Design, synthesis and biological evaluation of bivalent benzoxazolone and benzothiazolone ligands as potential anti-inflammatory/analgesic agents

Article abstract of DOI:10.1016/j.bmc.2015.04.057

Abstract Benzoxazolone and benzothiazolone were used as template blocks to develop two series of dimers as anti-inflammatory and analgesic agents based on the concept of bivalent ligands. The first series (I) involved varying the carbon chain lengths extending from the piperazine core to the nitrogen atom of the dibenzo[d]oxazol-2(3H)-one or dibenzo[d]thiazol-2(3H)-one. The second series (II) was designed by changing the attachment point. All compounds were screened for their in vitro anti-inflammatory activity in terms of the inhibition of inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NF-κB). Seventeen compounds inhibited both targets. Eleven of them exhibited IC₅₀ values below 3 μM while five compounds showed IC₅₀ values of 1 μM or below. Most of the compounds were found to be devoid of cytotoxicity against mammalian kidney and solid tumors cell lines up to 25 μg/mL. In vivo anti-inflammatory and antinociceptive studies revealed that compounds 3j, 5t and 8b have significant anti-inflammatory and analgesic activity comparable to that of indomethacin and ketorolac, respectively.

Full text of DOI:10.1016/j.bmc.2015.04.057

Accepted Manuscript
Design, synthesis and biological evaluation of bivalent benzoxazolone and ben-
zothiazolone ligands as potential anti-inflammatory/analgesic agents
Ahmed H. Abdelazeem, Shabana I. Khan, Stephen W. White, Kenneth J. Sufka,
Christopher R. McCurdy
PII:
S0968-0896(15)00365-X
http://dx.doi.org/10.1016/j.bmc.2015.04.057
BMC 12269
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 February 2015
9 April 2015
20 April 2015
Please cite this article as: Abdelazeem, A.H., Khan, S.I., White, S.W., Sufka, K.J., McCurdy, C.R., Design, synthesis
and biological evaluation of bivalent benzoxazolone and benzothiazolone ligands as potential anti-inflammatory/
analgesic agents, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.057
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Design, Synthesis and Biological Evaluation of Bivalent Benzoxazolone
and Benzothiazolone Ligands as Potential Anti-inflammatory/Analgesic
Agents
1
Ahmed H. Abdelazeem , Shabana I. Khan , Stephen W. White , Kenneth J. Sufka ,
2
3
2, 3
2, 4
and Christopher R. McCurdy*
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514,
2
Egypt. National Center for Natural Products Research, School of Pharmacy, The University of
3
Mississippi, MS 38677, USA. Department of Psychology, College of Liberal Arts, The University of
4
Mississippi, MS 38677, USA, Department of BioMolecular Sciences, School of pharmacy, The
University of Mississippi, MS 38677, USA.
Running title: Piperazine Dimers Targeting iNOS and NF-κB
*
To whom correspondence should be addressed: Department of BioMolecular Sciences,
School of Pharmacy, The University of Mississippi, MS 38677, USA
Tel.: +1 662 915 5882
Fax: +1 662 915 5638
E-mail address: cmccurdy@olemiss.edu (C.R. McCurdy, Ph.D.).
1

ABSTRACT
Benzoxazolone and benzothiazolone were used as template blocks to develop two series of
dimers as anti-inflammatory and analgesic agents based on the concept of bivalent ligands.
The first series (I) involved varying the carbon chain lengths extending from the piperazine
core to the nitrogen atom of the dibenzo[d]oxazol-2(3H)-one or dibenzo[d]thiazol-2(3H)-one.
The second series (II) was designed by changing the attachment point. All compounds were
screened for their in vitro anti-inflammatory activity in terms of the inhibition of inducible
nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-κB). Seventeen compounds
inhibited both targets. Eleven of them exhibited IC50 values below 3 µM while five
compounds showed IC50 values of 1 µM or below. Most of the compounds were found to be
devoid of cytotoxicity against mammalian kidney and solid tumors cell lines up to 25µg/mL.
In vivo anti-inflammatory and antinociceptive studies revealed that compounds 3j, 5t and 8b
have significant anti-inflammatory and analgesic activity comparable to that of indomethacin
and ketorolac, respectively.
Graphical abstract
Keywords:
Anti-inflammatory/analgesic, Nociception, iNOS, NF-κB, benzoxazolone, benzothiazolone,
piperazine, dimers
2

1
. Introduction
Inflammation is a multifactorial process caused by various stimuli including physical
1
damage, irradiation, microbial infections and immune reactions. There are several molecular
targets recognized from the inflammation cascades that could be antagonized to block the
2
output of these cascades. The major anti-inflammatory targets include cyclooxygenases
(
COXs), lipoxygenases (LOXs), nitric oxide synthases (NOS) enzymes, nuclear factor kappa
3
B (NF-κB), cytokines, cytokine receptors, tumor necrosis factor (TNF-α), chemokines,
4
interferons (IFNs) and many other targets. Over the past few decades, three isoforms of
nitric oxide synthase have been identified and found to catalyze the formation of nitric oxide
5
(
NO) from arginine. Of the three isoforms, inducible nitric oxide synthase (iNOS), which is
responsible for the overproduction of NO, has been involved in a number of inflammatory
diseases such as rheumatoid arthritis, asthma, inflammatory bowel disease and more recently,
6
,7
in neuropathic pain. Therefore, selective inhibition of iNOS would be a valid approach and
prime target for the reduction of inflammation and alleviation of the associated pain. On the
other hand, NF-κB represents a group of structurally related and evolutionarily conserved
proteins that are responsible for the regulation of the expression of several genes involved in
8
,9
inflammatory response, including iNOS and dynorphins at the transcriptional level. In
addition, it has been established that NF-kB signaling pathways promote the gene expression
1
0
of many proinflammatory cytokines as well as tightly controlling COX-2 expression.
Several studies have indicated the contribution of components of the NF-κB signaling
1
1
pathways in the pathogenesis of chronic inflammatory diseases especially, rheumatic ones.
Furthermore, there has been much evidence that a strong relationship exists between the
1
2
pathological processes of inflammation and cancer. NF-kB was found to be the central
molecular link between inflammation and carcinogenesis and its activation plays a pivotal
1
3
role in growth and progression of inflammation-induced cancer. Moreover, many
proinflammatory cytokines and chemokines in the tumor microenvironment which activate
the IKKβ-dependent NF-κB signaling pathway have been demonstrated to be associated with
1
4,15
cancer development and progression.
Based on the critical role of the inflammatory
microenvironment in progression of several cancer types, it was proposed that targeting the
inflammatory components rather than the malignant cell may offer a better therapeutic
1
2,16,17
approach that could prevent the quick mutation and acquired drug resistance.
Numerous
reports have demonstrated that NF-κB can stimulate cell proliferation, enhance angiogenesis,
suppress apoptosis, and promote metastasis via inducing the expression of diverse target
3

1
8
genes. Thus, it was suggested that the inhibition of NF-κB activation and its upstream
1
8
signaling pathway may warrant a potential cancer therapy strategy. However, several
challenges may counteract the using of NF-kB inhibitors in clinical practice. First, the
inhibition of NF-kB may impair immune functions since NF-kB activation is important for
1
9
maintaining both innate and acquired immunity. Also, the mechanisms that leads to the
activation of IKK-β and NF-kB in both malignant and the inflammatory components of
1
2
cancer need to be more clearly identified and fully understood.
To date, Non-steroidal anti-inflammatory drugs (NSAIDs) continue to be the most commonly
2
0
utilized medications for inflammation and pain. Concomitantly, NSAIDs are known to have
several serious adverse effects such as gastric ulceration, renal injury and recently,
2
1
cardiotoxicity. Therefore, there remains a substantial need for researchers to find new
compounds devoid of these side-effects. In an attempt to overcome these associated
liabilities, we herein, tried to introduce iNOS and NF-κB dual inhibitors as a potential
strategy for the development of anti-inflammatory/analgesic agents without serious side
effects. Our design of these agents was based on the previously reported compound (I), S-
1
4080, that has anti-inflammatory and antinociceptive activities superior to acetylsalicylic
acid and equivalent to glafenine. Compound (I) was found to inhibit the arachidonic acid
2
2
inflammatory cascade. Moreover, scaffold (II) was found to have a significant analgesic
2
3
activity. From the study of the structural features of the aforementioned compounds,
benzoxazolonone and benzothiazolonone heterocycles in addition to the piperazine core are
important for both anti-inflammatory and analgesic activities In this context, we decided to
incorporate these moieties in one template structure (III) utilizing the fragment-based drug
design concept (Fig. 1). Consequently, to investigate the effect of carbon linker length
between the piperazine core and the heterocyclic moiety on the activity, we synthesized two
series of dimers, homodimers and heterodimers, and varied the length of carbon chain from
two to six carbons.
2
. Results and discussion
2
.1. Chemistry
The synthesis of these compounds was facile and straightforward as outlined in Scheme 1.
The first series was designed as homodimers with equal length carbon chains around
piperazine ring. Commercially available 2(3H)-benzoxazolone and 2(3H)-benzothiazolone
were N-alkylated with various dibromoalkanes to give intermediates 2a-j. Then, reaction of
two equivalents of 2a-j with one equivalent of piperazine was performed to give the targeted
4

compounds 3a-j that finally separated as hydrochloride salts for biological testing. While the
second series was designed as heterodimers where the carbon chains around piperazine ring
were different. The series 3a-j will aid in understanding the structure activity relationship for
the inhibition of iNOS and NF-κB activities. The synthesis of these compounds is also
outlined in Scheme 1. N-alkylation of 2(3H)-benzoxazolone or 2(3H)-benzothiazolone by
various dibromoalkanes coupled with an equimolar amount of piperazine afforded
intermediates 4a-h. These intermediates were then treated with bromoalkyl benzoxazolones
or bromoalkyl benzthiazolones 2c-j to give the desired heterodimers 5a-t. Finally,
dihydrochloride salts were formed for biological testing.
To explore the role of the heterocycle attachment position, a series of S-alkyltaed compounds
were designed. Scheme 2 outlines the synthesis of the S-alkylated compounds wherein
dibromobutane was reacted with the previously thionated 2(3H)-benzoxazolone or 2(3H)-
benzothiazolone with Lawesson’s reagent to give intermediates 7a-b. The dibromobutane
was directed to the more reactive 2-thiocarbonyl. Subsequently, intermediates 7a-b were
utilized to alkylate the piperazine ring resulting in compounds 8a-b.
2
.2. Pharmacology
All the newly synthesized compounds, 3a-3j, 5a–5t and 8a-8b were screened through cellular
assays for their anti-inflammatory/analgesic potential based on their inhibitory activities on
two targets; iNOS and NF-κB. The results are expressed in terms of IC50 values (the
concentration that caused a 50% inhibition) and presented in Table 1. In addition, the
cytotoxicity towards a panel of mammalian cell lines was also determined. Based on the in
vitro results, three compounds 3j, 5t and 8b were selected for in vivo efficacy study. The
acetic acid induced-writhing test was used for testing the analgesic activity and the
carrageenan-induced assay was used for assessment of the anti-inflammatory activity.
2
.2.1. iNOS enzyme inhibitory assay
The iNOS inhibitory assay was performed in LPS-induced mouse macrophages (RAW264.7)
where the concentration of (NO) was determined by measuring the level of nitrite in the cell
2
4
culture supernatant using Griess reagent. L-N-monomethyl Arginine (L-NMMA) was used
as positive control. The homodimers 3h and 3j which contain benzothiazolone nucleus and
the longest alkyl chains displayed the most potent inhibition of iNOS in this series with IC50
values of 0.41 and 0.28 µM, respectively (Table 1). On the other hand, the corresponding
5

