Design and Synthesis of Novel Nonsteroidal Anti-Inflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis

Article abstract of DOI:10.1021/acs.jmedchem.6b01607

We report the synthesis of a series of hybrid compounds incorporating 6- and 7-substituted coumarins (carbonic anhydrase, CA inhibitors) derivatized with clinically used NSAIDs (indomethacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) as agents for the management of rheumatoid arthritis (RA). Most compounds were effective in inhibiting the RA overexpressed hCA IX and XII, with K_I values in the low nanomolar-subnanomolar ranges. The antihyperalgesic activity of such compounds was assessed by means of the paw-pressure and incapacitance tests using an in vivo RA model. Among all tested compounds, the 7-coumarine hybrid with ibuprofen showed potent and persistent antihyperalgesic effect up to 60 min after administration.

Full text of DOI:10.1021/acs.jmedchem.6b01607

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Article
Design and Synthesis of Novel Non Steroidal Anti-
Inflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids
(NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis
Silvia Bua, Lorenzo Di Cesare Mannelli, Daniela Vullo, Carla Ghelardini, Gian
Luca Bartolucci, Andrea Scozzafava, Claudiu T Supuran, and Fabrizio Carta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01607 • Publication Date (Web): 11 Jan 2017
Downloaded from http://pubs.acs.org on January 11, 2017
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Design and Synthesis of Novel Non Steroidal Anti-Inflammatory Drugs and Carbonic
Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis
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Silvia Bua,1 Lorenzo Di Cesare Mannelli,2 Daniela Vullo,3 Carla Ghelardini,2 Gianluca
Bartolucci,1 Andrea Scozzafava,3 Claudiu T. Supuran1* and Fabrizio Carta1*
1 University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff
6, 50019 Sesto Fiorentino (Florence), Italy
2
Dept. of Neuroscience, Psychology, Drug Research and Child Health ꢀ NEUROFARBA ꢀ
Pharmacology and Toxicology Section, University of Florence, Florence, Italy.
3
University of Florence, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via
della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
Keywords: Carbonic Anhydrase Inhibitors (CAIs), Non Steroidal AntiꢀInflammatory Drugs
(NSAIDs), Metalloenzymes, Rheumatoid Arthritis (RA), Hybrids.
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Abstract.
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We report the synthesis of a series of hybrid compounds incorporating 6ꢀ and 7ꢀsubstituted
coumarins (carbonic anhydrase, CA inhibitors) and clinically used NSAIDs (indomethacin,
sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) as agents for
the management of rheumatoid arthritis (RA). Most compounds were effective in inhibiting the RA
overꢀexpressed hCA IX and XII, with KIs in the low nanomolarꢀsubnanomolar ranges. The
antihyperalgesic activity of such compounds was assessed by means of the pawꢀpressure and
incapacitance tests using an in vivo RA model. Among all tested compounds, the 7ꢀcoumarine
hybrid with ibuprofen showed potent and persistent antihyperalgesic effect, up to 60 min after
administration.
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Introduction.
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Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease caused by a faulty
autoimmune response, which primary affects the lining of the joints, thus causing erosion of the
cartilage, bone damage and joints deformity at the latter stages. The dimension as well as the impact
of RA in the society are well reported within the Rochester Epidemiology Project.1 Data showed
that in the period 1995 – 2007, each year about 4.1 % of the American population was diagnosed
with RA. The incidence of the disease was strictly age dependent been present in 0.9 % among
patients aged 18ꢀ34 years, 5.4 % among those aged over 85 years, with the peak (8.9 %) comprised
in the population aged between 65ꢀ74 years. Data analysis of the same period among gender
showed that the incidence rates increased up to 2.5 % each year in women, conversely a decrease of
about 0.5 % was observed in men.1
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RA symptoms have high impacts on the life quality of the affected patients, which are progressively
unable to carry out activities in every domain of their lives such as work, social relations and
leisure.2
Stateꢀofꢀthe art on RA pharmacological treatments include two main classes: i) drugs acting to slow
or stop the course of the disease and to inhibit the joints tissutal damages and ii) those acting to ease
the symptoms.3,4 The first include the DiseaseꢀModifyingꢀAntirheumaticꢀDrugs (DMARDs),
biologics, JAKꢀinhibitors and corticosteroids, the latter are represented by the NonsteroidalꢀAntiꢀ
InflammatoryꢀDrugs (NSAIDs). Usually a therapeutic RA protocol accounts for the combination of
both drug classes along with the recommendation to conduct proper physical activities, with the
intent to tackle the symptoms as well as the progression of the pathology.3,4
Despite recent progresses in RA treatment, there is still no effective cure. Many research efforts are
constantly reported in the literature, thus offering innovative diagnostic tools, new biological targets
and a multitude of potential drug lead compounds.5,6
Recently, we reported that human (h) carbonic anhydrases (CAs, EC 4.2.1.1) isoforms IX and XII
are overꢀexpressed in inflamed synovium of patients affected from juvenile idiopathic arthritis
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(JIA), which is the most common RA disease in pediatric age.7 Previous studies also showed
abnormal expressions of the hCAs I, III and IV in specimens of RA affected patients, but only
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antibodies against these isoforms have been detected, withour measurements of the enzymatic
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activity in the synovium of the affected patients.
On the other hand, in the aboveꢀmentioned
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study,7 we have demonstrated overexpression of CA IX/XII (by using RTꢀPCR) as well as an
increased enzymatic activity due to these isoforms in the samples from JIA patients. In order to
better clarify the physioꢀ/pathological role of CAs in RA, we kept turning our attention towards the
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carbon dioxide hydration reaction (CO2 + H2O ↔ HCO3 + H+), which is the main transformation
catalyzed by these enzymes.12 According to the equilibrium reaction, CAs overꢀexpression
increases the ionic species concentrations. Since bicarbonate ions are vital building blocks in cells
life and are quickly recycled, a local extracellular acidosis is established. The dependence between
lowꢀpH values with the intensity of inflammation processes as well as their related pain symptoms,
were demonstrated in RA affected patients.13,14 In addition tissue acidosis was found to be
detrimental for both the humoral and cellular immunity processes.15 Considering thus the interplay
between various CA isoforms and the arthritisꢀlike diseases, we attempt here to target several of the
enzymes presumably involved in this condition by dual inhibitors incorporating both a CAꢀbinding
moiety (of the coumarin type) and a cyclooxygenase inhibitor of the NSAID type, the most widely
used painꢀrelief medication to date.16 Herein we report the design, synthesis of low molecular
weight hybrid compounds containing a selective hCA IX and/or XII inhibitor head, such as the
coumarin one,17 linked through a physiological cleavable linker to a commercially available,
clinically used NSAID tail, of the carboxylic acid type cyclooxygenase inhibitor (indomethacin,
sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc.). The ability of such compounds to
inhibit the relevant hCAs activity as well as to reduce the pain symptoms were assessed by mean of
in vitro kinetics and in vivo RA model respectively. Our scope was not to investigate
cyclooxygenase inhibition role in RA with the hybrid compounds, which is well documented for
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decades, but to show that CA IX/XII inhibition may represent an additional antiꢀinflammatory
mechanism, poorly investigated at this moment for the management of RA.
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Results and Discussion.
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Compounds Design and Synthesis.
The drug design rationale of this project is based upon the synthesis of small molecular hybrids
containing a selective hCA IX (XII) inhibitor head linked through a physiological cleavable linker
to a carboxylic acid NSAID (COX inhibitor) tail in a 1:1 ratio (Figure 1). As mentioned above, we
do not expect COX inhibitory activity of our compounds once that the COOH moiety essential for
this COX inhibition has been derivatized.17d
Figure 1. Rational design of the hybrids herein reported.
