Design and development of benzoxazole derivatives with toll-like receptor 9 antagonism

Article abstract of DOI:10.1016/j.ejmech.2017.03.086

Toll-like receptor 9 (TLR9) is a major therapeutic target for numerous inflammatory disorders. Development of small molecule inhibitors for TLR9 remains largely empirical due to lack of structural understanding of potential TLR9 antagonism by small molecules and due to the unusual topology of the ligand binding surface of the receptor. To develop a structural model for rational design of small molecule TLR9 antagonists, an enhanced homology model of human TLR9 (hTLR9) was constructed. Binding mode analysis of a series of molecules having characteristic molecular geometry, flexibility and basicity was conducted based on crystal structure of the inhibitory DNA (iDNA) bound to horse and bovine TLR9. Interaction with specific amino acid residues in four leucine rich repeat (LRR) regions of TLR9 was identified to be critical for antagonism by small molecules. The biological validation of TLR9 antagonism and its correlation with probe-receptor interactions led to a reliable model that could be used for development of novel small molecules with potent TLR9 antagonism (IC₅₀ 30–100?nM) with excellent selectivity against TLR7.

Full text of DOI:10.1016/j.ejmech.2017.03.086

Accepted Manuscript
Design and development of benzoxazole derivatives with toll-like receptor 9
antagonism
Swarnali Roy, Ayan Mukherjee, Barnali Paul, Oindrila Rahaman, Shounak Roy,
Gundaram Maithri, Bandaru Ramya, Sourav Pal, Dipyaman Ganguly, Arindam
Talukdar
PII:
S0223-5234(17)30254-4
10.1016/j.ejmech.2017.03.086
EJMECH 9346
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2017
Revised Date: 7 March 2017
Accepted Date: 31 March 2017
Please cite this article as: S. Roy, A. Mukherjee, B. Paul, O. Rahaman, S. Roy, G. Maithri, B. Ramya, S.
Pal, D. Ganguly, A. Talukdar, Design and development of benzoxazole derivatives with toll-like receptor
9 antagonism, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.086.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design and Development of Benzoxazole Derivatives with Toll-like Receptor 9 Antagonism
Ψ
Ψ
Swarnali Roy† , Ayan Mukherjee† , Barnali Paul†, Oindrila Rahaman‡, Shounak Roy‡,
Gundaram Maithri†, Bandaru Ramya‡, Sourav Pal, Dipyaman Ganguly‡* and Arindam
Talukdar†*
†Department of Organic and Medicinal Chemistry, CSIR-Indian Institute of Chemical Biology,
4 Raja S. C. Mullick Road, Kolkata 700032, WB (India).
‡Department of Cancer Biology and Inflammatory Disorders, CSIR-Indian Institute of Chemical
Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032 WB (India).
Keywords: TLR9, TLR7, inflammation, antagonist, homology model, drug design,
computational analysis
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Abstract
Toll-like receptor 9 (TLR9) is a major therapeutic target for numerous inflammatory disorders.
Development of small molecule inhibitors for TLR9 remains largely empirical due to lack of
structural understanding of potential TLR9 antagonism by small molecules and due to the
unusual topology of the ligand binding surface of the receptor. To develop a structural model for
rational design of small molecule TLR9 antagonists, an enhanced homology model of human
TLR9 (hTLR9) was constructed. Binding mode analysis of a series of molecules having
characteristic molecular geometry, flexibility and basicity was conducted based on crystal
structure of the inhibitory DNA (iDNA) bound to horse and bovine TLR9. Interaction with
specific amino acid residues in four leucine rich repeat (LRR) regions of TLR9 was identified to
be critical for antagonism by small molecules. The biological validation of TLR9 antagonism and
its correlation with probe-receptor interactions led to a reliable model that could be used for
development of novel small molecules with potent TLR9 antagonism (IC50 30-100 nM) with
excellent selectivity against TLR7.
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1.
Introduction
Toll-like receptors (TLRs) are germline-encoded pattern recognition molecules that are
major players for innate immunity in the body [1]. TLRs contain a ligand-binding ectodomain
(ECD) with 19-25 leucine-rich repeats (LRRs), a transmembrane domain and a characteristic
cytoplasmic domain called the TIR (Toll/IL‑1 receptor) domain for downstream signaling [1-2].
Major members of the TLR family are expressed on the cell surface of various immune cell
subsets, except for TLR3, TLR7, TLR8 and TLR9 that are expressed in endosomes [1-3]. These
endosomal TLRs are specialized for detecting non-self pathogen-derived nucleic acids that are
presented in the acidic (pH < 6.5) endolysosomal compartments after phagocytosis of the
pathogenic microbes. Cognate ligand binding initiates multiple downstream signaling pathways,
leading to activation of transcription factor NFκB or mitogen-activated protein kinases (MAPKs)
or recruitment of the IFN regulatory factors (IRFs) [1-3].
In addition to this role in protective immunity, recent studies by different groups,
including us, have implicated inadvertent activation of the endosomal TLRs in response to
endogenous nucleic acid ligands in pathogenesis of a large number of autoimmune and other
inflammatory disorders, as well as contexts of sepsis [4-5]. Evidence for such pathogenetic
involvement in contexts of sterile inflammation is particularly numerous in case of TLR9 [6-14],
that can recognize unmethylated CpG motifs on DNA molecules of both non-self and self-origin.
Pathogenetic role of TLR9 activation by self-DNA molecules have been documented in various
contexts of chronic inflammation and autoimmunity, e.g., systemic lupus erythematosus [5,11],
psoriasis [8, 12, 13], Sjogren‘s syndrome [10], scleroderma [14], type I diabetes [5], type II
diabetes [6,7] etc. Thus, TLR9 is an important therapeutic target in these clinical contexts and so
TLR9 antagonists are being sought for globally. In some clinical contexts TLR7 and TLR9
receptors may have opposing inflammatory and regulatory roles [15-17]. The divergent effects of
TLR7 and TLR9 emphasize the need for specific targeting to avoid unwanted immune reactions.
Most TLR9 antagonists are empirically derived as with other anti-inflammatory agents and
considerable efficiency is shown by DNA oligonucleotides that bind as usual TLR9 ligands but
fail to elicit activation and competitively inhibit agonistic DNA binding (inhibitory DNA
molecules or iDNAs) [15]. Due to the unusual topology of the ligand binding surface of TLR9
lacking conventional pockets, the functional mechanism of potential TLR9 antagonism by small
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molecules is not understood, consequently small molecule TLR9 antagonists have so far been
developed by empirical screening [18-22]. To do away with this empiricism we attempted to
identify the structural attributes in a molecule that confer hTLR9 antagonism and its correlation
with probe-receptor interactions for further development of new generation drugs with potential
use in relevant clinical contexts, where inadvertent TLR9 activation plays a major role. Our
results have identified selective hTLR9 antagonists showing IC50 value < 100 nM with 50-200
fold selectivity against hTLR7.
2.
Results and Discussion
We have designed and synthesized a set of small molecules constituting specific
geometry and with variable basicity (Supporting information) with an aim to examine their
potential for TLR9 antagonism. The design strategy necessitates the molecules to have an ideal
clogP value so that they can access the TLR9 receptor inside the endosomal compartment,
wherein the pH is 6-6.5. The biological validation for the humanTLR9 (hTLR9) antagonism was
performed with a reporter HEK293 cell line expressed with hTLR9 to report downstream
signaling through an enzymatic activity of an alkaline phosphatase that could be assessed
colorimetrically (Figure 3). To check potential humanTLR7 antagonism, similar assays were
conducted with another reporter HEK293 cell line expressed with hTLR7. To validate the TLR9
antagonism in a more physiological system, assays were also performed with human peripheral
mononuclear cells (PBMCs) and human plasmacytoid dendritic cells (pDCs) by stimulating them
with TLR9 ligand (CpGA), in presence of different concentrations of the candidate small
molecules. Assessment of hTLR9 antagonism by individual small molecules was based on IC50
values calculated from the reporter assays (Figure 3). None of the compounds showed any
cytotoxicity below 20.0 µM as measured by a tetrazolium dye reduction assay (MTT assay) on
hepatic and renal epithelial cells (Figure S4).
Syntheses of the compounds listed in table 1 are shown in schemes 1 and 2. The
formation of the benzoxazole core, from the necessary intermediates was achieved readily under
reflux in acetic acid. The obtained benzoxazole compounds (14-17) were then treated with 1-
bromo 3-chloropropane in presence of K2CO3 in dry DMF, and the resultant chloro derivatives
were treated with different in DMF to obtain the corresponding compounds 19, 20, 21, 23, 25,
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28, 29 and 30. Subsequently, we started the structural exploration of the hTLR9 antagonism with
the help of E6446 (compound 19) and 20 [19], both having 2-phenylbenzoxazole core that can
strongly inhibit TLR9 signaling triggered by agonistic CpG DNA. The structural features of both
19 and 20 are similar except that the side chain substitutions are pyrrolidine in 19 and piperidine
in 20. Despite showing potent TLR9 inhibitory activity, 19 and 20 have undesirable molecular
flexibility along with high clogP value for oral bioavailability, thus their limited drug-likeness
[23-25]. First, we tried to identify minimal structural requirements for inhibition of hTLR9
signaling. Accordingly, we synthesized compounds 25 and 28 lacking one of the flexible side
chains. Their hTLR9 antagonistic activity in terms of IC50 value, as compared to 19 and 20, was
substantially reduced as expected (Table 1). Interestingly, among themselves, 28 showed almost
nine-fold hTLR9 inhibition efficacy as compared to 25. A strong electron withdrawing group (–
CF3) was introduced in 29 to determine the effect of ring deactivation on hTLR9 inhibition.
Interestingly, 29 showed almost similar IC50 value of 1.1 µM as compared to 28 with 0.83 µM
showing negligible effect of ring deactivation. Next, we introduced specific groups with varying
basicity at both flexible side chains of benzoxazole core to corroborate the effect of early
endosomal pH on hTLR9 antagonism (Table 1). We replaced the basic nitrogen at the terminal
end of 28 with an imidazole moiety in 30. Excitedly, 30 showed an IC50 value of 2.1 µM as
compared to 0.83 µM for 28. For further exploration, 21 and 23 were synthesized that led to
another interesting observation. The activity of 21 and 23 with two nitrogen atoms at the terminal
end but with variable basicity differs by 85 fold. Compound 23 exhibited an IC50 of 60 nM,
whereas that of 21, with two imidazole groups, was only 5.1 µM. Presumably, the N-substitution
pattern of imidazole makes it difficult to protonate at early endosomal pH (6-6.5). On the other
hand, 19 showed an IC50 of 1.99 µM against hTLR7 with TLR9/TLR7 selectivity of 132 fold.
Remarkably, 23 presented improved selectivity of 180 fold with IC50 of 10.84 µM against
hTLR7. To identify the structural attributes in a molecule that confer human TLR9 (hTLR9)
antagonism and its correlation with probe-receptor interactions, we built a homology model of
the Ecto domain of hTLR9 in Discovery Studio 4.0 (supporting information). For the analysis for
potential ligand-binding region, the recently reported crystal structures of CpG DNA agonist, and
inhibitory DNA (iDNA) in horse and bovine TLR9 was taken into consideration [26]. The
binding area of iDNA on TLR9 was found to be quite large from LRR2–LRR11 by forming a
stem-loop structure in the interior of the ring structure of TLR9. Although the binding modes of
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agonistic DNA and iDNA are completely different, LRR4 and LRR5 are involved in the
overlapping binding site for agonistic DNA and iDNAs. Our model has 0.44 Å RMSD cut off
with that of template structure. The docking experiments for all the compounds were conducted
in C-DOCKER and Ligandfit to validate the best possible mode. To find the most probable
binding mode CDOCKER energy, CDOCKER interaction energy, PLP1, PMF04 and Ligscore1
were considered. During the analysis, CDOCKER interaction energy and PMF04 appeared to be
more correlating with IC50 value (Figure S3). The clogP value was determined by Discovery
Studio Calculate Molecular Property suite.
Scheme 1. Reagents and conditions: (a) CDI, MeCN, 10-12 h, rt; (b) BBr3, CH2Cl2, 0 ºC; (c)
AcOH, reflux, 48 h; (d) l-bromo-3-chloropropane, K2CO3, DMF, rt, 12 h; (e) DMF, 110 ºC,
sealed tube; (f) tert-butyl pyrrolidin-2-ylcarbamate, K2CO3, KI, DMF, sealed tube, 120 ºC; (g)
imidazole, NaH, DMF, 60 ºC; (h) TFA, CH2Cl2, 0 ºC to rt.
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Figure 1. A) Binding mode of 19 in hTLR9 depicting hydrogen bond interactions with Glu317,
Asp175, Lys207 and hydrophobic interaction with Phe173 and Phe343. Docking study of 28
(carbon atom in dark orange) and 25 (carbon atom in cyan) The docking study suggested that the
terminal side chain pyrrolidine group of both 25 and 28 orient in the same direction and form
hydrogen bond with Glu317.
Table 1. Minimal structural requirement for inhibition of TLR9 signaling
N
R2
O
R1
hTLR9:
IC50
Compa
R1
R2
cLogP
(µM)b
19c
20
0.015
4.57
5.48
0.098
7.0
3.93
3.93
25
28
0.83
2.1
1.1
3.07
4.87
30
29
5.1
2.85
21
23c
0.06
1.81
aAll compounds depicted in table were synthesized. bThe IC50 values were calculated via reporter
assay with HEK293 cells transfected with human TLR9 in our laboratory. cTested against hTLR7.
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The docking analysis suggested that both 19 and 20 share a similar interaction pattern as
iDNA at LRR5, LRR6 and LRR10. As depicted in figure 1A, the basic nitrogen atoms on both
the pyrrolidine ring of 19 were found to interact via salt bridge type hydrogen bonding network
