Discovery of novel 3-(hydroxyalkoxy)-2-alkylchromen-4-one analogs as interleukin-5 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.07.069

A series of novel chromen-4-one analogs 9a-d and 10a-u was designed, synthesized and evaluated for their IL-5 inhibitory activity. Most of the chromen-4-one analogs showed strong inhibitory activity in low micro molar potency. Among them, 5-(cyclohexylmet

Full text of DOI:10.1016/j.ejmech.2017.07.069

Accepted Manuscript
Discovery of novel 3-(hydroxyalkoxy)-2-alkylchromen-4-one analogs as interleukin-5
inhibitors
Pulla Reddy Boggu, Eeda Venkateswararao, Manoj Manickam, Youngsoo Kim, Sang-
Hun Jung
PII:
S0223-5234(17)30591-3
10.1016/j.ejmech.2017.07.069
EJMECH 9632
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 February 2017
Revised Date: 24 July 2017
Accepted Date: 28 July 2017
Please cite this article as: P.R. Boggu, E. Venkateswararao, M. Manickam, Y. Kim, S.-H. Jung,
Discovery of novel 3-(hydroxyalkoxy)-2-alkylchromen-4-one analogs as interleukin-5 inhibitors,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.069.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Discovery of novel 3-(hydroxyalkoxy)-2-alkylchromen-4-one analogs as interleukin-5
inhibitors

ACCEPTED MANUSCRIPT
Discovery of novel 3-(hydroxyalkoxy)-2-alkylchromen-4-one analogs as interleukin-5
inhibitors
Pulla Reddy Boggua, Eeda Venkateswararaoa, Manoj Manickama, Youngsoo Kimb, Sang-Hun
Junga*
a College of Pharmacy and Institute of Drug Research and Development, Chungnam
National University, Daejeon 34134, Republic of Korea.
b College of Pharmacy, Chungbuk National University, Cheongju 19421, Republic of Korea.
Abstract
A series of novel chromen-4-one analogs 9a-d and 10a-u was designed, synthesized and
evaluated for their IL-5 inhibitory activity. Most of the chromen-4-one analogs showed
strong inhibitory activity in low micro molar potency. Among them, 5-(cyclohexylmethoxy)-
3-(3-hydroxypropoxy)-2-isopropyl-4H-chromen-4-one (10t, 90.0% inhibition at 30 µM, IC50
=
5.5 µM, CLogP
=
4.76887) and 2-cyclohexyl-5-(cyclohexylmethoxy)-3-(3-
hydroxypropoxy)-4H-chromen-4-one (10u, 95.5% inhibition at 30 µM, IC50 = 3.0 µM,
CLogP = 5.96187) showed the best inhibition. The structure activity relationship reveals that
the hydrophobic cyclohexylmethoxy group at the position 5 of the chromen-4-one ring A is
preferable than at position 6 and the dual hydrogen bonding acceptor property on the
chromen-4-one ring should be important for the inhibitory activity. In addition, the optimum
length of the side chain at position 3 of chromen-4-one ring is critical for the donation of
hydrogen to the binding site and the 3-hydroxypropoxy group showed the best activity.
Moreover, the conformational restrictor (isopropyl, cyclohexyl group) at position 2 is much
more favorable for the formation of effective conformer of side chain with hydrogen bonding
donor property of these chromen-4-one analogs.
Key words: Interleukin-5; inhibitor; chromen-4-one; SAR

ACCEPTED MANUSCRIPT
*Corresponding author: jungshh@cnu.ac.kr
Present address: College of Pharmacy and Institute of Drug Research and Development,
Chungnam National University, Daejeon 34134, Republic of Korea.
1. Introduction
Allergic diseases, such as nasal rhinitis, asthma, idiopathic eosinophilic syndromes and atopic
dermatitis, have prominent inflammatory components that are characterized by eosinophilic
inflammation [1]. Thus, the major role of chronic pulmonary inflammation in the
pathophysiology of asthma has been studied widely in human and in animal models. In
asthma, pulmonary inflammation is characterized by edema, decreased mucociliary apparatus,
bronchoalveolar eosinophilia [1]. The eosinophil is one of the bone marrow-derived
leukocytes and is usually found in large numbers of tissues such as the bone marrow and
gastrointestinal tract. Its normal level represents less than 5% of leukocytes in the blood [2].
The unusual level of proliferation of eosinophil causes many diseases including allergies [3],
eosinophilic gastrointestinal disorders [4] and hyper-eosinophilic syndrome [5].
The development of eosinophil is being regulated mainly by the three cytokines such as
interleukin (IL)-3, IL-5 and granulocyte macrophage colony stimulating factor (GM-CSF)
[6–9]. These eosinophilopoietins likely provide permissive proliferation and differentiation
signals following the instructive signals specified by the transcription factors GATA-1, PU.1,
and C/EBPs. These cytokines are encoded by closely linked genes on chromosome 5q31. The
receptors that share a common beta chain and have unique alpha chains were bound to the
above cytokines [10,11]. Among them, IL-5 is one of the most specific to the eosinophil
lineage and is responsible for selective differentiation of eosinophils [12]. IL-5 also
stimulates the release of eosinophils from the bone marrow into the peripheral circulation

ACCEPTED MANUSCRIPT
[13]. The critical role of IL-5 in the production of eosinophils is best demonstrated by genetic
manipulation of mice. Overproduction and deletion of IL-5 in transgenic mice result in
profound eosinophilia and a marked reduction of eosinophils in the blood and lungs after
allergen challenge [14,15]. The overproduction of one or a combination of these three
cytokines occurs in humans with eosinophilia and diseases with selective eosinophilia are
often accompanied by overproduction of IL-5 [16]. Several clinical trials with humanized
anti-IL-5 antibody demonstrated the critical role of IL-5 in regulating eosinophils in humans.
These antibodies can reduce blood and bronchoalveolar eosinophilia caused by allergic
challenges or chronic diseases [17–19]. Therefore, exclusively inhibiting the actions of IL-5
can suppress at least one of the alleged causes of asthma, namely tissue damage due to the
eosinophil accumulation during pulmonary inflammation. Moreover, various clinical
investigations indicate that the IL-5-regulated eosinophilia plays a key role in the
pathogenesis of asthma.
The above reports clearly confirm that IL-5 is emerging as an important target for the
treatment of asthma. Among them, small organic isothiazolone analogs have been prepared
by the modification of Cys 66 residue in IL-5 and reported as its antagonists [20].
Nevertheless, the most significant discovery is the natural product sophoricoside and its
analogs (1, Figure 1) which showed a strong inhibitory effect in the IL-5 bioassay of mIL-5-
dependent Y16 proliferation [21]. Interestingly, it was also reported as a weak inhibition on
IL-3 and GM-CSF bioactivities [22]. However, sophoricoside contains the unstable glycoside
unit, thereby the structure activity relationship (SAR) studies are needed for the exploration
of more potent analog with drug-likeness property. Accordingly, a number of isoflavonoid
analogs (2, Figure 1) of sophoricoside [23,24] as well as chalcones [25] (3, Figure 1) were
investigated as potent IL-5 inhibitors by our group. The SAR studies of isoflavonoid analogs
revealed that planar chromen-4-one ring and a phenolic hydroxyl group at position 4 on

ACCEPTED MANUSCRIPT
phenyl ring B particularly play an important role in the inhibitory activity. However, the
glucopyranosyl component of sophoricoside may not be an essential motif for the innate
activity of these analogs as the weak glycosidic linkage of isoflavone glycoside is chemically
cleaved under metabolic conditions [24,26]. To further confirm the role of ring B, a number
of novel chromen-4-one analogs (4, Figure 1) with the insertion of one methylene motif
between chromen-4-one and phenyl ring were investigated for the IL-5 inhibitory activity
[27]. These modifications well retained the level of activity of isoflavones. In order to obtain
a deeper understanding of the effect of B-ring in isoflavone-type analogs, the amino alcohol
group was introduced as shown in 5 (Figure 1) at position 3 instead of phenyl ring [28]. The
outcome indicated that the replacement of the 4-hydroxyphenyl ring by
hydroxyethylaminomethyl motif was suitable for maintaining the inhibitory activity of
isoflavanone analogs. In addition, the results of this study also suggested that the existence of
a bulky hydrophobic cyclohexyl methyl group as a side chain in the amino ethanol moiety
(5a, Figure 1) is crucial for the IL-5 inhibitory activity [28]. Having understood some
principal structural characteristics that enhanced the inhibitory activity of IL-5, a series of
novel N-substituted hydroxyethylaminomethylchromone derivatives have been explored as
IL-5 inhibitors [29]. The results suggested that by increasing the hydrophobicity or bulkiness
at amino group the activity against IL-5 inhibition was intensified (6, Figure 1). In addition,
all N-benzyl analogs have presented the same level of activity regardless the type of
substituents at para-position. Furthermore, based on the conformational studies, the insertion
of a hydrophobic group on the nitrogen seems to enforce the more effective conformer of
hydroxyethylaminomethyl side chain, consequently increasing the activity of these analogs
[29]. These results proved that phenyl ring B of 1 is not necessary for the activity. Recently,
we investigated chromen-4-ones with a chalcone motif (hybrid chromen-4-one 7, Figure 1)

