Fabio Del Bello1, Alessandro Bonifazi1, Gianfabio Giorgioni1, Alessandro Piergentili1, Maria Giovanna Sabbieti2, Dimitrios Agas2, Marzia Dell'Aera3,4, Rosanna Matucci5, Marcin Górecki6,7, Gennaro Pescitelli6, Giulio Vistoli8, Wilma Quaglia1. 1. Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy. 2. Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, Via Gentile III da Varano, 62032 Camerino, Italy. 3. Istituto di Cristallografia IC-CNR, Via Amendola 122/o, 70126 Bari, Italy. 4. Dipartimento di Farmacia-Scienze del Farmaco, Università di Bari "A. Moro", Consorzio C.I.N.M.P.I.S., Via E. Orabona 4, I-70125 Bari, Italy. 5. Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Sezione di Farmacologia e Tossicologia, Università degli Studi di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy. 6. Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Moruzzi 13, 56124 Pisa, Italy. 7. Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52 Street, 01-224 Warsaw, Poland. 8. Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy.
Abstract
A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.
A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine33brevealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.
Muscarinic acetylcholine
receptors (mAChRs) are proteins with seven
transmembrane domains separated by intracellular and extracellular
loops. Acetylcholine binds to the extracellular region of mAChRs and
thereafter activates GTP-binding regulatory proteins in the intracellular
compartment. The mAChR family consists of five closely related members
(M1–M5). M1, M3, and M5 mAChRs are associated with Gq/11 proteins
to trigger phospholipase-C activation. Their activation increases
neuronal excitability through the opening of nonspecific cation channels,
mobilization of intracellularCa2+, or inhibition of small-conductance
Ca2+-activated K+ channels. M2 and
M4 subtypes couple to Gi/o proteins, inhibiting
adenylate cyclase and reducing the levels of intracellularadenosine
3′,5′-cyclic monophosphate (cAMP).[1] mAChRs mediate several functions in the central nervous
system (CNS), where they play a crucial role in cognitive functions[2] and pain circuits.[3] Moreover, in the periphery, M2 and/or M3 subtypes
are involved in smooth muscle contraction,[4] cardiovascular function,[5] and glandular
secretion.[6] Acetylcholine is not only a
neurotransmitter but can also act on non-neuronal cells, and the muscarinic
system is involved in the regulation of stem[7] and cancer cells,[8] in immunity and inflammation,[9] and in the mucocutaneous epithelial barrier.[10] Moreover, muscarinic signals have been demonstrated
to be transmitted by mesenchymal stem cells (MSCs) from different
tissues.[11,12]The 1,4-dioxanenucleus has been demonstrated
to be a versatile
scaffold for the development of compounds interacting with different
receptor systems,[13−20] including mAChRs.[21−25] We have demonstrated that the size of the substituent in the 6-position
affects the functional activity of 1,4-dioxane ligands directed to
mAChRs.[24] Indeed, a methyl group in this
position led to the effective agonist (2R,6S)-1,[23] whereas
aromatic rings characterized potent antagonists, such as the 6,6-diphenyl
derivative (S)-2[24] (Figure ). In in vivo studies in anesthetized rats, compared
to oxybutynin (Figure ), an antagonist clinically used for overactive bladder (OAB) treatment,[26] (S)-2 more efficaciously
reduced the volume-induced contractions of the urinary bladder.
Figure 1
Chemical structures
of (2R,6S)-1, (S)-2, oxybutynin,
and trospium.
Chemical structures
of (2R,6S)-1, n class="Gene">(S)-2, oxybutynin,
and trospium.
In the effort to obtain novel
potent mAChR antagonists preferentially
targeting peripheral M3 subtype and potentially useful
for the treatment of OAB, the diphenyl group in the 6-position of
compound 2 has been replaced by different lipophilic
groups (compounds 3–8, Figure ). Furthermore, the lipophilic moiety has
been moved from the 6- to 5-position (compounds 9–17, Figure ) or introduced
in both 5- and 6-positions of the 1,4-dioxane ring (compounds 18–19, Figure ). All the substituents are aromatic groups, except for the
aliphatic cyclohexyl ring in compounds 3 and 11, which has been selected because it is present in several potent
mAChR antagonists, including oxybutynin (Figure ).
Figure 2
Chemical structures of the new 1,4-dioxane derivatives 3–19. Only one enantiomer of the racemic mixture is
shown.
Chemical structures of the new 1,4-dioxane derivatives 3–19. Only one enantiomer of the racemic mixture is
shown.Owing to the lack of M3 subtype selectivity, the muscarinic
compounds used in therapy for OAB show cardiovascular side effects,
due to the interaction with peripheral M2 subtype, and/or
cognitive side effects, due to the blockade of central mAChRs.[27,28] Among these drugs, only trospium bears a hydrophilic quaternary
ammonium head (Figure ) that prevents the crossing of the blood–brain barrier (BBB),
thus minimizing CNS side effects.[29]Because of the high degree of amino acid sequence homology in the
orthosteric site of the M1–M5 mAChR subtypes,
it is very difficult to obtain orthosteric ligands selective for M3 mAChR over all the other subtypes. For this reason, the aim
of the present study was to confine the activity of the novel compounds
to peripheral tissues, minimizing CNS side effects and, hopefully,
to limit cardiovascular side effects by improving the M3/M2 selectivity ratio. Therefore, the new molecules have
been designed to have a quaternary ammonium function that should prevent
the crossing of the BBB.The novel compounds will provide further
information on the role
played by the lipophilic moiety in the interaction with the five mAChR
subtypes.Moreover, considering the pivotal role played by stereochemistry
in the interaction of both 1,4-dioxane agonists and antagonists with
the five mAChR subtypes,[22,24,30] the enantiomeric resolution of the most potent compound 3b was performed. The absolute configuration of the enantiomers of 3b was determined by quantum mechanical simulations of electronic
circular dichroism (ECD). To elucidate the binding mode of the described
compounds and to rationalize the biological results, docking simulations
on the M3 mAChR-resolved structure were performed.
Results
and Discussion
Chemistry
Compounds 3–8 were synthesized
following the procedure reported in Scheme and were obtained as racemates. 4-Benzylbenzaldehyde 20(31) was converted into the oxirane 22 by reaction with sodium hydride and trimethylsulfonium
iodide in dimethyl sulfoxide (DMSO), according to the procedure reported
by Corey and Chaykovsky.[32] The opening
of oxiranes 21,[33]22, and 23(34) with allyl alcohol
in the presence of Na gave alkenes 24–26, respectively.
The mixtures of diastereomers 28–30 were obtained
by the oxymercuration-reduction reaction with mercury(II) acetate
and subsequent treatment with an aqueous solution of potassium iodide
and iodine. The cis and trans isomers of 28 and 30 were separated by column chromatography, while attempts
to obtain the pure diastereomers of 29 failed. The iodo
derivatives 27a and 27b were synthesized
as previously reported.[16] The phenyl thioethers 30a and 30b were oxidized with meta-chloroperbenzoic acid (m-CPBA) to give the sulfoxides 31a and 31b after 30 min at room temperature
(r.t.) with one equivalent of m-CPBA or the sulfones 32a and 32b after 2 h at r.t. with 2 equivalents
of m-CPBA. Concerning the sulfoxide derivatives 31a and 31b, a further center of chirality was
introduced into the molecule. In both cases, only one of the two diastereomers
was obtained. The amination of the intermediate iodo derivatives 27–32 with dimethylamine afforded the corresponding
free amines 33–38, which were transformed into
the methiodides 3–8 by treatment with methyl iodide.
Only
one enantiomer of the racemic
mixture is shown.The relative configuration
between the substituents in 2- and 6-positions
of diastereomers 3a and 3b was determined
by X-ray diffraction analysis performed on 3b (Figure ), which confirmed
the structure of intermediates 27a and 27b previously assigned by 1HNMR studies.[16]
Figure 3
X-ray crystal structure of 3b. The X-ray coordinates
were deposited at Cambridge Crystallographic Data Centre (accession
number CCDC 1969353).
X-ray crystal structure of 3b. The X-ray coordinates
were deposited at Cambridge Crystallographic Data Centre (accession
number CCDC 1969353).The relative configuration
between the substituents in 2- and 6-positions
of the 1,4-dioxane ring of diastereoisomers 4a/b was
assigned based on the 1HNMR spectra of intermediates 28a/b (Figure A). Because of the steric bulk, one may suppose that both the substituents
in 2- and 6-positions of the cis isomers are equatorially oriented,
whereas in the trans isomers, only one of the two substituents adopts
the equatorial position and the other substituent is axially oriented.
In the 1HNMR spectrum of the iodo derivative 28b, precursor of the final methiodide4b, the protons
of the CH2I chain (3.55 ppm) are deshielded compared to
the same protons of diastereomer 28a (3.22 ppm), precursor
of methiodide 4a. This deshielding effect for CH2I protons of diastereomer 28b (see Supporting Information, Figure S4) suggests an
axial position for the side chain, as already evidenced in 1,4-dioxane
analogues bearing a CH2I chain[35] and, consequently, the relationship between the biphenyl substituent
and the chain is trans (Figure A).
Figure 4
Structure of (A) compounds 28a and 28b, precursors of 4a and 4b, respectively,
and of (B) compounds 52a and 52b, precursors
of 13a and 13b, respectively.
Structure of (A) compounds 28a and 2n class="Chemical">8b, precursors of 4a and 4b, respectively,
and of (B) compounds 52a and 52b, precursors
of 13a and 13b, respectively.