Since the 6ꢀ and 7ꢀsubstituted coumarins are usually reported selective inhibitors of the hCA IX/XII
18
over the other isoforms, we planned our hybrids up on these moieties and thus ensuring they are
committed with our recent findings on hCA IX and XII overꢀexpression in synovium specimen of
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JIA affected patients. For the scope of this work we considered a simple 2 carbon linear unit
spacer connecting the hCA IX (XII) inhibitor head and the NSAID terminal section. In particular,
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we focused on the ethanolamine fragment, which allows us: i) to insert a physiologically cleavable
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moiety, such as the amide, by making use of the NSAID carboxylic function; ii) to install the
biologically compatible and not cleavable ether moiety, thus virtually ensuring a single break point
on the hybrid; iii) all needed synthetic intermediates are easily accessible by means of efficient
procedures.
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As reported in scheme 1, the 6ꢀ and 7ꢀcoumarin substituted hybrids 4a-h and 5a-h respectively,
were obtained by coupling the commercially available NSAIDs 1a-h, by means of their
Nꢀ
hydroxysuccinamide (NHS) activated esters 2a-h, with the in situ liberated amines 3a and b
(Scheme 1).
O
O
O
O
O
EDCI.HCl
NHS
O
N
O
CF3COO
H3N
O
O
N
R
O
O
R
R
OH
DMF dry
DMF dry
O
NH
1a-h
3a=6-O-alchil
3b=7-O-alchil
2a-h
4a-h=6-O-alchil
5a-h=7-O-alchil
a= Indometacin
b= Sulindac
c= Ketoprofen
e= Dichlofenac
f= Flurbiprofen
g= Ketorolac
h= Naproxen
d= Ibuprofen
Scheme 1. General synthetic scheme of hybrid compounds 4a-h and 5a-h.
The key intermediates 3a,b were obtained by means of Williamson’s etherification of the
commercially available 6ꢀand 7ꢀhydroxycoumarins with the Bocꢀprotected 1ꢀbromoethylamine 6,
followed by deprotection with trifluoroacetic acid (TFA) (Scheme 2).
O
O
O
O
O
TFA
DCM
H
N
O
O
K2CO3/DMF
60°C
O
O
O
Br
HO
O
N
H3N
0°C
H
O
CF3COO
6-OH/7-OH Coumarin
6
7a,b
3a,b
Scheme 2. Synthesis of the key intermediates 3a,b.
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1
All final compounds (Figure 2) as well as their intermediates were characterized by means of H,
13C, 19FꢀNMR spectroscopy, and HRMS and were >95% pure, as determined by HPLC (see the
Experimental Protocols for details).
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O
O
O
O
O
O
O
O
O
O
O
O
O
F
HN
O
O
NH
NH
O
O
NH
N
O
S
O
Cl
O
4a-Indometacin
4b-Sulindac
4d-Ibuprofen
fen
topro
4c-Ke
O
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O
O
O
O
O
O
O
HN
O
N
NH
F
NH
NH
(S)
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O
O
NH
4f-Flurbiprofen
4g-Ketorolac
4h-Naproxen
Cl
Cl
4e-Dichlofenac
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O
O
O
O
O
O
O
O
O
O
O
O
O
F
HN
O
O
NH
NH
O
O
NH
N
O
S
O
Cl
O
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5a-Indometacin
5b-Sulindac
5d-Ibuprofen
n
profe
-Keto
5c
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
O
N
NH
F
NH
NH
(S)
O
O
O
O
NH
5f-Flurbiprofen
5g-Ketorolac
5h-Naproxen
Cl
Cl
5e-Dichlofenac
Figure 2. Structures of the compounds 4a-h and 5a-h.
Carbonic anhydrase inhibition.
All synthesized compounds, 4a-h and 5a-h, were tested in vitro for their inhibitory properties
against the physiological relevant hCA isoforms (I, II, IV, VII; IX and XII) by means of the
stoppedꢀflow carbon dioxide hydration assay,19 and their activities were compared to the standard
CAI acetazolamide (AAZ) (Table 1).
Table 1. hCA I, II, IV, VII, IX, and XII inhibition data with compounds 4a-h and 5a-h by a
StoppedꢀFlow, CO2 Hydrase Assay. 19
KI (nM)*
hCA I
>100
hCA II
>100
hCA IV
2.6
hCA VII
>100
hCA IX**
31.3
hCA XII
59.1
4a
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4b
4c
4d
4e
4f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
85.4
7.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.5
30.8
54.7
>100
28.9
23.5
31.8
>100
20.1
27.9
>100
>100
89.7
>100
>100
>100
25.1
81.7
>100
>100
92.6
5.9
4.4
0.44
5.6
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0.81
0.79
0.73
8.3
4g
4h
5a
5b
5c
5d
5e
5f
>100
>100
6.5
9.0
7.7
9.5
57.8
39.0
80.8
>100
>100
80.7
5.7
>100
>100
>100
>100
>100
12
9.1
9.1
8.8
5g
5h
9.4
9.8
AAZ 250
74.2
*Means from three different assays. Errors were within ±5−10% of the reported values (data not
shown).**Catalytic domain.
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Overall, the data reported in Table 1 clearly showed that all compounds were ineffective in
inhibiting the highly abundant cytosolic hCA I and II as well as the central nervous system (CNS)
expressed hCA VII (KIs >100 nM). The only exception was represented from the coumarine
substituted at 7ꢀposition with Ketoprofen (5c), which was a high nanomolar hCA II inhibitor (KI
85.4 nM).
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The following StructureꢀActivityꢀRelationships (SARs) for the remaining hCA isoforms are
reported:
i)
The membrane bound hCA IV, which is of interest since it seems to be overꢀexpressed in
RA (at least antibodies against this isoform were reported in the RA patients samples), 11 was highly
inhibited by the compounds included in this study. All compounds showed KIs in the low
nanomolar range and comprised between 0.44ꢀ9.8 nM, thus significantly more potent when
compared to the standard CAI Acetazolamide (KI 74.2 nM). The 6ꢀsubstituted coumarins (4a-h)
were 1.2 to 20.7 fold more potent than the 7ꢀsubstituted ones (5a-h) in inhibiting the hCA IV
isoform.
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Interesting data were obtained for the tumor associated 12 and RA overꢀexpressed 7 hCA IX
isoform, as the 6ꢀsubstituted compounds 4a, b and 4e-g showed KIs of 23.5 and 31.8, thus
comparable to the standard CAI AAZ (KI 25.1 nM). The introduction at the 6ꢀposition of the
coumarine scaffold of either Ibuprofen (4d) or Naproxen (4h) determined loss of the inhibitory
activity (KIs > 100 nM). Within the 7ꢀsubstituted series, only the compounds 5a and b were
effective inhibitors of hCA IX (KIs 20.1 and 27.9 nM respectively), whereas the Dichlofenac
counterpart (5e) was a high nanomolar inhibitor (KI 89.7 nM). All other compounds resulted
ineffective, thus demonstrating that the substitution position at the coumarine ring resulted in potent
effects on the inhibition potencies of these compounds.
iii)
The second RA relevant hCA isoform (i.e. XII) 12 was potently inhibited by the 6ꢀcoumarin
substituted Flurbiprofen derivative 4f (KI 5.9 nM), with a potency comparable to the standard CAI
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AAZ (5.7 nM). The introduction at the same position of the coumarin scaffold of all the NSAIDs
herein considered, determined either decrease (4a,b,e) or loss (4c,d,g and h) of the hCA XII
inhibition potency (see data in Table 1). Swap of the NSAID moieties from the 6ꢀ to the 7ꢀposition
of the coumarin ring determined a significant increase of the inhibition potency of the derivatives
5a-d with KIs comprised in the lowꢀmedium nM range (Table 1). Noteworthy the Indomethacin and
the Sulindac derivatives (5a, b) were particularly active in inhibiting the hCA XII, with KIs of 6.5
and 7.7 nM respectively, thus comparable to the standard CAI AAZ (5.7 nM). Conversely,
introduction of the Dichlofenac and Naproxen moieties at the 7ꢀposition of the coumarin scaffold
(5e and h) determined a slight increase of the inhibition potencies with KIs of 80.8 and 92.6 nM
respectively. Finally the inhibition activities of the Ketorolac 5g and Flurbiprofen 5f derivatives
resulted unvaried and worsened compared to their 6ꢀsubstituted counterparts (Table 1).