ACCEPTED MANUSCRIPT
and their saturated analogs (8, Figure 1). These results indicated that saturated alcohols 8
showed more potent than the unsaturated analogs 7 [30,31].
Therefore, considering all these structural characteristics of isoflavones and amino alcohol
motif of chromen-4-ones, we have rationally designed the target molecule by insertion of
additional oxygen atom near to the carbonyl group at position 3 of the chromen-4-one ring
with conformational restrictor at position 2 which can lead to more active analogs (Figure 2).
Additionally, we planned to explore the location of a hydrophobic group in chromen-4-one
ring A and study the role of phenyl ring B (Figure 2). Accordingly, a series of novel
chromen-4-one analogs 9 and 10 were synthesized and evaluated for their IL-5 inhibitory
activity.
Figure 1. The representative IL-5 inhibitors.

ACCEPTED MANUSCRIPT
Figure 2. Design of novel chromen-4-one analogs 9 and 10 with dual HBA as IL-5
inhibitors.
2. Chemistry
The novel series of chromen-4-one analogs (9a-d and 10a-u, Table 1) were synthesized from
commercially available 2ʹ,6ʹ-dihydroxyacetophenone (11a) and 2ʹ,5ʹ-dihydroxyacetophenone
(11b) in six steps as depicted in Schemes 1 - 5.
In brief, synthesis of the substituted acetophenone compounds 12 was accomplished by the
partial alkylation of 11a or 11b. Compounds 13 were prepared with excellent yields by the
aldol condensation of appropriate substituted acetophenone 12 with N, N-dimethylformamide
dimethylacetal (DMF-DMA) at reflux temperature, which were being converted to
appropriate cyclized chromen-4-one compounds 14 by treatment with concentrated
hydrochloric acid in dichloromethane at reflux temperature (Scheme 1). To obtain key
intermediate epoxide 15, initial treatment of 14a with H2O2 using 10% NaOH in ethanol/1,4-
dioxane yielded 16 as the result of oxidative cleavage of the chromen-4-one ring [32]. To
overcome this problem, mild oxidizing condition, viz., meta-chloroperbenzoic acid (m-
CPBA) in ethanol/dichloromethane (EtOH/DCM) (1:1) at room temperature was adopted to

ACCEPTED MANUSCRIPT
get the desired compounds 15. The epoxides 15 were reacted with concentrated hydrochloric
acid in acetone at room temperature to give 3-hydroxychromenones 9, which were further
reacted with the corresponding alkyl or benzyl halide in the presence of K2CO3 in N, N-
dimethylformamide (DMF) at 60 °C to obtain the final chromen-4-one compounds 10
(Schemes 1, 4 and 5). The intermediate bromoalcohol 17a was prepared from bromo ester 17
by reduction with diisobutylaluminium hydride (DIBAL-H) in high yield (Scheme 1).
O
O
OH
OH
(g)
16
O
O
O
O
O
HO
R1
O
R1
O
R1
O
R1
O
(a)
(b)
(c)
(d)
N
O
OH
O
OH
O
OH
11a-b
13a-d
14a-d
15a-d
12a-d
O
O
O
(h)
R1
O
R1
O
Br
Br
O
OH
(e)
(f)
R2
O
HO
O
O
17
17a
9a-d
10a, 10c, and 10e-q
Scheme 1. Synthesis of chromen-4-one derivatives 9a-d, 10a, 10c and 10e-q (substituents are
listed in Table 1). Reagents and Conditions: (a) R1-Br, K2CO3, CH3CN/DMF (1:1), 80 °C, 30
h, 70 - 76%; (b) DMF-DMA, reflux, 3 h, 85 - 90%; (c) DCM, Conc.HCl, reflux, 2 h, 85 -
92%; (d) mCPBA, EtOH/DCM (1:1), rt, 24 – 72 h, 35 - 45%; (e) Acetone, Conc.HCl, rt, 16
h, 70 - 78%; (f) R2-X, K2CO3, DMF, rt – 60 °C, 4-16 h, 25 - 80%; (g) 10% NaOH, 35%
H2O2, 1,4-dioxane/EtOH (1:1), 5 °C to rt, 16 h, 47%; (h) DIBAL-H, DCM, 0 °C to rt, 1 h,
75%.
In order to prepare compound 10b, methyl 4-((5-(cyclohexylmethoxy)-4-oxo-4H-chromen-3-
yloxy)methyl)benzoate (10c) was hydrolyzed with LiOH in THF/H2O mixture at reflux
temperature which gave the acid compound 10b in good yield (Scheme 2).

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of chromen-4-one derivative 10b. Reagents and Conditions: (a)
LiOH.H2O, THF/H2O (3:1), reflux, 5 h, 72%.
Analog 10d was prepared from the 4-hydroxybenzaldehyde 18 as depicted in Scheme 3. 18
was converted to intermediate 19 by reacting with chloromethyl methyl ether (MOM-Cl) in
the presence of sodium hydride (60%, dispersion in paraffin liquid) in DMF solution at 0 °C
for 30 min. Subsequently, 19 was transformed into alcohol 20 by reaction with NaBH4 in
methanol solution at 0 °C for 30 min followed by mesylation with methane sulfonyl chloride
and triethylamine in dichloromethane at 0 °C to room temperature resulted in 21. The
compound 21 was further reacted with 9a in the presence of K2CO3 and DMF at 20 °C to
afford 10d.
Scheme 3. Synthesis of chromen-4-one derivative 10d. Reagents and Conditions: (a) MOM-
Cl, 60% NaH, DMF, 0 °C, 30 min, 92%; (b) NaBH4, methanol, 0 °C, 30 min, 90%; (c)
CH3SO2Cl, TEA, DCM, 0 °C to rt, 1 h, 85%; (d) Compound 9a, K2CO3, DMF, 20 °C, 24 h,
55%.
Compound 10r with methyl substitution at position 2 was prepared as illustrated in Scheme
4. To obtain intermediate 24, 2ʹ,6ʹ-dihydroxyacetophenone (11a) was reacted with sodium

ACCEPTED MANUSCRIPT
metal in ethyl acetate to give 22, and then cyclized by treatment with concentrated
hydrochloric acid in methanol at reflux temperature followed by alkylation with
(bromomethyl)cyclohexane and K2CO3 in DMF at 60 °C. Compound 24 was converted to
chromen-4-one 10r in a manner similar to the preparation of 10 from chromen-4-one
intermediate 14.
Scheme 4. Synthesis of 2-methyl chromen-4-one derivative 10r. Reagents and Conditions:
(a) Na, EtOAc, rt, 18 h; (b) MeOH, Conc.HCl, 60 °C, 2 h, 75%; (c) Cy-CH2-Br, K2CO3,
DMF, 60 °C, 16 h, 65%; (d) mCPBA, EtOH/DCM (1:1), rt, 24 h, 32%; (e) Acetone,
Conc.HCl, rt, 16 h, 68%; (f) OH-(CH2)3-Br, K2CO3, DMF, 60 °C, 16 h, 55%.
2-Substituted chromen-4-one analogs 10s-u were prepared as shown in Scheme 5. Starting
with 1-(2-(cyclohexylmethoxy)-6-hydroxyphenyl)ethanone (12a), reaction with appropriate
acid chloride gave ester compounds 25a-c which were subsequently treated with NaH (60%,
dispersion in paraffin liquid) in tetrahydrofuran at reflux temperature yields diketone
compounds 26a-c respectively. The diketone compounds 26a-c were cyclized using
concentrated hydrochloric acid in methanol to give cyclized intermediates 27a-c accordingly,
which were in turn used for the preparation of 10s-u in a manner similar to the preparation of
10 from chromen-4-one intermediate 14.