with Glu317 (on LRR10) and Asp175 (on LRR5), whereas Lys207 (on LRR6) form hydrogen
bonding with phenolic oxygen atom of the benzoxazole core. The alkyl ring of both the
pyrrolidine may be stabilized by hydrophobic interaction with Phe173 (on LRR5) and Phe343
(on LRR11). Based on our binding mode analyses, we hypothesized that the interaction of
antagonists with Glu317, Asp175 and Lys207 amino acids along with hydrophobic interaction
with Phe173 and Phe343 is important for inhibition of hTLR9 signaling. The docking study of 25
and 28 (Figure 1B) suggested that the terminal side chain for both the compounds orient in the
same direction and form hydrogen bond with Glu317 along with hydrophobic interaction. This
led us to attribute a critical role to Glu317, along with hydrophobic interactions, for TLR9
inhibition.
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, rt, 12 h; (b) pyrrolidine, DMF, 110 ºC; (c)
imidazole, NaH, DMF, 60 ºC.
The initial results prompted us to incorporate two nitrogen atoms spatially present in the
next set of molecules (Table 2). Thus we considered substitution at the meta position of phenyl
ring of 2-phenylbenzoxazole core via amide, C-C bond and C-N bond linkages. We visualized
that these linkages will impart a favorable geometry enabling the molecules to interact with the
concave surface of the hTLR9. Given the apparent role of early endosomal pH, we also sought to
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systematically increase the basicity of terminal amino group while maintaining the hydrogen
bond network.
Syntheses of compounds from table 2 are shown in scheme 3. The formation of the
benzoxazole core was achieved in a similar fashion as described in Scheme 1. The nitro group
present in 41 was reduced to corresponding amine derivative 43 by hydrogenation using Pd/C.
HATU mediated coupling [27] of corresponding acids with 43 yielded 44, 46, 48, and 49 (Table
2). Further deprotection of the Boc group with TFA provided 48 and 49. Suzuki−Miyaura [28]
cross coupling of the bromo derivative 42 resulted in 50 and subsequent deprotection of the Boc
group in 50 gave compound 51. Reduction of the double bond in 51 with Pd/C provided with 52.
Compound 42 was subjected to Buchwald- Hartwig amination [29] with respective amine to
obtain 54, 55 and 56.
Interestingly, 46 with a primary amine group directly attached to phenyl ring showed very
poor hTLR9 with IC50 of 21.0 µM, whereas incorporating just a methylene bridge in 48
drastically increased the hTLR9 inhibition by 35-fold to 0.59 µM. Subsequently dimethylation of
the terminal amino function in 49 increased the hTLR9 inhibition by 450 folds to 48.0 nM as
compared to 46. The molecular docking experiment with 49 suggested that substitution at the
meta position of phenyl ring of 2-phenylbenzoxazole introduced a curvature which enabled these
compounds to maintain all the conserved hydrogen bonds in accordance with our binding mode
hypothesis. Compound 48, and 49 with an amide group showed excellent selectivity of 100 fold
against hTLR7 (table 2). The proposed inhibition of hTLR9 for 44, 46, 48, and 49 is shown in
figure 2A. For further validation of the binding mode hypothesis, we synthesized compounds 51,
52, 54, 55 and 56, taking into account the hitherto gained structural insight into hTLR9
antagonism.
In 51, the terminal piperidine group was attached directly to the main core via a C-C
bond. The direct attachment of piperidine group in 51 and 52 imparts a geometric rigidity in
these molecules. Accordingly, 51 and 52 showed similar IC50 value of 99 nM and 133 nM
respectively. The docking analysis (Figure 4) depicted a distinct characteristic for 51 and 52 with
the piperidine ring forming a skewed shape to form salt-bridge hydrogen bonding with Glu317.
Compound 51 (TLR9/TLR7 = 84-fold) showed better selectivity compared to 52 (TLR9/TLR7 =
38-fold) against hTLR7 with IC50 value of 8.30 µM and 4.99 µM.
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Table 2.Second generation compounds based on the binding mode
hTLR9: hTLR7:
Comp
R3
IC50
(µM)
IC50
(µM)
cLogP
4.71
44
2.56
21
NT
NT
3.96
46
48
0.59
66.17
4.71
3.82
4.78
49
0.048
51
52
0.099
0.133
8.30
4.99
3.97
4.08
54
55
56
0.308
7.55
3.31
0.13
8.09
4.57
5.43
3.85
0.037
All compounds depicted in table were synthesized and the IC50 values were calculated via
reporter assay with HEK293 cells transfected with human TLR9 and human TLR7 in our
laboratory. NT: not tested.
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Figure 2. A) TLR9 inhibition curve for 44, 46, 48, and 49. B) Structure of 44, 46, 48, and 49
showing the structural difference.
Scheme 3.aReagents and conditions: (a) CDI, MeCN, 10-12 h, rt; (b) BBr3, CH2Cl2, 0 ºC; (c)
AcOH, reflux, 48 h; (d) 1-bromo-3-chloropropane, K2CO3, DMF, rt, 12 h; (e) DMF, 110 ºC; (f)
Pd/C, H2, rt; (g) Substituted benzoic acids, HATU, DIPEA, DMF, rt, 12 h; (h) TFA, Dry DCM, 0
ºC to rt; (i) N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester, Pd(PPh3)4, Cs2CO3,
DMF:H2O(1:1), microwave, 110 ºC, 2 h; (j) 1-benzylpiperidin-4-amine/1-methylpiperidin-4-
amine, NatOBu, BINAP, Pd2(dba)3, toluene, reflux, 8-10 h.
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Figure 3. Representative TLR9 reporter assay results of synthesized TLR9 antagonists. The
compounds were assessed for dose (µM) dependent inhibition of TLR9, using a HEK- Blue
TLR9 cell line expressing a TLR9-responsive, NF-КB-inducible SEAP. Data shown are mean of
triplicate wells ± SD.
Figure 4. The characteristic docking poses of 52 (A) and 51 (B) (carbon atom in cyan) with
hydrogen bond depicted by red dotted line.
Further, we strategically introduced similar piperidine group in 54, as in 52, but with an
amino flexibility. The IC50 of 54 was found to be 308 nM. For further optimization aimed at
potent hTLR9 inhibition, we tried to exploit the hydrophobic interaction by introducing a benzyl
group in 55. The benzyl group did show π-π stacking with Phe343 and Glu317, and formed a
similar bridge-head type of hydrogen bond (Figure 5B). However, the other half of the molecule
cannot attain the required geometry to be able to interact with Lys207 and Asp175 on the
concave surface of hTLR9. Instead, the terminal side chain nitrogen on pyrrolidine can form
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hydrogen bond with Ser151 (on LRR4). Ser151 was also found to be an iDNA-interacting
residue.
B
A
C
Figure 5. Inhibition curve for compound 56. The compound was assessed for dose (µM)
dependent inhibition of TLR9 in A) PBMC B) pDCs. C) Reporter assay.
Figure 6. A) TLR9 inhibition curve for 19, 49, 51, 55 and 56. B) Docking study of 55 and 56
(carbon atom in cyan) showing different binding mode of 55 with π-π stacking with Phe343 as
well as missing interactions with Asp175, Lys207 and Phe173. C) Small molecule binding hot-
spot region shown in red as compared to large iDNA binding region in yellow color.
Having gained significant understanding, we synthesized 56, where the methyl protected
piperidine moiety allows formation of a hairpin bridge-head with Glu317 (figure 6B). Phe343
stabilizes this methyl group by hydrophobic interaction, while other important interactions are
maintained. Excitedly, the IC50 of 56 was found to be 37 nM as in comparison with 308 nM for
54, which strongly supports our binding mode hypothesis (Figure 6C). Figure 6 depicts dose
depended inhibition of TLR9 by 56 in PBMC assay, isolated human pDC assay and reporter
assay respectively. The importance of hydrophobicity at the terminal end can be further
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corroborated by comparing the IC50 value of 54, 55 and 56. The favorable geometry shown by 56
also explains the improvement in its hTLR9 antagonism as compared to 51 and 55. Interesting to
note that the most potent compound 56 provided excellent selectivity (TLR9/TLR7 = 125-fold)
against hTLR7 as compared to compound 55 (TLR9/TLR7 = 62-fold) and compound 54
(TLR9/TLR7 = 25-fold). Of note, the clogP value of compound 56 was 3.85, which is considered
to be ideal for cell membrane permeability, thus facilitating both endosomal access as well as
oral bioavailability.
3.
Conclusion
In conclusion, we have successfully illustrated a series of structural attributes responsible
for functional mechanism of selective hTLR9 antagonism by small molecules through these
studies. We biologically validated the computationally derived hypothesis using a series of
rationally designed molecules with different geometry and varying basicity. We found that
interaction with Glu317 (on LRR10), Asp175 (on LRR5) and Lys207 (on LRR6) to be of utmost
importance along with hydrophobic interaction with Phe343 (on LRR11) and Phe173 for hTLR9
antagonism (Figure 6C). Our activity-guided rational designing approach led to the development
of small molecules having hTLR9 antagonism property with IC50 values < 100 nM. These
hTLR9 antagonists were 50-200 fold selective against hTLR7. The selectivity is of special
significance as in some clinical contexts TLR7 and TLR9 receptors may have opposing
inflammatory and regulatory roles. From drug designing perspective these compounds had lesser
molecular flexibility. Validated correlation between our binding mode hypothesis and hTLR9
antagonistic activity of the newly synthesized molecules designed by us can be very useful as
benchmarks for further prediction of small molecule hTLR9 antagonists and development of new
generation drugs for a number of clinical contexts, where inadvertent TLR9 activation play a
major role.
4.
Methods
4.1. Chemistry
We have synthesized series of benzoxazole derivatives having characteristic molecular
geometry, flexibility and basicity for the development of hTLR9 antagonist. All starting
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materials and reagents were purchased from commercial suppliers and used without further
purification. TLC was performed on silica gel plates (Merck silica gel 60 F254) and the spots
were visualized under UV light (254 nm and 365nm). Microwave assisted reactions were run in a
CEM ExplorerTM microwave. For the compound purification flash chromatography was
performed with RediSepRf silica gel columns on Teledyne ISCO CombiFlashRf system. Melting
points were taken in Digital melting point apparatus. The structural analysis of all the
1
intermediate and target compounds was done by H NMR, 13C NMR, ESI and HRMS
spectrometer respectively. The purity of the compounds selected for the biological validation was
carried out by elemental analyses for C, H and N content. The purity of the selected compounds
was also analyzed by Waters HPLC using column Xtimate C18 (5.0 µm, 250 x 4.6 mm). All
compounds were of high purity of >95%. The synthetic protocol for all the compounds has been
detailed in the supplemental Information section.
4.1.1. General Procedure for the Synthesis of 6, 7, 8 and 9:
The acid (1, 2, 3; 3 mmol) and carbonyl diimidazole (3.5 mmol) were dissolved in dry
acetonitrile under a N2 atmosphere. The reaction mixture was stirred for 30 minutes and the
required aromatic amine (3.3 mmol) was added. The reaction mixture was stirred for 10-12 hrs.
Then acetonitrile was concentrated in vacuum. The residue was diluted with water and the
organic part was extracted using ethyl acetate. The combined organic layer was dried over
sodium sulfate and concentrated. The residue was purified by flash column chromatography
using ethyl acetate-hexane system to give the corresponding amide compounds.
4.1.1.1.
N-(2,4-Dimethoxyphenyl)-4-methoxybenzamide (6)
Yield 84% as white fluffy solid. Mp 107-108 ºC; 1H NMR (CDCl3, 300 MHz) δ 8.39 (d, J = 9.0
Hz, 1H), 8.26 (br s, 1H, -NH), 7.85 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 6.54-6.51 (m,
2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.81 (s, 1H); MS (FAB) m/z [M]+ 287.20.
4.1.1.2.
4-Methoxy-N-(2-methoxyphenyl)benzamide (7) [30]
Yield 73% as off white solid. Mp 88-89 ºC; 1H NMR (CDCl3, 300 MHz) δ 8.52 (dd, J = 9.0 Hz,
3.0 Hz, 1H), 8.49 (br s, -NH), 7.87 (d, J = 9.0 Hz, 2H), 7.10-6.90 (m, 5H), 3.93 (s, 3H), 3.87 (s,
3H); 13C NMR (CDCl3, 75 MHz) δ 164.7, 162.3, 148.0, 128.8, 127.9, 127.5, 123.5, 121.1, 119.7,
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113.9, 109.8, 55.7, 55.4. MS (ESI) m/z [M+Na]+ 280.29. HRMS (EI) m/z Calculated for
C15H15NO3 [M]+257.1052; found 257.1069.
4.1.1.3.
N-(2,4-Dimethoxyphenyl)benzamide (8) [31]
Yield 76% as off white solid. Mp 156-158 ºC; 1H NMR (CDCl3, 300 MHz) δ 8.44 (d, J = 9.0 Hz,
1H), 8.36 (br s, 1H, -NH), 7.90 (d, J = 6.0 Hz, 2H), 7.58-7.48 (m, 3H),6.58-6.54 (m, 2H), 3.92 (s,
3H), 3.84 (s, 3H); 13C NMR (CDCl3, 75Hz) δ 163.2, 151.8, 150.4, 146.7, 141.7, 129.9, 126.6,
122.3, 121.1, 118.6, 105.3, 46.2. MS (ESI) m/z [M+Na]+ 280.06. HRMS (EI) m/z Calculated for
C15H15NO3 [M]+257.1052; found 257.1054.
4.1.1.4.
4-(Trifluoromethyl)-N-(2,4-dimethoxyphenyl)benzamide (9)
1
Yield 88% as off white fluffy solid. Mp 114-116°C; H NMR (CDCl3, 300 MHz) δ 8.39 (d, J =
9.0 Hz, 1H), 8.33 (s, 1H), 7.99 (d, J = 6.0 Hz, 2H), 7.75 (d, J = 9.0 Hz, 2H), 6.56-6.53 (m, 2H),
3.91 (s, 3H), 3.83 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 163.6, 156.9, 149.5, 138.7, 133.4, 127.4,
125.7, 120.9, 103.9, 98.7, 55.8, 55.5. MS (ESI) m/z [M+Na]+ 348.07. HRMS (ESI) m/z
Calculated for C16H14F3NO3 [M+Na]+ 348.0824; found 348.0828.
4.1.2. General Procedure for the Synthesis of 10, 11 [32], 12 and 13
The amide (6, 7, 8, 9; 3 mmol) was dissolved in 10 mL of dry DCM in a two-necked R.B flask.
The reaction mixture was cooled to 0 ºC and BBr3 (8 mmol for 6 and 4 mmol for 7, 8 and 9) was
added carefully. The reaction was allowed to further stir at room temperature for 30 minutes. The
reaction mixture was again cooled to 0 ºC and crushed ice was added to the reaction mixture
followed by few drops of methanol. The precipitate obtained was filtered, dried and used directly
for the next step.
4.1.2.1.
N-(2,4-Dihydroxyphenyl)-4-hydroxybenzamide (10)
Yield 58% as ash color solid. Mp 157-159 ºC; 1H NMR (DMSO-d6, 300 MHz) δ 9.28 (brs, -NH),
7.83 (d. J = 9.0 Hz, 2H), 7.24 (d, J = 9.0 Hz, 1H), 6.84 (d, J = 6.0 Hz, 2H), 6.35 (d, J = 3.0 Hz,
1H), 6.23 (dd, J = 9.0 Hz, 3 Hz, 1H). MS (ESI) m/z [M+Na]+ 268.19.
4.1.2.2.
4-Hydroxy-N-(2-hydroxyphenyl)benzamide (11)
16

