Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and conjugation onto magnetic nanoparticles

Article abstract of DOI:10.1016/j.tet.2017.08.027

Methods for the construction of C3-amino substituted 2H-indazole motifs are scarce. While the Davis-Beirut reaction proved useful and versatile in the construction of alkoxy- and thia- C3-substituted 2H-indazoles under mild basic conditions, the same succ

Full text of DOI:10.1016/j.tet.2017.08.027

Tetrahedron xxx (2017) 1e9
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Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction
and conjugation onto magnetic nanoparticles
,
Mohammad H. El-Dakdouki a *, AbdulSattar Hussein a, Hiba Abdallah a, Rania Shatila b,
Youssef Mouneimne b
a Department of Chemistry, Beirut Arab University, P.O. Box 11-5020, Riad El Solh, 11072809, Beirut, Lebanon
b KAS CRSL, American University of Beirut, Beirut, Lebanon
a r t i c l e i n f o
a b s t r a c t
Article history:
Methods for the construction of C3-amino substituted 2H-indazole motifs are scarce. While the Davis-
Beirut reaction proved useful and versatile in the construction of alkoxy- and thia- C3-substituted 2H-
indazoles under mild basic conditions, the same success was not enjoyed when confronted with
nitrogen-based nucleophiles. Therefore, we set out to expand the scope of the Davis-Beirut reaction and
investigated the ability of amine nucleophiles to prompt the heterocyclization. In addition, a model 2H-
indazole analogue was successfully conjugated onto magnetic nanoparticles for the first time. The
toxicity of the synthesized analogues was estimated using in silico T.E.S.T software. This report provides a
reliable approach for the synthesis of indazole-loaded nanoparticles whose applications have not been
investigated before.
Received 5 May 2017
Received in revised form
4 August 2017
Accepted 14 August 2017
Available online xxx
Keywords:
2H-indazoles
Davis-Beirut reaction
Iron oxide nanoparticles
In silico toxicity
Hyaluronic acid
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
cyclization of 2-nitroaryl imine or 2-nitroarene precursors (Scheme
1D),8 and the Pd-catalyzed intramolecular carbonenitrogen bond
The indazole scaffold has recently received considerable atten-
tion due to its broad spectrum of biological activity that includes
anticancer, anti-inflammatory, antidepressant, anti-HIV, and con-
traceptive activities.1 Therefore, there is enormous potential in the
synthesis of novel indazole motifs to be used as building blocks for
the next generation of pharmaceuticals.2 Naturally occurring
indazoles are rare probably due to the difficulty of living organisms
to construct NeN bonds.3,4 This has enticed organic chemists to
develop new facile synthetic routes to access the indazole core and
optimize reported methods. While numerous synthetic approaches
were reported for the preparation of the 1H-indazole tautomer, we
are aware of only a handful of studies that have addressed the
synthesis of substituted-2H-indazoles derivatives. As depicted in
Scheme 1, routes to 2H-indazoles include DDQ oxidation of the
corresponding pyrazoles made from the Bayllis-Hillman adducts of
2-cyclohexen-1-one (Scheme 1A),5 copper-catalyzed intra-
molecular amination (Scheme 1B),6 [3 þ 2] dipolar cycloaddition of
sydnones and arynes (Scheme 1C),7 the indium-iodine reductive
formation (Scheme 1E).9 Recently, inorganic nanoparticles such as
copper (I) oxide10 and copper (II) oxide11 nanoparticles were
employed to catalyze the formation of 2H-indazoles. Despite the
achieved success, most of these methods still have limitations, and
new routes are still explored.
Most recently, the Davis-Beirut reaction has been introduced as
a simple, efficient, and versatile base-mediated N,N-bond forming
heterocyclization one-pot reaction for the preparation of a wide
variety of all-new 2H-indazoles and indazolones.12e15 The named
reaction led to the successful preparation of 3-alkoxy,13 3-thia,16
and 3-amino substituted 2H-indazoles which are the focus of this
study (Scheme 2).17 Substrate diversity in the Davis-Beirut reaction
presents itself in the numerous heterocycles that can easily be
fused to the 2H-indazole core. Such heterocycles would be difficult
to prepare by other methods.
Unfortunately, the success and versatility achieved in the
preparation of alkoxy- and thia-based 2H-indazoles under the
Davis-Beirut reaction conditions was not attained in the synthesis
of amino-based 2H-indazoles (i.e. using an alkylamine as the
nucleophile) which proved to be tedious. In this regard, only one
report described the synthesis of C3-amino-substituted-2H-inda-
zoles using aminobenzothiazole as the nucleophile and 1,8-
* Corresponding author.
E-mail address: m.eldakdouki@bau.edu.lb (M.H. El-Dakdouki).
http://dx.doi.org/10.1016/j.tet.2017.08.027
0040-4020/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
conjugation onto magnetic nanoparticles, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.08.027

2
M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
Synthesizing and characterizing the indazole-loaded magnetic
nanoparticles. This goal will be achieved by preparing hyaluronan-
coated superparamagnetic iron oxide nanoparticles, and the sub-
sequent conjugation of the 2H-indazole analogues via amide bond
formation; and (iii) Estimating the toxicity of the synthesized 2H-
indazoles in silico.
2. Results and discussion
2.1. Synthesis of benzylamine amine derivatives
The benzylamine derivatives 3 which act as the synthetic pre-
cursors for the desired 2H-indazoles were prepared as depicted in
Scheme 3. First, the Schiff bases were synthesized in excellent
yields by melting 6-methoxy-benzothiazol-2ylamine 1 with the
corresponding o-nitrobenzaldehyde derivative 2 at 120 ꢀC.17 The
as-prepared Schiff bases were then reduced to the corresponding
benzylamine derivatives 3 using sodium borohydride as reducing
agent. The reaction was complete when all orange Schiff bases were
converted into yellow benzylamine precipitate. The reduction was
highly efficient and the benzylamines required no further purifi-
cation as revealed by various spectroscopic techniques (Supporting
information).17
Scheme 1. Routes to 2H-indazole.
diazabicyclo[5.4.0]undec-7-ene (DBU) as a base.17 Thus, much work
is needed to extend the scope of the Davis-Beirut reaction.
2.2. Synthesis of the 2H-indazole analogues via the Davis-Beirut
reaction
Nanotechnology has emerged as a promising tool that has found
numerous applications in the medical field for the detection,
diagnosis, and treatment of diseases.18 While the indazole phar-
macophore has been investigated for diverse biological and in-
dustrial applications, no report has described the conjugation of
indazoles onto nanoparticles. Iron oxide nanoparticles have long
been used in diverse medical applications including drug delivery
and disease detection.19,20 It is crucial that the nanoparticles are
biocompatible and colloidally stable if they are to be administered
systemically in living organisms.21 In this regard, dextran-coated
superparamagnetic iron oxide nanoparticles (SPIONs) have been
used in biological systems.22 Two SPIONs agents, namely Feridex
and Resovist, are approved for clinical application.23 Hence, SPIONs
were selected as an ideal system to act as a carrier of the synthe-
sized 2H-indazole analogues. As the ultimate goal, which is beyond
the scope of this study, is to assess the biological activity of the
indazole-loaded nanoparticles in biological systems, we relied on
an in silico computer-based predictive model to estimate the po-
tential toxicity of the synthesized indazole analogues, and to direct
the future synthesis of biologically compatible molecules. There-
fore, in this study, we aim at: (i) Optimizing the Davis-Beirut re-
action conditions to yield the desired C3-amino substituted
indazoles analogues in good yields. Achieving this goal aims to-
wards avoiding the low synthetic productivity of the Davis-Beirut
reaction when confronted with nitrogen-based nucleophiles; (ii)
As our goal is to conjugate 2H-indazole analogues onto the
surface of iron oxide nanoparticles, we set out to synthesize inda-
zole derivatives equipped with a linker at the C-3 position bearing a
primary amino group to facilitate its linkage to carboxyl-
functionalized nanoparticles. We opted to access the target 2H-
indazole analogues 4 from the corresponding benzylamines via the
Davis-Beirut reaction using DBU as the base at room temperature as
depicted in Scheme 4.
