Bis-coumarins; non-cytotoxic selective urease inhibitors and antiglycation agents

Article abstract of DOI:10.1016/j.bioorg.2019.103170

The current study is concerned with the identification of lead molecules based on the bis-coumarin scaffold having selective urease inhibitory and antiglycation activities. For that purpose, bis-coumarins (1-44) were synthesized and structurally characterized by different spectroscopic techniques. Eight derivatives 4, 8-10, 14, 17, 34, and 40 demonstrated urease inhibition in the range of IC₅₀ = 4.4 ± 0.21–115.6 ± 2.13 μM, as compared to standard thiourea (IC₅₀ = 21.3 ± 1.3 μM). Especially, compound 17 (IC₅₀ = 4.4 ± 0.21 μM) was found to be five-fold more potent than the standard. Kinetic studies were also performed on compound 17 in order to identify the mechanism of inhibition. Kinetic studies revealed that compound 17 is a competitive inhibitor. Antiglycation activity was evaluated using glycation of bovine serum albumin by methylglyoxal in vitro. Compounds 2, 11-13, 16, 17, 19–22, 35, 37, and 42 showed good to moderate antiglycation activities with IC₅₀ values of 333.63–919.72 μM, as compared to the standard rutin (IC₅₀ = 294.46 ± 1.5 μM). Results of both assays showed that the compounds with urease inhibitory activity did not show any antiglycation potential, and vice versa. Only compound 17 showed dual inhibition potential. All compounds were also evaluated for cytotoxicity. Compounds 17, 19, and 37 showed a weak toxicity towards 3 T3 mouse fibroblast cell line. All other compounds were found to be non-cytotoxic. Urease inhibition is an approach to treat infections caused by ureolytic bacteria whereas inhibition of glycation of proteins is a strategy to avoid late diabetic complications. Therefore, these compounds may serve as leads for further research.

