment for infections caused by urease producing bacteria.
Indane-1,3-dione and its derivatives have attracted the considerable attention of organic chemists
due to their distinguishing features e.g. the presence of β-dicarbonyl moiety, and an active
methylene group capable of undergoing nucleophilic substitution. The enormous synthetic
potential of indane-1,3-diones often serves as a synthon for the preparation of more structurally
complex compounds via decomposition, condensation, cyclization, reduction, and
rearrangements. The molecule offers wide scope for the study of physiochemical properties such
as 1,3-indandione tautomerism, electrochemical redox properties, dual reactivity, unique
property of polycrystalline films, quantum mechanical calculations, and so forth. A wide range
of biological properties covering antimicrobial, antitumor, antiinflammatory, antiviral,
antihepatitis, anticoagulant, rodenticidal, herbicidal, and insecticidal properties are also
associated with indane-1,3-dione derivatives [10].
Previously, we had explored different classes of compounds for their potential as urease
inhibitors e.g. dihydropyrimidine, thiazolidinone, oxindole, arylidene N,N-dimethyl barbiturates,
thiobarbituric acid, 2-(2-pyridyl)benzimidazole, N,Nʹ-disubstituted thioureas, 1,3,4-oxadiazole,
1,2,4-triazoles,urea, and isatin [11-20]. Benzoquinone and its derivatives have already been
reported for their urease inhibitory potential [21]. It is worth noting that our synthesized
molecules 1-30 have structural resemblance with benzoquinone based urease inhibitors (Figure-
1). Therefore, in continuation of exploring new urease inhibitors, we decided to evaluate indane-
1,3-dione class for urease inhibitory studies for the first time. To the best of our knowledge,
except compounds 1, 5, 22, 26, 27, 28, 29, and 30, all derivatives are new [22].
2

Figure-1: Rationale of present study
Results and Discussion
Chemistry
Benzylidine derivatives of indane-1,3-dione (1-30) were synthesized by the condensation
reaction between indane-1,3-dione and substituted benzaldehydes. First, indane-1,3-dione was
treated with pyridine as base which abstracted one of its active methylene proton resulting in the
formation of enolate ion. Subsequent attack of enolate ion on electrophilic carbonyl carbon of
benzaldehyde resulted in the formation of benzylidine indane-1,3-diones [23] as shown in
Scheme-1 (Table-1). The progress of the reaction was observed by TLC. In all experiments,
product was afforded in the form of precipitates which were filtered and washed with excess of
hexane. The product thus obtained was dried under vacuum and crystallized with methanol. The
structures were determined by 1H-, 13C-NMR, EI-MS, and HREI-MS spectroscopic techniques.
Scheme-1: Synthesis of benzylidene indane-1,3-diones 1-30
3

Spectral characterization of most active compound (1)
1H-NMR Spectroscopy
1
The H-NMR was recorded in deuterated DMSO on 300 MHz instrument. The molecule
comprises of seven aromatic protons. A sharp signal of H-6′ appeared at δH 9.15 as doublet
showing ortho coupling (J6′,5′ = 9.0 Hz) with H-5′. The methine signal was resonated at δH 8.28
as singlet. An overlapping signal of four protons H-4, H-5, H-6, and H-7 of phenyl ring were
appeared at δH 7.89. H-3′ and H-5′ were resonated at δH 6.60 as an overlapped multiplete. The
most up field signal of the spectrum was appeared at δH 3.82 representing the presence of
1
methoxy group. The characteristic H-NMR chemical shifts of the active compounds are shown
in Figure-2A.
Figure 2: Representative 1H-(A) and 13C-NMR (B) chemical shifts of compound 1
13C-NMR broad-band decoupled spectrum (DMSO-d6) showed total seventeen carbon signals
including one methoxy, eight methine, and eight quarternary carbons. The most downfield
signals of carbonyl containing quartenary carbons C-1 and C-3 were appeared at C 190.4 and C
189.2, respectively. A sharp signal of methine resonated at δC 139.8. Quarternary carbons C-2′,
C-2, C-4′, C-4a, C-7a, and C-1′ resonated at C 166.3, 163.4, 141.5, 139.1, 123.4, and 114.1,
respectively. Signals of all remaining aromatic carbons appeared in the usual aromatic range of
C 100.2-122.4. The most upfield signal of methoxy carbon was resonated at C 55.5. The
characteristics 13C-NMR chemical shifts of the most active compound are shown in Figure-2B.
Mass Spectrometry
The EI-MS spectra of compound 1 showed the M+ at m/z 280 for molecular formula C17H12O4. A
fragment ion appeared at m/z 263 representing the loss of hydroxyl group. Further loss of
methoxy group from the previous fragment generates a new fragment ion at m/z 235. Another
4

fragment appeared at m/z 91 confirming the cleavage of carbon carbon double bond. The
characteristic fragmentation pattern of the representative compound is shown in Figure-3.
Figure-3: Representative fragmentation pattern of compound1
Table 1: Benzylidineindane-1,3-dione derivatives (1-30)
Compounds
1
Structures
IC50 ± SEMa (µM)
11.60 ± 0.3
20.11 ± 0.1
2
21.13 ± 0.8
25.06 ± 2.0
3
4
5

19.32 ± 1.3
5
33.12 ± 0.9
63.11 ± 1.0
19.10 ± 1.0
6
7
8
86.14 ± 0.1
97.04 ± 0.3
93.01 ± 1.5
9
10
11
6

62.20 ± 1.1
12
13
72.61 ± 1.8
47.23 ± 0.3
13.31 ± 0.1
14
15
60.50 ± 0.5
16
257.05 ± 0.7
17.41 ± 0.4
17
18
7

26.21 ± 1.6
23.10 ± 0.2
17.43 ± 1.5
51.00 ± 1.7
71.22 ± 10.6
18.23 ± 1.1
27.18 ± 0.5
19
20
21
22
23
24
25
8