As amine nucleophiles did not enjoy the same success as the
alkoxy- and thia-based counterparts, the reaction conditions were
optimized to yield the desired indazoles in good yields. We first
attempted to synthesize the indazole analogues with ethylene
diamine (EDA, 7a) at the C-3 position of the indazole ring. Moni-
toring the conversion of benzylamine 3a by MS confirmed the
formation of the desired product 5 (Fig. S38). Unfortunately, com-
pound 5 was obtained in very low yield (<5%) and its purification
by column chromatography or thick layer chromatography proved
to be tedious. Similar results were obtained when triethylene glycol
diamine (NH2-TEG-NH2) 7b was used as a linker (Fig. 1). Isolation
and analysis of an analytical sample by proton NMR spectroscopy
and MS confirmed the structure of the desired indazoles 6a and 6d
(Fig. 1, Fig. S39 and S40). However, the low yield of reactions (<5%)
hindered the applicability of the deployed approach.
To overcome this synthetic hurdle, we postulated that protect-
ing one of the primary amino groups of the diamine linkers 7a and
7b with a base-resistant protecting group such as tert-butylox-
ycarbonyl group (Boc), might afford the desired indazole analogues
in acceptable yields. Thus, the Boc-protected linkers namely Boc-
Scheme 2. Synthetic versatility of the Davis-Beirut reaction.
Scheme 3. Synthesis of the benzylamine derivatives.
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
conjugation onto magnetic nanoparticles, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.08.027

M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
3
Scheme 4. Synthesis of 2H-indazole derivatives via the Davis-Beirut reaction.
Fig. 2. Structures of Boc-protected 2H-indazoles.
intermediate.17 On the other hand, an electron-donating group has
the opposite effect. Furthermore, the optimal reaction conditions
were found to be stirring the reaction mixture at room temperature
for 48 h. Longer reaction times did not result in better yields, while
heating the reaction mixture adversely affected the outcome
especially under reflux conditions.
Fig. 1. Structures of 2H-indazoles 5, 6a and 6d prepared under Davis-Beirut reaction
conditions with deployed linkers.
2.3. Synthesis of indazole-loaded nanoparticles
EDA-NH2 8a and Boc-TEG-NH2 8b were prepared in excellent yields
by dropwise addition of Boc anhydride (Boc2O) solution in anhy-
drous DCM to five equivalents of the corresponding diamine
(Scheme 5).24
With their nanometer dimensions and unique magnetic prop-
erties, iron oxide nanoparticles have been used in a variety of
biological applications including bioseparation, biosensing, nonin-
vasive imaging, targeted drug delivery as well as disease treatment
based on hyperthermia.20 Dextran-coated superparamagnetic iron
oxide nanoparticles (SPIONs) have been selected as the carrier of
the synthesized 2H-indazole analogues due to its biocompatiblity
and colloidal stability. The dextran-coated SPIONs were synthe-
sized following the coprecipitation method.25,26 In brief, an
aqueous solution of ferric and ferrous ions was neutralized by
ammonium hydroxide in the presence of dextran at 80 ꢀC, followed
by crosslinking the dextran with epichlorohydrin to induce stability
to the surface coating. Amino groups were introduced into the
surface by stirring the crosslinked nanoparticles in an ammonium
hydroxide solution for 2 days at 37 ꢀC (Scheme 6). It is desirable that
the surface of the nanoparticles is functionalized with carboxyl
groups to ensure that the 2H-indazole derivatives with free primary
amine groups can be conjugated through amide bonds. To achieve
this goal, the biocompatible hyaluronic acid (HA) was selected as
the polymer of choice.27 HA coating provides stability against
agglomeration and opsonization. It also provides the necessary
carboxyl groups which serve as chemical handles for the conjuga-
tion of the synthesized analogues onto the nanoparticles by ami-
dation. A sonicated aqueous solution of hyaluronic acid was
activated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and
N-methylmorpholine (NMM) and the reaction was allowed to stir at
room temperature for 2 h after which the aqueous dispersion of
SPION was added and the mixture was stirred for 48 h to provide
SPION-HA (Scheme 6).
SPION-HA exhibited a hydrodynamic diameter of 88 nm as
measured by Dynamic Light Scattering (DLS) (Fig. 3A), and an iron
oxide core of 5e7 nm in diameter as revealed by TEM (Fig. S62). A
polydispersity index of 0.24 confirmed the monodispersity of
SPION-HA. Thermal gravimetric analysis (TGA) provided evidence
for the successful conjugation of HA onto SPION and demonstrated
that HA accounted for 44% weight of each SPION-HA (Fig. 3C). The
potential use of SPION-HA nanoparticles as negative MRI contrast
agents was assessed by measuring its ability to enhance the T2*
magnetic relaxation rate (r2* ¼ 1/T2*) of water in a magnetic field.
SPION-HA had excellent magnetic relaxivity (r2* ¼ 232 mMꢁ1sꢁ1 at
3T), thus are good contrast agents for T2* weighted MRI (Fig. 3D).
The 2H-indazole analogues were then conjugated to SPION-HA,
with indazole 9a chosen as the model molecule to run preliminary
experiments. To facilitate the conjugation of indazole 9a onto the
HA-coated nanoparticles via an amide bond, the Boc protecting
When benzylamine 3a and Boc-TEG-NH2 8b were reacted under
the Davis-Beirut reaction conditions, indazole 10a was obtained as
confirmed by MS (Fig. S47), but only in low yields (Fig. 2). Inter-
estingly, indazole 9a with Boc-EDA linker at C-3 was afforded in
good yield as confirmed by NMR spectroscopy and MS (Figs. S48-
S51). The poor outcome of the reaction with Boc-TEG compared
to that of Boc-EDA-NH2 can be rationalized by the reduced nucle-
ophilicity of the amino group due to the presence of a nearby
electron withdrawing oxygen. Thus, Boc-EDA was the linker of
choice for the synthesis of the proposed 2H-indazole derivatives
9a-e (Fig. 2). The successful synthesis of 2H-indazole derivatives
was confirmed by various spectroscopic techniques. The 1H NMR
spectroscopy revealed the disappearance of the peak around 5 ppm
of the benzylic methylene group of the benzylamine derivative, the
presence of eight to ten aromatic hydrogen atoms depending on the
degree of ring substitution, the presence of a sharp singlet around
1 ppm that counted for the nine hydrogens of the Boc moiety, and
the presence of two peaks at 3.5 and 3.9 ppm that integrated for
two protons each and accounted for the ethylene group in the
linker. Results were further confirmed by 13C NMR spectroscopy
that showed peaks around 27 and 28 ppm assigned to the methy-
lene carbons in the linker. Further evidence was collected by MS
(Supporting information).
As expected, the yield of the Davis-Beirut reactions appeared to
be influenced by the electronic properties of the substituent on the
benzene ring. While benzylamine derivatives with electron with-
drawing groups such as fluorine in 3d, chlorine in 3b, and bromine
in 3c afforded the highest yields for indazoles 9d (62%), 9b (51%),
and 9c (42%), those with electron donating groups such as dime-
thylamino in 3e produced the desired indazole 9e only in 14% yield.