Full text of DOI:10.1016/j.bioorg.2019.103170

Journal Pre-Proof
Bis-coumarins; Non-Cytotoxic Selective Urease Inhibitors and Antiglycation
Agents
Uzma Salar, Arsalan Nizamani, Fizza Arshad, Khalid Mohammed Khan,
Muhammed Imran Fakhri, Shahnaz Perveen, Nessar Ahmed, M. Iqbal
Choudhary
PII:
S0045-2068(19)30717-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103170
YBIOO 103170
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 May 2019
24 July 2019
30 July 2019
Please cite this article as: U. Salar, A. Nizamani, F. Arshad, K. Mohammed Khan, M. Imran Fakhri, S. Perveen, N.
Ahmed, M. Iqbal Choudhary, Bis-coumarins; Non-Cytotoxic Selective Urease Inhibitors and Antiglycation Agents,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103170
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JOURNAL PRE-PROOF
Bis-coumarins; Non-Cytotoxic Selective Urease Inhibitors and Antiglycation Agents
Uzma Salar,a,b Arsalan Nizamani,a Fizza Arshad,a Khalid Mohammed Khan,∗a,e Muhammed
Imran Fakhri,a Shahnaz Perveen,c Nessar Ahmed,d M. Iqbal Choudharya,b,f
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
cPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-
75280, Pakistan
dCentre for Biomedicine, School of Healthcare Science, Manchester Metropolitan University,
Manchester M1 5GD, United Kingdom
eDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia
fDepartment of Biochemistry, King Abdulaziz University, Jeddah-214412, Saudi Arabia
Abstract: The current study is concerned with the identification of lead molecules based on the
bis-coumarin scaffold having selective urease inhibitory and antiglycation activities. For that
purpose, bis-coumarins (1-44) were synthesized and structurally characterized by different
spectroscopic techniques. Eight derivatives 4, 8-10, 14, 17, 34, and 40 demonstrated urease
inhibition in the range of IC50 = 4.4 0.21 - 115.6 2.13 μM, as compared to standard thiourea
(IC50 = 21.3 1.3 μM). Especially, compound 17 (IC50 = 4.4 0.21 μM) was found to be five-
fold more potent than the standard. Kinetic studies were also performed on compound 17 in order
to identify the mechanism of inhibition. Kinetic studies revealed that compound 17 is a
competitive inhibitor. Antiglycation activity was evaluated using glycation of bovine serum
albumin by methylglyoxal in vitro. Compounds 2, 11-13, 16, 17, 19-22, 35, 37, and 42 showed
good to moderate antiglycation activities with IC50 values of 333.63 - 919.72 μM, as compared to
the standard rutin (IC50 = 294.46 1.5 μM). Results of both assays showed that the compounds
with urease inhibitory activity did not show any antiglycation potential, and vice versa. Only
compound 17 showed dual inhibition potential. All compounds were also evaluated for
cytotoxicity. Compounds 17, 19, and 37 showed a weak toxicity towards 3T3 mouse fibroblast
cell line. All other compounds were found to be non-cytotoxic. Urease inhibition is an approach
to treat infections caused by ureolytic bacteria whereas inhibition of glycation of proteins is a
strategy to avoid late diabetic complications. Therefore, these compounds may serve as leads for
further research.
*Corresponding Author: khalid.khan@iccs.edu; drkhalidhej@gmail.com; Tel. 00922134824910; Fax.
00922134819018
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Keywords: Bis-coumarins; urease; glycation; advanced glycation end products; peptic ulcer;
diabetes; non-cytotoxic.
Introduction
Bis-coumarins are biologically active pharmacophores, initially isolated from natural sources
[1,2]. Several biological activities are associated with bis-coumarins, such as α-glucosidase [3],
urease [4], nucleotide pyrophosphatases-1 [5], and DNA polymerase β-lyase inhibitory activities
[6]. Bis-coumarins are also reported to possess anticoagulant, and hemorrhagic properties [7].
However, there is still a need to explore this class for a wide spectrum of pharmacological
activities.
Urease (amidohydrolase EC 3.5.1.5) is a metalloenzyme, contains nickel in its active site. It
catalyzes the hydrolysis of urea into carbon dioxide and ammonia [8,9]. This enzyme synthesizes
by numerous plants, animals, bacteria, and other organisms [10]. Hyperactivity of urease is
harmful to human and animal health, as well as for the agricultural sector. Urease is a key
virulence in the pathogenesis of urolithiasis, urinary catheter encrustation, pyelonephritis, hepatic
coma, and hepatic encephalopathy [11,12]. It also participates in the pathologies caused by
ureolytic bacteria Helicobacter pylori (HP). It facilitates bacteria to survive in stomach at acidic
pH during initial colonization. Therefore, it plays an important role in the pathologies of ulcers
(gastric and peptic), and cancer [13-16]. In the agriculture sector, hyperactivity of urease leads to
considerable economic and environmental damage by liberating aberrantly large quantities of
ammonia into the atmosphere, during the process of urea fertilization [17]. Therefore, it is
important to develop strategies based on urease inhibition to solve the problems caused by urease
producing bacteria.
Glycation is a non-enzymatic reaction in which reducing sugars non-enzymatically bind with the
amino terminal of proteins via a nucleophilic addition reaction, ultimately giving rise to advanced
glycation end products (AGEs). Haemoglobin, serum albumin, collagen, elastin, and crystalline
are common proteins that undergo glycation. Changes in their structures and functions lead to
different abnormalities such as atherosclerosis, neuropathy, diabetic retinopathy, diabetic
nephropathy, etc. This process is cumulatively called glycation stress [18,19]. In this process,
reactive intermediates such as methylglyoxal (MG) are more prone to bind with amino groups as
compared to their carbohydrate precursors. Eighty percent (80%) of blood proteins is serum
albumin, which is more likely to be glycated [20]. As a result of complex rearrangements,
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substitution, and addition reactions of glycated proteins, AGEs are produced in the body which
change the functions of proteins, and accumulate with time in different tissues [21-23]. Many late
diabetic complications, such as retinopathy, nephropathy, cataracts, atherosclerosis, and
osteoporosis are due to the glycation of vital proteins, and accumulation of AGEs [24]. The
inhibition of glycation process plays a pivotal role in the prevention of many late diabetic
complications. Therefore, it is important to find inhibitors for glycation.
Bis-coumarins have not yet been reported for their antiglycation activity. Figure-1 showed that
chromone ring, a positional isomer of coumarin, is the main scaffold of rutin which encouraged
us to evaluate the compounds 1-44 for their antiglycation activity. Furthermore, we have
previously reported bis-coumarins for urease inhibitory activity [4]. New members of this series
were thus evaluated to identify more potent urease inhibitors [Figure-1]. In brief, forty-four
derivatives were synthesized and evaluated for their urease inhibitory, and antiglycation activities.
After knowing the selective potential of compounds, cytotoxicity was also checked. To the best
of our knowledge, except compounds 6, 14, 16, 17, 19, 20, 23-26, 28, and 30-32 [25-29], the rest
of the compounds were identified as new.
OH
B
HO
O
A
OH
OH
OH
C
HO
O
O
O
O
O
O
O
OH
O
O
Me
HO
OH
OH
OH
R
HO
Rutin (Standard) for Antiglycation Assay
(IC50 = 294.46 1.5 µM)
OH
Not active [4]
IC50 = 15.06-91.35 µM [4]
Coumarin ring
F or Cl group
O
O
O
O
R1
R1
OH
OH
R2
OH group
Aryl ring
Urease Inhibitory Activity (UIA) IC50 = 4.4-115.6 µM
⇒
Antiglycation Activity (AGA) IC50 = 333.63-919.72 µM
⇒
Non-Cytotoxic
Figure-1: Structural similarity between rutin and bis-coumarins 1-44 as a rationale of the
current study.
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Results and Discussion
Chemistry
Bis-coumarin derivatives 1-44 were synthesized by reacting 6-fluoro-4-hydroxy, 4-hydroxy, and
6-chloro-4-hydroxy coumarins with a variety of benzaldehydes in the presence of
tetraethylammonium bromide (TEAB) as a catalyst. Reactions were performed in distilled water
(Scheme-1) and checked periodically by TLC analysis. Precipitates of products were obtained in
good yields (Table-1). Compounds were structurally identified by various spectroscopic analyses
such as 1H- and 13C-NMR as well as FAB-, ESI-, and HRESI-MS.
O
O
O
O
O
O
O
TEAB, H2O
Reflux
+
R1
R2
H
R1
R1
OH
OH
OH
R2
1-44
1-15
16-31
32-44
R1 = F
R1 = H
R1 = Cl
Scheme-1: Synthesis of bis-coumarin derivatives 1-44.
Characteristic spectral features of representative compound 21
Structure elucidation of a new compound 21 is presented here as an example. 1H- and 13C-NMR
spectra of compound 21 were recorded in DMSO-d6. Characteristic signal of methine proton
resonated as a singlet at δH 5.60, also confirmed the formation of the bis-coumarin scaffold.
Amongst the protons of coumarin nucleus, H-7 and H-7′ resonated at δH 7.53 as triplet, and
showed ortho coupling (t, J7,6/7′,6′ = J7,8/7′,8′ = 6.9 Hz) with adjacent protons, H-6/H-6′ and H-8/H-
8′. Similarly, H-6 and H-6′ appeared as a triplet at δH 7.26, and also showed ortho coupling (t,
J6,5/6′,5′ = J6,7/6′,7′ = 7.5 Hz) with neighbouring protons. H-5 and H-5′ appeared as the most
downfield signal at δH 7.82 with ortho coupling (d, J5,6/5′,6′ = 6.3 Hz) with the adjacent H-6 and
H-6′. H-8 and H-8′ appeared at δH 7.27, ortho coupled (d, J8,7/8′,7′ = 8.1 Hz) with H-7 and H-7′. H-
2′′ and H-6′′ of ring R2 resonated as a broad singlet at δH 7.12 (Figure-2).
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7.27 (d, J8',7' = 8.1 Hz)
7.27 (d, J8,7 = 8.1 Hz)
H
8'
H
8
7.53 (t, J7',6' = J7',8' = 6.9 Hz)
7.53 (t, J7,6 = J7,8 = 6.9 Hz)
1
124.0
115.6
1'
H
O
124.0
115.6
164.2
131.3
164.2
O
O
167.2
148.1
O
O
167.2
148.1
H
O
O
O
2
2'
7'
7
6
130.2
123.1
130.2
123.1
3
3'
7.26 (t, J6',5' = J6',7' = 7.5 Hz)
7.26 (t, J6,5 = J6,7 = 7.5 Hz)
6'
131.3
H
H
152.4
4'
152.4
5
4
5'
6.16 (s)
35.0
OH
OH
H
H
H
OH
H
OH
7.82 (d, J5',6' = 6.3 Hz)
7.82 (d, J5,6 = 6.3 Hz)
111.7
119.3
1''
6''
5''
H
7.12 (bd.s)
2''
3''
119.3
102.9
7.12 (bd.s)
102.9
Br
Br
136.6 Br
4''
Br
OH
OH
Figure-2: 1H- and 13C-NMR chemical shifts of compound 21.
Total 16 signals of carbon (eleven methine and fourteen quarternary carbons) appeared in 13C-
NMR broad-band decoupled spectrum (DMSO-d6). Most downfield signal of C-2 and C-2′, which
are the quaternary carbons of lactone moiety, appeared at δC 167.2. Similarly, other quaternary
C-10/C-10′, and C-4/C-4′ resonated at δC 164.2 and 152.4 as the downfield signals due to the
adjacent electronegative oxygen atom. An upfield characteristic signal resonated at δC 35.0,
corresponding to the methine CH carbon.
The structure of compound 21 was further confirmed with the help of FAB (Neg.)-MS, ESI-MS,
and HRESI-MS. FAB (Neg.)-MS of compound 21 displayed the peaks at m/z = 584 [M-H]-1 586
,
[M+2-H]-1 and 588 [M+4-H]-1 which indicated the presence of two bromine atoms. Similarly,
ESI-MS of compound 21 displayed the [M+H]+, [M+2+H]+, and [M+4+H]+ at m/z 585, 587, and
589, respectively. HR-(ESI)MS showed the [M+H]+ at m/z 584.9208, corresponding to the
formula C25H15Br2O7 Calcd (584.9184). The UV spectrum of compound 21 showed absorption
at 298 nm, characteristic of coumarin moiety. The structure of new compound 21 was thus
deduced unambiguously.
Bioactivities in vitro
Forty-four bis-coumarin derivatives were evaluated for urease inhibitory, and antiglycation
activities (Table-1). Eight compounds 4, 8-10, 14, 17, 34, and 40, showed a good to moderate
urease inhibitory activity in the range of IC50 = 4.4 0.21-115.6 2.13 μM, as compared to
standard thiourea (IC50 = 21.3 1.3 μM). Whereas, compounds 2, 11-13, 16, 17, 19, 20-22, 35,
37, and 42 demonstrated good to moderate antiglycation activities in the range of IC50 = 333.63 -
919.72 μM, as compared to standard rutin (IC50 = 294.46 1.5 μM). Bis-coumarins have never
been reported before for its antiglycation activity. It is worth mentioning that the compounds
showed selective activity in both assays. Bis-coumarins which showed urease inhibitory activity
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did not give the antiglycation potential, and vice versa. All compounds were also checked for
their cytotoxicity and largely found to be non-cytotoxic.
Table-1: In vitro urease inhibitory activity, antiglycation activity, and cytotoxicity of bis-
coumarins (1-44).
Urease Inhibitory Antiglycation
Cytotoxicity
Activity
IC50 SEMa
Activity
IC50 SEMa IC50 SEMa
Compounds
R2
Category A (R1 = F)
OMe
OMe
6″
5″
1
2
NAb
NAb
NAb
NTc
>30
4″
F
6″
378.03 0.75
5″
3″
OMe
Br
6″
5″
3
NAb
NAb
NTc
3″
OMe
Cl
6″
5″
4
5
6
7
87.0 0.88
NAb
NAb
NAb
NAb
NTc
NTc
NTc
NTc
3″
Cl
OMe
6″
NAb
3″
Br
4″
6″
5″
2″
3″
NAb
Me
OMe
6″
NAb
3″
MeO
4″
6″
5″
2″
2′′′
5′′′
8
30.3 1.01
NAb
NTc
3′′′
O
OMe
4′′′
6′′′
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6″
5″
2″
9
79.8 1.01
NAb
NTc
3″
CF3
6″
5″
2″
10
11
12
193.7 0.66
NAb
NTc
>30
>30
CF3
OH
4″
6″
NAb
399.47 1.88
465.33 1.99
3″
F
4″
OH
6″
NAb
3″
Cl
4″
OH
6″
5″
13
14
15
NAb
187.3 1.75
NAb
443.64 0.57
>30
NTc
NTc
3″
OH
OMe
Br
6″
5″
2″
NAb
Br
OMe
6″
NAb
3″
MeO
Category B (R1 = H)
OH
OH
OH
OH
6″
5″
16
17
18
NAb
351.85 3.56
>30
4″
6²
5²
4.4 0.21
374.45 1.21 18.70 0.57
OH
Me
Me
6″
5″
NAb
NAb
NTc
4″
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OH
6″
19
20
NAb
397.07 2.21
>30
3″
Cl
4″
OH
6″
NAb
434.71 2.73 19.42 0.79
5″
3″
OH
6″
2″
21
NAb
432.58 0.76
>30
Br
Br
OH
6″
2″
22
23
24
NAb
NAb
NAb
428.84 2.44
>30
NTc
NTc
Cl
Cl
OMe
4″
6″
NAb
3″
MeO
4″
OMe
OMe
6″
5″
NAb
4″
6″
5″
2″
25
26
27
28
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NTc
NTc
NTc
NTc
OMe
OMe
OEt
6″
5″
3″
OMe
F
6″
5″
3″
Br
Br
6″
3″
MeO
OMe
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6″
5″
2″
2′′′
29
NAb
NAb
NTc
3′′′
O
OMe
4′′′
6′′′
2″
5′′′
6″
5″
30
31
NAb
NAb
NAb
NAb
NTc
NTc
OMe
OH
6″
5″
2″
3″
SMe
Category C (R1 = Cl)
6″
2″
32
33
34
NAb
NAb
NAb
NAb
NTc
NTc
NTc
5″
Br
OMe
6″
5″
2″
2′′′
5′′′
NAb
3′′′
O
4″
4′′′
6′′′
OH
6²
5²
115.6 2.13
OH
OH
6″
2″
35
36
37
38
NAb
NAb
NAb
NAb
744.82 5.64
>30
NTc
Br
Br
OH
8″
5″
OH
7″
6″
NAb
3″
4″
6″
2″
919.72 1.98 21.11 1.27
HO
OMe
OMe
I
6″
NAb
NTc
3″
Br
4″
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OH
6″
39
40
41
42
NAb
NAb
NAb
NTc
NTc
NTc
>30
3″
HO
4″
6″
5″
2″
36.5 1.37
Br
Br
OH
6″
NAb
NAb
3″
F
4″
6″
2″
NAb
333.63 1.98
MeO
OMe
Br
F
6″
43
NAb
NAb
NAb
NTc
NTc
5″
3″
OMe
2″
6″
44
NAb
Br
OMe
OH
Thiouread
Rutine
21.3 1.3
Standards
294 1.5
-
-
Cycloheximidef
0.26 0.1
SEMa (Standard error of mean); NAb (Not Active); NTc (Non Toxic); Thiouread (Standard inhibitor of urease
inhibitory activity); Rutine (Standard inhibitor of antiglycation activity); Cycloheximidef (Standard inhibitor for
cytotoxicity)
Structure-activity relationship (SAR)
All structural features of bis-coumarin derivatives such as coumarin ring, substitutions on
coumarin rings (R1), and aryl ring (R2) apparently playing their role in the inhibitory activity, and
variation in the activity can be attributed to the different substitution pattern on coumarin, and
aryl rings (Figure-3).
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Coumarin ring
F or Cl group
O
O
O
O
R1
R1
OH
OH
R2
OH group
Aryl ring
Figure-3: General structural features of bis-coumarin.
Results presented in Table-1 indicate that almost all compounds selectively exhibited their
potential in both assays, and found to be non-toxic as well.
Bis-coumarins 1-44 were divided into three categories in order to understand the structure-activity
pattern. In category “A”, fluoro groups are present at C-6 and C-6ꞌ of bis-coumarin i.e. 1-15.
Category “B” compounds 16-31 has no substitution on both coumarin rings, and category “C”
has chloro groups at C-6 and C-6ꞌ i.e. compounds 32-44.
In category “A”, non-cytotoxic compound 8 (IC50 = 30.3 1.01 μM) with 3ʺ-benzyloxy and 4ʺ-
methoxy substitutions showed comparable urease inhibitory activity with the standard thiourea
(IC50 = 21.3 1.3 μM), and did not show any antiglycation potential. The good urease inhibitory
activity of the compound 8 could be due to π-π stacking of the benzyloxy group with the active
site of urease enzyme. Activity of compound 8 was compared with the activity of non-cytotoxic
compound 14 (IC50 = 187.3 1.75 μM) which has a bromo group instead of a benzyloxy at C-3ʺ,
showed six times less urease inhibitory activity. This indicated the involvement of benzyloxy in
the inhibitory activity. Similarly, compound 14 did not show any antiglycation potential. It is
worth mentioning that both compounds 8 and 14 showed selective activity towards the urease, as
well as being non-cytotoxic. Other compounds with different positional combination of methoxy
and bromo groups such as compounds 3, 5, and 15, did not show any urease inhibitory and
antiglycation activities. However, all three were found to be non-cytotoxic. The activity of
compound 3 can be compared with compound 2 (IC50 = 378.03 0.75 μM) which has a fluoro
group, instead of a bromo at C-2ʺ, showed an antiglycation potential comparable to the standard
rutin (IC50 = 294.46 1.5 μM). It showed that fluoro group on aryl ring plays an important role
in the antiglycation activity (Figure-4).
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O O O O
O O O O
O O O O
OH OH
F
F
F
F
F
F
OH
OH
Br
OH
OH
Br
O
OMe
OMe
OMe
8
UIA IC50 = 30.3 1.01 µM
14
UIA IC50 = 187.3 1.75 µM
3
UIA Not Active
⇒
⇒
⇒
AGA Not Active
⇒
AGA Not Active
⇒
AGA
Not Active
⇒
O O O
OH
O
O O O O
O O O
OH
O
F
F
F
F
F
F
OH
OMe
OH
OH
OH
OMe
F
MeO
Br
OMe
Br
5
UIA Not Active
2
UIA Not Active
⇒
15
UIA Not Active
⇒
⇒
AGA
Not Active
⇒
AGA
Not Active
AGA
IC50 = 378.03 0.75 µM
⇒
⇒
Figure-4: Structure-activity relationship of compounds 2, 3, 5, 8, 14, and 15.
In the same category “A”, compounds with the halogen substitutions (4, 9, and 10), and the
compounds with combinations of halogen and hydroxy groups (11 and 12), showed a good
selectivity and non-cytotoxicity. For example, compound 4 (IC50 = 87.0 0.88 μM) with 2ʺ,4ʺ-
dichloro substitutions showed a selective urease inhibitory activity, and no antiglycation activity.
The two chloro groups on aryl ring might form some polar interactions with the active site of the
urease enzyme. Similarly, another compound 9 (IC50 = 79.8 1.01 μM) with a 4ʺ-trifluoromethyl
group, also showed urease inhibitory activity but no antiglycation activity. This might be that
trifluoromethyl group establish polar interaction with the active site of urease enzyme. However,
its positional isomer 10 (IC50 = 193.7 0.66 μM) with a 4ʺ-trifluoromethyl group, showed two
times lower activity. It showed that C-3′′ substitution is not favourable for urease inhibition
(Figure-5).
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O O O O
O O O O
O O O O
OH OH
F
F
F
F
F
F
OH
OH
OH
OH
Cl
CF3
Cl
CF3
4
UIA IC50 = 87.0 0.88 µM
10
UIA IC50 = 193.7 0.66 µM
9
UIA IC50 = 79.8 1.01 µM
⇒
⇒
⇒
AGA Not Active
⇒
AGA Not Active
⇒
AGA Not Active
⇒
Figure-5: Structure-activity relationship of compounds 4, 9, and 10.
Compounds with the combination of halogen and hydroxy, such as 11 (OH and F para to each
other) and 12 (OH and Cl para to each other) showed no urease inhibition, however, they did
show antiglycation activity. Similarly, compound 13 (IC50 = 443.64 0.57 μM) with 2ʺ,4ʺ-
dihydroxy substitutions also showed antiglycation potential (Figure-6). Thus hydroxy group is
apparently playing an important role in the antiglycation activity. Phenolic OH known to have
radical scavenging activity through phenolic OH groups, along with other structural features
which may contribute to the inhibition of protein glycation.
O O O O
O O O O
O O O O
F
F
F
F
F
F
OH
F
OH
OH
OH
Cl
OH
OH
OH
OH
OH
OH
12
UIA Not Active
11
UIA Not Active
13
UIA Not Active
⇒
⇒
⇒
AGA
IC50 = 465.33 1.99 µM
⇒
AGA
IC50 = 399.47 1.88 µM
⇒
AGA
IC50 = 443.64 0.57 µM
⇒
Figure-6: Structure-activity relationship of compounds 11-13.
Involvement of hydroxy groups in the antiglycation activity was further deduced by examining
antiglycation activity pattern of compounds 16 (IC50 = 351.85 3.56 μM), 17 (IC50 = 374.45
1.21 μM), and 20 (IC50 = 434.71 2.73 μM) belong to category “B”. Compound 16 (IC50 = 351.85
3.56 µM), having a 2,3-dihydroxy substitution, was selectively active against glycation but no
urease inhibitory activity. The antiglycation activity of compound 16 can be compared with
compound 20 (IC50 = 434.71 2.73 µM) which has a 2,4-dihydroxy group, instead of 2,3-
dihydroxy, and showed lower antiglycation activity. This shows that the positions of OH
substitution also plays a role in the activity. However, incorporation of one or more hydroxy group
in compound 17 (IC50 = 374.45 1.21 µM) enhanced the antiglycation potential to a considerable
13

