~ 38 ~
Chapter 4
Results and Discussions
During the work of my thesis I evaluated the pharmacological properties of compounds designed and synthetized for interacting with CB2r (see chapter 2). These compounds are characterized by different chemical structure:
1,2-Dihydro-6-methyl-2-oxopyridine-3-carboxamide (A):
A
Name R1 R2 R3
AD-6b Ethylmorpholino Cycloheptyl Br
AD-3 Ethylmorpholino Cycloheptyl H
EB-5b Ethylmorpholino Cycloheptyl Phenyl
FM-4b Ethylmorpholino Cycloheptyl p-Methoxyphenyl
MC-18a p-Fluorobenzyl Cycloheptyl H
AD-15b p-Fluorobenzyl Cycloheptyl Phenyl
FM-5b p-Fluorobenzyl Cycloheptyl p-Methoxyphenyl
FM-6b p-Fluorobenzyl Cycloheptyl Br N O O NH R2 R3 C H3 R1
~ 39 ~
6-Methyl-pyridine-3-carboxamide (B):B Biphenyl-3-carboxamide (C):
C
Name R1 R2 R3
AD-6a Ethylmorpholino Cycloheptyl Br
EB-5a Ethylmorpholino Cycloheptyl Phenyl
FM-4a Ethylmorpholino Cycloheptyl p-Methoxyphenyl
MC-18b p-Fluorobenzyl Cycloheptyl H
AD-15a p-Fluorobenzyl Cycloheptyl Phenyl
FM-5a p-Fluorobenzyl Cycloheptyl p-Methoxyphenyl
FM-6a p-Fluorobenzyl Cycloheptyl Br
N O R1 O NH R2 C H3 R3 O NH R2 O R1 R3 CH3
~ 40 ~
Name R1 R2 R3
SS-05 Methyl Cycloheptyl H
SS-025 n-Butyl Cycloheptyl Methoxy
GR-29a Benzyl Trans-4-methyl-cyclohexyl H
GR-28b Benzyl Cis-4-methyl-cyclohexyl H
GR-28c Benzyl Cis/trans-4-methyl-cycloexyl H
AC-22 n-Butyl Cycloheptyl F
AC-23a n-Butyl Trans-4-methyl-cyclohexyl F
AC-23b n-Butyl Cis-4-methyl-cyclohexyl F
AC-23c n-Butyl Cis/trans 4-methyl-cycloexyl F
AC-40 n-Butyl Cycloheptyl H
AC-42a n-Butyl Trans-4-methyl-cyclohexyl H
AC-42b n-Butyl Cis-4-methyl-cyclohexyl H
AC-42c n-Butyl Cis/trans 4-methyl-cycloexyl H
AC-17 p-fluorobenzyl Cycloheptyl Methoxy
AC-33a p-fluorobenzyl Trans-4-methyl-cyclohexyl Methoxy
AC-33b p-fluorobenzyl Cis-4-methyl-cyclohexyl Methoxy
AC-33c p-fluorobenzyl Cis/trans-4-methyl-cycloexyl Methoxy
AC-35 p-fluorobenzyl Cycloheptyl F
AC-64a p-fluorobenzyl Trans-4-methyl-cyclohexyl F
AC-64b p-fluorobenzyl Cis-4-methyl-cyclohexyl F
~ 41 ~
The synthesis of biphenyl-3-carboxamides (C) (biphenylic derivatives), 1,2-dihydro-6-methyl-2-oxopyridine-3-carboxamides A and 6-methyl-pyridine-3-carboxamides B (pyridine derivatives) are reported in work of thesis of Federica Mariani and in work of PhD thesis of Dott.ssa Chiara Arena. In my work of thesis I report the biological activity data obtained by testing all the above compounds on the different targets of ECs: cannabinoid receptors, MAGL, FAAH, ABHDs, COX-2 and EMT.
4.1 Cannabinoid receptor binding
An initial screening to evaluate the binding properties of the compounds A, B and C to CB1r and CB2r was performed at the concentration of 1 µM. The results are shown in Fig. 4.1 a, b.
~ 42 ~
a Biphenylic derivatives (C) AC -22 AC 23a AC 23b AC 23c AC 40 AC 42a AC 42b AC 42c AC 17 AC 33a AC 33b AC 33c AC 35 AC 64a AC 66 AC 64b SS-0 5 SS-0 25 GR -28b GR -28c GR -29a 0 50 100 150 CB1 CB2 [ 3 H ]C P 5 5 9 4 0 b o u n d ( % v eh ic le ) bPyridine derivatives (A and B)
AD 6b AD 3 AD 15a AD 15b EB5a EB5b MC-18a MC1 8b AD 6a FM -4a FM -4b FM -5a FM -5b FM -6a FM .6b 0 50 100 150 CB1 CB2 [ 3 H ]C P5 5 9 40 b o un d ( % v eh ic le )
Figure 4.1 a, b Binding competition against [3H]CP-55,904 on CB1r and CB2r at 1 µM of biphenylic derivatives and pyridine derivatives.
Compounds that showed a binding to either CB1r and/or CB2r higher than 50% at the screening concentration were selected and fully characterized by constructing concentration-dependent binding curves. The results showed that while only five biphenylic derivatives exhibit a significant binding to cannabinoid receptors,
most of all the pyridine derivatives are good binders at the concentration of 1 µM. These
~ 43 ~
results are in agreement with the recently published data (Lucchesi et al., 2014) showing that the 1,2-dihydro-2-oxopyridine-3-carboxamide derivatives generated potent and selective CB2r ligands.
