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NMR studies of buspirone (an anxiolytic drug) analogues
Journal of
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 385 (1996) 195-207
NMR studies of buspirone (an anxiolytic drug) analogues Zdzistaw
Chilmonczyk
a'*, J a c e k C y b u l s k i a, A g n i e s z k a Andrzej Leg b
Szelejewska-Wo~niakowska
a,
"Pharmaceutical Research Institute, 8 Rydygiera Street, 01-793 Warszawa, Poland bDepartment of Chemistry, University of Warsaw, 1 Pasteura Street, Warszawa, Poland
Received 1 April 1996; accepted 1 July 1996
Abstract
Conformations of piperazine rings in 8-{4-[4-(2-pyrimidyl)-l-piperazinyl]butyl}-8-azaspiro[4.5]-decane-7,9-dione (buspirone - - 1) and its two analogues 8-{4-[4-(2-quinolinyl)-l-piperazinyl]butyl}-8-azaspiro[4.5]-decane-7,9-dione (kaspar - - 2) and 4,4-dimethyl-l-{4-[4-(2-quinolinyl)-l-piperazinyl]butyl}-2,6-piperidinedione (mesmar - - 3) (Fig. 1) have been studied with the aid of 1H NMR and 13C NMR spectra. For free bases the two bands corresponding to piperazine hydrogen atoms in the spectra broaden considerably with a decrease in temperature to divide into four separate bands, indicating the presence of a dynamic exchange process. A similar dynamic process, but for higher temperatures, was observed for buspirone (1), kaspar (2) and mesmar (3) hydrochlorides. Proton and carbon atom resonance lines have been assigned with the aid of 2D COSY and 2D HETCOR two-dimensional spectra.
1. Introduction
The mechanism of activity of some anxiolytics has been suggested as being based mainly on an interaction with serotonin 5-HT1A receptor. Thus many research laboratories [1] have concentrated their work on the synthesis of the ligands of this receptor. One of such compounds, 8-{4-[4-(2-pyrimidyl)-lpiperazinyl]butyl }-8-azaspiro[4.5]-decane-7,9-dion hydrochloride (buspirone) (1), has been introduced into clinical practice [2]. This anxiolytic has been found to exhibit much smaller sedative effects than the widely used benzodiazepines. In our laboratory we have synthesized new * Corresponding author.
buspirone analogues, 8-{4-[4-(2-quinolinyl)-l-piperazinyl]butyl }-8-azaspiro[4.5]-decane-7,9-dione hydrochloride (kaspar) (2) and 4,4-dimethyl-l-{4-[4-(2quinolinyl)- 1-piperazinyl]butyl}-2,6-piperidinedione hydrochloride (mesmar) (3) [3]. Their affinity to the 5-HT1A receptor is close to that of buspirone (1) [4], suggesting that they might possess a similar profile of pharmacological action. In order to continue the search for new chemical structures of anxiety-relieving character it is necessary to examine the interaction occurring between the ligand and the 5-HTIA receptor binding site. It is supposed that the basic molecular features (basic pharmacophore) responsible for the binding of buspirone-like molecules to the 5-HTIA receptor are an aromatic ring and a basic nitrogen atom placed at a
0022-2860/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0022-2860(96)09404-5
196
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
specified distance (5.2-5.7 .~,) from the aromatic ring centroid. The pharmacophore was further differentiated for agonists and antagonists with respect to the nitrogen atom out of the aromatic ring plane deviation (0.2 A and 1.6 ,~ respectively) [5,6]. In buspirone (1) and its analogues 2 and 3 the basic nitrogen atom is located in the piperazine ring (N14). Thus the conformational dynamic of piperazine and aromatic moieties has a direct influence on the relative Nbasic-aromatic ring centroid distances included in the 5-HT 1A pharmacophore. In this paper we focus on the conformational analysis of the piperazine ring and identify additional protonation sites in buspirone (1) and its analogues in nonpolar (CD2C12, CDC13) and polar (DMSO) solutions with the aid of 1H NMR and 13C NMR spectra.
O
0
5
I.
4
I. 8
112 ~ 1
15
11 ~
14 '
17 16
19 i 18
'
12 13
\/
/'~"7",,' O 20 ~"
I "~' 23 zo
2. E x p e r i m e n t a l O
2.1. 11-1and 13C N M R data
The compounds were prepared and purified as described previously [3]. NMR spectra were recorded on a Bruker AM 500 NMR spectrometer (equipped with an Aspect 3000 computer using the DISNMR program version 890101.1.) operating at 500.13 and 126.76 MHz for 1H and 13C, respectively, and on a Varian Gemini 2000 spectrometer at a frequency of 199.98 MHz for ~H, equipped with a 5 mm 13C/1H dual probe. Approximately 10-20 mg of compounds 1-3 were dissolved in 0.6 ml CDCI3, CD2C12 or DMSO with TMS as internal standard. Conventional 1H NMR spectra at 500 MHz instrument were acquired using 7000-8000 Hz spectral widths digitized with 64 K data points with a pulse width of 8 #s (ca. 60 ° flip angle). Digital resolution for the real part was 0.254 Hz/PT. Conventional 13C NMR spectra (at 500 MHz) were acquired using a 30000 Hz spectral width digitized with 64 K data points with a pulse width of 3/xs (ca. 45 ° flip angle), 2 s delay between pulses and waltz 16. Standard pulse conditions were used for 1D NMR spectra at 200 MHz instrument with a sweep width of 3000 Hz digitized with 16 K data points (for 1H spectra) and 12500 Hz digitized with 32 K data points (for 13C spectra) and a pulse width of 18/~s (ca. 49 ° flip
O
C 3N 3 \
/
N---CH3
Fig. 1. Structures of buspirone (1), kaspar (2), mesmar (3), 1-(2quinolinyl)-4-methylpiperazine(4) and 1-(2-pyrimidyl)-4-methylpiperazine (5).
Z. Chilmonczyk et aL/Journal of Molecular Structure 385 (1996) 195-207
angle) and 20 #s (ca. 72 ° flip angle) for 1H and 13 C NMR spectra, respectively. The 2D COSY and HETCOR spectra in CDCI3 were measured with a Varian Gemini 2000 instrument. 2D homonuclear-correlated experiments were carded out using Jeenerm's two-pulse sequence. Typical spectral parameters were as follows: 1700 Hz in both
197
dimensions; 16 transients for each FID; 128 tl increments; recycling delay 1 s; and data matrix 512 x 512 for processing. 2D heteronuclear-correlated spectra were obtained using the sequence (90°, 1H)-t1/2(180°, 13C)-tl/2-rl-(90°, XH; 90°, 13C)-r2-(BB, 1H)acquire [7]. The fixed delays correspond to the coupling J (aac, 1H) = 140 Hz. Other parameter settings were 128 FIDs in the t~ domain and 4 K data points in the t2 domain, 400 transients for each tl increment and recycling delay of 1 s. 2.2. Computational quantum chemistry The ab initio RHF/6-31G** calculations of the optimal molecular structure of model compounds 1-(2-quinolinyl)-4-methylpiperazine (4) and 1-(2pyrimidyl)-4-methylpiperazine (5) (Fig. 1) were performed. The analytical first and second derivatives implemented in the Gaussian 92 code (installed on an IBM RISC 6000 workstation) with respect to the geometrical parameters, subject to certain constraints of molecular symmetry, were used. In compound 5 we used a symmetry plane passing through both piperazine nitrogen atoms and perpendicular to the aromatic plane of the pyrimidine ring. In order to reduce extensive computations in 4, the local symmetry planes were enforced also in the piperazine ring and in the quinoline aromatic ring. The present optimization procedure takes advantage of the assumption that the quinoline ring only weakly affects the piperazine symmetry.
300 K ...... ~ . ~ ..........
223 K . . . . . . . J " . . . . . .
J
........ J .....
3. Results and discussion
The temperature behaviour of the kaspar (2) free base and hydrochloride spectra is consistent with a dynamic process of conformation interconversions. Figs 2 and 3 show that four resonances from the aliphatic region of the 1H NMR spectra (500 MHz) (3.97, 3.05, 2.92, 2.11 ppm - - base; 4.55, 3.97, 3.54, 2.92 ppm - - hydrochloride) observed at low temperatures (153 K - base; room - - hydrochloride) progressively broaden and coalesce as the temperature is increasedk Similar
153 K , - ~ - ~
'
•
' 1
. . . .
4.0
i
. . . .
i
. . . .
3.0
i
. . . .
i
. . . .
2.0
i
. . . .
i
. . . .
1.0 PPM
Fig. 2. Effect of temperatureon the aliphatic region of IH NMR spectra (500 MHz): (a) kaspar (2) free base in CD2C12and CFCI3; (b) kaspar (2) hydrochloridein CDC13.
1In chloroformthe signals did not coalesceuntil 333 K. Coalescence was achievedin DMSO (200 MHz) at 363 -+ 1 K (for the geminal pair of protonsH13/13') and at 393 -+ 3 K (for the H12/ 12'e and H12/12'a protons).
198
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
-,-,'~'~
333 K
.~--'"-"~
~
~
.
~,
i
4.6
4.4
4.2
4.0 3.8 3.6 3.4 3.2 3.0 2.8 ppm Fig. 3. Effectof temperatureon the aliphaticregionof IH NMR spectra (500MI-lz)of kaspar (2) hydrochloridein CDCI3. spectral patterns were observed for buspirone (1) and mesmar (3) free bases and hydrochlorides. Two structural units i.e. the piperazine ring and the n-butyl spacer, have enough conformational freedom to be considered as responsible for this dynamic behaviour. The assignment of spectral resonances is thus necessary for identifying the processes occurring in the systems investigated.