JOURNAL PRE-PROOF
level. In addition, compound 17 was found to be a potent urease inhibitor, with five times more
activity than the standard thiourea (IC50 = 21.3 1.3 μM). Interestingly, this is the only compound
which showed dual inhibitory activity against urease and glycation (Figure-7).
O O O O
O O O O
OH OH
OH
O O O O
OH OH
OH
OH
OH
OH
OH
OH
OH
OH
16
UIA Not Active
20
UIA Not Active
17
UIA IC50 = 4.4 0.21 µM
⇒
⇒
⇒
AGA
IC50 = 351.85 3.56 µM
⇒
AGA
IC50 = 434.71 2.73 µM
⇒
AGA IC50 = 374.45 1.21 µM
⇒
Figure-7: Structure-activity relationship of compounds 16, 17, and 20.
Other compounds with the halogen substitutions such as compound 22 with 3ʺ,5ʺ-dichloro
substitutions, showed an antiglycation activity (IC50 = 428.84 2.44 µM) but no urease inhibitory
activity. Compounds having the combination of halogen (Cl and Br) with the hydroxy group such
as 19 and 21, did not display any urease inhibition, however, both compounds 19 (IC50 = 432.58
0.76 µM) and 21 (IC50 = 397.07 2.21 µM) showed antiglycation activity (Figure-8). All three
compounds were found to be non-cytotoxic.
O O O O
O O O O
O O O O
OH
Cl
OH
OH
Cl
OH
OH
OH
Br
OH
Cl
Br
OH
19
UIA Not Active
21
UIA Not Active
22
UIA Not Active
⇒
⇒
⇒
AGA
IC50 = 432.58 0.76 µM
AGA
IC50 = 428.84 2.44 µM
⇒
AGA
IC50 = 397.07 2.21 µM
⇒
⇒
Figure-8: Structure-activity relationship of compounds 19, 21, and 22.
In category “C”, compound 40 (IC50 = 36.5 1.37 μM) having 3ʺ-bromo-4ʺ-hydroxy
substitutions, showed a urease inhibitory activity comparable to standard thiourea (IC50 = 21.3
1.3 μM) and found non-cytotoxic. The hydroxy group is apparently making the hydrogen bonding
interaction as well as the bromo group involved in some polar interaction with the active site of
the enzyme. Activity of compound 40 can be compared with analogs 44 and 35 which have
additional methoxy and bromo group, respectively, at 5ʺ-position of R2, a complete loss of activity
14

JOURNAL PRE-PROOF
was observed. The inactivity of the compounds might be due to the additional groups which create
the steric hindrance for the hydroxy group to bind with the enzyme active site. However,
compound 35 (IC50 = 744.82 5.64 µM) showed selective antiglycation activity and also found
to be non-cytotoxic. Furthermore, non-cytotoxic compounds 42 (IC50 = 333.63 1.98 µM) and
37 (IC50 = 919.72 1.98 µM) showed a selective good to moderate antiglycation activity.
Compound 42 has a 3ʺ,5ʺ-dibromo-4ʺ-hydroxy substitution while compound 37 is with 4ʺ-bromo-
3ʺ,5ʺ-dimethoxy substitutions at phenyl ring R2. It is worthy to note that compounds 35, 37, and
42, are 3ʺ,4ʺ,5ʺ-trisubstituted analogs which demonstrated the selective antiglycation potential.
Another compound 34 (IC50 = 115.6 2.13 μM) having a 3ʺ,4ʺ,5ʺ-trihydroxy group, showed
selective but weak urease inhibitory activity. Its activity compared with the structurally similar
and most potent compound 17 (IC50 = 4.4 0.21 μM). Compound 34 might have a conformation
which does not fit well into the active site of enzyme. Similarly, additional chloro groups at C-6
and C-6′ positions are not playing their role in the activity (Figure-9).
O O O O
O O O O
O O O O
Cl
Cl
Cl
Cl
Cl
Cl
OH
Br
OH
OH
Br
OH
OH
Br
OH
OMe
Br
OH
OH
OH
40
UIA IC50 = 36.5 1.37 µM
44
UIA Not Active
⇒
35
UIA Not Active
⇒
⇒
AGA
Not Active
AGA
Not Active
⇒
⇒
AGA
Cl
IC50 = 744.82 5.64 µM
⇒
O O O O
O O O
OH
O
O O O O
OH OH
Cl
Cl
Cl
Cl
Cl
OH
HO
OH
OH
OH
OH
MeO
OMe
OMe
I
OH
Br
37
UIA Not Active
42
UIA Not Active
34
UIA IC50 = 115.6 2.13 µM
⇒
⇒
⇒
AGA
IC50 = 919.72 1.98 µM
⇒
AGA
Not Active
⇒
AGA
IC50 = 333.63 1.98 µM
⇒
Figure-9: Structure-activity relationship of compounds 34, 35, 37, 40, 42, and 44.
In brief, it was observed that all derivatives, except 17, were found to be selective and non-
cytotoxic. Our next goal is to place heterocycles on bis-coumarin skeleton in order to see their
effects on selectivity and cytotoxicity.
15

JOURNAL PRE-PROOF
Conclusion
Synthetic bis-coumarins 1-44 were evaluated for their urease inhibitory and antiglycation
activities. Seven derivatives 4, 8-10, 14, 34, and 40 showed selective urease inhibition, whereas,
twelve analogs 2, 11-13, 16, 17, 19-22, 35, 37, and 42 demonstrated antiglycation potential. Only
compound 17 showed dual inhibition. All compounds were largely found to be non-cytotoxic.
Newly identified compounds, based on bis-coumarin scaffold, may serve as leads for future
research for more powerful, non-cytotoxic, and selective agents against urease and protein
glycation.
Experimental
Materials and methods
Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates
(Kieselgel 60 F-254, 0.20 mm, Merck, Darmstadt, Germany). Chromatograms were visualized
by using a handhold UV lamp at 254, and 365 nm or placing in iodine vapors. Fast atom
bombardement mass spectra (FAB MS) were recorded on a Finnigan MAT-311A (Germany) (70
eV) spectrometers, electrospray ionization mass spectra (ESI-MS, HRESI-MS) were recorded on
a QSTAR XL LCMS-MS, and ABSciex (Germany) (50 kV) mass spectrometers, and the data are
tabulated as m/z. 1H- and 13C-NMR spectroscopic analysis was performed on an Avance Bruker
(Germany) AM spectrometer 300 MHz machine. Splitting patterns for 1H-NMR spectra were as
follows, s (singlet); d (doublet); t (triplet); m (multiplet). Chemical shifts are reported in δ (ppm)
and coupling constants are given in Hz. All solvents and reagents were of reagent grade, and used
directly without purification. Melting points of the compounds were determined on Büchi-M560
melting point apparatus.
General procedure for the syntheses of bis-coumarin derivatives 1-44
6-Fluoro-4-hydroxy /6-chloro-4-hydroxy /4-hydroxy coumarin (1 mmol), and a variety of
aromatic aldehydes (0.5 mmol), as well as 10 mol% of tetraethylammonium bromide (TEAB)
were dissolved in distilled water (15 mL) in a 100 mL round-bottommed flask. The reaction
mixture was refluxed for 2 h. Periodic TLC was taken to check the progress of reaction. Resulting
precipitates were filtered, and washed with distilled water. This afforded products 1-44 in high
yields.
16