4.1.1 Concentration-dependent binding curves of biphenylic derivatives
Biphenylic derivatives that presented a binding to either CB1r and/or CB2r higher than 50% at 1 µM were investigated deeply in a larger range of concentration and a precise Ki value was obtained for each one.
a b c
d e f
Figure 4.2 Concentration-dependent binding curves for (a) AC-35, (b) AC-40, (c) AC-64b,
(d) AC-66, (e) SS-05, (f) SS-025. -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 Log [AC-64b] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -11-10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [AC-66] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -11-10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB 2 Log [SS-05] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 Log [AC-35] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 Log [AC-40] (M) [ 3 H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 Log [SS-025] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le)
~ 44 ~
Table 4.1 Ki value and Confidence Interval for biphenylic compounds.
All the tested compounds showed Ki valuesfor CB2r in the nano-molar range, with AC-66 (C) being the most potent compound in the series (Ki = 75.2 nM) and selective (SI = 16.96, Selectivity index for CB2r calculated as Ki(CB1r)/Ki(CB2r) ratio). All the compounds showed Ki values forCB1r bigger than 1 µM.
These results suggest that the N-cycloheptyl group in position 3 and p-fluorobenzyl group in position 1 of the phenyl ring favor the binding interactions with CB2r. Furthermore, AC-35, AC-66 and SS-05 (general structure C), which show the best affinity on CB2r, were further evaluated with the [35S]GTPγS assay in order to elucidate the functional modulation of CB2r activity.
4.1.2. Concentration-dependent binding curves of pyridine derivatives
Pyridine derivatives that presented a binding to either CB1r and/or CB2r higher than 50% at 1 µM were investigated deeply in a larger range of concentration and a precise Ki value was obtained for each one.
Name Ki (mean and 95% CI, nM) CB1 Ki (mean and 95% CI, nM) CB2
AC-35 ˃ 1 µM 359 (170 – 787) AC-40 ˃ 1 µM 751 (518 – 1436) AC-64b ˃ 1 µM 670 (368 – 754) AC-66 1276 (859 – 3883) 75.2 (17.3 – 1779) SS-05 ˃ 1 µM 141,96 (81.8 – 246) SS-025 ˃ 1 µM 425 (239 – 777)
~ 45 ~
a b c d e f g h i -11 -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 CB2 CB1 Log [MC-18a] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB 2 Log [EB-5a] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [MC-18b] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -11-10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [AD-6b] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB 2 Log [AD-3] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le ) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [EB-5b] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 Log [AD-15a] (M) [ 3H ]C P 5 5 9 4 0 b o u n d ( % v e h ic le ) -11-10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [AD-15b] (M) [ 3H ]C P 5 5 9 4 0 b o u n d ( % v eh ic le) -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [FM-4a] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le)~ 46 ~
l m n
o p
Figure 4.3 Concentration-dependent binding curves for (a) MC-18a, (b) MC-18b, (c) EB-5a, (d) EB-5b, (e) AD-3, (f) AD-6b, (g) AD-1EB-5a, (h) AD-15b, (i) FM-4a, (l) FM-4b, (m) FM-5a, (n) FM-5b, (o) FM-6a, (p) FM-6b.
-10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [FM-4b] (M) [ 3H ]C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 -3 0 20 40 60 80 100 120 CB2 Log [FM-5a] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 CB2 CB1 Log [FM-5b] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 140 CB2 CB1 Log [FM-6a] (M) [ 3H] C P 5 5 9 4 0 b o u n d ( % v eh ic le) -12-11-10 -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 120 140 CB1 CB2 Log [FM-6b] (M) [ 3H ]C P 5 5 9 4 0 b o u n d ( % v eh ic le)
~ 47 ~
Table 4.2Ki value and Confidence Interval for pyridine derivatives (A and B).
The results showed that the insertion of the methyl group in position 6 of the pyridine ring does not alter the good cannabinoid receptor binding shown by the previous reported 1,2-dihydro-2-oxopyridine-3-carboxamide derivatives (Lucchesi et al., 2014). In addition, the pyridine derivatives clearly possess better binding properties to cannabinoid receptors compared to the biphenylic derivatives (Fig. 4.2/4.3). Indeed, several compounds showed Kivaluesin the low nano-molar range, such as
FM-6b (Fig. p), FM-5b (Fig. n), AD-15b (Fig. h) and MC18a (Fig. 4.3-a / T4.3-able 4.2).
Furthermore data suggest that the type of substituent present in position 1 of the pyridine ring significantly influences the binding properties of the molecule, especially for CB2r. In particular, among the different molecules tested, it was clear that the p-fluorobenzyl substituted ones show better binding properties compared to
Ki values (mean and 95% CI, nM)
Name CB1r CB2r MC-18a 14.2 (12 – 58) 9.40 (6 – 20) MC-18b 94.6 (62 – 257) 31.3 (25 – 98) EB-5a ˃ 1 µM 335 (152 – 687) EB-5b 336 (121 – 601) 36.0 (12 – 57) AD3 ˃ 1 µM 335 (121 – 540) AD6b 453 (235 – 730) 35.1 (20 – 54.2) AD-15a ˃ 1 µM 422 (278 – 978.2) AD-15b 176 (63 – 300.2) 5.60 (3.2 – 12.8) FM-4a 654 (254 – 1684) 621 (282 – 1368) FM-4b 306 (194.2 – 483.7) 177 (70.5 – 445.2) FM-5a ˃ 1 µM 919 (456 – 1349) FM-5b 9.80 (5.8 – 16.6) 26.2 (22 – 45.2) FM-6a 96.3 (41.8 – 222) 104.5 (98 – 337) FM-6b 8.87 (3.7 – 21.46) 2.82 (1.116 – 2.85)
~ 48 ~
the compounds bearing a 2-ethyilmorfolino group in the same position. Indeed the p-fluorobenzyl derivatives FM-6b, FM-5b, AD-15b, MC-18a showed the most potent binding to cannabinoid receptors displaying the lowest Kivalues (Table 4.2). When the substituent in position 1 was moved to the oxygen in position 2 of pyridine ring (general structure B), the binding affinity for CB1rand CB2r strongly decreased, as exemplified by the compounds FM-6a (fig. 4.3-o – Ki value of 104.53) and FM-6b (Fig. 4.3-p – Ki value of 2.82). These observations suggest that either the type or the position of the substituent group on the pyridine ring play a crucial role for the binding interactions to cannabinoid receptors. In particular, substituents in position 1 are more favorable for the receptor binding compared to shifting the same groups to position 2. All the most potent pyridine derivatives were tested with the [35S]GTPγS assay to evaluate the functional modulation of CB1r and CB2r receptor activity.