3.1. 1H and 13C NMR assignment 3.1.1. Kaspar (2) and mesmar (3) hydrochlorides 3.1.1.1. A. 1H NMR spectrum (Table 1). In the lowfield region only the signals corresponding to the quinoline ring protons were found. On the basis of the chemical shift and multiplicity [8] the following assignments were made: the doublet at 8 = 6.98 ppm (J = 8.60 Hz - - compound 2; J = 9.09 Hz - -
compound 3) - - H3, the doublet at/~ = 7.98 ppm (J = 9.08 Hz - - compound 2; J = 9.05 Hz - - compound 3) - - H4, the broad multiplet at ~i = 7.69-7.74 ppm - - H8, the doublet at/~ -- 7.65 ppm, J = 8.00 Hz - - H5 and the multiplets at 8 = 7.59 and t5 = 7.31 ppm to the H7 and H6 protons, respectively. The observed chemical shift values and splittings of respective multiplets resulting from the absorption of H3, H4, H5, H6, H7 and H8 protons are the same for compounds 2 and 3 (Table 1). The assignment of the signals corresponding to the protons from the imide fragment was based on comparison with the spectrum of the unsubstituted imide: 8 = 2.62 ppm in compound 2 and 8 = 2.54 ppm in compound 3 to H21/21', and ~ = 1.07 ppm (compound 3) to the H23/ 23' methyl group protons. The four-proton multiplets at t5 = 1.51 and 8 -- 1.71 ppm (compound 2) were assigned to H23/23' and H24/24' respectively. Taking into consideration the direction of changes
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
199
Table 1 IH NMR chemical shifts and splittings of hydrochlorides of compounds 2 and 3 a,b Proton No.
1H NMR compound 2
3 4 5 6 7 8 12,12'
6.98 7.98 7.65 7.31 7.59 7.74
3 d d m m m m
6.98 7.98 7.65 7.31 7.59 7.74
2D C O S Y c o m p o u n d
2D H E T C O R compound
2
2
3
d d m m m m 4.55 3.97 3.54 2.91
13,13' 15
16 17 18 21,21' 23,23' 24,24'
3.79 t 2.62 s
3
3.00 m 1.92 m
2.99 m 1.93 m
1.62 quint
1.63 quint
3.80 t 2.54 s 1.07 s
m m m m
4.56 3.97 3.55 2.92
m m m m
1.51 m 1.71 m
a tS(1H) in ppm f r o m T M S . b s = singlet, d = doublet, t = triplet, quint = quintuplet, m = multiplet.
of the chemical shift values tS(1H) for piperazine derivatives [9-11], and the fact that equatorial protons are deshielded by about 0.1-0.7ppm in comparison with axial ones, the H12/12' protons could be assigned two broad, diproton multiplets at 6 -- 4.55 ppm (for H12/12'e protons) and 6 = 3.97 ppm (for H12/12'a protons) (compound 2), and 6 = 4.56 ppm (for H12/12'e protons) and 6 = 3.97 ppm (for H12/12'a protons) (compound 3) and t5 = 3.54 ppm (for H13/13'e protons) and 6 = 2.91 ppm (for H13/13'a) (compound 2), 6 = 3.55 ppm (for H13/13'e protons) and 6 = 2.92 ppm (for H13/13'a) (compound 3) (the above assignments for the piperazine and imide moieties were further confirmed by the 2D HETCOR spectra). The diproton triplets at 6 = 3.79ppm, J = 6.93Hz (compound 2) and at 6 -- 3.80 ppm, J --- 6.94 Hz (compound 3) were assigned to the H18 protons on the basis of the characteristic deshielding effect exerted by the imide group [10,12]. According to the inductive substituent effect, the methylene protons H16 and H17 should give upfield signals as compared to H13/13' and H15 (at ot carbon atoms to N÷14). The signals at 6 = 1.92 ppm (compound 2), 6 = 1.93 ppm (compound 3) were assigned to the H16
'F2
"~
3-
i
dE
II
~
ii
•
11 5Z 6-"
8-"
p, "
8
7
6
5
F1
d
3
(ppm)
Fig. 4 . 2 D C O S Y spectrum of kaspar (2) hydrochloride in CDCI 3 at 200 MHz.
200
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
protons bonded to the ~ carbon atom with respect to the quaternary N14 nitrogen atom. The analysis of nondiagonal signals in 2D COSY spectra (Fig. 4) allowed for signal assignment to the protons bonded to C15 and C16 and C17 carbon atoms: ~ = 3.00 ppm - - H15 (compound 2), ~ = 2.99 ppm - H15 (compound 3); ~ = 1.92 ppm - H16 (compound 2), ~ = 1.93 ppm - - H16 (compound 3); 6 = 1.62 ppm - - H17 (compound 2), ~ = 1.63 ppm -H17 (compound 3). Application of the heteronuclear chemical shift correlation 1H-13C 2D HETCOR allowed us to assign the signals to the protons of the imide fragment ~ = 1.51ppm - - H23/23', 5 = 1.71 ppm - - H24/24' (compound 2) (Table 1). 3.1.1.2. B. 13C N M R (Table 2). The highest chemical
shifts were assigned to the carbon atoms of the carbonyl groups of the imide fragment: C20/20' ~ = 172.31ppm (compound 2), 5 = 172.02ppm (compound 3). By using empirical parameters for calculating chemical shifts in polyheteronuclear systems [13,14] the resonance lines were assigned to carbon atoms C2, C3, C4, C9 and C10 (Table 2).
Substitution of quinoline in position 2 by the piperazine system results in a strong paramagnetic effect on the C2 carbon atom (+4.70ppm with respect to unsubstituted quinoline), which is due to the induction effect of the nitrogen atom. The coupling effect of the lone electron pair of the nitrogen atom with the heteroaromatic ring is also visible in the form of a mesomeric effect, which results in the shielding of carbon atoms in positions ortho and para with respect to the substituent. Therefore the C3 carbon atom exhibits a diamagnetic effect of about -12.18 ppm (compounds 2 and 3), whereas for C10 the effect is weaker, being equal to - 5.47 ppm (with respect to the unsubstituted quinoline). The assignment of the resonance lines to carbon atoms C21/21', C22, C23/23' for compounds 2 and 3 and to C24/24' for compound 2 (for list of assignments see Table 2) was based on the comparison of 13C NMR spectra of compounds 2 and 3 with those of respective imides and the data for the spiro[4.5]decane system [15]. Signals of the carbon atoms C17, C16, C15, C13/13' and C12/12' can be assigned from the effect of the protonated N14
Table 2 13C NMR chemical shift of hydrochlorides of compounds 2 and 3 a Carbon No.
2 3 4 5 6 7 8 9 10 12,12' 13,13' 15 16 17 18 20,20' 21,21' 22 23,23' 24,24'
t3C NMR compound
2D HETCORcompound
2
3
155.60 109.32 138.32
155.56 109.32 138.33
146.76 123.24 42.28
172.31 44.62 39.34 37.41 24.02
a ~(13C)in ppm from TMS.
2
3
127.23 123.40 129.92 126.47
127.22 123.39 129.91 126.42
51.26 56.82 20.52 24.93 37.80
51.33 56.77 20.56 24.88 37.76
146.78 123.21 42.45
172.02 46.12 29.01 27.53 -
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
Table 3 Calculated data on the effect of protonation on chemical shift values ~3Cof carbon atoms of the butyl chain, substituted by amine group (-NRIR2) [17,18] Ci
8~÷
ASci= 83-80 N. N
C,~ C~ C~ C~
52.1 24.2 27.0 32.9
- 2.3 - 3.8 - 0.5 0.0
nitrogen atom. According to the known data [16,17], one could expect a different diamagnetic effect for the carbon atoms in the positions or, /3, 3, and ~ with respect to the protonated nitrogen atom. Thus the effect of the N14 nitrogen atom protonation on the chemical shift values was calculated [17,18] (Table 3). The size of the diamagnetic effect which appeared after the protonation of the N14 nitrogen atom was experimentally determined from the 13C N M R spectra for compounds 2 and 3 in the form of salt and free base (Table 4). A comparison of experimental and calculated values allowed us to differentiate between o~, /3 and 3' (but not 6) carbon atoms. Thus, signals at 8 = 56.82 and 51.26 ppm (compound 2) and 56.77 and 51.23 ppm (compound 3) were assigned to C15, C13/13' carbon atoms; the signals at 8 = 42.28 and 20.52 ppm (compound 2) and 6 = 42.45 and 20.56 ppm (compound 3) to the C12/12' and C16 carbon atoms (Ct~)
Table 4 The effect of protonation of the nitrogen atom on the chemical shift value aaC of the aliphatic carbon atoms of compounds 2 and 3 C~
~s+ (2)'
A8 s÷'~ (2) 6s+ (3)
ASN÷,N(3)
C~-C13 or C15 C~-C13 or C15 Ca-C12, C12' Ca-C16 Cv-C17 C~-C18 C21, C21' C22 C23, C23' C24, C24'
56.82 51.26 42.48 20.52 24.93 37.80 44.62 39.34 37.41 24.93
-
-
a Compound 2.