ACCEPTED MANUSCRIPT
O
O
O
O
O
O
O
O
O
O
O
O
O
(a)
(b)
(c)
(d)
R3
O
R3
OH
O
R3
OH
12a
25a-c
26a-c
27a-c
15f-h
O
R3
(g)
16
O
O
O
O
O
O
OH
OH
R3
(e)
(f)
R3
O
9f-h
10s-u
Scheme 5. Synthesis of 2-substituted chromen-4-one derivatives 10s-u (substituents are listed
in Table 1). Reagents and Conditions: (a) R3-CO-Cl, TEA, MC, 0 °C, 1 h, 90 - 95%; (b) 60%
NaH, THF, 60 °C, 2 h, 80 - 85%; (c) MeOH, Conc.HCl, 60 °C, 2 h, 70 - 86%; (d) mCPBA,
EtOH/DCM (1:1), rt, 24 – 72 h, 20 - 22%; (e) Acetone, Conc.HCl, rt, 16 h, 50 - 65%; (f) OH-
(CH2)3-Br, K2CO3, DMF, 60 °C, 4 -16 h, 45 - 55%; (g) 10% NaOH, 35% H2O2, 1,4-
dioxane/EtOH (1:1), 5 °C to rt, 16 h, 47%.
3. Pharmacology
The inhibitory activity of chromen-4-one analogs 9a-d and 10a-u against IL-5 was evaluated
using the IL-5 dependent pro-B Y16 cell line according to previously reported procedure
[21]. These cells were incubated with 3 units/mL mIL-5 for 48 h in the presence or absence
of the sample, and then cell metabolism was measured as an index of proliferation, using 2-
(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium sodium salt (WST-
1). The results of biological screening of chromen-4-one analogs against IL-5 are listed in
Table 1 as % inhibition at 30 µM and also IC50 values. The detailed assay protocols are
described in the experimental section.
Table 1. IL-5 inhibitory activity of chromen-4-one analogs 9a-d and 10a-u.
O
R1 O
O
R2
O
R3
9a-d
10a-u
and

ACCEPTED MANUSCRIPT
Substituents
IL-5
% Inhibition
at 30 µMb
68.2
aCLogP
Comp. No.
bIC50
(µM)
16.0
16.0
29.0
20.0
R1
R2
R3
5- CH2-Cyc
H
H
H
H
H
H
H
H
3.91755
3.25355
3.91755
3.25355
9a
9b
9c
9d
5-(CH2)2-CH(CH3)2
6- CH2-Cy
70.0
56.5
6-(CH2)2-CH(CH3)2
62.3
5- CH2-Cy
5- CH2-Cy
H
H
4.95087
5.73187
89.0
86.4
11.0
17.0
10a
10b
5- CH2-Cy
H
5.95787
84.0
16.0
10c
O
O
5- CH2-Cy
5- CH2-Cy
H
H
5.41447
5.90787
35.5
85.3
>30.0
20.0
10d
10e
5- CH2-Cy
5- CH2-Cy
5- CH2-Cy
H
H
H
5.98887
4.49187
6.70187
52.0
60.1
30.0
27.0
24.0
10f
10g
10h
>30.0
5- CH2-Cy
H
5.73187
86.0
13.0
10i
5- CH2-Cy
5- CH2-Cy
5- CH2-Cy
H
H
H
3.74187
3.34788
3.82088
90.6
79.2
15.5
10.0
17.0
10j
10k
10l
>30.0

ACCEPTED MANUSCRIPT
5- CH2-Cy
H
4.34987
3.07788
2.68387
3.74187
3.07788
4.24088
12.2
76.0
55.4
>30.0
14.0
10m
10n
10o
5-(CH2)2-CH(CH3)2
5-(CH2)2-CH(CH3)2
H
H
25.0
6- CH2-Cy
H
36.0
34.0
90.5
88.5
90.0
95.5
79.1d
70.2d
>30.0
>30.0
7.5
10p
10q
6-(CH2)2-CH(CH3)2
5- CH2-Cy
H
-CH3
10r
5- CH2-Cy
-(CH2)2CH3 5.29887
14.0
5.5
10s
5- CH2-Cy
-CH(CH3)2
-Cy
4.76887
5.96187
10t
5- CH2-Cy
3.0
10u
10.6
26.2
Sophoricoside
Budesonide
a ClogP values are calculated by Chemdraw® (ver. 11).
b % Inhibition and IC50 values are taken as a mean from three independent experiments.
c Cy = Cyclohexyl
d Inhibition at 50 µM.
4. Conformational analysis and alignment
Molecular models of the chromen-4-one analogs 10j and 10u were constructed using
SYBYL®-x2.0 program package (Tripos Associates Inc.) [33] and their geometry were
optimized (Powell conjugate gradient minimization, termination at a gradient of 0.0005
kcal/mol) [34] using the Tripos standard force field and Gasteiger-Huckel atomic partial
charges. All the molecules were aligned by an atom-by-atom least-square fit and used the
chromen-4-one structure as a template as represented. The 3D structures of the analyzed
chromen-4-one compounds were assumed to be a bioactive conformation and were aligned

ACCEPTED MANUSCRIPT
according to a chromen-4-one template as shown in Figure 3. The selected dihedral angles
and atomic distances are listed in Table 2 and 3 respectively.
Figure 3. (A) Alignment of 10j (Green color) and 10u (Salmon color); (B) Distance (Å)
calculation in 10j (Green color); (C) Distance (Å) calculation in 10u (Salmon color).
Table 2. Torsion angle (°) and total energy (kcal/mol) of 10j and 10u.
Compound 10j (14.620 kcal/mol) Compound 10u (18.086 kcal/mol)
∠C3-O8-C9-C10
∠C4-C3-O8-C9
∠C3-O8-C9-C10
∠C4-C3-O8-C9
179.8
180.3
179.6
359.8
a Numbers on the atoms of 10j and 10u are presented in Figure 3
Table 3. Distance (Å) in 10j and 10u.
Compound 10j
Compound 10u
2.74 O7-O8
O7-O8
2.91

ACCEPTED MANUSCRIPT
O7-O12
O8-O12
7.21 O7-O12
4.83 O8-O12
6.17
4.83
a Numbers on the atoms of 10j and 10u are presented in Figure 3
5. Results and discussion
For determining more detail structure activity relationship studies, we designed and
synthesized a number of novel chromen-4-one analogs (9a-d and 10a-u) by the introduction
of dual hydrogen bonding property on chromen-4-one ring and studied them for their IL-5
inhibitory activity as summarized in Table 1.
Initially, 3-hydroxychromenone analogs were prepared and studied for their IL-5 inhibitory
activity. Analog 9a (68.2% inhibition at 30 µM, IC50 = 16.0 µM) showed moderate inhibitory
activity. This result indicates that the addition of hydrogen bonding property (HBA and
HBD) on chromen-4-one ring should be favorable for the activity. Encouraged by these
results, our next effort was to confirm the importance of hydrophobic group on chromen-4-
one ring A. Therefore, we replaced cyclohexylmethoxy group with isopentoxy group as
shown in analog 9b (70.0% inhibition at 30 µM, IC50 = 16.0 µM) exhibited the same level of
activity. However, changing the position of the hydrophobic group on the chromen-4-one
ring A from 5 to 6 as shown in analogs 9c (56.5% inhibition at 30 µM, IC50 = 29.0 µM) and
9d (62.3% inhibition at 30 µM, IC50 = 20.0 µM) slightly decreases the activity. These results
suggest that both groups (cyclohexylmethoxy and isopentoxy) at position 5 are acceptable for
the hydrophobic interactions.
To get the better interactions with the binding site, we introduced alkyl groups with hydrogen
bonding property at position 3 of 9a as shown in analog 10a (4-hydroxymethylbenzyloxy
group, 89.0% inhibition at 30 µM, IC50 = 11.0 µM). Surprisingly, 10a showed the 2.5 fold
stronger inhibitory activity against IL-5 as compared to budesonide (70.2% inhibition at 30