Similar considerations can be made for diastereomers 30a/b, precursors of the final methiodides6a/b. Indeed,
the signals for CH2I protons of 30a and 30b are positioned at 3.18 and 3.52 ppm, respectively, indicating
a trans configuration between the substituents in the 1,4-dioxane
nucleus of 30b.The novel compounds 9–17 were prepared following
the procedure depicted in Scheme and were obtained as racemates. The opening of oxiranes 39,[36]21,[33]22, and 23(34) with allyl alcohol in the presence of perchloric
acid yielded compounds 40, 42, 43, and 44, respectively. The olefine 41 was
prepared starting from 3,3-diphenylpropane-1,2-diol (45),[37] whose primary hydroxyl group was
selectively protected with tert-butyldimethylsilyl
chloride (TBDMSCl) to give compound 46, which was treated
with allyl bromide in the presence of NaH affording olefine 47. The cleavage of the silyl ether with tetrabutylammonium
fluoride (TBAF) yielded the corresponding primary alcohol 41. The intermediates 48 and 49 were obtained
as previously described in the literature.[13]
Only one enantiomer of the racemic
mixture is shown.The mixtures of diastereomers 50–54 were obtained
starting from olefins 40–44 in the same reaction
conditions used for the preparation of 28–30.
The diastereomers were separated by column chromatography. The thioethers 54a and 54b were oxidized to give sulfoxides 55a and 55b, respectively, and sulfones 56a and 56b as above described for 31 and 32. Similarly to what was observed for 31a and 31b, also for 55a and 55b, only one of the two diastereomers was obtained. The amination of 48–56 with dimethylamine afforded the corresponding
amines 57–65, which were transformed into the
methiodides 9–17 by treatment with methyl iodide
(Scheme ).The
cis and trans configurations between the substituents in 2-
and 5-positions of diastereoisomers 9a and 9b, respectively, were assigned based on the previously published structures
of compounds 48a and 48b.[13] The structures of diastereoisomers 11–15 were assigned by 1HNMR spectroscopy. Because of the
steric bulk, it may be supposed that in the trans isomers both the
substituents in 2- and 5-positions of the 1,4-dioxane nucleus are
in the equatorial position, whereas in the cis isomers, only one of
the two substituents is equatorially oriented and the other is axially
oriented. Analogous to what occurs for the diastereomers 48a and 48b,[13] in the 1HNMR spectrum of the iodo derivative 52a, precursor
of the final methiodide13a, the protons of the CH2I chain (3.57 ppm) are deshielded compared to the same protons
of diastereomer 52b (3.19 ppm), precursor of methiodide13b (see Supporting Information, Figure S5). This deshielded effect for CH2I protons
of diastereomer 52a suggests an axial position for the
side chain, as also evidenced in 1,4-dioxane analogues bearing a 2-CH2I chain[35] and, therefore, a cis
configuration between the chain and the biphenyl substituent (Figure B).Similar
considerations can be made for diastereomers 51a/b, 53a/b, and 54a/b, precursors of the final
methiodides12a/b, 14a/b, and 15a/b. Indeed, the CH2I protons of diastereomers 51a, 53a, and 54a are more deshielded (3.38,
3.58, and 3.52 ppm, respectively) compared to those of diastereomers 51b, 53b, and 54b (3.08, 3.17, and
3.16 ppm, respectively), demonstrating a trans configuration between
the substituents in the 1,4-dioxane nucleus for 51a, 53a, and 54a.The relative orientation
between the CH2I fragment and
the 5-substituents of 11a and 11b was assigned
by 1HNMR analysis (NOESY studies, see Supporting Information, Figure S7). In particular, evident
NOEs were observed between the axial proton in the 3-position and
the hydrogen atoms of the phenyl ring in the 5-position and between
the axial protons in 2- and 6-positions (4.21 and 3.88 ppm, respectively)
of 11a, indicating that the 5-phenyl nucleus and the
2-side chain are axially and equatorially oriented, respectively.
Therefore, the relative configuration between the 2-side chain and
the 5-phenyl substituent is cis in 11a and, consequently,
trans in 11b (Figure ).
Figure 5
Structure of compounds 11a and 11b. The
arrows indicate the observed NOEs upon irradiation.
Structure of compounds 11a and n class="Chemical">11b. The
arrows indicate the observed NOEs upon irradiation.
Compounds 18a–c were synthesized following
the procedure described in Scheme and were obtained as racemates. The alcohol intermediates 68a and 68b, synthesized as previously described,[38] and 68c, obtained by treatment
of olefine 66(39) with m-CPBA and subsequent reaction of oxirane 67 with trifluoroacetic acid, were reacted with p-toluenesulfonyl
chloride followed by treatment with dimethylamine to give 69a, 69b, and 69c, whose reaction with methyl
iodide afforded the final methiodides18a, 18b, and 18c, respectively.
Only one enantiomer of the racemic
mixture is shown.The stereochemical relationship
among the substituents of the diastereomers 18a and 18b was determined based on the previously
assigned structure of the alcohol intermediates 68a and 68b.[38] The stereochemical relationship
among the substituents in 2-, 5-, and 6-positions of 18c was assigned by 1HNMR analysis (NOESY studies, see Supporting Information, Figure S8). In the 1HNMR spectrum of 18c, the axial hydrogen atom
in the 3-position at δ 3.69 ppm showed two large coupling constants
(J = 11.2 Hz and J = 10.0 Hz), one
with the geminal equatorial hydrogen atom and the other with the axial
hydrogen atom in the 2-position. Hence, the chain in the 2-position
is equatorially orientated. Moreover, NOEs were observed between the
axial proton in the 3-position and the proton in the 5-position at
3.69 and 5.22 ppm, respectively, and between the axial proton in the
2-position at 4.24 ppm and the phenyl ring in the 6-position, indicating
that the 2-side chain is trans oriented with both phenyl substituents
(Figure ).
Figure 6
Structure of
compound 18c. The arrows indicate the
observed NOEs upon irradiation.
Structure of
compound18c. The n class="Chemical">arrows indicate the
observed NOEs upon irradiation.
In the effort to obtain the fourth diastereomer, in which the stereochemical
relationship among the three substituents is cis, the olefine 66(39) was treated with mercury(II)
acetate, followed by an aqueous solution of potassium iodide and iodine.
However, also in this case, only one diastereomer (70) was obtained. The amination with dimethylamine and subsequent reaction
with methyl iodide yielded the same diastereomer (18c) obtained following the previously described procedure.Compounds 19a and 19b were prepared following
the procedure described in Scheme and were obtained as racemates. Olefine 72, obtained by reaction of the α-allyloxy ketone 71(40) with phenylmagnesium chloride, was
treated with m-CPBA in CH2Cl2, affording oxirane 73, whose treatment with trifluoroacetic
acid in CHCl3 led to alcohols 74a and 74b, which were separated by flash chromatography. Treatment
of the alcohols with p-toluenesulfonyl chloride followed
by reaction with dimethylamine afforded the amines75a and 75b, whose treatment with methyl iodide gave 19a and 19b, respectively.
Only one enantiomer of the racemic
mixture is shown.The relative configuration
between the 2-substituent and the 5-phenyl
group of the diastereomers 19a and 19b was
assigned by 1HNMR spectroscopy. In the 1HNMR
spectrum of the tertiary amine75a, precursor of 19a, the axial hydrogen atom in the 3-position at 3.78 showed
two large coupling constants (J = 11.2 Hz and J = 10.4 Hz), one with the geminal equatorially located
proton and one with the axial proton in the 2-position. Hence, the
CH2N(CH3)2 fragment in the 2-position
assumes the equatorial position. Analogously, as shown by the 1HNMR spectrum of 75b, precursor of 19b, the CH2N(CH3)2 fragment in the
2-position is equatorial because the axial proton in the 3-position
at 3.58 showed two large coupling constants (J =
11.5 Hz and J = 10.3 Hz), one with the geminal equatorially
positioned hydrogen atom and one with the axially oriented hydrogen
atom in the 2-position. Moreover, the proton in the 5-position of 75b (5.82 ppm) is deshielded compared to the same proton of 75a (4.95 ppm) (see Supporting Information, Figure S6). The observation that in the 1HNMR spectra
of the cis and trans diastereomers
of 5-phenyl-1,4-dioxane-2-carboxylic acid and 6-phenyl-1,4-dioxane-2-carboxylic
acid, whose structure had previously been determined by NOE measurements,[13] the equatorially oriented protons are deshielded
compared to the axially oriented protons allows us to hypothesize
that the proton in the 5-position is axially oriented in 75a and equatorially oriented in 75b. Therefore, the relative
configuration between the 2- CH2N(CH3)2 chain and the 5-phenyl ring is trans in 19a and cis
in 19b (Figure ).
Figure 7
Chemical structures of 19a and 19b.
Chemical structures of 19a and n class="Chemical">19b.
The enantiomers (+)-3b and (−)-3b were separated by preparative HPLC performed on the intermediate
amine (±)-33b using a Regis Technologies Whelk-O
1 (R,R) H (25 cm × 2 cm) column
as the chiral stationary phase and n-hexane/2-propanol
85/15 v/v as the mobile phase at a flow rate of 18 mL/min. The enantiomeric
excess (e.e.), determined by analytical HPLC using a Regis Technologies
Whelk-O 1 (R,R) H (25 cm ×
0.46 cm) column as the chiral stationary phase and n-hexane/2-propanol 85/15 v/v as the mobile phase at a flow rate of
1 mL/min, proved to be >99.5% for both enantiomers.The absolute
configuration of 33b was determined by
quantum mechanical simulations of ECD. The ECD spectra of the two
enantiomers of the tertiary amine33b (Figure ), measured on the oxalate
salt dissolved in acetonitrile, contain the typical bands of a benzene
chromophore attached to a chiral moiety:[41] the 1Lb band between 240 and 280 nm, which
is electric-dipole forbidden, shows the characteristic vibrational
fine structure, and the 1La band between 210
and 225 nm, which is electric-dipole allowed, is more intense.[42,43]
Figure 8
ECD
spectra of the oxalate salts of (+)-33b and (−)-33b measured in acetonitrile. The two regions 190–300
and 235–400 nm were measured with 0.05 and 1 cm cells, respectively.
ECD
spectra of the oxalate salts of (+)-n class="Chemical">33b and (−)-33b measured in acetonitrile. The two regions 190–300
and 235–400 nm were measured with 0.05 and 1 cm cells, respectively.