1.42 1.85 2.43 3.44 1.11 1.54 0.44 0.11 0.14 0.16
56.77 51.23 42.45 20.56 24.88 37.76 46.12 29.01 27.53 -
-
1.47 1.90 2.63 3.70 1.16 1.57 0.35 0.09 0.15
201
respectively 2, and the signals at 8 = 24.93 ppm (compound 2) and 24.88 ppm (compound 3) to the C17 carbon atoms (C.r). On the basis of the available data concerning the chemical shift values of N-substituted amides [21] the signals at <5 = 37.80 ppm (compound 2) and 6 = 37.76 ppm (compound 3) were assigned to the C18 carbon atoms (Ca). It should, however, be noted (Table 4) that the chemical shift values of the C18 carbon atoms in protonated compounds showed a diamagnetic effect of the order of -1.5 ppm. This effect is typical of the Ca atom whereas the C18 atom occupies a 8 position with respect to the protonated N14 nitrogen atom. Crosspeak signals at the intersection of the x- and y-axes of the contour diagram of the 2D H E T C O R spectra of compounds 2 and 3 allowed us to perform the following assignments: 8 = 127.23 ppm - - C5 (compound 2), d = 127.22ppm - - C5 (compound 3); 8 = 123.40 ppm - - C6 (compound 2), 8 = 123.39 ppm C6 (compound 3); (5 -- 129.92 ppm - - C7 (compound 2), 8 = 129.91ppm - - C7 (compound 3); 8 = 126.40 ppm - - C8 (compound 2), 8 = 126.46 ppm C8 (compound 3); 8 = 51.56 ppm - - C13/13' (compound 2), 8 = 51.33 ppm - - C13/13') (compound 3); 8 = 56.82 ppm - - C15 (compound 2), 8 = 56.77 ppm - C15 (compound 3). A comparative analysis of N M R spectra of the free base of buspirone (1) and its hydrochloride form has been performed [22]. For the assignment of the spectral resonances of kaspar (2) and mesmar (3) free bases, see Table 5. The above assignments allowed us to attribute the coalescing resonance lines in buspirone (1), kaspar (2) and mesmar (3) to the heterocyclic piperazine ring. Since chair conformations are supposed to be the lowest energy conformers of a piperazine ring [23] the four broadened resonances at 3.97, 3.05, 2.92 and 2.11 ppm in the kaspar (2) free base 1H N M R spectrum at 153 K (500 MHz) (Fig. 2) may be assigned to H12/ 12'e, H12/12'a, H13/13'e and H13/13'a piperazine protons respectively. This assignment is based on the data for piperazine derivatives with equatorial N-methyl substituents which show that the axial
2 Substitution at the nitrogen atom of the piperazine system with the alkyl chain should reduce the shielding effect for carbon atoms in position a with respect to the nitrogen atom, whereas the opposite effect was observed for the aromatic ring as substituent [3,19,20].
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
202
Table 5 1H and 13C NMR chemical shift and splittings of free bases of compounds 2and 3 a Proton No.
lH NMR compound 2
3 4 5 6 7 8 12,12' 13,13' 15 16 17 18 21,21' 23,23' 24,24' -
6.97 7,87 7.58 7.20 7.52 7.69 3.74 2.56 2.40 1.55 1.55 3.79 2.58 1.49 1.70
Carbon No.
13C NMR compound
3 db d d m m d t t t m m t s m m
6.97 7.87 7.58 7.21 7.52 7.69 3.74 2.56 2.40 1.56 1.56 3.80 2.49 1.07 -
d d d m m d t t t m m t s s
2 3 4 5 6 7 8 9 10 12,12' 13,13' 15 16 17 18 20,20' 21,21' 22 23,23' 24,24'
2
3
157.91 110.00 137.56 127.59 123.46 129.74 126.83 148.33 122.50 45.28 53.59 58.59 24.60 26.41 39.64 172.47 45.52 39.83 37.91 24.75
157.43 109.52 137.36 127.17 123.10 129.45 126.69 147.93 122.32 45.08 53.13 58.24 24.26 26.04 39.33 171.86 46.47 29.10 27.68 -
tS(tH) and ~(13C) in ppm from TMS. b s ffi singlet, d ffi doublet, t = triplet, m = multiplet.
protons in the piperazine ring are usually at a lower frequency than equatorial ones [24]. As the temperature is increased, the H12/12'a protons appear to exchange environments with the H12/12'e protons, and a similar interchange process occurs for the geminal H13/13' protons. At room temperature two triplets at 3.74 and 2.56 ppm (H12/12' and H13/13' respectively) for the interchanged axial and equatorial protons are observed. This spectral pattern reflects the conversion of piperazine ring low energy form back to itself after passing through an intermediate conformation in which axial and equatorial protons are interchanged. The possible conformers of a substituted piperazine ring and their interconversions by ring and nitrogen inversions, leading to the axial and equatorial protons interchange, were described by Cross et al. (Fig. 5) [25] and involve at least one ring inversion and as many nitrogen inversions as are required to return the nitrogen substituents to their previous positions. It was also assumed that the conformation with equatorial substituents at both piperazine nitrogen atoms was the lowest energy conformation. For buspirone (1), kaspar (2) and mesmar (3) we calculated the relative energies of the
piperazine chair conformations (with axial and equatorial substituents at N l l and N14 nitrogen atoms) with the use of the quantum mechanical ab initio RHF/ 3-21G** method applied for model compounds 1-(2quinolinyl)-4-methylpiperazine (4), a model for kaspar (2) and mesmar (3), and 1-(2-pyrimidyl)-4-methylpiperazine (5), a model for buspirone (1). The relative Ar
N --A~
Rin v
I
I,
N
I
Ninv
x
Ninv Ar
I
M e . _ ~ N ~
--Ar ~
I
Rinv
I Me
Fig. 5. Diagrammatic representation of the possible conformers of a substituted piperazine ring and their interconversion by ring and nitrogen inversion.
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
203
Table 6 O b s e r v e d and c a l c u l a t e d p y r a m i d i z a t i o n s o n N i l
and N 1 4 n i t r o g e n a t o m
Car
Calk a 3
~1
if2
~3
~
~1
~2
~3
1a
113.6 °
121.4 °
121.0 °
356.0 °
109.0 °
111.0 °
114.4 °
334.4 °
2"
110.7 °
120.7 °
118.1 °
349.5 °
109.0 °
113.5 °
110.0 °
332.5 °
3"
110.9 °
120.8 °
117.9 °
349.6 °
109.1 °
114.1 °
110.5 °
333.7 °
4b"
109.6 °
122.5 °
122.5 °
354.6 °
110.9 °
112.1 °
112.1 °
335.1 °
4 b**
109.6 °
122.6 °
122.6 °
354.8 °
110.9 °
112.1 °
112.1 °
335.1 °
5 b$
112.3 °
122.4 °
122.4 °
357.1 °
109.5 °
112.1 °
112.1 °
333.7 °
5 b$$
113.0 °
121.8 °
121.8 °
356.6 °
109.7 °
115.9 °
115.9 °
341.5 °
a X - r a y data [22]. b Theoretical calculations. * N-quinoline-axial, N-methyl-equatorial. ** N - q u i n o l i n e - e q u a t o r i a l , N - m e t h y l - e q u a t o r i a l . $ N-pyrimidyl-equatorial, N-methyl-equatorial. ss N - p y r i m i d y l - e q u a t o r i a l , N - m e t h y l - a x i a l .
positions of the aromatic and piperazine rings can be characterized by the pyramidization of the N l l nitrogen atom. Quantitatively, the pyramidization can be described in two ways: by the dihedral angle ~bbetween the aromatic ring plane and the plane passing through the central nitrogen atom and two nearest-neighbouring carbon atoms of the piperazine ring, or by the sum of three bond angles at the N11 nitrogen atom. The lowest total molecular energy in the 5 molecule was obtained for ~ = 164.5 °, corresponding to the equatorial position of the aromatic ring treated as a substituent of the nitrogen atom of the piperazine ring. Three bond angles at the nitrogen edge adopted the values ott -- 112.3 °, or2 = 122.4 °, ct3 = 122.4 ° (Table 6) that summed up to 357.1 °, characterizing a nearly planar (spZ-like) nitrogen atom attached to the pyrimidine ring. A search for another relative position that could be a stationary point on the molecular energy hypersurface was unsuccessful. In particular, we were unable to find any stable conformation corresponding to the axial position of the aromatic ring. We estimated only, by means of the ~b angle scanning, that the axial and equatorial positions should differ by some 2.1 kJ mo1-1. A slightly different situa-
tion occurs in compound 4, though the energy changes are even less significant. We found a stable axial position of the quinoline ring at ~b -- 200.9 ° and a slightly more stable (by about 1.0 kJ mo1-1) equatorial position at ~b -- 156.3 °, separated by a potential barrier of only 1.6 kJ mo1-1. The corresponding bond angles at the N l l nitrogen atom summed up to 354.6 ° and 354.8 ° (Table 6), exhibiting bigger deviations from planar configuration than for compound 5. In both cases (axial and equatorial) deviation from planarity was almost the same. It should be noted that a difference in pyramidization of the N11 nitrogen atom found by theoretical calculations was also observed experimentally in the solid state of compounds 1, 2 and 3, where the sum of the corresponding angles was found to be 356.0 °3, 349.5 ° and 349.6 ° respectively. These results show that pyramidization for compounds containing a quinoline aromatic ring is much bigger than for the pyrimidine analogue.