ACCEPTED MANUSCRIPT
µM, IC50 = 26.2 µM) and similar activity as compared to sophoricoside (79.1% inhibition at
30 µM, IC50 = 10.6 µM). Carboxylic acid compound 10b (86.4% inhibition at 30 µM, IC50 =
17.0 µM) and ester compound 10c (84.0% inhibition at 30 µM, IC50 = 16.0 µM) showed the
similar level of the activity. These results suggest that side chain with hydrogen bonding
enhances the activity.
Next, to find out the importance of hydrogen bonding property (HBD or HBA) on phenyl
ring, hydroxymethyl group in 10a was replaced with electron donating groups capable to be a
mild hydrogen bond acceptor such as methoxymethoxy analog 10d (35.5% inhibition at 30
µM, IC50 = >30.0 µM) and 4-methoxy analog 10e (85.3% inhibition at 30 µM, IC50 = 20.0
µM). Analog 10d dramatically reduces the activity and analog 10e showed less activity than
10a. This indicates that the methoxymethoxy group may be too large to fit into putative
binding pocket regardless of its hydrogen bond acceptor property.
Removal of hydroxymethyl group of 10a as shown in 10f (52.0% inhibition at 30 µM, IC50 =
27.0 µM) largely decreased the activity and the pyridine analog 10g (60.1% inhibition at 30
µM, IC50 = 24.0 µM) did not improve the activity. Chloro analog 10h (30.0% inhibition at 30
µM, IC50 = >30.0 µM) nearly abolished the activity. However, nitro analog 10i (86.0%
inhibition at 30 µM, IC50 = 13.0 µM) showed fairly strong inhibition. These results indicate
that the hydrogen bonding acceptor at the end of the side chain at position 3 of chromen-4-
one 9a is very much beneficial to the activity.
In the next set of experiments, to confirm the role of phenyl ring B of chromen-4-one analog
10a for their IL-5 inhibitory activity, we synthesized a series of simple hydroxyalkyl groups
having similar hydrogen bonding property. Accordingly, analog 10j (90.6% inhibition at 30
µM, IC50 = 10.0 µM, CLog P = 3.74187) gave an almost similar inhibitory activity as
compared to sophoricoside (79.1% inhibition at 30 µM, IC50 = 10.6 µM) and 10a (89.0%

ACCEPTED MANUSCRIPT
inhibition at 30 µM, IC50 = 11.0 µM). This suggests that the phenyl ring B of 10a can be
replaced by simple alkyl group to maintain the activity of chromen-4-one.
Next, to confirm the optimum length of the 3-hydroxypropyl moiety, methylene group units
were variated between oxygen (O) at position 3 and hydroxyl (OH) function in 10j. As a
result, decrement in the activity was observed in the case of two methylene unit analog 10k
(79.2% inhibition at 30 µM, IC50 = 17.0 µM) and complete loss of activity was observed in
four and five methylene unit analogs 10l (15.5% inhibition at 30 µM, IC50 = >30.0 µM) and
10m (12.2% inhibition at 30 µM, IC50 = >30.0 µM). This outcome supports that length of
hydroxyalkyl side chain plays a critical role in inhibitory activity against IL-5 of chromen-4-
one analogs and hydroxypropyl group is optimum.
Further, to confirm the importance of hydrophobic group on the chromen-4-one ring A with
hydroxyalkoxy at position 3, replacement of cyclohexylmethoxy group with the isopentyloxy
group as shown in 10n (76.0% inhibition at 30 µM, IC50 = 14.0 µM) and 10o (55.4%
inhibition at 30 µM, IC50 = 25.0 µM) was performed. However, these analogs did not
improve the IL-5 inhibitory activity. Thus, the cyclohexylmethoxy moiety on ring A of
chromen-4-one is an essential group for the activity.
In order to find out the suitable position of the hydrophobic group on the chromen-4-one ring
A of 10j and 10n, the location of these groups was changed from position 5 to 6 as shown in
analogs 10p (36.0% inhibition at 30 µM, IC50 = >30.0 µM) and 10q (34.0% inhibition at 30
µM, IC50 = >30.0 µM). This variation remarkably reduced the activity. Therefore, these
results proved that the hydrophobic group at position 5 of chromen-4-one ring is much more
preferable for the hydrophobic interactions.
Next objective of our work was to study on position 2 of chromen-4-one ring of 10j.
Introduction of the methyl group as a conformational restrictor at position 2 of 10j as shown

ACCEPTED MANUSCRIPT
in 10r (90.5% inhibition at 30 µM, IC50 = 7.5 µM) resulted in remarkable enhancement in the
activity. This implies that the conformational restrictor at position 2 should enforce the
formation of effective conformer of hydroxypropoxy side chain at position 3 of these
chromen-4-one analogs. Considering conformational restrictor at position 2 of the chromen-
4-one ring, we further investigated that the size of the group and therefore analogs 10s, 10t
and 10u were prepared. Among them, n-propyl analog 10s (88.5% inhibition at 30 µM, IC50
= 14.0 µM) decreases the activity. Isopropyl analog 10t (90.0 % inhibition at 30 µM, IC50 =
5.5 µM) and cyclohexyl analog 10u (95.5% inhibition at 30 µM, IC50 = 3.0 µM) showed
more potent activity against IL-5 inhibition. These results indicated that the bulky alkyl
groups at position 2 as a conformational restrictor is more favorable to IL-5 inhibitory
activity compared to a straight chain. Lipophilicity of analogs 10j-u as indicated with cLogP
(Table 1) does not correlate with the activity. Increasing the size of hydrophobic binding
ability of substituent at position 2 is important for the enhancement of the activity.
To investigate the effective conformations for the potent IL-5 inhibitor, we compared the 3D
structural sketches of unsubstituted analog 10j and highly active cyclohexyl analog 10u at
position 2 as shown in Figure 3 and Table 3 and observed a remarkable difference in the
region of side chain at position 3 of chromen-4-one. As mentioned in Table 2, the dihedral
angles (∠C4-C3-O8-C9) of analog 10j is 180.3°, which indicates that the side chain of
hydroxypropoxy group at position 3 of 10j is located away from the chromen-4-one carbonyl
group. The distance (O7-O12 = 7.21 Å, Figure 3 and Table 3) from the oxygen of chromen-4-
one ring carbonyl group to the oxygen of hydroxypropyl group of 10j also confirms this
conformation. The corresponding dihedral angle (∠C4-C3-O8-C9, Table 2) of 10u is 359.8°,
which depicts that the hydroxypropyl chain is close to carbonyl of chromen-4-one. Therefore
the shorter distance (O7-O12 = 6.17 Å, Figure 3 and Table 3) from the oxygen of chromen-4-

ACCEPTED MANUSCRIPT
one ring carbonyl group to the oxygen of hydroxypropyl group of 10u compared to that of
10j obviously indicates the folded conformation. Therefore, introduction of conformational
restrictor at position 2 might exert the favorable effect for the formation of more effective
conformer of hydrogen bonding capable hydroxypropyl side chain of these chromen-4-one
analogs and thus enhance the activity.
Figure 4. SAR of the novel chromen-4-one analogs.
6. Conclusion
In summary, a novel series of chromen-4-one analogs 9a-d and 10a-u were synthesized and
evaluated for their IL-5 inhibitory activity. Most of the chromen-4-one analogs showed
strong inhibitory activity in low micro molar potency. Among them, 5-(cyclohexylmethoxy)-
3-(3-hydroxypropoxy)-2-methyl-4H-chromen-4-one (10r, 90.5% inhibition at 30 µM, IC50 =
7.5 µM, CLogP = 4.24088), 5-(cyclohexylmethoxy)-3-(3-hydroxypropoxy)-2-isopropyl-4H-
chromen-4-one (10t, 90.0% inhibition at 30 µM, IC50 = 5.5 µM, ClogP = 4.76887) and 2-
cyclohexyl-5-(cyclohexylmethoxy)-3-(3-hydroxypropoxy)-4H-chromen-4-one (10u, 95.5%
inhibition at 30 µM, IC50 = 3.0 µM, ClogP = 5.96187) showed highly potent inhibitory
activity against IL-5. The structure activity relationship (Figure 4) revealed that the
hydrophobic cyclohexylmethoxy group at position 5 of the chromen-4-one ring A is more

ACCEPTED MANUSCRIPT
preferable than position 6 and the dual hydrogen bonding acceptor property on the chromen-
4-one ring should be important for the inhibitory activity. In addition to that, the optimum
length of the methylene unit (3-hydroxypropoxy group) is critical for the donation of
hydrogen to the binding site. Moreover, the conformational restrictor (isopropyl, cyclohexyl
group) at position 2 is much more favorable for the formation of effective conformer of the
side chain (3-hydroxypropoxy) hydrogen bonding donor property at position 3 of these
chromen-4-one analogs. These results strongly suggest that novel 3-alkoxychromen-4-one
analogs can be promising antagonists of IL-5 for the most efficacious treatment of allergic
inflammation like asthma.
7. Experimental section
7.1. Chemistry
Melting points (mp) were determined on an Electro thermal 1A 9100 MK2 apparatus and are
uncorrected. All commercial chemicals were used as obtained and all solvents were purified
by distillation prior to use applying the standard procedures [35]. Thin layer chromatography
(TLC) was performed on E Merck silica gel GF-254 pre-coated plates; identification was
performed under UV illumination (λ = 254 nm), and colorization with Iodine and KMnO4.
All compounds were purified by flash column chromatography which was performed on E
Merck silica gel (230–400 mesh). Infrared (IR) spectra were recorded on a Nicolet 380 model
1
13
FTIR. H NMR and C NMR spectra were measured against the peak of tetramethylsilane
using a Bruker Fourier 300 NMR (300 MHz) and JEOL, JNM-AL400 NMR (400 MHz)
spectrometer. High-resolution mass spectra (HRMS) were measured in ESI ionization using
AB Sciex Triple TOF 5600 LCMS instrument.
7.1.1. General synthetic procedure for the preparation of compounds 9.

