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

Article abstract of DOI:10.1021/acs.jmedchem.8b00420

Herein we report the design as well as the synthesis of a new series of dual hybrid compounds consisting of the therapeutically used nonsteroidal-anti-inflammatory drugs (NSAIDs; i.e., indometacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) and the carbonic anhydrase inhibitor (CAIs) fragments of the sulfonamide type. Such compounds are proposed as new tools for the management of ache symptoms associated with rheumatoid arthritis (RA) and related inflammation diseases. The majority of the hybrids reported were effective in inhibiting the ubiquitous human (h) CA I and II as well as the RA overexpressed hCAs IX and XII isoforms, with K_I values comprised of the low-medium nanomolar ranges. The antihyperalgesic activity of selected compounds was assessed by means of the paw-pressure and incapacitance tests using an in vivo RA model, and among them the hybrids 6B and 8B showed potent antinociceptive effects lasting up to 60 min after administration.

Full text of DOI:10.1021/acs.jmedchem.8b00420

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Article
Discovery of Novel Nonsteroidal Anti-Inflammatory Drugs
and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs
-CAIs) for the Management of Rheumatoid Arthritis
Ozlem Akgul, Lorenzo Di Cesare Mannelli, Daniela Vullo, Andrea Angeli, Carla Ghelardini, Gianluca
Bartolucci, Abdulmalik Saleh Alfawaz Altamimi, Andrea Scozzafava, Claudiu T Supuran, and Fabrizio Carta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00420 • Publication Date (Web): 10 May 2018
Downloaded from http://pubs.acs.org on May 10, 2018
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Discovery of Novel Nonsteroidal AntiꢀInflammatory Drugs and Carbonic Anhydrase
Inhibitors Hybrids (NSAIDs−CAIs) for the Management of Rheumatoid Arthritis
1,4
2
3
4
Ozlem Akgul, Lorenzo Di Cesare Mannelli, Daniela Vullo, Andrea Angeli, Carla
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Ghelardini, Gianluca Bartolucci, Abdulmalik Saleh Alfawaz Altamimi, Andrea
3
4*
4*
Scozzafava, Claudiu T. Supuran and Fabrizio Carta
1
Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry BornovaꢀĐzmirꢀ
Turkey 35100
2
Dept. of Neuroscience, Psychology, Drug Research and Child Health ꢀ NEUROFARBA ꢀ
Pharmacology and Toxicology Section, University of Florence, 50139 Florence, Italy.
3
University of Florence, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via
della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
4
University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff
6, 50019 Sesto Fiorentino (Florence), Italy
5
Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz
University, P.O. Box 173, Alkharj, 11942, Saudi Arabia.
Keywords: Carbonic Anhydrase Inhibitors (CAIs), Non Steroidal AntiꢀInflammatory Drugs
(
NSAIDs), Metalloenzymes, Rheumatoid Arthritis (RA), Hybrids.
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Abstract. Herein we report the design as well as the synthesis of a new series of dual hybrid
compounds consisting of the therapeutically used NonsteroidalꢀAntiꢀInflammatory Drugs (NSAIDs
;
i.e. indometacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase
inhibitors) and the Carbonic Anhydrase Inhibitor (CAIs) fragments of the sulfonamide type. Such
compounds are proposed as new tools for the management of ache symptoms associated to
rheumatoid arthritis (RA) and related inflammation diseases. The majority of the hybrids reported
were effective in inhibiting the ubiquitous human (h) CA I and II as well as the RA overꢀexpressed
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hCAs IX and XII isoforms, with K values comprised in the lowꢀmedium nanomolar ranges. The
antihyperalgesic activity of selected compounds was assessed by means of the pawꢀpressure and
incapacitance tests using an in vivo RA model, and among them the hybrids 6B and 8B showed
potent antinociceptive effects lasting up to 60 min after administration.
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Introduction.
We recently reported as proof-of-concept study that hybrid small molecules composed of the
NonsteroidalꢀAntiꢀInflammatoryꢀDrugs and CarbonicꢀAnhydraseꢀInhibitors (NSAIDs−CAIs)
fragments were more effective, in terms of potency and time efficacy when compared to the
reference drug ibuprofen, for the management of ache symptoms associated to inflammatory
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diseases such as rheumatoid arthritis (RA). This study was mainly supported by the data obtained
in an in vivo model of RA, which in turn was based on preliminary reports accounting for the
2
ꢀ5
contribution of several Carbonic Anhydrases (CAs, EC 4.2.1.1) to the inflammatory processes.
The serum of RA affected patients exhibited abnormal expressions of the human (h) CAs I, III and
2
ꢀ5
IV isoforms. Our group recently demonstrated the overꢀexpression of CA IX and XII isoforms in
6
the synovium obtained from patients affected by Juvenile Idiopathic Arthritis (JIA).
The main reaction catalyzed by CAs is the reversible hydration of carbon dioxide (CO +
2
+
ꢀ
7
H
2
O ⟺ H + HCO
3
; eq. 1). The involvement of this reaction in a plethora of physiological events
7
(both at the cellular as well as tissue level) is well established. However this transformation when
uncatalyzed is not able to meet the physiological needs of the cells, which is the reason why many
7
aꢀc
isoforms of the CAs have evolved.
Thus, it is not surprising that any disruption of this
1,7a
equilibrium is often associated to diseases, including RA.
In this context the CAs overꢀ
+
ꢀ
expression is expected to increase the ionic species concentrations (H and HCO ) at cellular level,
3
and since the bicarbonate ions are essential components in cells and are immediately recovered, a
local extracellular acidosis is immediately established. Pioneer studies demonstrated that intensity
of inflammation processes and the acheꢀrelated symptoms in RA affected patients were inversely
8
,9
correlated with tissutal pH values. In addition tissue acidosis, was found to be unfavorable for the
10
progression of both humoral and cellular immunity processes.
Despite the enormous progresses achieved in the treatment of RA, still no effective cure for the
disease was obtained. To date the pharmacological approaches against RA are based on the use of i)
DiseaseꢀModifyingꢀAntirheumaticꢀDrugs (DMARDs), JAKꢀinhibitors, corticosteroids and biologics
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which act slowing down the course of the disease, and ii) the NSAIDs which act to ease the
1
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symptoms of the inflammation.
As a result, many scientific reports account for the discovery of
1
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novel diagnostic tools, biological targets, as well as for a variety of lead compounds.
In this
context herein we report a large series of new small molecule hybrids of the NSAIDꢀCAI type,
which differ from the previously ones for the CAI fragment (of the sulfonamide type in this article,
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versus the coumarin chemotype in the previous report) as well as for the length of the linker
connecting the two fragments. We assessed the ability of the compounds reported to inhibit the
activity of the most relevant hCAs as well as to reduce the RA ache related symptoms by means of
in vitro enzymatic assays and in in vivo animal models of the disease, respectively. Our aim is to
further support our previous studies on hCAs as valid and robust pharmacological targets for the
treatment of RA inflammatory diseases, and for this reason no cyclooxygenase (COX) activities of
the hybrids were investigated in this study, considering the fact that the COOH moiety of the
NSAID is essential for their inhibition of COX (and in our reported compounds is transformed in an
15
amide).
Results and Discussion.
1
Design and Synthesis of Compounds. In analogy to our earlier article, we report the design and
synthetic procedures of NSAIDꢀCAI small molecule hybrids consisting of a clinically used NSAID
fragment connected to a CAI of the sulfonamide type, by means of a linear alkyl spacer (Figure 1).
Figure 1. Design of the NSAIDꢀCAI hybrids.
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In our synthetic strategy we made use of the NSAID carboxylic acid moiety in order to efficiently
install an amide bond, which also represents a virtually unique single break point within the entire
hybrid molecules. As for the linker, we focused on linear full carbon or ether type fragments which
are biocompatible and of easy access from the synthetic view point (Scheme 1).
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OH
O
O
R=
R=
R=
O
OH
OH
O
O
Naproxen; 2
Ibuprofen; 1
Ketoprofen; 3
O
O
F
OH
S
N
OH
O
R=
R=
R=
O
O
OH
Ketorolac; 5
Flurbiprofen; 4
F
Sulindac; 6
O
O
OH
N
Cl
OH
H
N
R=
Cl
O
R=
Cl
O
Indometacin; 7
Diclofenac; 8
Scheme 1. General synthetic procedure for the synthesis of NSAIDꢀCAIs.