ACCEPTED MANUSCRIPT
1
Yield 62% as white solid. Mp 165-167 ºC; H NMR (CD3OD, 300 MHz) δ 7.86 (d, J = 9.0 Hz,
2H), 7.78 (dd, J = 9.0 Hz, 3 Hz, 1H), 7.08-7.02 (m, 1H), 6.94-6.85 (m, 4H); 13C NMR (CD3OD,
75 MHz) δ 166.9, 161.1, 148.4, 129.2, 125.9, 125.4, 124.9, 122.5, 119.5, 115.8, 115.0. MS (ESI)
m/z [M+Na]+ 252.07. HRMS (EI) m/z Calculated for C13H11NO3 [M]+ 229.0739; found 229.0735.
4.1.2.3.
N-(2,4-Dihydroxyphenyl)benzamide (12)
Yield 64% as deep pink solid. Mp 208- 209 ºC; 1H NMR (CD3OD, 300 MHz) δ ppm 7.95 (d, J =
9.0 Hz, 2H), 7.62-7.50 (m, 3H), 7.44 (d, J = 9.0 Hz, 1H), 6.43 (d, J = 3.0 Hz, 1H), 6.35 (dd, J =
9.0 Hz, 3 Hz, 1H); 13C NMR (CD3OD, 75 MHz) δ 168.8, 157.5, 152.1, 135.9, 133.1, 129.9,
128.7, 126.1, 119.2, 107.8, 104.7. MS (ESI) m/z [M+Na]+ 252.17. HRMS (EI) m/z Calculated for
C13H11NO3 [M]+ 229.0739; found 229.0740.
4.1.2.4.
4-(Trifluoromethyl)-N-(2,4-dihydroxyphenyl)benzamide (13)
1
Yield 61% as ash color solid. Mp 225-226 ºC; H NMR (CD3OD, 300 MHz) δ 8.12 (d, J = 6.0
Hz, 2H), 7.83 (d, J = 6.0 Hz, 2H), 7.43 (d, J = 9.0 Hz, 1H), 6.44 (d, J = 3.0 Hz, 1H), 6.36 (dd, J =
9.0 Hz, 3 Hz, 1H); 13C NMR (CD3OD, 75 MHz) δ 167.4, 157.7, 152.3, 139.5, 134.5, 129.4,
126.7, 126.4, 118.8, 107.8, 104.6. MS (ESI) m/z [M+Na]+ 320.08. HRMS (EI) m/z Calculated for
C14H10F3NO3 [M]+ 297.0613; found 297.0607.
4.1.3. General Procedure for the Synthesis of 14, 15 [33], 16 [34] and 17 [35]
Acetic acid (2 mmol for 10 and 1 mmol for 11, 12 and 13) was added to 10, 11, 12 and 13; (1
mmol) independently, and the mixture was refluxed for 48 hours. The acetic acid was evaporated
under vacuum and the reaction mixture was diluted with water. The organic part was extracted
using ethyl acetate. Combined organic layer was dried over sodium sulfate and concentrated. The
residue was purified by flash chromatography using ethyl acetate-hexane system.
4.1.3.1.
2-(4-Hydroxyphenyl)benzo[d]oxazol-6-ol (14)
Yield 69% as pink solid. 1H NMR (CD3OD, 300 MHz) δ 8.02 (d, J = 9.0 Hz, 2H), 7.47 (d, J = 9.0
Hz, 1H), 7.03 (d, J = 3.0 Hz, 1H), 6.95 (d, J = 9.0 Hz, 2H), 6.86 (dd, J = 9.0 Hz, 3.0 Hz, 1H); MS
(EI) m/z [M]+ 227.
17