JOURNAL PRE-PROOF
3,3'-((2ʺ,3ʺ-Dimethoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one) (1)
White Solid; Yield: 82%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, Acetone-d6): δ 11.25 (s, 2H,
2OH), 7.67 (d, J5,6/5',6' = 8.7 Hz, 2H, H-5, H-5'), 7.50 (d, J7,8/7',8'/8,7/8',7' = 5.4 Hz, 4H, H-7, H-8, H-
7', H-8'), 7.01 (m, 3H, H-4′′, H-5′', H-6′′), 6.22 (s, 1H, -CH-), 3.81 (s, 3H, OCH3a), 3.49 (s, 3H,
OCH3b); 13C-NMR (75 MHz, DMSO-d6): δ 165.8, 165.8, 163.8, 168.3, 159.3, 159.3, 156.1, 152.3,
148.5, 146.4, 136.0, 124.4, 122.3, 121.1, 121.0, 120.9, 118.1, 117.8, 117.5, 117.3, 110.5, 109.3,
108.9, 104.5, 59.3, 55.4, 32.7; FAB (Pos.)-MS m/z = 509 [M+H]+; ESI-MS m/z = 509 [M+H]+;
HRESI-MS Calcd for C27H19F2O8 [M+H]+: m/z = 509.1047, found 509.1090.
3,3'-((2ʺ-Fluoro-4ʺ-methoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(2)
º
1
White Solid; Yield: 87%; M.p.: > 300 C dec.; H-NMR (300 MHz, DMSO-d6): δ 11.31 (bd.s,
2H, 2OH), 7.68 (d, J5,6F/5′,6F = 8.7 Hz, 2H, H-5, H-5′), 7.52 (d, J8,7/8′,7′/6′′,5′′ = 5.4 Hz, 3H, H-8, H-
8′, H-6′′), 7.32 (m, 1H, H-3''), 6.75 (m, 3H, H-7, H-7', H-5′′), 6.19 (bd.s, 1H, -CH-), 3.80 (s, 3H,
OCH3); 13C-NMR (75 MHz, DMSO-d6): δ 160.2, 159.9, 159.9, 159.2, 159.2, 156.7, 149.7, 148.5,
129.1, 120.0, 119.7, 119.3, 118.7, 118.4, 117.4, 117.3, 114.9, 114.8, 113.6, 112.1, 109.5, 109.2,
108.5, 101.5, 55.5, 28.3; FAB (Neg.)-MS m/z = 495 [M-H]-1; ESI-MS m/z = 477 [M+H-HF]+;
HRESI-MS Calcd for C26H15F2O7 [M+H-HF]+: m/z = 477.0785, Found 477.0787.
3,3'-((2ʺ-Bromo-4ʺ-methoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(3)
White Solid; Yield: 79%; M.p.: 230-232 ºC; 1H-NMR (300 MHz, Acetone-d6): δ 11.47 (bd.s, 2H,
2OH), 7.68 (d, J5,6F/5',6F = 8.1 Hz, 2H, H-5, H-5'), 7.52 (bd.s, 5H, H-7, H-8, H-7', H-8', H-3''), 7.32
(d, J6′′,5'' = 8.1 Hz, 1H, H-6′'), 7.02 (d, J5'',6'' = 8.1 Hz, 1H, H-5''), 6.15 (bd.s, 1H, -CH-), 3.86 (bd.s,
3H, OCH3); 13C-NMR (75 MHz, DMSO-d6): δ 160.1, 160.1, 159.5, 159.5, 156.9, 149.9, 148.8,
129.2, 125.2, 120.0, 119.8, 119.4, 118.9, 118.6, 117.6, 117.4, 114.8, 114.6, 113.7, 112.3, 109.4,
109.2, 108.4, 101.6, 55.7, 28.5; FAB (Neg.)-MS m/z = 556 [M-H]-1, 558 [M+2-H]-1; ESI-MS m/z
= 557 [M+H]+ 559 [M+2+H]+; HRESI-MS Calcd for C26H15BrF2O7 [M+H]+: m/z = 557.0047,
,
Found 557.0010.
17

JOURNAL PRE-PROOF
3,3'-((2ʺ,4ʺ-Dichlorophenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one) (4)
º
1
White Solid; Yield: 81%; M.p.: > 300 C dec.; H-NMR (300 MHz, Acetone-d6): δ 7.68 (dd,
J5,7//5',7' = 2.1 Hz, J5,6F/5',6F' = 7.8 Hz, 2H, H-5, H-5'), 7.57 (d, J6'',5'' = 8.4 Hz, 1H, H-6''), 7.49 (m,
4H, H-8, H-8', H-3'', H-5''), 7.39 (dd, J7,5/7',5' = 2.1 Hz, J7,8/7',8' = 6.6 Hz, 2H, H-7, H-7'), 6.24 (s,
13
1H, -CH-); C-NMR (75 MHz, DMSO-d6): δ 160.4, 160.1, 159.3, 157.1, 156.5, 156.1, 148.2,
148.0, 141.5, 135.4, 132.5, 131.7, 130.0, 126.6, 125.0, 124.9, 119.1, 119.0, 115.2, 115.0, 111.4,
111.3, 100.2, 100.0, 28.2; FAB (Neg.)-MS m/z = 516 [M-H]-1, 518 [M+2-H]-1; ESI-MS m/z = 517
[M+H]+; HRESI-MS Calcd for C25H13Cl2F2O6 [M+H]+: m/z = 517.0057, Found 517.0071.
3,3'-((5ʺ-Bromo-2ʺ-methoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(5)
White Solid; Yield: 77%; M.p.: 245-247 ºC; 1H-NMR (300 MHz, Acetone-d6): δ 11.21 (bd.s, 2H,
2OH), 7.67 (d, J5,6F/5',6F' = 8.7 Hz, 2H, H-5, H-5'), 7.51 (d, J8,7/8',7'/6'',4'' = 5.7 Hz, 3H, H-8, H-8', H-
6''), 7.43 (bd.s, 2H, H-7, H-7'), 6.96 (d, J3′′,4′′/4'',3'' = 8.7 Hz, 2H, H-3'', H-4''), 6.14 (s, 1H, -CH-),
3.61 (s, 3H, OCH3a); 13C-NMR (75 MHz, DMSO-d6): δ 165.8, 165.8, 163.5, 159.3, 157.5, 156.5,
156.2, 148.5, 148.5, 133.1, 131.1, 129.2, 124.2, 120.8, 120.7, 118.3, 117.9, 117.6, 117.4, 113.2,
111.4, 109.3, 109.0, 103.7, 55.8, 32.9; FAB (Neg.)-MS m/z = 556 [M-H]-1 558 [M+2-H]-1; ESI-
,
MS m/z = 557 [M+H]+ 559 [M+2+H]+; HRESI-MS Calcd for C26H16BrF2O7 [M+H]+: m/z =
,
557.0047, Found 557.0010.
3,3'-(p-Tolylmethylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one) (6)
White Solid; Yield: 76%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, Acetone-d6): δ 11.43 (s, 2H,
2OH), 7.68 (d, J5,6F/5',6F = 8.7 Hz, 2H, H-5, H-5'), 7.54 (d, J8,7/8′,7′/7,8 = 5.7 Hz, 3H, H-7, H-8, H-8'),
7.21 (d, J2′′,3′′/6′′,5′′/7′,8′ = 8.1 Hz, 3H, H-7′, H-2'', H-6''), 7.14 (d, J3′′,2′′/5′′,6′′ = 8.1 Hz, 2H, H-3'', H-5''),
6.10 (bd.s, 1H, -CH-), 2.29 (s, 3H, CH3); FAB (Neg.)-MS m/z = 461 [M-H]-1; ESI-MS m/z = 463
[M+H]+; HRESI-MS Calcd for C26H17F2O6 [M+H]+: m/z = 463.0993, Found 463.1022.
3,3'-((2ʺ,5ʺ-Dimethoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one) (7)
º
1
White Solid; Yield: 72%; M.p.: 240-242 C; H-NMR (300 MHz, Acetone-d6): δ 11.20 (s, 2H,
2OH), 7.67 (d, J5,6F/5',6F = 8.7 Hz, 2H, H-5, H-5'), 7.50 (d, J7,8/7',8'/8,7/8',7' = 5.7 Hz, 4H, H-7, H-8, H-
7', H-8'), 6.91 (m, 3H, H-3′′, H-4′', H-6''), 6.10 (s, 1H, -CH-), 3.68 (s, 3H, OCH3a), 3.52 (s, 3H,
CH3b); 13C-NMR (75 MHz, DMSO-d6): δ 165.1, 165.1, 163.7, 163.7, 159.3, 159.3, 156.2, 152.6,
18

JOURNAL PRE-PROOF
148.5, 146.3, 131.4, 125.3, 120.5, 120.4, 118.3, 118.0, 117.6, 117.5, 116.4, 111.9, 109.6, 109.3,
108.9, 104.4, 56.2, 55.0, 33.0; FAB (Neg.)-MS m/z = 507 [M-H]-1; ESI-MS m/z = 509 [M+H]+;
HRESI-MS Calcd for C27H19F2O8 [M+H]+: m/z = 509.1047, Found 509.1064.
3,3'-((3ʺ-(Benzyloxy)-4ʺ-methoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-
2-one) (8)
º
1
White Solid; Yield: 80%; M.p.: 200-202 C; H-NMR (300 MHz, Acetone-d6): δ 11.41 (s, 2H,
2OH), 7.66 (d, J5,6F/5',6F = 8.4 Hz, 2H, H-5, H-5'), 7.54 (bd.d, J8,7/8',7'/7,8/7',8' = 5.4 Hz, 4H, H-7, H-
8, H-7', H-8'), 7.27 (m, 5H, Ar-H), 6.96 (bd.s, 1H, H-2''), 6.93 (d, J5′′,6′' = 7.5 Hz, 1H, H-5′′), 6.87
(d, J6′′,5′' = 8.0 Hz, 1H, H-6′′), 6.05 (s, 1H, -CH-), 4.97 (s, 2H, -CH2-), 3.80 (s, 3H, OCH3); 13C-
NMR (100 MHz, DMSO-d6): δ 162.7, 162.7, 161.6, 161.6, 159.4, 159.4, 149.5, 148.2, 148.2,
146.9, 136.6, 135.4, 128.7, 128.7, 127.5, 127.2, 127.2, 125.3, 125.3, 122.0, 119.2, 119.2, 115.0,
115.0, 114.2, 112.4, 111.5, 111.5, 100.3, 100.3, 71.4, 56.2, 28.4; FAB (Neg.)-MS m/z = 582 [M-
H]-1; ESI-MS m/z = 585 [M+H]+; HRESI-MS Calcd for C33H23F2O8 [M+H]+: m/z = 585.1360,
Found 585.1332.
3,3'-((4ʺ-(Trifluoromethyl)phenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(9)
White Solid; Yield: 84%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, DMSO-d6): δ 7.53 (d, J5,6F/5',6F
= 8.4 Hz, 2H, H-5, H-5'), 7.49 (dd, J3'',5''/5'',3'' = 3.0 Hz, J3'',2''/5'',6'' = 6.0 Hz, 2H, H-3'', H-5''), 7.37
(m, 4H, H-8, H-8′, H-2′', H-6''), 7.29 (d, J7,8/7',8′ = 8.4 Hz, 2H, H-7, H-7'), 6.29 (bd.s, 1H, -CH-);
13C-NMR (100 MHz, DMSO-d6): δ 162.8, 162.8, 161.4, 161.4, 159.6, 159.6, 148.0, 148.0, 147.6,
128.9, 128.9, 128.4, 125.3, 125.3, 125.1, 125.1, 124.3, 119.4, 119.4, 115.5, 115.5, 111.6, 111.6,
100.9, 100.9, 28.4; FAB (Neg.)-MS m/z = 515 [M-H]-1; ESI-MS m/z = 517 [M+H]+; HRESI-MS
Calcd for C26H14F5O6 [M+H]+: m/z = 517.0710, Found 517.0755.
3,3'-((3ʺ-(Trifluoromethyl)phenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(10)
White Solid; Yield: 77%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, Acetone-d6): δ 11.52 (bd.s,
2H, 2OH), 7.69 (m, 4H, H-5, H-8, H-5', H-8'), 7.55 (m, 6H, H-7, H-7', H-2'', H-4'', H-5'', H-6''),
6.30 (s, 1H, -CH-); 13C-NMR (100 MHz, DMSO-d6): δ 162.8, 162.8, 161.4, 161.4, 159.6, 159.6,
148.0, 148.0, 142.7, 131.2, 130.7, 128.7, 127.3, 125.3, 125.3, 124.5, 122.2, 119.4, 119.4, 115.5,
19