benzoxazolone dimers 3g and 3i were not as potent (IC50, 4.4 µM and 4.2 µM) although they
have equal alkyl chains. The rest of the homodimer series, 3a-3f, with shorter alkyl chains did
not show any activity with the exception of 3d (IC50, 4.6 µM). Subsequently, structure-
activity relationship studies were performed to deduce how the variation of the alky chain
length could affect the activity. In this regard, a series of heterodimers were synthesized and
evaluated for their potential to inhibit iNOS activity. Out of the benzothiazolone
heterodimers, two compounds 5r and 5t were the most active with IC50 values of 0.51 and
0
.29 µM, respectively, while others (5h, 5l, 5n and 5p) with shorter alkyl chains were not as
effective (IC50 values in the range of 1.44 - 3.43 µM). On the other hand, the benzoxazolone
dimers 5q and 5s with the same alkyl chains length showed activity with IC50 values of 1.77
and 2.9 µM, respectively. However, other heterodimers were found to show weak activity
(
5e-5g, 5j-5k, 5m, 5o) or they were devoid of any activity (5a-5d and 5i). It was worth noting
that the difference in attachment point of the alkyl chain to the heterocycle significantly
affected the activity. Compounds 8a and 8b showed better iNOS inhibitory activities with
IC50 values 1.12 and 0.41 µM, respectively. On the contrary, compounds 3e and 3f with the
same alkyl chains length, attached to the nitrogen not to the sulfur atom, displayed no
activity.
2
.2.2. Inhibition of NF-κB transcriptional activity
The effect of compounds on the transcriptional activity of NF-κB was determined in PMA-
2
5
induced human chondrosarcoma (SW1353) cells through a reporter gene assay.
Parthenolide was used as a positive control. Interestingly, most compounds showing strong
inhibition of iNOS also showed a strong inhibition of NF-κB activity. The benzothiazolone
derivatives 3h and 3j exhibited the highest activity with IC50 values of 0.4 µM while, the
corresponding benzoxazolone dimer 3g was not as effective with an IC50 value of 3.43 µM,
and 3i was devoid of any activity (Table 1). Moreover, heterodimers 5p, 5r and 5t with long
alkyl chains showed acceptable inhibitory activity with IC50 values of 1.18, 0.83 and 1.06 µM
respectively. On the other hand, the benzoxazolone dimers 5q and 5s were equally active to
the corresponding benzothiazolone dimers 5r and 5t with IC50 values 1.24 and 1.40 µM,
respectively. Nevertheless, compounds 5h, 5j and 5n were moderately active while most of
the corresponding benzoxazolone dimers were devoid of activity except compound 5g which
displayed a weak activity with an IC50 value of 3.07 µM.
6

It should be mentioned that, changing the attachment point of the alkyl chains to the
heterocycle increases the activity similar to its effect on the iNOS inhibition. Compounds 8a
and 8b more potent than the corresponding dimers 3e and 3f where their alkyl chains attached
directly to the nitrogen atom of the heterocycle rather than the sulfur atom. In addition, it was
noted that the benzothiazolone dimer 8b has higher inhibitory activity than the
benzoxazolone dimer 8a with IC50 values of 0.34 and 1.40 µM, respectively.
These findings indicate that the heterodimers were, in general, more active than homodimers.
Furthermore, the benzothiazolone dimers that have long alkyl chains were more potent
inhibitors of both iNOS and NF-κB than the corresponding benzoxazolone dimers. None of
the compounds showed inhibition of Sp-1 activity which confirms that their activity towards
NF-κB is specific and is not related to cytotoxicity.
2
.2.3. Acetic acid-induced writhing test
Two compounds, 3j and 8b, which exhibited the highest inhibitory activities in vitro against
NF-κB and iNOS targets were selected to be evaluated for antinociceptive potential in vivo
using the well-known acetic acid-induced writhing assay. The effects of these ligands on the
acetic acid abdominal writhing assay are summarized in Fig. 2. Indomethacin was used as a
standard control for this assay. In vehicle treated mice, acetic acid produced robust writhing
responses during the observation period. Mice that received indomethacin displayed fewer
writhing responses. The test compounds 3j and 8b produced significant reduction in writhing
responses. However, the results revealed that the effect of the test compounds on the number
of writhing was markedly increased with the dose elevating from 3 mg/kg to 10 mg/kg.
2
.2.4. Carrageenan-induced rat paw edema assay
The anti-inflammatory activities of compounds 3j, 5t and 8b were tested using carrageenan-
2
6
induced rat paw edema assay where ketorolac was used as the reference drug. Mean
changes in paw edema thickness of animals pretreated with the test compounds were
measured after 1, 2, 3, and 4 hours from induction of inflammation. Data was analyzed by
one-way ANOVA, followed by Dunnett's multiple comparisons test (n = 5). All compounds
showed significant anti-inflammatory activity comparable to that of ketorolac (Table 2).
Results revealed that compound 8b was the most active dimer while compounds 3j and 5t
showed moderate activities compared to ketorolac. These results verified that the attachment
point of the alkyl chain significantly influences the activity in vivo as well as in vitro.
Another observation is the decline in activity of tested compounds after 3 hours. This could
7

be explained by the expected rapid degradation of test compounds via N-dealkylation or other
metabolic pathways.
2
.2.5. Cytotoxicity assay
All compounds were evaluated for cytotoxicity against a panel of four human solid tumor cell
lines (SK-MEL: malignant-melanoma; KB: oral epidermal carcinoma; BT-549: breast ductal
carcinoma, and SK-OV-3: ovary carcinoma) and two kidney cell lines (Vero: monkey kidney
2
7
fibroblasts and LLC-PK1: pig kidney epithelial cells). Most of the compounds were not
cytotoxic up to a concentration of 25 µg/mL and do not seem to have any significant anti-cell
proliferative activity towards cancer cells as shown in Table1.
2
.3. Docking study
In order to understand the structural basis for activity and the putative binding mode of our
ligands in the active site of iNOS, the best compounds 3j, 5t and 8b were docked by
2
8
employing LIGANDFIT embedded in Discovery Studio software. The X-ray structure of
iNOS complexed with the selective inhibitor AR-C120011 (pdb code: 3E65) was selected for
this approach. All compounds adopted a disposition and orientation similar to that of the
selective inhibitor AR-C120011. It has been reported that the correlated side-chain rotations
of Gln257, Arg260 and other residues expose a new specificity pocket extending from the
2
9
active-site heme pocket. This new, larger pocket may explain the accommodation of the
iNOS binding site for these bulky dimers and potentially their enhanced inhibitory activity.
All docked compounds showed three critical features and interactions within the binding
pocket of iNOS: 1) The heterocycle, benzoxazolone or benzothiazolone, made a stacking
interaction with heme propionate; 2) The piperazine core was enclosed by a polar cavity
consisting of Gln257, Val346, and Arg260 residues forming a network of hydrogen bonds
between the piperazine nitrogen atoms and the former amino acids; and 3) an additional
hydrogen bond tethered the sulfur or the carbonyl group of the extended inhibitor tails to
Asn115 residue, as shown in Fig. 3. It could also be appreciated from Fig. 3(A) how
compound 8b formed another hydrogen-bonding interaction with NH of Val346. In
conclusion, the results indicate contributions from heme stacking, hydrogen bonding and
hydrophobic interactions within the active site account for the binding mode of these
inhibitors and potentially explain their activity.
8

3
. Conclusion
Two series of benzoxazolone and benzothiazolone dimers were synthesized and evaluated in
vitro and in vivo for their anti-inflammatory and analgesic activities. The first series was
designed by varying the carbon chain length extending from the piperazine core to the
nitrogen atom of the dibenzo[d]oxazol-2(3H)-one or dibenzo[d]thiazol-2(3H)-one. The
second series was designed by changing the attachment point. Compounds 3j, 5t and 8b were
the most potent in inhibiting iNOS activity with IC50 values of 0.28, 0.29 and 0.41 µM
respectively. On the other hand, compounds 3h, 3j and 8b were found to be the most potent
in inhibiting NF-κB activity with IC50 values 0.43, 0.41 and 0.34 µM respectively. Based on
the overall results, we found that heterodimers with longer alkyl chains around the piperazine
core were more active than homodimers. Moreover, the attachment point on the heterocycle
is important and significantly affects the activity. Based on the potent in vitro activity
towards both iNOS and NF-κB, three compounds were selected for assessing their in vivo
anti-inflammatory/analgesic potential. Compound 8b exhibited promising in vivo activity
which was comparable to indomethacin and ketorolac. The docking simulation was carried
out for some active dimers to determine the probable binding modes and poses. The results
indicated that these active compounds fit nicely into the iNOS binding site, resulting in a
stable complex and acting as potential inhibitors of this enzyme.
4
. Experimental protocols
4
.1. Chemistry
Reagents and starting materials were obtained from commercial suppliers and were used
without purification. Precoated silica gel GF Uniplates from Analtech were used for thin-
layer chromatography (TLC). Column chromatography was done on silica gel 60 (Sorbent
1
13
technologies). H and C NMR spectra were obtained on a Bruker APX400 at 400 and 100
MHz, respectively. The high resolution mass spectra (HRMS) were recorded on a Waters
Micromass Q-Tof Micro mass spectrometer with a lock of spray source. The mass spectra
(
MS) were recorded on a Waters Acquity Ultra Performance LC with ZQ detector in ESI
mode. Elemental analysis (C, H, N) were recorded on an elemental analyzer, Perkin-Elmer
CHN/SO series II Analyzer. Chemical names were generated using ChemDraw Ultra
(
CambridgeSoft, version 12.0).
General Procedure A. Synthesis of bromoalkyl benzo[d]oxazol-2(3H)-one and bromoalkyl
benzo[d] thiazol-2(3H)-one derivatives. 3-(4-bromobutyl)benzo[d]oxazol-2(3H)-one (2e).
9