4.2 [35S]GTPγS assay performed for biphenylic derivatives (C) and pyridine derivatives (A and B) on CB1r and CB2r
The functional modulation of CB1r and CB2r activity was investigated using the [35S]GTPγS assay. In this assay, compounds (or vehicle) are incubated with membranes overexpressing the receptor of interest (CB1r or CB2r), GDP and the hydrolytically stable radiolabelled GTP analogue ([35S]GTPγS), that binds the G protein after the activation of the receptor, but which cannot be hydrolyzed. After the incubation time (necessary to reach the binding equilibrium) the radioactive signal is quantified (which corresponds to the amount of activated G-proteins) and compared with the signal of vehicle control samples. When the compound does not lead to any changes in the radioactive signal compared to control, it behaves as an antagonist, while a concentration-dependent increase of the radioactive signal implies a higher G-protein recruitment compared to vehicle control (agonist behavior). On the other hand, a reduction in the signal means a reduction of G-protein recruitment, thus indicating an inverse agonist behavior.
~ 49 ~
In these experimental settings, 2-AG and SR-144528 (SR-2) were selected as positive controls for agonist and inverse agonist (respectively) for CB2r. For CB1r, CP-55,940 and SR141716A (SR-1) were used as positive controls for agonist and inverse agonist, respectively.
4.2.1 Functional activity evaluation of biphenylic derivatives
Only the best three ligands among the biphenylic derivatives were chosen for the [35S]GTPγS assay on CB2r. The effects on CB1r were not assessed due the low binding affinity.
-9 -8 -7 -6 -5 -4 40 60 80 100 120 140 160 180 200 hCB2 receptors AC-66 AC-35 2-AG SR-2 SS-05 Log (M) [ 35 S ]G T P S b o u n d ( % o f v eh icl e)
EC50 (mean and 95% CI, nM) SS-05= 160 (37.2 - 688)
Figure 4.4 Concentration-dependent curves for [35S]GTPγS binding to CB2r induced by the
most potent biphenylic derivatives (general structure C). 2-AG and SR-2 were used as positive control for agonism and inverse agonism respectively.
Results showed an interesting difference between SS-05, that worked as an agonist, and AC-66 / AC-35 that showed inverse agonism behavior (general structure C).
SS-05 bears a methyl group in position 1 of the phenyl ring, while AC-66 and AC-35
~ 50 ~
be relevant for modulating the receptor binding affinity and to dictate the functional activity among the biphenylic derivatives. In particular more bulky substituents (i.e.
p-fluorobenzyl group) seems to confer weak inverse agonist properties at CB2r while
smaller groups (i.e. methyl group) led to agonistic behavior.
4.2.2 Functional activity of pyridine derivatives measured by for [35S]GTPγS
Figure 4.5 [35S]GTPγS assay performed on CB2r at 1 µM for the most potent pyridine derivatives (general structure A).
Initially, all best ligands of this class of compounds were tested at the screening concentration of 1 µM for CB2r modulation ([35S]GTPγS assay). In Fig. 4.5 the results show that most of the compounds behaved as antagonist/weak inverse agonist, unlike FM-6b which showed agonistic properties. Interestingly, FM-6b,
FM-5b and AD-15b, which share the same chemical structure and differ only for the
type of substituent present in position 5 of the pyridine ring (see Fig. 4.3-h, 4.3-n,
4.3-p) showed all the different possibility of receptor modulation (i.e FM-6b agonist,
AD-6b AD-3 MC1 8-a MC1 8-b EB -5a EB -5b AD1 5-a FM -4a FM -4b FM -5a FM -6a FM -6b FM -5b AD-15b 2-AG SR-2 50 100 150 200 hCB2 receptors [ 35 S ]G T P S b o u n d ( % o f v eh icl e)
~ 51 ~
FM-5b antagonist and AD-15b inverse agonist). As reported in Lucchesi et al.,
2014, the characteristic of the substituent group present in position 5 of the pyridine ring of 1,2-dihydro-2-oxopyridine-3-carboxamide general structure plays a crucial role to determine the functional modulation of CB2r. Indeed, it was shown that the presence of a p-methoxyphenyl group displayed pure antagonism whereas the presence of a phenyl group led as inverse agonist and finally, when a small substituent (a hydrogen group) is present in the same position, the molecule behaved as an agonist (Lucchesi et al., 2014). The molecules tested and characterized in my work of thesis differ from the 1,2-dihydro-2-oxopyridine-3-carboxamide derivatives previous studied (Lucchesi et al., 2014) only for the presence of a methyl group in position 6 of the pyridine ring. The results clearly show that this group (methyl group) does not influence the functional activity regulation exerted by the substituent in position 5. The obtained results from my work of thesis showed that when a bromide is present in position 5 (FM-6b) instead of a hydrogen, the molecule displayed the same agonistic activity on CB2r suggesting that the nature of the small group present in position 5 of pyridine ring might not be crucial for altering the agonist behavior.
FM-6b AD-15b FM-5b
Figure 4.6 Structures of pyridine derivatives with different functional activity. In blue is
highlighted the substituent responsible for the functional switch.