3 A v e r y g o o d a g r e e m e n t b e t w e e n the c a l c u l a t e d and e x p e r i m e n t a l value.
204
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
The pyramidization of another nitrogen atom in the piperazine ring becomes more prominent. The respective ab initio RHF/6-31G** calculations were performed for the 5 molecule, in which the N-CH3 moiety was placed in the equatorial and in the axial position with respect to the piperazine ring. The three bond angles at the N14 nitrogen edge summed up to 333.7 ° and 341.5 ° for equatorial and axial N-methyl substituent respectively, characterizing a rather large pyramidization of the nitrogen atom (sp3-1ike). The
393K
\ 388K
\
373K ~
363K ~
~
~/~ /
361 K_
353K
3 2 3 ~ .
sum of the calculated angles for the equatorial N CH3 group corresponded very well with the experimental value. The computed total molecular energy of the optimized 5 structure with the equatorial N-CH3 moiety was better, by some 28.5 kJ mo1-1, than the energy of the structure with the axial N-CH3 group. Therefore, one can expect that the equatorial relative position of the piperazine and butyl moieties should predominate over the axial position. The results of the ab initio quantum mechanical calculations and the experimental (X-ray) data suggest that although the relative position of the Nmethyl group in the piperazine ring should be equatorial, aromatic and piperazine rings can adopt almost equivalent axial and equatorial conformations (and a variety of conformations in between). This also suggests that their relative positions can be significantly affected by environmental effects. Therefore, although it is reasonable to assume that the lowest energy chair conformations of compounds 1-3 correspond to the equatorial Nil-equatorial N14 orientation, the conformations with axial Nil-equatorial N14 are expected to be almost equivalent. For monohydrochlorides of buspirone (1), kaspar (2) and mesmar (3) with a protonated N14 nitrogen atom (1H NMR spectra in CDC13 at 500 MHz) the interchange between piperazine axial and equatorial protons at room temperature was not observed, suggesting that at that temperature the ring inversion was NMR slow. The ring inversion can, however, be observed at elevated temperatures. A difference between free base and hydrochloride dynamic processes can be explained in terms of higher activation energy (caused by the hindering of the inversion on the protonated N14 nitrogen atom) for the conformational process of protonated species [25]. The Gibbs free energy of activation for the conformational process can be estimated from the observed coalescence temperature by Eq. (1) [25]: AG*(Jmol- 1) = 19.14Tc[9.97+log(Tc/Ap)]
(1)
Analysis of the variable-temperature 1H NMR (200 MHz) spectra for a solution of hydrochloride of compound 1 in DMSO 4 allowed us to determine the coalescence for the piperazine ring. The chemical Fig. 6. Effect of temperature on 200 MHz 1H NMR spectra of kaspar (2) hydrochloride in DMSO.
4 DMSO was used because of its high boiling point.
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
shift separation (Au) between H12/12'e and H12/12'a protons was determined to be 242 Hz from the spectrum at 292 K. Since protonation on the N14 nitrogen atom removes the lone pair, the chemical shift difference for the geminal pair of protons H13/13' should decrease substantially [26]; hence a value of 86 Hz was measured (at 292 K). Because of the difference between the values of the chemical shift separation for the geminal pairs of protons in the piperazine ring, two different coalescence temperatures (To) were observed: 363 _+ 2 K (for H13/13'e and H13/13'a protons) and 393 ___ 3 K (for H12/12'e and H12/12'a protons). Substitution of these values into Eq. (1) leads to an estimated barrier (AG*) of 73.5 _+ 0.4 kJ mol-I and 76.5 _+ 0.6 kJ mo1-1 for the geminal pair of protons H13/13' and H12/12', respectively; the error limit was based on the indicated error range at the coalescence temperature. Similar results were obtained for kaspar hydrochloride (2); the value of the energy barrier was 74.5 __+ 0.4 kJ mo1-1 (for H13/13' protons) and 75.8 - 0.4 kJ mo1-1 (for H12/ 12' protons) (Fig. 6). Independent variable-temperature IH NMR (500 MHz) spectra were recorded for the solution of free base of buspirone (1) in CD2C12. In that case the coalescence temperature (To) was 208 __+2 K, and an estimated Gibbs free energy activation (AG*) value derived from Eq. (1) was 38.9 _+ 0.4 kJ mo1-1. An experimentally determined AG* for the chairchair inversion of the piperazine ring of buspirone hydrochloride (1) was higher than some values reported for smaller molecules. For example, using the coalescence point method, Harris and Spragg [27] determined a n E a of 52.7 kJ mo1-1 (12.6 kcal mol -L) for the chair-chair inversion of N,N'dimethylpiperazine. By maintaining the sample pH at a low value, Sudmeier and Occupati [28] managed to study N,N'-dimethylpiperazine at a higher temperature and derived an activation energy value of E a ~ 61.1 kJ tool -1 (14.6 kcal mo1-1) for the chairchair conversion. However, for bigger molecules, such as dipiperazine compound Hoechst 32985 substituted with bulky methyl and aromatic groups, investigated by Cross et al. [25], the Gibbs free energy of activation (AG*) for the chair-chair inversion was found to be 69.4 _+ 0.4 kJ mol-1. In 1H NMR spectra of free bases and hydrochlorides of all compounds (1-3) the resonances
205
corresponding to piperazine protons were broadened in the whole range of temperatures investigated, suggesting that some additional exchange process can occur. When the temperature of a hydrochloride or base solution was decreased, no significant changes of the 500 MHz spectra (in CD2C12 solution) were observed except for the kaspar (2) hydrochloride spectrum at 183 K, where the H13/13'e proton resonance split into two lines separated by 10.6 Hz - - the value corresponding to the geminal coupling constant in a piperazine ring [26]. The broadening effect could be attributed to an inversion on the non-protonated N l l nitrogen atom and the subsequent changing of the relative position of the heteroaromatic ring with respect to the piperazine. In spite of the lower temperature the process remains NMR fast, although at 183 K the influence of the N i l nitrogen inversion on the H13/13'e protons most distant from the aromatic ring becomes relatively small (we were not able to reach spectra at a lower temperature because of solubility reasons). (We did not consider the rotation around the Nll-Caromatic bond because our previous calculations with the aid of the ab initio RHF/3-21G method have shown that in 1-(2-pyrimidyl)piperazine, which serves as the model of the buspirone (1) aromatic-piperazine part, the respective rotation barrier was 66.9 kJ mol-i high [22], being thus within the range of the chair-chair interconversion.) Another dynamic process observed for kaspar (2) hydrochloride corresponded to a broad resonance at 7.69-7.75ppm (1H NMR spectrum in CDC13 at 500 MHz, room temperature) attributed to the H8 aromatic proton. On lowering the temperature to 183 K the resonance transformed into a doublet with coupling constant 7.78 Hz. The observed dynamic effect can be explained in terms of hydrogen transfer from the environment (inter- or intramolecular) to the quinoline-N1 nitrogen atom, resulting in a broadened resonance line of the neighbouring H8 proton. IH NMR spectra (500 MHz) of buspirone (1) and kaspar (2) dihydrochlorides have shown four broadened resonances corresponding to piperazine protons, thus indicating NMR fast inversion on the piperazine N11 nitrogen atom and the presence of a (not protonated) lone pair on the atom. Thus the secondary protonation site can be assigned to the aromatic N1 (pyrimidine or quinoline) nitrogen atom.
206
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
expect that in the putative receptor binding site, where the protonated N14 nitrogen atom is expected to bind to aspartic acid residue (Asp 116 is located in the third transmembrane domain of the 5-HT tA receptor [29]) (Fig. 7), the piperazine ring adopts a rather stiff conformation with respect to the chair-chair interconversions. The aromatic substituent at Nll nitrogen atom exhibits, however, a relative freedom to change its configuration from axial to equatorial, with a very small preference for equatorial. Thus the active relative configuration of aromatic and piperazine moieties at the receptor binding site may be dependent only on the receptor structural requirements. The problem of the relative configuration of the aromatic and piperazine moieties, and thus the
4. Conclusions
In free bases of buspirone (1), kaspar (2) and mesmar (3) at room temperature the piperazine ring undergoes relatively fast chair-chair interconversions with an estimated energy barrier falling within the range of 40 kJ mo1-1 (38.9 _+ 0.4 kJ mo1-1 for compound 1). In the protonated species the inversion on the N14 nitrogen atom is hindered and the respective energy barrier is considerably higher, ca. 75 kJ mo1-1 (76.5 +__0.6 kJ mo1-1 for compound 1), making the process NMR slow. The protonated molecules, however, still have the possibility of inversion on the Nll nitrogen atom, allowing permanent change of the N14-aromatic ring relative distance. One may thus
I
Cys 1g4 o
"l______2j
i~rtCO2H ~
Ext III
f
~ 2 ~ O
3~ 106
NIt2 Fig. 7. Putative interactions of aspartic acid residues with N1 and N14 nitrogen atoms of compound 2.
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
relative distance between the aromatic ring centroid and the N14 nitrogen atom, corresponds to agonisticantagonistic activity of the 5-HTIA receptor ligands since it was suggested [5,6] that for agonists the distance should fall in the range of 5.2 A and for antagonists 5.6 ,~. Another important observation stems from the fact that in kaspar (2) monohydrochloride the hydrogen atom, formerly assumed to be entirely connected to the N14 nitrogen atom (which further is assumed to bind to Asp 116 residue), can be exchanged at room temperature between two protonation sites - - piperazine N14 and aromatic N1 nitrogen atoms. The open question is whether a similar hydrogen exchange can occur at the receptor binding site and the protonated N1 nitrogen atom can bind to Asp 116 or to the other aspartic acid residue - - Asp 82 - - located in the second transmebrane domain. It should be noted that both aspartic acid residues (Asp 82 and Asp 116) were expected to be involved in binding the N14 nitrogen atom (corresponding to dipropylamino moiety in 8hydroxydipropylaminoteralin, a potent 5-HTIA agonist). We believe that all possibilities discussed here can be used for the refinement of interactions for the known homology models of the 5-HT~A receptor with its ligands (for a review see Refs. [30,31]).
Acknowledgements We thank Dr S. Szymariski for valuable discussions. This study was supported by the Polish State Committee for Scientific Research (KBN), Grant No. 6 6378 92 03 (1992-1995).
References [1] J. Traber and T. Glaser, Trends Pharmacol. Sci., 8 (1987) 432. [2] D.L. Murphy, K.P. Leach, C.S. Aulakh and T.A. Pigott, Pharmacol. Rev., 43 (1991) 527. [3] J. Cybulski, Z. Chilmonczyk, W. Szelejewski, K. Wojtasiewicz and J.T. Wr6bel, Arch. Pharm. (Weinheim), 325 (1992) 313.