Time-dependent density functional theory (TDDFT)
calculations have
been shown to be practical means to simulate the CD spectra of this
series of ligands, especially with reference to the 1La band.[43] In fact, the 1Lb band is more problematic because of some known issues
of TDDFT for aromatic hydrocarbons[44] and
because vibronic calculations are needed to reproduce the vibrational
pattern.[45] This fact practically limits
the comparison between experiment and calculation to a single band,
namely, 1La. To exclude possible pitfalls, the
present computational protocol based on DFT calculations[46,47] was first validated on the tertiary amines of (S)-2 and (R)-2, whose absolute
configuration is known.[24]Conformational
analysis and DFT geometry optimizations run on the
ammonium ion of (2R,6R)-33b led to only two populated conformers at r.t. (Figure ). They mainly differ in the rotation around
the cyclohexyl-C2 bond, while the rest of the structure is preserved.
The preferential orientation of the 2-side chain is dictated by an
intramolecularNH–O1hydrogen bond.
Figure 9
Two populated conformers
of the ammonium ion of (2R,6R)-33b.
Two populated conformers
of the ammonium ion of n class="Chemical">(2R,6R)-33b.
TDDFT calculations were run with several different DFT functionals
and basis sets (see ECD andNMR Calculations), either in vacuo or including a solvent model
for acetonitrile. All explored combinations predicted a negative rotational
strength for the 1La band of both conformers,
in a very consistent way. Thus, the prediction of the diagnostic ECD
band is very robust. In Figure , the experimental spectrum is compared with the spectrum
calculated at the CAM-B3LYP/def2-SVP/PCM level. As can be seen, the
relative energy of the 1Lb band is overestimated
by calculations and the vibrational pattern is missing. Still, the
correct negative rotational strength is reproduced for this band too.
The agreement between experimental and calculated ECD spectra is satisfactory.
Therefore, the absolute configuration is (2R,6R) for (+)-33b and (2S,6S) for (−)-33b.
Binding Studies
The pharmacological profile of methiodides 3–19 was assessed by radioligand binding assays with
human recombinant hM1–hM5 receptor subtypes
stably expressed in Chinese hamsterovary (CHO) cell lines using [3H]N-methylscopolamine ([3H]NMS)
as a radioligand to label mAChRs, following previously described protocols.[48,49] The affinities, expressed as pKi, are
shown in Table along
with those of compound 2, oxybutynin and trospium, which
are included for useful comparison.
Table 1
Equilibrium Binding
Affinity of 2–19, Oxybutynin, and Trospium
pKia
compd
hM1
hM2
hM3
hM4
hM5
2
9.10b
8.24b
8.44b
8.58b
8.36b
3a
8.03 ± 0.09
7.36 ± 0.12
7.84 ± 0.13
7.51 ± 0.08
7.26 ± 0.05
3b
9.28 ± 0.19
7.91 ± 0.13
9.07 ± 0.03
9.03 ± 0.16
8.41 ± 0.1
4a
<5
5.06 ± 0.02
<5
<5
<5
4b
<5
5.27 ± 0.12
<5
<5
<5
5a/b
<5
5.31 ± 0.05
<5
<5
5.13 ± 0.10
6a
5.50 ± 0.05
5.47 ± 0.07
<5
5.43 ± 0.05
5.58 ± 0.13
6b
5.51 ± 0.01
6.71 ± 0.01
5.77 ± 0.05
5.70 ± 0.02
5.95 ± 0.13
7a
<5
5.52 ± 0.02
<5
<5
5.21 ± 0.08
7b
5.17 ± 0.08
6.38 ± 0.10
5.88 ± 0.14
5.42 ± 0.02
5.74 ± 0.16
8a
5.08 ± 0.09
5.13 ± 0.01
<5
<5
5.21 ± 0.14
8b
5.29 ± 0.07
6.70 ± 0.01
5.63 ± 0.14
5.20 ± 0.07
5.80 ± 0.06
9a
5.63 ± 0.07
5.06 ± 0.10
5.14 ± 0.10
5.10 ± 0.07
<5
9b
<5
<5
<5
<5
<5
10
7.44 ± 0.12
6.75 ± 0.09
6.87 ± 0.04
6.81 ± 0.06
6.80 ± 0.06
11a
8.01 ± 0.06
7.26 ± 0.08
7.43 ± 0.09
7.15 ± 0.09
7.45 ± 0.08
11b
7.81 ± 0.04
7.22 ± 0.11
7.36 ± 0.10
7.23 ± 0.10
7.32 ± 0.09
12a
6.28 ± 0.08
5.87 ± 0.09
5.77 ± 0.03
5.60 ± 0.06
5.49 ± 0.06
12b
6.08 ± 0.07
6.06 ± 0.08
5.62 ± 0.11
5.52 ± 0.06
5.20 ± 0.01
13a
<5
<5
<5
<5
<5
13b
<5
5.49 ± 0.02
<5
<5
<5
14a
<5
5.65 ± 0.05
5.65 ± 0.12
<5
5.53 ± 0.13
14b
<5
<5
<5
<5
<5
15a
5.43 ± 0.09
6.70 ± 0.13
6.01 ± 0.11
5.70 ± 0.01
6.00 ± 0.14
15b
5.57 ± 0.07
5.86 ± 0.04
5.35 ± 0.06
5.14 ± 0.01
5.40 ± 0.15
16a
<5
5.52 ± 0.02
<5
<5
5.21 ± 0.08
16b
5.17 ± 0.09
6.38 ± 0.10
5.88 ± 0.14
5.42 ± 0.02
5.72 ± 0.15
17a
<5
6.39 ± 0.04
5.14 ± 0.10
<5
5.05 ± 0.08
17b
<5
<5
<5
<5
<5
18a
5.85 ± 0.09
5.72 ± 0.09
5.33 ± 0.12
5.25 ± 0.08
5.34 ± 0.07
18b
6.30 ± 0.09
5.65 ± 0.09
5.81 ± 0.10
5.40 ± 0.09
5.52 ± 0.08
18c
7.19 ± 0.07
6.62 ± 0.08
6.45 ± 0.01
6.40 ± 0.06
6.21 ± 0.01
19a
6.17 ± 0.16
5.36 ± 0.08
5.70 ± 0.11
5.41 ± 0.01
5.40 ± 0.19
19b
6.26 ± 0.19
5.67 ± 0.04
5.96 ± 0.12
5.93 ± 0.03
5.84 ± 0.23
oxybutyninb
8.62
7.93
8.82
8.44
7.85
trospiumc
8.46
8.94
8.99
8.84
8.22
Inhibition binding constants (pK) for hM1–hM5 mAChRs expressed in CHO-K1 cell membranes. The values represent
the arithmetic mean ± S.E.M. of at least three independent experiments,
each one performed in duplicate.
Taken from ref (24).
Taken from ref (50).
Inhibition binding constants (pK) for hM1–hM5 mAChRs expressed in CHO-K1 cell membranes. The values represent
the arithmetic mean ± S.E.M. of at least three independent experiments,
each one performed in duplicate.Taken from ref (24).Taken from ref (50).The analysis of data reveals that among all the modifications,
the replacement of one of the two phenyl rings of 2 with
a cyclohexyl group, affording 3, proved to be the most
favorable for the interaction with mAChRs. In particular, the diastereomer 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the
cyclohexyl fragment in the 6-position of the 1,4-dioxane ring, shows
pKi values for all mAChR subtypes, except
for M2, higher than those of the 6,6-diphenyl derivative 2. Compound 3b displays a selectivity profile
analogous to that of the clinically approved drug oxybutynin, with
affinities for M1, M3, and M4 higher
than those for M2 and M5 subtypes. Interestingly,
the M3/M2 selectivity ratio of 3b (14.5) is significantly higher than those of the lead 2 and trospium (1.6 and 1.1, respectively). The M3/M2 selectivity profile of 3b is noteworthy because
the presence of a quaternary ammonium head, enhancing the charge transfer
interactions that it elicits with the surrounding aromatic residues,
generally increases the pKi values for
all muscarinic subtypes at the expense of the selectivity ratios.
Indeed, these aromatic side chains, and in particular four tyrosine
residues, represent a structural signature which is completely conserved
by all mAChR subtypes.The trans configuration between the substituents
in 2- and 6-positions
of the diastereomer 3a is detrimental for the binding
affinity for all the mAChR subtypes, confirming that stereochemistry
plays a crucial role in the interaction of 1,4-dioxane derivatives
with the mAChRs.[22,24,30]The replacement of the 6,6-diphenyl group of 2 with
a para-biphenyl group, affording the diastereomers 4a and 4b, induces a dramatic decrease in affinity
for all the mAChR subtypes. The higher flexibility of the terminal
phenyl group of 4 obtained by introducing a methylene
button (mixture 5a/b) or a sulfur atom (diastereomers 6a and 6b) between the two phenyl groups does
not improve mAChR affinity. Similar results are obtained by oxidizing
the sulfur atom of 6 to sulfoxide and sulfone, affording
compounds 7 and 8, respectively. In the
pairs of diastereomers 6a/6b, 7a/7b, and 8a/8b, the trans isomers show pKi values slightly higher than those of the corresponding cis isomers.The shift of the diphenyl group from the 6- to 5-position of the
1,4-dioxane ring of 2, affording compound 10, is also detrimental for the binding to the five mAChR subtypes.
The removal of one aromatic group of 10, obtaining the
diastereomers 9a and 9b, further decreases
the mAChR affinity. Similar to what was observed for the 6-substituted
ligands, the replacement of an aromatic group of 10 with
a cyclohexyl ring is favorable for the binding to the five mAChRs.