2 3
K CO (9.2 g, 66.6 mmol) and 1,4-dibromobutane (21.0 mL, 177.6 mmol) were added, under
stirring, to a solution of benzo[d]oxazol-2(3H)-one (3.0 g, 22.2 mmol) in anhydrous DMF (30
°
mL). The reaction mixture was heated at 60 C for 3 h. After cooling, the reaction mixture
was poured into 100 mL water and extracted with ethyl acetate (3 x 70 mL). The combined
organic layers were washed with saturated aqueous NaCl and dried over sodium sulfate. The
solvent was removed in vacuo, and the residue was purified by flash column chromatography
(
1
SiO
2
) using hexane/ ethyl acetate (8: 2) as eluent to give 3.6 g (62%) of 2e as a white solid.
H NMR (CDCl ): δ 7.30 – 7.05 (m, 3H), 7.00 (d, J = 7.4, 1H), 3.86 (t, J = 5.1, 2H), 3.44 (t, J
10.6, 2H), 1.95 (m, 4H). C NMR (CDCl
3
1
3
=
3
): δ 154.54, 142.67, 130.94, 123.89, 122.49,
+
1
10.09, 108.25, 41.28, 32.74, 29.41, 26.31. MS (EI) m/z 292 (M +23).
3
-(2-bromoethyl)benzo[d]oxazol-2(3H)-one (2a). General Procedure A, yield 58%, yellow
1
oil. H NMR (400 MHz, CDCl
3
) δ 7.38 – 6.79 (m, 4H), 4.21 (t, J = 6.2, 2H), 3.67 (t, J = 6.1,
1
3
2
4
H). C NMR (101 MHz, CDCl
3
) δ 154.20, 142.54, 130.83, 123.96, 122.75, 110.17, 108.54,
+
3.88, 27.73. MS (EI) m/z 242 (M +1).
3
-(2-bromoethyl)benzo[d]thaizol-2(3H)-one (2b). General Procedure A, yield 57%, white
1
solid. H NMR (400 MHz, CDCl
3
) δ 7.64 – 6.99 (m, 4H), 4.37 (t, J = 6.2, 2H), 3.82 (t, J = 6.1,
1
3
2
5
H). C NMR (101 MHz, CDCl
3
) δ 166.07, 154.40, 142.70, 135.83, 134.62, 122.03, 120.40,
+
5.74, 39.59. MS (EI) m/z 258 (M +1).
3
-(3-bromopropyl)benzo[d]oxazol-2(3H)-one (2c). General Procedure A, yield 59%,
1
yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.41 – 6.87 (m, 4H), 4.00 (t, J = 6.8, 2H), 3.46 (t, J
1
3
=
6.3, 2H), 2.94 – 1.89 (m, 2H). C NMR (101 MHz, CDCl
3
) δ 154.45, 142.65, 131.13,
+
1
23.97, 122.59, 110.14, 108.27, 40.61, 30.75, 29.87. MS (EI) m/z 279 (M +23).
3
-(3-bromopropyl)benzo[d]thaizol-2(3H)-one (2d). General Procedure A, yield 61%,
1
yellow solid. H NMR (400 MHz, CDCl
3
) δ 7.54 – 6.95 (m, 4H), 4.11 (t, J = 7.5, 2H), 3.46 (t,
1
3
J = 6.4, 2H), 2.35 – 2.29 (m, 2H). C NMR (101 MHz, CDCl
3
) δ 156.76, 144.96, 133.45,
+
1
26.29, 124.91, 112.46, 110.59, 42.93, 33.06, 32.18. MS (EI) m/z 272 (M +1).
3
-(4-bromobutyl)benzo[d]thaizol-2(3H)-one (2f). General Procedure A, yield 63%, yellow
1
oil. H NMR (400 MHz, CDCl
3
) δ 7.56 – 6.74 (m, 4H), 3.94 (t, J = 6.6, 2H), 3.41 (t, J = 6.0,
1
3
2
1
H), 2.14 – 1.63 (m, 4H). C NMR (101 MHz, CDCl
3
) δ 169.79, 136.85, 126.44, 123.15,
+
22.72, 122.66, 110.56, 41.67, 33.07, 29.58, 26.16. MS (EI) m/z 308 (M +23).
3
-(5-bromopentyl)benzo[d]oxazol-2(3H)-one (2g). General Procedure A, yield 65%, yellow
1
oil. H NMR (400 MHz, CDCl
3
) δ 7.25 – 6.95 (m, 4H), 3.84 (t, J = 7.2, 2H), 3.39 (t, J = 6.7,
1
0

1
3
2
H), 1.97 – 1.87 (m, 2H), 1.85-1.78 (m, 2H), 1.63 – 1.45 (m, 2H). C NMR (101 MHz,
CDCl
3
) δ 154.54, 142.68, 131.08, 123.82, 122.39, 110.07, 108.21, 42.00, 33.21, 32.09, 26.96,
+
2
5.21. MS (EI) m/z 306 (M +23).
3
-(5-bromopentyl)benzo[d]thaizol-2(3H)-one (2h). General Procedure A, yield 64%,
1
yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.50 – 6.86 (m, 4H), 3.89 (t, J = 7.3, 2H), 3.33 (t, J
1
3
=
6.7, 2H), 1.88 – 1.81 (m, 2H), 1.74 – 1.67 (m, 2H), 1.52 – 1.44 (m, 2H). C NMR (101
MHz, CDCl
3
) δ 169.67, 136.95, 126.37, 123.05, 122.68, 122.66, 110.55, 42.42, 33.48, 32.20,
+
2
6.76, 25.32. MS (EI) m/z 323 (M +23).
3
-(6-bromohexyl)benzo[d]oxazol-2(3H)-one (2i). General Procedure A, yield 65%, yellow
1
oil. H NMR (400 MHz, CDCl
3
) δ 7.39 – 6.73 (m, 4H), 3.82 (t, J = 7.2, 2H), 3.38 (t, J = 6.7,
1
3
2
1
3
H), 1.95 – 1.66 (m, 4H), 1.66 – 1.23 (m, 4H). C NMR (101 MHz, CDCl ) δ 154.56,
42.67, 131.14, 123.79, 122.33, 110.01, 108.25, 42.10, 33.65, 32.48, 27.66, 27.61, 25.82. MS
+
(
EI) m/z 298 (M +1).
3
-(6-bromohexyl)benzo[d]thaizol-2(3H)-one (2j). General Procedure A, yield 63%, yellow
1
oil. H NMR (400 MHz, CDCl
3
) δ 7.52 – 6.77 (m, 4H), 3.91 (t, J = 7.2, 2H), 3.36 (t, J = 6.7,
1
3
2
1
1
H), 1.94 – 1.60 (m, 4H), 1.56 – 1.24 (m, 4H). C NMR (400 MHz, CDCl ) δ 206.96,
3
98.73, 196.03, 195.20, 195.13, 195.11, 192.07, 174.98, 172.76, 172.45, 171.25, 171.17,
+
70.79. MS (EI) m/z 314 (M +1).
General Procedure B. Synthesis of piperazine dibenzo[d]oxazol-2(3H)-one Homodimers.
,3'-(piperazine-1,4-diylbis(butane-4,1-diyl))bis(benzo[d]oxazol-2(3H)-one) (3e). K CO
0.76 g, 5.55 mmol) and piperazine (0.1 g, 0.92 mmol) were added, under stirring, to a
3
2
3
(
solution of 2e (0.5 g, 1.85 mmol) in anhydrous DMF (10 mL). The reaction mixture was
°
heated at 60 C for 1.5 h. After cooling, the reaction mixture was poured into 50 mL water
and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with
saturated aqueous NaCl and dried over sodium sulfate. The solvent was removed in vacuo,
and the residue was purified by flash column chromatography (SiO
2
) using dichlormethane/
1
methanol (9.5: 0.5) as eluent to give 0.3 g (70%) of 3e as a white solid. H NMR (CDCl
3
): δ
7
4
1
4
.26-6.96 (m, 8H), 3.82 (t, J = 7.0, 4H), 2.39 (s, 8H), 2.37 – 2.29 (m, 4H), 1.78 -1.75 (m,
1
3
H), 1.57-1.52 (m, 4H). C NMR (CDCl
3
): δ 154.51, 142.65, 131.12, 123.73, 122.27,
+
09.97, 108.30, 57.66, 53.13, 42.08, 25.61, 23.88. HRMS calcd for C26
64.2424, found 465.2509. Anal. calcd for C26
H
32
N
4
O [M+H]
4
H
34Cl
2
N
4
O
4
: C, 58.10; H, 6.38; N, 10.42.
Found: C, 58.34; H, 6.87; N, 10.21.
1
1

3
,3'-(piperazine-1,4-diylbis(ethane-2,1-diyl))bis(benzo[d]oxazol-2(3H)-one) (3a). General
1
Procedure B, yield 51%, white solid. H NMR (400 MHz, CDCl
3
) δ 7.48 – 7.00 (m, 8H), 4.06
) δ
69.87, 137.08, 126.27, 123.03, 122.75, 122.66, 110.57, 54.85, 53.27, 40.40. HRMS calcd
13
(
t, J = 7.1, 4H), 2.72 – 2.62 (t, J = 7.2, 4H), 2.56 (s, 8H). C NMR (101 MHz, CDCl
3
1
+
for C22
H
24
N
4
O
4
[M+H] 408.1798, found 409.1891. Anal. calcd for C22
H
26Cl
2
N
4
O
4
: C, 54.89;
H, 5.44; N, 11.64. Found: C, 55.14; H, 5.87; N, 11.28.
3
,3'-(2,2'-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))dibenzo[d]thaizol-2(3H)-one
(3b).
1
General Procedure B, yield 52%, white solid. H NMR (400 MHz, DMSO) δ 7.85 – 6.92 (m,
1
3
8
1
H), 4.32 (t, J = 6.7, 4H), 3.30 – 3.16 (m, 12H). C NMR (101 MHz, DMSO) δ 169.53,
36.88, 127.15, 123.82, 123.40, 122.06, 112.00, 52.59, 49.59, 37.54. HRMS calcd for
+
C
22
H
24
N
4
O
2
S
2
[M+H] 440.1431, found 441.1432. Anal. calcd for C22
H
26Cl
2
N
4
O
2
2
S : C,
5
1.46; H, 5.10; N, 10.91. Found: C, 51.31; H, 5.47; N, 10.55.
3
,3'-(piperazine-1,4-diylbis(propane-3,1-diyl))bis(benzo[d]oxazol-2(3H)-one)
(3c).
1
General Procedure B, yield 53%, white solid. H NMR (400 MHz, D
2
O) δ 7.27 – 7.03 (m,
1
3
8
H), 3.86 (t, J = 6.5, 4H), 3.19 (s, 8H), 3.00 (t, J = 6.8, 4H), 2.07 (m, 4H). C NMR (101
MHz, CDCl
3
) δ 154.28, 142.41, 131.79, 124.19, 122.40, 109.97, 109.62, 55.25, 52.99, 24.24.
+
HRMS calcd for C24
H
28
N
4
O
4
[M+H] 436.2111, found 437.2173. Anal. calcd for
24 2 4 4
C H30Cl N O : C, 56.58; H, 5.94; N, 11.00. Found: C, 56.91; H, 5.43; N, 11.29.
3
,3'-(piperazine-1,4-diylbis(propane-3,1-diyl))bis(benzo[d]thiazol-2(3H)-one)
(3d).
1
General Procedure B, yield 50%, white solid. H NMR (400 MHz, DMSO) δ 7.81 – 7.02 (m,
1
3
8
H), 4.03 (t, J = 6.8, 4H), 3.69 – 3.03 (m, 12H), 2.27 – 1.96 (m, 4H). C NMR (101 MHz,
DMSO) δ 169.35, 137.18, 127.11, 123.67, 123.36, 122.06, 111.89, 48.47, 22.61, 17.88.
+
HRMS calcd for C24
H
28
N
4
O
2
S
2
[M+H] 468.1735, found 469.1736. Anal. calcd for
24 2 4 2 2
C H30Cl N O S : C, 53.23; H, 5.58; N, 10.35. Found: C, 53.59; H, 5.12; N, 10.76.
3
,3'-(piperazine-1,4-diylbis(butane-4,1-diyl))bis(benzo[d]thiazol-2(3H)-one) (3f). General
1
Procedure B, yield 51%, yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.70 – 6.46 (m, 8H), 3.92
1
3
(
(
t, J = 6.9, 4H), 2.42 - 2.32 (m, 12H), 2.02 – 1.60 (m, 4H), 1.56 – 1.54 (m, 4H). C NMR
101 MHz, CDCl
3
) δ 169.81, 137.05, 126.26, 122.97, 122.73, 122.61, 110.69, 57.64, 53.06,
+
4
2.48, 25.37, 23.81. HRMS calcd for C26
Anal. calcd for C26
.65.
H
32
N
4
O
2
S
2
[M+H] 496.1967, found 497.2057.
H34Cl
2
N
4
O
2
S : C, 54.82; H, 6.02; N, 9.84. Found: C, 54.45; H, 6.51; N,
2
9
1
2