83.13 ± 0.1
85.23 ± 1.5
95.21 ± 1.9
26
27
28
121.61 ± 1.3
29
30
74.15 ± 1.6
27.0 ± 0.5
Acetohydroxamic Acid
SEMa (Standard error of mean); NAb (Not active); Acetohydroxamic Acid (Standard inhibitor for urease enzyme)
Structure-activity relationship (SAR)
Synthetic benzylidene indane-1,3-diones (1-30) were evaluated for their urease inhibitory
activity. Off thirty derivatives, thirteen compounds 1-5, 8, 15-16, 18-21, and 24 showed excellent
inhibitory potential in the range of (IC50 = 9.2 ± 1.1 – 26.21 ± 1.6 µM) as compared to the
standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 µM). Compound 25 (IC50 = 27.18 ± 0.5 µM)
showed comparable activity with the standard, however, sixteen compounds 6-7, 9-14, 17, 22-23,
and 26-30 exhibited moderate inhibitory activity having IC50 values in the range of 33.12 –
257.05 µM. Limited structure-activity relationship (SAR) of synthetic compounds suggests that
9

the variation in activity of compounds depends on different substituents and their positions on
aryl part (Figure-4).
Figure-4: General Structural Features of Synthetic Compounds
Compound 1 (IC50 = 11.60 ± 0.3 μM) having hydroxy and methoxy substituents at ortho and
para positions was found to be the most active compound of this library might be due to the
better interactions of both groups with the active site of enzyme. However, shifting the position
of hydroxy from ortho to para and methoxy from para to meta resulted in decreased activity as
in compound 2 (IC50 = 20.11 ± 0.1 μM). Addition of another methoxy group at meta position in
compound 3 (IC50 = 21.13 ± 0.8 μM) also displayed less activity as compared to compound 1,
nevertheless, it showed comparable activity to compound 2. So, it can be said that the addition of
another methoxy had no impact on the activity. Compound 4 (IC50 = 25.06 ± 2.0 μM) having two
hydroxy groups at ortho and meta positions, respectively, also exhibited decreased activity as
compared to the compounds 1-3 with mixed substitutions of hydroxy and methoxy. Based on
above observations, it can be concluded that hydroxy and methoxy groups at certain positions are
playing a vital role in the activity of compounds. Replacement of methoxy with hydroxy,
addition of another methoxy, and shifting the position of hydroxy and methoxy substituents
resulted in the decreased activity. However, all these derivatives were found to have good
inhibitory potential as compared to the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM)
(Figure-5).
10

Figure-5: Structure-activity relationship of compounds 1-4
Among methoxy derivatives, the dimethoxy compound 5 (IC50 = 19.32 ± 1.3 μM) having
methoxy groups at ortho and meta positions showed potent activity, however, its structurally
similar compound 6 (IC50 = 33.12 ± 0.9 μM) having methoxy groups at ortho and para positions
was found to be less active. Similarly, shifting of both methoxy groups to ortho positions in
compound 7 (IC50 = 63.11 ± 1.0 μM) also displayed weak activity as compared to the compound
5 as well as standard. Compound 8 (IC50 = 19.10 ± 1.0 μM) with three methoxy groups at ortho,
meta, and para positions exhibited potent activity as compared to the standard and displayed
comparable activity with compound 5. This showed that the substitution of methoxy groups at
ortho and meta positions are mainly contributing in the activity might be the compounds with
these positions fit best in the enzyme pocket. Shifting of methoxy at para position resulted in
decreased activity, likewise addition of another methoxy group at para position did not have any
impact on the activity (Figure-6).
Figure-6: Structure-activity relationship of compounds 5-8
Compounds 9 (IC50 = 86.14 ± 0.1 μM), 10 (IC50 = 97.04 ± 0.3 μM), and 11 (IC50 = 93.01 ± 1.5
μM), with para ethoxy, para butoxy, and ortho butoxy groups, respectively, displayed weak
activity as compared to the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM) which shows
that the methoxy group is contributing in the activity and increase in alkyl chain length i.e.,
11

ethoxy and butoxy resulted in decreased activity. Increasing the hydrophobic part might result in
increased steric hindrance to fit well into the active site of enzyme. Compound 12 (IC50 = 62.20
± 1.1 μM) having benzyloxy and methoxy groups at meta and para positions, respectively,
displayed good activity as compared to ethoxy and butoxy substituted analogs 9, 10, and 11.
Removal of methoxy from meta position as in compound 13 (IC50 = 72.61 ± 1.8 μM) resulted in
decreased activity which again indicated that methoxy group is actively participating in the
activity and binds well within the enzyme’s active site (Figure-7).
Figure-7: Structure-activity relationship of compounds 9-13
Among halogen substituted derivatives, compound 14 (IC50 = 47.23 ± 0.3 μM) with two chloro
groups at ortho positions of the phenyl ring exhibited weak activity as compared to the standard
acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM). Compound 15 (IC50 = 13.31 ± 0.1 μM) having
methoxy and chloro substituents at meta and ortho positions, was found to be the second most
active compound of the series which again showed that replacement of one chloro group with
methoxy resulted in the three folds increased inhibitory activity. Compound 16 (IC50 = 60.50 ±
0.5 μM) bearing hydroxy and chloro groups at ortho and meta positions was less active. The
replacement of chloro with bromo and hydroxy with methoxy in compound 17 (IC50 = 257.05 ±
0.7 μM) showed ten folds decreased activity as compared to the standard, and is the least active
12

compound of the series. Compound 18 (IC50 = 17.41 ± 0.4 μM) having similar groups with
shifting of bromo from meta to ortho showed potent activity might be bromo group at ortho
position is actively participating in the inhibition. Compounds 19 (IC50 = 26.21 ± 1.6 μM) and 20
(IC50 = 23.10 ± 0.2 μM) with two methoxy and bromo groups showed comparable activity with
that of standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM). Interestingly, compound 21 (IC50
= 17.43 ± 1.5 μM) having hydroxy and bromo substituents at meta and para positions showed
potent activity as compared to the standard as well as its methoxy substituted derivatives 17, 18,
19, and 20 (Figure-8).
Figure-8: Structure-activity relationship of compounds 14-21
Compound 22 (IC50 = 51.00 ± 1.7 μM) with fluoro group at para position showed weak
inhibitory activity as compared to the standard. The addition of hydroxy group at ortho position
and shifting the position of fluoro from para to meta as in compound 23 (IC50 = 71.22 ± 0.6 μM)
exhibited decreased activity. Compound 24 (IC50 = 18.3 ± 1.1 μM) with triflouro methyl group
showed potent activity as compared to the standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM)
as well as other flouro substituted derivatives 22 and 23. Compound 25 (IC50 = 27.18 ± 0.5 μM)
bearing iodo, methoxy, and hydroxyl groups at meta and ortho positions, respectively, showed
comparable activity with that of standard (Figure-9).
13