207
[4] J. Cybulski, K. Wojtasiewicz, Z. Chilmonczyk, M. Bogdal and A. Wo~niakowska, Pol. Pat. Appl. 299147 (1993). [5] M.F. Hibert, M.W. Gittos, D.N. Middlemiss, A.K. Mir and J.R. Fozard, J. Med. Chem., 31 (1988) 1087. [6] M.F. Hibert, I. McDermott, D.N. Middlemiss, A.K. Mir and J.R. Fozard, Eur. J. Med. Chem., 24 (1989) 31. [7] A. Bax and G. Morris, J. Magn. Res., 64 (1981) 501. [8] P.J. Black, R.D. Brown and M.L. Heffernan, Aus. J. Chem., 17 (1964) 558. [9] K. Nukada, O. Yamamato, T. Suzuki, M. Takeuchi and M. Ohinishi, Anal. Chem., 35 (1963) 1892. [10] N.F. Chamberlain, F.C. Stehling, K.W. Bartz and J.J.R. Reed, Nuclear Magnetic Resonance. Data for hydrogen-l, Esso Research and Engineering Company, Box 4255, Babytown, TX 77520, USA (1965). [11] M.C. Caserio, R.E. Pratt and R.J. Holland, J. Am. Chem. Soc., 88 (1966) 5747. [12] A.J.R. Bourn, D.G. Gillies and E.W. Randall, Tetrahedron, 22 (1966) 1825. [13] R.J. Pugmire, DM. Grant, M.J. Robins and R.K. Robins, J. Am. Chem. Soc., 91 (1969) 6381. [14] J.T. Clerc, E. Pretsch and S. Sternhell, 13C-Kernresonanzspektroskopie, Akademische Verlagsgesellschaft, Frankfurt, 1973. [15] R. Kutschan, Tetrahedron, 33 (1977) 1833. [16] I. Morishima, K. Yoshikawa, K. Okada, T. Yonezawa and K. Goto, J. Am. Chem. Soc., 95 (1973) 165. [17] J.G. Batchelor, J. Feeney and G.C.K. Roberts, J. Magn. Res., 20 (1975) 19. [18] H. Eggert and C. Djerasii, J. Am. Chem. Soc., 95 (1973) 3710. [19] G. Ellis and R.G. Jones, J. Chem. Soc., Perkin Trans. II (1972) 437. [20] I. Morishima, K. Yoshikawa, K. Okada, T. Yonezawa and K. Goto, J. Am. Chem. Soc., 93 (1971) 3922. [21] R.A. Newmark and J.R. Hill, J. Magn. Res., 21 (1976) 1. [22] Z. Chiimonczyk and A. Le~ A. Wo~niakowska, J. Cybulski, A.E,. Koziot and M. Gdaniec, J. Med. Chem., 38 1701 (1995). [23] F.A.L. Anet and I. Yavari, Tetrahedron Lett., 25 (1976) 2093. [24] J.B. Lambert and R.G. Keske, J. Am. Chem. Soc., 88 (1966) 620. [25] K.J. Cross, K.J. Embrey, M. Coles, W. Bicknel and D.J. Craik, Magn. Res. Chem., 32 (1994) 509. [26] J.B. Lambert, R.G. Keske, R.E. Carhart and A.P. Jovanovich, J. Am. Chem., 89 (1967) 3761. [27] R.K. Harris and R.A. Spragg, Chem. Comm., 12 (1966) 314. [28] J.L. Sudmeier and G. Occupati, J. Am. Chem. Soc., 90 (1968) 154. [29] J. Sylte, O. Edvardson and S.G. Dahl, Prot. Eng., 6 (1993) 691. [30] W.C. Probst, L.A. Snyder, D.I. Schuster, J. Brosius and S.C. Sealfon, DNA and Cell Biol., 11 (1992) 1. [31] Z. Chilmonczyk, J. Pharm. Pharmacol., 47 (1995) 791.
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 385 (1996) 195-207
NMR studies of buspirone (an anxiolytic drug) analogues Zdzistaw
Chilmonczyk
a'*, J a c e k C y b u l s k i a, A g n i e s z k a Andrzej Leg b
Szelejewska-Wo~niakowska
a,
"Pharmaceutical Research Institute, 8 Rydygiera Street, 01-793 Warszawa, Poland bDepartment of Chemistry, University of Warsaw, 1 Pasteura Street, Warszawa, Poland
Received 1 April 1996; accepted 1 July 1996
Abstract
Conformations of piperazine rings in 8-{4-[4-(2-pyrimidyl)-l-piperazinyl]butyl}-8-azaspiro[4.5]-decane-7,9-dione (buspirone - - 1) and its two analogues 8-{4-[4-(2-quinolinyl)-l-piperazinyl]butyl}-8-azaspiro[4.5]-decane-7,9-dione (kaspar - - 2) and 4,4-dimethyl-l-{4-[4-(2-quinolinyl)-l-piperazinyl]butyl}-2,6-piperidinedione (mesmar - - 3) (Fig. 1) have been studied with the aid of 1H NMR and 13C NMR spectra. For free bases the two bands corresponding to piperazine hydrogen atoms in the spectra broaden considerably with a decrease in temperature to divide into four separate bands, indicating the presence of a dynamic exchange process. A similar dynamic process, but for higher temperatures, was observed for buspirone (1), kaspar (2) and mesmar (3) hydrochlorides. Proton and carbon atom resonance lines have been assigned with the aid of 2D COSY and 2D HETCOR two-dimensional spectra.
1. Introduction
The mechanism of activity of some anxiolytics has been suggested as being based mainly on an interaction with serotonin 5-HT1A receptor. Thus many research laboratories [1] have concentrated their work on the synthesis of the ligands of this receptor. One of such compounds, 8-{4-[4-(2-pyrimidyl)-lpiperazinyl]butyl }-8-azaspiro[4.5]-decane-7,9-dion hydrochloride (buspirone) (1), has been introduced into clinical practice [2]. This anxiolytic has been found to exhibit much smaller sedative effects than the widely used benzodiazepines. In our laboratory we have synthesized new * Corresponding author.
buspirone analogues, 8-{4-[4-(2-quinolinyl)-l-piperazinyl]butyl }-8-azaspiro[4.5]-decane-7,9-dione hydrochloride (kaspar) (2) and 4,4-dimethyl-l-{4-[4-(2quinolinyl)- 1-piperazinyl]butyl}-2,6-piperidinedione hydrochloride (mesmar) (3) [3]. Their affinity to the 5-HT1A receptor is close to that of buspirone (1) [4], suggesting that they might possess a similar profile of pharmacological action. In order to continue the search for new chemical structures of anxiety-relieving character it is necessary to examine the interaction occurring between the ligand and the 5-HTIA receptor binding site. It is supposed that the basic molecular features (basic pharmacophore) responsible for the binding of buspirone-like molecules to the 5-HTIA receptor are an aromatic ring and a basic nitrogen atom placed at a
0022-2860/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0022-2860(96)09404-5
196
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
specified distance (5.2-5.7 .~,) from the aromatic ring centroid. The pharmacophore was further differentiated for agonists and antagonists with respect to the nitrogen atom out of the aromatic ring plane deviation (0.2 A and 1.6 ,~ respectively) [5,6]. In buspirone (1) and its analogues 2 and 3 the basic nitrogen atom is located in the piperazine ring (N14). Thus the conformational dynamic of piperazine and aromatic moieties has a direct influence on the relative Nbasic-aromatic ring centroid distances included in the 5-HT 1A pharmacophore. In this paper we focus on the conformational analysis of the piperazine ring and identify additional protonation sites in buspirone (1) and its analogues in nonpolar (CD2C12, CDC13) and polar (DMSO) solutions with the aid of 1H NMR and 13C NMR spectra.
O
0
5
I.
4
I. 8
112 ~ 1
15
11 ~
14 '
17 16
19 i 18
'
12 13
\/
/'~"7",,' O 20 ~"
I "~' 23 zo
2. E x p e r i m e n t a l O
2.1. 11-1and 13C N M R data
The compounds were prepared and purified as described previously [3]. NMR spectra were recorded on a Bruker AM 500 NMR spectrometer (equipped with an Aspect 3000 computer using the DISNMR program version 890101.1.) operating at 500.13 and 126.76 MHz for 1H and 13C, respectively, and on a Varian Gemini 2000 spectrometer at a frequency of 199.98 MHz for ~H, equipped with a 5 mm 13C/1H dual probe. Approximately 10-20 mg of compounds 1-3 were dissolved in 0.6 ml CDCI3, CD2C12 or DMSO with TMS as internal standard. Conventional 1H NMR spectra at 500 MHz instrument were acquired using 7000-8000 Hz spectral widths digitized with 64 K data points with a pulse width of 8 #s (ca. 60 ° flip angle). Digital resolution for the real part was 0.254 Hz/PT. Conventional 13C NMR spectra (at 500 MHz) were acquired using a 30000 Hz spectral width digitized with 64 K data points with a pulse width of 3/xs (ca. 45 ° flip angle), 2 s delay between pulses and waltz 16. Standard pulse conditions were used for 1D NMR spectra at 200 MHz instrument with a sweep width of 3000 Hz digitized with 16 K data points (for 1H spectra) and 12500 Hz digitized with 32 K data points (for 13C spectra) and a pulse width of 18/~s (ca. 49 ° flip
O
C 3N 3 \
/
N---CH3
Fig. 1. Structures of buspirone (1), kaspar (2), mesmar (3), 1-(2quinolinyl)-4-methylpiperazine(4) and 1-(2-pyrimidyl)-4-methylpiperazine (5).