ACCEPTED MANUSCRIPT
To a solution of the corresponding epoxide compound 15 (1.46 mmol) in acetone (1 mL),
conc. HCl (2 mL) was added and the resulting mixture was stirred for 16 h at room
temperature. The reaction mixture was diluted with water, and then extracted with EtOAc.
The combined organic layers were washed with saturated aqueous NaHCO3 solution, dried
over anhydrous Na2SO4, filtered and then concentrated to afford 3-hydroxy chromenone
compound 9. The residue was subjected to flash silica gel (230-400 mesh) column
chromatography (eluting with 0-5% ethyl acetate in hexanes) to afford the title compounds 9.
7.1.1.1. 5-(Cyclohexylmethoxy)-3-hydroxy-4H-chromen-4-one (9a). Yield 75%; White solid;
mp 112 - 115 °C; IR (neat) 3296, 2923, 2852, 1620, 1457, 1354, 1236, 1151, 1071, 843, 804,
1
772 cm-1; H NMR (400 MHz, CDCl3)
δ 7.85 (s, 1H), 7.52 (t, J = 8.41 Hz, 1H), 7.00 (d, J =
8.54 Hz, 1H), 6.75 (d, J = 8.29 Hz, 1H), 6.42 (br. s., 1H), 3.90 (d, J = 6.10 Hz, 2H), 1.93 -
13
2.06 (m, 3H), 1.69 - 1.85 (m, 3H), 1.31 - 1.43 (m, 3H), 1.12 - 1.30 (m, 2H); C NMR (75
MHz, CDCl3)
δ 172.8, 159.6, 158.4, 142.0, 135.5, 133.8, 110.0, 109.5, 105.8, 74.7, 37.5,
29.8, 26.5, 25.8; HRMS (ESI) Calcd. for C16H18O4 [M+H]+ 275.1283, found 275.1303.
7.1.1.2. 3-Hydroxy-5-(isopentyloxy)-4H-chromen-4-one (9b). Yield 78%; White solid; mp
116 - 118 °C; IR (neat) 3282, 2955, 2868, 1621, 1460, 1355, 1241, 1151, 1067, 966, 847, 804
1
772 cm-1; H NMR (300 MHz, CDCl3)
δ 7.85 (s, 1H), 7.53 (t, J = 8.38 Hz, 1H), 7.01 (d, J =
8.57 Hz, 1H), 6.77 (d, J = 8.20 Hz, 1H), 6.42 (s, 1H), 4.16 (t, J = 6.71 Hz, 2H), 1.92 - 2.08
(m, 1H), 1.86 (q, J = 6.71 Hz, 2H), 1.02 (d, J = 6.52 Hz, 6H); 13C NMR (75 MHz, CDCl3)
δ
172.7, 159.4, 158.4, 142.0, 135.5, 133.8, 112.8, 110.1, 105.9, 67.8, 37.6, 25.0, 22.6; HRMS
(ESI) Calcd. for C14H16O4 [M+H]+ 249.1127, found 249.1151.
7.1.1.3. 6-(Cyclohexylmethoxy)-3-hydroxy-4H-chromen-4-one (9c). Yield 71%; off white
solid; mp 126 - 129 °C; IR (neat) 3246, 2921, 2855, 1603, 1489, 1403, 1276, 1199, 1163,
1
982, 850, 800, 784 cm-1; H NMR (300 MHz, CDCl3)
δ 8.01 (s, 1H), 7.54 (d, J = 2.89 Hz,

ACCEPTED MANUSCRIPT
1H), 7.39 - 7.46 (m, 1H), 7.27 - 7.33 (m, 1H), 6.20 (br. s., 1H), 3.86 (d, J = 5.96 Hz, 2H),
13
1.66 - 1.95 (m, 6H), 1.17 - 1.41 (m, 3H), 1.08 (d, J = 11.92 Hz, 2H); C NMR (75 MHz,
CDCl3) δ 173.0, 156.2, 151.3, 141.2, 138.2, 124.7, 122.3, 119.8, 104.5, 74.2, 37.6, 29.8, 26.5,
25.7; HRMS (ESI) Calcd. for C16H18O4 [M+H]+ 275.1283, found 275.1305.
7.1.1.4. 3-Hydroxy-6-(isopentyloxy)-4H-chromen-4-one (9d). Yield 70%; off white solid; mp
118 - 119 °C; IR (neat) 3250, 3082, 2947, 2868, 1602, 1488, 1471, 1407, 1183, 1148, 976,
887, 799 cm-1; 1H NMR (300 MHz, CDCl3)
δ 8.01 (s, 1H), 7.56 (d, J = 2.98 Hz, 1H), 7.38 -
7.46 (m, 1H), 7.30 (m, 1H), 6.22 (s, 1H), 4.09 (t, J = 6.61 Hz, 2H), 1.86 (m, 1H), 1.73 (q, J =
13
6.86 Hz, 2H), 0.99 (d, J = 6.61 Hz, 6H); C NMR (75 MHz, CDCl3)
δ 173.0, 156.1, 151.3,
141.3, 138.3, 124.7, 122.3, 119.8, 104.4, 67.2, 37.8, 25.1, 22.6; HRMS (ESI) Calcd. for
C14H16O4 [M+H]+ 249.1127, found 249.1153.
7.1.1.5. 5-(Cyclohexylmethoxy)-3-hydroxy-2-methyl-4H-chromen-4-one (9e). Yield 68%; Off
white solid; mp 147 - 149 °C; IR (neat) 3295, 2921, 2852, 1619, 1457, 1236, 1152, 1071,
843, 804, 772 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.48 (t, J = 8.38 Hz, 1H), 6.97 (d, J = 8.57
Hz, 1H), 6.73 (d, J = 8.20 Hz, 1H), 3.89 (d, J = 6.05 Hz, 2H), 2.43 (s, 3H), 1.92 - 2.08 (m,
13
3H), 1.68 - 1.85 (m, 3H), 1.10 - 1.41 (m, 5H); C NMR (100 MHz, CDCl3)
δ 172.0, 159.5,
157.7, 145.6, 138.8, 133.2, 112.2, 109.7, 105.7, 74.6, 37.4, 29.8, 26.5, 25.7, 14.7; HRMS
(ESI) Calcd. for C17H20O4 [M+H]+ 289.144, found 289.1463.
7.1.1.6. 5-(Cyclohexylmethoxy)-3-hydroxy-2-propyl-4H-chromen-4-one (9f). Yield 50%; Off
white solid; mp 89 - 92 °C; IR (neat) 3294, 2922, 2852, 1621, 1457, 1236, 1152, 1071, 843,
1
804, 772 cm-1; H NMR (300 MHz, CDCl3)
δ 7.48 (t, J = 8.38 Hz, 1H), 6.99 (d, J = 8.57 Hz,
1H), 6.73 (d, J = 8.20 Hz, 1H), 6.47 (br. s., 1H), 3.90 (d, J = 6.15 Hz, 2H), 2.76 (t, J = 7.50
Hz, 2H), 1.93 - 2.10 (m, 3H), 1.72 - 1.88 (m, 5H), 1.28 - 1.46 (m, 3H), 1.07 - 1.25 (m, 3H),
13
1.02 (t, J = 7.40 Hz, 3H); C NMR (100 MHz, CDCl3)
δ 172.2, 159.4, 157.7, 149.2, 138.6,