Figure-9: Structure-activity relationship of compounds 22-25
Compound 26 (IC50 = 83.13 ± 0.1 μM) with phenyl ring exhibited weak activity as compared to
the standard. Replacing phenyl ring with pyridine as in compound 27 (IC50 = 85.13 ± 1.5 μM)
also showed similar activity. Compound 28 (IC50 = 95.21 ± 1.9 μM) having furan ring was found
to have less activity as compared to compounds 26 and 27, however, compound 29 (IC50 =
121.61 ± 1.3 μM) with methyl group at furan ring resulted in the further decreased activity which
might be due to the steric factor of methyl group. Compound 30 (IC50 = 74.15 ± 1.6 μM) with di
methyl aniline group at the para position also displayed weak activity as compared to the
standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 μM) (Figure-10).
Figure-10: Structure-activity relationship of compounds 26-30
Thus, from the above observations it can be rationalized that the hydroxy and methoxy groups on
phenyl ring are actively participating in the activity of compounds. However, other substituents
14

have different impact on the basis of positions on phenyl ring. The molecular docking studies of
these compounds were performed to understand the plausible ligand (synthetic compounds)
binding mechanism.
Molecular Docking Study
The docking studies were carried out using the crystal structure of Jack Bean Urease (PDB
4GYZ). All the thirty benzylidene indane-1,3-dionederivatives (1-30) were docked using the
Glide Standard Precision protocol and the resulting poses were analyzed visually to understand
the interaction pattern.
As previously mentioned, the substitution on the aryl part is the prime factor governing the
observed variation in the activity of the compounds. Among the hydroxy and methoxy
substitutes (1-4), compound 1 is the most active derivative (IC50 = 11.60 ± 0.3 μM). The analysis
of top-ranked docking pose of compound 1 suggests that the hydroxyl group is involved in
hydrogen bonding with the neighboring amino acid, Glu-493 and Asp-494, and a special
bidentate interaction with Arg-609. Moreover, the aromatic rings of the ligands present π-
stacking interactions with His-492 and His-593 (Figure-11).
15

Figure-11: The docked pose of the compound 1 in the active site of the Jack Bean Urease
(4GY7). The hydrogen bonds are presented as blue dashed lines. Some of the residues have been
ghosted for visual clarity.
The analysis of binding poses of the other members of the series 2-4, suggests that shifting the
position of hydroxy and methoxy groups result in the depletion of hydrogen bonding network
observed in case of compound 1. In case of compounds 2 and 3, the interaction between the
enzyme and the ligands is mediated by the hydrogen bonding between the hydroxyl group at the
para position and residues; Alu-493 and Arg-609 (Figure-12).
16

Figure-12: The docking pose of compound 2, 3, 4 in the active site of Jack Bean Urease
Among, the methoxy derivatives (5-8), compound 8 (IC50 = 19.10 ± 1.0 μM) bearing three
methoxy positions is the most active representative, followed by the compound 5 (IC50 = 19.32 ±
1.3 μM). The docked pose of compound 8, highlights the formation of a bidentate bond between
Arg-609 and the hydroxyl group of the indane moiety (Figure-13). The π-cation interaction
between the indane ring and His-519 facilitates the anchorage of the ligand in the active site of
protein. The visual analysis suggests the aryl moiety is stacked against the imidazole ring of His-
593.
17

Figure-13: The docked pose of the compound 8 in the active site of the Jack Bean Urease
(4GY7). The hydrogen bonds are presented as blue dashed lines. Some of the residues have been
ghosted for visual clarity.
The compound 6, mediate hydrophobic and π-stacking interactions with Ala-440, Trp-495, and
His-593 (Figure-14). We hypothesize that the decrease in the activity of compound 6, is
attributed to the lack of hydrogen bonding observed in case of both 5 and 8.
Figure-14. The docking pose of compound 5and6in the active site of Jack Bean Urease
To rationalize the observed decrease in activity upon the substitution of longer aliphatic chains,
the docked poses of compounds 9-13 were visually analyzed. The analysis of binding pose of
18

compound 9 suggests formation of hydrophobic contact between the ethoxy group and Ala-636.
The aromatic rings of the compound interact with His-492 and His-593 which partially explains
the observed urease inhibitory activity (Figure-15).
Figure-15: The docked pose of the compound 9 in the active site of the Jack Bean Urease
(4GY7). The hydrogen bonds are presented as blue dashed lines. The indane ring can be seen
staked against His-593. Some of the residues have been ghosted for visual clarity.
The observation is consistent in case of compound 11, which mediates hydrophobic contacts
with Ala-436, His-593 and Trp-495. Another weak inhibitor, compound 12 (IC50 = 62.20 ± 1.1
μM), demonstrate hydrophobic contacts with the surrounding residues (Figure-16).
19

Figure-16: The docking pose of compound 11 and 12 in the active site of Jack Bean Urease
The compound 15 with methoxy and chloro substituents was the second most active compound
of the series. The docking studies suggest that the compound interacts with the modified cysteine
residue (CME-592) present in the histidine rich region. It has been reported that this particular
cysteine is essential for enzymatic activity [28]. The indane ring of the compound is involved in
hydrophobic interaction with Ala-436 and Trp-495 (Figure-17).
20