Z. Chilmonczyk et aL/Journal of Molecular Structure 385 (1996) 195-207
angle) and 20 #s (ca. 72 ° flip angle) for 1H and 13 C NMR spectra, respectively. The 2D COSY and HETCOR spectra in CDCI3 were measured with a Varian Gemini 2000 instrument. 2D homonuclear-correlated experiments were carded out using Jeenerm's two-pulse sequence. Typical spectral parameters were as follows: 1700 Hz in both
197
dimensions; 16 transients for each FID; 128 tl increments; recycling delay 1 s; and data matrix 512 x 512 for processing. 2D heteronuclear-correlated spectra were obtained using the sequence (90°, 1H)-t1/2(180°, 13C)-tl/2-rl-(90°, XH; 90°, 13C)-r2-(BB, 1H)acquire [7]. The fixed delays correspond to the coupling J (aac, 1H) = 140 Hz. Other parameter settings were 128 FIDs in the t~ domain and 4 K data points in the t2 domain, 400 transients for each tl increment and recycling delay of 1 s. 2.2. Computational quantum chemistry The ab initio RHF/6-31G** calculations of the optimal molecular structure of model compounds 1-(2-quinolinyl)-4-methylpiperazine (4) and 1-(2pyrimidyl)-4-methylpiperazine (5) (Fig. 1) were performed. The analytical first and second derivatives implemented in the Gaussian 92 code (installed on an IBM RISC 6000 workstation) with respect to the geometrical parameters, subject to certain constraints of molecular symmetry, were used. In compound 5 we used a symmetry plane passing through both piperazine nitrogen atoms and perpendicular to the aromatic plane of the pyrimidine ring. In order to reduce extensive computations in 4, the local symmetry planes were enforced also in the piperazine ring and in the quinoline aromatic ring. The present optimization procedure takes advantage of the assumption that the quinoline ring only weakly affects the piperazine symmetry.
300 K ...... ~ . ~ ..........
223 K . . . . . . . J " . . . . . .
J
........ J .....
3. Results and discussion
The temperature behaviour of the kaspar (2) free base and hydrochloride spectra is consistent with a dynamic process of conformation interconversions. Figs 2 and 3 show that four resonances from the aliphatic region of the 1H NMR spectra (500 MHz) (3.97, 3.05, 2.92, 2.11 ppm - - base; 4.55, 3.97, 3.54, 2.92 ppm - - hydrochloride) observed at low temperatures (153 K - base; room - - hydrochloride) progressively broaden and coalesce as the temperature is increasedk Similar
153 K , - ~ - ~
'
•
' 1
. . . .
4.0
i
. . . .
i
. . . .
3.0
i
. . . .
i
. . . .
2.0
i
. . . .
i
. . . .
1.0 PPM
Fig. 2. Effect of temperatureon the aliphatic region of IH NMR spectra (500 MHz): (a) kaspar (2) free base in CD2C12and CFCI3; (b) kaspar (2) hydrochloridein CDC13.
1In chloroformthe signals did not coalesceuntil 333 K. Coalescence was achievedin DMSO (200 MHz) at 363 -+ 1 K (for the geminal pair of protonsH13/13') and at 393 -+ 3 K (for the H12/ 12'e and H12/12'a protons).
198
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
-,-,'~'~
333 K
.~--'"-"~
~
~
.
~,
i
4.6
4.4
4.2
4.0 3.8 3.6 3.4 3.2 3.0 2.8 ppm Fig. 3. Effectof temperatureon the aliphaticregionof IH NMR spectra (500MI-lz)of kaspar (2) hydrochloridein CDCI3. spectral patterns were observed for buspirone (1) and mesmar (3) free bases and hydrochlorides. Two structural units i.e. the piperazine ring and the n-butyl spacer, have enough conformational freedom to be considered as responsible for this dynamic behaviour. The assignment of spectral resonances is thus necessary for identifying the processes occurring in the systems investigated.
3.1. 1H and 13C NMR assignment 3.1.1. Kaspar (2) and mesmar (3) hydrochlorides 3.1.1.1. A. 1H NMR spectrum (Table 1). In the lowfield region only the signals corresponding to the quinoline ring protons were found. On the basis of the chemical shift and multiplicity [8] the following assignments were made: the doublet at 8 = 6.98 ppm (J = 8.60 Hz - - compound 2; J = 9.09 Hz - -
compound 3) - - H3, the doublet at/~ = 7.98 ppm (J = 9.08 Hz - - compound 2; J = 9.05 Hz - - compound 3) - - H4, the broad multiplet at ~i = 7.69-7.74 ppm - - H8, the doublet at/~ -- 7.65 ppm, J = 8.00 Hz - - H5 and the multiplets at 8 = 7.59 and t5 = 7.31 ppm to the H7 and H6 protons, respectively. The observed chemical shift values and splittings of respective multiplets resulting from the absorption of H3, H4, H5, H6, H7 and H8 protons are the same for compounds 2 and 3 (Table 1). The assignment of the signals corresponding to the protons from the imide fragment was based on comparison with the spectrum of the unsubstituted imide: 8 = 2.62 ppm in compound 2 and 8 = 2.54 ppm in compound 3 to H21/21', and ~ = 1.07 ppm (compound 3) to the H23/ 23' methyl group protons. The four-proton multiplets at t5 = 1.51 and 8 -- 1.71 ppm (compound 2) were assigned to H23/23' and H24/24' respectively. Taking into consideration the direction of changes
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
199
Table 1 IH NMR chemical shifts and splittings of hydrochlorides of compounds 2 and 3 a,b Proton No.
1H NMR compound 2
3 4 5 6 7 8 12,12'
6.98 7.98 7.65 7.31 7.59 7.74
3 d d m m m m
6.98 7.98 7.65 7.31 7.59 7.74
2D C O S Y c o m p o u n d
2D H E T C O R compound
2
2
3
d d m m m m 4.55 3.97 3.54 2.91
13,13' 15
16 17 18 21,21' 23,23' 24,24'
3.79 t 2.62 s
3
3.00 m 1.92 m
2.99 m 1.93 m
1.62 quint
1.63 quint
3.80 t 2.54 s 1.07 s
m m m m
4.56 3.97 3.55 2.92
m m m m
1.51 m 1.71 m
a tS(1H) in ppm f r o m T M S . b s = singlet, d = doublet, t = triplet, quint = quintuplet, m = multiplet.
of the chemical shift values tS(1H) for piperazine derivatives [9-11], and the fact that equatorial protons are deshielded by about 0.1-0.7ppm in comparison with axial ones, the H12/12' protons could be assigned two broad, diproton multiplets at 6 -- 4.55 ppm (for H12/12'e protons) and 6 = 3.97 ppm (for H12/12'a protons) (compound 2), and 6 = 4.56 ppm (for H12/12'e protons) and 6 = 3.97 ppm (for H12/12'a protons) (compound 3) and t5 = 3.54 ppm (for H13/13'e protons) and 6 = 2.91 ppm (for H13/13'a) (compound 2), 6 = 3.55 ppm (for H13/13'e protons) and 6 = 2.92 ppm (for H13/13'a) (compound 3) (the above assignments for the piperazine and imide moieties were further confirmed by the 2D HETCOR spectra). The diproton triplets at 6 = 3.79ppm, J = 6.93Hz (compound 2) and at 6 -- 3.80 ppm, J --- 6.94 Hz (compound 3) were assigned to the H18 protons on the basis of the characteristic deshielding effect exerted by the imide group [10,12]. According to the inductive substituent effect, the methylene protons H16 and H17 should give upfield signals as compared to H13/13' and H15 (at ot carbon atoms to N÷14). The signals at 6 = 1.92 ppm (compound 2), 6 = 1.93 ppm (compound 3) were assigned to the H16
'F2
"~
3-
i
dE
II
~
ii
•
11 5Z 6-"
8-"
p, "
8
7
6
5
F1
d
3
(ppm)
Fig. 4 . 2 D C O S Y spectrum of kaspar (2) hydrochloride in CDCI 3 at 200 MHz.
200
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
protons bonded to the ~ carbon atom with respect to the quaternary N14 nitrogen atom. The analysis of nondiagonal signals in 2D COSY spectra (Fig. 4) allowed for signal assignment to the protons bonded to C15 and C16 and C17 carbon atoms: ~ = 3.00 ppm - - H15 (compound 2), ~ = 2.99 ppm - H15 (compound 3); ~ = 1.92 ppm - H16 (compound 2), ~ = 1.93 ppm - - H16 (compound 3); 6 = 1.62 ppm - - H17 (compound 2), ~ = 1.63 ppm -H17 (compound 3). Application of the heteronuclear chemical shift correlation 1H-13C 2D HETCOR allowed us to assign the signals to the protons of the imide fragment ~ = 1.51ppm - - H23/23', 5 = 1.71 ppm - - H24/24' (compound 2) (Table 1). 3.1.1.2. B. 13C N M R (Table 2). The highest chemical
shifts were assigned to the carbon atoms of the carbonyl groups of the imide fragment: C20/20' ~ = 172.31ppm (compound 2), 5 = 172.02ppm (compound 3). By using empirical parameters for calculating chemical shifts in polyheteronuclear systems [13,14] the resonance lines were assigned to carbon atoms C2, C3, C4, C9 and C10 (Table 2).