3
,3'-(piperazine-1,4-diylbis(pentane-5,1-diyl))bis(benzo[d]oxazol-2(3H)-one)
(3g).
1
General Procedure B, yield 53%, yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.23 – 6.92 (m,
8
H), 3.80 (t, J = 7.2, 4H), 2.42 (s, 8H), 2.29 (t, J = 7.3, 4H), 1.86 – 1.71 (m, 4H), 1.56 -1.48
1
3
(
m, 4H), 1.44 – 1.31 (m, 4H). C NMR (101 MHz, CDCl
3
) δ 154.55, 142.67, 131.17, 123.73,
1
22.27, 110.01, 108.23, 58.30, 53.19, 42.21, 27.64, 26.38, 24.63. HRMS calcd for
+
C
28
H
36
N
4
O
4
[M+H] 492.2737, found 493.2814. Anal. calcd for C28
H
38Cl
2
N
4
O
4
: C, 59.47; H,
6
.77; N, 9.91. Found: C, 59.34; H, 6.97; N, 10.33.
3
,3'-(piperazine-1,4-diylbis(pentane-5,1-diyl))bis(benzo[d]thiazol-2(3H)-one)
(3h).
1
General Procedure B, yield 52%, yellow oil. H NMR (400 MHz, D
2
O) δ 8.04 – 6.96 (m,
8
H), 4.24 (t, J = 6.9, 4H), 3.83 (s, 8H), 3.52 – 3.36 (m, 4H), 2.14 – 1.91 (m, 8H), 1.65 – 1.58
1
3
(
m, 4H). C NMR (101 MHz, CDCl
3
) δ 155.45, 143.57, 132.07, 124.63, 123.17, 110.90,
+
1
5
09.13, 59.20, 54.09, 43.11, 28.54, 27.28, 25.53. HRMS calcd for C28
24.2280, found 525.2373. Anal. calcd for C28
H
36
N
4
O
2
S [M+H]
2
H
38Cl
2
N
4
O
2
S
2
: C, 56.27; H, 6.41; N, 9.37.
Found: C, 56.62; H, 6.81; N, 9.26.
3
,3'-(piperazine-1,4-diylbis(hexane-6,1-diyl))bis(benzo[d]oxazol-2(3H)-one) (3i). General
1
Procedure B, yield 52%, yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.20 – 6.95 (m, 4H), 3.80
(
t, J = 7.2, 2H), 2.45 (s, 4H), 2.30 (t, J = 7.3, 2H), 1.80 – 1.73 (m, 2H), 1.51 -1.44 (m, 2H),
1
3
1
1
3
.42 – 1.34 (m, 4H). C NMR (101 MHz, CDCl ) δ 154.56, 142.68, 131.20, 123.72, 122.26,
10.00, 108.24, 58.45, 53.07, 42.22, 27.68, 27.08, 26.58, 26.56. HRMS calcd for C30
H
40
N
4
O
4
+
[
M+H] 520.3050, found 521.3132. Anal. calcd for C30
H
42Cl
2
N
4
4
O : C, 60.70; H, 7.13; N,
9
.44. Found: C, 60.43; H, 7.17; N, 9.55.
3
,3'-(piperazine-1,4-diylbis(hexane-6,1-diyl))bis(benzo[d]thiaazol-2(3H)-one)
(3j).
1
General Procedure B, yield 53%, yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.68 – 6.71 (m,
8
H), 3.89 (t, J = 6.8, 4H), 2.84 – 1.99 (m, 12H), 1.70 (t, J = 6.2, 4H), 1.45 – 1.33 (m, 12H).
1
3
C NMR (101 MHz, CDCl
3
) δ 169.76, 137.09, 126.25, 122.93, 122.75, 122.62, 110.56,
+
5
5
8.44, 53.02, 42.70, 27.50, 27.14, 26.66, 26.57. HRMS calcd for C30
52.2593, found 553.2695. Anal. calcd for C30
40 4 2 2
H N O S [M+H]
H
42Cl
2
N
4
O
2
S
2
: C, 57.59; H, 6.77; N, 8.95.
Found: C, 57.14; H, 6.28; N, 9.41.
General Procedure C. Piperazine benzo[d]oxazol-2(3H)-one derivatives. 3-(4-(piperazin-1-
yl)butyl)benzo[d]oxazol-2(3H)-one (4e). K CO (2.3 g, 16.71 mmol) and piperazine (2.4 g,
7.85 mmol) were added, under stirring, to a solution of 2e (1.5 g, 5.57 mmol) in anhydrous
2
3
2
°
DMF (20 mL). The reaction mixture was heated at 60 C for 0.5 h. After cooling, the reaction
1
3

mixture was poured into 60 mL water and extracted with dichloromethane (3 x 50 mL). The
combined organic layers were washed with saturated aqueous NaCl and dried over sodium
sulfate. The solvent was removed in vacuo, and the residue was purified by flash column
chromatography (SiO
2
) using dichloromethane/ methanol: NH OH (9: 1) as eluent to give 1.2
4
1
g (78%) of 4e as a white solid. H NMR (D
2
O): δ 7.16 – 7.06 (m, 4H), 3.72 (t, J = 6.0, 2H),
1
3
3
1
2
.62 (s, 8H), 3.27 (t, J = 6.3, 2H), 1.76 -1.68 (m, 4H). C NMR (101 MHz, D O) δ 156.03,
42.10, 130.26, 124.44, 123.05, 110.02, 109.36, 56.63, 48.47, 41.27, 40.81, 24.17, 20.65. MS
+
(
EI) m/z 276 (M +1).
3
-(2-(piperazin-1-yl)ethyl)benzo[d]oxazol-2(3H)-one (4a). General Procedure C, yield
1
6
7%, white solid. H NMR (400 MHz, D
2
O) δ 7.31 – 7.13 (m, 4H), 4.30 (t, J = 5.9, 2H), 3.77
1
3
(
s, 4H), 3.72 (t, J = 6.3, 2H), 3.64 (s, 4H). C NMR (101 MHz, D
2
O) δ 156.20, 142.47,
+
1
29.53, 124.69, 123.70, 110.46, 109.22, 54.25, 48.86, 40.72, 36.50. MS (EI) m/z 248 (M +1).
3
-(2-(piperazin-1-yl)ethyl)benzo[d]thaizol-2(3H)-one (4b). General Procedure C, yield
1
6
8%, white solid H NMR (400 MHz, D
2
O) δ 7.69 – 6.78 (m, 4H), 4.29 (t, J = 6.0, 2H), 3.68
1
3
(
s, 4H), 3.64 – 3.40 (m, 6H). C NMR (101 MHz, D
2
O) δ 173.78, 135.44, 127.08, 124.30,
+
1
23.12, 122.16, 111.08, 54.24, 48.90, 40.69, 36.85. MS (EI) m/z 264 (M +1).
3
-(3-(piperazin-1-yl)propyl)benzo[d]oxazol-2(3H)-one (4c). General Procedure C, yield
1
6
6%, white solid H NMR (400 MHz, D
2
O) δ 7.27 – 7.01 (m, 4H), 3.88 (t, J = 6.7, 2H), 3.62
1
3
(
s, 8H), 3.37 (t, J = 6.5, 2H), 2.35 – 2.13 (m, 2H). C NMR (101 MHz, D
2
O) δ 156.00,
1
42.21, 130.12, 124.54, 123.26, 110.19, 109.23, 54.50, 48.65, 40.84, 39.08, 22.26. MS (EI)
+
m/z 262 (M +1).
3
-(3-(piperazin-1-yl)propyl)benzo[d]thiazol-2(3H)-one (4d). General Procedure C, yield
1
6
3%, white solid H NMR (400 MHz, D
2
O) δ 7.17 – 7.06 (m, 4H), 3.72 (t, J = 6.8, 2H), 3.62
1
3
(
s, 8H), 3.29 (t, J = 6.5, 2H), 1.79 – 1.76 (m, 2H). C NMR (101 MHz, D
2
O) δ 156.03,
1
42.10, 130.26, 124.44, 123.05, 110.02, 109.36, 56.63, 48.47, 41.27, 40.81, 24.17. MS (EI)
+
m/z 277 (M ).
3
6
3
-(4-(piperazin-1-yl)butyl)benzo[d]thaizol-2(3H)-one (4f). General Procedure C, yield
1
5%, yellow oil H NMR (400 MHz, D
2
O) δ 7.67 – 6.90 (m, 4H), 3.95 (t, J = 5.9, 2H), 3.46 –
1
3
.38 (m, 8H), 3.20 – 2.86 (m, 2H), 1.87 – 1.58 (m, 2H), 1.22 – 1.17 (m, 2H). C NMR (101
MHz, D
2
O) δ 156.91, 142.99, 131.15, 125.33, 123.93, 110.91, 110.25, 57.52, 49.36, 42.16,
+
4
1.70, 25.06, 21.54. MS (EI) m/z 292 (M +1).
1
4