In this case, stereochemistry seems not to play a role in the binding
at mAChRs, both diastereomers 11a and 11b showing similar pKi values, with a preference
for the M1 subtype. The increased distance between the
diphenyl lipophilic moiety and the ammonium head of 10, yielding the diastereomers 12a and 12b, decreases the pKi values for all the
mAChRs. Analogous to what was observed for the corresponding 6-substituted
derivatives, all the other modifications performed on the 6,6-diphenyl
group of 10 (i.e., its replacement with
C6H4-4-C6H5, C6H4-4-CH2-C6H5, C6H4-4-S-C6H5, C6H4-4-SO-C6H5, and C6H4-4-SO2-C6H5), affording 13–17, are detrimental for the affinity for mAChRs.
Though with low affinity, the diphenylsulfone17a shows
selectivity for M2 over the other subtypes. This selectivity
profile agrees with what was reported for other muscarinic derivatives
bearing the diphenylsulfone moiety.[51]Compared to the 5-mono-phenyl derivatives 9a and 9b and the previously described 6-mono-phenyl derivatives,[24] the presence of a phenyl substituent in both
5- and 6-positions of the 1,4-dioxane ring (18a, 18b and 18c) seems to be advantageous, especially
when the two phenyl groups are in a cis stereochemical relationship
(18c). Instead, the insertion of a phenyl substituent
in the 5-position of the 6,6-diphenyl derivative 2, affording 19a and 19b, markedly reduces the binding affinities.The well-established influence of chirality on the biological activity
of mAChR ligands[22,24,30] prompted us to prepare and study the enantiomers of the most interesting
ligand 3b. Moreover, considering that the basic function
of mAChR antagonists can also be a tertiary amine,[24] the racemic 33b and its enantiomers were included
in this study.The pKi values of
(±)-3b, (±)-n class="Chemical">33b and their enantiomers
(2R,6R)-(+)-3b and
(2S,6S)-(−)-3b, (2R,6R)-(+)-33b e
(2S,6S)-(−)-33bare reported in Table together with those
of the lead compound (±)-2 and its enantiomers (R)-(+)-2 and (S)-(−)-2.
Table 2
Equilibrium Binding Affinity of (±)-2, (±)-3b, (±)-33b, and
Their Enantiomers
pKia
Compd
hM1
hM2
hM3
hM4
hM5
(±)-2
9.10b
8.24b
8.44b
8.58b
8.36b
(R)-(+)-2
7.79b
7.48b
7.21b
6.82b
6.97b
(S)-(−)-2
9.30b
8.55b
8.83b
8.83b
8.77b
ER
32
12
42
102
63
(±)-3b
9.28 ± 0.22
7.91 ± 0.13
9.07 ± 0.03
9.03 ± 0.16
8.41 ± 0.36
(2R,6R)-(+)-3b
8.30 ± 0.25
7.86 ± 0.14
7.51 ± 0.12
7.45 ± 0.11
7.68 ± 0.35
(2S,6S)-(−)-3b
9.52 ± 0.19
8.22 ± 0.10
9.05 ± 0.10
9.17 ± 0.19
9.18 ± 0.18
ER
17
2
35
52
24
(±)-33b
8.86 ± 0.16
7.88 ± 0.08
8.72 ± 0.10
8.62 ± 0.12
8.62 ± 0.17
(2R,6R)-(+)-33b
7.68 ± 0.16
7.17 ± 0.11
6.73 ± 0.06
7.01 ± 0.08
7.16 ± 0.27
(2S,6S)-(−)-33b
9.10 ± 0.28
8.10 ± 0.10
9.02 ± 0.10
9.09 ± 0.15
8.83 ± 0.10
ER
26
9
195
120
47
See footnote
a in the legend of Table .
Taken from ref (24).
See footnote
a in the legend of Table .Taken from ref (24).As expected, the data reveal how the racemic tertiary
amine (±)-33b shows high affinity for all mAChRs,
though with pKi values slightly lower
than those of the corresponding
ammonium salt (±)-3b. Moreover, it maintains the
interesting selectivity for M3 over M2 subtype
(M3/M2 = 7.0) already observed with methiodide
(±)-3b (M3/M2 = 14.5). Between
the enantiomers of the tertiary amine [(2R,6R)-(+)-33b and (2S,6S)-(−)-33b] as well as those of the
quaternary ammonium salt [(2R,6R)-(+)-3b and (2S,6S)-(−)-3b], the eutomers are the ones in which
the absolute configuration of the carbon atom in position 2 is S [(2S,6S)-(−)-33b and (2S,6S)-(−)-3b, respectively]. Such a configuration is the same of the
eutomer (S)-(−)-2, suggesting
that these derivatives bind to the same mAChR sites. The eudismic
ratios (ERs) between the enantiomers of the tertiary amineare significantly
higher than those between the corresponding enantiomers of the methiodide
for all mAChR subtypes, especially for M3, for which the
eutomer (2S,6S)-(−)-33b shows a pKi value 195-fold
higher than that of the distomer (2R,6R)-(+)-33b.
Docking Studies
To investigate the
factors influencing
the observed enantioselectivity of the three pairs of enantiomers
(R)-2/(S)-2, (2R,6R)-3b/(2S,6S)-3b, and (2R,6R)-33b/(2S,6S)-33b, docking simulations were carried out
on the human M3 mAChR structure in complex with a selective
antagonist (PDB Id: 5ZHP).[52]Figure A, showing the putative complex as computed
for compound (2S,6S)-3b, endowed with the highest affinity, reveals the following set of
interactions: (a) the charged ammonium head is engaged by a set of
contacts comprising the key ion-pair with Asp1473.32 plus
several charge transfer interactions with surrounding aromatic side
chains (e.g., Tyr1483.33, Trp5036.48, Tyr5066.51, Tyr5297.39, and Tyr5337.43); (b) the O4 dioxane atom is involved in a key H-bond with Asn5076.52, while the O1 atom is shielded by the close ammonium head
and cannot elicit significant interactions; (c) the phenyl ring can
stabilize π–π stacking interactions with a set
of surrounding aromatic residues such as Tyr1483.33, Trp1994.57 and Trp5036.48; (d) the cyclohexyl ring is
accommodated within a subpocket in which it can contact alkyl side
chains such as Leu225ECL2, Ala2355.43, and Ala2385.46. On these grounds, one may argue that the observed enantioselectivity
can be ascribed to four moieties, the arrangement of which is influenced
by the chiral centers: (a) the O4 dioxane atom, a feature which involves
all three pairs of enantiomers; (b) the cyclohexyl and (c) the phenyl
rings which concern only the compounds 3b and 33b; (d) the ammonium head which seems to play a marginal role for 2 and 3 reasonably due to the symmetry of the
trimethyl ammonium group, while the need to properly arrange the proton
toward Asp1473.32, and the N-methyl groups
toward the aromatic residues, may impact on the enantioselectivity
of 33b.
Figure 10
Main interactions stabilizing the putative complex for
(2S,6S)-3b with the
M3 mAChR structure (PDB Id: 5ZHP) (A). Comparison between the complexes
for the two
enantiomers of 2 (B), 3b (C), and 33b (D). In all comparisons, the eutomer is in lime and the
distomer in azure.
Main interactions stabilizing the putative complex for
(2S,6S)-3b with the
M3 mAChR structure (PDB Id: 5ZHP) (A). Comparison between the complexes
for the two
enantiomers of 2 (B), 3b (C), and 33b (D). In all comparisons, the eutomer is in lime and the
distomer in azure.Hence, inspection of Figure B, comparing the
computed poses for the two enantiomers
of compound 2, reveals that they suitably and similarly
arrange the phenyl rings and the ammonium head, while the pose of
the dioxane ring differs in the two complexes. In detail, Figure B shows that the
eutomer (S)-2 is able to establish a
strong H-bond with Asn5076.52, while the distomer (R)-2 less suitably arranges the O4 atom (as
defined by both N–H···O distance, 2.1 Å vs 2.7 Å, and corresponding angle, 171.5 vs 103.6, for (S)-2 and (R)-2, respectively) which, therefore, weakly contacts
Asn5076.52.Similarly, Figure C, comparing the best obtained complexes
for the two enantiomers
of compound 3b, shows that both of them are able to conveniently
accommodate the dioxane ring (e.g., the N–H···O4
distance with Asn5076.52 is equal to 2.3 Å in both
complexes) and the ammonium head but unavoidably differ for the arrangements
of the two rings in the 6-position. Indeed, while the eutomer (2S,6S)-3b properly accommodates
the phenyl and the cyclohexyl rings as described above, the distomer
(2R,6R)-3b is constrained
to approach the phenyl ring toward the alkyl side chains with the
cyclohexyl ring completely surrounded by aromatic residues. Notably,
the capacity of both enantiomers of 3b to stabilize similar
H-bonds with Asn5076.52 suggests that the greater (despite
always restricted) flexibility of the cyclohexyl ring with respect
to the phenyl one allows the distomer (2R,6R)-3b to minimize the configurational effects
on the pose of the dioxane ring.Finally, Figure D, comparing the best poses
as computed for the two enantiomers of 33b, highlights
that they differ for the arrangement of both
the O4 dioxane atom and the cyclohexyl/phenyl rings. In detail, while
the eutomer (2S,6S)-33b can elicit the key H-bond with Asn5076.52 (N–H···O4
distance with Asn5076.52 is equal to 2.6 Å) and to
insert the cyclohexyl and phenyl rings within the suitable subpockets,
the distomer (2R,6R)-33b cannot contact Asn5076.52 (N–H···O4
distance with Asn5076.52 is equal to 3.8 Å) and accommodates
the two rings in the 6-position within the wrong subpockets. Notably,
the unique difference between 3b and 33b involves the ammonium head which is a quaternary salt only in the
former. Figure D
indicates that both enantiomers of 33bare able to properly
arrange the ammonium head even though the lack of the symmetric trimethyl
group in 33b increases the relevance of the C2 configuration
and can explain why the enantiomers of 33bare constrained
to differ for the arrangement of the O4 dioxane atom, while both enantiomers
of 3b are able to properly accommodate the dioxane ring
by minimizing the effects of the C2 configuration.These observations
find encouraging confirmations in the reported
ERs, thus allowing for some meaningful considerations. First, the
observed differences in the dioxanearrangement exert a conceivably
greater impact on affinity compared to those in the cyclohexyl/phenyl
rings as seen when comparing the ER values of 2 and 3b. Again, the combination of both factors (dioxane and cyclohexyl/phenyl
rings) reveals a synergistic effect by showing an ER value for 33b markedly higher than the previous ones. Such a synergistic
effect can be explained at an atomic level by considering that, while
both enantiomers of 2 are able to stabilize the H-bond
with Asn5076.52 even though the distomer elicits weaker
interactions (as seen in the reported geometrical parameters), the 33b distomer is substantially unable to approach Asn5076.52, thus missing this key interaction. Finally, similar trends
can also be seen when analyzing the corresponding affinity values
and, in particular, the affinities of the distomers. Indeed, while
the eutomers show comparable affinity values with 3b and 33b which reveal slightly higher values probably due to the
favorable hydrophobic interaction stabilized by the cyclohexyl ring,
the distomers show greater differences in affinity which are ascribable
to their reduced interactions. Hence, (2R,6R)-3b which only fails in properly arranging
the rings in the 6-position reveals the greatest affinity, followed
by (R)-2 which elicits a weak H-bond
with Asn5076.52. The lowest affinity is shown by (2R,6R)-33b, which does not
stabilize the mentioned H-bond and unsuitably arranges the rings in
the 6-position.For completeness and even though the affinity
values of the single
enantiomers were not measured, docking simulations also involved other
proposed derivatives by focusing attention on those with pKi values on M3 mAChR greater than
6. While avoiding systematic analyses, the docking results allow for
some general considerations. The lower affinity values of the ligands
bearing cyclohexyl/phenyl rings in 5 (instead of 6, e.g., 10, 11a, and 11b) can be
ascribed to the steric hindrance exerted by these rings on the O4
dioxane atom which weakens the key H-bond with Asn5076.52. In contrast, the reduced steric hindrance exerted on the O1dioxane
atom allows this to be engaged in additional H-bonds as seen for (2S,6S)-11a with Tyr1483.33. The low affinity of ligands bearing a 4-(phenylthio)phenyl
moiety (15a and 15b) and similar diphenyl
groups is explainable by considering that these bulky substituents
constrain the ligands to assume inconvenient poses, where even the
ammonium head assumes suboptimal arrangements, without adding any
additional contacts. Finally, the lower affinity values of the ligands
with substituents in both 5- and 6-positions (e.g., 18c) is ascribable to the same factors affecting the
binding of compounds substituted only in 5, namely, the greater steric
hindrance on the O4 dioxane atom which weakens the H-bond with Asn5076.52.