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As above reported the compound series was obtained by means of well established amide
coupling reactions using the sulfanilamide A and 4ꢀ(2ꢀaminoꢀethyl)ꢀbenzenesulfonamide B for the
1Aꢀ8A and 1Bꢀ8B series respectively, whereas the intermediates CꢀD and E were obtained
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according to the synthetic procedure depicted in Scheme 2.
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Scheme 2. Synthetic procedure for the synthesis of C, D and E intermediates.
1
All final compounds obtained and their intermediates were properly characterized by means of H,
13
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C, FꢀNMR spectroscopy, HRMS and were ≥ 95% HPLC pure (see Experimental Section for
details).
Carbonic Anhydrase Inhibition Studies. All compounds herein reported and the standard CAI
acetazolamide (AAZ) were assessed for their inhibition properties against the relevant hCA
isoforms (i.e. I, II, IV, IX and XII) using the stoppedꢀflow carbon dioxide hydration assay (Table 1).
1
7
Table 1. hCA I, II, IV, IX, and XII in vitro inhibition data with NSAIDꢀCAI compounds by the
1
7
StoppedꢀFlow CO hydrase assay.
2
K (nM)*
I
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**
Compound
hCA I
hCA II
hCA IV
hCA IX
hCA XII
1
2
3
4
A
A
A
A
40.4
7.9
32.4
4.2
218.1
653.2
49.1
13.9
12.6
5.1
33.0
28.5
2.9
1
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0
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0
19.4
58.5
16.1
73.3
208.6
68.2
79.3
19.1
8.1
4.6
5.5
717.7
1495
519.1
4375
179.3
7298
6028
3005
3053
3877
6857
8365
8654
59.9
9.2
40.0
24.5
27.2
40.8
7.0
5A
3.6
49.1
61.3
58.7
5.3
6
7
8
A
A
A
23.3
92.5
89.2
9.2
1B
2B
72.2
12.9
39.3
38.1
94.9
62.5
62.5
43.3
26.2
21.2
76.5
93.5
91.9
54.0
92.8
90.3
94.4
92.7
90.2
7.0
5.2
3
4
5
B
B
B
6.5
42.5
534.4
64.9
494.0
68.4
267.9
73.8
21.3
7.8
78.9
27.3
95.8
43.7
7.4
6B
7
8
1
2
B
B
C
C
7.8
73.4
7.5
3C
6.7
77.7
8.6
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C
C
C
C
233.8
87.0
375.9
769.7
59.4
57.6
7.1
7.0
354.7
5996
1598
5483
63.7
244.4
103.2
177.2
144.4
10.4
18.3
12.5
17.5
18.3
141.0
202.4
137.1
14.0
16.3
8.0
68.6
65.0
44.0
57.6
6.5
7.5
4.2
0
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0
186.8
25.2
5.8
1D
2
3
4
5
D
D
D
D
219.8
77.2
19.4
5.2
5.0
54.2
44.4
4.9
6.3
301.3
295.9
811.0
522.6
672.8
2804
2693
92.8
43.3
7.3
5.0
6D
5.4
35.0
84.1
59.2
6.6
7
8
1
D
D
E
81.7
63.4
48.4
71.4
20.9
242.7
93.8
86.5
341.8
250.0
84.3
46.2
284.7
47.1
6.1
2E
4.2
3
4
6
7
E
E
E
E
4.1
86.7
66.4
8.1
7264
7737
8136
8023
74.0
45.5
22.0
7.7
94.1
5.0
64.0
93.8
5.7
8E
78.8
12.1
51.9
25.8
AAZ
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*
Means from three diverse assays. The errors were comprised within ±5−10% of the values reported
**
(data not shown). Catalytic domain.
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On the basis of the kinetic data reported in Table 1 on the NSAIDꢀCAIs, the following Structureꢀ
ActivityꢀRelationships (SARs) can be drawn.
Compounds 1Aꢀ8A.
i) The in vitro kinetic data showed that the Naproxen derivative 2A was the most potent within the
series against the hCA I isoform (K
I
7.9 nM), followed by the ketorolac 5A and ketoprofen 3A (K
I
s
16.1 and 19.4 nM respectively). Medium inhibition potencies, with K
I
values spanning between
40.4ꢀ73.3 nM, were obtained for the ibuprofen 1A, flurbiprofen 4A, sulindac 6A and diclofenac 8A,
whereas the indometacin benzenesulfonamide 7A resulted far the less potent (K 208.6 nM) and
I
similar to the standard CAI AAZ (K
ii) As for the ubiquitous hCA II isoform, better inhibition results were obtained for the 1Aꢀ8A series
when compared to the hCA I. As reported in Table 1, compounds 2Aꢀ5A showed K values
comprised between 5.5 and 3.6 nM, thus up to 3.4 fold stronger when compared to AAZ (K 12.1
nM). The ibuprofen 1A and sulindac 6A derivatives were far less potent (K s 32.4 and 23.3 nM
I
250 nM).
I
I
I
I
respectively) followed by the indometacin 7A and diclofenac 8A derivatives (K s 92.5 and 89.2 nM
respectively).
iii) The hCA IV isoform, which is of particular interest as it is widely expressed in the kidneys and
heart tissues, showed that only the ketoprofen derivative 3A was a valuable inhibitor, with a K
I
value 1.51 fold lower when compared to the reference CAI AAZ (K s 49.1 and 74.0 nM
I
respectively). Conversely all the other compounds of the series showed weak inhibitory properties
(
K
I
comprised between 179.3 and 4375 nM).
iv) Compounds 3A, 4A and 8A were very potent inhibitors of the tumor associated hCA IX isoform
with K values in the low nanomolar range (K s 5.1, 9.2 and 5.3 nM respectively), thus more potent
I
I
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when compared to AAZ (K 25.8 nM). Weaker inhibition potencies were reported for the ibuprofen
I
1A and naproxen 2A derivatives (K s 13.9 and 12.6 nM respectively), followed by the ketorolac 5A,
I
sulindac 6A and indometacin 7A (K
I
s 49.1, 61.3 and 58.7 nM respectively). The latter were also
weaker in inhibiting the hCA IX isoform when compared to the standard AAZ (K
I
25.8 nM).
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v) Among the 1Aꢀ8A series, the ketoprofen 3A and the diclofenac 8A derivatives showed low
nanomolar inhibition values against the second tumor associated hCA (i.e. isoform XII) and
comparable to AAZ. As reported in Table 1 the first resulted 1.97 fold stronger when compared to
I
AAZ (K s 2.9 and 5.7 nM respectively), whereas the latter showed a K
I
inhibition value slightly
higher (K
I
7.0 nM). As for the other compounds, similar inhibition values and ranging between 27.2
and 33.0 nM were obtained for the ibuprofen 1A, naproxen 2A, ketorolac 5A and the sulindac 6A.
Finally the flurbiprofen 4A and the indometacin 7A derivatives resulted the less potent inhibiting
the hCA XII, and again with closeꢀmatching K s (40.0 and 40.8 nM respectively).
I
Compounds 1Bꢀ8B.
i) Among the ethylaminobenzene sulfonamide series, the hCA I isoform resulted potently inhibited
I I
from the ketoprofen 3B derivative (K 8.1 nM) followed by the naproxen 2B compound (K 19.1
nM) and thus they resulted more effective in comparison to the CAI AAZ (K 250.0 nM). Medium
I
inhibition values were obtained for the ibuprofen 1B, flurbiprofen 4B, sulindac 6B and diclofenac
I
8B derivatives (K s 79.3, 42.5, 64.9 and 68.4 nM respectively). The ketorolac 5B and indometacin
I
7B compounds resulted ineffective against the hCA I (K s 534.4 and 494.0 nM respectively).
ii) Better inhibition results were obtained for the hCA II isoform. As reported in Table 1,
compounds 1Bꢀ4B were all low nanomolar inhibitors of this isoform with K s spanning between 5.2
I
and 9.2 nM, and among them the naproxen 3B derivative was the most potent as being 2.33 fold
more potent as compared to the CAI AAZ (K
I
12.1 nM). Within the 1Bꢀ8B series the sulindac 6B
s 27.3 and 43.7 nM respectively),
and the diclofenac 8B resulted medium potency inhibitors (K
I
whereas the remaining ketorolac 5B and indometacin 7B were the less potent.