JOURNAL PRE-PROOF
115.5, 111.6, 111.6, 100.9, 100.9, 28.5; FAB (Neg.)-MS m/z = 515 [M-H]-1; ESI-MS m/z = 517
[M+H]+; HRESI-MS Calcd for C26H14F5O6 [M+H]+: m/z = 517.0710, Found 517.0755.
3,3'-((5ʺ-Fluoro-2ʺ-hydroxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(11)
White Solid; Yield: 73%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, DMSO-d6): δ 7.88 (m, 1H,
H-5), 7.75 (m, 1H, H-5'), 7.61 (m, 2H, H-8, H-8'), 7.46 (m, 3H, H-7, H-7', H-6''), 7.19 (m, 1H, H-
13
4''), 6.98 (m, 1H, H-3''), 5.69 (s, 1H, -CH-); C-NMR (100 MHz, DMSO-d6): δ 162.2, 161.9,
160.5, 160.1, 159.5, 159.0, 155.2, 151.6, 149.5, 148.8, 125.6, 125.1, 122.5, 119.9, 119.5, 117.6,
117.2, 117.0, 114.8, 114.7, 109.7, 109.4, 101.6, 101.4, 28.5; FAB (Neg.)-MS m/z = 462 [M-H-
HF]-1; ESI-MS m/z = 465 [M+H-H2O]+; HRESI-MS Calcd for C25H12F3O6 [M+H-H2O]+: m/z =
465.0585, Found 465.0549.
3,3'-((5ʺ-Chloro-2ʺ-hydroxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(12)
White Solid; Yield: 79%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, Acetone-d6): δ 7.84 (m, 2H,
H-5, H-5'), 7.50 (m, 7H, H-7, H-8, H-7', H-8', H-3'', H-4'', H-6''), 5.79 (s, 1H, -CH-); 13C-NMR
(100 MHz, DMSO-d6): δ 162.5, 162.2, 160.4, 160.0, 159.6, 159.3, 151.4, 149.5, 148.9, 126.7,
125.7, 125.2, 122.6, 119.8, 119.4, 117.7, 117.4, 117.1, 114.7, 114.3, 109.8, 109.3, 101.7, 101.5,
28.6; FAB (Neg.)-MS m/z = 497 [M-H]-1; 499 [M+2-H]-1; ESI-MS m/z = 499 [M+H]+; HRESI-
MS Calcd for C25H13ClF2O7 [M+H]+: m/z = 499.0318, Found 499.0336.
3,3'-((2ʺ,4ʺ-Dihydroxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one) (13)
White Solid; Yield: 81%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, Acetone-d6): δ 7.84 (dd, J5,7
= 2.1 Hz, J5,6F = 8.7 Hz, 1H, H-5), 7.76 (d, J5',7' = 2.4 Hz, J5',6F = 8.7 Hz, 1H, H-5'), 7.52 (m, 4H,
H-7, H-8, H-7', H-8'), 7.10 (d, J6'',5'' = 8.4 Hz, 1H, H-6''), 6.82 (bd.s, 1H, H-3''), 6.65 (m, 1H, H-
5''), 5.62 (bd.s, 1H, -CH-); 13C-NMR (75 MHz, DMSO-d6): δ 160.2, 159.9, 159.5, 157.3, 156.7,
156.3, 148.5, 148.3, 129.1, 119.9, 119.6, 119.5, 119.2, 118.7, 118.6, 118.4, 118.3, 115.0, 114.9,
113.1, 111.9, 108.6, 108.3, 102.9, 28.2; FAB (Neg.)-MS m/z = 461 [M-H-H2O]-1; ESI-MS m/z =
463 [M+H-H2O]+; HRESI-MS Calcd for C25H13F2O7 [M+H-H2O]+: m/z = 463.0629, Found
463.0616.
20

JOURNAL PRE-PROOF
3,3'-((3ʺ-Bromo-4ʺ-methoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-2-one)
(14)
º
1
White Solid; Yield: 85%; M.p.: 210-212 C; H-NMR (300 MHz, Acetone-d6): δ 11.47 (s, 2H,
2OH), 7.69 (d, J5,6F/5',6F = 8.7 Hz, 2H, H-5, H-5'), 7.54 (d, J7,8/8,7/7′,8′/8′,7′ = 6.6 Hz, 4H, H-7, H-8, H-
7', H-8'), 7.52 (bd.s, 1H, H-2''), 7.32 (bd.d, J6'',5'' = 8.1 Hz, 1H, H-6''), 7.02 (d, J5'',6'' = 8.4 Hz, 1H,
H-5''), 6.14 (s, 1H, -CH-), 3.87 (s, 3H, OCH3); 13C-NMR (75 MHz, DMSO-d6): δ 166.5, 166.4,
164.1, 159.4, 156.2, 153.1, 148.7, 135.4, 130.8, 127.2, 120.8, 120.7, 118.6, 118.2, 117.7, 117.6,
112.3, 110.0, 109.4, 109.1, 103.7, 56.1, 35.2; FAB (Neg.)-MS m/z = 555 [M-H]-1, 557 [M+2-H]-
1; ESI-MS m/z = 557 [M+H]+; HRESI-MS Calcd for C26H16BrF2O7 [M+H]+: m/z = 557.0047,
Found 557.0046.
3,3'-((4ʺ-Bromo-2ʺ,5ʺ-dimethoxyphenyl)methylene)bis(6-fluoro-4-hydroxy-2H-chromen-
2-one) (15)
º
1
White Solid; Yield: 83%; M.p.: 205-207 C; H-NMR (300 MHz, Acetone-d6): δ 11.30 (s, 2H,
2OH), 7.67 (d, J8,7/8',7' = 8.4 Hz, 2H, H-8, H-8'), 7.49 (d, J7,8/8,7/7′,8′/8′,7′ = 5.4 Hz, 4H, H-7, H-8, H-
7′, H-8′), 7.14 (s, 1H, H-3''), 7.10 (s, 1H, H-6''), 6.13 (s, 1H, -CH-), 3.72 (s, 3H, OCH3b), 3.57 (s,
3H, OCH3a); 13C-NMR (75 MHz, DMSO-d6): δ 165.7, 165.6, 163.6, 163.5, 159.3, 159.2, 151.8,
149.6, 148.8, 148.5, 131.3, 125.3, 125.2, 120.8, 120.7, 118.3, 117.9, 117.6, 117.4, 116.0, 114.4,
109.3, 109.0, 107.5, 104.0, 56.5, 56.5, 33.0; FAB (Neg.)-MS m/z = 586 [M-H]-1, 588 [M+2-H]-1;
ESI-MS m/z = 587 [M+H]+, 589 [M+2+H]+; HRESI-MS Calcd for C27H18BrF2O8 [M+H]+: m/z =
587.0153, Found 587.0111.
3,3'-((2ʺ,3ʺ-Dihydroxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (16)
White Solid; Yield: 82%; M.p.: 110-112 ºC; 1H-NMR (300 MHz, Acetone-d6): δ 8.64 (bd.s, 1H,
OH), 8.36 (d, J5,6 = 8.1 Hz, 1H, H-5), 8.06 (d, J5',6' = 8.1 Hz, 1H, H-5'), 7.72 (t, J7,6 = J7,8 = 8.4 Hz,
1H, H-7), 7.61 (t, J7′,6′ = J7′,8′ = 7.2 Hz, 1H, H-7′), 7.49 (t, J6,5 = J6,7 = 7.2 Hz, 1H, H-6), 7.39 (d,
J8,7 = 8.7 Hz, 1H, H-8), 7.39 (t, J6′,5′ = J6′,7′ = 8.7 Hz, 1H, H-6′), 7.24 (d, J8',7' = 8.1 Hz, 1H, H-8'),
6.98 (t, J5'',4'' = J5'',6'' = 7.8 Hz, 1H, H-5''), 6.88 (d, J6'',5'' = 6.6 Hz, 1H, H-6''), 6.76 (d, J4'',5'' = 7.5 Hz,
13
1H, H-4''), 5.67 (s, 1H, -CH-); C-NMR (75 MHz, DMSO-d6): δ 160.5, 152.1, 151.9, 151.9,
145.0, 145.0, 132.3, 132.3, 132.0, 132.0, 125.0, 124.3, 124.3, 123.9, 123.9, 123.2, 123.2, 118.2,
116.3, 116.3, 116.1, 116.1, 115.4, 114.0, 28.7; FAB (Neg.)-MS m/z = 425 [M-H-H2O]-1; ESI-MS
21