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Pain relieve efficacy tests
The pain relieve efficacy of compounds 4a, 4f and 5aꢀg (10 mg kgꢀ1 p.o.) was evaluated in a rat
model of rheumatoid arthiritis induced by intraꢀarticular (i.a.) Complete Freund’s Adjuvant (CFA)
injection. The effects of compounds herein reported were compared with 100 mg kgꢀ1 ibuprofen, the
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classical NSAID administered at the active dosage usually employed in this model.
hypersensitivity to a mechanical noxious stimulus (Paw pressure test) is reported in figure 3.
The
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Figure 3. Acute pain relieving effect of 4a, 5b, 5d, 5f, 5g and ibuprofen in a rat model of
rheumatoid arthritis induced by CFA i.a. injection. Compounds were administered p.o. at the dose
of 10 mg kgꢀ1 while ibuprofen (p.o.) at the dose of 100 mg kgꢀ1. The Paw pressure test was
performed to evaluate the hypersensitivity to noxious mechanical stimuli. Measurements were
performed on day 14 after CFA intraꢀarticular injection. Compounds were suspended in 1% CMC
and orally administered, evaluating the pain threshold over time. Control animals were treated with
vehicle. The values represent the mean of 8 rats performed in 2 different experimental sets.
^^P<0.01 vs vehicle + vehicle treated animals. *P<0.05 and **P<0.01 vs time 0 min of the same
group.
Data collection at day 14 after CFA injection, showed that the reference group (vehicle + vehicle;
1% CMC), had a nociceptive threshold stable over the time course of the experiments and that the
pain threshold of the ipsilateral paw of the CFA + vehicle group was significantly decreased when
compared to the reference (47.6 ± 1.2 g and 65.3 ± 1.0 g respectively). A single oral administration
of the 7ꢀsusbstituted coumarin ibuprofen derivative (5d) was able to significantly increase the
weight tolerated on the ipsilateral paw at 15 min (58.7 ± 1.3 g) and with a maximum effect between
30 and 45 min (63.8 ± 1.3 g and 61.3 ± 1.3 g respectively) after treatment. The antinociceptive
effect completely disappeared at 75 min (Figure 1). Interestingly, the effect of ibuprofen per se
administered at a 10 fold higher dosage peaked 15 min after administration lasting up to 30 min.
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The sulindac derivative 5b increased the pain threshold of the ipsilateral paw starting 15 min after
administration (56.7 ± 0.8 g), the antiꢀhypersensitivity effect was still significant after 30 and 45
min. Compounds 4a, 5f and 5g induced a significant reduction of hypersensitivity showing
comparable efficacy (about 56 g). The effect peaked at 15 min and disappeared 45 min after
treatment (figure 1). Compounds 4f, 5a, 5c and 5e were not able to counteract the hypersensitivity
induced by CFA i.a. injection (data not shown). Moreover, none of the compounds herein
considered altered the pain threshold of the contralateral paw (See Table 1 in Supporting
information), thus suggesting that these molecules do not influence the normal pain sensitivity.
The compound efficacies were also evaluated by means of the incapacitance test, against the
spontaneous pain. We measured the hind limb weight bearing alterations caused by unilateral
damage (figure 4).
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Figure 4. Acute pain relieving effect of 4a, 5b, 5d, 5f and 5g, and ibuprofen in a rat model of
rheumatoid arthritis induced by CFA i.a. injection. Compounds were administered p.o. at the dose
of 10 mg kgꢀ1 while ibuprofen (p.o.) at the dose of 100 mg kgꢀ1. The Incapacitance test was
performed to evaluate the hind limb weight bearing alterations measured as postural imbalance
related to pain. Data are expressed as the difference between the weight applied on the contralateral
and ipsilateral limb (D Weight). Measurements were performed on day 14 after CFA i.a. injection.
Compounds were suspended in 1% CMC and orally administered, evaluating the postural balance
over time. Control animals were treated with vehicle. The values represent the mean of 8 rats
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performed in 2 different experimental sets. ^^P<0.01 vs vehicle + vehicle treated animals. *P<0.05
and **P<0.01 vs time 0 min of the same group.
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As above reported, the difference between the weight burdened on the contralateral and the
ipsilateral paw (∆ Weight) was significantly increased in the CFA + vehicle treated animals
compared to the control group (45.6 ± 3.5 g and 2.5 ± 3.4 g respectively). Such results obtained
after compounds acute treatments matched with those recorded with the paw pressure test
previously reported in figure 3. The best performing compound was the 7ꢀcoumarin substituted
ibuprofen (5d) at a concentration of 10 mg kgꢀ1, which was able to fully reduce the ꢁ Weight
between 30 and 45 min after treatment (7.9 ± 1.3 g and 11.1 ± 1.6 g respectively). The effect lasted
up to 60 min. On the contrary compounds 4a, 5b and 5f partially reduced the postural imbalance
starting from 15 min until 30 min after administration. The effect of ibuprofen (100 mg kgꢀ1) per se
was comparable to that induced from these latter compounds.
Conclusions
Herein we report for the first time the design and synthesis of a series of low molecular weight
NSAIDꢀCAI hybrids as agents for the treatment of RA and related inflammatory diseases. Most
compounds showed in vitro potency as inhibitors of the RA overꢀexpressed hCA IV, IX and XII,
with KIs in the low nanomolar range. The ability of the obtained compounds to affect the
inflammation process was assessed by means on the pawꢀpressure and incapacitance tests on a CFA
induced arthritis model in rats. Among the compounds considered, the 7ꢀcoumarine substituted
ibuprofen derivative 5d showed a potent antihyperalgesic effect, which was maintained for 60 min
after administration.The hybrid 5d, which is the most relevant compound in this research, showed
interesting antihyperalgesic effects and its inhibitory activity on hCAs was limited to the isoforms
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IV and XII and not IX. Such a result is in apparent contradiction with our expectations, which
accounted for the fully engagement of both hCA IX and XII in the RA inflammation processes.7 In
our previous investigation we reported, by means of quantitative real time PCR experiments, that
JIA derived fibroblasts possessed significantly higher hCA XII mRNA levels when compared to
healthy synovial fibroblasts. Opposite data were obtained for the mRNA hCA IX. Since the
densitometric experiments showed that both hCA IX and XII protein levels were highly increased in
JIA affected patients, we postulated and in agreement with the literature12a, 32 that the hypoxic
condition present in closed compartments, such as the joints, is the main cause sustaining the hCA
IX overꢀexpression. Among the compounds here reported the dual hybrid 5d was the only one in
the series able to selectively inhibit the hCA IV and XII and to show spectacular antihyperalgesic
effects. Inhibitors of the hCA IV, IX and XII, such as 4a, 5b and d, failed in the in vivo RA model.
This might suggest that the role of the hCA IX in the RA inflammation process is somehow
different as initially postulated. Future work on dual NSAIDꢀCA hybrids will be directed toward a
better definition of the role of the hCA IX in RA diseases. It should be however mentioned that the
hCA IV activity was not measured in the synovial samples from RA or JIA patients so far, and only
antibodies against this isoform were detected in patients with RA.11 Thus, our results suggest that
probably CA IV and eventually other membraneꢀassociated CAS (e.g., CA XIV and/or CA XV)
may be involved in inflammatory conditions. Work is in progress in our laboratories to assess
whether indeed CA IV may be the most relevant isoform in such conditions.
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We demonstrated for the first time that NSAIDꢀCAI hybrids are highly promising tools for the
treatment of symptoms, such as pain, originated from RA and related diseases in an animal model
of this disease. Furthermore, the inhibition of the RA overꢀexpressed hCA isoforms IV, IX and XII
can significantly contribute to reduction of the joint local acidosis, and thus to restore the humoral
and cellular immunity processes which resulted are hampered in such pathologies.
Experimental protocols.
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Chemistry. Anhydrous solvents and all reagents were purchased from SigmaꢀAldrich, Alfa Aesar
and TCI. All reactions involving airꢀ or moistureꢀsensitive compounds were performed under a
nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear
magnetic resonance (1HꢀNMR, 13CꢀNMR, 19FꢀNMR) spectra were recorded using a Bruker
Advance III 400 MHz spectrometer in DMSOꢀd6. Chemical shifts are reported in parts per million
(ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated
as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; brs, broad singlet; dd, doublet of
doublets. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of
D2O. Analytical thinꢀlayer chromatography (TLC) was carried out on Merck silica gel Fꢀ254 plates.