ACCEPTED MANUSCRIPT
133.2, 112.2, 109.8, 105.6, 74.6, 37.4, 30.4, 29.7, 26.4, 25.7, 19.9, 13.6; HRMS (ESI) Calcd.
for C19H24O4 [M+H]+ 317.1753, found 317.1774.
7.1.1.7. 5-(Cyclohexylmethoxy)-3-hydroxy-2-isopropyl-4H-chromen-4-one (9g). Yield 60%;
White solid; mp 104 - 106 °C; IR (neat) 3293, 2921, 2852, 1620, 1457, 1236, 1152, 1072,
842, 804, 772 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.48 (t, J = 8.38 Hz, 1H), 7.00 (d, J = 7.73
Hz, 1H), 6.72 (d, J = 8.10 Hz, 1H), 6.49 (s, 1H), 3.89 (d, J = 6.15 Hz, 2H), 3.38 (spt, J = 6.91
Hz, 1H), 1.91 - 2.07 (m, 3H), 1.70 – 1.82 (m, 3H), 1.10 - 1.40 (m, 11H); 13C NMR (100
MHz, CDCl3)
δ 172.4, 159.4, 157.7, 152.6, 137.1, 133.1, 112.2, 109.8, 105.6, 74.7, 37.4,
29.8, 27.7, 26.5, 25.7, 19.3; HRMS (ESI) Calcd. for C19H24O4 [M+H]+ 317.1753, found
317.1776.
7.1.1.8. 2-Cyclohexyl-5-(cyclohexylmethoxy)-3-hydroxy-4H-chromen-4-one (9h). Yield 65%;
White solid; mp 160 - 162 °C; IR (neat) 3292, 2922, 2852, 1620, 1458, 1235, 1152, 1072,
842, 804, 772 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.48 (t, J = 8.38 Hz, 1H), 7.00 (d, J = 8.48
Hz, 1H), 6.72 (d, J = 8.10 Hz, 1H), 6.52 (s, 1H), 3.89 (d, J = 6.24 Hz, 2H), 3.05 (tt, J = 3.15,
13
11.89 Hz, 1H), 1.68 - 2.05 (m, 13H), 1.10 - 1.47 (m, 9H); C NMR (100 MHz, CDCl3)
δ
172.4, 159.4, 157.7, 152.2, 137.3, 133.1, 112.2, 109.8, 105.5, 74.6, 37.5, 37.4, 29.8, 29.2,
26.5, 25.9, 25.7, 25.7; HRMS (ESI) Calcd. for C22H28O4 [M+H]+ 357.2066, found 357.2092.
7.1.2. General synthetic procedure for the preparation of compounds (10a, 10c and 10e-u).
To a solution of the corresponding 3-hydroxy chromenone compound 9 (0.547 mmol) in
DMF (3 mL), K2CO3 (0.601 mmol) and alkyl halide (0.601 mmol) were added. The resulting
mixture was stirred for 4 – 16 h at 60 °C. After cooling to room temperature, the reaction
mixture was diluted with water, and then extracted with EtOAc. The combined organic layers
were washed with water, brine solution, dried over anhydrous Na2SO4, filtered and then
concentrated under reduced pressure. The residue was subjected to flash silica gel (230-400

ACCEPTED MANUSCRIPT
mesh) column chromatography (eluting with 0-15% ethyl acetate in hexanes) to afford the
title compounds.
7.1.2.1. 5-(Cyclohexylmethoxy)-3-(4-(hydroxymethyl)benzyloxy)-4H-chromen-4-one (10a).
Yield 48%; White solid; mp 131 - 133 °C; IR (neat) 3435, 2928, 2843, 1613, 1482, 1457,
1
1166, 1067, 1015, 839, 807, 772 cm-1; H NMR (300 MHz, CDCl3)
δ 7.49 (s, 1H), 7.32 -
7.47 (m, 5H), 6.90 (d, J = 8.57 Hz, 1H), 6.73 (d, J = 8.20 Hz, 1H), 5.10 (s, 2H), 4.70 (br. s.,
2H), 3.88 (d, J = 6.24 Hz, 2H), 1.90 - 2.10 (m, 3H), 1.64 - 1.84 (m, 3H), 1.04 - 1.43 (m, 5H);
13C NMR (75 MHz, CDCl3)
δ 173.3, 160.0, 157.8, 143.4, 141.4, 140.8, 135.8, 133.4, 128.2,
127.1, 115.1, 109.5, 106.3, 74.8, 72.4, 65.0, 37.5, 29.7, 25.8; HRMS (ESI) Calcd. for
C24H26O5 [M+H]+ 395.1858, found 395.1886.
7.1.2.2. Methyl 4-((5-(cyclohexylmethoxy)-4-oxo-4H-chromen-3-yloxy)methyl)benzoate (10c).
Yield 60%; White solid; mp 117 - 119 °C; IR (neat) 2924, 2852, 1712, 1638, 1602, 1456,
1
1432, 1267, 1238, 1182, 1068, 1027, 760 cm-1; H NMR (300 MHz, CDCl3)
δ 8.04 (d, J =
8.29 Hz, 2H), 7.44 - 7.56 (m, 4H), 6.92 (d, J = 8.47 Hz, 1H), 6.75 (d, J = 8.20 Hz, 1H), 5.17
(s, 2H), 3.92 (s, 3H), 3.89 (d, J = 6.24 Hz, 2H), 1.91 - 2.10 (m, 3H), 1.66 - 1.85 (m, 3H), 1.04
- 1.44 (m, 5H); 13C NMR (100 MHz, CDCl3)
δ 173.3, 166.9, 160.1, 157.9, 143.5, 141.8,
141.7, 133.5, 129.9, 127.7, 115.2, 109.6, 106.5, 74.8, 72.1, 52.1, 37.4, 29.7, 26.4, 25.7;
HRMS (ESI) Calcd. for C25H26O6 [M+H]+ 423.1807, found 423.1834.
7.1.2.3. 5-(Cyclohexylmethoxy)-3-(4-methoxybenzyloxy)-4H-chromen-4-one (10e). Yield
72%; White solid; mp 136 - 139 °C; IR (neat) 2925, 2859, 1650, 1516, 1456, 1232, 1168,
1
1007, 827, 805, 777 cm-1; H NMR (300 MHz, CDCl3)
δ 7.42 - 7.51 (m, 2H), 7.34 (d, J =
8.66 Hz, 2H), 6.84 - 6.94 (m, 3H), 6.73 (d, J = 8.20 Hz, 1H), 5.05 (s, 2H), 3.88 (d, J = 6.24
13
Hz, 2H), 3.81 (s, 3H), 1.92 - 2.11 (m, 3H), 1.67 - 1.85 (m, 3H), 1.14 - 1.44 (m, 5H); C
NMR (75 MHz, CDCl3)
δ 173.4, 160.1, 159.6, 157.9, 143.4, 141.8, 133.3, 129.8, 128.6,