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iii) Compared to the 1Aꢀ8A compounds previously discussed, the series 1Bꢀ8B was ineffective in
inhibiting the hCA IV isoform. The corresponding K values are reported in Table 1 and were all
I
comprised between 3.0 and 8.7 µM.
iv) The transmembrane and tumor related hCA IX was efficiently inhibited only from the naproxen
1
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derivative 2B being 2 fold more potent of the standard CAI AAZ (K
respectively). Then the ketoprofen 3B, the flurbiprofen 4B and the diclofenac 8B containing
compounds showed medium nanomolar inhibition values (K s 39.3, 38.1 and 43.3 nM respectively),
I
s 12.9 and 25.8 nM
I
whereas all remaining hybrids in the series resulted low active in inhibiting this isoform (K
comprised between 62.5 and 94.9 nM).
I
s
v) Among the compound series 1Bꢀ8B, the ketoprofen 3B derivative was the most potent as hCA
XII inhibitor and also resulted 9.5 fold less potent when compared to the reference AAZ (K s 54.0
I
and 5.7 nM respectively). The remaining derivatives were all high nanomolar hCA XII inhibitors
I
with K s spanning between 90.2 and 94.4 nM.
Compounds 1Cꢀ4C and 6Cꢀ8C.
i) Data in table 1 showed that only the ketoprofen 3C derivative was potent against the hCA I
isoform with a K value 10 fold higher when compared to AAZ (K s 21.3 and 250 nM
I
I
respectively), followed by the naproxen 2C and the sulindac 6C which resulted both high
nanomolar inhibitors of this isoform (K s 73.8 and 87.0 nM). All the remaining compounds in the
I
I
series were inactive in inhibiting the hCA I with K values comparable or higher to the reference
AAZ.
ii) In analogy to the compound series previously discussed, also in this case the hCA II resulted
particularly inhibited. Data in table 1 showed that among this series only the diclofenac 8C was
ineffective against the hCA II (K
s ranging between 4.2 and 7.8 nM, thus slightly more potent when compared to the AAZ (K
nM).
I
186.8 nM) whereas all the others were nanomolar inhibitors with
K
I
I
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iii) As for hCA IV the Ibuprofen 1C resulted the most potent inhibitor within the series with a KI
being slightly more potent (1.24 fold) when compared to the reference AAZ (K s 59.9 and 74.0 nM
I
respectively). The naproxen 2C and the ketoprofen 3C derivatives (73.4 and 77.7 nM respectively)
showed each other similar inhibition potencies whose values were analogous to the reference
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compound AAZ (K 74.0 nM). Finally, all the other compounds in the series resulted inactive
against the hCA IV (K s comprised between 354.7 and 5996 nM).
I
iv) The in vitro kinetic data reported in Table 1 showed that the hCA IX isoform was efficiently
inhibited from the ibuprofen 1C and naproxen 2C derivatives whose K
the reference AAZ (K s 26.2, 21.2 and 25.8 nM respectively). Then the ketoprofen derivative 3C
resulted 2.97 fold less potent in inhibiting the hCA IX isoform when compared to the standard CAI
K s 76.5 and 25.8 nM respectively), followed by all the remaining hybrids which were weak hCA
I
values were quite similar to
I
(
I
IX inhibitors with K values ranging between 103.2 and 244.4 nM.
I
v) The second tumor associated hCA isoform (XII) in analogy to the previously discussed one
I
resulted potently inhibited from the ibuprofen 1C and the naproxen 2C derivatives with K values
quite similar each other (7.0 and 7.5 nM) and slightly less effective when compared to the reference
AAZ (K 5.7 nM). Again the Ketoprofen 3C showed lower inhibition potency (K 8.6 nM) followed
I
I
by the remaining compounds in the series with K values comprised between 44.0 and 68.6 nM.
I
Compounds 1Dꢀ8D.
i) Within the 1Dꢀ8D series tested, the ketoprofen 3D and the sulindac 6D were highly potent in
I
inhibiting the hCA I isoform with K s values comprised within the low nanomolar range (7.1 and
4.9 nM respectively), and thus more potent when compared to the reference AAZ (K 250 nM). All
I
the other compounds in the series resulted medium potency inhibitors of this isoform. Among them
the ketorolac 5D derivative was the most potent (K
naproxen 2D and the ibuprofen 1D (K s 54.2, 57.6 and 59.4 nM respectively). The least potent
inhibitors of the hCA I isoform were the dichlofenac 8D and the indometacin 7D which showed K
I
44.4 nM) followed by the flurbiprofen 4D, the
I
I
s
in the high nanomolar (K s 63.4 and 81.7 nM respectively).
I
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ii) The highly abundant hCA II isoform was efficiently inhibited from the naproxen 2D, the
ketoprofen 3D, the flurbiprofen 4D, the ketorolac 5D and the sulindac 6D derivatives with low
nanomolar K
potent compared to AAZ (K
for the ibuprofen 1D (K 25.2 nM), the diclofenac 8D (K
4.3 nM) derivatives.
I
s and spanning between 5.4 and 6.3 nM, thus being in the average of 2 fold higher
12.1 nM). Significant worsening of the hCA I inhibition was observed
46.2 nM) and the indometacin 7D (K
I
I
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iii) As reported in Table 1 the hCA IV isoform was efficiently inhibited from the ibuprofen 1D and
the ketoprofen 3D derivatives. In particular the first one resulted 1.16 fold more potent of the
reference CAI AAZ (K
I
s of 63.7 and 74.0 nM respectively), whereas the latter was slightly less
potent (K 77.2 nM). All the remaining compounds in the series resulted ineffective in inhibiting the
I
hCA IV isoform with K values comprised in within 219.8 and 811.0 nM.
I
iv) As for the tumor associated hCA IX, the sulindac 6D, the indometacin 7D and the diclofenac 8D
I
derivatives (K s 141.0, 202.4 and 137.1 nM respectively) resulted lower potent inhibitors in
comparison to the standard CAI (K
I
25.8 nM). More interesting results were obtained for the
remaining compounds 1Dꢀ5D, which showed low nanomolar inhibition values and almost flat SAR.
Among them, the ibuprofen 1D and the ketoprofen 3D derivatives resulted the most potent CAIs
with K s of 10.4 and 12.5 nM respectively, thus being 2.5 and 2.0 fold respectively more potent
I
when compared to the reference AAZ.
v) Remarkable data were obtained for the second tumor correlated hCA (the XII). The ketoprofen
3D derivative was the most potent within the series and being also slightly more potent of the CAI
AAZ (K s of 5.2 and 5.7 nM respectively). Similarly, the ibuprofen 1D and the ketorolac 5D
I
resulted quite effective inhibitors of the hCA XII (K values of 6.5 and 7.3 nM respectively). As
I
reported in table 1, a definite SAR was obtained for the remaining compounds in the series as
I
increasing K values against the hCA XII were obtained for the naproxen 2D (19.4 nM), the
sulindac 6D (35.0 nM), the flurbiprofen 4D (43.3 nM), the diclofenac 8D (59.2 nM) and for the
indometacin 7D (84.1 nM) derivatives.
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Compounds 1Eꢀ4E and 6Eꢀ8E
i) In analogy to the three compound series previously discussed, also in this case the ketoprofen 3E
derivative resulted the most effective inhibitors of the hCA I isoform with K value of 20.9 nM, thus
being 12.0 fold more potent of the reference CAI AAZ (K 250 nM). Increasing K values were
I
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observed for the other compounds in the series such as the ibuprofen 1E, the naproxen 2E, the
indometacin 7E and the sulindac 6E which showed K values comprised between 48.4 and 93.8 nM.
I
Finally the flurbiprofen 4E and the diclofenac 8E were the least active in the series (K s 242.7 and
I
341.8 nM).
ii) The hCA II isoform was efficiently inhibited from the ketoprofen 3E and indometacin 7E
derivatives being 1.98 and 1.50 fold respectively more potent when compared to the standard AAZ
(
K s 6.1, 8.1 and 12.1 nM respectively). Medium potency inhibition against the hCA II was shown
I
from the naproxen 2E and the sulindac 6E (K s of 47.1 and 66.6 nM) followed by the diclofenac 8E
I
and the flurbiprofen 4E derivatives which were high nanomolar K
Finally the ibuprofen 1E derivative was ineffective (K 284.7 nM).
I I
values (K s 78.8 and 86.7 nM).