ACCEPTED MANUSCRIPT
4.1.3.2.
4-(Benzo[d]oxazol-2-yl)phenol (15)
1
Yield 67% as white solid. Mp 275-276 ºC; H NMR (CD3OD, 300 MHz) δ 8.09 (d, J = 9.0 Hz,
13
2H), 7.69-7.63 (m, 2H), 7.40-7.37 (m, 2H), 6.98 (d, J = 9.0 Hz, 2H); C NMR (acetone-d6, 75
MHz) δ 164.8, 162.4, 152.3, 144.1, 131.1, 126.2, 126.0, 121.1, 120.2, 117.6, 111.9. MS (ESI)
m/z [M+H]+ 212.12. HRMS (ESI)m/z Calculated for C13H9NO2 [M+Na]+234.0531; found
234.0531.
4.1.3.3.
2-Phenylbenzo[d]oxazol-6-ol (16)
1
Yield 65% as pink solid. Mp 110-112 ºC; H NMR (300 MHz, CD3OD) δ 8.18-8.14 (m, 2H),
13
7.57-7.54 (m, 4H), 7.51 (s, 1H), 7.06 (d, J = 3 Hz, 1H), 6.89 (dd, J = 9.0 Hz, 3.0 Hz, 1H); C
NMR (CD3OD, 75 MHz) δ 163.4, 158.1, 153.1, 135.6, 132.6, 130.3, 128.4, 128.2, 120.6, 114.9,
98.4. MS (ESI) m/z [M+Na]+ 234.06. HRMS (ESI)m/z Calculated for C13H9NO2 [M+Na]+
234.0531; found 234.0533.
4.1.3.4.
2-(4-(Trifluoromethyl)phenyl)benzo[d]oxazol-6-ol (17)
1
Yield 68% as white crystal. Mp 238-240 °C; H NMR (CDCl3, 300MHz) δ 8.33 (d, J = 6.0 Hz,
2H), 7.86 (d, J = 9.0 Hz, 2H), 7.56 (d, J = 6.0 Hz, 1H), 7.08 (d, J = 3.0 Hz, 1H), 6.92 (dd, J = 9.0
Hz, 3Hz, 1H); 13C NMR (CD3OD, 75 MHz) δ 161.7,158.6, 153.4, 135.7, 133.5, 132.1, 128.7,
127.3, 121.2, 115.3, 98.3. MS (FAB)m/z [M+H]+ 280.10. HRMS (EI)m/z Calculated for
C14H8F3NO2 [M]+ 279.0507; found 279.0503.
4.1.4. 6-(3-Chloropropoxy)-2-(4-(3-chloropropoxy)phenyl)benzo[d]oxazole (18
)
14 (1 mmol) was dissolved in dry DMF under nitrogen and K2CO3 (10 mmol) was added. The
reaction mixture was allowed to stir for 15 minutes. Thereafter, 1-bromo 3-chloro propane (6
mmol) was introduced and reaction mixture further stirred for 12 hours at room temperature. The
reaction mixture was diluted with excess amount of water and organic layer was extracted using
ethyl acetate. Combined organic layer was dried over sodium sulfate and concentrated under
vacuum. Column chromatography was done in ethyl acetate-hexane system to get the purified
product 18 as white fluffy solid (70%). Mp 59-60 ºC; 1H NMR (CDCl3, 300 MHz) δ 8.14 (d, J =
6.0 Hz, 2H), 7.61 (d, J = 9.0 Hz, 1H), 7.11 (d, J = 3.0 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.94 (dd,
18