3
-(5-(piperazin-1-yl)pentyl)benzo[d]oxazol-2(3H)-one (4g). General Procedure C, yield
1
6
7%, white solid H NMR (400 MHz, D
2
O) δ 7.22 - 7.11 (m, 4H), 3.75 (t, J = 6.8, 2H), 3.61
13
(
s, 8H), 3.22 (t, J = 6.5, 2H), 1.86 – 1.63 (m, 4H), 1.41 -1.33 (m, 2H). C NMR (101 MHz,
D
2
O) δ 156.26, 142.21, 130.51, 124.40, 123.00, 110.05, 109.50, 57.07, 48.40, 41.70, 40.79,
+
2
6.45, 22.85, 22.81. MS (EI) m/z 290 (M +1).
3
-(5-(piperazin-1-yl)pentyl)benzo[d]thaizol-2(3H)-one (4h). General Procedure C, yield
1
6
6%, white solid H NMR (400 MHz, D
2
O) δ 7.89 – 6.53 (m, 4H), 3.79 – 3.31 (m, 10H), 3.13
1
3
(
t, J = 7.3, 2H), 1.72 – 1.57 (m, 2H), 1.54 – 1.52 (m, 2H), 1.24 – 1.23 (m, 2H). C NMR (101
MHz, D
2
O) δ 136.36, 136.32, 126.81, 123.73, 122.64, 121.80, 111.64, 100.00, 56.98, 48.32,
+
4
2.16, 40.71, 26.29, 22.81. MS (EI) m/z 306 (M +1).
General Procedure D. Synthesis of piperazine dibenzo[d]oxazol-2(3H)-one Heterodimmers.
3
2
-(5-(4-(4-(2-oxobenzo[d]oxazol-3(2H)-yl)butyl)piperazin-1-yl)pentyl)benzo[d]oxazol-
(3H)-one (5o). K CO (0.75 g, 5.45 mmol) and 4e (0.5 g, 1.82 mmol) were added, under
2
3
stirring, to a solution of 2g (0.49 g, 1.82 mmol) in anhydrous DMF (20 mL). The reaction
°
mixture was heated at 60 C for 2 h. After cooling, the reaction mixture was poured into 60
mL water and extracted with ethyl acetate (3 x 40 mL). The combined organic layers were
washed with saturated aqueous NaCl and dried over sodium sulfate. The solvent was
removed in vacuo, and the residue was purified by flash column chromatography (SiO )
2
using dichlormethane/ methanol (9.5: 0.5) as eluent to give 0.58 g (73%) of 5o as a white
1
solid. H NMR (400 MHz, D
2
O) δ 7.42 – 6.82 (m, 8H), 3.74 (t, J = 6.7, 2H), 3.69 (t, J = 6.9,
2
H), 3.57 (s, 8H), 3.22 (t, J = 7.8, 2H), 3.19 – 3.12 (m, 2H), 1.84 – 1.57 (m, 8H), 1.34 – 1.27
1
3
(
m, 2H). C NMR (101 MHz, D
2
O) δ 156.21, 156.20, 142.17, 142.13, 130.41, 130.28,
1
4
4
24.37, 124.32, 123.03, 122.92, 110.04, 109.97, 109.40, 109.30, 56.65, 56.20, 48.60, 48.56,
+
1.59, 41.14, 26.38, 24.07, 22.88, 22.71, 20.66. HRMS calcd for C27
H
34
N
4
4
O [M+H]
78.2580, found 479.3266. Anal. calcd for C27
H
36Cl
2
N
4
4
O : C, 58.80; H, 6.58; N, 10.16.
Found: C, 58.47; H, 6.13; N, 10.39.
3
2
7
3
1
4
-(3-(4-(2-(2-oxobenzo[d]oxazol-3(2H)-yl)ethyl)piperazin-1-yl)propyl)benzo[d]oxazol-
1
(3H)-one (5a). General Procedure D, yield 67%, white solid. H NMR (400 MHz, D
2
O) δ
.36 – 7.10 (m, 8H), 4.25 (t, J = 5.9, 2H), 3.93 (t, J = 6.6, 2H), 3.67 – 3.46 (m, 10H), 3.37 –
1
3
2
.26 (m, 2H), 2.29 – 2.11 (m, 2H). C NMR (101 MHz, D O) δ 156.40, 156.28, 142.53,
30.27, 124.59, 124.50, 123.58, 123.28, 110.44, 110.30, 109.18, 109.16, 54.10, 53.91, 49.35,
+
9.09, 39.01, 22.27. HRMS calcd for C23
H
26
N
4
O
4
[M+H] 422.1954, found 423.2025. Anal.
2 4 4
calcd for C23H28Cl N O : C, 55.76; H, 5.70; N, 11.31. Found: C, 56.14; H, 5.24; N, 11.01.
1
5

3
-(3-(4-(2-(2-oxobenzo[d]thiazol-3(2H)-yl)ethyl)piperazin-1-yl)propyl)benzo[d]thiazol-2
1
(
3H)-one (5b). General Procedure D, yield 63%, white solid. H NMR (400 MHz, CDCl
3
) δ
7
1
1
3
.24 – 6.87 (m, 8H), 3.97 – 3.79 (m, 4H), 2.66 (t, J = 6.5, 2H), 2.46 – 2.32 (m, 10 H), 1.95 –
1
3
.82 (m, 2H). C NMR (101 MHz, CDCl ) δ 154.55, 142.63, 142.60, 131.43, 131.17, 123.69,
3
23.67, 122.26, 122.17, 109.91, 109.86, 108.47, 108.44, 55.14, 54.93, 53.15, 52.95, 40.38,
+
9.81, 24.58. HRMS calcd for C23
H
26
N
4
O
2
S
2
[M+H] 454.1497, found 455.1564. Anal. calcd
for C23
H
28Cl
2
N
4
O
2
S
2
: C, 52.37; H, 5.35; N, 10.62. Found: C, 52.59; H, 5.12; N, 10.11.
3
2
7
4
-(2-(4-(4-(2-oxobenzo[d]oxazol-3(2H)-yl)butyl)piperazin-1-yl)ethyl)benzo[d]oxazol-
1
(3H)-one (5c). General Procedure D, yield 64%, yellow oil. H NMR (400 MHz, CDCl
3
) δ
.28 – 6.79 (m, 8H), 3.91 (t, J = 6.4, 2H), 3.82 (t, J = 7.0, 2H), 2.68 (t, J = 6.4, 2H), 2.52 (s,
1
3
H), 2.39 – 2.32 (m, 6H), 1.88 – 1.67 (m, 2H), 1.63 – 1.46 (m, 2H). C NMR (101 MHz,
) δ 154.59, 154.54, 142.67, 131.20, 131.11, 123.74, 123.68, 122.30, 122.28, 110.00,
09.96, 108.44, 108.29, 57.55, 55.18, 53.20, 53.02, 42.08, 39.85, 25.60, 23.85. HRMS calcd
CDCl
3
1
+
for C24
H
28
N
4
O
4
[M+H] 436.2111, found 437.2196. Anal. calcd for C24
2 4 4
H30Cl N O : C, 56.58;
H, 5.94; N, 11.00. Found: C, 56.90; H, 5.88; N, 10.91.
3
2
7
4
-(2-(4-(4-(2-oxobenzo[d]thiazol-3(2H)-yl)butyl)piperazin-1-yl)ethyl)benzo[d]thiazol-
1
(3H)-one (5d). General Procedure D, yield 61%, yellow oil. H NMR (400 MHz, CDCl
3
) δ
.54 – 6.98 (m, 8H), 4.06 (t, J = 7.2, 2H), 3.96 (t, J = 7.4, 2H), 2.71 – 2.63 (m, 2H), 2.57 (s,
1
3
H), 2.45 (s, 4H), 2.41 – 2.34 (m, 2H), 1.80 -1.73 (m, 2H), 1.61 - 1.54 (m, 2H). C NMR
) δ 169.84, 137.10, 137.09, 126.25, 123.01, 122.97, 122.82, 122.77,
(
101 MHz, CDCl
3
1
22.65, 122.64, 110.66, 110.56, 57.58, 54.92, 53.31, 53.03, 42.51, 40.42, 29.68, 25.38, 23.82.
+
HRMS calcd for C24
H
28
N
4
O
2
S
2
[M+H] 468.1654, found 469.1735. Anal. calcd for
C
24
H
30Cl
2
N
4
O
2
2
S : C, 53.23; H, 5.58; N, 10.35. Found: C, 53.31; H, 5.17; N, 10.83.
3
2
7
3
1
9
-(5-(4-(2-(2-oxobenzo[d]oxazol-3(2H)-yl)ethyl)piperazin-1-yl)pentyl)benzo[d]oxazol-
1
(3H)-one (5e). General Procedure D, yield 62%, yellow oil. H NMR (400 MHz, D
2
O) δ
.57 – 7.43 (m, 8H), 4.53 (t, J = 6.0, 2H), 4.08 (t, J = 6.8, 2H), 3.91 – 3.75 (m, 10H), 3.51 –
1
3
2
.36 (m, 2H), 2.15 – 1.87 (m, 4H), 1.66 – 1.59 (m, 2H). C NMR (101 MHz, D O) δ 165.86,
42.69, 131.02, 130.10, 124.95, 124.70, 123.93, 123.31, 110.79, 110.46, 109.84, 109.52,
9.99, 56.99, 54.19, 49.35, 49.28, 41.98, 37.36, 34.96, 26.58, 23.04. HRMS calcd for
+
C
25
H
30
N
4
O
4
[M+H] 450.2267, found 451.2339. Anal. calcd for C25
H
32Cl
2
N
4
O
4
: C, 57.36; H,
6
.16; N, 10.70. Found: C, 57.14; H, 5.94; N, 10.26.
1
6

