Synthesis of novel dansyl-labeled Celecoxib derivatives

Article abstract of DOI:10.1016/j.tetlet.2013.09.025

Four novel dansyl-labeled derivatives of Celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, were designed and synthesized. To realize the fluorophore-linker-approach divergent and convergent synthetic strategies were applied. Therefore Celecoxib p-benzoic acid, 8, was synthesized in a new and convenient way. The yield and the synthetic route to Celecoxib, 1, its pyrazolylic acid, 7, and its pyrazolylic methyl ester, 6, were improved. Through a convenient synthesis 1,11-diamino-3,6,9-trioxundecane, 19, was obtained in high yield and purity and used as a linker for the dansyl moiety.

Full text of DOI:10.1016/j.tetlet.2013.09.025

Tetrahedron Letters 54 (2013) 6682–6686
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of novel dansyl-labeled Celecoxib derivatives
Andreas Lill a, Klaus Scholich b, Holger Stark a,c,
⇑
a Johann Wolfgang Goethe University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
b Johann Wolfgang Goethe University, Institute of Clinical Pharmacology, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany
c Heinrich-Heine-Universität Düsseldorf, Institute of Pharmaceutical and Medicinal Chemistry, Universitätsstraße 1, 40225 Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2013
Revised 5 September 2013
Accepted 10 September 2013
Available online 19 September 2013
Four novel dansyl-labeled derivatives of Celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, were
designed and synthesized. To realize the fluorophore-linker-approach divergent and convergent
synthetic strategies were applied. Therefore Celecoxib p-benzoic acid, 8, was synthesized in a new and
convenient way. The yield and the synthetic route to Celecoxib, 1, its pyrazolylic acid, 7, and its pyrazol-
ylic methyl ester, 6, were improved. Through a convenient synthesis 1,11-diamino-3,6,9-trioxundecane,
19, was obtained in high yield and purity and used as a linker for the dansyl moiety.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescence labeling
Celecoxib
Dansyl
COX
NSAR
Linker
Introduction
The visualization of COX-2 by molecular imagining has been
shown by Marnett et al.11 The unselective COX inhibitor indometh-
Visualization of molecules can help to understand the interac-
tions and molecular pathways of pharmacologically active sub-
stances. Labeled substances are tools to identify unknown
binding partners and to clarify the details of the mechanism of ac-
tion. They can be used as a diagnostic or therapeutic marker to de-
sign assay systems for distinct binding partners as well, for
example an enzyme or receptor associated with a disease. Aside
from radiolabels or imaging agents for complex optical photon
techniques, the introduction of a fluorescence moiety is the easiest
method to visualize a molecule.1,2
acin has been derivatized to a diagnostic and therapeutic fluores-
cent marker for COX-2.12–14 Other groups have studied
radiolabeled Celecoxib derivatives that are suitable for molecular
imaging—for example introduction of 123I and fluorination.11,15,16
Recently Marnett et al. described Celecoxib derivatives that are
linked to a fluorophore.12,13 Here we report the synthesis of novel
fluorescent labeled Celecoxib derivatives with a different design
approach. The final compounds were tested for their COX-2 inhib-
itory activity and absorptions and emission spectra in different sol-
vents were recorded.
It is well known that cyclooxygenase 2 (COX-2), the inducible
form of two isoenzymes that catalyze the conversion of arachi-
donic acid to prostaglandin PGH2, is overexpressed in inflamed
and tumerous tissue, distinct from most normal tissue. Through
the produced prostaglandins, COX-2 is a major contributor in can-
cer progression and might also be important in some neurological
diseases.3–6
Celecoxib, 1, a nonsteroidal anti-inflammatory drug (NSAID), is
a selective COX-2 inhibitor. COX-2-dependent and independent
anti-carcinogenic effects are observed independent of its concen-
tration, but they are far from being understood.7–9 Visualization
of COX-2 and/or Celecoxib is one way to get better insights in
the molecular actions involving Celecoxib.6,10
Results
Modeling
For the design of fluorescence-labeled Celecoxib derivatives a
linker approach was chosen to maintain the pharmacophore and
introduce the labeling element (Fig. 1).
Known structure–activity-relationship (SAR) studies,17 X-ray
structure of COX-2 with bond ligands18 and molecular docking-
experiments on Celecoxib and its derivatives19,20 revealed that
the sulfonamide moiety is essential for biological activities.
Whereas the trifluoromethyl group at the heterocycle points to-
ward the entry tunnel of the active site and should not alter the
affinity. The SAR showed that a pyrazole-3-carboxamide is still ac-
tive on COX-2.17 Derivatization of the tolyl group could serve as an
⇑
Corresponding author. Tel.: +49 211 81 10478; fax: +49 211 81 13359.
E-mail address: stark@hhu.de (H. Stark).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.09.025