ACCEPTED MANUSCRIPT
J = 9.0 Hz, 3 Hz, 1H), 4.23-4.17 (m, 4H), 3.81-3.76 (m, 4H), 2.33-2.25 (m, 4H). MS (ESI) m/z
[M+Na]+ 401.94.
4.1.5. 6-(3-(Pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl)benzo[d]oxazole
(19)
18 (1 mmol) and pyrrolidine (6 mmol) was dissolved in dry DMF in a sealed tube under a N2
atmosphere. The reaction mixture was heated at 110 ºC for 8 hours and diluted with excess
amount of water and the organic part was extracted with 5% Methanol/CHCl3 system. Column
chromatography was done using CHCl3/Methanol system to get the pure product 19 as white
solid (66%). Mp 107-109 °C; 1H NMR (CD3OD, 300 MHz) δ 8.11 (d, J = 9 Hz, 2H), 7.56 (d, J =
9.0 Hz, 1H), 7.25 (d, J = 3.0 Hz, 1H), 7.09 (d, J = 9.0 Hz, 2H), 6.99 (dd, J = 9.0 Hz, 3.0 Hz, 1H),
4.16-4.09 (m, 4H), 2.75-2.62 (m, 12H), 2.11-2.04 (m, 4H), 1.86-1.84 (m, 8H); MS (ESI) m/z
[M+H]+ 450.39. HRMS (ESI) m/z Calculated for C27H35N3O3 [M+H]+ 450.2758; found
450.2759. HPLC Purity 95.7%.
4.1.6. 6-(3-(Piperidin-1-yl)propoxy)-2-(4-(3-(piperidin-1-yl)propoxy)phenyl)benzo[d]oxazole
(20)
18 (1 mmol) and piperidine (6 mmol) was dissolved in dry DMF in a sealed tube under a N2
atmosphere. The reaction mixture was heated to 110 ºC for 8 hours and diluted with excess
amount of water and organic part was extracted with 5% Methanol/CHCl3 system. Column
chromatography was done using CHCl3/Methanol system to get the pure product 20 as off white
solid (69%). Mp 269-270 °C; 1H NMR (CD3OD, 300 MHz) δ 8.14 (d, J = 9.0 Hz, 2H), 7.59 (d, J
= 6.0 Hz, 1H), 7.30 (d, J = 3.0 Hz, 1H), 7.15 (d, J = 9.0 Hz, 2H), 7.03 (dd, J = 9.0 Hz, 3.0 Hz,
1H), 4.23-4.16 (m, 4H), 3.17-3.07 (m, 12H), 2.28-2.21 (m, 4H), 1.83-1.66 (m, 12H). MS (ESI)
m/z [M+H]+ 478.20. HRMS (FAB) m/z Calculated for C29H39N3O3 [M+H]+478.3070; found
478.3069. HPLC Purity 98.9%.
4.1.7. 6-(3-(1H-imidazol-1yl)propoxy)-2-(4-(3-(1H-imidazol-1-yl)propoxy)phenyl)benzo[d]-
oxazole (21
)
NaH (1.6 mmol) was added to the solution of imidazole (1.2 mmol) in dry DMF at 0 °C and the
reaction mixture allowed to stir for 45 minutes at room temperature. The reaction mixture was
19

ACCEPTED MANUSCRIPT
cooled to 0 °C and compound 18 (0.2 mmol) was added under a N2 atmosphere. The reaction
mixture was heated to 60 °C for 10 hours. The organic part was extracted with 5%
Methanol/CHCl3system. Flash column chromatography was done using CHCl3/Methanol system
to get the pure product 21 as off-white solid (70%). Mp 56-58 °C; 1H NMR (CDCl3, 300 MHz) δ
8.13 (d, J = 9.0 Hz, 2H), 7.70 (s, 2H), 7.58 (d, J = 6.0 Hz, 1H), 7.25 (d, J = 3.0 Hz, 1H), 7.19 (s,
2H), 7.12 (d, J = 9.0 Hz, 2H), 7.02 (dd, J = 9.0 Hz, 3 Hz, 3H), 4.32-4.28 (m, 4H), 4.06-4.01 (m,
13
4H), 2.36-2.28 (m, 4H); C NMR (CDCl3, 75 MHz) δ 163.9, 162.9, 158.8, 152.7, 138.7, 136.8,
130.1, 129.3, 120.9, 120.4, 116.1, 114.5, 97.4, 66.4, 65.9, 44.9, 31.8. MS (ESI) m/z [M+Na]+
466.22. HRMS (FAB) m/z Calculated for C25H25N5O3 [M+H]+ 444.2037; found 444.2020. HPLC
Purity 99.3%.
4.1.8. A sealed tube was charged with 18, (0.4 mmol), K2CO3 (4 mmol) and KI (1.6 mmol)
under a N2 atmosphere in dry DMF. 3-Boc amino pyrrolidine (1.6 mmol) was added and the
reaction mixture was heated to 120 ºC for 10 hours. The reaction mixture was diluted with excess
amount of water and organic part was extracted with 5% Methanol/CHCl3 system. The residue
was purified by column chromatography using CHCl3/Methanol system to get 22 as yellow
gummy (72%) semisolid. 1H NMR (CDCl3, 300 MHz) δ 8.08 (d, J = 6.0 Hz, 2H), 7.57 (d, J = 9.0
Hz, 1H), 7.06 (s, 1H), 6.99 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 6.0 Hz, 1H), 4.47 (s, 2H), 4.14 (d, J
= 3.0 Hz, 4H), 3.55-3.38 (m, 12H), 2.53-2.14 (m, 9H), 1.42 (s, 18H); 13C NMR (CDCl3, 75 MHz)
δ 162.3, 161.3, 157.1, 155.4, 151.4, 135.9, 128.8, 119.8, 119.4, 114.7, 112.9, 96.1, 79.2, 66.9,
66.2, 61.1, 52.8, 52.6, 52.5, 49.7, 32.4, 28.4, 28.2. MS (ESI) m/z [M+Na]+ 702.26. HRMS (ESI)
m/z Calculated for C37H53N5O7 [M+Na]+ 702.3843; found 702.3845.
4.1.9. 1-(3-(4-(6-(3-(3-Aminopyrrolidin-1-yl)propoxy)benzo[d]oxazol-2-yl)phenoxy)propyl)
pyrrolidin-3-amine (23
)
22 (1 mmol) was dissolved in dry DCM and trifluoroacetic acid (1 mL) was added to the solution
under ice-cold condition and allowed to stir at room temperature for 2 hours. The solution was
neutralized with 1N NaOH solution keeping it under ice-cold condition. The organic part was
extracted with 10% Methanol/CHCl3 system. Flash column chromatography was done using
1
CHCl3/Methanol/NH3 system to get the pure product 23 as yellow gummy semisolid (72%). H
NMR (CDCl3, 600 MHz) δ 8.12 (d, J = 12.0 Hz, 2H), 7.59 (d, J = 6.0 Hz, 1H), 7.10 (d, J = 6.0
20