3
-(5-(4-(2-(2-oxobenzo[d]thiazol-3(2H)-yl)ethyl)piperazin-1-yl)pentyl)benzo[d]thiazol-2
1
(
3H)-one (5f). General Procedure D, yield 59%, yellow oil. H NMR (400 MHz, CDCl
3
) δ
7
4
.60 – 6.89 (m, 8H), 4.04 (t, J = 7.2, 2H), 3.92 (t, J = 7.4, 2H), 2.64 (t, J = 7.1, 2H), 2.56 (s,
H), 2.42 (s, 4H), 2.30 (t, J = 7.3, 2H), 1.77 – 1.70 (m, 2H), 1.55 – 1.48 (m, 2H), 1.45 – 1.32
1
3
(
m, 2H). C NMR (101 MHz, CDCl
3
) δ 169.80, 169.79, 137.09, 126.25, 122.99, 122.95,
1
2
22.80, 122.73, 122.64, 110.57, 110.54, 58.23, 54.92, 53.33, 53.12, 42.70, 40.42, 27.45,
+
6.40, 24.71. HRMS calcd for C25
H
30
N
4 2 2
O S [M+H] 482.1810, found 483.1908. Anal. calcd
for C25
H
32Cl
2
N
4
O
2
S
2
: C, 54.05; H, 5.81; N, 10.08. Found: C, 54.39; H, 6.17; N, 9.78.
3
2
7
1
1
5
-(6-(4-(2-(2-oxobenzo[d]oxazol-3(2H)-yl)ethyl)piperazin-1-yl)hexyl)benzo[d]oxazol-
1
(3H)-one (5g). General Procedure D, yield 57%, colorless oil. H NMR (400 MHz, D
2
O) δ
.34 – 6.83 (m, 8H), 4.25 (t, J = 7.3, 2H), 3.82 – 3.42 (m, 12H), 3.27 – 3.06 (m, 2H), 1.70 -
13
2
.64 (m, 4H), 1.45 – 1.20 (m, 4H). C NMR (101 MHz, D O) δ 156.34, 156.33, 142.47,
42.22, 130.58, 129.57, 124.59, 124.32, 123.60, 122.91, 110.41, 110.02, 109.52, 109.14,
6.85, 56.75, 53.94, 48.99, 48.72, 45.46, 41.86, 36.77, 36.74, 31.44, 26.58, 23.11, 23.09.
+
HRMS calcd for C26
H
32
N
4
O
4
[M+H] 464.2424, found 465.2507. Anal. calcd for
C
26
H
34Cl
2
N
4
4
O : C, 58.10; H, 6.38; N, 10.42. Found: C, 57.88; H, 6.10; N, 10.11.
3
2
7
1
1
5
-(6-(4-(2-(2-oxobenzo[d]thiazol-3(2H)-yl)ethyl)piperazin-1-yl)hexyl)benzo[d]thiazol-
1
(3H)-one (5h). General Procedure D, yield 55%, yellow oil. H NMR (400 MHz, DMSO) δ
.57 – 7.06 (m, 8H), 4.33 – 4.17 (m, 2H), 3.81 (t, J = 7.0, 2H), 3.74 – 3.01 (m, 12H), 1.82 –
1
3
2
.57 (m, 4H), 1.48 – 1.24 (m, 4H). C NMR (101 MHz, D O) δ 153.69, 153.67, 139.82,
39.57, 127.92, 126.92, 121.94, 121.66, 120.95, 120.25, 107.75, 107.36, 106.87, 106.48,
4.09, 51.28, 46.34, 46.06, 42.80, 39.21, 34.11, 28.78, 23.93, 20.43.. HRMS calcd for
+
C
26
H
32
N
4
O
2
S
2
[M+H] 496.1967, found 497.2053. Anal. calcd for C26
H
34Cl
2
N
4
O
2
2
S : C,
5
4.82; H, 6.02; N, 9.84. Found: C, 54.30; H, 5.77; N, 10.23.
3
2
7
2
-(3-(4-(4-(2-oxobenzo[d]oxazol-3(2H)-yl)butyl)piperazin-1-yl)propyl)benzo[d]oxazol-
1
(3H)-one (5i). General Procedure D, yield 53%, colorless oil. H NMR (400 MHz, D
2
O) δ
.44 - 7.34 (m, 8H), 4.12 (t, J = 6.7, 2H), 4.02 (t, J = 6.9, 2H), 3.88 (s, 8H), 3.66 – 3.56 (m,
1
3
2
H), 3.52 (t, J = 7.5, 2H), 2.59 – 2.35 (m, 2H), 2.01-2.00 (m, 4H). C NMR (101 MHz, D O)
δ 142.56, 142.55, 124.81, 124.71, 123.54, 123.34, 122.83, 110.52, 110.50, 110.40, 110.37,
1
09.66, 109.64, 109.47, 56.58, 54.49, 49.01, 48.84, 41.49, 39.31, 24.33, 22.51, 20.91. HRMS
+
calcd for C23
H
26
N
4
O
4
[M+H] 422.1954, found 423.2025. HRMS calcd for C25
H
30
N
4
O
4
+
[
M+H] 450.2267, found 451.2359. Anal. calcd for C25
H
32Cl
2
N
4
4
O : C, 57.36; H, 6.16; N,
1
0.70. Found: C, 57.01; H, 5.84; N, 10.31.
1
7

3
-(3-(4-(4-(2-oxobenzo[d]thiazol-3(2H)-yl)butyl)piperazin-1-yl)propyl)benzo[d]thiazol-2
1
(
3H)-one (5j). General Procedure D, yield 52%, yellow oil. H NMR (400 MHz, D
2
O) δ 7.79
–
2
1
1
7.44 (m, 8H), 4.28 (t, J = 6.6, 2H), 4.20 (t, J = 6.2, 2H), 3.76 (s, 8H), 3.50 – 3.44 (m, 4H),
13
.50 – 2.32 (m, 2H), 2.09 – 1.87 (m, 4H). C NMR (101 MHz, D O) δ 173.23, 173.17,
2
36.39, 136.16, 126.98, 126.87, 124.03, 123.89, 122.97, 122.84, 122.06, 122.04, 111.64,
11.35, 56.19, 54.20, 48.74, 48.69, 41.58, 39.43, 23.83, 22.21, 20.58. HRMS calcd for
+
C
25
H
30
N
4
O
2
S
2
[M+H] 482.1810, found 483.1900. Anal. calcd for C25
H
32Cl
2
N
4
O
4
S
2
: C,
5
4.05; H, 5.81; N, 10.08. Found: C, 54.14; H, 6.12; N, 10.45.
3
-(3-(4-(5-(2-oxobenzo[d]oxazol-3(2H)-yl)pentyl)piperazin-1-yl)propyl)benzo[d]oxazol-2
1
(
3H)-one (5k). General Procedure D, yield 53%, yellow oil. H NMR (400 MHz, DMSO) δ
7
2
1
9
.85 – 6.76 (m, 8H), 4.02 (t, J = 6.3, 2H), 3.93 (t, J = 6.6, 2H), 3.68 – 3.08 (m, 12H), 2.11 (m,
1
3
3
H), 1.73 – 1.64 (m, 4H), 1.33 (m, 2H). C NMR (101 MHz, CDCl ) δ 158.95, 158.87,
26.46, 126.21, 115.40, 115.33, 112.08, 112.04, 111.88, 111.79, 111.76, 111.65, 99.93,
9.68, 47.44, 44.22, 42.35, 42.19, 31.82, 30.09, 16.59, 15.55, 13.96, 13.86. HRMS calcd for
+
C
26
H
32
N
4
O
4
[M+H] 464.2424, found 465.2491. Anal. calcd for C26
H
34Cl
2
N
4
O
4
: C, 58.10; H,
6
.38; N, 10.42. Found: C, 58.65; H, 6.05; N, 9.97.
3
2
-(3-(4-(5-(2-oxobenzo[d]thiazol-3(2H)-yl)pentyl)piperazin-1-yl)propyl)benzo[d]thiazol-
1
(3H)-one (5l). General Procedure D, yield 51%, colorless oil. H NMR (400 MHz, CDCl
3
)
δ 7.57 – 6.82 (m, 8H), 3.95 (t, J = 6.9, 2H), 3.88 (t, J = 7.1, 2H), 2.72 – 2.13 (m, 12H), 1.90 -
1
3
1
.82 (m, 2H), 1.74 – 1.67 (m, 2H), 1.62 – 1.44 (m, 2H), 1.39 – 1.32 (m, 2H). C NMR (101
MHz, CDCl
3
) δ 169.80, 169.72, 137.31, 137.06, 126.25, 126.18, 122.93, 122.89, 122.73,
1
2
22.64, 122.61, 122.51, 110.78, 110.54, 58.29, 55.07, 53.20, 53.04, 42.67, 40.94, 27.44,
+
6.41, 24.81, 24.71. HRMS calcd for C26
H
32
N
4
O
2
S [M+H] 496.1967, found 497.2055.
2
Anal. calcd for C26
H34Cl
2
N
4
O
2
2
S : C, 54.82; H, 6.02; N, 9.84. Found: C, 55.19; H, 5.94; N,
1
0.11.
3
2
-(3-(4-(6-(2-oxobenzo[d]oxazol-3(2H)-yl)hexyl)piperazin-1-yl)propyl)benzo[d]oxazol-
1
(3H)-one (5m). General Procedure D, yield 56%, yellow oil. H NMR (400 MHz, DMSO)
δ 7.85 – 6.76 (m, 8H), 4.02 (t, J = 6.3, 2H), 3.93 (t, J = 6.6, 2H), 3.67 – 3.04 (m, 12H), 2.17 –
1
3
2
1
1
2
3
.05 (m, 2H), 1.69 – 1.61 (m, 4H), 1.33 (m, 4H). C NMR (101 MHz, CDCl ) δ 165.67,
65.59, 133.29, 133.06, 122.10, 122.03, 118.77, 118.75, 118.72, 118.64, 118.50, 118.40,
06.64, 106.40, 54.36, 50.99, 49.10, 49.04, 48.96, 38.62, 36.89, 23.41, 23.04, 22.56, 22.53,
+
0.72.HRMS calcd for C27
H
34
N
4
O
4
[M+H] 478.2580, found 479.2675. Anal. calcd for
27 2 4 4
C H36Cl N O : C, 58.80; H, 6.58; N, 10.16. Found: C, 58.48; H, 6.13; N, 9.85.
1
8