Figure-17: The docked pose of the compound 15 in the active site of the Jack Bean Urease
(4GY7). The hydrogen bonds are presented as blue dashed lines. The Cme-592 has been
presented in ball and stick model for visual clearance.
The table of activities shows that the compounds bearing bromo groups presented potent urease
inhibitory activity. Compound 21 bearing hydroxyl and bromo groups was found to be more
potent than compounds 17-20, bearing methoxy moiety. The analysis of docking pose suggests
that the bromo group of the compound 21 demonstrate halogen bond with Gly-550. The potency
of the compound might be attributed to the hydrogen bonding interactions between the hydroxyl
group and Gly-493, His-519, and Arg-609 (Figure-18).
Figure-18: The docking pose of compound 21 in the active site of Jack Bean Urease.
Conclusion
Evaluation of synthesized benzylidene indane-1,3-diones (1-30) as novel urease inhibitors
revealed that thirteen compounds 1-5, 8, 15-16, 18-21, and 24 showed excellent and potent
inhibitory activity in the range of (IC50 = 9.2 ± 1.1 – 26.21 ± 1.6 µM) as compared to the
standard acetohydroxamic acid (IC50 = 27.0 ± 0.5 µM). Other compounds were moderately
active against urease enzyme. Structure-activity relationship suggested that compounds bearing
hydroxy and methoxy substituents were more active. In silico study was performed to determine
the binding interactions between enzyme and the ligand which also supported the experimental
data. The newly identified urease inhibitors might assist as lead candidates for further research in
order to get anti-urease agents for the treatment of peptic ulcers and other biomedical
applications.
21

Materials and Methods
13
1H- and C-NMR spectra were recorded on Bruker Avance 300 and 400 MHz spectrometers.
Mass experiments were carried out on a Finnigan MAT-311A (Germany) mass spectrometer.
Thin-layer chromatography (TLC) was monitored on pre-coated silica gel aluminum plates
(Kieselgel 60, 254, E. Merck, Germany). Visualization of TLC chromatograms was performed at
wavelengths of 254 and 365 nm. Dichloromethane of analytical grade was used as received from
supplier RCI Labscan Limited, Thailand. Indane-1,3-dione, pyridine and benzaldehyes of
analytical grade were used as received from the supplier Wako, USA.
General procedure for the synthesis of compounds 1-30
In a round bottomed flask, indane-1,3-dione (1 mmol) was dissolved in pyridine (5 mL) and
stirred for 5-10 minutes. Then substituted benzaldehyde (1 mmol) was added and the reaction
°
mixture was refluxed for 1 h at 100 C. The reaction progress was monitored by TLC. After
completion, the reaction mixture was brought to room temperature and then poured onto ice cold
water. The resulting precipitates were filtered and washed thoroughly with hexane. The
precipitates were crystallized from methanol.
Molecular docking assay
The molecular docking studies were performed to predict the molecular basis of the observed
urease inhibition demonstrated by the newly synthesized indane derivatives. In this connection,
the crystal structure of the Jack Bean Urease under the accession code 4GYZ was retrieved. The
Protein Preparation Wizard implemented in Schrodinger software suite was implemented to fill
in the missing loops and atoms, assign bond orders, and for the treatment of formal charges using
OPLS-3 force field [24]. The prepared structure was further used for grid receptor generation.
The scaling factor for the Vander Waals radius scaling value of the nonpolar parts of the receptor
and the partial charge cut off was set to 1.0 and 0.25, respectively. A grid box of (20 x 20 x
20ꢀ Å) was generated using the centroids of the residues Kcx-490, His-492, His-519, His-545,
Asp-633, Ni-901, and Ni-902. All the other parameters were set as default.
The compounds were sketched using the 2D-builder module. The Lig-Prep module was used to
assign the protonation and tautomeric states at neutral pH and energy was minimized using the
OPLS-3 force field [25].
22

The molecular docking studies of the newly synthesized indane molecules were performed using
the Glide SP docking protocol implemented in Schrodinger (Maestro 11.1.011) using the default
parameters [26]. The top-ranked binding pose of each compound was analyzed visually using the
Protein-Ligand Interaction Profiler (PLIP) web server [27]. All the visuals were rendered using
Chimera [28].
Urease inhibition assay
Reaction mixtures comprising 25 μL of enzyme (Jack bean and Bacillus pasteuriiureases)
solution and 55 μL of buffers containing urea (2–24 mM for jack bean and Bacillus pasteurii
ureases) were incubated with 5 μL of test compounds at 30 ˚C for 4.15 min in 96-well plates. The
increasing absorbance at 560 nm was measured after 10 min, using a microplate reader
(Molecular Devices, USA). All reactions were performed in triplicate in a final volume of 200
μL. The results (change in absorbance per min.) were processed by using SoftMax Pro software
(Molecular Devices, USA). All the assays were performed at pH 6.8 (3ꢀ mM sodium phosphate
buffer) and 7 μg of phenol red per mL as indicator [29]. Thiourea was used as the standard
inhibitor of urease. Percentage inhibitions were calculated from the formula:
100 − (ODtestwell/ODcontrol) × 100.
Spectral data of benzylidine of indane-1,3-diones (1-30)
2-(2-Hydroxy-4-methoxybenzylidene)-1H-indene-1,3(2H)-dione (1)
Yield: 80%, M.p.: 176-180°C, 1H-NMR (300 MHz, DMSO-d6): δ 9.15 (d, J6′,5′ = 9.0 Hz, 1H, H-
6′), 8.28 (s, 1H, =CH), 7.89 (m, 4H, H-5, H-4, H-6, H-7), 6.60 (m, 1H, H-5′), 6.50 (d, J3′,5′ = 2.1
13
Hz, 1H, H-3′), 3.82 (s, 3H, OCH3), C-NMR (100 MHz, DMSO-d6):  190.4, 189.2, 166.3,
163.4, 141.5, 139.8, 139.1, 135.3, 135.3, 135.1, 123.4, 122.5, 122.4, 114.1, 107.0, 100.2, 55.5,
EI-MS m/z (% rel. abund.): 280 (M+, 100), 263 (33), 235 (64), 175 (31), 152 (68), 140 (13), 105
(48), 91 (12), 76 (81), HREI-MS Calcd for C17H12O4 280.0736, Found 280.0742.
2-(4-Hydroxy-3-methoxybenzylidene)-1H-indene-1,3(2H)-dione (2)
1
Yield: 60%, M.p.: 176-180°C, H-NMR (300 MHz, DMSO-d6): δ 10.58 (s, 1H, OH), 8.69 (d,
J2′,6′ = 1.8 Hz, 1H, H-2′), 7.97 (m, 5H, H-5, H-4, H-6, H-7, H-6′), 7.75 (s, 1H, =CH), 6.95(d, J5′,6′
23