It seems that presence of an electron-withdrawing group enhances
the electrophilicity of the nitro group rendering it more susceptible
to deoxygenation or reduction, forming the nitroso aldehyde
Scheme 5. Synthesis of Boc-protected linkers.
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
conjugation onto magnetic nanoparticles, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.08.027

4
M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
especially the LUMO (Fig. 4B). The concentration of the indazole
loaded onto the nanoparticles was 0.071 mg/ml. Further evidence
was obtained by comparing the difference in the effective hydro-
dynamic diameters of the nanoparticles. While the hydrodynamic
diameter of SPION-HA was measured to be 88 nm, the conjugation
of the 2H-indazole 11a onto the nanoparticles resulted in an in-
crease in the size of the nanoparticles yielding SPION-HA-IND with
diameter of 139 nm (Fig. 3B). The polydispersity index of the SPION-
HA-IND was found to be 0.2, highlighting the monodispersity of the
nanoparticles and reflecting its potential safe application in bio-
logical systems (Fig. 3B).
2.4. In silico toxicity prediction for the synthesized 2H-indazole
analogues
As the synthesized 2H-indazole analogues (free and conjugated
onto nanoparticles) are to be evaluated for diverse biological ac-
tivities in living organisms, we set out to assess its potential toxicity
using the Toxicity Estimation Software Tool (T.E.S.T.; version
4.2.1).28 T.E.S.T is an in silico computer-based predictive model
developed by the USA Environmental Protection Agency (EPA) to
screen untested compounds in order to establish priorities for
expensive and time-consuming traditional bioassays designed to
establish toxicity levels. The consensus method was used to assess
developmental toxicity, rat lethal dose (LD50), and Ames mutage-
nicity. Developmental toxicity predicts whether a chemical in-
terferes with normal development of humans and animals both
before and after birth. A compound is classified Ames positive if it
significantly induces revertant colony growth in at least one of out
of five strains. The oral rat LD50 endpoint represents the amount of
the chemical (mass of the chemical per body weight of the rat)
Scheme 6. Preparation of indazole-loaded magnetic nanoparticles.
group was removed under acidic conditions to yield the indazole
11a with a free amino group (Scheme 6). HA was activated using
EDC/NHS coupling chemistry in MES buffer. Indazole 11a in
MES:DMSO was then added, and the reaction was allowed to stir in
the dark over 48 h to provide the SPION-HA-IND that was purified
by centrifugation.
The loading of analogue 11a onto the surface of the nano-
particles was revealed by UVevis spectroscopy. The absorbance
pattern of indazole-loaded nanoparticle SPION-HA-IND was
compared to that of 6-benzothiazolamine 1 and the indazole 9a
(Fig. 4A). Precursor 1 showed an absorbance maximum at 220
assigned to
265 nm (n/
(aromatic ring) due to lengthening of a
band at 326 nm attributed to n/ * transition. The successful
conjugation of the indazole onto SPION-HA was inferred via
absorbance at 230 nm ( *). Interestingly, the absorption band
at 326 nm (n/ *) disappeared. This has been attributed to the
p
p
/
p
* transition (benzene ring) and another band at
*). The 2H-indazole 9a showed a red shift to 230 nm
-conjugated system, and a
p
p
p/p
p
possible protonation of the lone pair (n) of electrons on nitrogen
under the acidic coupling reaction conditions. As a result, the lone
pair (n) becomes a bonding pair (s). Furthermore, all electrons are
now held more tightly and all MO energy levels are lowered
Fig. 3. Hydrodynamic diameter of SPION-HA (A) and SPION-HA-IND (B). (C) Ther-
mogravimetric analysis of SPION and SPION-HA showing 44% weight difference in
organic coating resulting from the successful conjugation of HA onto the nanoparticles.
(D) Determination of magnetic relaxivity (r2* ¼ 232 mMꢁ1 sꢁ1) of SPION-HA.
Fig. 4. (A) UVevis spectra of benzothiazole 1, 2H-indazole 9a, and SPION-HA-IND. (B)
Spectral red shift of
p/
p* transition upon protonation.
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conjugation onto magnetic nanoparticles, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.08.027

M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
5
Table 1
In silico toxicity prediction for some 2H-indazole analogues.
Entry
R1
R2
Oral Rat LD50 (mg/kg)
Mutagenicitya (a.u.)
Developmental Toxicityb (a.u.)
1
2
3
4
5
6
7
8
H
F
H
H
H
H
1047.50
1561.09
633.56
1921.91
764.03
1616.70
1250.98
1487.11
0.79 (Positive)
0.85 (Positive)
0.84 (Positive)
0.75 (Positive)
0.58 (Positive)
0.94 (Positive)
0.70 (Positive)
0.47 (Negative)
0.48 (Non-toxicant)
0.60 (Toxicant)
0.54 (Toxicant)
0.82 (Toxicant)
0.55 (Toxicant)
0.31 (Non-toxicant)
0.31 (Non-toxicant)
0.32 (Non-toxicant)
Cl
Br
H
CH3
H
N(CH3)2
H
CH3
C(CH3)3
h
a
Chemicals with predicted value > 0.5 are considered mutagenicity positive.
Chemicals with predicted value > 0.5 are considered toxicants.
b
which when orally ingested kills half of the sample rats.
chloride hexahydrate (FeCl3$6H2O), NH4OH (36%), ferrous chloride
tetrahydrate (FeCl2$4H2O), and dextran (13e20 kDa) were obatined
from Sigma-Aldrich. 6-methoxy-benzothiazol-2-ylamine, 5-
chloro-2-nitrobenzaldehyde, DBU, NMM, CDMT, anhydrous DCM,
anhydrous methanol, and anhydrous THF were purchased from
Across Organics. Di-tert-butyldicarbonate was obtained from Fluka.
DCM reagent grade was attained from Fischer Scientific. Instru-
mental analysis was carried out at Beirut Arab University unless
stated otherwise.
NMR data were recorded with a Bruker 300 MHz spectrometer
and chemical shifts were reported as ppm downfield from TMS as
internal standard. Melting points were recorded with Gallenkamp
digital Melting-point apparatus and were not corrected. FTIR
spectra was attained on a Nicolet Avatar 360 FT-IR spectrometer in
KBr discs at the American University of Beirut. Dynamic Light
Scattering (DLS) analysis was collected on a NanoBrook 90Plus
particle size analyser in the Department of Chemical and Petroleum
Engineering at the American University of Beirut. Mass spectros-
copy spectra were obtained on the Waters Quattro micro API LC/
MS/MS at Michigan State University (USA), or on AGILENT 1100
Series Quaternary Pump HPLC, with Agilent LC/MSD Trap XCP Mass
Spectrometry Detector at the American University of Beirut Spec-
trophotometry was attained using a Jasco V-670 spectrophotom-
eter. Concentration refers to rotary evaporation of volatiles.
Thermogravimetric analysis (TGA) was carried on a Thermal
Advantage (TA-Instruments-Waters LLC) TGA-Q500 series and the
samples were burned under nitrogen. Relaxivity measurements
were carried out on a GE 3T Signa® HDx MR scanner (GE Health-
care, Waukesha, WI) at Michigan State University (USA).
The toxicity parameters were assessed for the acetamide ana-
logues of the 2H-indazoles to mimic the amide linkage between
hyaluronic acid on the nanoparticles and the synthesized indazoles.