ACCEPTED MANUSCRIPT
124.7, 113.9, 109.6, 106.4, 74.9, 72.5, 55.3, 37.5, 29.8, 26.5, 25.9; HRMS (ESI) Calcd. for
C24H26O5 [M+H]+ 395.1858, found 395.1882.
7.1.2.4. 3-(Benzyloxy)-5-(cyclohexylmethoxy)-4H-chromen-4-one (10f). Yield 75%; White
solid; mp 118 - 120 °C; IR (neat) 2927, 2844, 1640, 1599, 1456, 1238, 1179, 1067, 1022,
1
829, 805, 779, 757, 738 cm-1; H NMR (300 MHz, CDCl3)
δ 7.50 (s, 1H), 7.31 - 7.50 (m,
6H), 6.91 (d, J = 8.48 Hz, 1H), 6.73 (d, J = 8.20 Hz, 1H), 5.11 (s, 2H), 3.89 (d, J = 6.15 Hz,
2H), 1.91 - 2.10 (m, 3H), 1.67 - 1.84 (m, 3H), 1.06 - 1.43 (m, 5H) 13C NMR (75 MHz,
CDCl3)
δ 173.3, 160.1, 157.8, 143.5, 141.5, 136.5, 133.3, 128.5, 128.2, 128.0, 115.2, 109.6,
106.4, 74.8, 72.8, 37.5, 29.8, 26.5, 25.9; HRMS (ESI) Calcd. for C23H24O4 [M+H]+ 365.1753,
found 365.1776.
7.1.2.5. 5-(Cyclohexylmethoxy)-3-(pyridin-4-ylmethoxy)-4H-chromen-4-one (10g). Yield
80%; Light yellow solid; mp 106 - 108 °C; IR (neat) 2925, 2851, 1635, 1603, 1477, 1456,
1
1364, 1241, 1183, 1067, 1035, 836, 803, 772 cm-1; H NMR (300 MHz, CDCl3)
δ 8.61 (d, J
= 5.87 Hz, 2H), 7.62 (s, 1H), 7.49 (t, J = 8.38 Hz, 1H), 7.37 (d, J = 5.59 Hz, 2H), 6.94 (d, J =
8.48 Hz, 1H), 6.76 (d, J = 8.29 Hz, 1H), 5.14 (s, 2H), 3.89 (d, J = 6.15 Hz, 2H), 1.92 - 2.08
(m, 3H), 1.73 - 1.84 (m, 3H), 1.05 - 1.44 (m, 5H); 13C NMR (75 MHz, CDCl3)
δ 173.1,
160.1, 157.9, 149.9, 145.8, 143.5, 142.1, 133.6, 122.0, 115.2, 109.6, 106.6, 74.9, 71.2, 37.4,
29.8, 26.5, 25.8; HRMS (ESI) Calcd. for C22H23NO4 [M+H]+ 366.1705, found 366.1731.
7.1.2.6. 3-(4-Chlorobenzyloxy)-5-(cyclohexylmethoxy)-4H-chromen-4-one (10h). Yield 65%;
Off White solid; mp 155 - 157 °C; IR (neat) 3076, 2919, 2851, 1639, 1605, 1474, 1456,
1231, 1179, 1068, 981, 855, 804, 772 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.52 (s, 1H), 7.48
(t, J = 8.38 Hz, 1H), 7.31 - 7.40 (m, 4H), 6.92 (d, J = 8.01 Hz, 1H), 6.74 (d, J = 8.20 Hz, 1H),
5.08 (s, 2H), 3.89 (d, J = 6.24 Hz, 2H), 1.92 - 2.10 (m, 3H), 1.67 - 1.84 (m, 3H), 1.05 - 1.44
(m, 5H) 13C NMR (75 MHz, CDCl3)
δ 173.3, 160.1, 157.9, 143.4, 141.9, 135.0, 134.0, 133.4,

ACCEPTED MANUSCRIPT
129.5, 128.7, 115.2, 109.6, 106.5, 74.9, 72.1, 37.5, 29.8, 26.5, 25; HRMS (ESI) Calcd. for
C23H23ClO4 [M+H]+ 399.1363, found 399.1391.
7.1.2.7. 5-(Cyclohexylmethoxy)-3-(4-nitrobenzyloxy)-4H-chromen-4-one (10i). Yield 56%;
Yellow solid; mp 111 – 112 °C; IR (neat) 2930, 2907, 2854, 1650, 1605, 1516, 1456, 1340,
1
1244, 1182, 1046, 839, 766, 734 cm-1; H NMR (300 MHz, CDCl3)
δ 8.24 (d, J = 8.66 Hz,
2H), 7.64 (t, J = 4.19 Hz, 3H), 7.50 (t, J = 8.38 Hz, 1H), 6.95 (d, J = 8.48 Hz, 1H), 6.77 (d, J
= 8.29 Hz, 1H), 5.22 (s, 2H), 3.90 (d, J = 6.24 Hz, 2H), 2.04 (d, J = 11.18 Hz, 3H), 1.67 -
1.85 (m, 3H), 1.06 - 1.44 (m, 5H); 13C NMR (75 MHz, CDCl3)
δ 173.2, 160.1, 159.9, 157.9,
144.0, 143.3, 142.3, 133.7, 128.2, 123.8, 115.2, 109.6, 106.6, 74.8, 71.7, 37.4, 29.8, 26.5,
25.8; HRMS (ESI) Calcd. for C23H23NO6 [M+H]+ 410.1603, found 410.1624.
7.1.2.8. 5-(Cyclohexylmethoxy)-3-(3-hydroxypropoxy)-4H-chromen-4-one (10j). Yield 62%;
off white solid; mp 99 - 101 °C; IR (neat) 3456, 2928, 2845, 1624, 1604, 1478, 1458, 1240,
1
1176, 1067, 1025, 806, 770 cm-1; H NMR (300 MHz, CDCl3)
δ 7.75 (s, 1H), 7.49 (t, J =
8.38 Hz, 1H), 6.96 (d, J = 8.10 Hz, 1H), 6.74 (d, J = 8.20 Hz, 1H), 4.08 (t, J = 5.73 Hz, 2H),
3.83 - 3.96 (m, 4H), 3.55 (t, J = 6.10 Hz, 1H), 1.88 - 2.06 (m, 5H), 1.69 - 1.84 (m, 3H), 1.05 -
1.41 (m, 5H); 13C NMR (75 MHz, CDCl3)
δ 173.3, 160.1, 158.0, 144.3, 142.3, 133.6, 115.2,
109.6, 106.5, 74.9, 69.3, 59.3, 37.5, 32.0, 29.8, 26.5, 25.8; HRMS (ESI) Calcd. for C19H24O5
[M+H]+ 333.1702, found 333.1723.
7.1.2.9. 5-(Cyclohexylmethoxy)-3-(2-hydroxyethoxy)-4H-chromen-4-one (10k). Yield 25%;
off white solid; mp 101 - 103 °C; IR (neat) 3376, 2928, 2849, 1624, 1603, 1478, 1456, 1242,
1
1177, 1068, 1033, 807, 776 cm-1; H NMR (300 MHz, CDCl3)
δ 7.77 (s, 1H), 7.51 (t, J =
8.38 Hz, 1H), 6.97 (d, J = 8.48 Hz, 1H), 6.75 (d, J = 8.29 Hz, 1H), 4.01 - 4.10 (m, 2H), 3.85 -
13
3.93 (m, 4H), 1.88 - 2.06 (m, 3H), 1.65 - 1.84 (m, 3H), 1.03 - 1.42 (m, 5H); C NMR (75
MHz, CDCl3)
δ 174.0, 160.1, 158.0, 144.4, 142.1, 133.7, 115.0, 109.6, 106.6, 74.9, 74.4,

ACCEPTED MANUSCRIPT
61.4, 37.5, 29.8, 26.5, 25.8; HRMS (ESI) Calcd. for C18H22O5 [M+H]+ 319.1545, found
319.1569.
7.1.2.10. 5-(Cyclohexylmethoxy)-3-(4-hydroxybutoxy)-4H-chromen-4-one (10l). Yield 55%;
Pale yellow solid; mp 65 – 68 °C; IR (neat) 3397, 2921, 2845, 1635, 1604, 1475, 1455, 1239,
1178, 1067, 826, 805, 768 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.61 (s, 1H), 7.47 (t, J = 8.38
Hz, 1H), 6.94 (d, J = 8.47 Hz, 1H), 6.72 (d, J = 8.20 Hz, 1H), 3.97 (t, J = 6.15 Hz, 2H), 3.86
(d, J = 6.33 Hz, 2H), 3.73 (t, J = 6.19 Hz, 2H), 1.70 - 2.09 (m, 10H), 1.03 - 1.42 (m, 5H); 13C
NMR (75 MHz, CDCl3)
δ 173.0, 160.0, 157.8, 144.4, 138.9, 133.3, 109.5, 106.1, 74.7, 70.3,
62.4, 37.4, 29.7, 29.3, 26.5, 25.8, 25.8; HRMS (ESI) Calcd. for C20H26O5 [M+H]+ 347.1858,
found 347.1882.
7.1.2.11. 5-(Cyclohexylmethoxy)-3-(5-hydroxypentyloxy)-4H-chromen-4-one (10m). Yield
58%; Pale Yellow solid; mp 55 – 58 °C IR (neat) 3441, 2919, 2851, 1644, 1601, 1476, 1456,
1238, 1177, 1068, 1030, 834, 805, 774 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.59 (s, 1H), 7.47
(t, J = 8.38 Hz, 1H), 6.94 (d, J = 8.47 Hz, 1H), 6.72 (d, J = 8.20 Hz, 1H), 3.92 (t, J = 6.47 Hz,
2H), 3.86 (d, J = 6.33 Hz, 2H), 3.68 (t, J = 6.19 Hz, 2H), 1.51 - 2.09 (m, 12H), 1.04 - 1.42
(m, 5H); 13C NMR (75 MHz, CDCl3)
δ 173.0, 160.0, 157.8, 152.1, 138.7, 133.3, 109.5,
106.0, 74.7, 70.3, 62.6, 37.5, 32.3, 29.7, 28.9, 26.5, 25.8, 22.1; HRMS (ESI) Calcd. for
C21H28O5 [M+H]+ 361.2015, found 361.2041.
7.1.2.12. 3-(3-Hydroxypropoxy)-5-(isopentyloxy)-4H-chromen-4-one (10n). Yield 60%; Light
yellow oil; IR (neat) 3414 (br. peak, OH), 2954, 2871, 1640, 1605, 1460, 1239, 1179, 1066,
807, 768 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.77 (s, 1H), 7.51 (t, J = 8.38 Hz, 1H), 6.98 (d,
J = 8.57 Hz, 1H), 6.77 (d, J = 8.29 Hz, 1H), 4.04 - 4.19 (m, 4H), 3.92 (t, J = 5.54 Hz, 2H),
3.57 (br. s., 1H), 1.91 - 2.06 (m, 3H), 1.83 (q, J = 6.77 Hz, 2H), 0.99 (d, J = 6.61 Hz, 6H); 13C
NMR (75 MHz, CDCl3)
δ 174.0, 159.9, 158.1, 144.2, 142.9, 133.6, 115.3, 109.8, 106.7, 69.6,