= 8.1 Hz, 1H, H-5′), 3.92 (s, 3H, OCH3), EI-MS m/z (% rel. abund.): 279 (M-1, 100), 263 (50),
249 (49), 237 (55), 209 (22), 181 (18), 165 (8), 152 (26), 140 (12), HREI-MS Calcd for
C17H12O4 280.0736, Found, 280.0730.
2-(4-Hydroxy-3,5-dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (3)
Yield: 68%, M.p.: 156-160°C, 1H-NMR (300 MHz, DMSO-d6): δ 10.01 (s, 1H, OH), 8.17 (s, 2H,
H-6′/H-2′), 7.94 (m, 4H, H-5, H-4, H-6, H-7), 7.78 (s, 1H, =CH), 3.89 (s, 6H, OCH3), 13C-NMR
(100 MHz, DMSO-d6):  147.6, 147.2, 139.1, 135.4, 135.3, 125.0, 123.5, 122.6, 121.6, 112.9,
112.6, 106.4, 97.4, 56.1, EI-MS m/z (% rel. abund.): 310 (M+, 100), 295 (8), 279 (45), 267 (22),
252 (9), 235 (9), 224 (5), 207 (7), 196 (11), 180 (7), 152 (7), 139 (11), HREI-MS Calcd for
C18H14O5 310.0841, Found 310.0827.
2-(2,3-Dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (4)
1
Yield: 58%, M.p.: 158-162°C, H-NMR (300 MHz, DMSO-d6): δ 9.80 (s, 2H, OH), 8.39 (dd,
J6′,5′ = 8.0 Hz, J6′,4′ = 1.2 Hz, 1H, H-6′), 8.36 (s, 1H, CH), 7.97 (m, 4H, H-5, H-4, H-6, H-7), 7.03
(dd, J4′,5′ = 7.6 Hz, J4′,6′ = 1.2 Hz, 1H, H-4′), 6.77 (t, J5′(4′, 6′) = 8.0 Hz, 1H, H-5′), 13C-NMR (75.0
MHz, DMSO-d6):  189.9, 188.7, 149.1, 145.3, 141.8, 140.2, 139.2, 135.7, 135.5, 126.8, 123.2,
122.8, 122.8, 120.5, 118.5, EI-MS m/z (% rel. abund.): 266 (M+, 100), 249 (35), 238 (54), 222
(93), 194 (61), 181 (84), 152 (95), 127 (23), 126 (16), 105 (87), 76 (82), HREI-MS for C16H10O4
266.0579, Found, 266.0575.
2-(2,5-Dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (5)
Yield: 75%, M.p.: 117-120°C, 1H-NMR (300 MHz, DMSO-d6): δ 8.64 (d, J6′,2′ = 2.8 Hz, 1H, /H-
6′), 8.24 (s, 1H, =CH), 8.00 (m, 4H, H-5, H-4, H-6, H-7), 7.25 (m, 1H, H-3′), 7.13 (d,J4′,5′ = 8.8
13
Hz, 1H, H-4′), 3.90 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), C-NMR (100 MHz, DMSO-d6): 
189.6, 188.8, 154.9, 152.3, 141.8, 139.3, 138.8, 135.7, 128.1, 123.0, 122.9, 122.6, 121.3, 116.5,
112.7, 56.4, 55.5, EI-MS m/z (% rel. abund.): 294 (M+, 46), 263 (100), 220 (8), 152 (7), 104 (3),
HREI-MS Calcd for C18H14O4 294.0892, Found, 294.0875.
24