Flash chromatography purifications were performed on Merck Silica gel 60 (230ꢀ400 mesh ASTM)
as the stationary phase and ethyl acetate/nꢀhexane were used as eluents. Melting points (m.p.) were
measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are
uncorrected. HPLC was performed by using a Waters 2690 separation module coupled with a
photodiode array detector (PDA Waters 996) and as column a NovaꢀPak C18 4 ꢂm 3.9 mm × 150
mm (Waters), silicaꢀbased reverse phase column. Sample was dissolved in acetonitrile 10%, and an
injection volume of 45 ꢂL was used. The mobile phase, at a flow rate of 1 mL/min, was a gradient
of water + trifluoroacetic acid (TFA) 0.1% (A) and acetonitrile + TFA 0.1% (B), with steps as
follows: (A%:B%), 0−10 min 90:10, 10−25 min gradient to 60:40, 26:28 min isocratic 20:80,
29−35 min isocratic 90:10. TFA 0.1% in water as well in acetonitrile was used as counterion. All
compounds reported here were >96% HPLC pure. The solvents used in MS measures were acetone,
acetonitrile (Chromasolv grade), purchased from SigmaꢀAldrich (Milan ꢀ Italy), and mQ water 18
Mꢃ, obtained from Millipore's Simplicity system (MilanꢀItaly). The mass spectra were obtained
using a Varian 1200L triple quadrupole system (Palo Alto, CA, USA) equipped by Electrospray
Source (ESI) operating in both positive and negative ions. Stock solutions of analytes were prepared
in acetone at 1.0 mg mLꢀ1 and stored at 4°C. Working solutions of each analyte were freshly
prepared by diluting stock solutions in a mixture of mQ H2O/ACN 1/1 (v/v) up to a concentration of
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1.0 ꢂg mLꢀ1 The mass spectra of each analyte were acquired by introducing, via syringe pump at 10
/L minꢀ1, of the working solution. Rawꢀdata were collected and processed by Varian Workstation
Vers. 6.8 software.
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General procedure for the synthesis of acids 1d, 1e, 1g and 1h.
O
O
HCl/MeOH
H2O
R
OH
R
O
X
1d, 1e, 1g, 1h
1d; R= Ibuprofene; X=Na
1e; R= Diclofenac; X= Na
1g; R= Ketorolac; X= Trometamina
1h; R= Naprossene; X= Na
The appropriate carboxylic acid salt was treated with a 6.0 M or 12.0 M aqueous solution of
hydrochloric acid. The reaction was vigorously stirred at 0°C, warmed up to r.t. until starting
material was consumed (TLC monitoring). The formed white precipitate was collected by filtration,
dried under vacuo to afford the title compounds 1d, 1e, 1g and 1h as white solids.
Synthesis of (±)-2-(4-isobutylphenyl)propanoic acid ((±)-Ibuprofen) 1d. Compound 1d was
obtained according to the general procedure earlier reported using a 6.0 M aqueous solution of
hydrochloric acid and (±)ꢀIbuprofen sodium salt (0.5 g, 1.0 eq ): 89.3% yield; silica gel TLC Rf 0.81
(Ethyl acetate/nꢀhexane 60% v/v ); δH (400 MHz, DMSOꢀd6) 0.89 (6H, d, J 6.8, 2 x CH3), 1.37 (3H,
d, J 6.8, CH3), 1.83 (1H, m, CH3CH), 3.36 (2H, s, CH2), 3.65 (1H, m, (CH3)2CH), 7.13 (2H, d, J
8.0, ArꢀH), 7.22 (2H, d, J 8.0, ArꢀH), 12.29 (1H, m, exchange with D2O, COOH).
Synthesis of 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetic acid (Diclofenac) 1e. Compound 1e
was obtained according to the general procedure earlier reported using a 12.0 M aqueous solution of
hydrochloric acid and Diclofenac sodium salt (0.5 g, 1.0 eq): 70% yield; silica gel TLC Rf 0.45
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(Ethyl acetate/nꢀhexane 30% v/v ); δH (400 MHz, DMSOꢀd6) 3.74 (2H, s, COCH2), 6.32 (1H, d, J
7.2, ArꢀH), 6.89 (1H, m, ArꢀH), 7.10 (1H, m, ArꢀH), 7.24 (3H, m, ArꢀH), 7.57 (2H, d, J 8.0, ArꢀH
and NH), 12.70 (1H, m, exchange with D2O, COOH).
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Synthesis of (±)-5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid ((±)-Ketorolac) 1g.
Compound 1g was obtained according to the general procedure earlier reported using a 12.0 M
aqueous solution of hydrochloric acid and (±)ꢀKetorolac tris salt (0.3 g, 1.0 eq): 83.6% yield; silica
gel TLC Rf 0.31 (Ethyl acetate/nꢀhexane 50% v/v ); δH (400 MHz, DMSOꢀd6) 2.78 (2H, q, J 7.2, 2 x
CHHax), 4.12 (1H, t, J 7.4, COCH), 4.33 (1H, m, CHHeq), 4.44 (1H, m, CHHeq), 6.12 (1H, d, J 3.8,
pyrroleꢀH), 6.81 (1H, d, J 3.8, pyrroleꢀH), 7.55 (2H, t, J 7.6, ArꢀH), 7.64 (1H, t, J 7.2, ArꢀH), 7.79
(2H, d, J 7.6, ArꢀH), 12.65 (1H, m, exchange with D2O, COOH).
Synthesis of (S)-2-(6-methoxynaphthalen-2-yl)propanoic acid ((S)-(+)-Naproxen) 1h. Three
tablets of Momendol® containing 0.220 g each of Naproxen sodium salt were crushed and the
obtained powder was dissolved in EtOAc and treated with a 6.0 M aqueous solution of hydrochloric
acid. The mixture was vigorously stirred at r.t. until consumption of starting materials (TLC
monitoring). The mixture was filteredꢀoff and the filtrate was extracted with EtOAc (3 x 10 mL).
The combined organic layers were washed with H2O (3 x 20 mL), dried over Na2SO4, filteredꢀoff
and concentrated under vacuo to give a pale yellow oil that was purified by silica gel column
chromatography eluting with EtOAc/nꢀhexane 30% v/v to afford the titled compound 1h as a white
solid: 82.2% yield; silica gel TLC Rf 0.61 (Ethyl acetate/nꢀhexane 50% v/v ); δH (400 MHz, DMSOꢀ
d6) 1.48 (3H, d, J 7.6, CH3), 3.83 (1H, q, J 7.2, CH3CH), 3.90 (3H, s, OCH3), 7.18 (1H, dd, J 8.8
3.8, ArꢀH), 7.32 (1H, s, ArꢀH), 7.44 (1H, d, J 7.2, ArꢀH), 7.74 (1H, s, ArꢀH), 7.82 (2H, t, J 9.6, Arꢀ
H), 12.34 (1H, m, exchange with D2O, COOH).
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General procedure for the synthesis of 2-((2-oxo-2
H
-chromen-6-yl)oxy)ethan-1-amonium
6
7
8
9
trifluoroacetate salt 3a and 2-((2-oxo-2
salt 3b.
H-chromen-7-yl)oxy)ethan-1-amonium trifluoroacetate
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O
O
O
O
O
TFA
H
N
O
O
K CO /DMF
2
3
O
O
O
Br
HO
O
N
H N
DCM 0°C
3
60°C
H
O
CF COO
3
6-OH/7-OH Coumarin
6
7a,b
3a,b
Step 1: tertꢀButyl (2ꢀbromoethyl)carbamate 6 (1.0 g, 1.0 eq) was treated with 6ꢀhydroxyꢀ2Hꢀ
chromenꢀ2ꢀone (6-OH) and K2CO3 (3.0 eq) in acetone or alternatively with 7ꢀhydroxyꢀ2Hꢀ
chromenꢀ2ꢀone (7-OH) (1.0 eq) was treated in the same conditions
using dry N,Nꢀ
dimethylformamide (5.0 mL) as solvent and under N2 atmospheres. The reaction mixtures were
stirred at 60 °C O.N. until consumption of starting materials (TLC monitoring), then cooled down
to r.t. and treated respectively as follows.
i)
The white precipitate was filteredꢀoff and the obtained filtrate was concentrated under vacuo
to afford the 7a as an orange residue;
ii)
The reaction was quenched with a 3.0 M aqueous hydrochloric acid solution to give a
precipitate which was collected by filtration and triturated with diethyl ether to afford the 7b
as a white solid.