Substitution of quinoline in position 2 by the piperazine system results in a strong paramagnetic effect on the C2 carbon atom (+4.70ppm with respect to unsubstituted quinoline), which is due to the induction effect of the nitrogen atom. The coupling effect of the lone electron pair of the nitrogen atom with the heteroaromatic ring is also visible in the form of a mesomeric effect, which results in the shielding of carbon atoms in positions ortho and para with respect to the substituent. Therefore the C3 carbon atom exhibits a diamagnetic effect of about -12.18 ppm (compounds 2 and 3), whereas for C10 the effect is weaker, being equal to - 5.47 ppm (with respect to the unsubstituted quinoline). The assignment of the resonance lines to carbon atoms C21/21', C22, C23/23' for compounds 2 and 3 and to C24/24' for compound 2 (for list of assignments see Table 2) was based on the comparison of 13C NMR spectra of compounds 2 and 3 with those of respective imides and the data for the spiro[4.5]decane system [15]. Signals of the carbon atoms C17, C16, C15, C13/13' and C12/12' can be assigned from the effect of the protonated N14
Table 2 13C NMR chemical shift of hydrochlorides of compounds 2 and 3 a Carbon No.
2 3 4 5 6 7 8 9 10 12,12' 13,13' 15 16 17 18 20,20' 21,21' 22 23,23' 24,24'
t3C NMR compound
2D HETCORcompound
2
3
155.60 109.32 138.32
155.56 109.32 138.33
146.76 123.24 42.28
172.31 44.62 39.34 37.41 24.02
a ~(13C)in ppm from TMS.
2
3
127.23 123.40 129.92 126.47
127.22 123.39 129.91 126.42
51.26 56.82 20.52 24.93 37.80
51.33 56.77 20.56 24.88 37.76
146.78 123.21 42.45
172.02 46.12 29.01 27.53 -
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
Table 3 Calculated data on the effect of protonation on chemical shift values ~3Cof carbon atoms of the butyl chain, substituted by amine group (-NRIR2) [17,18] Ci
8~÷
ASci= 83-80 N. N
C,~ C~ C~ C~
52.1 24.2 27.0 32.9
- 2.3 - 3.8 - 0.5 0.0
nitrogen atom. According to the known data [16,17], one could expect a different diamagnetic effect for the carbon atoms in the positions or, /3, 3, and ~ with respect to the protonated nitrogen atom. Thus the effect of the N14 nitrogen atom protonation on the chemical shift values was calculated [17,18] (Table 3). The size of the diamagnetic effect which appeared after the protonation of the N14 nitrogen atom was experimentally determined from the 13C N M R spectra for compounds 2 and 3 in the form of salt and free base (Table 4). A comparison of experimental and calculated values allowed us to differentiate between o~, /3 and 3' (but not 6) carbon atoms. Thus, signals at 8 = 56.82 and 51.26 ppm (compound 2) and 56.77 and 51.23 ppm (compound 3) were assigned to C15, C13/13' carbon atoms; the signals at 8 = 42.28 and 20.52 ppm (compound 2) and 6 = 42.45 and 20.56 ppm (compound 3) to the C12/12' and C16 carbon atoms (Ct~)
Table 4 The effect of protonation of the nitrogen atom on the chemical shift value aaC of the aliphatic carbon atoms of compounds 2 and 3 C~
~s+ (2)'
A8 s÷'~ (2) 6s+ (3)
ASN÷,N(3)
C~-C13 or C15 C~-C13 or C15 Ca-C12, C12' Ca-C16 Cv-C17 C~-C18 C21, C21' C22 C23, C23' C24, C24'
56.82 51.26 42.48 20.52 24.93 37.80 44.62 39.34 37.41 24.93
-
-
a Compound 2.
1.42 1.85 2.43 3.44 1.11 1.54 0.44 0.11 0.14 0.16
56.77 51.23 42.45 20.56 24.88 37.76 46.12 29.01 27.53 -
-
1.47 1.90 2.63 3.70 1.16 1.57 0.35 0.09 0.15
201
respectively 2, and the signals at 8 = 24.93 ppm (compound 2) and 24.88 ppm (compound 3) to the C17 carbon atoms (C.r). On the basis of the available data concerning the chemical shift values of N-substituted amides [21] the signals at <5 = 37.80 ppm (compound 2) and 6 = 37.76 ppm (compound 3) were assigned to the C18 carbon atoms (Ca). It should, however, be noted (Table 4) that the chemical shift values of the C18 carbon atoms in protonated compounds showed a diamagnetic effect of the order of -1.5 ppm. This effect is typical of the Ca atom whereas the C18 atom occupies a 8 position with respect to the protonated N14 nitrogen atom. Crosspeak signals at the intersection of the x- and y-axes of the contour diagram of the 2D H E T C O R spectra of compounds 2 and 3 allowed us to perform the following assignments: 8 = 127.23 ppm - - C5 (compound 2), d = 127.22ppm - - C5 (compound 3); 8 = 123.40 ppm - - C6 (compound 2), 8 = 123.39 ppm C6 (compound 3); (5 -- 129.92 ppm - - C7 (compound 2), 8 = 129.91ppm - - C7 (compound 3); 8 = 126.40 ppm - - C8 (compound 2), 8 = 126.46 ppm C8 (compound 3); 8 = 51.56 ppm - - C13/13' (compound 2), 8 = 51.33 ppm - - C13/13') (compound 3); 8 = 56.82 ppm - - C15 (compound 2), 8 = 56.77 ppm - C15 (compound 3). A comparative analysis of N M R spectra of the free base of buspirone (1) and its hydrochloride form has been performed [22]. For the assignment of the spectral resonances of kaspar (2) and mesmar (3) free bases, see Table 5. The above assignments allowed us to attribute the coalescing resonance lines in buspirone (1), kaspar (2) and mesmar (3) to the heterocyclic piperazine ring. Since chair conformations are supposed to be the lowest energy conformers of a piperazine ring [23] the four broadened resonances at 3.97, 3.05, 2.92 and 2.11 ppm in the kaspar (2) free base 1H N M R spectrum at 153 K (500 MHz) (Fig. 2) may be assigned to H12/ 12'e, H12/12'a, H13/13'e and H13/13'a piperazine protons respectively. This assignment is based on the data for piperazine derivatives with equatorial N-methyl substituents which show that the axial
2 Substitution at the nitrogen atom of the piperazine system with the alkyl chain should reduce the shielding effect for carbon atoms in position a with respect to the nitrogen atom, whereas the opposite effect was observed for the aromatic ring as substituent [3,19,20].
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
202
Table 5 1H and 13C NMR chemical shift and splittings of free bases of compounds 2and 3 a Proton No.
lH NMR compound 2
3 4 5 6 7 8 12,12' 13,13' 15 16 17 18 21,21' 23,23' 24,24' -
6.97 7,87 7.58 7.20 7.52 7.69 3.74 2.56 2.40 1.55 1.55 3.79 2.58 1.49 1.70
Carbon No.
13C NMR compound
3 db d d m m d t t t m m t s m m
6.97 7.87 7.58 7.21 7.52 7.69 3.74 2.56 2.40 1.56 1.56 3.80 2.49 1.07 -
d d d m m d t t t m m t s s
2 3 4 5 6 7 8 9 10 12,12' 13,13' 15 16 17 18 20,20' 21,21' 22 23,23' 24,24'
2
3
157.91 110.00 137.56 127.59 123.46 129.74 126.83 148.33 122.50 45.28 53.59 58.59 24.60 26.41 39.64 172.47 45.52 39.83 37.91 24.75
157.43 109.52 137.36 127.17 123.10 129.45 126.69 147.93 122.32 45.08 53.13 58.24 24.26 26.04 39.33 171.86 46.47 29.10 27.68 -
tS(tH) and ~(13C) in ppm from TMS. b s ffi singlet, d ffi doublet, t = triplet, m = multiplet.
protons in the piperazine ring are usually at a lower frequency than equatorial ones [24]. As the temperature is increased, the H12/12'a protons appear to exchange environments with the H12/12'e protons, and a similar interchange process occurs for the geminal H13/13' protons. At room temperature two triplets at 3.74 and 2.56 ppm (H12/12' and H13/13' respectively) for the interchanged axial and equatorial protons are observed. This spectral pattern reflects the conversion of piperazine ring low energy form back to itself after passing through an intermediate conformation in which axial and equatorial protons are interchanged. The possible conformers of a substituted piperazine ring and their interconversions by ring and nitrogen inversions, leading to the axial and equatorial protons interchange, were described by Cross et al. (Fig. 5) [25] and involve at least one ring inversion and as many nitrogen inversions as are required to return the nitrogen substituents to their previous positions. It was also assumed that the conformation with equatorial substituents at both piperazine nitrogen atoms was the lowest energy conformation. For buspirone (1), kaspar (2) and mesmar (3) we calculated the relative energies of the
piperazine chair conformations (with axial and equatorial substituents at N l l and N14 nitrogen atoms) with the use of the quantum mechanical ab initio RHF/ 3-21G** method applied for model compounds 1-(2quinolinyl)-4-methylpiperazine (4), a model for kaspar (2) and mesmar (3), and 1-(2-pyrimidyl)-4-methylpiperazine (5), a model for buspirone (1). The relative Ar
N --A~
Rin v
I
I,
N
I
Ninv
x
Ninv Ar
I
M e . _ ~ N ~
--Ar ~
I
Rinv
I Me
Fig. 5. Diagrammatic representation of the possible conformers of a substituted piperazine ring and their interconversion by ring and nitrogen inversion.