Functional Studies on MSCs from Mouse Bone
Marrow
It
is well established that bone marrow MSC behavior is influenced by
a variety of signaling systems. In context, cholinergic intramural
stimuli and, in particular, muscarinic signals orchestrate MSCs viability
and commitment.[11,12] Considering also the pluripotent
MSC nature and their contribution to bone, blood, and systemic homeostasis,[53] viability studies on MSCs were performed to
determine the functional profile of 3b, the most interesting
compound in this series. Namely, the effect of this compound was similar
to that of the well-known mAChR antagonist atropine because it was
able to down-regulate MSCs viability when used at high concentration
(10–4 M), while increased cell viability when used
at low concentration (10–10 M) (Figure A). Successively, the efficacy
of 3b in contrasting cell viability induced by the well-known
mAChR agonist carbachol was evaluated. The data reported in Figure B indicate that,
analogous to atropine, the new compound 3b is able to
contrast the increase of carbachol-induced MSC viability, confirming
its mAChR antagonist profile. Further research on the intracellular
mechanistic outcomes of 3b on MSCs remains mandatory.
Figure 11
(A)
Dose–response effect of 3b and atropine
on the metabolic activity of viable MSCs. The graphic represents the
mean ± SEM of four independent experiments; *p < 0.05 vs controls (Untreated MSCs and DMSO).
(B) Effects of carbachol (10–10 M) on the metabolic
activity of MSCs in the absence or in the presence of different doses
of 3b or atropine. The graphic represents the mean ±
SEM of four independent experiments; *p < 0.05 vs controls; #p < 0.05 vs carbachol.
(A)
Dose–response effect of 3b andatropine
on the metabolic activity of viable MSCs. The graphic represents the
mean ± SEM of four independent experiments; *p < 0.05 vs controls (Untreated MSCs and DMSO).
(B) Effects of carbachol (10–10 M) on the metabolic
activity of MSCs in the absence or in the presence of different doses
of 3b or atropine. The graphic represents the mean ±
SEM of four independent experiments; *p < 0.05 vs controls; #p < 0.05 vs carbachol.
Conclusions
In
the present study, the 6,6-diphenyl structural element of the
potent mAChR antagonist 2 was replaced by lipophilic
substituents in 5- and/or 6-position of the 1,4-dioxane nucleus. Among
the novel compounds, the 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl
ring in the 6-position, showed pKi values
for all mAChR subtypes, except for M2, higher than those
of 2. Moreover, its selectivity profile is similar to
that of the therapeutically used drug oxybutynin, with pKi values for M1, M3, and M4 subtypes higher than those for M2 and M5 subtypes.
The study of the enantiomers of 3b and those of the corresponding
tertiary amine33b, whose absolute configuration was
determined by quantum mechanical simulations of ECD, provided useful
information about the role played by chirality in the interaction
with mAChRs. In particular, the absolute configuration of the carbon
atom in the 2-position of the eutomers (2S,6S)-(−)-3b and (2S,6S)-(−)-33b is the same as (S)-(−)-2, suggesting that these derivatives bind
to the same mAChR sites. The ERs between the enantiomers of the tertiary
amine33b proved to be higher than those between the
corresponding enantiomers of methiodide 3b for all mAChR
subtypes, especially for M3. Docking studies on the M3 mAChR-resolved structure allowed us to shed light on the
binding mode of the proposed compounds. In particular, while the enantiomers
of 33b differ for the arrangement of O4 dioxane atom,
both enantiomers of 3b are able to properly accommodate
the dioxane ring by minimizing the effect of the C2 configuration.
Finally, the assays on MSCs from mouse bone marrow showed for 3b a functional profile similar to that of the mAChR antagonist
atropine concerning both the dose–response effect produced
on the metabolic activity of viable MSCs and the effect in contrasting
the increase of carbachol-induced MSC viability.Compared to
the tertiary amine drugs clinically used for the treatment
of OAB, 3b presents a quaternary ammonium function that
should prevent the crossing of BBB, minimizing central anticholinergic
activity and, therefore, limiting CNS side effects. The prediction
by SwissADME that 3b is a potential P-gp substrate makes
the profile of such a compound more and more interesting.[54] Not to mention that the transformation into
a quaternary amine markedly enhances the metabolic stability of this
compound. Indeed, the metabolic prediction based on the similarity
analysis using the MetaQSAR database on the tertiary amine indicates
the oxidation in alpha to the N atom as a truly probable metabolic
reaction which is largely inhibited by the presence of a permanent
positive charge.[55] Moreover, the M3/M2 selectivity ratio of 3b (14.5),
which is significantly higher than those of the quaternary ammonium
compounds 2 and trospium (1.6 and 1.1, respectively),
might limit cardiovascular side effects. Therefore, the methiodide 3b might represent a valuable lead compound for the design
of novel antagonists potentially useful in peripheral diseases in
which M3 receptors are involved.
Experimental
Section
General
Melting points (mp) were
taken in glass capillary
tubes on a Büchi SMP-20 apparatus and are uncorrected. 1HNMR and 13CNMR spectra were recorded on Varian
GEM200, Varian Mercury AS400, or Bruker 500 MHz instruments, and chemical
shifts (ppm) are reported relative to tetramethylsilane. Spin multiplicities
are given as s (singlet), d (doublet), dd (double doublet), t (triplet),
or m (multiplet). IR spectra were recorded on a PerkinElmer 297 instrument,
and spectral data (not shown because of the lack of unusual features)
were obtained for all compounds reported and are consistent with the
assigned structures. The microanalyses were recorded on a FLASH 2000
instrument (Thermo Fisher Scientific). The elemental composition of
the compounds agreed to within ±0.4% of the calculated value.
Optical activity was measured at 20 °C with a PerkinElmer 241
polarimeter. Analytical chiral HPLC was performed on a Shimadzu chromatography
system using a Regis Technologies (R,R)-Whelk-O 1 (25 cm × 0.46 cm) column. Preparative chiral HPLC
was performed on a Shimadzu chromatography system using a Regis Technologies
(R,R)-Whelk-O 1 (25 cm × 2
cm). Mass spectra were obtained using a Hewlett Packard 1100 MSD instrument
utilizing electron-spray ionization (ESI). The compounds were detected,
and a purity of >95% was confirmed by UV absorption at 220 nm.
All
reactions were monitored by thin-layer chromatography using silica
gel plates (60 F254; Merck), visualizing with ultraviolet light. Chromatographic
separations were performed on silica gel columns (Kieselgel 40, 0.040–0.063
mm, Merck) by flash chromatography. Compounds were named following
IUPAC rules as applied by ChemBioDraw Ultra (version 11.0) software
for systematically naming organic chemicals. The purity of the novel
compounds was determined by combustion analysis and was ≥95%.
This compound was prepared starting from (2S,6S)-(−)-33b following
the procedure described for 3a: a white solid was obtained,
which was recrystallized from 2-PrOH (88% yield). [α]D20 = −42.5
(c 1, CH3OH); mp and 1HNMR spectrum were identical
to those of racemic compound (±)-3b. Anal. Calcd
(C20H32INO2) C, H, N. C, 53.94; H,
7.24; N, 3.14. Found: C, 54.06; H, 7.41; N, 3.29.