Step 2: tertꢀButyl (2ꢀ((2ꢀoxoꢀ2Hꢀchromenꢀ6ꢀyl)oxy)ethyl)carbamate 7a (1.0 eq) and tertꢀbutyl (2ꢀ
((2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)ethyl)carbamate 7b (1.0 eq) were dissolved in DCM and TFA (6.0
eq) was added dropꢀwise to the suspension. The solution was stirred at 0°C and then at r.t. until
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starting materials were consumed (TLC monitoring). The solvent was evaporated and the obtained
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residue dried under vacuo to afford the titled compounds 3a and 3b as white solids.
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2-((2-Oxo-2H-chromen-6-yl)oxy)ethan-1-aminium 2,2,2-trifluoroacetate 3a: 20.0% yield; silica
gel TLC Rf 0.55 (Ethyl acetate/nꢀhexane 50% v/v); δH (400 MHz, DMSOꢀd6) 3.30 (2H, q, J 5.2,
CH2NHꢀ), 4.25 (2H, t, J 5.0, CH2O), 6.54 (1H, d, J 9.2, ArꢀH), 7.29 (1H, d, J 3.2, ArꢀH), 7.36 (1H,
d, J 2.8, ArꢀH), 7.41 (1H, d, J 8.8, ArꢀH), 8.06 (1H, d, J 9.2, ArꢀH); δC (100 MHz, DMSOꢀd6) 39.3,
66.0, 112.7, 117.7, 118.5, 120.2, 121.0, 144.9, 149.2, 155.1, 161.1; δF (376 MHz, DMSOꢀd6) ꢀ
74.84.
2-((2-Oxo-2H-chromen-7-yl)oxy)ethan-1-aminium 2,2,2-trifluoroacetate 3b: 61.3% yield; silica
gel TLC Rf 0.50 (Ethyl acetate/nꢀhexane 50% v/v ); δH (400 MHz, DMSOꢀd6) 3.31 (2H, q, J 5.2,
CH2NHꢀ), 4.31 (2H, t, J 5.16, CH2O), 6.36 (1H, d, J 9.02, ArꢀH), 7.03 (1H, dd, J 8.6, 2.5, ArꢀH),
7.07 (1H, d, J 2.3, ArꢀH), 7.70 (1H, d, J 8.66, ArꢀH), 8.05 (1H, d, J 9.2, ArꢀH); δC (100 MHz,
DMSOꢀd6) 38.2, 65.2, 101.5, 112.8, 112.9, 113.0, 129.7, 144.4, 115.3, 160.3, 160.9; δF (376 MHz,
DMSOꢀd6) ꢀ74.23.
General procedure for the synthesis of tert-butyl (2-((2-oxo-2
H
-chromen-6-
yl)oxy)ethyl)carbamates 4a-h and tert-butyl (2-((2-oxo-2
H-chromen-7-yl)oxy)ethyl)
carbamates 5a-h.
O
O
O
O
O
EDCI.HCl
NHS
O
N
O
CF3COO
H3N
O
O
N
R
O
O
R
R
OH
DMF dry
DMF dry
NH
O
1a-h
3a=6-O-alchil
3b=7-O-alchil
2a-h
4a-h=6-O-alchil
5a-h=7-O-alchil
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NꢀHydroxysuccinimide (NHS) (1.5 eq) and EDCI.HCl (1.5 eq) were added to a solution of the
appropriate carboxylic acid 1a-h (0.5 g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The
reaction was stirred at r.t. until starting materials were consumed (TLC monitoring), followed by
addition of 2ꢀ((2ꢀoxoꢀ2Hꢀchromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀammoniun trifluoroacetate salt 3a (1.0 eq) or
2ꢀ((2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)ethanꢀ1ꢀammonium trifluoroacetate salt 3b (1.0 eq) and
triethylamine (Et3N) (1.0 eq). The mixture was stirred at 60°C until starting materials were
consumed (TLC monitoring), then quenched with H2O (10 mL) to afford a precipitate which was
collected by filtration or alternatively the reaction solution was extracted with EtOAc (3 x 10 mL).
The combined organic layers were washed with H2O (3 x 20 mL), dried over Na2SO4, filteredꢀoff
and concentrated under vacuo to give a solid that was purified by silica gel column chromatography
eluting with the appropriate mixture of EtOAc/nꢀhexane to afford the desired compounds.
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Synthesis
of
2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(2-((2-oxo-2H-
chromen-6-yl)oxy)ethyl)acetamide 4a. Compound 4a was obtained according to the general
procedure earlier reported using NHS (1.5 eq) and EDCI.HCl (1.5 eq) in a solution of Indometacin
1a (0.3 g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was stirred at
r.t. until the formation of the activated ester intermediate A (TLC monitoring, not isolated). Then 2ꢀ
((2ꢀoxoꢀ2Hꢀchromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3a and triethylamine (Et3N)
were added to the mixture, which was stirred at 60 °C until starting materials were consumed (TLC
monitoring), followed by quenching with H2O (10 mL). The formed brown precipitate was filteredꢀ
off, dried under vacuo to give a residue that was purified by silica gel column chromatography
eluting with EtOAc/nꢀhexane 50% v/v to afford the titled compound 4a as a white solid: 65.8%
yield; m.p.181ꢀ183 °C; silica gel TLC Rf 0.35 (Ethyl acetate/nꢀhexane 90% v/v ); δH (400 MHz,
DMSOꢀd6) 2.24 (3H, s, CH3), 3.51 (2H, q, J 6.3, CH2NH-), 3.58 (2H, s, CH2CO), 3.78 (3H, s,
OCH3), 4.09 (2H, t, J 5.2, OCH2), 6.53 (1H, d, J 9.4, ArꢀH), 6.73 (1H, dd, J 8.0 2.4, ArꢀH), 6.98
(1H, d, J 8.8, ArꢀH), 7.15 (1H, d, J 2.0, ArꢀH), 7.19 (1H, dd, J 8.0 2.4, ArꢀH), 7.29 (1H, d, J 2.8,
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ArꢀH), 7.36 (1H, d, J 8.8, ArꢀH), 7.65 (2H, d, J 8.4, ArꢀH), 7.71 (2H, d, J 8.4, ArꢀH), 8.01 (1H, d, J
9.4, ArꢀH), 8.34 (1H, t, J 6.3, exchange with D2O, CONH); δC (100 MHz, DMSOꢀd6) 13.5, 31.2,
38.5, 55.5, 67.1, 102.0, 111.3, 111.6, 114.3, 114.6, 116.7, 117.4, 119.3, 119.9, 129.1, 130.4, 130.9,
131.2, 134.3, 135.3, 137.6, 144.1, 148.0, 154.8, 155.7, 160.2, 167.9, 169.9; m/z (ESI positive) 545.1
[M+H]+.