3
-(3-(4-(6-(2-oxobenzo[d]thiazol-3(2H)-yl)hexyl)piperazin-1-yl)propyl)benzo[d]thiazol-2
1
(
3H)-one (5n). General Procedure D, yield 49%, colorless oil. H NMR (400 MHz, CDCl
3
)
δ 7.54 – 6.94 (m, 8H), 3.98 (t, J = 7.0, 2H), 3.90 (t, J = 7.1, 2H), 2.66 – 2.13 (m, 12H), 1.92 –
1
3
1
1
1
2
.85 (m, 2H), 1.76 – 1.68 (m, 2H), 1.62 – 1.27 (m, 6H). C NMR (101 MHz, CDCl ) δ
3
69.78, 169.69, 137.39, 137.16, 126.20, 126.14, 122.88, 122.85, 122.83, 122.74, 122.66,
22.60, 122.50, 110.75, 110.51, 58.46, 55.10, 53.21, 53.06, 42.73, 40.99, 27.51, 27.15, 26.67,
+
6.64, 24.83. HRMS calcd for C27
H
34
N
4
O
2
S
2
[M+H] 510.2123, found 511.2226. Anal. calcd
for C27
H
36Cl
2
N
4
O
2
S
2
: C, 55.56; H, 6.22; N, 9.60. Found: C, 55.19; H, 6.57; N, 10.03.
3
-(5-(4-(4-(2-oxobenzo[d]thiazol-3(2H)-yl)butyl)piperazin-1-yl)pentyl)benzo[d]thiazol-2
1
(
3H)-one (5p). General Procedure D, yield 54%, yellow oil. H NMR (400 MHz, CDCl
3
) δ
7
1
1
4
5
.57 – 6.85 (m, 8H), 3.94 – 3.88 (m, 4H), 2.64 – 2.19 (m, 12H), 1.74 – 1.69 (m, 4H), 1.63 –
1
3
3
.44 (m, 4H), 1.40 – 1.34 (m, 2H). C NMR (101 MHz, CDCl ) δ 169.71, 169.68, 137.13,
26.21, 122.89, 122.80, 122.78, 122.60, 122.57, 110.66, 110.50, 58.29, 57.61, 53.20, 53.10,
+
2.70, 42.52, 27.42, 26.40, 25.38, 24.72, 23.85. HRMS calcd for C27
11.2123, found 511.2203. Anal. calcd for C27
34 4 2 2
H N O S [M+H]
H
36Cl
2
N
4
O
2
S
2
: C, 55.56; H, 6.22; N, 9.60.
Found: C, 56.14; H, 6.17; N, 9.69.
3
2
7
3
1
1
2
-(6-(4-(4-(2-oxobenzo[d]oxazol-3(2H)-yl)butyl)piperazin-1-yl)hexyl)benzo[d]oxazol-
1
(3H)-one (5q). General Procedure D, yield 48%, yellow oil. H NMR (400 MHz, D
2
O) δ
.18 (m, 8H), 3.84 (t, J = 6.1, 2H), 3.76 (t, J = 6.7, 2H), 3.53 (s, 8H), 3.21 (t, J = 7.3, 2H),
13
2
.15 (t, J = 7.5, 2H), 1.90 – 1.54 (m, 8H), 1.31 (m, 4H). C NMR (101 MHz, D O) δ 156.44,
42.36, 142.31, 130.66, 130.46, 124.41, 124.34, 123.11, 122.94, 110.18, 110.07, 109.56,
09.38, 56.78, 56.76, 56.22, 48.71, 48.66, 41.88, 41.18, 26.59, 25.17, 25.09, 24.09, 23.17,
+
0.73. HRMS calcd for C28
H
36
N
4
O
4
[M+H] 492.2737, found 493.2828. Anal. calcd for
C
28
H
38Cl
2
N
4
4
O : C, 59.47; H, 6.77; N, 9.91. Found: C, 59.14; H, 6.53; N, 10.26.
3
2
-(6-(4-(4-(2-oxobenzo[d]thiazol-3(2H)-yl)butyl)piperazin-1-yl)hexyl)benzo[d]thiazol-
1
(3H)-one (5r). General Procedure D, yield 50%, colorless oil. H NMR (400 MHz, CDCl
3
)
δ 7.59 – 6.82 (m, 8H), 3.98 – 3.76 (m, 4H), 2.80 – 2.06 (m, 12H), 1.75 – 1.65 (m, 4H), 1.61 –
1
3
1
1
2
3
.24 (m, 8H). C NMR (101 MHz, CDCl ) δ 169.73, 137.08, 137.05, 126.24, 122.93, 122.91,
22.74, 122.72, 122.60, 122.58, 110.69, 110.54, 58.49, 57.61, 53.15, 53.03, 42.69, 42.48,
+
7.51, 27.14, 26.66, 26.62, 25.36, 23.81. HRMS calcd for C28
H
36
N
4
O
2
S
2
[M+H] 524.2280,
found 525.2384. Anal. calcd for C28
6.31; H, 6.66; N, 9.01.
H
38Cl
2
N
4
O
2
2
S : C, 56.27; H, 6.41; N, 9.37. Found: C,
5
1
9

3
2
7
8
1
4
-(5-(4-(6-(2-oxobenzo[d]oxazol-3(2H)-yl)hexyl)piperazin-1-yl)pentyl)benzo[d]oxazol-
1
(3H)-one (5s). General Procedure D, yield 55%, colorless oil. H NMR (400 MHz, D
2
O) δ
.22 – 7.22 (m, 8H), 3.87 – 3.67 (m, 4H), 3.55 (s, 8H), 3.18 -3.14 (m, 4H), 1.81 – 1.52 (m,
13
H), 1.35 – 1.30 (m, 6H). C NMR (101 MHz, D O) δ 156.49, 142.39, 142.37, 130.73,
2
30.67, 124.41, 124.39, 123.04, 122.99, 110.16, 110.13, 109.62, 109.55, 56.74, 48.59, 48.55,
1.93, 41.67, 34.61, 26.62, 26.40, 25.20, 25.12, 23.18, 22.93, 22.76. HRMS calcd for
+
C
29
H
38
N
4
O
4
[M+H] 506.2893, found 507.2983. Anal. calcd for C29
H
40Cl
2
N
4
O
4
: C, 60.10; H,
6
.96; N, 9.67. Found: C, 59.84; H, 6.81; N, 9.29.
3
-(5-(4-(6-(2-oxobenzo[d]thiazol-3(2H)-yl)hexyl)piperazin-1-yl)pentyl)benzo[d]thiazol-2
1
(
3H)-one (5t). General Procedure D, yield 56%, yellow oil. H NMR (400 MHz, DMSO) δ
7
1
1
3
.74 – 7.08 (m, 8H), 3.96 – 3.91 (m, 4H), 3.70 – 3.06 (m, 12H), 1.91 – 1.48 (m, 8H), 1.45 –
1
3
.18 (m, 6H). C NMR (101 MHz, D
2
O) δ 156.45, 142.31, 142.30, 130.65, 130.58, 124.33,
24.32, 122.97, 122.92, 110.08, 110.05, 109.55, 109.48, 56.76, 48.52, 48.50, 41.85, 41.59,
38 4 2 2
4.51, 34.46, 26.57, 26.35, 25.14, 25.05, 23.12, 22.87, 22.68. HRMS calcd for C29H N O S
+
[
M+H] 538.2436, found 539.2540. Anal. calcd for C29
H
40Cl
2
N
4
O
2
S
2
: C, 56.94; H, 6.59; N,
9
.16. Found: C, 57.15; H, 6.09; N, 9.52.
General Procedure E. Synthesis of benzo[d]thiazole-2(3H)-thione (6b). Lawesson’s reagent
3.56 g, 8.82 mmol) was added, under stirring, to a solution of benzo[d]thiazol-2(3H)-one (2
(
g, 13.24 mmol) in anhydrous toluene (80 mL). The reaction mixture was heated under reflux
for 5 h. The solvent was removed in vacuo, and the residue was purified by flash column
chromatography (SiO
2
) using hexane/ethyl acetate (7: 3) as eluent to give 1.4 g (63%) of 6b
1
13
as a white solid. H NMR (400 MHz, DMSO) δ 13.72 (s, SH, 1H), 7.76 – 7.06 (m, 4H). C
NMR (101 MHz, DMSO) δ 190.28, 141.70, 129.80, 127.54, 124.60, 122.14, 112.86. MS (EI)
+
m/z 168 (M +1).
1
Benzo[d]oxazole-2(3H)-thione (6a). General Procedure E, yield 65%, white solid. H NMR
1
3
(
400 MHz, DMSO) δ 13.16 (s, SH, 1H), 7.10 – 6.67 (m, 4H). C NMR (101 MHz, DMSO) δ
+
1
87.52, 138.94, 127.04, 124.78, 121.84, 119.39, 110.10. MS (EI) m/z 152 (M +1).
General Procedure F. Synthesis of 2-(4-bromobutylthio)benzo[d]thiazole (7b). K
2 3
CO
(
2.47 g, 17.94 mmol) and 1,4-dibromobutane (5.65 mL, 47.84 mmol) were added, under
stirring, to a solution of 6b (1.0 g, 5.98 mmol) in anhydrous DMF (20 mL). The reaction
°
mixture was heated at 60 C for 2 h. After cooling, the reaction mixture was poured into 70
mL water and extracted with ethyl acetate (3 x 40 mL). The combined organic layers were
2
0

washed with saturated aqueous NaCl and dried over sodium sulfate. The solvent was
2
removed in vacuo, and the residue was purified by flash column chromatography (SiO )
1
using hexane/ ethyl acetate (8: 2) as eluent to give 1.2 g (67%) of 7b as yellow oil. H NMR
(
CDCl ): δ 7.95 – 7.24 (m, 4H), 3.47 (t, J = 5.3, 2H), 3.40 (t, J = 6.7, 2H), 2.16 – 1.90 (m,
3
1
3
4
3
H). C NMR (CDCl
3
) δ 166.58, 153.23, 135.21, 126.06, 124.25, 121.51, 120.98, 32.83,
+
2.49, 31.52, 27.88. MS (EI) m/z 302 (M +1).
2
-(4-bromobutylthio)benzo[d]oxazole (7a). General Procedure F, yield 69%, dark yellow
1
oil. H NMR (400 MHz, CDCl
3
) δ 7.43 – 6.80 (m, 4H), 3.89 (t, J = 6.4, 2H), 3.37 (t, J = 5.9,
1
3
2
1
H), 1.85 – 1.81 (m, 4H). C NMR (101 MHz, CDCl
3
) δ 169.71, 136.82, 126.45, 123.14,
+
22.70, 122.59, 110.59, 41.66, 33.16, 29.59, 26.17. MS (EI) m/z 286 (M +1).
General Procedure G. Synthesis of 1,4-bis(4-(benzo[d]thiazol-2-ylthio)butyl)piperazine
8b). K CO (0.68 g, 4.98 mmol) and piperazine (0.07 g, 0.83 mmol) were added, under
(
2
3
stirring, to a solution of 7b (0.5 g, 1.66 mmol) in anhydrous DMF (10 mL). The reaction
°
mixture was heated at 60 C for 1.5 h. After cooling, the reaction mixture was poured into 50
mL water and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were
washed with saturated aqueous NaCl and dried over sodium sulfate. The solvent was
2
removed in vacuo, and the residue was purified by flash column chromatography (SiO )
using dichlormethane/ methanol (9.5: 0.5) as eluent to give 0.53 g (61%) of 8b as a yellow
1
oil. H NMR (CDCl3):δ 7.26 (m, 8H), 3.29 (t, J = 7.2, 4H), 2.41 (s, 8H), 2.32 (t, J = 7.2, 4H),
1
3
1
1
5
.82 (m, 4H), 1.62 (m, 4H). C NMR (CDCl
3
) δ 165.01, 151.75, 141.96, 124.18, 123.72,
+
18.30, 109.76, 57.80, 53.16, 32.17, 27.31, 25.81. HRMS calcd for C26
28.1567, found 529.1573. Anal. calcd for C26
32 4 4
H N S [M+H]
H
34Cl
2
N
4
S
4
: C, 51.90; H, 5.70; N, 9.31. Found:
C, 52.11; H, 6.17; N, 9.29.
1
,4-bis(4-(benzo[d]oxazol-2-ylthio)butyl)piperazine (8a). General Procedure G, yield 63%,
1
colorless oil. H NMR (400 MHz, CDCl
3
) δ 8.05 – 7.01 (m, 8H), 3.33 (t, J = 7.2, 4H), 2.43
1
3
(
3
s, 8H), 2.33 (t, J = 10.2 , 4H), 1.82 (m, 4H), 1.63 (m, 4H). C NMR (101 MHz, CDCl ) δ
1
67.07, 153.32, 135.16, 125.98, 124.11, 121.44, 120.91, 57.86, 53.17, 33.48, 27.31, 25.94.
+
HRMS calcd for C26
H
32
N
4
O
2
S
2
[M+H] 496.2032, found 497.2035. Anal. calcd for
26 2 4 2 2
C H34Cl N O S : C, 54.82; H, 6.02; N, 9.84. Found: C, 54.97; H, 6.52; N, 10.07.
4
4
.2. Pharmacology
.2.1. Assay for iNOS inhibition:
2
1