The in silico toxicity predictions are summarized in Table 1. Only the
unsubstituted 2H-indazole (entry 1) was predicted not to exhibit
developmental toxicity, and all synthesized analogues (entries 1e5)
were predicted to induce mutagenicity. The predicted oral rat LD50
values for the prepared analogues (entries 1e5) ranged from
633.56 mg/kg for the 5-chloro-substituted analogue to 1921.91 mg/
kg for the 5-bromo-substituted analogue, indicating that the
former is the most toxic while the latter is the least. To set the stage
for future design and synthesis of novel non-toxic 2H-indazole
analogues, we estimated the toxicity of several analogues. The
software predicted that the presence of an electron donating group
such a methyl (entries 6e7) lowered developmental toxicity but
not mutagenicity. Interestingly, the presence of a bulky tert-butyl
group (entry 8) yielded an analogue that is neither a developmental
toxicant nor mutagenic.
3. Conclusion
The novelty of the current study stems not only from the
preparation of novel heterocyclic 2H-indazoles compounds via the
Davis-Beirut reaction, but also from extending the scope of the
named reaction by overcoming a major limitation represented in
the introduction of an amine substituent on position C-3 of the
heterocycle. The reactions were conducted under mild conditions
without the need of special reagents or catalysts. In addition, the
successful conjugation of a model 2H-indazole analogue onto
magnetic nanoparticles has not been reported before. The in silico
toxicity predictions provide a tool for designing future potentially
safe 2H-indazoles analogues. The synthetic accessibility and feasi-
bility of indazole-loaded nanoparticles opens new avenues for
assessing the previously unexplored biomedical applications of this
novel formulation.
4.1. Synthesis of Benzylidene amine derivatives
An equimolar mixture of 6-methoxy-benzothiazol-2-ylamine 1
and an o-nitrobenzaldehyde derivative 2 was melted in an oil bath
set around 120 ꢀC to ensure complete melting and removal of water
as it forms. The reaction proceeded until TLC showed consumption
of the starting materials. Purification as required is given for each
synthesis.
4. Experimental section
Reagents were purchased from local suppliers and used without
further purification. o-nitrobenzaldehyde, 4-dimethylamino-2-
nitrobenzaldehyde, 4-fluoro-2-nitrobenz-aldehyde, 4-bromo-2-
nitrobenzaldehyde, methanol, sodium borohydride, trifluoroacetic
acid, ethylene diamine, and diaminated triethylene glycol, ferric
4.1.1. N-(6-methoxy-benzothiazol-2-yl)-2-nitrobenzylidene amine
(6-Methoxy-benzothiazol-2yl)-amine 1 (0.54 g, 3 mmol) and o-
nitrobenzaldehyde 2a (0.5 g, 3 mmol) were used and the corre-
sponding Schiff base was obtained as a yellow solid in 92% yield
following purification on a silica gel column using DCM as an
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
conjugation onto magnetic nanoparticles, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.08.027

6
M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
eluent. M.p. 178e180 ꢀC; 1H NMR (300 MHz, DMSO) dH 3.98 (s, 3H),
7.09 (dd, J ¼ 6.12, 3.05, 1H), 7.30 (d, J ¼ 6.24, 1H), 7.70e7.77 (m, 2H),
7.90 (d, J ¼ 6.81, 1H), 8.11 (d, J ¼ 6.34, 1H), 8.45 (d, J ¼ 6.67, 1H), 9.49
(s, 1H); 13C NMR (100 MHz, DMSO) dC 55.81, 104.24, 116.13, 124.35,
124.85, 129.50, 130.05, 132.61, 133.96, 136.16, 146.05, 149.79, 158.15,
160.5, 168.01; IR (KBr) ymax: 755 (s), 1122 (m), 1530 (s), 1647 (m),
3411 (s); MS: Theoretical mass: 313.05 calculated for C15H11N3O3S;
Found: m/z [MþH]þ: 314.06;
4.2. Synthesis of Benzylamines derivatives
Benzylidene derivatives were pestled and suspended in a min-
imal amount of methanol, followed by the addition of sodium
borohydride (2 equivalents). Effervescence indicated the advance-
ment of the reaction. The reaction proceeded at ambient temper-
ature until TLC showed consumption of the starting material, and a
change in color of the crystals occurred. Reduced solids were
collected on a Hirsch funnel and washed with cold methanol. No
further purification was required.
4.1.2. N-(6-methoxy-benzothiazol-2-yl)-5-chloro-2-nitrobenzyli-
dene amine
4.2.1. N-(6-methoxy-benzothiazol-2-yl)-2-nitrobenzylamine (3a)
The corresponding Schiff base (0.62 g, 2 mmol) was reduced,
and provided 3a as a yellow solid in 95% yield; M.p. 140-143ꢀC; 1H
NMR (300 MHz, CDCl3) dH 2.17 (s, 2H), 3.81 (s, 3H), 4.79 (s, 1H), 6.90
(dd, J ¼ 6.58, 3.46,1H), 7.10 (d, J ¼ 3.48,1H), 7.46 (dd, J ¼ 12, 6.56,1H),
(6-Methoxy-benzothiazol-2yl)-amine 1 (0.54 g, 3 mmol) and 5-
chloro-o-nitrobenzaldehyde 2b (0.55 g, 3 mmol) were used, and the
corresponding Schiff base was obtained as a reddish solid 94% yield
following purification by column chromatography using DCM as an
eluent. M.p. 165-166ꢀC; 1H NMR (300 MHz, DMSO) dH 3.85 (s, 3H),
7.15 (dd, J ¼ 6.97, 6.32,1H), 7.80 (dd, J ¼ 6.36, 5.72,1H), 7.92e7.94 (m,
2H), 8.21e8.23 (m, 2H), 9.42 (s, 1H); 13C NMR (100 MHz, DMSO) dC
55.02, 105.08, 105.59, 116.32, 123.86, 126.46, 127.01, 129.01, 130.82,
132.64, 133.55, 135.97, 138.69, 145.41, 145.87, 147.24, 147.99, 154.81,
157.71, 161.19, 162.49, 167.37, 188.67; IR (KBr) ymax: 827 (s), 1225 (s),
1525 (s), 1605 (s), 2940 (w);
7.62 (t, J ¼ 9.19, 1H), 7.80 (d, J ¼ 3.48, 1H), 8.11 (d, J ¼ 3.81, 1H); 13
C
NMR (100 MHz, CDCl3) dC 30.94, 46.55, 55.89, 105.46, 113.83, 119.14,
125.36, 128.88, 130.63, 131.28, 133.32, 134.10, 155.59, 165.03, 171.79;
IR (KBr) ymax 821(s), 1220 (s), 1521(s), 1625 (s), 2904 (w), 3107 (s);
MS: Theoretical mass: 315.07 calculated for C15H13N3O3S; Found:
m/z [MþH]þ: 316.08.
4.2.2. N-(6-methoxy-benzothiazol-2-yl)-5-chloro-2-nitrobenzyl-
amine (3b)
4.1.3. N-(6-methoxy-benzothiazol-2-yl)-5-bromo-2-nitrobenzyli-
dene amine
The corresponding benzylidene derivative (0.54 g, 1.5 mmol)
was reduced, and 3b was obtained as a yellow solid in 93% yield.