ACCEPTED MANUSCRIPT
68.1, 59.3, 37.6, 32.0, 25.0, 22.6; HRMS (ESI) Calcd. for C17H22O5 [M+H]+ 307.1545, found
307.1569.
7.1.2.13. 3-(2-Hydroxyethoxy)-5-(isopentyloxy)-4H-chromen-4-one (10o). Yield 40%; Light
yellow oil; IR (neat) 3355 (br. peak, OH), 2953, 2871, 1632, 1605, 1461, 1240, 1182, 1068,
807, 768 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.78 (s, 1H), 7.52 (t, J = 8.38 Hz, 1H), 6.99 (d,
J = 8.48 Hz, 1H), 6.78 (d, J = 8.29 Hz, 1H), 4.13 (t, J = 6.75 Hz, 2H), 4.03 - 4.09 (m, 2H),
3.87 - 3.94 (m, 2H), 1.92 - 2.07 (m, 1H), 1.83 (q, J = 6.80 Hz, 2H), 0.99 (d, J = 6.52 Hz, 6H);
13C NMR (75 MHz, CDCl3)
δ 172.7, 159.9, 158.0, 144.3, 142.5, 133.7, 109.8, 109.4, 106.8,
74.6, 68.0, 61.4, 37.6, 25.0, 22.6; HRMS (ESI) Calcd. for C16H20O5 [M+H]+ 293.1389, found
293.1409.
7.1.2.14. 6-(Cyclohexylmethoxy)-3-(3-hydroxypropoxy)-4H-chromen-4-one (10p). Yield
68%; off white solid; mp 76 – 79 °C; IR (neat) 3401 (br. peak, OH), 2920, 2850, 1644, 1611,
1
1485, 1460, 1264, 1203, 1182, 1062, 1016, 863, 806 cm-1; H NMR (300 MHz, CDCl3)
δ
7.91 - 7.97 (m, 1H), 7.57 (d, J = 2.89 Hz, 1H), 7.36 - 7.44 (m, 1H), 7.23 - 7.31 (m, 2H), 4.14
(t, J = 5.68 Hz, 2H), 3.94 (t, J = 5.54 Hz, 2H), 3.85 (d, J = 6.05 Hz, 2H), 2.00 (t, J = 5.59 Hz,
13
2H), 1.67 - 1.94 (m, 6H), 1.00 - 1.40 (m, 5H); C NMR (75 MHz, CDCl3)
δ 174.3, 156.5,
150.8, 145.5, 143.2, 124.9, 124.6, 119.5, 105.2, 74.2, 69.8, 59.2, 37.6, 32.0, 29.8, 26.5, 25.7;
HRMS (ESI) Calcd. for C19H24O5 [M+H]+ 333.1702, found 333.1728.
7.1.2.15. 3-(3-Hydroxypropoxy)-6-(isopentyloxy)-4H-chromen-4-one (10q). Yield 75%; Light
yellow solid; mp 86 - 87 °C; IR (neat) 3399, 2929, 2867, 1614, 1487, 1474, 1391, 1267,
1204, 1067, 983, 943, 851, 809 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.95 (s, 1H), 7.59 (d, J =
3.07 Hz, 1H), 7.41 (d, J = 9.22 Hz, 1H), 7.27 (dd, J = 3.03, 9.17 Hz, 2H), 4.14 (t, J = 5.68
Hz, 2H), 4.08 (t, J = 6.61 Hz, 2H), 3.95 (t, J = 5.54 Hz, 2H), 2.61 (br. s., 2H), 2.00 (quin, J =
5.61 Hz, 2H), 1.85 (tt, J = 6.54, 13.34 Hz, 1H), 1.72 (q, J = 6.74 Hz, 2H), 0.98 (d, J = 6.52

ACCEPTED MANUSCRIPT
Hz, 6H); 13C NMR (75 MHz, CDCl3)
δ
174.3, 156.3, 150.8, 145.5, 143.3, 124.9, 124.6,
119.5, 105.1, 69.9, 67.2, 59.2, 37.8, 32.1, 25.1, 22.6; HRMS (ESI) Calcd. for C17H22O5
[M+H]+ 307.1545, found 307.1568.
7.1.2.16. 5-(Cyclohexylmethoxy)-3-(3-hydroxypropoxy)-2-methyl-4H-chromen-4-one (10r).
Yield 55%; Off white solid; mp 85 - 87 °C; IR (neat) 3478, 2924, 2850, 1632, 1604, 1456,
1
1230, 1203, 1055, 809, 770 cm-1; H NMR (300 MHz, CDCl3)
δ 7.48 (t, J = 8.38 Hz, 1H),
6.95 (d, J = 8.47 Hz, 1H), 6.74 (d, J = 8.29 Hz, 1H), 4.62 (t, J = 6.85 Hz, 1H), 4.05 (t, J =
5.63 Hz, 2H), 3.97 (q, J = 5.62 Hz, 2H), 3.87 (d, J = 6.05 Hz, 2H), 2.41 (s, 3H), 1.88 - 2.05
(m, 5H), 1.66 - 1.85 (m, 3H), 1.04 - 1.42 (m, 5H); 13C NMR (100 MHz, CDCl3)
δ 174.3,
159.9, 157.6, 157.4, 140.6, 133.4, 115.0, 109.5, 106.7, 74.9, 68.9, 58.7, 37.3, 32.1, 29.7, 26.5,
25.7, 15.0; HRMS (ESI) Calcd. for C20H26O5 [M+H]+ 347.1858, found 347.1879.
7.1.2.17. 5-(Cyclohexylmethoxy)-3-(3-hydroxypropoxy)-2-propyl-4H-chromen-4-one (10s).
Yield 51%; Colorless oil; IR (neat) 3425 (br. peak, OH), 2923, 2852, 1632, 1605, 1475, 1458,
1
1241, 1189, 1056, 808, 770 cm-1; H NMR (400 MHz, CDCl3)
δ 7.48 (t, J = 8.32 Hz, 1H),
6.96 (d, J = 8.54 Hz, 1H), 6.74 (d, J = 8.29 Hz, 1H), 4.69 (t, J = 7.07 Hz, 1H), 4.04 (t, J =
5.24 Hz, 2H), 3.97 (q, J = 5.77 Hz, 2H), 3.88 (d, J = 5.85 Hz, 2H), 2.73 (t, J = 7.44 Hz, 2H),
13
1.88 - 2.03 (m, 5H), 1.67 - 1.84 (m, 5H), 1.08 - 1.37 (m, 5H), 1.02 (t, J = 7.32 Hz, 3H); C
NMR (100 MHz, CDCl3)
δ 174.5, 160.6, 159.9, 157.6, 140.5, 133.3, 115.0, 109.5, 106.7,
74.9, 69.2, 58.7, 37.3, 32.2, 30.4, 29.7, 26.5, 25.7, 20.3, 13.7; HRMS (ESI) Calcd. for
C22H30O5 [M+H]+ 375.2171, found 375.2199.
7.1.2.18.
5-(Cyclohexylmethoxy)-3-(3-hydroxypropoxy)-2-isopropyl-4H-chromen-4-one
(10t). Yield 55%; off white solid; mp 65 – 69 °C; IR (neat) 3393, 2924, 2851, 1628, 1603,
1476, 1456, 1232, 1195, 1101, 1069, 811, 780 cm-1; 1H NMR (300 MHz, CDCl3)
δ
7.49 (t, J
= 8.38 Hz, 1H), 6.98 (d, J = 8.48 Hz, 1H), 6.74 (d, J = 8.29 Hz, 1H), 4.79 (br. s., 1H), 4.04 (t,