2-(2,4-Dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (6)
1
Yield: 55%, M.p.: 117-120°C, H-NMR (300 MHz, DMSO-d6): δ 9.16(d, J6′,5′ = 9.0 Hz, 1H, H-
6′), 8.24 (s, 1H, =CH), 7.82 (m, 4H, H-5, H-4, H-6, H-7), 6.75 (m, 2H, H-5′, H-3′), 3.96 (s, 3H,
OCH3), 3.92 (s, 3H, OCH3), 13C-NMR (100 MHz, DMSO-d6): 190.2, 189.0, 166.6, 162.9,
141.6, 139.1, 138.9, 135.5, 135.5, 135.4, 124.8, 122.7, 122.7, 114.7, 106.7, 56.3, 56.0, EI-MS
m/z (% rel. abund.): 294 (M+, 81), 277 (8), 263 (100), 235 (4), 220 (9), 178 (2), 165 (9), 149 (8),
140 (2), 76 (8), HREI-MS Calcd for C18H14O4 294.0892, Found, 294.0895.
2-(2,6-Dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (7)
1
Yield: 65%, M.p.: 201-205°C, H-NMR (300 MHz, DMSO-d6): δ 7.99 (m, 4H, H-5, H-4, H-6, H-
7), 4), 8.50 (s, 1H, =CH), 7.47 (t, J4′(3′,5′) = 8.4 Hz, 1H, H-4′), 6.74 (d, J3′,4′ = 5′,4′ = 8.4 Hz, 2H, H-
13
3′/H-5′), 3.76 (s, 6H, OCH3), C-NMR (100 MHz, DMSO-d6):  189.1, 187.2, 159.2, 142.0,
139.6, 135.9, 135.5, 135.2, 133.2, 130.7, 123.0, 122.7, 111.1, 103.6, 55.7, EI-MS m/z (% rel.
abund.): 294 (M+, 83), 279 (4), 263 (100), 248 (61), 220 (14), 205 (5), 180 (4), 147 (2), 140 (6),
HREI-MS for C18H14O4 Calcd 294.0892, Found, 294.0877.
2-(2,3,4-Trimethoxybenzylidene)-1H-indene-1,3(2H)-dione (8)
1
Yield: 65%, M.p.: 200-204°C, H-NMR (300 MHz, DMSO-d6): δ 8.95 (d, J6′,5′ = 9.3 Hz, 1H, H-
6′), 8.11 (s, 1H, =CH), 7.97 (m, 4H, H-5, H-4, H-6, H-7), 7.06 (d, J5′,6′ = 9.3 Hz, 1H, H-5′), 3.95
(s, 6H, OCH3), 3.78 (s, 3H, OCH3), EI-MS m/z (% rel. abund.): 324 (M+, 23), 293 (18), 278 (6),
249 (6), 195 (2), 139 (4), HREI-MS for C19H16O5 324.0939, Found, 324.1000.
2-(4-Ethoxybenzylidene)-1H-indene-1,3(2H)-dione (9)
1
Yield: 75%, M.p.: 154-158°C, H-NMR (300 MHz, DMSO-d6): δ 8.60 (d, J3′,2′ = J5′,6′ = 8.8 Hz,
2H, H-3′, H-5′), 7.96 (m, 4H, H-5, H-4, H-6, H-7), 7.79 (s,1H, =CH), 7.11 (d, J6′,5′ = J2′,3′ = 8.8
Hz, 2H, H-2′, H-6′), 4.20 (m, 2H, OCH2), 1.37 (s, 3H, CH3), 13C-NMR (100 MHz, DMSO-d6): 
189.7, 188.9, 163.1, 145.7, 141.7, 139.2, 137.0, 135.6, 135.5, 126.1, 125.7, 122.8, 122.7, 114.8,
63.8, 14.4, EI-MS m/z (% rel. abund.): 278 (M+, 85), 249 (100), 233 (20), 222 (7), 194 (5), 165
(9), 149 (3), 104 (3), HREI-MS for C18H14O3 278.0943, Found, 278.0942.
25

2-(4-Butoxybenzylidene)-1H-indene-1,3(2H)-dione (10)
Yield: 55%, M.p.: 164-168°C, 1H-NMR (300 MHz, DMSO-d6): δ 8.60 (d, J2′,3′ = 6′,5′ = 8.8 Hz, 2H,
H-2′, H-6′), 7.96 (m, 4H, H-5, H-4, H-6, H-7), 7.79 (s, 1H, CH), 7.12 (d, J3′,2′ = 5′,6′ = 8.8 Hz, 2H,
H-3′, H-5′), 4.13 (m, 2H, OCH2), 1.76 (m, 2H, CH2), 1.47 (m, 2H, CH2), 0.97 (s, 3H, CH3), 13C-
NMR (100 MHz, DMSO-d6):  189.8, 188.9, 163.3, 145.8, 141.7, 139.3, 137.0, 135.6, 135.5,
126.2, 125.7, 122.8, 122.8, 114.9, 67.8, 30.5, 18.6, 13.6, EI-MS m/z (rel. abund. %): 306 (M+,
65), 249 (100), 233 (17), 222 (8), 194 (4), 176 (2), 165 (9), 104 (3), 76 (2), HREI-MS Calcd for
C20H18O3 306.1256, Found, 306.1241.
2-(3-Butoxybenzylidene)-1H-indene-1,3(2H)-dione (11)
1
Yield: 75%, M.p.: 198-202°C, H-NMR (300 MHz, DMSO-d6): δ 8.61(d, J5′,6′ = J4′,3′= 8.8 Hz,
2H, H-5′, H-4′), 7.96 (m, 4H, H-5, H-4, H-6, H-7), 7.80 (s, 1H, =CH), 7.13 (d, J2′,3′ = J6′,5′ = 8.8
Hz, 2H, H-2′, H-6′), 4.14 (t, 3H, OCH2), 1.76 (m, 2H, CH2), 1.49 (m, 2H, CH2), 0.95 (s, 3H,
OCH3), EI-MS m/z (% rel. abund.): 306 (M+, 80), 249 (100), 233 (40), 222 (18), 194 (8), 176 (6),
165 (24), 104 (10), 76 (8), HREI-MS for C20H18O3 306.1256, Found, 306.1240.
2-(4-(Benzyloxy)-3-methoxybenzylidene)-1H-indene-1,3(2H)-dione (12)
Yield: 75%, M.p.: 196-200°C, 1H-NMR (300 MHz, DMSO-d6): δ 8.69 (d, J2′,4′ = 1.2 Hz, 1H, H-
2′), 8.03 (m, 4H, H-5, H-4, H-6, H-7), H-6′), 7.80 (s, 1H, =CH), 7.48 (d, J2′′,3′′ = J5′′,6′′ = 7.2 Hz,
2H, H-2′′, H-5′′), 7.43 (t, J = 7.5 Hz, 2H, H-3′′, H-4′′), 7.37 (d, J6′′,5′′ = 7.2 Hz, 1H, H-6′), 7.26 (d,
J5′′,6′′ = 6.6 Hz, 1H, H-5′′), 5.25 (s, 2H, OCH2), 3.91(s, 3H, OCH3), EI-MS m/z (% rel. abund.):
370 (M+, 32), 279 (12), 251 (6), 152 (4), 91 (100), HREI-MS Calcd for C24H18O4 370.1205,
Found, 370.1187.
2-(4-(Benzyloxy)benzylidene)-1H-indene-1,3(2H)-dione (13)
1
Yield: 85%, M.p.: 164-168°C, H-NMR (300 MHz, DMSO-d6): δ 8.62 (d, J6′,5′ = J2′,3′ = 8.8 Hz,
2H, H-2′, H-6′), 7.97 (m, 4H, H-5, H-4, H-6, H-7), 7.81 (s, 1H, =CH), 7.49 (d, J3′,4′ = J5′,6′ = 7.2
Hz, 2H, H-3′, H-5′), 7.39 (t, J = 7.2 Hz, 2H, H-5′, H-3′) 7.36 (d, J6′′,5′′ = 7.2 Hz, 1H, H-6′′), 7.22
(d, J2′′,3′′ = 3′′,4′′ = 8.8 Hz, 2H, H-2′′, H-3′′), 5.26 (s, 2H, OCH2), EI-MS m/z (% rel. abund.): 249
(M+, 14), 233 (2), 221 (6), 207 (1), 193 (3), 182 (6), 165 (18), 139 (7), 91 (100), HREI-MS Calcd
for C23H16O3 340.1099, Found, 340.1092.
26