M.p. 138-140ꢀC; 1H NMR (300 MHz, DMSO) dH 3.73 (s, 3H), 4.89 (s,
2H), 6.82 (dd, J ¼ 8.73, 6.09, 1H), 7.28 (d, J ¼ 8.76, 1H), 7.34 (d,
J ¼ 2.58, 1H), 7.62e7.65 (m, 2H), 8.12 (d, J ¼ 8.43, 1H), 8.40 (s, 1H);
13C NMR (100 MHz, DMSO) dC 41.38, 55.61, 106.55, 113.33, 118.67,
126.68, 127.87, 130.45, 132.89, 135.43, 136.76, 147.47, 154.74, 162.96;
IR (neat) ymax: 829 (s), 1340 (s), 1434 (s), 1603 (s), 2901 (w), 3101
(w). MS: Theoretical mass: 349.0 calculated for C15H12ClN3O3S;
Found: m/z [M-H]-: 347.8;
The product was obtained as reddish solid from (6-methoxy-
benzothiazol-2yl)-amine 1 (0.54 g, 3 mmol) and 5-bromo-o-nitro-
benzaldehyde 2c (0.69 g, 3 mmol) in 96% yield following recrys-
tallization from DCM. M.p. 155-157ꢀC; 1H NMR (300 MHz, DMSO)
dH 3.85 (s, 3H), 6.80 (dd, J ¼ 8.73, 6.06, 2H), 7.23 (d, J ¼ 8.79, 1H), 7.70
(d, J ¼ 2.52, 1H), 7.90 (d, J ¼ 8.94, 1H), 8.41 (d, J ¼ 1.83, 1H), 9.37 (s,
1H); 13C NMR (100 MHz, DMSO) dC 55.61, 109.38, 114.11, 122.52,
124.78, 127.83, 130.08, 133.41, 134.16, 135.31, 146.35, 150.23, 155.94,
156.25, 157.38; IR (KBr) ymax: 829 (s), 1228 (s), 1597 (s), 1642 (s),
3086 (w). MS: Theoretical mass: 390.96 calculated for
C
15H10BrN3O3S; Found: m/z [MþNa]þ: 413.9.
4.2.3. N-(6-methoxy-benzothiazol-2-yl)-5-bromo-2-nitrobenzyl-
amine (3c)
4.1.4. N-(6-methoxy-benzothiazol-2-yl)-4-fluoro-2-nitrobenzyli-
dene amine
The corresponding benzylidene adduct (0.5 g, 1.27 mmol) was
reduced, and provided 3c as a yellow solid in 91% yield; M.p. 137-
139ꢀC; 1H NMR (300 MHz, CDCl3) dH 3.82 (s, 3H), 4.92 (s, 2H), 6.02
(s, 1H), 6.90 (dd, J ¼ 6.21, 2.61, 1H), 7.12 (d, J ¼ 2.58, 1H), 7.42 (d,
J ¼ 8.82, 1H), 7.68e7.75 (m, 1H), 8.22 (s, 1H); 13C NMR (100 MHz,
CDCl3) dC 44.38, 55.61, 106.54, 113.33, 118.67, 121.37, 127.33, 132.16,
(6-Methoxy-benzothiazol-2yl)-amine 1 (0.54 g, 3 mmol) and 4-
fluoro-o-nitrobenzaldehyde 2d (0.51 g, 3 mmol) were reacted to
provide the corresponding Schiff base as a reddish solid in 95% yield
following purification by column chromatography using DCM as an
eluent. M.p. 150-151ꢀC; 1H NMR (300 MHz, CDCl3) dH 3.90 (s, 3H),
7.11 (dd, J ¼ 6.25, 3.83, 1H), 7.31e7.38 (m, 2H), 7.91 (d, J ¼ 3.42, 1H),
8.14e8.21 (m, 2H), 9.45 (s, 1H); 13C NMR (100 MHz, CDCl3) dC 55.83,
104.19, 116.35, 116.55, 116.75, 119.35, 119.54, 125.53, 127.97, 145.99,
158.29, 159.11, 166.07, 167.44; MS: Theoretical mass: 331.04 calcu-
lated for C15H10FN3O3S; Found. m/z [MþH]þ: 332.05.
132.98, 133.10, 138.45, 146.08, 149.12, 154.73, 162.96; IR (KBr) ymax
:
829 (s), 1340 (s), 1473 (s), 1609 (s), 2902 (mb), 3102 (mb). MS:
Theoretical mass: 392.97.0 calculated for C15H12BrN3O3S; Found: m/
z [MþH]þ: 393.9.
4.2.4. N-(6-methoxy-benzothiazol-2-yl)-4-fluoro-2-nitrobenzyl-
amine (3d)
4.1.5. N-(6-methoxy-benzothiazol-2-yl)-4-dimethylamino-2-nitro-
benzylidene amine
The corresponding Schiff base (0.5 g, 1.5 mmol) was reduced to
3d which was obtained as a yellow solid in 91% yield; M.p. 140-
142ꢀC; 1H NMR (300 MHz, CDCl3) dH 3.80 (s, 3H), 4.98 (s, 2H), 6.88
(dd, J ¼ 6.56, 3.04, 1H), 7.09e7.11 (m, 2H), 7.41 (d, J ¼ 3.51, 1H), 7.51
(dd, J ¼ 6.59, 3.08, 1H), 8.18 (dd, J ¼ 6.28, 3.46, 1H); 13C NMR
(100 MHz, CDCl3) dC 46.32, 55.83, 105.38, 113.71, 115.52, 115.71,
117.90, 118.09, 119.54, 128.89, 128.37, 131.33, 137.83, 137.90, 144.06,
145.76,155.52,164.30,164.73,166.35; MS: Theoretical mass: 333.06
calculated for C15H12FN3O3S; Found: m/z [MþH]þ: 334.07.
(6-Methoxy-benzothiazol-2yl)-amine 1 (0.54 g, 3 mmol) and 4-
dimethylamino-o-nitrobenzaldehyde 2e (0.58 g, 3 mmol) were
used, and the corresponding Schiff base was obtained as a reddish-
brown solid in 92% yield. No further purification was required. M.p.
195-197ꢀC; 1H NMR (300 MHz, CDCl3) dH 3.16 (s, 6H), 3.89 (s, 3H),
6.92 (dd, J ¼ 8.97, 6.39, 1H), 7.07 (dd, J ¼ 8.94, 6.36, 1H), 7.13e7.30
(m, 2H), 7.85 (d, J ¼ 8.91, 1H), 8.41 (d, J ¼ 9.03, 1H), 9.26 (s, 1H); 13
C
NMR (100 MHz, CDCl3) dC 40.78, 55.61, 109.38, 112.72, 114.10,
120.09, 124.77, 129.05, 131.66, 134.16, 144.02, 150.22, 153.24, 155.94,
156.22, 157.38, IR (KBr) ymax: 825 (s), 1221 (s), 1619 (s), 2934 (w).
MS: Theoretical mass: 331.04 calculated for C17H16N4O3S; Found. m/
z [MþH]þ: 332.05.
4.2.5. N-(6-methoxy-benzothiazol-2-yl)-4-dimethylamino-2-nitro-
benzylamine (3e)
The corresponding Schiff base (0.5 g, 1.40 mmol) was reduced,
and 3e was obtained as a red-orange solid in 93% yield; M.p. 162-
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
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M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
7
164ꢀC; 1H NMR (300 MHz, CDCl3) dH 3.16 (s, 6H), 3.89 (s, 3H), 5.30
4.4.3. N-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-4-fluoro-2-(6-metho
xybenzo[d]thiazol-2-yl)-2H-indazol-3-amine (6d)
Benzylamine 3d (0.3 g; 0.89 mmol) and TEG-NH2 (0.38 g;
2.04 mmol) were dissolved in THF (10 mL), and provided 6d as a
yellow solid in very low yield (<5%); MS: Theoretical mass: 445.16
calculated for C21H24FN5O3S; Found: m/z [MþH]þ 446.12.