In order to get a precise EC50 value, a dose-concentration curve was obtained from FM-6b, FM-5b and AD-15b (Fig. 4.7) for CB1rand CB2r.
~ 52 ~
a b
c d
e f
Figure 4.7 [35S]GTPγS binding curves for (a) FM-6b, (c) AD-15b, (e) FM-5b on CB1
receptors and (b) FM-6b, (d) AD-15b, (f) FM-5b on CB2r. 2-AG and SR-2 were used as positive control for agonism and inverse agonism at CB2r, respectively while CP-55,940 and SR-1 for agonism and inverse agonism at CB1r, respectively
-10 -9 -8 -7 -6 -5 50 100 150 200 250 hCB1 receptors Vehicle CP-55,940 SR-1 Log [FM-6b] (M) [ 35S ]G T P S b o u n d ( % o f v eh icl e) -11 -10 -9 -8 -7 -6 -5 50 100 150 200 250 Vehicle 2-AG SR-2 hCB2 receptors Log [FM-6b] (M) [ 35S ]G T P S b o u n d ( % o f v eh icl e) -11 -10 -9 -8 -7 -6 -5 50 100 150 200 hCB1 receptors Vehicle CP-55,940 SR-1 Log [AD-15b] (M) [ 35 S ]G T P S b o u n d ( % o f v e h ic le ) -10 -9 -8 -7 -6 -5 0 50 100 150 200 Vehicle 2-AG SR-2 hCB2 receptors Log [AD-15b] (M) [ 35S ]G T P S b o u n d ( % o f v eh icl e) -10 -9 -8 -7 -6 -5 50 100 150 200 250 hCB1 receptors Vehicle CP-55,940 SR-1 Log [FM-5b] (M) [ 35 S ]G T P S b o u n d ( % o f v eh icl e) -10 -9 -8 -7 -6 -5 50 100 150 200 Vehicle 2-AG AM-630 SR-2 hCB2 receptors Log [FM-5b] (M) [ 35S ]G T P S b o u n d ( % o f v eh icl e)
~ 53 ~
Table 4.3 EC50 values for FM-6b, AD-15b and FM-5b on CB1r and CB2r obtained with
[35S]GTPγS assay.
EC50 values (mean and 95% CI, nM)
Name CB1r CB2r
FM-6b 55.2 (19.9 – 159) 11.6 (4.40 – 30.7)
AD-15b n.a. 19.4 (9.80 – 38.2)
FM-5b n.a. n.a.
Results showed that FM-6b works as full agonist at both receptors, while AD-15b behaved as inverse agonist at CB2r and pure antagonist at CB1r(with a tendency towards of weak agonist activity). FM-5b did not modify the activation state of CB2r thus behaving as pure antagonist, while it weakly but significantly increased the G-protein recruitment upon CB1r activation at micro-molar. In following experiments, the agonist FM-6b and inverse agonist AD-15b were incubated with CB2r in presence of the pure antagonist FM-5b at 300 nM.
Figure 4.8 Concentration-dependent curve for [35S]GTPγS binding of FM-6b alone (black
line) or after 30 min pre-incubation with 300 nM of FM-5b (red line) on CB2r. 2-AG and
SR-2 were used as positive control for agonism and inverse agonism respectively.
-11-10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 160 180 200 hCB2 receptors FM-6b plus vehicle FM-6b plus FM-5b 2-AG SR-2 FM-5b Log [FM-6b] (M) [ 35 S ]G T P S b o u n d ( % o f v eh ic le )
~ 54 ~
The concentration curve of FM-6b in presence of the antagonist was right-shifted compared to vehicle treated samples indicating a competition between the two ligands. This is also confirmed by the increase of the EC50 value as shown in Table 4.4 reported below. By definition of competitive antagonism, the antagonist
competes with the agonist for the same receptor binding site, therefore by increasing the concentration of the agonist it is still possible to reach the maximal receptor activation as in the absence of antagonist. In order to confirm the competitive antagonism the [35S]GTPγS assay was performed in presence of two other concentrations of FM-5b (100 nM and 1 µM).
Figure 4.9 Concentration-dependent curve for [35S]GTPγS of FM-6b alone (black curve)
and in competition with 100 nM, 300 nM and 1 µM of antagonist pre-incubated for half hour.
Table 4.4 EC50 values and maximal efficacy of [
35S]GTPγS binding upon CB2r activation
induced by FM-6b in presence of different concentrations of FM-5b or vehicle.
FM-6b EC50 values (mean and
95% CI, nM)
Maximal receptor activation (mean and 95% CI)
Plus vehicle 11.6 (4.40 – 30.7) 210 (194 – 227) Plus FM-5b (100 nM) 81.2 (36.2 – 182) 219 (200 – 238) Plus FM-5b (300 nM) 192 (55.4 – 663) 209 (180 – 238) Plus FM-5b (1 µM) 711 (150 – 3371) 208 (168 – 249) -11 -10 -9 -8 -7 -6 -5 0 50 100 150 200 250 FM-6b plus FM-5b 100 nM FM-6b plus FM-5b 1M FM-6b plus vehicle FM-6b plus FM-5b 300 nM hCB2 receptors Log [FM-6b] (M) [ 35S ]G T P S b o u n d ( % o f v eh icl e)
~ 55 ~
As shown in Fig. 4.9, increasing concentrations of FM-5b determined a more pronounced right-shift of the agonist-induced [35S]GTPγS binding quantified by a corresponding increase of the EC50 values, without any change in the maximal effect
(see Table 4.4).
The same experiments were performed using AD-15b in presence of 300 nM of the antagonist FM-5b. As shown in Fig. 4.10 the [35S]GTPγS binding was upward shifted
.