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Synthesis of (
Z)-2-(5-fluoro-2-methyl-1-(3-(methylsulfinyl)benzylidene)-1H-inden-3-yl)-N-(2-
((2-oxo-2 -chromen-6-yl)oxy)ethyl)acetamide 4b. Compound 4b was obtained according to the
H
general procedure earlier reported using NHS (1.5 eq) and EDCI.HCl (1.5 eq) in a solution of
Sulindac 1b (0.3 g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was
stirred at r.t. until the formation of the activated ester intermediate B (TLC monitoring, not
isolated). 2ꢀ((2ꢀoxoꢀ2Hꢀchromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3a and
triethylamine (Et3N) were added to the mixture, which was stirred at 60 °C O.N. followed by 80 °C
for 3hrs. Then the reaction mixture was quenched with H2O (10 mL) and the formed precipitate was
filteredꢀoff and concentrated under vacuo to give a yellow precipitate that was triturated with
acetone to afford the titled compound 4b as a yellow solid: 54.7% yield; m.p. 152ꢀ153°C; silica gel
TLC Rf 0.19 (Ethyl acetate 100%); δH (400 MHz, DMSOꢀd6) 2.21 (3H, s, CH3), 2.85 (3H, s,
SOCH3), 3.51 (4H, m, CH2CO and NHCH2), 4.09 (2H, t, J 5.4, OCH2), 6.53 (1H, d, J 9.6, ArꢀH),
6.71 (1H, dt, J 8.2 8.0, ArꢀH), 7.18 (3H, m, ArꢀH), 7.31 (1H, s, Arꢀ H), 7.36 (2H, s, ArꢀH), 7.75
(2H, d, J 8.1, ArꢀH), 7.82 (2H, d, J 8.1, ArꢀH), 8.02 (1H, d, J 9.6, ArꢀH), 8.46 (1H, t, J 5.0,
exchange with D2O, CONH); δC (100 MHz, DMSOꢀd6) 10.5, 32.8, 38.6, 43.2, 67.1, 106.2, 110.4
(J2 23), 111.6, 116.7, 117.4, 119.3, 119.9, 123.1 (J2C-F9), 124.0, 129.3, 129.5 (J3 3), 130.0,
C-F
C-F
133.7, 137.9, 138.7, 140.6, 144.1, 146.3, 147.3 (J2 9), 148.0, 154.9, 160.2, 162.6 (J1 243),
C-F
C-F
169.3; δF (376 MHz, DMSOꢀd6) ꢀ113.49 (1F, s); m/z (ESI positive) 544.1 [M+H]+.
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Synthesis of (±)-2-(3-benzoylphenyl)-N-(2-((2-oxo-2H-chromen-6-yl)oxy)ethyl)propanamide
4
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4c. Compound 4c was obtained according to the general procedure earlier reported using NHS (1.5
eq) and EDCI.HCl (1.5 eq) in a solution of (±)ꢀKetoprofen 1c (0.3 g, 1.0 eq) in dry DMF (3.0 mL)
under a N2 atmosphere. The reaction mixture was stirred at r.t. until the formation of the activated
ester intermediate C (TLC monitoring, not isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀchromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀ
amonium trifluoroacetate salt 3a and triethylamine (Et3N) were added to the mixture and that was
stirred at 60 °C until starting materials were consumed (TLC monitoring), followed by quenching
with H2O (10 mL) and the mixture was extracted with EtOAc (3 x 10 mL). The combined organic
layers were washed with H2O (3 x 20 mL), dried over Na2SO4, filteredꢀoff and concentrated under
vacuo to give a yellow bright semisolid that was purified by silica gel column chromatography
eluting with an increasing amount of EtOAc/nꢀhexane from 50% to 70% v/v to afford the titled
compound 4c as a white solid: 38.4% yield; m.p. 101ꢀ103°C; silica gel TLC Rf 0.23 (Ethyl
acetate/nꢀhexane 90% v/v ); δH (400 MHz, DMSOꢀd6) 1.39 (3H, d, J 6.8 CH3), 3.46 (2H, q, J 5.6,
CH2NH), 3.79 (1H, q, J 6.8), 4.04 (2H, t, J 5.6, OCH2), 6.51 (1H, d, J 9.6, ArꢀH), 7.15 (1H, dd, J
9.2 3.2, ArꢀH), 7.27 (1H, d, J 2.8 ArꢀH), 7.32 (1H, d, J 9.2, ArꢀH), 7.51 (1H, t , J 7.6, ArꢀH), 7.56
(1H, s, ArꢀH), 7.59 (2H, dd, J 8.0 3.0, ArꢀH), 7.67 (1H, t, J 6.8, ArꢀH), 7.70 (1H, s, ArꢀH), 7.71
(3H, d, J 1.2 ArꢀH), 7.99 (1H, d, J 9.6, ArꢀH), 8.38 (1H, t, J 5.4 exchange with D2O, CONH). δC
(100 MHz, DMSOꢀd6) 18.6, 38.4, 44.8, 67.1, 111.7, 116.7, 117.4, 119.3, 119.9, 128.2, 128.5,
128.60, 128.63, 129.7, 131.7, 132.8, 136.9, 137.1, 142.8, 144.1, 147.9, 154.8, 160.2, 173.4, 195.8;
m/z (ESI positive) 442.1 [M+H]+.
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Synthesis of (±)-2-(4-isobutylphenyl)-N-(2-((2-oxo-2H-chromen-6-yl)oxy)ethyl)propanamide
4d. Compound 4d was obtained according to the general procedure earlier reported using NHS (1.5
eq) and EDCI.HCl (1.5 eq) in a solution of (±)ꢀIbuprofen 1d (0.58 g, 1.0 eq) in dry DMF (3.0 mL)
under a N2 atmosphere. The reaction mixture was stirred at r.t. until the formation of the activated
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ester intermediate D (TLC monitoring, not isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀchromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀ
amonium trifluoroacetate salt 3a and triethylamine (Et3N) were added to the mixture which was
stirred at 60°C until starting materials were consumed (TLC monitoring), followed by quenching
with H2O (10 mL), extracted with EtOAc (3 x 10 mL) and the combined organic layers were
washed with H2O (3 x 20 mL), dried over Na2SO4, filteredꢀoff and concentrated under vacuo to give
a white semisolid that was purified by silica gel column chromatography eluting with EtOAc/nꢀ
hexane 50% v/v to afford the titled compound 4d as a white solid: 25.3% yield; m.p. 99ꢀ101°C;
silica gel TLC Rf 0.24 (Ethyl acetate/nꢀhexane 50% v/v); δH (400 MHz, DMSOꢀd6) 0.85 (6H, d, J
6.8, 2 x CH3), 1.33 (3H, d, J 7.2, CH3), 1.78 (1H, sept, CH(CH3)2), 2.40 (2H, d, J 7.2, CH2), 3.46
(2H, q, J 5.6, NHCH2), 3.62 (1H, q, J 7.0, CHCH3), 4.04 (2H, t, J 5.6, OCH2), 6.52 (1H, d, J 9.6,
ArꢀH), 7.05 (2H, d, J 8.0, ArꢀH), 7.16 (1H, dd, J 8.8 3.0, ArꢀH), 7.23 (2H, d, J 8.0, ArꢀH), 7.29 (1H,
d, J 2.8, ArꢀH), 7.35 (1H, d, J 8.8, Arꢀ H), 8.02 (1H, d, J 9.6, ArꢀH), 8.25 (1H, t, J 5.6 exchange
with D2O, CONH); δC (100 MHz, DMSOꢀd6) 18.6, 22.1, 22.2, 29.7, 38.4, 44.3, 44.7, 67.1, 111.6,
116.6, 117.4, 119.3, 119.9, 127.0, 128.8, 139.2, 139.5, 144.1, 147.9, 154.8, 160.2, 173.9; m/z (ESI
positive) 394.2 [M+H]+.