(s, 2H), 6.92 (dd, J ¼ 8.97, 6.39, 1H), 7.07 (dd, J ¼ 8.94, 6.36, 1H),
7.13e7.30 (m, 2H), 7.85 (d, J ¼ 8.91, 1H), 8.41 (d, J ¼ 9.03, 1H), 9.26 (s,
1H); 13C NMR (100 MHz, CDCl3) dC 40.78, 44.38, 55.61, 106.55,
113.33, 115.84, 118.51, 118.67, 129.13, 132.89, 138.41, 142.64, 149.12,
152.78, 154.47, 162.96; IR (KBr) ymax: 826 (s), 1366 (s), 1471 (s), 1599
(s), 2906 (w). MS: Theoretical mass: 358.1 calculated for
C
17H18N4O3S; Found: m/z [M-H]-: 356.7.
4.4.4. tert-Butyl(2-((2-(6-methoxybenzo[d]thiazol-2-yl)-2H-
indazol-3-yl)amino)ethyl)-carbamate (9a)
Benzylamine 3a (0.3 g, 0.68 mmol) and EDA-Boc 8a (0.45 g,
2.85 mmol) were reacted, and indazole 9a was obtained as yellow
solid in 38% yield. M.p. 217-219ꢀC; 1H NMR (300 MHz, CDCl3) dH
1.43 (s, 9H), 3.55e3.58 (m, 2H), 3.90 (s, 3H), 3.91e3.96 (m, 2H), 5.04
(s,1H); 6.79 (t, J ¼ 6,1H), 7.03e7.08 (m,1H); 7.32 (d, J ¼ 1.8,1H); 7.44
4.3. Synthesis of Boc-protected linkers
Boc-protected linkers were prepared by the dropwise addition
of a diluted solution of di-tert-butyl dicarbonate in anhydrous DCM
to the corresponding diamine precursor dissolved in DCM in an ice-
chilled round bottom flask. The reaction mixture was stirred at
room temperature for 18 h after which the volatiles were evapo-
rated. The as-formed residue was extracted dissolved in aqueous
NaHCO3 and extracted with DCM. The organic aliquot was
collected, dried over anhydrous MgSO4, and concentrated in vaccuo.
(d, J ¼ 6, 1H); 7.66 (d, J ¼ 6, 1H); 7.83 (d, J ¼ 15, 1H); 8.75 (s, 1H); 13
C
NMR (100 MHz, CDCl3) dC 27.23, 28.35, 40.68, 44.69, 55.85, 104.31,
115.56, 116.19, 118.74, 122.30, 122.78, 129.91, 133.63, 144.71, 150.90,
156.08, 157.51; IR (KBr) ymax: 621(m), 1116 (m), 1521 (m), 1676 (s),
3459 (s); MS: Theoretical mass: 439.17 calculated for C22H25N5O3S;
Found: m/z [MþH]þ 440.20.
4.3.1. NH2-EDA-Boc (8a)24
4.4.5. tert-Butyl(2-((5-chloro-2-(6-methoxybenzo[d]thiazol-2-yl)-
2H-indazol-3yl) amino)-ethyl)carbamate (9b)
To a solution of di-tert-butyl dicarbonate (1.09 g, 5 mmol) in
anhydrous DCM (80 mL) was added a solution of EDA 7a (1.80 g,
30 mmol) in anhydrous DCM (10 mL). 8a was obtained as a colorless
viscous oil in 94% yield; 1H NMR (300 MHz, CDCl3) dH 1.43 (s, 9H),
1.59 (s, 2H), 2.79 (t, J ¼ 6.13, 2H), 3.16 (d, J ¼ 3.35, 2H), 4.94 (s, 1H);
13C NMR (100 MHz, CDCl3) dC 20.38,41.79, 43.23, 76.7, 77.01, 77.23,
156.21; MS: Theoretical mass: 160.12 calculated for C7H16N2O2;
Found: m/z [MþH]þ 162.21.
Benzylamine 3b (0.3 g, 0.85 mmol) and EDA-Boc 8a (0.45 g,
2.85 mmol) were used, and 9b was collected as a yellow solid in 51%
yield; M.p. 238-241ꢀC; 1H NMR (300 MHz, CDCl3) dH 1.45 (s, 9H),
3.58 (t, J ¼ 11.97, 2H), 3.88 (t, J ¼ 12.45, 2H), 3.92 (s, 3H), 7.08e7.14
(m, 2H), 1.34e7.37 (m, 3H), 7.64 (s, 1H), 7.85 (d, J ¼ 9.42, 1H), 8.54 (t,
J ¼ 9.72, 1H); 13C NMR (100 MHz, CDCl3) dC 28.30, 40.77, 46.38,
55.61, 79.52, 106.73, 110.65, 113.33, 118.82, 121.43, 121.62, 124.83,
127.77, 133.31, 137.46,145.78,149.49, 154.74,156.81,158.06; IR (KBr)
ymax: 912 (s), 1355 (s), 1470 (s), 1678 (s), 2980 (w). MS: Theoretical
mass: 474.1 calculated for C22H24ClN5O3S; Found: m/z [MþNa]þ
497.1.
4.3.2. NH2-TEG-Boc (8b)29
To solution of di-tert-butyl dicarbonate (1.09 g, 5 mmol) in
anhydrous DCM (80 mL) was added a solution of TEG-NH2 7b
(4.44 g, 30 mmol) in anhydrous DCM (10 mL). The desired product
8b was afforded as a colorless viscous oil in 98% yield; 1H NMR
(300 MHz, CDCl3) dH 1.43 (s, 9H), 2.80e2.95 (m, 2H), 3.28e3.34 (m,
2H), 3.50e3.65 (m, 10H); 13C NMR (100 MHz, CDCl3) dC 28.4, 40.31,
41.50, 41.70, 53.26, 70.20, 72.71, 73.35; MS: Theoretical mass:
248.17 calculated for C11H24N2O3; Found: m/z [MþH]þ: 249.18.
4.4.6. tert-Butyl(2-((5-bromo-2-(6-methoxybenzo[d]thiazol-2-yl)-
2H-indazol-3-yl)amino)-ethyl)carbamate (9c)
Benzylamine 3c (0.3 g, 0.75 mmol) and EDA-Boc 8a (0.45 g,
2.85 mmol) were used, and 9c was collected as a yellow solid in 43%
yield; M.p. 224-226ꢀC; 1H NMR (300 MHz, CDCl3) dH 1.18 (s, 9H),
3.46 (t, J ¼ 12.3, 2H), 3.61e3.71 (m, 5H), 4.93 (s, 1H), 6.71 (dd,
J ¼ 9.09, 7.47, 1H), 7 (dd, J ¼ 6.36, 2.55, 1H), 7.25 (d, J ¼ 2.52, 1H),
7.49e7.53 (m, 2H), 7.55 (d, J ¼ 16.14, 1H), 7.47e7.86 (m, 1H); 13C
NMR (100 MHz, CDCl3) dC 28.30, 40.77, 46.83, 55.61, 79.52, 106.73,
111.08, 113.3, 114.83,118.8, 121, 122.68,131.25,133.31, 137.46,145.08,
149.49, 154.74, 156.81, 158.06. IR (KBr) ymax: 750 (m), 1220 (s), 1498
(m), 1650 (s), 3450 (s); MS: Theoretical mass: 473.6 calculated for
4.4. Synthesis of the 2H-Indazoles via the Davis-Beirut reaction
Benzylamine derivatives and a linker (EDA, TEG-NH2, NH2-EDA-
Boc, or NH2-TEG-Boc) were dissolved in anhydrous THF in a round
bottom flask wrapped in aluminum foil. Five drops of DBU were
then added, and the reaction mixture was stirred at room tem-
perature for 48 h. The dark solution was concentrated under vac-
uum and the oily residue was washed with DCM. The obtained
precipitate was filtered through a Hirsch funnel and washed with
DCM. No further purification was required.