This compound was prepared stn class="Chemical">arting from (2R,6R)-(+)-33b following the procedure
described for 3a: a white solid was obtained, which was
recrystallized from 2-PrOH (85% yield). [α]D20 = +42.9 (c 1, CH3OH); mp and 1HNMR spectrum were identical to those of
racemic compound (±)-3b. Anal. Calcd (C20H32INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 36b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (89% yield); mp 197–198 °C. 1HNMR (DMSO):
δ 3.02–3.98 (m, 14, N(CH3)3, CH2N, dioxane), 4.22 (dd, J = 9.6, 2.7 Hz, 1H,
dioxane), 4.48 (m, 1H, dioxane), 5.11 (dd, J = 13.4,
10.0 Hz, 1H, dioxane), 7.21–7.42 (m, 9H, ArH). ESI/MS m/z: 344.2 [M]+. Anal. Calcd
(C20H26INO2S) C, H, N, S.
This compound was prepared stn class="Chemical">arting
from 37a following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (82% yield); mp 166–167 °C. 1HNMR (DMSO):
δ 3.03–3.59 (m, 13H, CH2N, N(CH3)3 and dioxane), 3.78 (m, 1H, dioxane), 3.93 (dd, J = 10.2, 3.3 Hz, 1H, dioxane), 4.45 (m, 1H, dioxane), 4.88
(dd, J = 10.2, 2.5 Hz, 1H, dioxane), 7.42–7.81
(m, 9H, ArH). ESI/MS m/z: 360.2
[M]+. Anal. Calcd (C20H26INO3S) C, H, N, S.
This compound was prepared stn class="Chemical">arting
from 37b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (79% yield); mp 173–174 °C. 1HNMR (DMSO):
δ 3.10 (m, 9H, N(CH3)3), 3.24–3.98
(m, 5H, CH2N, dioxane), 4.22 (dd, J =
13.9, 9.7 Hz, 1H, dioxane), 4.46 (m, 1H, dioxane), 5.15 (dd, J = 9.3, 2.9 Hz, 1H, dioxane), 7.42–7.80 (m, 9H,
ArH). ESI/MS m/z: 360.2 [M]+. Anal. Calcd (C20H26INO3S) C, H, N, S.
This compound was prepared stn class="Chemical">arting
from 57a following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (92% yield); mp 204–205 °C. 1HNMR (DMSO):
δ 3.12 (s, 9H, N(CH3)3), 3.42 (m, 2H,
CH2N), 3.70–3.96 (m, 3H, dioxane), 4.13–4.42
(m, 2H, dioxane), 4.66 (dd, J = 8.2, 3.0 Hz, 1H,
dioxane), 7.31–7.47 (m, 5, ArH). ESI/MS m/z: 236.2 [M]+, 599.2 [2M + I]+. Anal.
Calcd (C14H22INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 59b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (79% yield); mp 175–176 °C. 1HNMR (DMSO):
δ 0.40–2.24 (m, 11H, cyclohexyl), 3.02–3.82 (m,
14H, N(CH3)3, CH2N, dioxane), 4.18
(m, 1H, dioxane), 4.45 (d, J = 12.3 Hz, 1H, dioxane),
7.18–7.42 (m, 5H, ArH). ESI/MS m/z: 318.2 [M]+, 763.4 [2M + I]+. Anal. Calcd
(C20H32INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 60a following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (74% yield); mp 218–219 °C. 1HNMR (DMSO):
δ 2.90–3.68 (m, 15H, N(CH3)3, CH2N, dioxane), 3.92 (m, 1H, dioxane), 4.28 (d, 1H, CH(Ar)2), 4.56 (m, 1H, dioxane), 7.05–7.62 (m, 10H, ArH).
ESI/MS m/z: 326.2 [M]+ Anal. Calcd (C21H28INO2) C, H,
N.
This compound was prepared stn class="Chemical">arting
from 60b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
MeOH (81% yield); mp 266–267 °C. 1HNMR (DMSO):
δ 2.95–3.46 (m, 14H, N(CH3)3, CH2N, dioxane), 3.70 (m, 1H, dioxane), 3.91 (d, 1H, CH(Ar)2), 4.12 (m, 1H, dioxane), 4.39 (d, J = 10.6,
2.8 Hz, 1H, dioxane), 7.08–7.46 (m, 10H, ArH). ESI/MS m/z: 326.2 [M]+. Anal. Calcd
(C21H28INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 61b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
MeOH (91% yield); mp 300–301 °C. 1HNMR (DMSO):
δ 3.05–3.68 (m, 14H, N(CH3)3, CH2N, dioxane), 3.91 (m, 1H, dioxane), 4.31 (m, 1H, dioxane),
4.60 (d, J = 10.2, 2.6 Hz, 1H, dioxane), 7.25–7.72
(m, 9H, ArH). ESI/MS m/z: 312.2
[M]+, 751.3 [2M + I]+. Anal. Calcd (C20H26INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 62a following the procedure described for 3a: a white solid was obtained, which was recrystallized from
MeOH (88% yield); mp 161–162 °C. 1HNMR (DMSO):
δ 3.00–3.92 (m, 14H, N(CH3)3, CH2N, dioxane), 3.94 (s, 2H, CH2Ar), 4.12 (m, 1H,
dioxane), 4.38 (m, 1H, dioxane), 4.60 (m, 1H, dioxane), 7.08–7.42
(m, 9H, ArH). ESI/MS m/z: 326.2
[M]+. Anal. Calcd (C21H28INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 63b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (91% yield); mp 173–175 °C. 1HNMR (DMSO):
δ 3.01–3.62 (m, 14H, N(CH3)3, CH2N, dioxane), 3.90 (dd, J = 11.6, 3.4 Hz,
1H, dioxane), 4.27 (m, 1H, dioxane), 4.58 (dd, J =
10.4, 2.6 Hz, 1H, dioxane), 7.20–7.46 (m, 9H, ArH). ESI/MS m/z: 344.2 [M]+. Anal. Calcd
(C20H26INO2S) C, H, N, S.
This compound was prepared stn class="Chemical">arting
from 64a following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (91% yield); mp 78–80 °C. 1HNMR (DMSO):
δ 2.95–3.94 (m, 14H, N(CH3)3, CH2N, dioxane), 4.10 (dd, J = 13.9, 9.9 Hz,
1H, dioxane), 4.38 (m, 1H, dioxane), 4.72 (dd, J =
7.6, 3.5 Hz, 1H, dioxane), 7.38–7.83 (m, 9H, ArH). ESI/MS m/z: 360.2 [M]+. Anal. Calcd
(C20H26INO3S) C, H, N, S.
This compound was prepared stn class="Chemical">arting
from 64b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (91% yield); mp 175–176 °C. 1HNMR (DMSO):
δ 2.91–3.59 (m, 14H, CH2N, N(CH3)3, dioxane), 3.86 (m, 1H, dioxane), 4.25 (m, 1H, dioxane),
4.60 (dd, J = 10.4, 2.6 Hz, 1H, dioxane), 7.40–7.81
(m, 9H, ArH). ESI/MS m/z: 360.2
[M]+. Anal. Calcd (C20H26INO3S) C, H, N, S.
This compound was prepared stn class="Chemical">arting
from 65b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
EtOH (90% yield); mp 192–193 °C. 1HNMR (DMSO):
δ 2.96–3.60 (m, 13H, N(CH3)3, CH2N, dioxane), 3.79–4.00 (m, 2H, dioxane), 4.29 (m, 1H,
dioxane), 4.64 (dd, J = 10.1, 2.9 Hz, 1H, dioxane),
7.48–8.00 (m, 9H, ArH). ESI/MS m/z: 376.2 [M]+. Anal. Calcd (C20H26INO4S) C, H, N, S.
This compound was prepared stn class="Chemical">arting
from 69a following the procedure described for 3a: a white solid
was obtained, which was recrystallized from 2-PrOH (90% yield); mp
190–191 °C. 1HNMR (DMSO): δ 3.07–3.68
(m, 12H, N(CH3)3, CH2N, dioxane),
3.90 (m, 1H, dioxane), 4.60 (m, 2H, dioxane), 5.06 (d, J = 11.0 Hz, 1H, dioxane), 6.85–7.37 (m, 10H, ArH). ESI/MS m/z: 312.2 [M]+, 751.3 [2M +
I]+. Anal. Calcd (C20H26INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 75a following the procedure described for 3a: a white solid was obtained, which was recrystallized from
2-PrOH (80% yield); mp 275–276 °C. 1HNMR (DMSO):
δ 2.89–3.51 (m, 11H, N(CH3)3, CH2N), 3.62–4.18 (m, 3H, dioxane), 5.01 (s, 1H, dioxane),
6.62–7.62 (m, 15H, ArH). ESI/MS m/z: 388.2 [M]+. Anal. Calcd (C26H30INO2) C, H, N.
This compound was prepared stn class="Chemical">arting
from 75b following the procedure described for 3a: a white solid was obtained, which was recrystallized from
2-PrOH (82% yield); mp 262–263 °C. 1HNMR (DMSO):
δ 3.01–3.52 (m, 11H, N(CH3)3, CH2N), 3.81–4.20 (m, 3H, dioxane), 6.15 (s, 1H, dioxane),
6.87–7.80 (m, 15H, ArH). ESI/MS m/z: 388.2 [M]+. Anal. Calcd (C26H30INO2) C, H, N.
A solution of 27a(16) (0.19 g, 0.5 mmol) and dimethylamine (10 mL) in dry benzene (20
mL) was heated in a sealed tube for 72 h at 110 °C. After evaporation
of the solvent, the residue was dissolved in CHCl3, which
was washed with NaOH 2 N and dried over Na2SO4. The solvent was concentrated in vacuo to give
a residue, which was purified by column chromatography, eluting with
CHCl3/CH3OH (9.5:0.5). An oil was obtained (90%
yield). 1HNMR (CDCl3): δ 0.60–1.92
(m, 11H, cyclohexyl), 2.33 (s, 6H, N(CH3)2),
2.34–2.54 (m, 2H, CH2N), 3.21 (dd, 1H, dioxane),
3.40 (d, 1H, dioxane), 3.89 (dd, 1H, dioxane), 4.19 (m, 1H, dioxane),
4.46 (d, 1H, dioxane), 7.21–7.32 (m, 5H, ArH).