A. Lill et al. / Tetrahedron Letters 54 (2013) 6682–6686
6683
glycol and an alkyl linker were chosen. Despite their different
lipophilicity the alkyl linker was selected for simplicity reasons
and it can serve as a negative control as well. 5-(Dimethyl-
amino)naphthalene-1-sulfonamide (Dansyl) was used as a fluo-
rophore because it is
a small fluorophore with similar
physicochemical properties compared to Celecoxib. In addition
to that the spectral properties of dansyl conjugates are sensitive
to their immediate environment which could be helpful in
whole cell studies.21–23
Figure 1. Linker approach for fluorescence-labeled derivatives with marked sites
for derivatization (bold), Celecoxib, 1 (4-(5-p-tolyl-3-(trifluoromethyl)-1H-pyrazol-
1-yl)benzenesulfonamide).
Chemistry
Starting from 4-methylacetophenon, 2, the Celecoxib acid
derivatives 7 and 8 were synthesized in good overall yields
(7:53%, 8:74%). In both cases the corresponding 1,3-diketones 3
and 4 were obtained by Claisen condensation (Scheme 1) using
sodium hydride24 in THF since sodium methylate17 resulted in
poorer yields (30–40% lower) and longer reaction times. For
the ester 4 ultrasound aided synthesis was applied. The dike-
tones were reacted with 4-hydrazinyl benzenesulfonamide hydro
chloride to give Celecoxib, 1, and the methyl ester 6, which were
both separated from their corresponding 3,5-regioisomers by col-
umn chromatography. For the pyrazol substituted Celecoxib
additional approach because it is unpredictable in which way fluo-
rescence-labeled Celecoxib analogues may bind in the binding
pocket.18
The physicochemical properties (e.g., lipophilicity) of the
fluorescence-labeled Celecoxib derivative should be at best in
a comparable range to that of Celecoxib, to avoid great changes
in the pharmacological behavior, for example cell permeability.
Molecular docking-experiments pointed out that the linker
should have a length of about 14 atoms to allow the fluores-
cence moiety to stay out of the binding pocket. A tetraethylene
Scheme 1. Synthesis of linkable Celecoxib derivatives 7 and 8: (i) NaH, THF, 30 min, 0 °C; (ii) F3CCOOEt, THF, 5 h, rt; (iii) (COOMe)2, THF, 16 h, 45 °C, ultrasound; (iv) EtOH,
20 h (1)/3h (4), reflux; (v) KMnO4, 2M NaOH, H2O, 6 h, reflux; (vi) 1.1 M NaOH, THF, 20 h, reflux.
Scheme 2. Linear synthesis of dansyl-labeled Celecoxib derivatives 11 and analogues: (i) HOBtÁH2O, EDC, DMF, 1 h, rt, then 1,10-diaminodecane, 14, DMF, 22 h, rt; (ii) DsCl,
NEt3, dioxane, 24 h, rt.

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A. Lill et al. / Tetrahedron Letters 54 (2013) 6682–6686
Scheme 3. Synthesis of dansyl-labeled linkers 15 and 20: (i) 0.33 equiv DsCl,
dioxane, 1 h, rt +30 min, 90 °C: (ii) MsCl, NEt3, DCM, 45 min, À5 to 0 °C; (iii) KPht,
DMF, 18 h, 80 °C; (iv) N2H4ÁH2O, THF/MeOH (1:1), 12 h, rt.
Scheme 4. Synthesis of tolyl ether Celecoxib derivatives 28: (i) K2CO3, MeCN, 3 d,
reflux; (ii) NaH, THF, 30 min, rt; (iii) F3CCOOEt, rt, 1d (quant.); (iv) 4-Hydrazinyl
benzensulfonamide hydrochlorid, EtOH, 20 h, reflux; (v) N2H4ÁH2O, THF, 6 h, reflux.
derivative 6 was hydrolyzed to pyrazolic acid 7.17 Celecoxib, 1,
was oxidized to 8 with potassium permanganate under basic
conditions. This simple one step synthesis offers direct availabil-
ity of 8 in good yield (66%), whereas the literature17 describes a
three step synthesis—tolyl-bromination of Celecoxib, conversion
into the alcohol followed by Jones oxidation—with an overall
yield of 11% for 8.
Following a simple linear synthetic approach, the carboxylic
acid 7 was activated with 1H-benzo[d][1,2,3]triazol-1-ol hydrate
(HOBtÁH2O) and N1-((ethylimino)methylene)-N3,N3-dimethyl pro-
pane-1,3-diamine (EDC) and treated with commercially available
1,10-diamino-decane, 14. The amidation yielded the desired Cele-
coxib-linker-amine 9 (25%) besides the Celecoxib-dimere 10 and
the diaminated product 11, which had to be separated by column
chromatography (Scheme 2). Mono-BOC-14 was not used, because
it only would have prevented the formation of 11—at the cost of
two more steps. Hence the primary amine 9 was reacted with dan-
syl chloride to give the dansyl-labeled Celecoxib derivative 12 as
well as the dilabeled product 13. 12 and 13 could be isolated and
purified through repeated column chromatography from the crude
1:1 mixture.
To circumvent the formation of the dimeric products 10 and 13
that is caused by the high reactivity of the sulfonamide, a
convergent synthesis was applied. At first 1,10-diaminodecane,
14, and the synthesized terminal diaminated tetraethylene glycol
derivative 19 were mono-dansylated25 to yield the dansyl-labeled
linker amine building blocks 15 and 20 (Scheme 3). 19 was ob-
tained in an overall yield of 74% by employing a new straightfor-
ward strategy avoiding corrosive reagents like phosphorus
halides, explosive azides, hydrogenation or the use of ammonia.
Scheme 5. Synthesis of fluorescence-labeled Celecoxib derivatives 12, 21, 22, and 29: (i) HOBtÁH2O, EDC, DMF, 1 h, rt, then 15 or 20, DMF, 22 h, RT; (ii) DsCl, dioxane,
24 h, rt ? 90 °C.