I
iii) In analogy to the compounds series previously discussed the hCA IV was the least inhibited
among those considered in the present study. As reported in table 1 the ketoprofen 3E derivative
was the only compound in the series which showed inhibition potency, although of modest entity,
I
being 1.25 fold less potent when compared to the reference CAI AAZ (K s of 92.8 and 74.0 nM
respectively). All the remaining derivatives in the series resulted not active with K
comprised within the low micromolar range.
I
s values
iv) Conversely to the hCA IV, the IX isoform was potently inhibited from the majority of the
compounds herein reported. In particular, the indometacin 7E and the ketoprofen 3E derivatives
were the most potent, having K
reference CAI AAZ (table 1). Slight higher inhibition values were obtained for the ibuprofen 1E
and for the naproxen 2E derivatives (K s of 14.0 and 16.3 nM respectively), followed by the
I
values 3.35 and 3.23 fold respectively lower when compared to the
I
sulindac 6E whose inhibition value was comparable to the AAZ (K s of 22.0 and 25.8 nM
I
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respectively). The remaining compounds in the series, the flurbiprofen 4E and the diclofenac 8E
derivatives, were the least potent with K s of 45.5. and 51.9 nM respectively.
I
v) Interesting results were also obtained for the second tumor associated hCA (the XII isoform). The
flurbiprofen 4E and the diclofenac 8E derivatives were both ineffective in inhibiting the enzyme
1
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being 16.5 fold less potent when compared to the reference CAI AAZ (K s of 94.1, 93.8 and 5.7 nM
respectively), followed by the indometacin 7E derivative which was a medium potency inhibitor (KI
value of 64.0 nM). Better results were obtained for the remaining compounds which showed
comparable inhibition values to the standard CAI (such as the ibuprofen 1E and the sulindac 6E
I
derivatives whose K s were of 6.6 and 5.0 nM) or even lower as in the case of the naproxen 2E and
the ketoprofen 3E derivatives (K
I
s 4.2 and 4.1 nM respectively).
In summary the effects on the in vitro kinetic activity due to the linker lengths connecting the
NSAID and the CAI fragments within the compound series here reported, afforded quite complex
SARs which are not clearly defined. Among each CA isoform we observed: i) the naproxen 2A, the
ketoprofen 3B and its longer derivative 3D, were the most potent inhibitors of the ubiquitous hCA I
I
isoform (K s of 7.9, 8.1 and 7.1 nM respectively); the hCA II isoform was potently inhibited by
many compounds, with K s in the low nanomolar range regardless the nature of the NSAID or the
I
linker. Among them the ketorolac 5A, the naproxen 2A, the ketoprofen 3A and the indometacin 7C
derivatives resulted the most effective hCA II inhibitors (K
respectively); iii) The membrane associated hCA IV was potently inhibited by the ketoprofen 3A
(K 49.1 nM) followed by the ibuprofen 1C and 1D derivatives (K s of 59.9 and 63.7 nM) which
I
s of 3.6, 4.2, 4.6 and 4.2 nM
I
I
were slightly more potent when compared to AAZ (K 74.0 nM); iv) The hCA XII isoform was
I
potently inhibited from the ketoprofen 3A and the diclofenac 8A with K s of 5.1 and 5.3 nM
I
respectively, thus being 5.1 and 4.9 fold respectively more potent of the reference CAI AAZ (K
I
of
25.8 nM); amongst all compounds tested the ketoprofen 3A derivative was also the most potent
I
inhibitor the last hCA here considered (XII) with a K value of 2.9 nM, thus being nearly 2 fold
more potent of the standard CAI AAZ (K of 5.7 nM). Slightly less potent inhibition values against
I
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this isoform were obtained for the naproxen 2E, the ketoprofen 3E, the sulindac 6E and the
ketoprofen 3D derivatives (K
I
s of 4.2, 4.1, 5.0 and 5.2 nM respectively), which behaved anyhow as
very potent inhibitors.
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ꢀ
Pain relieving tests. The acute pain relieving effect of hybrids 2B, 3B, 6B and 8B (0.1–10.0 mg kg
1
p.o.) was assessed in an in vivo model of RA which was induced by complete Freund’s adjuvant
(CFA) i.a. treatment. Efficacy was compared with NSAIDs contained in the different hybrids
(naproxen, ketoprofen, sulindac and diclofenac). For this purpose, NSAIDs were administered p.o.
using a dosage equimolar to the highest dose of composite compounds (10.0 mg/kg).
Figure 2. Acute pain reliever effect of 2B, 3B, 6B and 8B in a CFA i.a. injection induced RA model
in rats. The Paw pressure test was used to assess the hypersensitivity towards noxious mechanical
stimuli. NSAIDs (naproxen, ketoprofen, sulindac and diclofenac) contained in the different hybrids
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were used as references tested at a dosage equimolar to 10 mg/kg of the corresponding NSAID–
CAI. The measurements were accomplished on day 14 after CFA injection. The hybrids were
suspended in 1% carboxymethyl cellulose (CMC) and administered orally. The values reported are
the mean of 8 rats performed in 2 distinct experiments. ^^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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In Figure 2, the hypersensitivity to a mechanical noxious stimulus (Paw pressure test) is
shown. After 14 days from i.a. CFA injection, the tolerated weight on the ipsilateral paw decreased
to 43.5 ± 0.5 g (CFA + vehicle group in figure 2), thus considerably lower to the value of 63.3 ± 1.0
g relative to vehicle treated animals (1% CMC), which in turn displayed a stable threshold to
nociceptive stimuli over the time course of the tests. Among the compounds tested, the naproxen
derivative 2B at 0.1 mg/kg concentration resulted ineffective in determining any antinociceptive
effect, whereas at 1.0 mg/kg concentration was able to increase the pain threshold in a dose
dependent manner. As reported in Figure 2 it was observed a sensible effect after 15 min
administration, peaking at 30 min and lasting up to 60 min. The administration of 2B at 10.0 mg/kg
resulted only in a slight increase of the observed values at 30 and 45 min after administration when
compared to the previous experiment at 10 fold lower concentration. Interestingly no extension of
the antinociceptive effect over the time was observed. The naproxen alone (5.6 mg/kg) resulted
completely ineffective. As for the ketoprofen 3B, a dosage concentration of 0.1 mg/kg determined a
slight pain relieving effect at 15 min which remained constant up to 30 min (50.0 ± 0.3 and 50.0 ±
ꢀ1
0.2 g respectively) and lasted for 45 min. An increase of the of the dosage of 3B to 1.0 mg kg ,
showed higher effects. As reported in Figure 1 at 15 minutes the antinociceptive response evoked
from 3B was significative (51.7 ± 1.7 g), picking at 30 min (53.3 ± 0.8 g) and lasting up to 60 min
after administration. The higher dosage of 3B (10 mg/kg) resulted in further increase of the pain
relieve effects, which reached the highest value at 15 min and remained constant up to 30 (56.7 ±
0.8 g). In this case the antinociceptive effect was also prolonged up to 75 min from the treatment.
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The ketoprofen alone (5.8 mg/kg) increased the pain threshold only at 15 min with value
comparable to its corresponding hybrid 3B at the lowest concentration (Figure 2).
In analogy to 3B also the sulindac 6B hybrid, evoked in a dose dependent manner the antiꢀ
hypersensitive effect. As reported in Figure 2 the compound resulted completely ineffective at 0.1
0
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0
ꢀ1
ꢀ1
mg/kg dosage, whereas a significant efficacy at 1.0 mg/kg dosage was reported and lasted up to
0 min after administration. As shown in Figure 2 the maximum effect was reached at 15 min and
was constant up to 30 min (55.0 ± 2.5 and 55.0 ± 0.4 g respectively). Finally, the treatment with 6B
6
ꢀ1
at 10 fold higher concentrations (10 mg/kg ) fully counteracted the CFAꢀinduced hypersensitivity
of the ipsilateral paw at 15 min (62.5 ± 2.0 g) and picked at 30 min (64.2 ± 0.8 g) and the effect
lasted up to 75 min after administration. Again the sulindac di per se (6.6 mg/kg) resulted inactive
when considered in our experiments.