JOURNAL PRE-PROOF
m/z = 427 [M+H-H2O]+; HRESI-MS Calcd for C25H16O8 [M+H-H2O]+: m/z = 427.0845, Found
427.0868.
3,3'-((2ʺ,3ʺ,4ʺ-Trihydroxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (17)
White Solid; Yield: 77%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, Acetone-d6): δ 8.36 (d, J5,6
8.1 Hz, 1H, H-5), 8.36 (bd.s, 1H, OH), 8.08 (bd.s, 1H, OH), 8.05 (d, J5',6' = 6.6 Hz, 1H, H-5'),
7.72 (t, J7,6 = J7,8 = 8.7 Hz, 1H, H-7), 7.60 (t, J7',6' = J7',8' = 7.2 Hz, 1H, H-7'), 7.49 (t, J6,5 = J6,7
=
=
7.2 Hz, 1H, H-6), 7.40 (d, J6'',5'' = 8.7 Hz, 1H, H-6''), 7.38 (t, J6',5' = J6',7' = 8.1 Hz, 1H, H-6'), 7.24
(d, J5'',6'' = 8.1 Hz, 1H, H-5''), 6.66 (m, 2H, H-8, H-8'), 5.60 (s, 1H, -CH-); 13C-NMR (100 MHz,
DMSO-d6): δ 160.5, 160.5, 156.1, 152.0, 152.0, 151.8, 151.8, 145.5, 145.5, 138.7, 132.9, 132.9,
132.2, 131.9, 124.3, 124.3, 123.8, 123.4, 117.3, 116.2, 116.1, 114.1, 112.2, 28.3; FAB (Neg.)-MS
m/z = 441 [M-H-H2O]-1; ESI-MS m/z = 443 [M+H-H2O]+; HRESI-MS Calcd for C25H15O8 [M+H-
H2O]+: m/z = 443.0766, Found 443.0744.
3,3'-((2ʺ,3ʺ-Dimethylphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (18)
White Solid; Yield: 73%; M.p.: 203-205 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.81 (d, J5,6/5',6'
7.5 Hz, 2H, H-5, H-5'), 7.49 (t, J7,6 = J7,8 = J7',6' = J7',8' = 6.9 Hz, 2H, H-7, H-7'), 7.24 (d, J8,7/8',7'
=
=
8.1 Hz, 2H, H-8, H-8'), 7.24 (t, J6,5 = J6,7 = J6′,5′ = J6',7' = 8.1 Hz, 2H, H-6, H-6'), 7.14 (d, J4'',5'' = 8.4
Hz, 1H, H-4''), 6.80 (m, 2H, H-5′′, H-6′'), 6.02 (s, 1H, -CH-), 2.16 (s, 3H, CH3), 2.01 (s, 1H, CH3);
13C-NMR (100 MHz, DMSO-d6): δ 162.2, 162.2, 161.4, 161.4, 152.6, 152.6, 136.8, 135.7, 135.4,
128.2, 128.2, 127.5, 125.4, 125.3, 125.3, 123.4, 123.4, 121.4, 116.6, 116.6, 112.2, 112.2, 100.0,
100.0, 28.5, 19.3, 17.2; FAB (Neg.)-MS m/z = 439 [M-H]-1; ESI-MS m/z = 441 [M+H]+; HRESI-
MS Calcd for C27H21O6 [M+H]+: m/z = 441.1338, Found 441.1328.
3,3'-((5ʺ-Chloro-2ʺ-hydroxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (19)
White Solid; Yield: 78%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, DMSO-d6): δ 8.10 (d, J5,6 =
7.5 Hz, 1H, H-5), 8.04 (bd.d, 1H, H-5'), 7.73 (t, J7,6 = J7,8 = 8.7 Hz, 1H, H-7), 7.62 (t, J7',6' = J7',8'
= 8.4 Hz, 1H, H-7'), 7.51 (m, 6H, H-6, H-8, H-6', H-8', H-3'', H-4''), 7.18 (s, 1H, H-6''), 5.70 (bd.s,
1H, -CH-); FAB (Neg.)-MS m/z = 461 [M-H]-1, 463 [M+2-H]-1; ESI-MS m/z = 445 [M+H-H2O]+;
HRESI-MS Calcd for C25H14ClO6 [M+H-H2O]+: m/z = 445.0478, Found 445.0453.
22

JOURNAL PRE-PROOF
3,3'-((2ʺ,4ʺ-Dihydroxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (20)
º
1
White Solid; Yield: 82%; M.p.: > 300 C dec.; H-NMR (300 MHz, DMSO-d6): δ 9.68 (s, 1H,
OH), 8.07 (d, J5,6 = 6.9 Hz, 1H, H-5), 7.95 (bd.s, 1H, H-5'), 7.69 (t, J7,6 = J7,8 = 7.2 Hz, 1H, H-7),
7.52 (t, J7′,6′ = J7′,8′ = 7.2 Hz, 1H, H-7′), 7.48 (t, J6,5 = J6,7 = 8.1 Hz, 1H, H-6), 7.43 (d, J8,7 = 7.5 Hz,
1H, H-8), 7.25 (m, 2H, H-6′, H-8'), 7.00 (d, J6'',5'' = 8.4 Hz, 1H, H-6''), 6.67 (d, J3'',5'' = 2.1 Hz, 1H,
H-3''), 6.55 (dd, J5'',3'' = 2.1 Hz, J5'',6'' = 6.0 Hz, 1H, H-5''), 5.57 (s, 1H, -CH-); FAB (Neg.)-MS m/z
= 425 [M-H-H2O]-1; ESI-MS m/z = 427 [M+H-H2O]+; HRESI-MS Calcd for C25H15O7 [M+H-
H2O]+: m/z = 427.0817, Found 427.0816.
3,3'-((3ʺ,5ʺ-Dibromo-4-hydroxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (21)
White Solid; Yield: 87%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, DMSO-d6): δ 7.82 (d, J5,6/5',6'
= 6.3 Hz, 2H, H-5, H-5'), 7.53 (t, J7,6 = J7,8 = J7',6' = J7',8' = 6.9 Hz, 2H, H-7, H-7'), 7.27 (d, J8,7/8',7'
= 8.1 Hz, 2H, H-8, H-8'), 7.26 (t, J6,5 = J6,7 = J6',5' = J6',7' = 7.5 Hz, 2H, H-6, H-6'), 7.12 (bd.s, 2H,
13
H-2'', H-6''), 6.16 (s, 1H, -CH-); C-NMR (75 MHz, DMSO-d6): δ 167.2, 167.2, 164.2, 164.2,
152.4, 152.4, 148.1, 148.1, 136.6, 131.3, 131.3, 130.2, 130.2, 124.0, 124.0, 123.1, 123.1, 119.3,
119.3, 115.6, 115.6, 111.7, 102.9, 102.9, 35.0; FAB (Neg.)-MS m/z = 584 [M-H]-1 586 [M+2-H]-
,
1; ESI-MS m/z = 585 [M+H]+ 587 [M+2+H]+ 589 [M+4+H]+; HRESI-MS Calcd for C25H15Br2O7
,
,
[M+H]+: m/z = 584.9184, Found 584.9208.
3,3'-((3ʺ,5ʺ-Dichlorophenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (22)
White Solid; Yield: 82%; M.p.: 232-234 ºC; 1H-NMR (300MHz, Acetone-d6): δ 8.14 (d, J5,6 = 6.3
Hz, 1H, H-5), 8.08 (d, J5',6' = 6.6 Hz, 1H, H-5'), 7.76 (t, J7,6 = J7,8 = 8.4 Hz, 1H, H-7), 7.62 (t, J7′,6′
= J7′,8′ = 7.5 Hz, 1H, H-7′), 7.55 (t, J6,5 = J6,7 = 7.5 Hz, 1H, H-6), 7.52 (d, J2′',6′'/6′′,2′′ = 2.4 Hz, 1H,
H-2′', H-6′′), 7.43 (t, J6',5' = J6',7' = 8.4 Hz, 1H, H-6'), 7.38 (d, J8,7 = 7.2 Hz, 1H, H-8), 7.32 (d,
J
4′′,2′′/4′′,6′′ = 2.4 Hz, 1H, H-4′′), 7.27 (d, J8',7' = 8.1 Hz, 1H, H-8'), 5.74 (s, 1H, -CH-); 13C-NMR (100
MHz, DMSO-d6): δ 162.7, 162.7, 161.6, 161.6, 152.4, 152.4, 145.2, 135.7, 135.7, 128.4, 128.4,
126.8, 126.8, 126.3, 125.3, 125.3, 123.4, 123.4, 116.5, 116.5, 116.3, 116.3, 100.1, 100.1, 28.4;
FAB (Neg.)-MS m/z = 476 [M-H]-1, 478 [M+2-H]-1; ESI-MS m/z = 479 [M+H]+, 481 [M+2+H]+;
HRESI-MS Calcd for C25H15Cl2O6 [M+H]+: m/z = 479.0089, Found 479.0105.
23

JOURNAL PRE-PROOF
3,3'-((2ʺ,5ʺ-Dimethoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (23)
White Solid; Yield: 74%; M.p.: 181-183 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.87 (dd, J5,7/5',7'
= 1.2 Hz, J5,6/5',6' = 6.6 Hz, 2H, H-5, H-5'), 7.56 (t, J7,6 = J7,8 = J7',6' = J7',8' = 8.4 Hz, 2H, H-7, H-7'),
7.31 (d, J8,7/8',7' = 7.8 Hz, 2H, H-8, H-8'), 7.30 (t, J6,7 = J6,8 = J6',7' = J6',8' = 7.5 Hz, 2H, H-6, H-6'),
6.79 (d, J4'',3'' = 8.4 Hz, 1H, H-4''), 6.71 (s, 1H, H-6′′), 6.71 (d, J3'',4'' = 8.7 Hz, 1H, H-3''), 6.19 (s,
1H, -CH-), 3.60 (s, 3H, OCH3a), 3.48 (s, 3H, OCH3b); 13C-NMR (75 MHz, DMSO-d6): δ 165.1,
163.9, 152.7, 152.1, 151.6, 131.1, 131.1, 123.7, 123.7, 123.2, 123.2, 118.5, 116.2, 115.6, 115.6,
111.8, 109.6, 104.2, 56.1, 55.0, 32.9; FAB (Neg.)-MS m/z = 470 [M-H]-1; ESI-MS m/z = 473
[M+H]+; HRESI-MS Calcd for C27H20O8 [M+H]+: m/z = 473.1236, Found 473.1198.
3,3'-((2ʺ,3ʺ-Dimethoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (24)
White Solid; Yield: 76%; M.p.: 190-192 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.85 (dd, J5,7/5',7'
= 1.2 Hz, J5,6/5',6' = 6.9 Hz, 2H, H-5, H-5'),7.53 (t, J7,6 = J7,8 = J7',6' = J7',8' = 8.4 Hz, 2H, H-7, H-7'),
7.28 (d, J8,7/8',7' = 8.4 Hz, 2H, H-8, H-8'), 7.27 (t, J6,7 = J6,8 = J6',7' = J6',8' = 7.5 Hz, 2H, H-6, H-6'),
6.89 (m, 3H, H-4'', H-5'', H-6''), 6.29 (s, 1H, -CH-), 3.71 (s, 3H, OCH3b), 3.44 (s, 3H, OCH3a);
13C-NMR (75 MHz, DMSO-d6): δ 165.0, 164.0, 152.2, 152.0, 146.5, 135.1, 131.2, 131.2, 123.7,
123.7, 123.3, 123.3, 122.5, 120.8, 118.5, 115.7, 115.7, 110.7, 104.6, 59.2, 55.4, 32.7; FAB (Neg.)-
MS m/z = 471 [M-H+]-1. FAB (Neg.)-MS m/z = 471 [M-H]-1; ESI-MS m/z = 473 [M+H]+; HRESI-
MS Calcd for C27H21O8 [M+H]+: m/z = 473.1236, Found 473.1207.
3,3'-((4ʺ-Ethoxy-3ʺ-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (25)
White Solid; Yield: 78%; M.p.: 195-197 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.81 (dd, J5,7/5',7'
= 1.5 Hz, J5,6/5',6' = 6.3 Hz, 2H, H-5, H-5'), 7.50 (t, J7,6 = J7,8 = J7',6' = J7',8' = 6.9 Hz, 2H, H-7, H-7'),
7.25 (d, J8,7/8',7' = 8.1 Hz, 2H, H-8, H-8'), 7.23 (t, J6,7 = J6,8 = J6',7' = J6',8' = 7.2 Hz, 2H, H-6, H-6'),
6.73 (d, J5'',6'' = 8.7 Hz, 1H, H-5''), 6.64 (s, 1H, H-2′′), 6.60 (d, J6'',5'' = 8.7 Hz, 1H, H-6''), 6.18 (s,
1H, -CH-), 3.92 (q, 2H, OCH2-), 3.50 (s, 3H, OCH3), 1.29 (t, 3H, CH3); FAB (Neg.)-MS m/z =
485 [M-H]-1; ESI-MS m/z = 487 [M+H]+; HRESI-MS Calcd for C28H23O8 [M+H]+: m/z =
487.1392, Found 487.1385.
24