This compound was prepared starting from 27b(16) following the procedure described for 33a: an oil was obtained (91% yield). 1HNMR (CDCl3): δ 0.61–1.89 (m, 11H, cyclohexyl), 2.15–2.46
(m, 8H, CH2N, N(CH3)2), 3.28 (t, J = 11.3 Hz, 1H, dioxane), 3.60–3.85 (m, 3H, dioxane),
4.52 (d, J = 12.1 Hz, 1H, dioxane), 7.20–7.52
(m, 5H, ArH). The free base was transformed into the oxalate salt,
which was crystallized from EtOH: mp 142–143 °C. 1HNMR (DMSO): δ 0.65–1.83 (m, 11H, cyclohexyl),
2.76 (s, 6H, N(CH3)2), 2.90–3.11 (m,
2H, CH2N), 3.16 (t, J = 10.9 Hz, 1H, dioxane),
3.55 (m, 2H, dioxane), 3.78 (m, 1H, dioxane), 4.62 (d, J = 12.2 Hz, 1H, dioxane), 7.21–7.48 (m, 5H, ArH), 8.21 (br
s, 2H, COOH). 13CNMR (DMSO): δ 26.4, 26.4, 26.5,
26.5, 27.2, 44.1 (cyclohexyl); 47.6 (N(CH3)2); 57.8, 65.5, 68.2, 69.5, 80.0 (CH2N and dioxane); 127.4,
128.3, 128.5 (ArH); 140.0 (Ar); 164.5 (COOH). ESI/MS m/z: 304.2 [M + H]+, 326.2 [M + Na]+ Anal. Calcd (C19H29NO2.C2H2O4) C, H, N.
Enantiomeric
Resolution of (±)-33b
The enantiomers of
(±)-33b were separated by chiral
HPLC by using a Regis Technologies Whelk-O 1 (R,R) H (25 cm × 2 cm, 10 μm particle size) column;
mobile phase: n-hexane/2-propanol 85/15% v/v; flow
rate 18 mL/min; detection was monitored at a wavelength of 220 nM.
Retention times: 5.6 min for compound (−)-33b and
11.4 min for compound (+)-33b. ee >99.5% for both
enantiomers.(2S,6S)-(−)-33b: [α]D20 = −31.2 (c 1, n class="Chemical">CHCl3). The 1HNMR spectrum
was identical to that of racemic compound (±)-33b. The free base was transformed into the oxalate salt, which was
recrystallized from EtOH: [α]D20 = +47.7 (c 1, CH3OH), mp 142–143
°C. Anal. Calcd (C21H31NO6)
C, H, N.
(2R,6R)-(+)-n class="Chemical">33b:
[α]D20 = +31.5 (c 1, CHCl3). The 1HNMR spectrum
was identical to that of racemic compound (±)-33b. The free base was transformed into the oxalate salt, which was
recrystallized from EtOH: [α]D20 = +46.9 (c 1, CH3OH), mp 142–143
°C. Anal. Calcd (C21H31NO6)
C, H, N.
This compound was prepared stn class="Chemical">arting from 28b following the procedure described for 33a: an oil was
obtained (93% yield). 1HNMR (CDCl3): δ
2.29 (s, 6H, N(CH3)2), 2.63 (m, 2H, CH2N), 3.66–4.00 (m, 5H, dioxane), 4.89 (dd, 1H, dioxane), 7.32–7.62
(m, 9H, ArH).
1-((2R*,6S*)-6-(4-Benzylphenyl)-1,4-dioxan-2-yl)-N,N-dimethylmethanamine and 1-((2R*,6R*)-6-(4-benzylphenyl)-1,4-dioxan-2-yl)-N,N-dimethylmethanamine (35a/b)
This mixture of cis/trans (6:4) diastereomers was prepared
stn class="Chemical">arting from 29a/b following the procedure described
for 33a: an oil was obtained (91% yield). 1HNMR (CDCl3): δ 2.28 (s, 6H trans, N(CH3)2), 2.32 (s, 6H cis, N(CH3)2),
2.47 (m, 2H cis, CH2N), 2.67 (m, 2H trans, CH2N), 3.28–4.05 (m, 7H cis + 7H trans, dioxane and CH2Ar), 4.65 (dd, 1H cis, dioxane), 4.80 (dd, 1H trans, dioxane), 7.08–7.39
(m, 9H cis + 9H trans, ArH).
This compound was prepared stn class="Chemical">arting from 31b following the procedure described for 33a: an oil was
obtained (93% yield). 1HNMR (CDCl3): δ
2.22 (s, 6H, N(CH3)2), 2.58 (m, 2H, CH2N), 3.60–3.98 (m, 5H, dioxane), 4.82 (dd, 1H, dioxane), 7.41–7.68
(m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 32b following the procedure described for 33a: an oil was
obtained (90% yield). 1HNMR (CDCl3): δ
2.24 (s, 6H, N(CH3)2), 2.60 (m, 2H, CH2N), 3.61–3.98 (m, 5H, dioxane), 4.86 (dd, 1H, dioxane), 7.44–7.99
(m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 48b(13) following the procedure described for 33a: an oil was obtained (85% yield). 1HNMR (CDCl3): δ 2.19–2.53 (m, 8H, CH2N, N(CH3)2), 3.54 (dd, 1H, dioxane), 3.75–4.03 (m,
4H, dioxane), 4.58 (dd, 1H, dioxane), 7.30–7.40 (m, 5H, ArH).
This compound
was prepared stn class="Chemical">arting from 50b following the procedure
described for 33a: an oil was obtained (85% yield). 1HNMR (CDCl3): δ 0.58–1.87 (m, 11H,
cyclohexyl), 2.18–2.80 (m, 8H, N(CH3)2, CH2N), 3.58–3.82 (m, 4H, dioxane), 4.42 (d, 1H,
dioxane), 7.21–7.40 (m, 5H, ArH).
This compound was prepared stn class="Chemical">arting from 51a following
the procedure described for 33a: an oil was
obtained (85% yield). 1HNMR (CDCl3): δ
2.19–2.73 (m, 8H, CH2N, N(CH3)2), 3.57–3.78 (m, 5H, dioxane), 4.30 (m, 1H, dioxane), 4.42
(d, 1H, CH(Ar)2), 7.14–7.40 (m, 10H, ArH).
This compound was prepared stn class="Chemical">arting from 51b following the procedure described for 33a: an oil was
obtained (82% yield). 1HNMR (CDCl3): δ
2.10–2.47 (m, 8H, CH2N, N(CH3)2), 3.38 (m, 2H, dioxane), 3.60–3.92 (m, 4H, dioxane), 4.28
(m, 1H, dioxane), 7.12–7.40 (m, 10H, ArH).
This compound was prepared stn class="Chemical">arting from 52a following the procedure described for 33a: an oil was
obtained (85% yield). 1HNMR (CDCl3): δ
2.28–2.85 (m, 8H, CH2N, N(CH3)2), 3.72–4.08 (m, 5H, dioxane), 4.69 (dd, 1H, dioxane), 7.31–7.63
(m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 53a following the procedure described for 33a: an oil was
obtained (86% yield). 1HNMR (CDCl3): δ
2.30–2.92 (m, 8H, CH2N, N(CH3)2), 3.68–4.02 (m, 7H, dioxane, ArCH2Ar), 4.62 (dd,
1H, dioxane), 7.17–7.38 (m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 53b following the procedure described for 33a: an oil was
obtained (81% yield). 1HNMR (CDCl3): δ
2.18–2.52 (m, 8H, CH2N, N(CH3)2), 3.52 (m, 2H dioxane), 3.81–4.02 (m, 5H, dioxane, ArCH2Ar), 4.54 (dd, 1H, dioxane), 7.16–7.36 (m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 54a following the procedure described for 33a: an oil was
obtained (86% yield). 1HNMR (CDCl3): δ
2.32–2.88 (m, 8H, CH2N, N(CH3)2), 3.65–3.99 (m, 5H, dioxane), 4.62 (dd, 1H, dioxane), 7.20–7.40
(m, 9H, ArH).
This compound
was prepared stn class="Chemical">arting from 54b following the procedure
described for 33a: an oil was
obtained (87% yield). 1HNMR (CDCl3): δ
2.17–2.52 (m, 8H, CH2N, N(CH3)2), 3.50 (m, 2H, dioxane), 3.72–4.02 (m, 3H, dioxane), 4.56
(dd, 1H, dioxane), 7.22–7.38 (m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 55a following the procedure described for 33a: a solid
was obtained (84% yield). 1HNMR (CDCl3): δ
2.22–2.82 (m, 8H, N(CH3)2, CH2N), 3.60–3.95 (m, 5H, dioxane), 4.62 (dd, 1H, dioxane), 7.41–7.69
(m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 64b following
the procedure described for 3a: a white solid
was obtained (84% yield); mp 96–99 °C. 1HNMR
(CDCl3): δ 2.16–2.47 (m, 8H, CH2N, N(CH3)2), 3.44 (m, 2H, dioxane), 3.77 (m,
1H, dioxane), 3.92 (m, 2H, dioxane), 4.55 (dd, 1H, dioxane), 7.38–7.65
(m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 56a following
the procedure described for 33a: an oil was
obtained (85% yield). 1HNMR (CDCl3): δ
2.28–2.81 (m, 8H, CH2N, N(CH3)2), 3.60–3.98 (m, 5H, dioxane), 4.66 (dd, 1H, dioxane), 7.46–7.99
(m, 9H, ArH).
This compound was prepared stn class="Chemical">arting from 56b following
the procedure described for 33a: an oil was
obtained (84% yield). 1HNMR (CDCl3): δ
2.16–2.50 (m, 8H, CH2N, N(CH3)2), 3.50 (m, 2H, dioxane), 3.70–4.02 (m, 3H, dioxane), 4.60
(dd, 1H, dioxane), 7.43–7.95 (m, 9H, ArH).