2-(2,6-Dichlorobenzylidene)-1H-indene-1,3(2H)-dione (14)
Yield: 65%, M.p.: 156-160°C, 1H-NMR (300 MHz, DMSO-d6) δ 8.08 (m, 4H, H-4, H-5, H-6, H-
13
7), 7.84 (s, 1H, =CH), 7.59 (d, J3′,4′ = J5′,4′ = 6.6 Hz, 2H, H-3′, H-5′), 7.51 (m, 1H, H-4′), C-
NMR (100 MHz, DMSO-d6):  187.7, 187.0, 141.6, 140.5, 136.7, 136.6, 136.6, 134.0, 133.2,
131.3, 128.1, 123.6, 123.3, 114.5, EI-MS m/z (% rel. abund.): 267 (M+-Cl, 100), 239 (4), 176
(14), 133 (3), HREI-MS Calcd for C16H8Cl2O2 300.9823, Found, 300.9839.
2-(2-Chloro-3-methoxybenzylidene)-1H-indene-1,3(2H)-dione (15)
1
Yield: 65%, M.p.: 208-212°C, H-NMR (300 MHz, DMSO-d6): δ 8.07 (s, 1H, =CH), 8.04 (m,
2H, H-7, H-4′), 7.98 (m, 3H, H-4, H-5, H-6), 7.47 (t, J5ʹ(4ʹ,6ʹ)= 8.0 Hz, 1H, H-5′), 7.37 (d, J6′,5′
=
13
7.5 Hz, 1H, H-6′), 3.91(s, 1H, OCH3), C-NMR (100 MHz, DMSO-d6):  188.8, 187.6, 154.7,
142.0, 139.3, 136.0, 131.3, 131.2, 127.1, 124.6, 123.4, 123.2, 116.0, 56.4, EI-MS m/z (% rel.
abund.): 263 (M+-Cl, 100), 248 (56), 220 (58), 192 (5), 176 (12), 163 (40), 123(9), 104 (15),
HREI-MS Calcd for C17H11ClO 298.0397, Found, 298.0372.
2-(5-Chloro-2-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (16)
Yield: 65%, M.p.: 185-189°C, 1H NMR (300 MHz, DMSO-d6): δ 11.14 (s, 1H, OH), 8.98 (d, J 6′,
4′ = 2.8 Hz, 1H, H-6′), 8.18 (s, 1H, =CH), 8.00 (m, 4H, H-5/H-4/H-6/H-7), 7.50(d, J3′,4′ = 6.6 Hz,
13
J3′,5′ = 2.1 Hz, 1H, H-3′), 7.02 (d, J4′,3′ = 8.8 Hz, 1H, H-4′), C-NMR (100 MHz, DMSO-d6): 
158.6, 141.9, 139.4, 137.9, 135.9, 135.8, 135.0, 131.7, 128.3, 123.1, 123.0, 122.4, 121.0, 117.7,
EI-MS: m/z (rel. abund. %) 286 (M+2, 31), 284 (M+, 100), 267 (16), 255 (34), 239 (21), 228
(10), 221 (30), 205 (5), 165 (15), 105 (17). HREI-MS for C16H9ClO3 284.0240, Found,
284.0255.
2-(5-Bromo-2-methoxybenzylidene)-1H-indene-1,3(2H)-dione (17)
Yield: 65%, M.p.: 196-200°C, 1H NMR (300 MHz, DMSO-d6): δ 9.01 (d, J6′, 2′ = 2.4 Hz, 1H, H-
6′), 8.10 (s, 1H, =CH), 8.01 (m, 4H, H-5, H-4, H-6, H-7), 7.79 (dd, J4′,3′ = 8.1 Hz, J4′,2′ = 2.1 Hz,
13
1H, H-4′), 7.18 (d, J3′, 4′ = 9.0 Hz ,1H, H-3′), 3.94 (s, 3H, OCH3), C-NMR (100 MHz, DMSO-
d6):  189.2, 188.5, 159.0, 141.9, 139.4, 137.3, 136.8, 135.9, 134.7, 129.5, 123.0, 122.8, 114.0,
27