Figure 4.10 Concentration-dependent curve for [35S]GTPγS binding of AD-15b alone (black
line) or after 30 min pre-incubation with 300 nM of FM-5b (red line) on CB2r. 2-AG and SR-2 were used as positive control for agonism and inverse agonism respectively.
The results suggest that the inverse agonist AD-15b and the antagonist FM-5b interact with the CB2r binding site in different ways. Indeed, FM-5b induced a significantly lower maximal effect given by AD-15b compared to vehicle, while not significantly changing the potency (Table 4.5), indicating a non-competitive antagonism between these two CB2r ligands. By definition, non-competitive antagonism can be associated to the irreversible binding of the antagonist to the receptor that does not allow to reach the maximum efficacy expressed by the ligand alone. Alternatively, non-competitive antagonism can be determined by different interactions with the binding site of the receptor by the two ligands.
-10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 160 AD-15b plus FM-5b AD-15b plus vehicle hCB2 receptors FM-5b 2-AG SR-2 Log [AD-15b] (M) [ 35S ]G T P b o u n d ( % o f co n tr o l)
~ 56 ~
In order to confirm the non-competitive mechanism, the [35S]GTPγS assay was performed in presence of 100 nM and 1 µM of the antagonist as showed above for
FM-6b.
Figure 4.11 Concentration-dependent curve for [35S]GTPγS of AD-15b alone (black curve)
and in competition with 100 nM, 300 nM and 1 µM of antagonist pre-incubated for half hour.
Table 4.5 EC50 values and maximal efficacy of [
35S]GTPγS binding upon CB2r activation
induced by FM-6b in presence of different concentrations of FM-5b or vehicle.
AD-15b EC50 values (mean and
95% CI, nM)
Maximal effect (mean and 95% CI)
Plus vehicle 19.4 (9.80 – 38.2) 50.1 (44.3 – 55.9) Plus FM-5b (100 nM) 102 (20.1 – 500) 63.7 (52.5 – 75.0) Plus FM-5b (300 nM) 62.4 (14.4 – 270) 71.4 (62.4 – 80.5) Plus FM-5b (1 µM) 103 (16.8 – 656) 75.1 (63.6 – 86.7)
As shown in Fig. 4.11, increasing concentrations of the antagonist induced an upward shift of the curve obtained in presence of AD-15b alone. The calculated EC50
values do not show a significant difference, while the maximal effect is statistically significantly reduced by the two higher concentrations of the antagonist compared to
-10 -9 -8 -7 -6 -5 0 50 100 150 200 AD-15b plus FM-5b 300 nM AD-15b plus vehicle AD-15b plus FM-5b 100 nM AD15-b plus FM-5b 1M hCB2 receptors Log [AD-15b] (M) [ 35 S ]G T P S b o u n d ( % o f v eh ic le )
~ 57 ~
inverse agonist alone (Table 4.6), thus confirming the non-competitive nature of interaction between the two ligands.
The data obtained by the different concentrations of antagonist in presence of the agonist or the inverse agonist were further analyzed and linearized to generate a Schild Plot. This analysis allows to investigate the type of antagonism behavior between two ligands. When the slope is equal to 1 indicates a competitive antagonism while a slope different from the unit suggests a non-competitive antagonism. The graph was constructed by plotting the logarithm of the concentrations of the antagonist FM-5b on x axis and the logarithm of [DR – 1] on y axis. The dose ratio (DR) was calculated by diving the EC50 values calculated from
the curves generated by the agonist (or inverse agonist) alone and in presence of the related fix concentration of the antagonist. As shown in Fig. 4.12, the slope of the line generated for FM-6b is 1.002 confirming the functional competitive antagonism with FM-5b. On the contrary, the line generated by the inverse agonist AD-15b is parallel to the x axis with a slope of 0, thus indicating a functional non-competitive behavior between these two ligands.
Figure 4.12 Schild Plot for the [35S]GTPγS binding calculated for the CB2r agonist FM-6b
and the inverse agonist AD-15b in presence of different concentrations of the pure antagonist
FM-5b.
Schild plot
-8 -7 -6 -5 0.0 0.5 1.0 1.5 2.0 2.5 FM-6b Slope = 1.002 0.09930 AD-15b Slope = 0.0 0.3326 Log (FM-5b) L o g ( DR -1 )~ 58 ~
Altogether these results suggest that these three compounds show different types of interaction with the cannabinoid binding site on CB2r. As shown by the radiometric binding assays, all three compounds can fully and potently displace [3H]CP55,940 from CB2r, while in the functional assay, only the agonist and the antagonist show competitive effects. Interestingly, the inverse agonist and the antagonist differentially modulate the activation state of CB2r.
4.3 Inhibition activity on MAGL, FAAH, ABHDs and hCOX-2 activity
The effect of pyridine derivatives (general structure A and B) and biphenylic derivatives (general structure C) on the activity of main enzymes involved in the degradation of endocannabinoids was investigated at the screening concentration of 10 µM (see Fig. 4.13).
.
4.3.1 Inhibition activity on MAGL
Monoacylglycerol Lipase (MAGL) is the most efficient enzyme involved in the hydrolysis of 2-AG and accounts for almost 80-85% of 2-AG hydrolysis in the brain (Blankman et al., Chem Biol, 2007) As shown in Fig. 4.13 a, b, none of the tested compounds induced a significant inhibition of 2-AG hydrolysis at 10 µM, with only a 20-25 % inhibition exerted by AC-64a, AC-66, AD-3 and MC-18a.
~ 59 ~
a
b
Figure 4.13 MAGL inhibition induced by biphenylic derivatives (a) and pyridine
derivatives (b) derivatives tested at 10 µM. JZL-184 (1 µM) and MAFP (10 µM) were used as positive controls for MAGL and full 2-AG hydrolysis inhibition, respectively (see section Materials and Methods).