ꢀ1
The last compound tested (i.e. the diclofenac derivative 8B) at a dosage of 0.1 mg/kg was able to
induce partial antinociceptive effect which lasted up to 45 min after administration and peaked at 15
min with a value of 52.5 ± 2.5 g. The antinociceptive response for compound 8B resulted further
ꢀ1
enhanced at the dose of 1.0 mg kg . In this case the maximum effect was reached at 30 min (56.7 ±
ꢀ
1
0
.8 g) and completely vanished at 60 min. The administration of 8B at 10.0 mg kg dose resulted
both in increase of the pain threshold of the ipsilateral paw (peaking at 30 min with a value of 66.3
1.0 g) as well as of the efficacy time frame (extended up to 75 min). The NSAID diclofenac alone
±
ꢀ1
(
6.2 mg/kg ) induced a significant pain relief only at 15 min after administration with a value
ꢀ1
perfectly matching with the hybrid 8B at 1.0 mg/kg dosage.
Interestingly any of the tested compounds changed the pain threshold of the contralateral paw, thus
suggesting that such molecules do not influence the normal pain sensitivity (data not shown).
The efficacy of hybrids 2B, 3B, 6B and 8B was also evaluated against the spontaneous pain, which
was measured as the hind limb weight bearing alterations originated by unilateral damage
(Incapacitance test, Figure 3).
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Figure 3. Acute pain reliever effect of 2B, 3B, 6B and 8B in a CFA i.a. injection induced RA model
in rats. The incapacitance test was performed in order to assess the hind limb weight bearing
alterations, which were measured as postural imbalance related to pain stimuli. The data values are
the mathematical difference between the weight applied on the contralateral and ipsilateral limb
(ꢀ Weight). NSAIDs (naproxen, ketoprofen, sulindac and diclofenac) contained in the different
ꢀ1
hybrids were used as references tested at a dosage equimolar to 10.0 mg/kg of the corresponding
NSAID–CAI. The measurements were done 14 days after CFA injection. All hybrids were
suspended in 1% CMC and administered orally. The values are the mean of 8 rats and obtained in 2
different experiments. ^^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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The difference values between the weight burdened on the contralateral and the ipsilateral
ꢀ weight) was increased in CFA treated animals when compared to the values of the control
paw (
group (54.2 ± 5.1 g vs 2.5 ± 3.4 g respectively). Compound 2B was inactive at the lowest
ꢀ1
concentration (0.1 mg/kg ), whereas it showed activity when administered at higher dosages of 1.0
0
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ꢀ1
and 10.0 mg/kg . In both cases the maximum effect was reached at 30 min after administration
34.3 ± 5.6 g and 29.3 ± 0.4 g respectively) and lasted up to 60 min. As for the hybrid 3B, its
administration at the lowest concentration slightly reduced the weight with a peaking value at 15
(
ꢀ
min of 42.3 ± 0.7 g. Significative effects were observed when 3B was administered at 1.0 and 10.0
ꢀ1
mg/kg . In both cases both the intensity as well as the duration of the effect were dose dependent.
ꢀ1
As shown in figure 3, the compound 3B at 1.0 mg/kg peaked at 30 min with a value of 36.8 ± 2.9 g
ꢀ1
and lasted up to 60 min. A 10 fold increase of the dose administered (10.0 mg/kg ) resulted in a
maximum weight value at 30 min of 28.6 ± 4.8 g and enhancement of the effect up to 75 min. The
ꢀ
experiments conducted on the sulindac hybrid 6B revealed a complete ineffectiveness on reducing
ꢀ1
the postural imbalance when administered at the lowest dosage (0.1 mg/kg ). Conversely
significant effects were observed when 6B was administered at the concentrations of 1.0 and 10.0
ꢀ1
mg/kg . As reported in figure 3, the acute pain reliever effect followed a dose dependent trend after
a single administration, and peaked at 15 and 30 min respectively (22.3 ± 3.5 g and 8.6 ± 1.1 g). In
both cases the effect lasted up to 60 min. The final compound considered (i.e. the diclofenac hybrid
ꢀ1
8
B) showed a weak effect at the lowest concentration (0.1 mg/kg ) with a peaking value of 39.0 ±
1
.8 g and lasting up to 45 min after single dose administration. More intense effects were observed
ꢀ1
when the compound 8B was administered at 1.0 and 10.0 1 mg/kg dosages. As reported in figure 3
the dosage increase resulted both in widening of the time frame efficacy as well as of the magnitude
with peaking values of 20.6 ± 3.9 g and 14.7 ± 2.2 g respectively at 30 min after administration. The
acute pain reliever effects of the hybrid compounds 2B, 3B, 6B and 8B tested were compared with
their relative NSAIDs counterparts (i.e. naproxen, ketoprofen, sulindac and diclofenac). As reported
in figure 3, only the ketoprofen and diclofenac were the able to evoke significant relief effects even
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if their efficacy and potency were significantly lower when compared to their hybrid 3B and 8B
respectively (Figure 3).
Conclusions. We report new low molecular weight NSAIDꢀCAI hybrid molecules as potential drug
lead compounds for the treatment of acheꢀrelated symptoms associated to inflammatory diseases
such as the RA. All compounds in the obtained series consisted of a clinically used NSAID
fragment (acting as COX inhibitors) linked to a sulfonamide based CAI moiety. Unlike our
previously reported NSAIDꢀCAI hybrids, which showed remarkable inhibition properties of the
1
1
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0
I
hCA IV, moderate K s against the IX and XII isoforms and no activity on the ubiquitous hCAs I and
II, the ones herein reported were ineffective inhibitors of hCA IV and effective inhibitors of the
remaining enzymatic isoforms considered here (hCA I, II, IX and XII). Such a complementary in
vitro kinetic activity between the two hybrid series is certainly due to the nature of the CAI
1
fragment, which is of the coumarin type in the first series and of the benzenesulfonamide in the
latter one. This has been demonstrated by comparison of analogous derivatives between the two
series having the same tether length. Even if it was not possible to draw a straightforward SAR from
the obtained in vitro kinetic data, we were able to demonstrate that the most potent hybrid
derivatives acting as efficient inhibitors against the hCAs herein considered showed remarkable in
vivo efficacy. More importantly, in this study we carried out for the first time a complete kinetic
profiling of a large series of NSAIDꢀCAI hybrids potentially useful for the development of drug
leads for the treatment of RA related diseases. Such a data which will be particularly useful for the
selection of the compounds out of the pool having the proper selectivity index against the hCA
isoforms involved in RA. The in vivo data obtained by using a CFA induced arthritis model in rats
with selected NSAIDꢀCAI hybrids, showed potent antihyperalgesic effects in a dose dependent
ꢀ1
manner, starting already at 1 mg kg . In particular, compounds 6B and 8B resulted to be the best
performing ones in terms of intensity and longꢀlasting effects of the antihyperalgesic action, both in
the in vivo tests considered.
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In conclusion we gave further evidence to our previous hypothesis that NSAID−CAI hybrid
compounds are indeed valuable leads for the management of ache symptoms typical of
inflammatory pathologies such as the RA. Moreover, the high affinity showed by these new hybrid
molecules on the ubiquitous and erythrocyte expressed hCAs I and II represents an interesting
feature which can be of particular advantage both for the systemic distribution of the drugs into the
organism as well as for enhancement of their halfꢀlife, since their binding to blood present CA
isoforms may lead to a depot effect. In this context it is worth mentioning that RA symptoms highly
affect the life quality of patients, which are progressively unable to carry out any activities such as
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30
at work, in social relations and leisures. Therefore an ideal pharmacological approach should
consist of long lasting drugs or with extended time clearances. Studies in this direction are currently
being developed in our laboratories.
Experimental protocols.
Chemistry. All anhydrous solvents and reagents used in this study were purchased from Alfa
Aesar, TCI and SigmaꢀAldrich. The synthetic reactions involving airꢀ or moistureꢀsensitive
chemicals were carried out under a nitrogen atmosphere using dried glassware and syringes
1
13
19
techniques in order to transfer the solutions. Nuclear magnetic resonance ( HꢀNMR, CꢀNMR, Fꢀ
NMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer using DMSOꢀd as
6
solvent. The chemical shifts are reported in parts per million (ppm) and the coupling constants (J)
are expressed in Hertz (Hz). The splitting patterns are designated as: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; brs, broad singlet; dd, doublet of doublets. The correct assignment of
exchangeable protons (i.e. OH and NH) was carried out by means of the addition of D O.