111.5, 56.5, EI-MS m/z (rel. abund. %): 345 (M+2, 3), 343 (M+, 4), 311 (100), 263 (3), 248 (4),
220 (4), 163 (8), HREI-MS Calcd for C17H11BrO3 341.9892, Found, 341.9915.
2-(6-Bromo-2-methoxybenzylidene)-1H-indene-1,3(2H)-dione (18)
1
Yield: 75%, M.p.: 196-200°C, H NMR (300 MHz, DMSO-d6): δ 9.01 (d, J4′,6′ = 2.4 Hz, 1H, H-
4′), 8.10 (s, 1H, =CH), 7.98 (m, 4H, H-5, H-4, H-6, H-7), 7.79 (d, J6′,4′ = 2.3 Hz, 1H, H-6′), 7.18
(d, J3′,2′ = 9.0 Hz, 1H, H-3′), 3.94 (s, 3H, OCH3), EI-MS m/z (rel. abund. %): 346 (M+2, 16), 344
(M+, 18), 311 (100), 176 (5), 163 (3), 131.6 (9), HREI-MS for C17H11BrO3 341.9892, Found,
341.9879.
2(2-Bromo-4,5-dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (19)
Yield: 55%, M.p.: 204-208°C, 1H-NMR (300 MHz, DMSO-d6): δ 8.51 (s, 1H, H-2′), 8.45 (s, 1H,
=CH), 8.02 (m, 4H, H-5, H-4, H-6, H-7), 7.81 (s, 1H, H-6′), 3.91 (s, 6H, OCH3), EI-MS m/z (%
rel. abund.): 293 (M+-Br, 100), 277 (47), 249 (62), 232 (23), 207 (19), 179 (6), 151 (28), 125 (6),
104 (8), 75 (35), HREI-MS Calcd for C18H13BrO4 371.9997, Found 372.0033.
2-(3-Bromo-4, 5-dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (20)
Yield: 65%, M.p.: 204-208°C, 1H NMR (300 MHz, DMSO-d6): δ 8.51 (s, 1H, H-6′), 8.45 (s, 1H,
=CH), 8.02 (m, 4H, H-5, H-4, H-6, H-7), 7.81 (s, 1H, H-2′), 3.95 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 13C-NMR (100 MHz, DMSO-d6),  152.7, 149.5, 143.9, 141.9, 139.4, 136.0, 135.9,
130.0, 129.1, 123.2, 123.1, 117.7, 116.4, 113.8, 102.5, 60.5, 56.3, EI-MS: m/z (rel .abund%) 374
(M+2, 100), 372 (M+, 100), 357 (9), 293 (9), 278 (32), 250 (23), 207 (16), 176 (4), 147 (3),
HREI-MS for C18H13BrO4 371.9997, Found, 371.9973.
2-(3-Bromo-4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (21)
Yield: 55%, M.p.: 190-194°C, 1H NMR (300 MHz, DMSO-d6): δ 11.64 (s, 1H, OH), 9.10 (d, J2′,6′
= 1.8 Hz, 1H, H-2′), 8.33 (dd, J6′,5′ = 8.7 Hz, J6′,2′ = 2.1 Hz, 1H, H-6′), 7.98 (m, 4H, H-5, H-4, H-
6, H-7), 7.73 (s, 1H, =CH), 7.10 (d, J5′,6′ = 8.7 Hz, 1H, H-5′), 13C-NMR (100 MHz, DMSO-d6): 
189.6, 189.0, 159.2, 144.6, 141.7, 139.3, 138.8, 136.6, 135.7, 135.6, 126.5, 125.8, 122.9, 116.2,
109.8, EI-MS m/z (rel. abund. %): 331 (M+2, 92) 329 (M+, 90), 311 (13), 300 (10), 274 (7), 249
28

(43), 221 (25), 205 (4), 193 (10), 163 (7), 104 (30), 83 (29), HREI-MS for C16H9BrO3 327.9735,
Found, 327.9752
2-(4-Fluorobenzylidene)-1H-indene-1,3(2H)-dione (22)
1
Yield: 75%, M.p.: 168-172°C, H-NMR (400 MHz, DMSO-d6): δ 8.64 (m, 2H, H-3′, H-5′), 8.01
(m, 4H, H-5, H-4, H-6, H-7), 7.94 (s, 1H, =CH), 7.44 (m, 2H, H-2′, H-6′), 13C-NMR (100 MHz,
DMSO-d6):  204.5, 189.3, 186.7, 144.1, 141.9, 139.5, 136.8, 136.7, 136.0, 135.9, 129.6, 128.9,
123.1, 116.1, 115.8, EI-MS m/z (% rel. abund.): 252 (M+, 70), 251 (100), 223 (12), 196 (22), 147
(5), 104 (14), 76 (10), HREI-MS for C16H9FO2 252.0587, Found, 252.0599.
2-(5-Fluoro-2-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (23)
Yield: 85%, M.p.: 188-192°C,1H-NMR (300 MHz, DMSO-d6): δ 10.88 (s, 1H, OH), 8.80 (dd,
J4ʹ,6ʹ = 3.3 Hz, J4ʹ,5ʹ = 10.6 Hz, 1H, H-4′), 8.23 (s, 1H,=CH), 7.99 (m, 4H, H-5, H-4, H-6, H-7),
7.38 (m, 1H, H-6′), 7.02 (m, 1H, H-3′), EI-MS m/z (% rel. abund.): 268 (M+, 100), 251 (29), 239
(75), 212 (25), 183 (35), 163 (13), 157 (4), 134 (1), 105 (25), HREI-MS for C16H9FO3 268.0536,
Found 268.0534.
2-(2-(Trifluoromethyl) benzylidene)-1H-indene-1,3(2H)-dione (24)
Yield: 55%, M.p.: 138-142°C, 1H-NMR (300 MHz, DMSO-d6): δ 8.23 (d, J3′,4′ = 7.2 Hz, 1H, H-
3′), 8.06 (m, 4H, H-5, H-4, H-6, H-7), 7.98 (s, 1H, =CH), 7.90 (d, J4′,5′ = 7.8 Hz, 1H, H-4′), 7.82
(m, 2H, H-6′, H-5′), EI-MS m/z (%rel. abund.): 301 (M+, 9), 233 (100), 205 (9), 176 (13), 151
(15), 125 (2), 104 (55), 76 (71), HREI-MS for C17H9F3O2 302.0555, Found, 302.0546.
2-(4-Hydroxy-5-iodo-3-methoxybenzylidene)-1H-indene-1,3(2H)-dione (25)
1
Yield: 79%, M.p.: 164-168°C, H-NMR (300 MHz, DMSO-d6): δ 11.06 (s, 1H, OH), 8.63 (d, J
6′,2′ = 1.2 Hz, 1H, H-6′), 8.50 (s, 1H, H-2′), 7.97 (m, 4H, H-5, H-4, H-6, H-7), 7.71 (s, 1H, =CH),
3.94 (s, 3H, OCH3), 13C-NMR (100 MHz, DMSO-d6),  189.7, 189.1, 152.5, 146.5 145.1, 141.7,
139.2, 138.9, 135.5, 126.4, 125.9, 122.8, 122.7, 116.8, 84.9, 56.2, EI-MS m/z (% rel. abund.):
406 (M+, 100), 391 (8), 375 (16), 363 (7), 279 (17), 264 (25), 236 (17), 208 (10), 180 (5), 152
(9), 140 (4), HREI-MS for C17H11IO4 406.9780, Found, 406.9778.
29