ACCEPTED MANUSCRIPT
Hz, 1H), 7.01 (d, J = 12.0 Hz, 2H), 6.93 (dd, J = 12.0 Hz, 6.0 Hz, 1H), 4.12-4.07 (m, 4H), 3.55-
3.51 (m, 2H), 2.77-2.72 (m, 4H), 2.68-2.60 (m, 4H), 2.50-2.46 (m, 2H), 2.39-2.36 (m, 2H), 2.24-
2.18 (m, 2H), 2.05-1.99 (m, 5H), 1.52-1.50 (m, 2H); 13C NMR (150 MHz, CDCl3) δ 162.4,
161.5, 157.3, 151.5, 135.9, 128.9, 119.5, 114.8, 112.9, 96.2, 67.1, 66.4, 64.0, 53.4, 52.9, 50.9,
35.1, 28.5. MS (ESI) m/z [M+H]+ 480.33. HRMS (ESI) m/z Calculated for C27H37N5O3 [M+H]+
480.2976; found 480.2973. HPLC Purity 97.6%
4.1.10. 2-(4-(3-Chloropropoxy)phenyl)benzo[d]oxazole (24
)
15 (1 mmol) was dissolved in dry DMF and K2CO3 (6mmol) was added under nitrogen. The
reaction mixture was allowed to stir for 15 minutes. Thereafter, 1-bromo 3-chloro propane (3
mmol) was introduced and reaction mixture stirred for 10 hours at room temperature. The
reaction mixture was diluted with excess amount of water and organic layer was extracted using
ethyl acetate. Combined organic layer was dried over sodium sulphate and concentrated under
vacuum. Column chromatography was done in ethyl acetate-hexane system to get the purified
product 24 as white fluffy solid (72%). Mp 73-75 °C; 1H NMR (CDCl3, 300 MHz) δ 8.19 (d, J =
9.0 Hz, 2H), 7.70-7.64 (m, 2H), 7.41-7.38 (m, 2H), 7.15 (d, J = 9.0 Hz, 2H), 4.25 (t, J = 6.0 Hz,
2H), 3.80 (t, J = 6.0 Hz, 2H), 2.32-2.24 (m, 2H); 13C NMR (CDCl3,75 MHz) δ 163.0, 161.4,
150.6, 142.2, 129.3, 124.6, 124.4, 119.9, 119.6, 114.8, 110.3, 64.4, 41.3, 32.0. MS (FAB) m/z
[M+H]+288.20. EI-HRMS m/z Calculated for C16H14ClNO2 [M]+ 287.0713; found 287.0710.
4.1.10. 2-(4-(3-Pyrrolidin-1-yl)propoxy)phenyl)benzo[d]oxazole (25
)
24 (1 mmol) and pyrrolidine (6 mmol) were dissolved in dry DMF in a sealed tube under a N2
atmosphere. The reaction mixture was heated to 110 °C for 8 hours and diluted with excess
amount of water and the organic part was extracted with 5% Methanol/CHCl3 system. Column
chromatography was done using CHCl3/Methanol system to get the pure product 25 as light
brown solid (68%). Mp 86-88 °C; 1H NMR (CDCl3, 300 MHz) δ 8.19 (d, J = 9.0 Hz, 2H), 7.75-
7.72 (m, 1H), 7.57-7.54 (m, 1H), 7.34 (t, J = 4.5 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H), 4.12 (t, J =
13
7.5 Hz, 2H), 2.65 (t, J = 6.0 Hz, 2H), 2.54 (m, 4H), 2.06-2.00 (m, 2H), 1.83-1.78 (m, 4H); C
NMR (CDCl3, 75 MHz) δ 163.2, 161.7, 150.6, 142.2, 129.3, 124.5, 124.3, 119.5, 114.8, 110.3,
66.5, 54.2, 52.9, 28.5, 23.4. MS (ESI) m/z [M+H]+ 323.20. HRMS (EI) m/z Calculated for
C20H22N2O2 [M]+ 322.1681; found 322.1683. HPLC Purity 95.6%.
21

ACCEPTED MANUSCRIPT
4.1.10. 6-(3-Chloropropoxy)-2-phenylbenzo[d]oxazole (26
)
16 (1 mmol) was dissolved in dry DMF and K2CO3 (6mmol) was added under nitrogen. The
reaction mixture was allowed to stir for 15 minutes. Thereafter, 1-bromo 3-chloro propane (3
mmol) was introduced and reaction mixture stirred for 10 hours at room temperature. The
reaction mixture was diluted with excess amount of water and organic layer was extracted using
ethyl acetate. Combined organic layer was dried over sodium sulfate and concentrated under
vacuum. Column chromatography was done in ethyl acetate-hexane system to get the purified
1
product 26 as white fluffy solid (76%). Mp 61-63°C; H NMR (300 MHz, CDCl3) δ 8.22-8.20
(m, 2H), 7.65 (d, J = 6.0 Hz, 1H), 7.52 (t, J = 3.0 Hz, 3H), 7.14 (d, J = 3.0 Hz, 1H), 6.97 (dd, J =
9.0 Hz, 3Hz, 1H), 4.20 (t, J = 3.0 Hz, 2H), 3.79 (t, J = 3.0 Hz, 2H), 2.32-2.28 (m, 2H); 13C NMR
(75 MHz, CDCl3) δ 162.4, 157.3, 151.6, 136.1, 131.1, 128.9, 127.2, 120.0, 113.3, 96.2, 65.1,
41.4, 32.2. MS (FAB) m/z [M+H]+ 288.1. HRMS (EI) m/z Calculated for C16H14ClNO2 [M]+
287.0713; found 287.0707.
4.1.11. 6-(3-Chloropropoxy)-2-(4-trifluoromethyl)phenyl)benzo[d]oxazole (27
)
17 (1 mmol) was dissolved in dry DMF and K2CO3 (6mmol) was added under nitrogen. The
reaction mixture was allowed to stir for 15 minutes. Thereafter, 1-bromo 3-chloro propane (3
mmol) was introduced and reaction mixture stirred for 10 hours at room temperature. The
reaction mixture was diluted with excess amount of water and organic layer was extracted using
ethyl acetate. Combined organic layer was dried over sodium sulfate and concentrated under
vacuum. Column chromatography was done in ethyl acetate-hexane system to get the purified
1
product 27 as white solid (66%). Mp 68-70 °C; H NMR (CDCl3, 300 MHz) δ 8.32 (d, J = 6.0
Hz, 2H), 7.78 (d, J = 9.0 Hz, 2H), 7.68 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 3.0 Hz, 1H), 7.00 (dd, J =
9.0 Hz, 3 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.80 (t, J = 7.5 Hz, 2H), 2.34-2.26 (m, 2H); 13C NMR
(CDCl3, 75 MHz) δ 160.7, 157.8, 151.7, 135.8, 132.7, 130.5, 127.3, 125.9, 125.5, 120.4, 113.8,
96.1, 65.0, 41.4, 32.1. MS (FAB) m/z [M+H]+356.10. HRMS (FAB)m/z Calculated for
C17H13ClF3NO2 [M+H]+ 356.0667; found 356.0673.
4.1.12. 6-(3-(Pyrrolidin-1-yl)propoxy)-2-phenylbenzo[d]oxazole (28
)
22

ACCEPTED MANUSCRIPT
26 (1 mmol) and pyrrolidine (6 mmol) was dissolved in dry DMF in a sealed tube under a N2
atmosphere. The reaction mixture was heated to 110 °C for 8 hours and diluted with excess
amount of water and the organic part was extracted with 5% Methanol/CHCl3 system. Column
chromatography was done using CHCl3/Methanol system to get the pure product 28 as pale solid
(65%). Mp 65-68 °C; 1H NMR (300 MHz, CD3OD) δ 8.22-8.18 (m, 2H), 7.64-7.57 (m, 4H), 7.31
(d, J = 3.0 Hz, 1H), 7.04 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 4.15 (t, J = 6.0 Hz, 2H), 2.85-2.72 (m,
13
6H), 2.13-2.06 (m, 2H), 1.92-1.87 (m, 4H); C NMR (75 MHz, CD3OD) δ 163.9, 159.6, 153.1,
132.8, 130.3, 128.3, 120.8, 115.1, 97.4, 68.1, 55.3, 54.4, 29.4, 24.3. MS (EI) m/z [M]+ 322.
HRMS (EI) m/z Calculated for C20H22N2O2 [M]+ 322.1681; found 322.1682. HPLC Purity
97.0%.
4.1.13. 6-(3-(Pyrrolidin-1-yl)propoxy)-2-(4-(trifluoromethyl)phenyl)benzo[d]oxazole (29
)
27 (1 mmol) and pyrrolidine (6 mmol) was dissolved in dry DMF in a sealed tube under a N2
atmosphere. The reaction mixture was heated to 110°C for 8 hours and diluted with excess
amount of water and the organic part was extracted with 5% Methanol/CHCl3 system. Column
chromatography was done using CHCl3/Methanol system to get the pure product 29 as brown
solid (71%). Mp 80-82°C; 1H NMR (CD3OD, 300 MHz) δ 8.37 (d, J = 9.0 Hz, 2H), 7.88 (d, J =
6.0 Hz, 2H), 7.65 (d, J = 6.0 Hz, 1H), 7.31 (d, J = 3.0 Hz, 1H), 7.05 (dd, J = 9.0 Hz, 3 Hz, 1H),
4.14 (t, J = 6.0 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.69 (m, 4H), 2.14-2.05 (m, 2H), 1.88 (m, 4H);
13C NMR (150 MHz, CDCl3) δ 160.6, 158.2, 151.8, 135.6, 132.6, 132.4, 130.6, 127.4, 125.9,
120.4, 113.9, 96.1, 67.1, 54.3, , 53.1, 28.6, 23.5. MS (ESI) m/z [M+H]+ 391.14. HRMS (EI) m/z
Calculated for C21H21F3N2O2 [M]+ 390.1555; found 390.1556. HPLC Purity 97.1%.
4.1.14. 6-(3-(1H-imidazol-1-yl)propoxy)-2-phenylbenzo[d]oxazole (30
)
NaH (0.8 mmol) was added to the solution of imidazole (0.4 mmol) in dry DMF at 0 °C and the
reaction mixture allowed to stir for 45 minutes at room temperature. The reaction mixture was
cooled to 0 °C and 26 (0.2 mmol) was added under a N2 atmosphere. The reaction mixture was
heated to 60 °C for 10 hours. The organic part was extracted with 5% Methanol/CHCl3 system.
Column chromatography was done using CHCl3/Methanol system to get the pure product as pale
color solid (64%). Mp 113-115 °C; 1H NMR (CDCl3, 300 MHz,) δ 8.22-8.19 (m, 2H), 7.65 (d, J
= 9.0 Hz, 1H), 7.53-7.51 (m, 4H), 7.08 (s, 2H), 6.97-6.94 (m, 2H), 4.24 (t, J = 7.5 Hz, 2H), 3.98
23