2
Analytical thinꢀlayer chromatography (TLC) was done on Merck silica gel Fꢀ254 plates. Flash
chromatography was performed on Merck Silica gel 60 (230ꢀ400 mesh ASTM) as the stationary
phase and appropriate mixtures of ethyl acetate/nꢀhexane were the eluents. Melting points (m.p.)
were measured in open capillary tubes with a Gallenkamp MPD350.BM3.5 apparatus and are
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uncorrected. The HPLC was performed by using a Waters 2690 separation module coupled with a
photodiode array detector (PDA Waters 996) using a NovaꢀPak C18 4 ꢁm 3.9 mm × 150 mm
(
Waters) silicaꢀbased reverse phase column. The sample was dissolved in 10% acetonitrile/H
an injection volume of 45 ꢁL. The mobile phase (flow rate 1.0 mL/min) was a gradient of H
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
2
O and
2
O +
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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5
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5
6
0
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2
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8
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0
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0
1
2
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8
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0
1
2
3
4
5
6
7
8
9
0
(
isocratic 90:10. TFA 0.1% in water as well in acetonitrile was used as counterion. All compounds
reported here were ≥ 95% HPLC pure. The solvents used in MS measures were acetone, acetonitrile
(Chromasolv grade) and mQ water 18 Mꢂ. The high resolution mass spectrometry (HRMS)
analysis were performed with a Thermo Finnigan LTQ Orbitrap mass spectrometer coupled with an
+
electrospray ionization source (ESI). Analysis were carried out in positive ion mode [M+H] , and it
was used a proper dwell time acquisition to achieve 60,000 units of resolution at Full Width at Half
Maximum (FWHM). Elemental composition of compounds were calculated on the basis of their
measured accurate masses, accepting only results with an attribution error less than 5 ppm and a not
3
1
integer RDB (double bond/ring equivalents) value.
Stock solutions of analytes were prepared
ꢀ1)
using acetone (1.0 mg mL and stored at 4 °C. Then working solutions of each analyte were
prepared by dilution of the stock solutions using mQ H O/ACN 1/1 (v/v) up to a concentration of
2
ꢀ1
1
.0 ꢁg mL The HRMS analysis was performed by introducing the analyte working solution via
ꢀ1
syringe pump at 10 ꢁL min .
18
a) General Procedure for synthesis of compounds 1Aꢀ8A, 1Cꢀ4C and 6Cꢀ8C, 1Dꢀ8D, 1Eꢀ8E.
The appropriate NSAID 1ꢀ8 (1.0 eq.), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
.
hydrochloride salt (EDCI HCl, 2.0 eq.) and 1ꢀhydroxyꢀ7ꢀazabenzotriazole (HOAT, 2.0 eq.), were
dissolved in dry DMF or DMA solvents (3.0 ml) and stirred for 10 minutes at r.t., followed by
addition of the amine A, C, D or E (2.0 eq.) and N,Nꢀdiisopropylethylamine (DIPEA, 4.0 eq.) in the
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6
same solvent system (1.5 ml). The reaction was stirred until the reaction stops (TLC monitoring),
quenched with a 3.0 M aqueous HCl solution at 0 °C and extracted with ethyl acetate (3 x 15 ml).
The combined organic layers were washed with 3.0 M aqueous HCl solution, H
2
O (3 x 20 ml), sat.
aqueous NaHCO (3 x 20 ml), dried over Na SO and evaporated under vacuo to give a residue
3
2
4
0
1
2
3
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2
3
4
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8
9
0
which was purified by silica gel column chromatography eluting with ethyl acetate/nꢀhexane or
MeOH/DCM, crystallization or trituration from the appropriate solvent to afford the titled
compounds.
18
b) General Procedure for synthesis of compounds 1Bꢀ8B.
The appropriate NSAID 1ꢀ8 (1.0 eq.) and N,N'ꢀdicyclohexylcarbodiimide (DCC, 1.1 eq.) were
dissolved in dry dimethylformamide (DMF, 3.0 ml) and stirred at r.t. under a nitrogen atmosphere
for 10 minutes until a white precipitate occurs, then Nꢀhydroxysuccinimide (NHS, 1.1 eq.)
dissolved in dry DMF (1.5 ml) was added and the reaction mixture was stirred at r.t. until starting
material was consumed (TLC monitoring) followed by addition of the 4ꢀ
aminoethylbenzenesulfonamide B (2.0 eq.) dissolved in the same solvent. The reaction mixture was
stirred until the reaction stops (TLC monitoring), quenched with a 3.0 M aqueous HCl solution at 0ꢀ
5 °C and extracted with ethyl acetate (3 x 15 ml). The precipitate formed during the extraction was
filteredꢀoff and the combined organic layers were washed with H
2
O (5 x 15 ml), sat. aqueous
NaHCO (3 x 20 ml), dried over Na SO and evaporated under vacuo to give a deposit that was
3
2
4
purified by silica gel chromatography eluting with MeOH/DCM 5% v/v followed by crystallization
from ethyl acetate/petroleum ether to afford the title compounds.
2
ꢀ(±)ꢀ(4ꢀIsobutylphenyl)ꢀ
N
ꢀ(4ꢀsulfamoylphenyl)propanamide 1A. Ibuprofen 1 (1.0 eq.), 1ꢀethylꢀ
.
3
ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride salt (EDCI HCl, 2.0 eq.) and 1ꢀhydroxyꢀ7ꢀ
azabenzotriazole (HOAT, 2.0 eq.), sulfonilamide A (2.0 eq.) and N,Nꢀdiisopropylethylamine
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(
DIPEA, 4.0 eq.) in DMF (4.5 ml) were treated according to the general procedure a previously
reported. The crude product was purified by silica gel column chromatography eluting with ethyl
acetate/nꢀhexane 50% v/v and crystallized from ethyl acetate/petroleum ether to afford the title
compound 1A as a white solid in 19 % yield, silica gel TLC R
v/v); mp 194.8 ºC; δ (400 MHz, DMSOꢀd ) 0.88 (6H, d, J 6.6, ꢀCH(CH
CHCH ), 1.83 (1H, p, J 6.8, CH(CH ) ), 2.43 (2H, d, J 7.1, ꢀCH CH(CH ) ), 3.85 (1H, p, J 6.8, ꢀ
f
0.68 (Ethyl acetate/nꢀhexane 50%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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0
1
2
3
4
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7
8
9
0
H
6
3 2
)
), 1.43 (3H, d, J 7.0, ꢀ
3
3 2
2
3 2
CHCH ), 7.14 (2H, d, J 8.0, ArꢀH), 7.26 (2H, s, SO NH , exchange with D O), 7.32 (1H, d, J 8.0,
3
2
2
2
ArꢀH), 7.77 (4H, s, 4 x ArꢀH), 10.39 (1H, s, ꢀCONH, exchange with D
2 C
O); δ (100 MHz, DMSOꢀ
d
6
) 174.0 (C=O), 143.1, 140.7, 139.8, 139.3, 130.0 (2 x ArꢀC), 128.0 (2 x ArꢀC), 127.7 (2 x ArꢀC),
1
3 2
19.7 (2 x ArꢀC), 46.7, 45.3, 30.6, 23.2 (ꢀCH(CH ) ), 19.7.
19
Experimental in agreement with reported data.
2ꢀ(±)ꢀ(4ꢀIsobutylphenyl)ꢀNꢀ(4ꢀsulfamoylphenethyl)propanamide 1B. Ibuprofen 1 (1.0 eq.), N,N'ꢀ
dicyclohexylcarbodiimide (DCC, 1.1 eq.), Nꢀhydroxysuccinimide (NHS, 1.1 eq.) and 4ꢀ
aminoethylbenzenesulfonamide B (2.0 eq.), dissolved in dry DMF (1.5 ml), were treated according
to the general procedure b previously reported to afford the titled compound 1B as a white solid in
2
3% yield, silica gel TLC R
.90 (6H, d, J 6.6, ꢀCH(CH
CH(CH
ꢀ), 3.55 (1H, d, J 7.2, ꢀCHCH
3
f
0.19 (MeOH/DCM 5 % v/v); mp 149.3 ºC; δ
), 1.32 (3H, d, J 7.0, ꢀCHCH ), 1.85 (1H, p, J 6.8, ꢀCH(CH
), 2.77 (2H, t, J 7.1, ꢀCONHCH CH ꢀ), 3.33ꢀ3.26 (2H, m, ꢀ
), 7.12 (2H, d, J 8.1, ArꢀH), 7.22(2H, d, J 8.1, ArꢀH),
H
(400 MHz, DMSOꢀd
6
)
0
3
)
2
3
3 2
)
), 2.46
(2H, d, J 7.1, ꢀCH
2
3
)
2
2
2
CONHCH CH
2
2
7
.30 (2H, d, J 8.3, ArꢀH), 7.32 (2H, s, SO NH , exchange with D O), 7.72 (2H, d, J 8.3, ArꢀH), 8.03
2
2
2
(1H, t, J 5.6, CONH, exchange with D
2
O); δ
C
6
(100 MHz, DMSOꢀd ) 174.4 (C=O), 144.7, 143.0,
1
4
40.4, 140.2, 130.2 (2 x ArꢀC), 129.8 (2 x ArꢀC), 128.0 (2 x ArꢀC), 126.6 (2 x ArꢀC), 45.7, 45.2,
0.8 (ꢀCONHCH CH ꢀ), 35.7 (ꢀCONHCH CH ꢀ), 30.7, 23.2(ꢀCH(CH ), 19.4; ESIꢀHRMS (m/z)
S = 389.1893, found 389.1894.