JOURNAL PRE-PROOF
3,3'-((2ʺ,4ʺ-Dimethoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (26)
White Solid; Yield: 72%; M.p.: 197-199 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.82 (d, J5,6/5',6'
6.9 Hz, 2H, H-5, H-5'), 7.50 (t, J7,6 = J7,8 = J7',6' = J7',8' = 8.4 Hz, 2H, H-7, H-7'), 7.25 (d, J8,7/8',7'
=
=
8.1 Hz, 2H, H-8, H-8'),7.22 (t, J6,7 = J6,8 = J6',7' = J6',8' = 7.8 Hz, 2H, H-6, H-6'), 7.06 (d, J6'',5'' = 8.1
Hz, 1H, H-6''), 6.39 (m, 2H, H-3'', H-5''), 6.12 (s, 1H, -CH-), 3.68 (s, 3H, OCH3b), 1.29 (t, 3H,
13
OCH3a); C-NMR (100 MHz, DMSO-d6): δ 164.4, 163.9, 158.8, 158.0, 152.0, 131.2, 131.2,
128.7, 123.6, 123.6, 123.3, 123.3, 120.9, 118.2, 115.7, 115.7, 104.6, 103.8, 98.5, 55.5, 55.0, 32.2;
FAB (Neg.)-MS m/z = 470 [M-H]-1; ESI-MS m/z = 473 [M+H]+; HRESI-MS Calcd for C27H21O8
[M+H]+: m/z = 473.1236, Found 473.1230.
3,3'-((4ʺ-Bromo-2ʺ-fluorophenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (27)
White Solid; Yield: 83%; M.p.: 245-247 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.81 (d, J5,6/5',6'
=
6.6 Hz, 2H, H-5, H-5'), 7.52 (t, J7,6 = J7,8 = J7',6' = J7',8' = 8.4 Hz, 2H, H-7, H-7'), 7.27 (m, 7H, H-6,
H-8, H-6', H-8', H-3'', H-5'', H-6′′), 6.24 (s, 1H, -CH-); 13C-NMR (100 MHz, DMSO-d6): δ 162.9,
162.5, 162.2, 160.4, 160.0, 151.4, 150.9, 132.6, 128.4, 127.2, 125.7, 125.2, 121.5, 120.5, 119.8,
119.4, 117.7, 117.4, 114.7, 114.3, 109.8, 109.3, 101.7, 101.5, 28.6; FAB (Neg.)-MS m/z = 507
[M-H]-1 509 [M+2-H]-1; ESI-MS m/z = 509 [M+H]+ 511 [M+2+H]+; HRESI-MS Calcd for
,
,
C25H15BrFO6 [M+H]+: m/z = 509.0036, Found 509.0032.
3,3'-((2ʺ-Bromo-4ʺ,5ʺ-dimethoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (28)
White Solid; Yield: 85%; M.p.: 200-202 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.83 (d, J5,6/5',6'
6.6 Hz, 2H, H-5, H-5'), 7.26 (t, J7,6 = J7,8 = J7',6' = J7',8' = 7.2 Hz, 2H, H-7, H-7'), 7.26 (d, J8,7/8',7'
=
=
8.7 Hz, 2H, H-8, H-8'), 7.25 (t, J6,7 = J6,8 = J6',7' = J6',8' = 8.1 Hz, 2H, H-6, H-6'), 7.01 (s, 1H, H-3''),
6.97 (s, 1H, H-6''), 5.97 (s, 1H, -CH-), 3.70 (s, 3H, OCH3a), 1.29 (t, 3H, OCH3b); 13C-NMR (100
MHz, DMSO-d6): δ 190.1, 165.3, 163.5, 152.2, 147.6, 147.1, 133.0, 131.2, 131.2, 123.7, 123.7,
123.3, 123.3, 118.4, 116.0, 115.7, 115.7, 114.4, 113.4, 110.5, 104.1, 55.7, 55.6, 37.9; FAB (Neg.)-
MS m/z = 549 [M-H]-1 551 [M+2-H]-1; ESI-MS m/z = 551 [M+H]+ 553 [M+2+H]+; HRESI-MS
,
,
Calcd for C27H20BrO8 [M+H]+: m/z = 551.0341, Found 551.0327.
25

JOURNAL PRE-PROOF
3,3'-((3ʺ-(Benzyloxy)-4ʺ-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one)
(29)
º
1
White Solid; Yield: 81%; M.p.: 210-212 C; H-NMR (300 MHz, Acetone-d6): δ 11.44 (s, 1H,
OH), 8.01 (d, J5,6/5',6' = 6.9 Hz, 2H, H-5, H-5'), 7.74 (t, J7,6 = J7,8 = J7',6' = J7',8' = 7.2 Hz, 2H, H-7,
H-7'), 7.48 (t, J6,7 = J6,8 = J6',7' = J6',8' = 8.1 Hz, 2H, H-6, H-6'), 7.48 (d, J8,7/8',7' = 7.8 Hz, 2H, H-8,
H-8'), 7.26 (m, 2H, H-2''', H-6'''), 7.20 (m, 3H, H-3''', H-4''', H-6'''), 6.97 (bd.s, 1H, H-2'''), 6.93 (d,
J6'',5'' = 7.2 Hz, 1H, H-6''), 6.87 (d, J5'',6'' = 7.2 Hz, 1H, H-5''), 6.04 (s, 1H, -CH-), 4.96 (s, 2H, -
CH2-), 3.80 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6): δ 162.9, 162.9, 161.7, 161.7, 152.6,
152.6, 149.8, 146.6, 136.5, 135.7, 128.7, 128.7, 128.4, 128.4, 127.4, 127.2, 127.2, 125.5, 125.5,
123.2, 123.2, 122.4, 116.7, 116.7, 116.2, 116.2, 114.2, 112.4, 100.4, 100.4, 71.2, 28.7, 56.2; FAB
(Neg.)-MS m/z = 546 [M-H]-1; ESI-MS m/z = 549 [M+H]+; HRESI-MS Calcd for C33H25O8
[M+H]+: m/z = 549.1549, Found 549.1550.
3,3'-((4ʺ-Hydroxy-3ʺ-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (30)
º
1
White Solid; Yield: 84%; M.p.: 255-257 C; H-NMR (300 MHz, Acetone-d6): δ 11.45 (s, 2H,
2OH), 8.02 (d, J5,6/5',6' = 7.2 Hz, 2H, H-5, H-5'), 7.75 (t, J7,6 = J7,8 = J7',6' = J7',8' = 7.5 Hz, 2H, H-7,
H-7'), 7.51 (m, 4H, H-6, H-8, H-6', H-8'), 6.95 (s, 1H, H-2''), 6.77 (s, 2H, H-5'', H-6''), 6.08 (s,
1H, -CH-), 3.67 (s, 3H, OCH3); FAB (Neg.)-MS m/z = 457 [M-H]-1; ESI-MS m/z = 459 [M+H]+;
HRESI-MS Calcd for C26H19O8 [M+H]+: m/z = 459.1079, Found 459.1053.
3,3'-((4ʺ-(Methylthio)phenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (31)
º
1
White Solid; Yield: 77%; M.p.: 260-262 C; H-NMR (300 MHz, Acetone-d6): δ 11.50 (s, 2H,
2OH), 8.03 (d, J5,6/5',6' = 8.4 Hz, 2H, H-5, H-5'), 7.76 (t, J7,6 = J7,8 = J7',6' = J7',8' = 6.9 Hz, 2H, H-7,
H-7'), 7.49 (m, 4H, H-6, H-8, H-6', H-8'), 7.29 (d, J2'',3''/6'',5'' = 8.1 Hz, 2H, H-2'', H-6''), 7.23 (d,
J3'',2''/5'',6'' = 8.7 Hz, 2H, H-3'', H-5''), 6.10 (s, 1H, -CH-), 2.46 (s, 3H, SCH3); FAB (Neg.)-MS m/z
= 457 [M-H]-1; ESI-MS m/z = 459 [M+H]+; HRESI-MS Calcd for C26H19O6S [M+H]+: m/z =
459.0902, Found 459.0922.
26

JOURNAL PRE-PROOF
3,3'-((3ʺ-Bromo-4ʺ-methoxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(32)
White Solid; Yield: 85%; M.p.: 204-206 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.73 (d, J5,7/5',7'
=
2.4 Hz, 2H, H-5, H-5'), 7.55 (dd, J7,5/7',5' = 2.4 Hz, J7,8/7',8' = 9.0 Hz, 2H, H-7, H-7'), 7.32 (d, J8,7/8',7'
= 8.7 Hz, 2H, H-8, H-8'), 7.15 (bd.s, 1H, H-2''), 7.04 (bd.d, J6'',5'' = 8.4 Hz, 1H, H-6''), 6.92 (d, J5'',6''
= 8.7 Hz, 1H, H-5''), 6.15 (s, 1H, -CH-), 3.76 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6): δ
166.3, 163.9, 153.1, 151.0, 135.3, 130.8, 130.8, 130.7, 127.2, 127.2, 123.2, 123.2, 121.1, 117.7,
117.7, 112.3, 110.0, 103.8, 56.0, 32.2; FAB (Neg.)-MS m/z = 587 [M-H]-1; 589 [M+2-H]-1; 591
[M+4-H]-1; ESI-MS m/z = 599 [M+H]+; HRESI-MS Calcd for C26H15BrCl2O7 [M+H]+: m/z =
588.9456, Found 588.9479.
3,3'-((3ʺ-(Benzyloxy)phenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one) (33)
White Solid; Yield: 80%; M.p.: 192-194 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.72 (d, J5,7/5',7'
=
2.4 Hz, 2H, H-5, H-5'), 7.54 (dd, J7,5/7',5' = 2.4 Hz, J7,8/7',8' = 6.3 Hz, 2H, H-7, H-7'), 7.31 (m, 4H,
H-8, H-8', H-3''', H-5'''), 7.24 (m, 3H, H-2''', H-4''', H-6''' ), 7.09 (t, J5'',4'' = J5'',6'' = 8.1 Hz, 1H, H-
5''), 6.74 (d, J6'',5'' = 8.1 Hz, 1H, H-6''), 6.64 (m, 2H, H-2'', H-4''), 6.18 (s, 1H, -CH-), 4.94 (s, 3H,
OCH2); 13C-NMR (75 MHz, DMSO-d6): δ 166.1, 166.1, 164.0, 164.0, 158.3, 151.0, 151.0, 143.2,
137.2, 130.8, 130.8, 128.8, 128.2, 128.2, 127.7, 127.6, 127.6, 127.2, 127.2, 123.2, 123.2, 121.1,
119.3, 119.3, 117.7, 117.7, 113.7, 110.9, 104.0, 104.0, 69.1, 36.2; FAB (Neg.)-MS m/z = 585 [M-
H]-1, 587 [M+2-H]-1; ESI-MS m/z = 587 [M+H]+; HRESI-MS Calcd for C32H21Cl2O7 [M+H]+:
m/z = 587.0664, Found 587.0666.
3,3'-((2ʺ,3ʺ,4ʺ-Trihydroxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(34)
White Solid; Yield: 86%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, DMSO-d6): δ 9.26 (bd.s, 1H,
OH), 9.06 (bd.s, 1H, OH), 8.56 (d, J5,7 = 2.7 Hz, 2H, H-5), 7.95 (bd.s, 1H, H-5′), 7.72 (d, J7,8 =
8.7 Hz, 1H, H-7), 7.58 (d, J7',8' = 9.0 Hz, 1H, H-7'), 7.47 (d, J8,7 = 9.0 Hz, 1H, H-8), 7.31 (d, J8',7'
= 8.4 Hz, 1H, H-8'), 6.55 (d, J6'',5'' = 8.4 Hz, 1H, H-6''), 6.46 (d, J5'',6'' = 8.4 Hz, 1H, H-5''), 5.56 (s,
1H, -CH-); 13C-NMR (100 MHz, DMSO-d6): δ 166.0, 165.7, 164.1, 163.6, 151.4, 151.0, 148.6,
147.4, 133.9, 131.3, 130.9, 129.5, 129.2, 126.7, 126.4, 124.5, 122.9, 122.5, 118.9, 118.6, 116.7,
100.5, 100.2, 108.6, 30.6; FAB (Neg.)-MS m/z = 509 [M-H-H2O]-1; ESI-MS m/z = 511 [M+H-
H2O]+; HRESI-MS Calcd for C25H14Cl2O9 [M+H-H2O]+: m/z = 511.0093, Found 511.0062.
27