ACCEPTED MANUSCRIPT
(t, J = 6.0 Hz, 2H), 2.33-2.24 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 162.5, 156.9, 151.6, 136.3,
131.2, 129.8, 128.9, 127.3, 120.2, 118.9, 113.1, 96.3, 64.6, 43.4, 30.8. MS (EI) m/z [M+H]+319.
HRMS (FAB) m/z Calculated for C19H17N3O2[M+H]+ 320.1401; found 320.1407. HPLC Purity
96.2%.
4.1.15.1.
N-(2,4-Dimethoxyphenyl)-3-nitrobenzamide (33)
31 (3 mmol) and carbonyl diimidazole (4.5 mmol) was dissolved in dry acetonitrile under a N2
atmosphere. The reaction mixture was stirred for 30 minutes and the amine 6 (3.3 mmol) was
added. The reaction mixture was stirred for 12 hrs. Then acetonitrile was concentrated in
vacuum. The residue was diluted with water and the organic part was extracted using ethyl
acetate. The combined organic layer was dried over sodium sulfate and concentrated. The residue
was purified by flash column chromatography using ethyl acetate-hexane system to give 33 as
1
yellow crystal (73%). Mp 146-148 °C; H NMR (CDCl3, 300 MHz) δ 8.71 (s, 1H), 8.41-8.34
(m, 3H), 8.24 (d, J = 9.0 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 6.56-6.54 (m, 2H), 3.92 (s, 3H), 3.83
13
(s, 3H); C NMR (CDCl3, 150 MHz) δ 162.5, 157.1, 149.6, 148.4, 137.1, 133.1, 129.9, 126.1,
121.9, 121.0, 120.6, 103.9, 98.7, 55.9, 55.6. MS (ESI) m/z [M+Na]+ 325.26. HRMS (EI) m/z
Calculated for C15H14N2O5 [M]+ 302.0903; found 302.0906.
4.1.15.2.
3-Bromo-N-(2,4-dimethoxyphenyl)benzamide (34)
32 (3 mmol) and carbonyl diimidazole (4.5 mmol) were dissolved in dry acetonitrile under a N2
atmosphere. The reaction mixture was stirred for 30 minutes and the amine 6 (3.3 mmol) was
added. The reaction mixture was stirred for 12 hrs. Then acetonitrile was concentrated in
vacuum. The residue was diluted with water and the organic part was extracted using ethyl
acetate. The combined organic layer was dried over sodium sulfate and concentrated. The residue
was purified by flash column chromatography using ethyl acetate-hexane system to give the
compound 34 as off white solid (64%). Mp 100-101 °C; 1H NMR (CD3OD, 300 MHz) δ 8.36 (d,
J = 9.0 Hz, 1H), 8.25 (s, 1H), 8.01 (s, 1H), 7.78 (d, J = 6.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H),
7.36 (t, J = 7.5 Hz, 1H), 6.54-6.51 (m, 2H), 3.90 (s, 3H), 3.82 (s, 3H); 13C NMR (CDCl3, 75
MHz) δ 163.4, 156.7, 149.6, 137.4, 134.4, 130.2, 125.4, 122.9, 120.8, 103.8, 98.6, 55.8, 55.5. MS
(ESI) m/z [M+Na]+ 358.13. HRMS (EI) m/z Calculated for C15H14BrNO3 [M]+ 335.0157; found
335.0154.
24

ACCEPTED MANUSCRIPT
4.1.15.3.
N-(2,4-Dihydroxyphenyl)-3-nitrobenzamide (35)
The amide 33 (3 mmol) was dissolved in 10 mL of dry DCM in a two-necked R.B flask. BBr3 (8
mmol) was added carefully under ice-cold condition. The reaction was allowed to stir at room
temperature for 30 minutes. Ice crystal was added to the reaction mixture followed by few drops
of methanol under ice-cold condition. The precipitate obtained was filtered and dried to get 35 as
yellow solid (62%). Mp 219-220 °C;1H NMR (CD3OD, 300 MHz) δ 8.81 (s, 1H), 8.45 (d, J = 9.0
Hz, 1H), 8.35 (d, J = 9.0 Hz, 1H), 7.79 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 6.44 (d, J =
3.0 Hz, 1H), 6.35 (dd, J = 9.0 Hz, 3.0 Hz, 1H); 13C NMR (CD3OD, 150Hz) δ 164.9, 156.5, 151.3,
148.3, 136.2, 133.1, 129.7, 125.7, 125.5, 122.3, 116.9, 106.2, 102.9. MS (ESI) m/z [M+Na]+
297.23. HRMS (EI) Calculated for C13H10N2O4 [M]+ 274.0590; found 274.0588.
4.1.15.4.
3-Bromo-N-(2,4-dihydroxyphenyl)benzamide (36)
The amide 34 (3 mmol) was dissolved in 10 mL of dry DCM in a two-necked R.B flask. BBr3 (8
mmol) was added carefully under ice-cold condition. The reaction was allowed to stir at room
temperature for 30 minutes. Crushed ice was added to the reaction mixture followed by few
drops of methanol under ice-cold condition. The precipitate obtained was filtered and dried to get
36 as light pink solid (58%). Mp 235 °C.1H NMR (CD3OD, 300 MHz) δ 8.13 (s, 1H), 7.93 (d, J
= 9.0 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 6.43 (d,
J = 3.0 Hz, 1H), 6.35 (dd, J = 9.0 Hz, 3.0 Hz, 1H); 13C NMR (CD3OD, 75 MHz) δ 167.9, 158,4,
153.1, 139.8, 138.7, 136.6, 132.4, 130.8, 130.1, 128.1, 127.2, 124.5, 119.5, 108.5, 105.4. MS ESI
m/z [M+Na]+ 332.26. HRMS (EI) m/z Calculated for C13H10BrNO3 [M]+ 306.9844; found
306.9842.
4.1.15.5.
2-(3-Nitrophenyl)benzo[d]oxazol-6-ol (37)
Acetic acid (1 mmol) was added to 35 (1 mmol) and the mixture was refluxed for 24 hours. The
acid was evaporated and the reaction mixture was diluted with water and organic part was
extracted using ethyl acetate. Combined organic layer was dried over sodium sulfate and
concentrated. Flash chromatography was done using ethyl acetate-hexane system to get the pure
1
product 37 as yellow solid (64%). Mp 251-553 °C; H NMR (CD3OD, 300 MHz) δ 8.97 (t, J =
3.0 Hz, 1H), 8.55 (d, J = 9.0 Hz, 1H), 8.42 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H),
25

ACCEPTED MANUSCRIPT
13
7.59 (d, J = 9.0 Hz, 1H), 7.11 (d, J = 3.0 Hz, 1H), 6.93 (dd, J = 9.0 Hz, 3.0 Hz, 1H); C NMR
(CD3OD, 75 MHz) δ 167.5, 160.8, 158.5, 153.3, 149.8, 136.5, 134.1, 131.2, 128.3, 125.4, 122.6,
121.2, 115.3, 98.3. MS (ESI) m/z [M+Na]+ 279.23. HRMS (EI) m/z Calculated for C13H8N2O4
[M]+ 256.0484; found 256.0474.
4.1.15.6.
2-(3-Bromophenyl)benzo[d]oxazol-6-ol (38)
Acetic acid (1 mmol) was added to the compound 36 (1 mmol) and the mixture was refluxed for
48 hours. The acid was evaporated and the reaction mixture was diluted with water and organic
part was extracted using ethyl acetate. Combined organic layer was dried over sodium sulfate and
concentrated. Flash chromatography was done using ethyl acetate-hexane system to get the pure
1
product 38 as off white crystal (67%): mp 268-269 °C; H NMR (CD3OD, 300 MHz) δ 8.32 (s,
1H), 8.15 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 6.0 Hz, 1H), 7.56-7.47 (m, 2H), 7.08 (d, J = 3.0 Hz,
1H), 6.91 (dd, J = 9.0 Hz, 3 Hz, 1H); 13C NMR (Acetone-d6, 75 MHz) δ 159.7, 156.5, 151.8,
134.9, 133.7, 131.0, 129.5, 129.3, 125.6, 122.4, 120.2, 113.6, 97.11. MS (ESI) m/z [M+H]+
290.06. HRMS (EI) m/z Calculated for C13H8BrNO2 [M]+ 288.9738; found 288.9737.
4.1.15.7.
6-(3-Chloropropoxy)-2-(3-nitrophenyl)benzo[d]oxazole (39)
37 (1 mmol) was dissolved in dry DMF and K2CO3 (6mmol) was added under nitrogen. The
reaction mixture was allowed to stir for 15 minutes. Thereafter, 1-bromo 3-chloro propane (3
mmol) was introduced and reaction mixture stirred for 10 hours at room temperature. The
reaction mixture was diluted with excess amount of water and organic layer was extracted using
ethyl acetate. Combined organic layer was dried over sodium sulfate and concentrated under
vacuum. Flash column chromatography was done in ethyl acetate-hexane system to get the
1
purified product 39 as yellow solid (66%). Mp 113-115 °C; H NMR (CDCl3, 300 MHz) δ 9.06
(t, J = 3.0 Hz, 1H), 8.55 (d, J = 9.0 Hz, 1H), 8.38 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 7.76-7.68 (m,
2H), 7.18 (d, J = 3.0 Hz, 1H), 7.03 (dd, J = 9.0 Hz, 3.0 Hz, 1H), 4.23 (t, J = 6.0 Hz, 2H), 3.82 (t,
J = 7.5 Hz, 2H), 2.36-2.28 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 159.8, 157.9, 151.7, 148.6,
135.7, 132.5, 130.0, 128.9, 125.3, 121.9, 120.5, 113.9, 96.1, 65.1, 41.4, 32.1. MS (ESI) m/z
[M+Na]+ 355.28. HRMS (ESI) m/z Calculated for C16H13ClN2O4 [M+Na]+ 355.0462; found
355.0451.
26