Biphenylic derivatives (C) AC -22 AC 23a AC 23b AC 23c AC 40 AC 42a AC 42b AC 42c AC 17 AC 33a AC 33b AC 33c AC 35 AC 64a AC 66 AC 64b SS-0 5 SS-0 25 GR -28b GR -28c GR -29a JZL -184 MA FP 0 50 100 150 [ 3H] 2 -O G h y d ro ly si s (% o f v eh icl e)
Pyridine derivatives (A and B)
AD 6b AD 3 AD 15a AD 15b EB 5a EB 5b MC-18a MC-18b AD 6a FM -4a FM -4b FM -5a FM -5b FM -6a FM -6b JZL-18 4 MAF P 0 50 100 150 [ 3H] 2 -O G h y d ro ly si s (% o f v eh icl e)
~ 60 ~
4.3.2 Inhibition activity on ABHDs
Recently, α,β hydrolase-6 and -12 (ABHD-6 and -12) have been reported to significantly contribute to the hydrolysis of 2-AG in the brain and peripheral tissues (Blankman et al., Chem Biol, 2007). As shown in Fig. 4.14, none of the tested compounds significantly affected the activity of ABHDs at 10 µM, apart for compound AD-15b, AC-66, AC-22 and AC-64b that induced a 25% inhibition of 2-OG hydrolysis.
~ 61 ~
a
b
Figure 4.14 ABHDs inhibition induced by biphenylic derivatives (a) and pyridine
derivatives (b) tested at 10 µM. Orlistat (20 µM), WWL70 (10 µM) and the combination of two inhibitors were used as positive controls for ABHD-12, ABHD-6 and ABHD-6 plus ABHD-12 inhibition, respectively (see section Materials and Methods). . Biphenylic derivatives (C) AC -22 AC 23a AC 23b AC 23c AC 40 AC 42a AC 42b AC 42c AC 17 AC 33a AC 33b AC 33c AC 35 AC 64a AC 66 AC 64b SS-0 5 SS-0 25 GR -28b GR -28c GR -29a OR LIS TA T WW7 0 OR LIS TA T + WW70KT 172 0 50 100 150 [ 3H] 2 -O G h y d ro ly si s (% o f v eh icl e)
Pyridine derivatives (A and B)
AD -6b AD3 AD 15a AD 15b EB5a EB 5b MC -18a MC -18b AD6a FM -4a FM -4b FM -5a FM -5b FM -6a FM -6b OR LIS TA T WW7 0 OR LIS TA T + WW70KT 172 0 50 100 150 [ 3H] 2 -O G h y d ro ly si s (% o f v eh icl e)
~ 62 ~
4.3.3 Inhibition activity on FAAHFAAH is a member of the serine hydrolase family of enzymes and plays a crucial role in hydrolyzing AEA, being the most important degradating enzyme.
As reported in fig. 4.15, any of the tested compounds showed a significant enzyme inhibition at 10 µM.
a
b
Figure 4.15 FAAH inhibition induced by biphenylic derivatives (a) and pyridine derivatives
(b) tested at 10 µM. URB-597 (1 µM) was used as positive controls for FAAH inhibition (see section Materials and Methods).
Biphenylic derivatives (C) AC -22 AC 23a AC 23b AC 23c AC 40 AC 42a AC 42b AC 42c AC 17 AC 33a AC 33b AC 33c AC 35 AC 64a AC 66 AC 64b SS-0 5 SS-0 25 GR -28b GR -28c GR -29a UR B 597 0 50 100 150 [ 3 H ]A E A h y d ro ly si s (% o f v e h ic le )
Pyridine derivatives (A and B)
AD 6b AD 3 AD 15a AD 15b EB 5a EB 5b MC -18a MC -18b AD6a FM -4a FM -4b FM -5a FM -5b FM -6a FM -6b UR B 597 0 50 100 150 [ 3H ]A E A h y d ro ly si s (% o f v eh ic le )
~ 63 ~
4.3.4 Inhibition activity on hCOX-2The main degradation pathway of endocannabinoids is the hydrolysis, but due to the arachidonoyl chain, AEA and 2-AG can also undergo oxygenation catalyzed by cyclooxygenase-2 (COX-2), lypooxygenase-15 (LOX-15) and cytochrome (cyp450). In certain conditions, such as inflammation and highly COX-2 expressing tissues (i.e. kidney) the oxygenation pathway plays a relevant role in endocannabinoid degradation (Urquhart et al., Biochim Biophys Acta, 2014) Moreover, COX-2 mediated oxygenation has been shown to generate bioactive compounds, such as prostaglandin ethanolamides and prostaglandin glycerol esters deriving from AEA and 2-AG, respectively (Alhouayek and Muccioli, Trends Pharmacol Sci, 2014). Several COX-2 inhibitors have shown to selectively inhibit endocannabinoid oxygenation while other not. Here we tested the ability of pyridine derivatives and biphenylic derivatives to inhibit COX-2 oxygenation of the classic substrate arachidonic acid and alternative 2-AG. As reported in fig. 4.16, all compounds showed an IC50 value ˃ 10 µM, with only a 25% of inhibition induced by AD-15a,
~ 64 ~
a
b
Figure 4.16 COX-2 inhibition induced by biphenylic derivatives (a) and pyridine
derivatives (b) tested at 10 µM. DuP-697 (0.1 µM) was used as positive controls for non-selective COX-2 inhibition (see section Materials and Methods).
In conclusion, all the compounds belonging to the two chemical scaffolds showed a negligible modulation of the main degrading enzymes for endocannabinoids at the tested concentration of 10 µM (see Table 4.6 for the summary the results).