JOURNAL PRE-PROOF
3,3'-((3ʺ,5ʺ-Dibromo-4ʺ-hydroxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-
2-one) (35)
White Solid; Yield: 82%; M.p.: > 300 ºC dec.; 1H-NMR (300 MHz, DMSO-d6): δ 7.74 (d, J5,7/5',7'
= 2.4 Hz, 2H, H-5, H-5'), 7.55 (dd, J7,5/7',5' = 2.4 Hz, J7,8/7',8' = 6.3 Hz, 2H, H-7, H-7'), 7.32 (d,
13
J
8,7/8',7' = 8.7 Hz, 2H, H-8, H-8'), 7.13 (s, 2H, H-2'', H-6'' ), 6.13 (s, 1H, -CH-); C-NMR (100
MHz, DMSO-d6): δ 166.4, 166.4, 164.3, 164.3, 151.3,150.8, 150.8, 139.0, 134.5, 134.5, 131.0,
131.0, 129.5, 129.5, 126.8, 126.8, 122.9, 122.9, 118.8, 118.8, 110.3, 110.3, 100.2, 100.2, 35.2;
FAB (Neg.)-MS m/z = 651 [M-H]-1; 653 [M+2-H]-1; ESI-MS m/z = 653 [M+H]+; HRESI-MS
Calcd for C25H12Br2Cl2O7 [M+H]+: m/z = 652.8405, Found 652.8436.
3,3'-((2ʺ-Hydroxynaphthalen-1ʺ-yl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(36)
º
1
White Solid; Yield: 86%; M.p.: 252-254 C; H-NMR (300 MHz, DMSO-d6): δ 10.83 (s, 2H,
2OH), 9.13 (s, 1H, OH), 8.66 (d, J5′′,6′′/8′',7′' = 8.4 Hz, 2H, H-5′′, H-8′'), 8.32 (d, J4'',3'' = 9.3 Hz, 1H,
H-4''), 8.11 (d, J3'',4'' = 7.8 Hz, 1H, H-3''), 7.79 (t, J6'',5'' = J6'',7'' = 7.5 Hz, 1H, H-6''), 7.67 (m, 4H, -
CH-, H-5, H-5', H-7′′), 7.51 (dd, J7,5/7',5' = 2.4 Hz, J7,8/7',8' = 8.7 Hz, 2H, H-7, H-7'), 6.94 (d, J8,7/8',7'
13
= 8.7 Hz, 2H, H-8, H-8'); C-NMR (75 MHz, DMSO-d6): δ 190.4, 158.1, 156.9, 154.3, 139.6,
139.6, 135.1, 135.1, 134.2, 134.2, 129.9, 129.4, 129.4, 129.2, 129.0, 129.0, 128.8, 128.8, 127.5,
126.4, 125.7, 122.9, 122.5, 122.5, 118.9, 118.9, 116.5, 112.7, 28.6; FAB (Neg.)-MS m/z = 440
[M-H-2H2O-2Cl]-1; ESI-MS m/z = 529 [M+H-H2O]+; HRESI-MS Calcd for C29H16Cl2O7 [M+H-
H2O]+: m/z = 529.0245, Found 529.0268.
3,3'-((4ʺ-Hydroxy-3ʺ-iodo-5ʺ-methoxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-
chromen-2-one) (37)
White Solid; Yield: 80%; M.p.: 234-236 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.74 (d, J5,7/5',7'
=
2.7 Hz, 2H, H-5, H-5'), 7.54 (dd, J7,5/7',5' = 2.7 Hz, J7,8/7',8' = 6.3 Hz, 2H, H-7, H-7'), 7.31 (d, J8,7/8',7'
= 9.0 Hz, 2H, H-8, H-8'), 6.90 (bd.s, 1H, H-2''), 6.64 (bd.s, 1H, H-6''), 6.12 (s, 1H, -CH-), 3.57 (s,
3H, OCH3); 13C-NMR (75 MHz, DMSO-d6): δ 166.1, 166.1, 163.8, 163.8, 150.9, 150.9, 146.7,
143.9, 134.4, 130.7, 130.7, 127.8, 127.8, 127.2, 127.2, 123.1, 123.1, 121.0, 117.7, 117.7, 111.2,
104.0, 104.0, 84.1, 55.9, 35.4; FAB (Neg.)-MS m/z = 651 [M-H]-1, 653 [M+2-H]-1; ESI-MS m/z
= 653 [M+H]+; HRESI-MS Calcd for C26H16Cl2IO8 [M+H]+: m/z = 652.9267, Found 652.9280.
28

JOURNAL PRE-PROOF
3,3'-((5ʺ-Bromo-2ʺ-methoxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(38)
White Solid; Yield: 81%; M.p.: 251-253 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 7.73 (d, J5,7/5',7'
=
2.4 Hz, 2H, H-5, H-5'), 7.52 (dd, J7,5/7',5' = 2.7 Hz, J7,8/7',8' = 6.0 Hz, 2H, H-7, H-7'), 7.29 (d, J8,7/8',7'
= 8.7 Hz, 2H, H-8, H-8'), 7.23 (d, J3'',4'' = 6.9 Hz, 1H, H-3''), 7.21 (s, 1H, H-6''), 6.80 (d, J4'',3'' = 8.4
Hz, 1H, H-4''), 6.15 (s, 1H, -CH-), 3.53 (s, 1H, OCH3); 13C-NMR (100 MHz, DMSO-d6): δ 166.2,
166.0, 163.7, 163.5, 157.7, 150.7, 150.5, 134.6, 131.2, 131.0, 129.8, 129.6, 126.7, 126.5, 129.4,
118.8, 118.6, 115.5, 113.6, 100.3, 100.1, 123.4, 122.8, 122.6, 56.2, 29.7; FAB (Neg.)-MS m/z =
587 [M-H]-1, 589 [M+2-H]-1, 591 [M+4-H]-1; ESI-MS m/z = 599 [M+H]+; HRESI-MS Calcd for
C26H15BrCl2O7 [M+H]+: m/z = 588.9456, Found 588.9478.
3,3'-((2ʺ,5ʺ-Dihydroxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one) (39)
White Solid; Yield: 76%; M.p.: 274-276 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 9.37 (s, 1H, OH),
8.07 (d, J5,7 = 2.1 Hz, 1H, H-5), 8.00 (bd.s, 1H, H-5'), 7.74 (dd, J7,5 = 2.4 Hz, J7,8 = 6.3 Hz, 1H,
H-7), 7.62 (d, J7',8' = 7.5 Hz, 1H, H-7'), 7.49 (d, J8,7 = 8.7 Hz, 1H, H-8), 7.35 (d, J8',7' = 8.7 Hz, 1H,
H-8'), 7.21 (d, J3'',4'' = 8.7 Hz, 1H, H-3''), 6.67 (d, J4'',3'' = 6.0 Hz, 1H, H-4''), 6.58 (bd.s, 1H, H-6''),
13
5.62 (bd.s, 1H, -CH-); C-NMR (100 MHz, DMSO-d6): δ 166.2, 165.9, 164.3, 164.0, 151.3,
151.0, 149.2, 148.7, 131.4, 131.1, 129.3, 129.0, 126.5, 126.2, 124.3, 122.7, 122.5, 118.7, 118.4,
117.2, 116.7, 114.6, 104.6, 104.3, 30.1; FAB (Neg.)-MS m/z = 493 [M-H-H2O]-1; 495 [M+2-H]-
1; ESI-MS m/z = 495 [M+H]+; HRESI-MS Calcd for C25H14Cl2O8 [M+H]+: m/z = 495.0038,
Found 495.0065.
3,3'-((3ʺ-Bromo-4ʺ-hydroxyphenyl)methylene)bis(6-chloro-4-hydroxy-2H-chromen-2-one)
(40)
White Solid; Yield: 75%; M.p.: 248-250 ºC; 1H-NMR (300 MHz, DMSO-d6): δ 9.84 (bd.s, 2H,
2OH),7.73 (d, J5,7/5',7' = 2.4 Hz, 2H, H-5, H-5'), 7.55 (dd, J7,5/7',5' = 2.7 Hz, J7,8/7',8' = 6.0 Hz, 2H,
H-7, H-7'), 7.31 (d, J8,7/8',7' = 8.7 Hz, 2H, H-8, H-8'), 7.06 (bd.s, 1H, H-2''), 6.88 (d, J6'',5'' = 8.7 Hz,
1H, H-6''), 6.76 (d, J5'',6'' = 8.4 Hz, 1H, H-5''), 6.11 (s, 1H, -CH-); 13C-NMR (100 MHz, DMSO-
d6): δ 166.1, 166.1, 163.9, 163.9, 151.5, 151.5, 151.0, 133.5, 133.5, 130.8, 130.5, 127.2, 127.2,
127.1, 123.2, 123.2, 121.0, 121.0, 117.7, 117.7, 116.0, 108.7, 103.9, 103.9, 35.2; FAB (Neg.)-MS
m/z = 573 [M-H]-1, 575 [M+2-H]-1; ESI-MS m/z = 575 [M+H]+; HRESI-MS Calcd for
C25H13BrCl2O7 [M+H]+: m/z = 574.9300, Found 574.9326.
29