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Synthesis
of
2-(2-((2,6-dichlorophenyl)amino)phenyl)-N-(2-((2-oxo-2H-chromen-6-
yl)oxy)ethyl)acetamide 4e. Compound 4e was obtained according to the general procedure earlier
reported using NHS (1.5 eq) and EDCI.HCl (1.5 eq) in a solution of Diclofenac 1e (0.3 g, 1.0 eq) in
dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was stirred at r.t. until the
formation of the activated ester intermediate E (TLC monitoring, not isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀ
chromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3a and triethylamine (Et3N) were added to
the mixture which was stirred at 60°C until starting materials were consumed (TLC monitoring),
followed by quenching with H2O (10 mL). The formed precipitate was filteredꢀoff and dried under
vacuo to give a white residue which was triturated with acetone to afford the title compound 4e as a
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white solid: 34.8 % yield; m.p. 176ꢀ178 °C; silica gel TLC Rf 0.33 (Ethyl acetate/nꢀhexane 50%
v/v); δH (400 MHz, DMSOꢀd6) 3.52 (2H, q, J 4.8, NHCH2), 3.65 (2H, s, CH2), 4.11 (2H, t, J 5.0,
OCH2), 6.32 (1H. d, J 8.0, ArꢀH), 6.53 (1H, d, J 9.6, ArꢀH), 6.87 (1H, appt, J 7.4, ArꢀH), 7.07 (1H,
appt, J 8.2, ArꢀH), 7.20 (3H, m, ArꢀH), 7.35 (2H, m, ArꢀH), 7.52 (2H, d, J 8.0, ArꢀH), 8.02 (1H, d, J
9.6, ArꢀH), 8.34 (1H, s, exchange with D2O, NH), 8.71 (1H, t, J 4.8, exchange with D2O, CONH);
δC (100 MHz, DMSOꢀd6) 39.5, 40.4, 67.9, 112.6, 116.9, 117.6, 118.3, 120.2, 120.9, 121.6, 126.0,
126.3, 128.2, 130.1, 130.4, 131.4, 138.1, 143.9, 144.9, 148.9, 155.7, 161.1, 172.9; m/z (ESI
positive) 483.0 [M+H]+.
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Synthesis
of
(±)-2-(2-fluoro-[1,1'-biphenyl]-4-yl)-N-(2-((2-oxo-2H-chromen-6-
yl)oxy)ethyl)propanamide 4f. Compound 4f was obtained according to the general procedure
earlier reported using NHS (1.5 eq) and EDCI.HCl (1.5 eq) in a solution of (±)ꢀFlurbiprofen 1f (0.2
g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was stirred at r.t. until
the formation of the activated ester intermediate F (TLC monitoring, not isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀ
chromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3a and triethylamine (Et3N) were added to
the mixture which was stirred at r.t. until starting materials were consumed (TLC monitoring),
followed by quenching with H2O (10 mL) and the mixture was extracted with EtOAc (3 x 10 mL).
The combined organic layers were washed with H2O (3 x 20 mL), dried over Na2SO4, filteredꢀoff
and concentrated under vacuo to give an orange oil that was purified by silica gel column
chromatography eluting with EtOAc/nꢀhexane 60% v/v to afford the titled compounds 4f as a white
solid: 61.4% yield; m.p. 130ꢀ131 °C; silica gel TLC Rf 0.25 (Ethyl acetate/nꢀhexane 50% v/v ); δH
(400 MHz, DMSOꢀd6) 1.39 (3H, d, J 6.8, CH3), 3.46 (2H, q, J 5.4, NHCH2), 3.75 (1H, q, J 7.1,
CHCH3), 4.06 (2H, t, J 5.4, OCH2), 6.51 (1H, d, J 9.6, ArꢀH), 7.20 (2H, dd, J 8.8 4.0, ArꢀH), 7.28
(2H, m, ArꢀH), 7.35 (1H, d, J 8.8, ArꢀH), 7.41 (2H, d, J 6.4, ArꢀH), 7.44 (1H, d, J 4.0, ArꢀH), 7.47
(2H, d, J 4.0, ArꢀH), 7.51 (1H, t, J 7.6, ArꢀH), 8.01 (1H, d, J 9.6, ArꢀH), 8.36 (1H, t, J 5.4, exchange
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with D2O, CONH); δC (100 MHz, DMSOꢀd6) 19.3, 39.3, 45.4, 67.9, 112.5, 115.7 (J2C-F 23), 117.5,
118.3, 120.2, 120.8, 124.7 (J5 3), 127.2 (J5 3), 128.7, 129.5, 129.6 (J5 3), 131.4, 135.9,
C-F
C-F
C-F
6
145.0, 145.1 ( J4C-F 8), 148.9, 155.7, 159.0 (J1C-F 245), 161.1, 174.1; δF (376 MHz, DMSOꢀd6) ꢀ
118.75 (1F, s); m/z (ESI positive) 432.0 [M+H]+.
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Synthesis
of
(±)-5-benzoyl-N-(2-((2-oxo-2H-chromen-6-yl)oxy)ethyl)-2,3-dihydro-1H-
pyrrolizine-1-carboxamide 4g. Compound 4g was obtained according to the general procedure
earlier reported using NHS (1.5 eq) and EDCI HCl (1.5 eq) in a solution of (±)ꢀKetorolac 1g (0.17
g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was stirred at r.t. until
the formation of the activated ester intermediate G (TLC monitoring, not isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀ
chromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3a and triethylamine (Et3N) were added to
the mixture which was stirred at 60°C until starting materials were consumed (TLC monitoring),
followed by quenching with H2O (10 mL), extracted with EtOAc (3 x 10 mL) and the combined
organic layers were washed with H2O (3 x 20 mL), dried over Na2SO4, filteredꢀoff and concentrated
under vacuo to give a brown residue that was purified by silica gel column chromatography eluting
with EtOAc/nꢀhexane 80% v/v to afford the titled compound 4g as a white solid: 33.9% yield; m.p.
198ꢀ199 °C; silica gel TLC Rf 0.25 (Ethyl acetate/nꢀhexane 80% v/v); δH (400 MHz, DMSOꢀd6)
2.75 (2H, m, 2 x CHHax), 3.54 (2H, q, J 4.9, NHCH2), 4.03 (1H, dd, J 8.8 4.8, CH), 4.11 (2H, t, J
5.6, OCH2), 4.39 (2H, m, 2 x CHHeq), 6.02 (1H, dd, J 4.0, pyrroleꢀH), 6.53 (1H, d, J 9.2, ArꢀH),
6.75 (1H, d, J 4.0, pyrroleꢀH), 7.26 (2H, dd, J 9.0 4.0, ArꢀH), 7.35 (1H, d, J 3.2, ArꢀH), 7.40 (1H, d,
J 8.8, ArꢀH), 7.54 (2H, m), 7.62 (1H, m), 7.76 (1H, d, J 8.0, ArꢀH), 8.05 (1H, d, J 9.6, ArꢀH), 8.58
(1H, t, J 6.0, exchange with D2O, CONH); δC (100 MHz, DMSOꢀd6) 38.6, 42.8, 47.9, 67.0, 102.4,
111.7, 116.7, 117.5, 119.31, 119.33, 120.0, 124.5, 126.1, 128.4, 128.5, 131.5, 139.0, 144.1, 145.5,
148.0, 154.9, 160.2, 170.8, 183.5; m/z (ESI positive) 443.1 [M+H]+.
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Synthesis
of
(
S)-(+)-2-(6-methoxynaphthalen-2-yl)-N-(2-((2-oxo-2H-chromen-6-
4
5
6
7
8
yl)oxy)ethyl)propanamide 4h. Compound 4h was obtained according to the general procedure
earlier reported using NHS (1.5 eq) and EDCI.HCl (1.5 eq) in a solution of (S)ꢀ(+)ꢀNaproxen free
acid 1h (0.23 g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was
stirred at r.t. until the formation of the activated ester intermediate H (TLC monitoring, not
isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀchromenꢀ6ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3a and
triethylamine (Et3N) were added to the mixture which was stirred at 60°C until starting materials
were consumed (TLC monitoring), followed by quenching with H2O (10 mL) and extracted with
EtOAc (3 x 10 mL). The combined organic layers were washed with H2O (3 x 20 mL), dried over
Na2SO4, filteredꢀoff and dried under vacuo to give an oil which was triturated with diethyl ether.