2
2
2
2
3 2
)
+
29 2 3
calculated for [M+H] ion species C21H N O
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2
ꢀ(±)ꢀ(4ꢀIsobutylphenyl)ꢀ
N
ꢀ(2ꢀ(4ꢀsulfamoylphenoxy)ethyl)propanamide 1C. Ibuprofen 1 (1.0
.
eq.), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride salt (EDCI HCl, 2.0 eq.) and 1ꢀ
hydroxyꢀ7ꢀazabenzotriazole (HOAT, 2.0 eq.), 4ꢀ((3ꢀhydroxyethyl)amino)benzenesulfonamide C
(2.0 eq.) and N,Nꢀdiisopropylethylamine (DIPEA, 4.0 eq.) in dry DMA (4.5 ml) were treated
according to the general procedure b previously reported. The crude product was purified by silica
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
gel column chromatography using Ethyl acetate/nꢀhexane 66% v/v and crystallized from EtOH/H O
2
to afford the title compound 1C as a white solid in 25% yield, silica gel TLC R
acetate/nꢀhexane 66% v/v); mp 152 ºC; δ (400 MHz, DMSOꢀd ) 0.88 (6H, d, J 6.4, ꢀCH(CH
1.34 (3H, d, J 7.2, ꢀCHCH ), 1.82 (1H, p, J 6.8, ꢀCH(CH ), 2.43 (2H, d, J 7.2, ꢀCH CH(CH
3.45 (2H, m, ꢀCONHCH CH ), 3.59 (1H, q, J 6.8, ꢀCHCH ), 4.08 (2H, t, J 5.6, ꢀCONHCH
f
0.36 (Ethyl
H
6
3
3
)
)
2
2
),
),
),
3
3
)
2
2
2
2
2
3
2
CH
7
8
1
6
.08 (4H, d, J 8.8, ArꢀH), 7.24ꢀ7.23(4H, m, SO NH , exchange with D O, 2 x ArꢀH), 7.76 (2H, d, J
2
2
2
.8, ArꢀH), 8.21 (1H, t, J 5.6, ꢀCONH, exchange with D
2
O); δ
C
6
(100 MHz, DMSOꢀd ) 174.7 (C=O),
61.7, 140.3, 140.1, 137.2, 129.6 (2 x ArꢀC), 128.5 (2 x ArꢀC), 127.8 (2 x ArꢀC), 115.4 (2 x ArꢀC),
7.6 (ꢀCONHCH
2
CH
2
), 45.5, 45.0, 39.0, 30.5, 23.1, 23.1, 19.5; ESIꢀHRMS (m/z) calculated for
S = 405.1842, found 405.1841.
+
[M+H] ion species C21
H
29
N
2
O
4
2ꢀ(±)ꢀ(4ꢀIsobutylphenyl)ꢀ
N
ꢀ(3ꢀ(4ꢀsulfamoylphenoxy)propyl)propanamide 1D. Ibuprofen 1 (1.0
.
eq.), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride salt (EDCI HCl, 2.0 eq.) and 1ꢀ
hydroxyꢀ7ꢀazabenzotriazole (HOAT, 2.0 eq.), 4ꢀ((3ꢀhydroxypropyl)amino)benzenesulfonamide D
(2.0 eq.) and N,Nꢀdiisopropylethylamine (DIPEA, 4.0 eq.) in dry DMA (4.5 ml) were treated
according to the general procedure b previously reported. The crude product purified by silica gel
column chromatography using Ethyl acetate/nꢀhexane 50% v/v and crystallized from ethyl
acetate/petroleum ether to yield the title compound 1D as a white solid in 28% yield, silica gel TLC
f H 6
R 0.10 (Ethyl acetate/nꢀhexane 50% v/v); mp 127.1 ºC; δ (400 MHz, DMSOꢀd ) 0.87ꢀ0.85 (8H,
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m, ꢀCH(CH ) , ꢀCONHCH CH CH ), 1.32 (3H, d, J 7.0, ꢀCHCH ), 1.88ꢀ1.77 (1H, m, ꢀ
3 2
2
2
2
3
CH(CH3)2), 2.41 (2H, d, J 7.1, ꢀCH CH(CH3)2), 3.23ꢀ3.18 (2H, m, ꢀCONHCH CH CH ), 3.56
2
2
2
2
(
(
(
(
1H, d, J 7.0, ꢀCHCH3 ), 4.00ꢀ3.97 (2H, m, ꢀCONHCH
2
CH
2
CH
2
), 7.02 (2H, d, J 8.9, ArꢀH), 7.08
NH , exchange with D O), 7.75
O); δ (100 MHz, DMSOꢀd ) 173.9
C=O), 161.3, 140.1, 139.6, 136.6, 129.2 (2 x ArꢀC), 128.0 (2 x ArꢀC), 127.3 (2 x ArꢀC), 114.8 (2 x
2H, d, J 8.0, ArꢀH), 7.23( 2H, d, J 7.6, ArꢀH), 7.23 (2H, s, SO
2H, d, J 8.9, ArꢀH), 8.05 (1H, m, ꢀCONH, exchange with D
2
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
C
6
ArꢀC), 66.0 (ꢀCONHCH CH CH ), 45.2, 44.6, 35.8, 30.0, 29.0, 22.6, 19.0; ESIꢀHRMS (m/z)
2
2
2
+
31 2 4
calculated for [M+H] ion species C22H N O S = 419.1999, found 419.2006.
2
ꢀ(±)ꢀ(4ꢀIsobutylphenyl)ꢀ
N
ꢀ(4ꢀ(4ꢀsulfamoylphenoxy)butyl)propanamide 1E. Ibuprofen 1 (1.0
.
eq.), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride salt (EDCI HCl, 2.0 eq.) and 1ꢀ
hydroxyꢀ7ꢀazabenzotriazole (HOAT, 2.0 eq.), 4ꢀ((3ꢀhydroxybuthyl)amino)benzenesulfonamide E
(2.0 eq.) and N,Nꢀdiisopropylethylamine (DIPEA, 4.0 eq.) in dry DMA (4.5 ml) were treated
according to the general procedure b previously reported. The crude product was purified by silica
gel column chromatography using Ethyl acetate/nꢀhexane 50% v/v and crystallized from ethyl
acetate/petroleum ether to afford the title compound 1E as a white solid in 23% yield, silica gel
TLC R
J 6.8, ꢀCH(CH
(2H, p, J 7.0, ꢀCONHCH
CH CH(CH ), 3.12 (2H, q, J 6.4, ꢀCONHCH
2H, t, J 6.4, ꢀCONHCH CH CH CH ), 7.11ꢀ7.06 (4H, m, 4 x ArꢀH), 7.22 (2H, s, SO NH ,
f
0.5 (Ethyl acetate/nꢀhexane 66% v/v); mp 101.8 ºC; δ
), 1.34 (3H, d, J 7.2, ꢀCHCH ), 1.55 (2H, p, J 7.1, ꢀCONHCH
CH CH CH ), 1.84 (1H, sep., J 6.8, ꢀCH(CH ), 2.43 (2H, d, J 6.8, ꢀ
CH CH CH ), 3.58 (1H, q., J 6.8, ꢀCHCH ), 4.03
H
(400 MHz, DMSOꢀ d
6
) 0.88 (6H, d,
3
)
2
3
2
CH CH
2
2
CH ), 1.70
2
2
2
2
2
3 2
)
2
3
)
2
2
2
2
2
3
(
2
2
2
2
2
2
exchange with D
2
O), 7.24 (2H, d, J 8.0, 2 x ArꢀH), 7.77 (2H, d, J 8.8, 2 x ArꢀH), 7.96 (1H, t, J 5.4,
ꢀ
CONH, exchange with D O), δ (100 MHz, DMSOꢀ d ) 174.2 (C=O), 161.9, 140.6, 140.0, 137.0,
2
C
6
129.6 (2 x ArꢀC), 128.5 (2 x ArꢀC), 127.8 (2 x ArꢀC), 115.3 (2 x ArꢀC), 68.5, 45.7, 45.1, 39.0, 30.5,
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2
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2
2
2
2
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5
5
5
5
5
5
5
5
6
+
2
6.8, 26.5, 23.1 (ꢀCH(CH ) ), 19.5; ESIꢀHRMS (m/z) calculated for [M+H] ion species
3 2
C H N O S = 433.2155, found 433.2163.