C
22H24BrN5O3S; Found: m/z [MþNa]þ 497.1.
4.4.7. tert-Butyl(2-((4-fluoro-2-(6-methoxybenzo[d]thiazol-2-yl)-
2H-indazol-3-yl)amino)-ethyl)carbamate (9d)
Benzylamine 3d (0.3 g, 0.65 mmol) and EDA-Boc 8a (0.45 g,
2.85 mmol) were mixed. Indazole 9d was afforded as a yellow solid
in 62% yield; 1H NMR (300 MHz, CDCl3) dH 1.45 (s, 9H), 3.58 (t,
J ¼ 12.05, 2H), 3.85 (m, 5H), 5 (s, 1H), 7.07 (d, J ¼ 15.24, 1H), 7.35 (dd,
4.4.1. N1-(2-(6-methoxybenzo[d]thiazol-2-yl)-2H-indazol-3-yl)
ethane-1,2-diamine (5)
Benzylamine 3a (0.3 g; 0.68 mmol) and EDA (0.12 g; 2.04 mmol)
were dissolved in THF (10 mL), and provided 5 as a yellow solid in
very low yield (<5%); MS: Theoretical mass: 339.12 calculated for
J ¼ 6.32, 12.34, 1H), 7.4 (m, 2H), 7.83 (d, J ¼ 12.42, 1H), 8.5 (s, 1H); 13
C
NMR (100 MHz, CDCl3) dC 28.33, 40.76, 44.69, 55.87, 104.36, 115.63,
117.92, 121.30, 122.83, 133.62, 144.70, 148.42, 154.96, 157.63; MS:
Theoretical mass: 457.16 calculated for C22H24FN5O3S; Found: m/z
[MþH]þ: 458.26.
C
15H13N3O3S; Found: m/z [MþH]þ 340.12.
4.4.2. N-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-2-(6-methoxybenzo
[d]thiazol-2-yl)-2H-indazol-3-amine (6a)
Benzylamine 3a (0.3 g; 0.68 mmol) and TEG-NH2 (0.38 g;
2.04 mmol) were dissolved in THF (10 mL), and provided 6a as a
yellow solid in very low yield (<5%); MS: Theoretical mass: 427.17
calculated for C21H25N5O3S; Found: m/z [MþH]þ 428.17.
4.4.8. tert-Butyl(2-((6-(dimethylamino)-2-(6-methoxybenzo[d]
thiazol-2-yl)-2H-indazol-3-yl)amino)ethyl)carbamate (9e)
Benzylamine 3e (0.3 g, 0.65 mmol) and EDA-Boc 8a (0.45 g,
2.85 mmol) were reacted, and 9e was obtained as a red-orange
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
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8
M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
solid in 14% yield; M.p. 171-173ꢀC; 1H NMR (300 MHz, CDCl3) dH
1.61 (s, 6H), 3.02 (s, 9H), 3.82 (s, 3H), 4.78 (s, 2H), 5.94 (s, 1H), 6.90
(d, J ¼ 7.98, 2H), 7.12 (s, 1H), 7.33 (s, 2H), 7.45 (d, J ¼ 8.55, 1H), 7.57
(d, J ¼ 8.28, 1H); 13C NMR (100 MHz, CDCl3) dC 28.30, 40.78, 46.38,
55.61, 79.52, 106.73, 110.87, 112.84, 113.33, 118.82, 120.17, 122.46,
133.31, 137.46, 138.86, 149, 149.5, 154.74, 156.81, 158.06; IR (KBr)
ymax: 833 (s), 1358 (s), 1471 (s), 1607 (s), 2905 (mb), 3105 (mb). MS:
Theoretical mass: 482.2 calculated for C24H30N6O3S; Found: m/z
[M-H]-: 481.0.
allowed to stir over 2 h after which time a sonicated solution of
indazole 9a (10 mg, 0.0029 mmol) in MES: DMSO (5:1) was added
and the reaction was allowed to stir in the dark at room tempera-
ture for 48 h. The nanoparticle solution was diluted with double
distilled water and purified by dialysis and centrifugation. The
hydrodynamic diameter of the nanoparticles was found to be
139 nm and the polydispersity index was 0.2.
4.6.4. Spectrophotometric analysis
To evaluate the amount of indazole 9a loaded on the nano-
particles, a standard linear-fit curve of free indazole was created by
plotting the UVeVis absorbance at 230 nm of several indazole
standard solutions in methanol. The stock solution was prepared by
dissolving 2 mg of 9a in 10 mL methanol for a concentration of
0.2 mg/mL. The absorbance of standards of serial dilutions of 9a
(0.2; 0.1; 0.05; 0.025; 0.0125 mg/mL) against methanol blank
showed a linear correlation between the concentration gradient
and the corresponding absorbencies at 230 nm.
The slope gives the value of the molecular absorptivity (ε) for
indazole 9a and was calculated to be 12.4. SPION-HA-IND aqueous
sample of concentration 0.16 mg/mL was prepared by diluting a
2 mL aliquot of nanoparticle stock solution of initial concentration
0.8 mg/mL to a final volume of 10 mL dd-water. The absorbance at
230 nm was measured against indazole-free SPIONs blank which
4.4.9. tert-Butyl(2-(2-(2-((2-(6-methoxybenzo[d]thiazol-2-yl)-2H-
indazol-3yl)amino)ethoxy)-ethoxy)ethyl)carbamate (10a)
Required benzylamine 3a (0.3 g, 0.68 mmol) and TEG-Boc 8b
(0.66 g, 2.85 mmol), and provided as yellow solid in very low yield
(<5%); MS: Theoretical mass: 527.22 calculated for C26H33N5O4S;
Found: m/z [MþH]þ 528.22.
4.5. Synthesis of N1-(2-(6-methoxybenzo[d]thiazol-2-yl)-2H-
indazol-3yl)ethane-1,2-diamine (11a)
Indazole 3a (0.2 g, 0.45 mmol) was dissolved in ice-chilled DCM
(10 mL) and TFA (2 mL) was added. The reaction mixture was
stirred for 3 h at room temperature after which it was concentrated
in vaccuo. The as-formed yellowish solid was washed with hexane
to remove excess TFA. 11a was obtained as a yellowish solid in 92%
yield; M.p. 117-121ꢀC; 1H NMR (300 MHz, CDCl3) dH 3.46 (t, J ¼ 12.3,
2H), 3.61e3.71 (m, 5H), 4.93 (s, 1H), 6.71 (dd, J ¼ 9.09, 7.47, 1H), 7
(dd, J ¼ 6.36, 2.55, 1H), 7.25 (d, J ¼ 2.52, 1H), 7.49e7.53 (m, 2H), 7.55
(d, J ¼ 16.14, 1H), 7.47e7.86 (m, 1H); 13C NMR (100 MHz, CDCl3) dC
28.30, 40.77, 46.83, 55.61, 79.52, 106.73, 111.08, 113.3, 114.83, 118.8,
121, 122.68, 131.25, 133.31, 137.46, 145.08, 149.49, 154.74, 156.81,
158.06. IR (KBr) ymax: 722 (m), 805 (m), 1198 (s), 1540 (m), 1619 (s),
2917 (m), 3443 (s); MS: Theoretical mass: 339.11 calculated for
was prepared by diluting a 266 mL aliquot of SPION-HA aqueous
solution of initial concentration 6 mg/mL to a final volume of 10 mL.