Biphenylic derivatives (C) AC -22 AC -23a AC -23b AC -23c AC -40 AC -42a AC -42b AC -42c AC -17 AC -33a AC -33b AC -33c AC -35 AC -64a AC -66 AC -64bSS-0 5 SS-0 25 GR -28b GR -28c GR -29a DU P-69 7 0 50 100 150 AA 2-AG C O X -2 a c ti v it y ( % o f c o n tr o l)
Pyridine derivatives (A and B)
AD 6b AD 3 AD -15a AD -15b EB -5a EB -5b MC -18a MC -18b AD -6a FM -4a FM -4b FM -5a FM -5b FM -6a FM -6b DU P-69 7 0 50 100 150 AA 2-AG C O X -2 a c ti v it y ( % o f c o n tr o l)
~ 65 ~
Table 4.6 Summary of enzymatic inhibition activity of biphenylic derivatives (a) and
pyridine derivatives (b) expressed as % of enzymatic activity at 10 µM.
a
Biphenylic derivatives
% of enzymatic activity at 10 µM
MAGL ABHDs FAAH hCOX-2
AC-22
80
75
100
100
AC-23a
100
100
100
100
AC-23b
100
100
100
100
AC-23c
100
100
100
100
AC-40
100
100
100
100
AC-42a
100
100
100
100
AC-42b
100
100
100
100
AC-42c
100
100
100
100
AC-17
100
80
90
100
AC-33a
100
100
100
100
AC-33b
100
100
100
100
AC-33c
100
100
90
100
AC-35
100
100
100
100
AC-64a
75
100
100
100
AC-66
75
75
100
100
AC-64b
85
75
100
100
SS-05
85
100
100
100
SS-025
100
100
100
100
GR-29a
100
100
100
100
GR-28b
100
100
100
100
GR-28c
100
100
100
100
~ 66 ~
b
Pyridine derivatives
% of enzymatic activity at 10 µM
MAGL ABHDs FAAH hCOX-2
AD-6b
100
100
90
100
AD-3
80
100
100
100
AD-15a
100
100
100
75
AD-15b
100
75
90
100
EB-5a
100
100
100
80
EB-5b
100
90
100
100
MC-18a
80
100
100
100
MC-18b
100
100
90
100
AD-6a
100
100
90
100
FM-4a
100
100
100
100
FM-4b
100
100
100
75
FM-5a
100
100
100
100
FM-5b
100
90
100
100
FM-6a
100
100
100
75
FM-6b
100
90
100
100
~ 67 ~
4.4 Uptake inhibitionThe EMT (Endocannabinoid membrane transporter) is a putative membrane transporter responsible for the bidirectional passive movement of AEA and 2-AG across plasma membrane (Chicca et al.,. 2012-a). The first screening of biphenylic derivatives and pyridine derivatives was performed at concentration of 10 µM.
a
b
Figure 4.17 Inhibition of [3H]AEA uptake into U937 cells induced by biphenylic derivatives (a) and pyridine derivatives (b) at 10 µM. UCM-707 (10 µM) was used as positive control. AC -22 AC 23a AC 23b AC 23c AC 40 AC 42a AC 42b AC 42c AC 17 AC 33a AC 33b AC 33c AC 35 AC 64a AC 66 AC 64b SS-0 5 SS-0 25 GR -28b GR -28c GR -29a UC M-70 7 0 50 100 150 [ 3 H ] A E A u p ta k e ( % o f co n tr o l)
Pyridine derivatives (A and B)
AD -6b AD3 AD 15a AD 15b EB5a EB5b MC-18a MC-18b AD 6a FM -4a FM -4b FM -5a FM -5b FM -6a FM -6b UC M-707 0 50 100 150 Hit compounds [ 3H ] A E A u p ta k e ( % o f c o n tr o l)
~ 68 ~
As shown in Fig. 4.17, all biphenylic derivatives showed a negligible inhibition of AEA uptake, apart from AC-17 which gave a 48% reduction of intracellular AEA compared to control (Fig. 4.17-a). On the contrary, most of the pyridine derivatives showed significant inhibition of AEA uptake (ranging from 30% to 70% at 10 µM).
AD-15b, MC-18b, FM-4a, FM-5b and FM-6b induced a reduction of intracellular
accumulation of AEA higher than 50% compared to controls, therefore were selected for further concentration-dependent effects.
a b c
d e
Figure 4.18 Concentration-dependent inhibition of [3H]AEA uptake into U937 cells induced by biphenylic derivatives (a) FM-6b, (b) FM-5b, (c) AD-15b, (d) MC-18b and (e) FM-4a. UCM-707 (10 µM) was used as positive control.
-10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 AEA UCM-707 Log [FM-5b] (M) A E A u p ta k e ( % o f c o n tr o l) -11-10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 AEA UCM-707 Log [FM-6b] (M) A E A u p ta k e ( % o f c o n tr o l) -11-10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 AEA UCM-707 Log [AD-15b] (M) A E A u p ta k e ( % o f c o n tr o l) -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 UCM-707 AEA Log [MC-18b] (M) A E A u p ta k e ( % o f c o n tr o l) -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 AEA UCM-707 Log [FM-4a] (M) A E A u p ta k e (% o f c o n tro l)
~ 69 ~
Table 4.7 EC50 value for [3
H]AEA uptake inhibition induced by FM-6b, FM-5b, AD-15b,
MC-18b and FM-4a in U937 cells.
Name EC50 value (mean and 95% CI, nM)
FM-6b 71.0 (10.8 – 466.3)
FM-5b 83.4 (23.8 – 292.1)
AD-15b 3.01 (0.6 – 10.5)
MC-18b 21.1 (72.1 – 618.8)
FM-4a n.d.