A. Lill et al. / Tetrahedron Letters 54 (2013) 6682–6686
6685
Figure 2. Absorption and emission spectra of the dansyl-labeled Celecoxib derivatives 12, 21, 22, and 29: (A 0.1 mM in ethanol, B 0.1 mM in Dulbecco’s PBS buffer prepared
from 1 mM stock in ethanol).
After converting the mesylated tetraethylene glycol, 17, into the
diphthalide, 18, the amine was obtained by treatment with hydra-
zine monohydrate.26 The diamine 19 could be isolated in good
yield and high purity by distillation.
Subsequently the fluorescent amine building blocks 15 and 20
were coupled with the Celecoxib acids 7 and 8 to yield the dan-
syl-labeled Celecoxib derivatives 12, 21, and 22 (Scheme 5). 7
and 8 were preactivated with EDC and HOBT and then treated with
the primary amines 15 and 20, respectively. The lower yields in the
amidation step can be explained by side reactions of the activated
acid moiety with the sulfonamide group. In all cases Celecoxib di-
mers could be detected.
For the tolyl ether linked dansyl-labeled Celecoxib derivative 29
a linear strategy had to be applied (Scheme 4), because it was
found that the sulfonamide causes solubility problems and the for-
mation of side products occurred in alkylation reactions.
Therefore 11-bromoundecylphthalimide, 24, which was ob-
tained by monosubsitution of 1,11-dibromo-undecane,27 was
alkylated with 4-hydroxy-acetophenone, 23,.28 Ketone 25 was
analogously17 condensed to diketone 26. After the cyclization to
the Celecoxib derivative 27, the phthalimide moiety was cleaved
with hydrazine. The free amine 28 was dansylated to 29
(Scheme 5).25 Again, the lower yield in this step is caused by the
formation of dansylated-sulfonamide products.
Spectral properties
Absorption and emission spectra of the final compounds 12, 21,
22, and 29 were recorded in ethanolic (Fig. 2A ) and buffer solution
(Fig. 2B). The emission maxima shift to a 50 nm shorter wave-
length in buffer, whereas the intensity remains almost the same.
Biological activity
Final compounds 12, 21, 22, and 29 were tested for COX-1
and COX-2 activity in a commercially available assay (Cayman

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A. Lill et al. / Tetrahedron Letters 54 (2013) 6682–6686
Catalog.No.: 560131) measuring the produced amount of prosta-
Supplementary data
glandin PGF2 in comparison to a 100% control.18 All compounds
a
were tested in concentrations ranging from 0.001 to 10 lM with
Supplementary data (experimental details for the synthesis as
well as analytical data, exemplified biological results and images
from staining experiments) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.tetlet.2013.09.025.
Celecoxib as a reference. The biological screening showed no
significant reduction of prostaglandin production for both COX
isoforms, therefore only exemplified data for 29 are shown in
Supplementary data.
Staining experiments with HeLa cells showed that all com-
pounds are able to pass the cell membrane and accumulate in
the cytoplasm. It might be speculated that they enrich in the endo-
plasmatic reticulum (cp. fluorescence pictures in Supplementary
data).
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We have successfully designed and synthesized four new Cele-
coxib derivatives with a differently linked dansyl moiety as fluoro-
phore (12, 21, 22, and 29). These substances represent a useful
addition to the currently known Celecoxib fluorescent labels.
To overcome the specificity problem and side reactions—forma-
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synthetic route, a convergent one was followed. Hence the novel
and reasonably designed dansyl-labeled Celecoxib derivatives
were synthesized in higher yields by classic HOBt/EDC amide bond
formation of acids 7 and 8 with the amine-dansyl-building blocks
15 and 20. The linkable Celecoxib building blocks 7 and 8 were ob-
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from Celecoxib by employing the known method of tolyl oxidation
with potassium permanganate. For the synthesis of the tolyl-
labeled derivative 29 a linear strategy had to be followed, because
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side reactions in a direct alkylation of the corresponding phenolic
Celecoxib derivative. In the course of the convergent synthesis
we have established a simple, straightforward, and high yield syn-
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Support by the LOEWE Lipid Signaling Forschungszentrum
Frankfurt (LiFF) and Onkogene Signaltransduktion Frankfurt (OSF)
is gratefully acknowledged.