The concentration of indazole 9a loaded onto the nanoparticles was
then calculated to be: [IND] ¼ 0.88/12.4 ¼ 0.071 mg/ml
4.6.5. Determining the r2* values for HA-NP
Serial dilutions of the HA-NP were prepared to a final volume of
5 mL in 15 mL-centrifuge tubes (Corning). The tubes were placed on
a polystyrene tube holder. All MRI experiments were carried out on
a GE 3T Signa® HDx MR scanner (GE Healthcare, Waukesha, WI). To
evaluate the r2* characteristics of the nanoparticles, the following
parameters were used: head coil, 3D fast spoiled gradient recalled
(FSPGR) sequence, flip angle ¼ 15ꢀ, 16 echo times (TEs) ¼ 2.1 ms, 4.6
ms, 7.0 ms, 9.4 ms, 11.8 ms, 14.3 ms, 16.7 ms, 19.1 ms, 21.5 ms, 24.0
ms, 26.4 ms, 28.8 ms, 31.2 ms, 33.7 ms, 36.1 ms, and 38.5 ms, time of
repetition (TR) ¼ 41.9 ms, receiver bandwidth (rBW) ¼ 62.5 kHz,
field of view (FOV) ¼ 16 cm, slice thickness ¼ 1.5 mm, number of
slices ¼ 16, acquisition matrix ¼ 256 ꢂ 256, number of excitation
(NEX) ¼ 1, and scan time ¼ 1 min 55 s [Fe] was plotted against (1/
T2*), and r2* is the slope of the generated straight line.
C
17H17N5OS; Found: m/z [MþH]þ 340.12.
4.6. Synthesis of indazole-loaded nanoparticles
4.6.1. Synthesis of dextran-coated aminated nanoparticles (SPION)
Dextran (4.5 g, 9e11 kDa) was dissolved by sonication in double
distilled water (20 mL) and then filtered through 0.22 mm syringe
filter. FeCl3.6H2O (0.32 g, 1.2 mmol) was added and the mixture was
stirred vigorously at 0 ꢀC under nitrogen for 2 h FeCl2.4H2O (0.13 g,
0.65 mmol) in 0.22 mm filtered degassed water (0.9 mL) was then
added slowly. Chilled 30% NH4OH solution (0.9 mL) was added
dropwise with rapid stirring resulting in a greenish suspension that
was heated at ~85 ꢀC for 90 min. The mixture was then cooled
forming black superparamagnetic iron oxide nanoparticles
(SPIONs). Ammonium chloride and excess dextran were removed
by extensive dialysis (14 kDa cutoff), followed by ultrafiltration
(30 kDa cutoff membrane). After several washes, the colloidal
product was concentrated by ultrafiltration and filtered through a
Acknowledgments
This work was supported by The National Council for Scientific
Research-Lebanon (Grant reference: 01-09-2013). We are grateful
for Dr. Walid Saad for running the DLS measurements, and for Dr.
David Zhu from Michigan State University for determining mag-
netic relaxivity.
0.22 mm membrane filter to a final concentration of 6 mg-NP/mL.
4.6.2. Synthesis of hyaluronan-coated nanoparticles (SPION-HA)
Acetonitrile (2 mL) and NMM (50 l) were dropped into HA
Appendix A. Supplementary data
m
(70 mg) dissolved in double distilled water (3 mL) by sonication.
CDMT (10 mg) was added to the solution of HA and the reaction was
allowed to stir at room temperature for 2 h. SPION (2 mL) were then
added and the mixture was allowed to at room temperature for
36 h. SPION-HA were purified by ultrafiltration. The hydrodynamic
diameter of the nanoparticles was found to be 88.3 nm.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2017.08.027.
References
1. Ali NA, Dar BA, Pradhan V, Farooqui M. Mini Rev Med Chem. 2013;13:
1792e1800.
2. Thangadurai A, Minu M, Wakode S, Agrawal S, Narasimhan B. Med Chem Res.
2012;21:1509e1523.
3. Yuan T, Nahar P, Sharma M, et al. J Nat Prod. 2014;77:2316e2320.
4. Blair LM, Sperry J. J Nat Prod. 2013;76:794e812.
4.6.3. Synthesis of indazole-loaded nanoparticles (SPION-HA-IND)
SPION-HA (2 mL), EDC (0.15 mg; 1 mmol), and NHS (0.46 mg,
4 mmol) were dissolved in MES buffer (10 mL) and the solution was
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
conjugation onto magnetic nanoparticles, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.08.027

M.H. El-Dakdouki et al. / Tetrahedron xxx (2017) 1e9
9
5. Lee KY, Gowrisankar S, Kim JN. Tetrahedron Lett. 2005;46:5387e5391.
6. Hu J, Cheng Y, Yang Y, Rao Y. Chem Comm. 2011;47:10133e10135.
7. Fang Y, Wu C, Larock RC, Shi F. J Org Chem. 2011;76:8840e8851.
8. Ahn GH, Lee JJ, Jun YM, Lee BM, Kim BH. Org Biomol Chem. 2007;5:2472e2485.
9. Song JJ, Yee NK. Org Lett. 2000;2:519e521.
20. El-Dakdouki MH, El-Boubbou K, Xia J, Kavunja H, Huang X. In: Narain R, ed.
Chemistry of Bioconjugates. John Wiley & Sons; 2014:281e314.
21. Silva AKA, Espinosa A, Kolosnjaj-Tabi J, Wilhelm C, Gazeau F. In: Iron Oxides.
Wiley-VCH Verlag GmbH & Co. KGaA; 2016:425e472.
22. Tassa C, Shaw SY, Weissleder R. Acc Chem Res. 2011;44:842e852.
23. Wang YX. Quant Imaging Med Surg. 2011;1:35e40.
10. Sharghi H, Aberi M. Synlett. 2014;25:1111e1115.
11. Khatun N, Gogoi A, Basu P, Das P, Patel BK. RSC Adv. 2014;4:4080e4084.
12. Conrad WE, Fukazawa R, Haddadin MJ, Kurth MJ. Org Lett. 2011;13:3138e3141.
13. Mills AD, Nazer MZ, Haddadin MJ, Kurth MJ. J Org Chem. 2006;71:2687e2689.
14. Avila B, Solano DM, Haddadin MJ, Kurth MJ. Org Lett. 2011;13:1060e1063.
15. Haddadin MJ, Conrad WE, Kurth MJ. Mini-Rev Med Chem. 2012;12:1293e1300.
16. Farber KM, Haddadin MJ, Kurth MJ. J Org Chem. 2014;79:6939e6945.
17. Avila B, El-Dakdouki MH, Nazer MZ, et al. Tetrahedron Lett. 2012;53:
6475e6478.
24. Riva E, Comi D, Borrelli S, et al. Bioorg Med Chem. 2010;18:8660e8668.
25. El-Dakdouki MH, Xia J, Zhu DC, et al. ACS Appl Mater Interfaces. 2014;6:
697e705.
26. El-Dakdouki MH, Zhu DC, El-Boubbou K, et al. Biomacromolecules. 2012;13:
1144e1151.
ꢀ
ꢀ
ꢀ
27. Lapcík L, Lapcík L, De Smedt S, Demeester J, Chabrecek P. Chem Rev. 1998;98:
2663e2684.
28. Abdallah H, Arnaudguilhem C, Abdulrahim H, Lobinski R, Jaber F. Food Chem.
2016;192:212e227.
29. Jha S, Ramadori F, Quarta S, et al. Bioconjug Chem. 2017;28:222e229.
18. Petros RA, Desimone JM. Nat Rev Drug Discov. 2010;9:615e627.
19. Reiss G, Hutten A. Nat Mater. 2005;4:725e726.
Please cite this article in press as: El-Dakdouki MH, et al., Synthesis of novel 2H-indazole analogues via the Davis-Beirut reaction and
conjugation onto magnetic nanoparticles, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.08.027