As reported in Table 4.7, the compounds showed EC50 values in the low nano-molar
range, with the exception of FM-4a. Compound AD-15b resulted the most potent inhibitor with an EC50 value of 3.0 nM. Interestingly, some of the most potent uptake
inhibitors showed also the best binding properties to cannabinoid receptors (see Table 4.2). These data suggest that the pyridine general structure (A and B) might provide potent and selective compounds that interact with the ECs unlike the biphenylic general structure C, which provides only few moderately potent CB2r ligands. Interestingly, among of the pyridine derivatives some showed similar receptor binding properties while significantly differing for the AEA uptake inhibition. For example MC-18a and MC-18b showed similar affinity for CB2r binding (Ki of 9.4 nM and 31.3 nM for MC-18a and MC-18b, respectively), while having clear differences in the potency of AEA uptake inhibition (EC50 > 10 µM and
21.1 nM for MC-18a and MC-18b, respectively). The more potent MC-18b bears a
p-fluorobenzyl on the oxygen in position 2, while the ineffective derivative MC-18a
has the p-fluorobenzyl group in position 1 of the pyridine ring (see general structures
A and B). Several other compounds showed an inverted tendency in terms of the
positioning of the R1 group. When it is present in position 1 of the pyridine ring the
inhibition of AEA uptake is more pronounced compared than when it is shifted to the oxygen in position 2 (i.e. FM-6b/FM-6a and FM-5b/FM-5a in Fig. 4.17-b and Table 4.7). In addition, the type of substituent in R1 affects the potency of AEA
~ 70 ~
uptake inhibition, in fact the p-fluorobenzyl group (FM-5b) displays a better inhibition profile than ethylmorpholino (FM-4b) (see Fig. 4.17-b). Altogether these data suggest that both the position and the type of the R1 group on the pyridine ring
(general structures A and B) might play a crucial role for the interaction with the EMT. In addition, data also suggest that the type of R3 group present in position 5 of
the pyridine ring does not play a role as important as R1 in the inhibition of AEA
uptake, indeed all the best compounds FM-6b, FM-5b, AD-15b, FM-4a bear different substituent in position 5, and particularly compound MC-18b has just a hydrogen in R3. These data suggest that the presence of substituent R3 is not crucial
for inhibition of AEA uptake. These preliminary data constitute an interesting and promising base for a complete structure-activity relationship investigation of the pyridine scaffold in terms of the interaction with several components of the ECs. AEA uptake and hydrolysis are two intrinsically related processes and therefore any FAAH inhibitor would affect the AEA uptake despite not (or partially) directly interacting with the EMT (Chicca et al., 2012-a). In particular, in the [3H]AEA uptake assay performed in U937 cells, a FAAH inhibitor induces an indirect inhibition of the [3H]AEA uptake which is indistinguishable from a pure EMT inhibitor (Chicca et al., 2012-a). In order to characterize the function of the hit compounds as EMT inhibitor more experiments were carried out in collaboration with Dr. Andrea Chicca and Dr. Simon Nicolussi at the University of Bern. A further evaluation of the FAAH activity was performed in U937 cell homogenate in order to allow a direct comparison with the AEA uptake assay (as reported in Nicolussi et al., 2014) in addition to the data herein presented on pig brain homogenate (see Fig.
4.15). Preliminary data confirmed the inactivity of several compounds, while others
showed a stronger FAAH inhibition in U937 cell homogenate than in pig brain homogenates (data not shown). When the experiments will be completed, a more detailed evaluation about the selectivity of the pyridine derivatives towards the EMT over FAAH will be possible. The three most potent AEA uptake inhibitors FM-5b,
FM-6b and AD-15b were also tested for [3H]2-AG uptake inhibition. Several reports suggest that AEA and 2-AG share the same mechanism of trafficking across the plasma membrane despite differing for the intracellular targets and degrading enzymes. As shown in Fig. 4.19 all three compounds inhibited [3H]2-AG uptake.
~ 71 ~
Interestingly, compound AD-15b showed a biphasic inhibition curve, with a significant inhibition of [3H]2-AG uptake a low nanomolar concentrations (0.1-10 nM), while having a weak loss of inhibitory potency at 10 nM- 1 µM and recovering the full effect at the highest concentration tested (10 µM). This effect might be due to a partial inhibition of 2-AG hydrolysis which in this type of assay would counteract the [3H]2-AG uptake as shown before (Chicca et al., 2012-a). Interestingly, AD-15b is the only compound among the three tested here that shows a significant inhibition of 2-AG hydrolysis (ABHDs). An intriguing hypothesis to explain this biphasic effect might be an early inhibition of the EMT which is then partially counteracted by the ABHDs inhibition at high nano-molar concentrations, while at higher micro-molar concentrations the EMT inhibition becomes again predominant. Further experiments are necessary to confirm or disprove this hypothesis, but it is noteworthy to mention that a similar biphasic effect was already shown for the natural product β-amyrin for [3H]2-AG uptake (Chicca et al., 2012-b).
a b c
Figure 4.19 Concentration-dependent inhibition of [3H]2-AG uptake into U937 cells induced by pyridine derivatives (a) FM-6b, (b) FM-5b and (c) AD-15b. UCM-707 (10 µM) was used as positive control.
-10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 2-AG UCM-707 Log [FM-5b] (M) 2 -A G u p ta k e ( % o f c o n tr o l) -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 2-AG UCM-707 Log [FM-6b] (M) 2 -A G u p ta k e ( % o f c o n tr o l) -11-10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 UCM-707 2-AG Log [AD-15b] (M) 2 -A G u p ta k e ( % o f c o n tr o l)
~ 72 ~
Table 4.8 EC50 value for [3
H]2-AG uptake inhibition induced by FM-6b, FM-5b and
AD-15b in U937 cells.
Name EC50 value (mean and 95% CI, nM)
FM-6b 91.35 (51.17 – 163.1)
FM-5b 538.7 (182.3 – 1592)