The residue obtained was purified by silica gel column chromatography eluting with EtOAc/nꢀ
hexane 60% v/v to afford the titled compound 4h as a white solid: 43.2% yield; m.p. 126ꢀ128 °C;
silica gel TLC Rf 0.22 (Ethyl acetate/nꢀhexane 60% v/v); δH (400 MHz, DMSOꢀd6) 1.43 (3H, d, J
6.8, CH3), 3.72 (2H, q, J 5.3, NHCH2), 3.80 (1H, q, J 6.9, CH), 3.88 (3H, s, OCH3), 4.04 (2H, t, J
5.6, OCH2), 6.51 (1H, d, J 9.6, ArꢀH), 7.15 (2H, m, ArꢀH), 7.28 (3H, m), 7.46 (2H, d, J 8.8, ArꢀH),
7.74 (2H, m, ArꢀH), 7.97 (1H, d, J 9.6, ArꢀH), 8.31 (1H, t, J 5.3, exchange with D2O, CONH); δC
(100 MHz, DMSOꢀd6) 18.6, 38.4, 45.0, 55.2, 67.1, 105.7, 111.7, 116.6, 117.4, 118.6, 119.2, 119.9,
125.3, 126.5, 126.7, 128.4, 129.1, 133.2, 137.4, 144.1, 147.9, 154.8, 157.1, 160.2, 173.9; m/z (ESI
positive) 418.1 [M+H]+.
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Synthesis
of
2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(2-((2-oxo-2H-
chromen-7-yl)oxy)ethyl)acetamide 5a. Compound 5a was obtained according to the general
procedure earlier reported using NHS (1.5 eq) and EDCI.HCl (1.5 eq) in a solution of Indometacin
1a (0.5 g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was stirred at
r.t. until the formation of the activated ester intermediate A (TLC monitoring, not isolated). 2ꢀ((2ꢀ
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Oxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3b and triethylamine (Et3N) were
added to the mixture, which was stirred at 60°C until starting materials were consumed (TLC
monitoring), followed by quenching with H2O (10 mL) and the formed precipitate was filteredꢀoff,
dried under vacuo to afford the titled compound 5a as a yellow solid: 79.2% yield; m.p. 157ꢀ158
°C; silica gel TLC Rf 0.26 (Ethyl acetate/nꢀhexane 70% v/v); δH (400 MHz, DMSOꢀd6) 2.24 (3H, s,
CH3), 3.50 (2H, q, J 5.4, NHCH2), 3.57 (2H, s, COCH2), 3.74 (3H, s, OCH3), 4.15 (2H, t, J 5.0,
OCH2), 6.33 (1H, d, J 9.6, ArꢀH), 6.73 (1H, dd, J 9.0 3.87, ArꢀH), 6.93 (1H, dd, J 8.8 3.73, ArꢀH),
6.99 (2H, m, ArꢀH), 7.14 (1H, d, J 2.4, ArꢀH), 7.64 (1H, d, J 7.2, ArꢀH), 7.65 (2H, d, J 8.72, ArꢀH),
7.71 (2H, d, J 8.6, ArꢀH), 8.02 (1H, d, J 9.6, ArꢀH), 8.32 (1H, t, J 5.8, exchange with D2O CONH);
δc (100 MHz, DMSOꢀd6) 13.5, 31.2, 38.3, 55.5, 67.1, 101.3, 102.0, 111.2, 112.5, 112.6, 112.7,
114.3, 114.6, 129.1, 129.6, 130.4, 131.0, 131.2, 134.3, 135.3, 137.6, 144.4, 155.4, 155.6, 160.4,
161.7, 168.0, 170.0; m/z (ESI positive) 545.0 [M+H]+.
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Synthesis of (
Z)-2-(5-fluoro-2-methyl-1-(3-(methylsulfinyl)benzylidene)-1H-inden-3-yl)-N-(2-
((2-oxo-2 -chromen-7-yl)oxy)ethyl)acetamide 5b. Compound 5b was obtained according to the
H
general procedure earlier reported using NHS (1.5 eq) and EDCI.HCl (1.5 eq) in a solution of
Sulindac 1b (0.5 g, 1.0 eq) in dry DMF (3.0 mL) under a N2 atmosphere. The reaction mixture was
stirred at r.t. until the formation of the activated ester intermediate B (TLC monitoring, not
isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)ethanꢀ1ꢀamonium trifluoroacetate salt 3b and
triethylamine (Et3N) were added to the mixture which was stirred at 60°C until starting materials
were consumed (TLC monitoring), followed by quenching with H2O (10 mL) and the formed
precipitate was filteredꢀoff, dried under vacuo to give a residue was purified by silica gel column
chromatography eluting with EtOAc/nꢀhexane 80% v/v to afford the titled compound 5b as a yellow
solid: 51.4% yield; m.p. 152ꢀ153 °C; silica gel TLC Rf 0.15 (Ethyl acetate/nꢀhexane 80% v/v ); δH
(400 MHz, DMSOꢀd6) 2.21 (3H, s, CH3), 2.86 (3H, s, SOCH3), 3.52 (4H, m, CH2CO and NHCH2),
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Journal of Medicinal Chemistry
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4.17 (2H, t, J 5.2, OCH2), 6.33 (1H, d, J 9.6, ArꢀH), 6.71 (1H, dt, J 9.1 9.0, ArꢀH), 6.97 (1H, dd, J
8.8 3.7, ArꢀH), 7.01 (1H, d, J 2.4, ArꢀH), 7.13 (1H, dd, J 9.30 3.9, ArꢀH), 7.19 (1H, q, J 4.4, ArꢀH),
7.38 (1H, s, ArꢀH), 7.66 (1H, d, J 8.8, ArꢀH), 7.75 (2H, d, J 8.2, ArꢀH), 7.82 (2H, d, J 8.2, ArꢀH),
8.03 (1H, d, J 9.6, ArꢀH) 8.44 (1H, t, J 5.2, exchange with D2O, CONH); δC (100 MHz, DMSOꢀd6)
10.5, 32.8, 38.4, 43.2, 67.1, 101.4, 112.6, 112.7, 112.7, 124.0, 129.3, 129.5, 129.6, 129.6, 130.0,
133.6, 133.6, 133.6, 137.9, 138.7, 140.6, 144.4, 146.3, 146.3, 147.3, 147.4, 147.4, 155.5, 160.4,
161.7, 169.3; δF (376 MHz, DMSOꢀd6) ꢀ113,52(1F, s); m/z (ESI positive) 544.0 [M+H]+.
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Synthesis of (±)-2-(3-benzoylphenyl)-N-(2-((2-oxo-2H-chromen-7-yl)oxy)ethyl)propanamide
5c. Compound 5c was obtained according to the general procedure earlier reported using NHS (1.5
eq) and EDCI.HCl (1.5 eq) in a solution of (±)ꢀKetoprofen 1c (0.5 g, 1.0 eq) in dry DMF (3.0 mL)
under a N2 atmosphere. The reaction mixture was stirred at r.t. until the formation of the activated
ester intermediate C (TLC monitoring, not isolated). 2ꢀ((2ꢀOxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)ethanꢀ1ꢀ
amonium trifluoroacetate salt 3b and triethylamine (Et3N) were added to the mixture which was
stirred at 60°C until starting materials were consumed (TLC monitoring), followed by quenching
with H2O (10 mL), extracted with EtOAc (3 x 10 mL) and the combined organic layers were
washed with H2O (3 x 20 mL), dried over Na2SO4, filteredꢀoff and dried under vacuo to give a
white residue that was purified by silica gel column chromatography eluting with EtOAc/nꢀhexane
50% v/v to afford the titled compound 5c as a white solid: 44.7% yield; m.p. 116ꢀ118 °C; silica gel
TLC Rf 0.16 (Ethyl acetate/nꢀhexane 50% v/v); δH (400 MHz, DMSOꢀd6) 1.39 (3H, d, J 6.8, CH3),
3.46 (2H, J 5.4, NHCH2), 3.79 (1H, q, J 6.8, CH), 4.11 (2H, t, J 5.4, OCH2), 6.31 (1H, d, J 9.6, Arꢀ
H), 6.90 (1H, dd, J 8.6 3.6, ArꢀH), 6.97 (1H, d, J 2.4, ArꢀH), 7.66 (1H, d, J 7.2, ArꢀH), 7.68 (9H, m,
ArꢀH), 7.99 (1H, d, J 9.6, ArꢀH), 8.35 (1H, t, J 5.22, exchange with D2O, CONH); δC (100 MHz,
DMSOꢀd6) 18.6, 38.2, 44.8, 67.1, 101.4, 112.5, 112.6, 112.7, 128.2, 128.5, 128.6, 128.7, 129.6,
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