The assay was performed in mouse macrophages (RAW264.7, obtained from ATCC))
cultured in phenol red free RPMI medium supplemented with 10% bovine calf serum and 100
U/mL penicillin G sodium, and 100 µg/mL streptomycin at 37 °C in an atmosphere of 5%
CO and 95% humidity. Cells were seeded in 96-well plates (50,000 cells/well) and incubated
2
for 24 hrs for a confluency of 75% or more. Test samples diluted in serum free medium were
added and after 30 minutes of incubation LPS (5 µg/mL) was added and cells were further
incubated for 24 hrs. The concentration of nitric oxide (NO) was determined by measuring
2
4
the level of nitrite in the cell culture supernatant by using Griess reagent. Percent inhibition
of nitrite production by the test compound was calculated in comparison to the vehicle
control. IC50 values were obtained from dose curves. L-NMMA was used as positive control.
4
.2.2. Assay for NF-κB inhibition:
The assay was performed in human chondrosarcoma (SW1353, obtained from ATCC) cells
2
6
as described earlier. Cells were cultured in 1:1 mixture of DMEM/F12 supplemented with
1
0% FBS, 100 U/mL penicillin G sodium, and 100 µg/mL streptomycin at 37 °C in an
7
atmosphere of 5% CO2 and 95% humidity. Cells (1.2 x 10 ) were washed once in an
antibiotic and FBS-free DMEM/F12, and then resuspended in 500 µL of antibiotic-free
DMEM/F12 containing 2.5% FBS. NF-κB luciferase plasmid construct was added to the cell
suspension at a concentration of 50 µg/mL and incubated for 5 min at room temperature. The
cells were electroporated at 160 V and one 70-ms pulse using BTX disposable cuvettes model
6
40 (4-mm gap) in a BTX Electro Square Porator T 820 (BTX I, San Diego, CA). After
5
electroporation, cells were plated to the wells of 96-well plates at a density of 1.25 X 10
cells per well. After 24 h, cells were treated with different concentrations of test compound
for 30 min prior to the addition of PMA (70 ng/mL) and incubated for 8 h. Luciferase activity
was measured using the Luciferase Assay kit (Promega). Light output was detected on a
SpectraMax plate reader. Percent inhibition of luciferase activity was calculated as compared
to vehicle control and IC50 values were obtained from dose curves. Parthenolide was used as
positive control. Sp-1 was used as a control transcription factor which is unresponsive to
inflammatory mediators (such as PMA). This is useful in detecting agents that
nonspecifically inhibit luciferase expression due to cytotoxicity or inhibition of luciferase
enzyme activity.
4
.2.3. Assay for in vitro cytotoxicity:
Cytotoxicity was determined against four human tumor cell lines [SK-MEL (malignant
melanoma); KB (epidermal carcinoma, oral); BT-549 (ductal carcinoma, breast); SK-OV-3
2
2

(
ovary carcinoma)] and two noncancerous kidney cell lines [Vero cells (African green
monkey kidney fibroblasts) and LLC-PK11 (pig kidney epithelial cells)] as described
1
8
earlier. All the cell lines were obtained from ATCC and cultured in RPMI-1640 medium
supplemented with bovine calf serum (10%) and amikacin (60 mg/L), at 37°C, 95%
humidity, 5% CO
2
. Cells were seeded at a density of 25,000 cells/well and grown for 24 h.
Samples, diluted appropriately in serum free medium, were added to the cells and again
3
0
incubated for 48 h. The number of viable cells was determined by Neutral Red assay.
Percent decrease in cell viability was calculated in comparison to vehicle control. IC50 (the
concentration of the test compound that caused a 50% decrease in cell viability) was
calculated from the dose curves. Doxorubicin was used as positive control. All assays were
performed in triplicate and the mean values are given in Table 1.
4
.2.4. In vivo assays: (Acetic acid-induced writhing in mice)
Male Swiss mice (20-30g, Harlan, Indianapolis, IN, USA) were grouped housed (n=3) in
polycarbonate cages (20x35x12 cm). Food (Purina 5001 Laboratory Rodent Chow, St Louis,
MO, USA) and water were available ad libitum. Room temperature was maintained at 22 +/-
o
1
C and overhead illumination was maintained on a 12-h light-dark cycle.
3
1
Procedure: Acetic acid-induced writhing assay was performed as mentioned before.
Briefly, mice received IP injections of test compounds or vehicle 15 min before nociceptive
testing. The positive control 3 mg/kg indomethacin was administered IP 30 min before
nociceptive testing. Subjects received 15 min of apparatus habituation in a 15 cm diameter
acrylic observation enclosure immediately before i.p. injection of 0.9% acetic acid (1 ml/ 0.1
kg). The number of writhing responses was counted for 20 minutes. In this assay, it is typical
3
2
for 10 – 20% of vehicle treated animals to be non-responders. In the present study 5 of the
6 subjects (i.e. 13.9%) did not respond to acetic acid injections and their data were omitted
3
prior to statistical analyses; this yielded groups sizes of n = 4-6. Data were analyzed one-way
ANOVA followed by Dunnett Multiple Comparisons Test. These procedures were conducted
under the ethical standards of the International Association for the Study of Pain and in
accordance with the principles of laboratory animal care as detailed in the National Institutes
of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised
1
985) and were approved by the University of Mississippi IACUC (Protocol # 10-007).
4
.2.5. Carrageenan-induced rat paw edema assay
Adult albino rats of both sexes weighing between 120–150 g were used. Rats were uniformly
hydrated by giving 3 ml water/rat through gastric inoculation to reduce variability to edema
2
3

response. Animals were divided into groups each of five animals. The control group was
given saline containing few drops of Tween 80. Test compounds or standard anti-
inflammatory compound, ketorolac tromethamine, were suspended in distilled water by the
aid of few drops of Tween 80 and administrated intraperitoneally one hour before induction
of inflammation. Induction of inflammation was performed by S.C. injection of 50 ml of 1%
carrageenan-sodium gel (Sigma–Aldrich, USA), into the subplantar region of the right hind
paw. The dorso-ventral diameter (thickness) of the right and left hind paws of each rat was
measured using a pair of dial thickness gauge calipers accurate to 0.0001 cm 1 h, 2 h, 3 h and
4
h after induction of inflammation. The left hind paw diameter served as a control for the
degree of inflammation in the right hind paw. The percentage of anti-inflammatory activity
% inhibition of inflammation) was calculated according to the following equation:
inhibition= (1 – L /L ) X 100
(
%
t
c
Lt is the mean increase in paw thickness in rats treated with the tested compounds.
Lc is the mean increase in paw thickness in control group.
Data were analyzed and results were presented in Table 2.
5
.2. Docking study
The binding site was generated from the co-crystallized ligand (AR-C120011) within iNOS
protein (PDB code: 3E65). Selected active ligands were docked using the following docking
configuration: (i) number of Monte Carlo search trials = 30000, search step for torsions with
polar hydrogens = 30°. (ii) The Root Mean Square Difference (RMS) threshold for ligand-to-
binding site shape match was set to 2.0 employing a maximum of 1.0 binding site partitions
and 1.0 site partition seed. (iii) The interaction energies were assessed employing Consistent
Force Field (CFF) force field with a non-bonded cutoff distance of 10.0 Å and distance-
dependent dielectric. An energy grid extending 3.0 Å from the binding site was implemented.
(
iv) Rigid body ligand minimization parameters were: 10 iterations of steepest descend (SD)
minimization followed by 20 Broyden–Fletcher–Goldfarb–Shanno (BFGS) iterations applied
to every successful orientation of the docked ligand. (v) A maximum of 10 diverse docked
conformations/poses of optimal interaction energies were saved. (vi) The saved
conformers/poses were further energy-minimized within the binding site for a maximum of
1
000 rigid-body iterations. (vii) Six scoring functions were used to score the high ranking
3
3-35
34,35
36
37,38
docked poses/conformers: Jain,
LigScore1, LigScore2,
PLP1, PLP2 and PMF.
2
4

Acknowledgments
This work was supported in part by NIGMS P20 GM104932 (CRM). The authors wish to
thank Prof. Dr. Mutasem O. Taha, Jordan University for his help and valuable discussion in
performing the docking study. Also, we deeply thank Mr. Paul Bates and Ms. Katherine
Martin for excellent technical help in carrying out the in vitro bioassays.
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2
6

Scheme, Table and Figure legends:
Figure 1: Design strategy for the proposed dimers
o
Scheme 1: Reagents and conditions: (a) dibromoalkane, K
2
CO
3
, DMF, 60 C, 2 h; (b) piperazine,
o
o
o
K
2
CO
3
, DMF, 60 C, 1.5 h; (c) piperazine, K
2
CO
3
, DMF, 60 C, 0.5 h; (d) K CO , DMF, 60
2 3
C, 2 h.
Scheme 2: Reagents and conditions: (a) Lawesson's reagent, toluene, reflux, 5 h; (b) 1,4
o
o
2 3 2 3
dibromobutane, K CO , DMF, 60 C, 2 h; (c) Piperazine, K CO , DMF, 60 C, 1.5 h.
Table 1: Inhibition of iNOS and NF-κB activity and cytotoxicity evaluation.
Positive controls: L-NMMA for iNOS inhibition, Parthenolide for NF-κB inhibition
and Doxorubicin for cytotoxicity. NA = no activity up to 25 µg/ml. IC50 = the test
concentration that caused a 50% inhibition.
Four human cancer cell lines were used: SK-MEL (Melanoma), KB (epidermal
carcinoma), BT-549 (breast carcinoma) and SK-OV-3 (ovarian carcinoma). Two non-
cancerous kidney cell lines were also used: Vero (Monkey kidney fibroblast) and
LLC-PK11 (Pig kidney epithelial cells).
Figure 2: Acetic acid-induced writhing test. Writhing responses for 20 minutes were
measured in mice pretreated with vehicle, Indomethacin (Indo) or test compounds
after i.p. injection of 0.9% acetic acid (1 ml/ 0.1 kg). The observations are mean ± SD.
*
p <0.05, ** p <0.01 (n = 4-6), compared to control.
Table 2: Edema thickness and inhibition percent of control, ketorolac and tested
compounds. The observations are mean ± SD. * p <0.01 (n = 5), compared to control.
Figure 3: A) Docking and binding mode of compound 8b into iNOS active site (PDB code:
3
E65), B) Docking and binding mode of compound 5t into iNOS active site (PDB
code: 3E65),C) Docking and binding mode of compound 3j into iNOS active site
PDB code: 3E65), D) The superimposition of the docked poses 3j (blue), 5t (pink),
b (green) and the crystallographic ligand, AR-C120011 (yellow) within active site of
(
8
iNOS. Hydrogen bonds are represented by dashed green lines. All hydrogens are
removed for the purposes of clarity. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article).
2
7

Figure 1
2
8

Scheme 1
2
9