23
33
2
4
0
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9
0
2ꢀ(±)ꢀ(6ꢀMethoxynaphthalenꢀ2ꢀyl)ꢀ
N
ꢀ(4ꢀsulfamoylphenyl)propanamide 2A. Naproxen 2 (1.0
.
eq.), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride salt (EDCI HCl, 2.0 eq.) and 1ꢀ
hydroxyꢀ7ꢀazabenzotriazole (HOAT, 2.0 eq.), sulfonilamide
A
(2.0 eq.) and N,Nꢀ
diisopropylethylamine (DIPEA, 4.0 eq.) in DMF (4.5 ml) were treated according to the general
procedure a previously reported to afford the title compound 2A as a white solid in 8% yield, silica
gel TLC R
3H, d, J 7.0, ꢀCHCH
naphtalenꢀH ), 7.26 (2H, s, SO
1H, dd, J 1.7/8.6, naphtalenꢀH), 7.77ꢀ7.85 (7H, m, 3 x naphtalenꢀH, 4 x ArꢀH), 10.46 (1H, s, ꢀ
CONH, exchange with D O); δ (100 MHz, DMSOꢀd ) 173.5 (C=O), 157.7, 142.7, 138.9, 137.2,
f
0.17 (Ethyl acetate/nꢀhexane 50% v/v); mp 202.5 ºC; δ
), 3.89 (3H, s,ꢀOCH ), 4.02 (1H, d, J 7.0, ꢀCHCH
NH , exchange with D O), 7.32 (1H, d, J 2.5, naphtalenꢀH), 7.54
H
(400 MHz, DMSOꢀd
6
) 1.54
(
3
3
3
), 7.18 (1H, dd, J 2.6/8.9,
2
2
2
(
2
C
6
1
33.9, 129.7, 129.0, 127.5 (2 x ArꢀC), 127.2, 126.8, 126.1, 119.4 (2 x ArꢀC), 119.3, 106.3, 55.8 (ꢀ
+
OCH
3
), 46.6 (ꢀCHCH
3
), 19.3 (ꢀCHCH
3
); ESIꢀHRMS (m/z) calculated for [M+H] ion species
C H N O S = 385.1216, found 385.1209.
20
21
2
4
2ꢀ(±)ꢀ(6ꢀMethoxynaphthalenꢀ2ꢀyl)ꢀ
N
ꢀ(4ꢀsulfamoylphenethyl)propanamide 2B. Naproxen 2 (1.0
eq.), N,N'ꢀdicyclohexylcarbodiimide (DCC, 1.1 eq.), Nꢀhydroxysuccinimide (NHS, 1.1 eq.) and 4ꢀ
aminoethylbenzenesulfonamide B (2.0 eq.) dissolved in dry DMF (1.5 ml) were treated according to
the general procedure b previously reported to afford the titled compound 2B as a white solid in
5
5% yield, silica gel TLC R 0.14 (MeOH/DCM 5 % v/v); mp 161.3 °C; δ (400 MHz, DMSOꢀd )
f
H
6
1.42 (3H, d, J 7.0, ꢀCHCH
CONHCH CH ), 3.72 (1H, q, J 7.0, ꢀCHCH
naphtalenꢀH), 7.31ꢀ7.30 (5H, m, naphtalenꢀH, 2 x ArꢀH, SO
3
), 2.79ꢀ2.76 (2H, m, ꢀCONHCH
), 3.90 (3H, s,ꢀOCH
NH , exchange with D
2
2
CH
), 7.18(1H, dd, J 2.6/8.9,
O), 7.43 (1H,
2
), 3.32ꢀ3.28 (2H, m, ꢀ
2
2
3
3
2
2
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dd, J 1.8/8.5, naphtalenꢀH), 7.69 (2H, d, J 8.3, 2 x naphtalenꢀH), 7.82ꢀ7.72 (3H, m, naphtalenꢀH, 2 x
ArꢀH), 8.09 (1H, t, J 5.6, ꢀCONH, exchange with D O); δ (100 MHz, DMSOꢀd ) 174.0 (C=O),
2
C
6
1
1
57.6, 144.3, 142.6, 138.0, 133.8, 129.7 (2 x ArꢀC), 129.0, 127.22, 127.1, 126.2 (2 x ArꢀC), 125.9,
19.2, 106.3, 55.8 (ꢀOCH
);
Experimental in agreement with reported data.
3
), 45.7 (ꢀCHCH
3
), 40.8 (ꢀCONHCH
2
CH
2
), 35.3 (ꢀCONHCH
2
CH
2
), 19.0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(ꢀCHCH
3
20
2ꢀ(±)ꢀ(6ꢀMethoxynaphthalenꢀ2ꢀyl)ꢀ
N
ꢀ(2ꢀ(4ꢀsulfamoylphenoxy)ethyl)propanamide
2C.
Naproxen 2 (1.0 eq.), 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride salt
.
(
EDCI HCl, 2.0 eq.) and 1ꢀhydroxyꢀ7ꢀazabenzotriazole (HOAT, 2.0 eq.), 4ꢀ((3ꢀ
hydroxyethyl)amino)benzenesulfonamide C (2.0 eq.) and N,Nꢀdiisopropylethylamine (DIPEA, 4.0
eq.) in dry DMA (4.5 ml) were treated according to the general procedure b previously reported.
2
The crude product was crystallized from EtOH/H O to afford the title compound 2C as a white
solid in 19 % yield, silica gel TLC R 0.23 (EtOAc/nꢀhexane 66% v/v); mp 170.3 ºC; δ (400 MHz,
f
H
DMSOꢀd
CHCH
XnaphtalenꢀH), 7.16 (1H, dd, J 2.4/8.8, naphtalenꢀH), 7.24( 2H, s, SO
6
) 1.44 (3H, d, J 6.8, ꢀCHCH
3
), 3.47 (2H, m, ꢀCONHCH
2
CH
CH
NH
2
), 3.80 (1H, d, J 6.8, ꢀ
3
), 3.89 (3H, s, ꢀOCH
3
), 4.09 (2H, t, J 5.6, ꢀCONHCH
2
2
), 7.07 (2H, d, J 8.8,
, exchange with D O),
2
2
2
2
7.29 (1H, d, J 2.8, naphtalenꢀH), 7.46 (1H, dd, J 1.6/8.8, naphtalenꢀH), 7.77ꢀ7.73 (5H, m,
naphtalenꢀH, 4 x ArꢀH), 8.30ꢀ8.27 (1H, t, J 5.4, ꢀCONH, exchange with D O); δ (100 MHz,
2
C
DMSOꢀd
27.3, 126.1, 119.4, 115.4 (2 x ArꢀC), 106.6, 67.6 (ꢀCONHCH
CHCH ), 39.1 (ꢀCONHCH CH ), 19.4 (ꢀCHCH
species C22 S = 429.1479 found 429.1484.
6
) 174.7 (C=O), 161.7, 158.0, 138.2, 137.2, 134.0, 130.0, 129.3, 128.6 (2 x ArꢀC), 127,5,
1
2
CH
2
), 56.1 (ꢀOCH
3
), 45.8 (ꢀ
+
3
2
2
3
); ESIꢀHRMS (m/z) calculated for [M+H] ion
25 2 5
H N O
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