Tosyl chloride
(1.8 g, 9.4 mmol) was added to a stirred solution of 68a(38) (2 g, 7.4 mmol) in pyridine (5 mL)
at 0 °C over 30 min. After 3 h at 0 °C, the mixture was
left for 20 h at 4 °C in the freezer. Then, it was poured into
ice and concentrated HCl (5 mL) and extracted with CHCl3. The organic layers were washed with 2 NHCl (15 mL), NaHCO3 saturated solution (15 mL), and H2O (15 mL) and
then dried over Na2SO4. The evaporation of the
solvent afforded the intermediate tosyl derivative, which was used
in the next step without further purification. Dimethylamine (10 mL)
was added to a solution of tosyl derivative in dry benzene (20 mL),
and the mixture was heated in a sealed tube for 72 h at 110 °C.
After evaporation of the solvent, the residue was dissolved in CHCl3, which was washed with NaOH 2 N and dried over Na2SO4. The solvent was concentrated in vacuo to give a residue, which was purified by column chromatography,
eluting with CHCl3/CH3OH (9.5:0.5). An oil was
obtained (85% yield). 1HNMR (CDCl3): δ
2.32 (s, 6H, N(CH3)2), 2.52 (m, 2H, CH2N), 3.69 (dd, 1H, dioxane), 4.05–4.20 (m, 2H, dioxane), 4.37
(d, 1H, dioxane), 4.52 (d, 1H, dioxane), 6.96–7.23 (m, 10H,
ArH).
This compound was prepared stn class="Chemical">arting from 74b following the procedure described for 69a: an oil was
obtained (75% yield). 1HNMR (CDCl3): δ
2.23–2.72 (m, 8H, CH2N, N(CH3)2), 3.40 (dd, 1H, dioxane), 3.58 (dd, J = 11.5 Hz
and J = 10.3 Hz, 1H, dioxane), 3.99 (m, 1H, dioxane),
5.82 (s, 1H, dioxane), 6.87–7.72 (m, 15H, ArH).
ECD and
NMR Calculations
Merck molecular force field
(MMFF) and DFT calculations were run with Spartan’18 (Wavefunction,
Inc., Irvine CA, 2014), with standard parameters and convergence criteria.
DFT and TDDFT calculations were run with Gaussian’16 (Rev.
B.02, Gaussian, Inc., Wallingford CT, 2016),[56] with default grids and convergence criteria. The calculations were
run on the N-protonated forms of 2 and 33b (charge +1). Conformational searches were run with the Monte Carlo
algorithm implemented in Spartan’18 using MMFF. All structures
thus obtained were first optimized with the DFT method using ωB97X-D
functional and 6-31G(d) basis set in vacuo and then
re-optimized using ωB97X-D functional and 6-31G+(d) basis set,
first in vacuo then using the SMD solvent model for
acetonitrile. TDDFT calculations were run using several combinations
of functionals (ωB97X-D, B3LYP, CAM-B3LYP, wB97X-D, BH&HLYP,
M11), basis sets (def2-SVP and def2-TZVP), either in vacuo or using IEF-PCM solvent model for acetonitrile; they included at
least 16 excited states (roots). Boltzmann populations were estimated
at 300 K from internal energies. ECD spectra were generated using
the program SpecDis,[57,58] by applying a Gaussian band shape
with 0.25 eV exponential half-width, shifted by 15 nm, scaled by a
factor 2, from dipole-length rotational strengths.
Cell
Culture and Membrane Preparation
CHO-K1 cells
stably transfected with the human muscarinic receptor subtypes (hM1–5) were grown in Dulbecco’s modified Eagle’s
medium (DMEM) with nutrient mixture F12 (DMEM/F12, 50/50), containing
10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 U/mL), l-glutamine (4 mM), and geneticin (G-418, 50 μg/mL) at
37 °C in a 5% CO2 humidified incubator. In order to
harvest the cells, the culture medium was removed;the cells were washed
with PBS and then trypsinized by trypsin–EDTA treatment for
2–3 min. Serum (0.7 mL) was added to inactivate the trypsin,
and the cells were spun down by centrifuging at 300g for 5 min. The cells were then resuspended in ice-cold 25 mM sodium
phosphate buffer containing 5 mM MgCl2, pH 7.4 (binding
buffer) and homogenized using a cell disrupter (Ultra-Turrax, setting
3, 30 s). The homogenate was sedimented by centrifugation (17,000g, 15 min). The supernatant was discarded, and the resulting
membrane pellets were resuspended with Ultra-Turrax in the same buffer
to give a final protein concentration of 1–2 mg/mL. The protein
content was determined by the method of Bradford (1976) with bovine
serum albumin (Sigma) as a standard and stored at −80 °C.
Inhibition Radioligand Binding Assay
Inhibition radioligand
binding assays were conducted as previously described[48,49] with 0.2 nM [3H]NMS in binding buffer in a final volume
of 250 μL. Nonspecific binding was defined in the presence of
10 μM atropine. Briefly, membrane fractions (about 25–70
μg/mL of protein) were incubated with radioligand and unlabeled
test compounds for 2 h at r.t. Bound and free radioactivity were separated
by filtering the assay mixture through UniFilter GF/B plates using
a FilterMate Cell Harvester (PerkinElmer Life and Analytical Science).
The filter bound radioactivity was counted by a TopCount NXT Microplate
Scintillation Counter (PerkinElmer Life and Analytical Science). Data
(cpm) were normalized to percentage-specific binding and analyzed
using a four-parameter logistic equation in GraphPad Prism 5.02; IC50 values were determined, and Ki values were calculated.[59] The values reported in Tables and 2 represent the
arithmetic mean ± S.E.M. of at least three independent experiments,
each one performed in duplicate.Docking simulations involved the ligands
with pKi values on M3 mAChR
greater than 6 and the recently resolved M3 mAChR structure
in complex with a selective antagonist (PDB Id: 5ZHP).[52] The protein structure was completed by adding hydrogen
atoms, and the ionizable groups were set to be compatible to physiological
pH using the VEGA suite of programs.[60] The
prepared structure was finally minimized by using the NAMD program[61] and keeping fixed the backbone atoms to retain
the experimental folding. The structure of the considered ligands
was optimized by PM7-based semi-empirical calculations.[62] Docking simulations were performed by PLANTS[63] by focusing the searches within a 8.0 Å
radius around the bound resolved antagonist. The simulations were
carried out using the ChemPLP primary score with speed equal to 1
and 10 poses were generated for each ligand. The obtained complexes
were optimized by using NAMD and by keeping fixed all atoms outside
a 10 Å radius sphere around the docked ligand and then rescored
by ReScore+.[64]
Functional Studies on MSCs
from Mouse Bone Marrow
MSC Collection and Culture
The in vitro studies were performed by using bone marrow MSCs
as a model. Male
BALB/c mice (Harlan Italy SrL, Milano, Italy) (8 weeks old; body weight
∼24.5 g; n = 4) were kept in a laminar-flow
cage in a standardized environmental condition. Food (Harlan, Italy),
and water was supplied ad libitum. Mice were sacrificed by CO2narcosis and cervical dislocation in accordance with the
recommendations of the Italian Ethical Committee and under the supervision
of authorized investigators. Long bones (femurs and tibiae) were dissected
and cleaned from skin, muscle, and connective tissues as much as possible.
Bones were placed in a culture dish containing sterile PBS. Then,
the bone cavity was flushed in DMEM with a syringe in order to collect
the bone marrow cells into a 50 mL sterile tube. The procedure was
repeated until all marrow was removed. Cell suspension was filtered
through a cell strainer (70 μm size) to remove cell clumps or
bone debris. Then, bone marrow cells were plated in 100 mm culture
dishes in DMEM containing 10% heat-inactivated-fetal calf serum (HIFCS),
penicillin, and streptomycin. In order to obtain a population of bone
marrow MSCs, the protocol by Solimani and Nadri[65] was followed. Cells were incubated at 37 °C with 5%
CO2 in a humidified chamber. After 3 h, the nonadherent
cells that accumulate on the surface of the dish were removed by changing
the medium and replacing it with a fresh complete medium. After 8
h of culture, the medium was further replaced with fresh complete
medium. The last step was repeated every 8 h for up to 72 h of initial
culture. Then, the adherent cells were washed with sterile PBS and
added with a fresh medium every 3–4 days. After 2 weeks of
initiating culture, cells were washed with PBS, detached by trypsinization,
counted, and plated at the density of 5,000 cells/well in 96 culture
plates (Costar Corp., Milano, Italy) in DMEM containing 10% HIFCS,
penicillin, and streptomycin.
Experimental Protocol
MSCs were treated with compound 3b (from 10–4 to 10–10 M) for 24 h. Control cultures were performed
by incubating the cells
with the only vehicle (DMSO) or by untreated cells. Parallel other
cultures were incubated with 3b from 10–4 to 10–10 M for 1 h, and then, the culture medium
was replaced with a fresh medium. The MSCs were maintained in the
presence of carbachol at 10–10 M for 24 h. At the
end of each procedure, the MSCs viability was measured by MTS assay.
Specifically, cells were incubated with Cell Titer 96 Aqueous One
Solution Reagent (Promega Italia, Milano, Italy) for 2 h in a humidified
5% CO2 atmosphere. The quantity of the formazan product
was directly proportional to the number of living cells in culture.
The colored formazan was measured by reading the absorbance at 490
nm using a 6-well plate reader.
Authors: Hongtao Liu; Josefa Hofmann; Inbar Fish; Benjamin Schaake; Katrin Eitel; Amelie Bartuschat; Jonas Kaindl; Hannelore Rampp; Ashutosh Banerjee; Harald Hübner; Mary J Clark; Sandra G Vincent; John T Fisher; Markus R Heinrich; Kunio Hirata; Xiangyu Liu; Roger K Sunahara; Brian K Shoichet; Brian K Kobilka; Peter Gmeiner Journal: Proc Natl Acad Sci U S A Date: 2018-11-07 Impact factor: 11.205
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