ACCEPTED MANUSCRIPT
4.1.15.8.
2-(3-Bromophenyl)-6-(3-chloropropoxy)benzo[d]oxazole (40)
38 (1 mmol) was dissolved in dry DMF and K2CO3 (6 mmol) was added under nitrogen. The
reaction mixture was allowed to stir for 15 minutes. Thereafter, 1-bromo 3-chloro propane (3
mmol) was introduced and reaction mixture stirred for 10 hours at room temperature. The
reaction mixture was diluted with excess amount of water and organic layer was extracted using
ethyl acetate. Combined organic layer was dried over sodium sulfate and concentrated under
vacuum. Flash column chromatography was done in ethyl acetate-hexane system to get the
purified product 40 as white crystal (69%). Mp 61-63 °C; 1H NMR (CDCl3, 300 MHz) δ 8.35 (t,
J = 3.0 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.65-7.61 (m, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.12 (d, J =
3.0 Hz, 1H), 6.97 (dd, J = 9.0 Hz, 3 Hz, 1H), 4.18 (t, J = 6.0 Hz, 2H), 3.79 (t, J = 6.0 Hz, 2H),
2.42-2.25 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 160.7, 157.6, 151.6, 135.8, 133.9, 130.4, 130.0,
129.1, 125.6, 122.9, 120.2, 113.6, 96.1, 65.0, 41.5, 32.2. MS (ESI) m/z [M+H]+ 366.09. HRMS
(ESI) m/z Calculated for C16H13BrClNO2 [M+H]+ 365.9898; found 365.9898.
4.1.15.9.
2-(3-Nitrophenyl)-6-(3-(pyrrolidin-1-yl)propoxy)benzo[d]oxazole (41)
39 (1 mmol) and pyrrolidine (6 mmol) was dissolved in dry DMF in a sealed tube under nitrogen
atmosphere. The reaction mixture was heated to 110 °C for 8 hours and diluted with excess
amount of water and the organic part was extracted with 5% Methanol/CHCl3 system. Column
chromatography was done using CHCl3/Methanol system to get the pure product 41 as yellow
solid (67%). Mp 234-235 °C; 1H NMR (CDCl3, 300 MHz) δ 9.03 (t, J = 3.0 Hz, 1H), 8.52 (d, J =
6.0 Hz, 1H), 8.36 (dd, J = 9.0 Hz, 3 Hz, 1H), 7.74-7.66 (m, 2H), 7.14 (d, J = 3.0 Hz, 1H), 6.98
(dd, J = 9.0 Hz, 3 Hz, 1H), 4.16 (t, J = 6 Hz, 2H), 3.10-3.05 (m, 6H), 2.37-2.29 (m, 2H), 2.05(m,
4H); 13C NMR (CDCl3, 75 MHz) δ 159.7, 158.1, 151.7, 148.6, 132.5, 130.0, 129.0, 125.3, 121.9,
120.4, 113.9, 96.0, 73.9, 66.7, 54.1, 53.0, 27.9, 23.4. MS (ESI) m/z [M+H]+ 368.27. HRMS (ESI)
m/z Calculated for C20H21N3O4 [M+H]+ 368.1612; found 368.1609.
4.1.15.10.
2-(3-Bromophenyl)-6-(3-(pyrrolidin-1-yl)propoxy)benzo[d]oxazole (42)
40 (1 mmol) and pyrrolidine (6 mmol) was dissolved in dry DMF in a sealed tube under nitrogen
atmosphere. The reaction mixture was heated to 110 °C for 8 hours and diluted with excess
amount of water and the organic part was extracted with 5% Methanol/CHCl3 system. Column
chromatography was done using CHCl3/Methanol system to get the pure product 42 as light
27

ACCEPTED MANUSCRIPT
brown solid (71%). Mp 77-78 °C; 1H NMR (CD3OD, 300 MHz) δ 8.33-8.32 (m, 1H), 8.16 (d, J
= 6.0 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.31 (d,
J = 3.0 Hz, 1H), 7.04 (dd, J = 9.0 Hz, 3 Hz, 1H), 4.15 (t, J = 6.0 Hz, 2H), 2.84-2.72 (m, 6H),
13
2.15-2.06 (m, 2H), 1.91-1.88 (m, 4H); C NMR (CDCl3, 75 MHz) δ 160.6, 157.9, 151.6, 135.6,
133.8, 130.4, 130.0, 129.2, 125.6, 122.9, 120.1, 113.6, 96.1, 67.1, 54.2, 53.1, 28.5, 23.4. MS ESI
m/z [M+H]+ 400.89. HRMS (ESI) m/z Calculated for C20H21BrN2O2 [M+H]+ 401.0866; found
401.0864.
4.1.15.11.
3-(6-(3-(Pyrrolidin-1-yl)propoxy)benzo[d]oxazole-2-yl)aniline (43)
Pd/C was added to a solution of compound 41 in methanol. The reaction mixture was allowed to
stir at room temperature under hydrogen atmosphere for 2 hours. After completion of reaction,
the reaction mixture was filtered through celite bed using methanol. Flash column
chromatography was done using CHCl3/Methanol system to get the pure product 43 as yellow
solid (63%). Mp 90-92 °C; 1H NMR (CDCl3, 300 MHz) δ 7.60 (t, J = 7.5 Hz, 2H), 7.53 (m, 1H),
7.30(d, J = 6.0 Hz, 1H), 7.09 (d, J = 3.0 Hz, 1H), 6.93 (dd, J = 9.0 Hz, 3 Hz, 1H), 6.82 (dd, J =
9.0 Hz, 3 Hz, 1H), 4.11 (t, J = 6.0 Hz, 2H), 2.92-2.84 (m, 6H), 2.27-2.16 (m, 2H), 1.94 (m, 4H);
13C NMR (CD3OD, 75 MHz) δ 164.4, 159.2, 152.8, 150.1, 136.5, 130.9, 128.8, 120.5, 119.4,
117.6, 114.8, 114.3, 97.2, 67.9, 55.2, 54.2, 29.1, 24.3. MS (ESI) m/z [M+H]+ 338.36. HRMS
(ESI) m/z Calculated for C20H23N3O2 [M+ H]+ 338.1870; found 338.1868.
4.1.16. General Procedure for the Synthesis of 44, 45, 46 and 47
The acid (2 mmol) and HATU (1.5 mmol) were dissolved in dry DMF and DIPEA (4 mmol) was
added it. After 15 minutes respective amine (1 mmol) was added and the reaction mixture stirred
at room temperature for 4-5 hours. The organic part was extracted with 5% Methanol/CHCl3
system. Column chromatography was done using CHCl3/Methanol system to get the pure
product.
4.1.16.1.
N-(3-(6-(3-(Pyrrolidin-1-yl)propoxy)benzo[d]oxazol-2-yl)phenyl)benzamide (44)
1
Yellow gummy semisolid (72%). H NMR (CD3OD, 300 MHz) δ 8.63 (s, 1H), 8.01-7.96 (m,
3H), 7.89 (d, J = 9.0 Hz, 1H), 7.65-7.53 (m, 5H), 7.31 (s, 1H), 7.05 (d, J = 3.0 Hz, 1H), 4.19 (t, J
= 6.0 Hz, 2H), 3.28 (m, 6H), 2.28-2.20 (m, 2H), 2.10-2.06 (m, 4H); 13C NMR (CD3OD, 75 MHz)
28