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
203
Table 6 O b s e r v e d and c a l c u l a t e d p y r a m i d i z a t i o n s o n N i l
and N 1 4 n i t r o g e n a t o m
Car
Calk a 3
~1
if2
~3
~
~1
~2
~3
1a
113.6 °
121.4 °
121.0 °
356.0 °
109.0 °
111.0 °
114.4 °
334.4 °
2"
110.7 °
120.7 °
118.1 °
349.5 °
109.0 °
113.5 °
110.0 °
332.5 °
3"
110.9 °
120.8 °
117.9 °
349.6 °
109.1 °
114.1 °
110.5 °
333.7 °
4b"
109.6 °
122.5 °
122.5 °
354.6 °
110.9 °
112.1 °
112.1 °
335.1 °
4 b**
109.6 °
122.6 °
122.6 °
354.8 °
110.9 °
112.1 °
112.1 °
335.1 °
5 b$
112.3 °
122.4 °
122.4 °
357.1 °
109.5 °
112.1 °
112.1 °
333.7 °
5 b$$
113.0 °
121.8 °
121.8 °
356.6 °
109.7 °
115.9 °
115.9 °
341.5 °
a X - r a y data [22]. b Theoretical calculations. * N-quinoline-axial, N-methyl-equatorial. ** N - q u i n o l i n e - e q u a t o r i a l , N - m e t h y l - e q u a t o r i a l . $ N-pyrimidyl-equatorial, N-methyl-equatorial. ss N - p y r i m i d y l - e q u a t o r i a l , N - m e t h y l - a x i a l .
positions of the aromatic and piperazine rings can be characterized by the pyramidization of the N l l nitrogen atom. Quantitatively, the pyramidization can be described in two ways: by the dihedral angle ~bbetween the aromatic ring plane and the plane passing through the central nitrogen atom and two nearest-neighbouring carbon atoms of the piperazine ring, or by the sum of three bond angles at the N11 nitrogen atom. The lowest total molecular energy in the 5 molecule was obtained for ~ = 164.5 °, corresponding to the equatorial position of the aromatic ring treated as a substituent of the nitrogen atom of the piperazine ring. Three bond angles at the nitrogen edge adopted the values ott -- 112.3 °, or2 = 122.4 °, ct3 = 122.4 ° (Table 6) that summed up to 357.1 °, characterizing a nearly planar (spZ-like) nitrogen atom attached to the pyrimidine ring. A search for another relative position that could be a stationary point on the molecular energy hypersurface was unsuccessful. In particular, we were unable to find any stable conformation corresponding to the axial position of the aromatic ring. We estimated only, by means of the ~b angle scanning, that the axial and equatorial positions should differ by some 2.1 kJ mo1-1. A slightly different situa-
tion occurs in compound 4, though the energy changes are even less significant. We found a stable axial position of the quinoline ring at ~b -- 200.9 ° and a slightly more stable (by about 1.0 kJ mo1-1) equatorial position at ~b -- 156.3 °, separated by a potential barrier of only 1.6 kJ mo1-1. The corresponding bond angles at the N l l nitrogen atom summed up to 354.6 ° and 354.8 ° (Table 6), exhibiting bigger deviations from planar configuration than for compound 5. In both cases (axial and equatorial) deviation from planarity was almost the same. It should be noted that a difference in pyramidization of the N11 nitrogen atom found by theoretical calculations was also observed experimentally in the solid state of compounds 1, 2 and 3, where the sum of the corresponding angles was found to be 356.0 °3, 349.5 ° and 349.6 ° respectively. These results show that pyramidization for compounds containing a quinoline aromatic ring is much bigger than for the pyrimidine analogue.
3 A v e r y g o o d a g r e e m e n t b e t w e e n the c a l c u l a t e d and e x p e r i m e n t a l value.
204
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
The pyramidization of another nitrogen atom in the piperazine ring becomes more prominent. The respective ab initio RHF/6-31G** calculations were performed for the 5 molecule, in which the N-CH3 moiety was placed in the equatorial and in the axial position with respect to the piperazine ring. The three bond angles at the N14 nitrogen edge summed up to 333.7 ° and 341.5 ° for equatorial and axial N-methyl substituent respectively, characterizing a rather large pyramidization of the nitrogen atom (sp3-1ike). The
393K
\ 388K
\
373K ~
363K ~
~
~/~ /
361 K_
353K
3 2 3 ~ .
sum of the calculated angles for the equatorial N CH3 group corresponded very well with the experimental value. The computed total molecular energy of the optimized 5 structure with the equatorial N-CH3 moiety was better, by some 28.5 kJ mo1-1, than the energy of the structure with the axial N-CH3 group. Therefore, one can expect that the equatorial relative position of the piperazine and butyl moieties should predominate over the axial position. The results of the ab initio quantum mechanical calculations and the experimental (X-ray) data suggest that although the relative position of the Nmethyl group in the piperazine ring should be equatorial, aromatic and piperazine rings can adopt almost equivalent axial and equatorial conformations (and a variety of conformations in between). This also suggests that their relative positions can be significantly affected by environmental effects. Therefore, although it is reasonable to assume that the lowest energy chair conformations of compounds 1-3 correspond to the equatorial Nil-equatorial N14 orientation, the conformations with axial Nil-equatorial N14 are expected to be almost equivalent. For monohydrochlorides of buspirone (1), kaspar (2) and mesmar (3) with a protonated N14 nitrogen atom (1H NMR spectra in CDC13 at 500 MHz) the interchange between piperazine axial and equatorial protons at room temperature was not observed, suggesting that at that temperature the ring inversion was NMR slow. The ring inversion can, however, be observed at elevated temperatures. A difference between free base and hydrochloride dynamic processes can be explained in terms of higher activation energy (caused by the hindering of the inversion on the protonated N14 nitrogen atom) for the conformational process of protonated species [25]. The Gibbs free energy of activation for the conformational process can be estimated from the observed coalescence temperature by Eq. (1) [25]: AG*(Jmol- 1) = 19.14Tc[9.97+log(Tc/Ap)]
(1)
Analysis of the variable-temperature 1H NMR (200 MHz) spectra for a solution of hydrochloride of compound 1 in DMSO 4 allowed us to determine the coalescence for the piperazine ring. The chemical Fig. 6. Effect of temperature on 200 MHz 1H NMR spectra of kaspar (2) hydrochloride in DMSO.
4 DMSO was used because of its high boiling point.
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
shift separation (Au) between H12/12'e and H12/12'a protons was determined to be 242 Hz from the spectrum at 292 K. Since protonation on the N14 nitrogen atom removes the lone pair, the chemical shift difference for the geminal pair of protons H13/13' should decrease substantially [26]; hence a value of 86 Hz was measured (at 292 K). Because of the difference between the values of the chemical shift separation for the geminal pairs of protons in the piperazine ring, two different coalescence temperatures (To) were observed: 363 _+ 2 K (for H13/13'e and H13/13'a protons) and 393 ___ 3 K (for H12/12'e and H12/12'a protons). Substitution of these values into Eq. (1) leads to an estimated barrier (AG*) of 73.5 _+ 0.4 kJ mol-I and 76.5 _+ 0.6 kJ mo1-1 for the geminal pair of protons H13/13' and H12/12', respectively; the error limit was based on the indicated error range at the coalescence temperature. Similar results were obtained for kaspar hydrochloride (2); the value of the energy barrier was 74.5 __+ 0.4 kJ mo1-1 (for H13/13' protons) and 75.8 - 0.4 kJ mo1-1 (for H12/ 12' protons) (Fig. 6). Independent variable-temperature IH NMR (500 MHz) spectra were recorded for the solution of free base of buspirone (1) in CD2C12. In that case the coalescence temperature (To) was 208 __+2 K, and an estimated Gibbs free energy activation (AG*) value derived from Eq. (1) was 38.9 _+ 0.4 kJ mo1-1. An experimentally determined AG* for the chairchair inversion of the piperazine ring of buspirone hydrochloride (1) was higher than some values reported for smaller molecules. For example, using the coalescence point method, Harris and Spragg [27] determined a n E a of 52.7 kJ mo1-1 (12.6 kcal mol -L) for the chair-chair inversion of N,N'dimethylpiperazine. By maintaining the sample pH at a low value, Sudmeier and Occupati [28] managed to study N,N'-dimethylpiperazine at a higher temperature and derived an activation energy value of E a ~ 61.1 kJ tool -1 (14.6 kcal mo1-1) for the chairchair conversion. However, for bigger molecules, such as dipiperazine compound Hoechst 32985 substituted with bulky methyl and aromatic groups, investigated by Cross et al. [25], the Gibbs free energy of activation (AG*) for the chair-chair inversion was found to be 69.4 _+ 0.4 kJ mol-1. In 1H NMR spectra of free bases and hydrochlorides of all compounds (1-3) the resonances
205
corresponding to piperazine protons were broadened in the whole range of temperatures investigated, suggesting that some additional exchange process can occur. When the temperature of a hydrochloride or base solution was decreased, no significant changes of the 500 MHz spectra (in CD2C12 solution) were observed except for the kaspar (2) hydrochloride spectrum at 183 K, where the H13/13'e proton resonance split into two lines separated by 10.6 Hz - - the value corresponding to the geminal coupling constant in a piperazine ring [26]. The broadening effect could be attributed to an inversion on the non-protonated N l l nitrogen atom and the subsequent changing of the relative position of the heteroaromatic ring with respect to the piperazine. In spite of the lower temperature the process remains NMR fast, although at 183 K the influence of the N i l nitrogen inversion on the H13/13'e protons most distant from the aromatic ring becomes relatively small (we were not able to reach spectra at a lower temperature because of solubility reasons). (We did not consider the rotation around the Nll-Caromatic bond because our previous calculations with the aid of the ab initio RHF/3-21G method have shown that in 1-(2-pyrimidyl)piperazine, which serves as the model of the buspirone (1) aromatic-piperazine part, the respective rotation barrier was 66.9 kJ mol-i high [22], being thus within the range of the chair-chair interconversion.) Another dynamic process observed for kaspar (2) hydrochloride corresponded to a broad resonance at 7.69-7.75ppm (1H NMR spectrum in CDC13 at 500 MHz, room temperature) attributed to the H8 aromatic proton. On lowering the temperature to 183 K the resonance transformed into a doublet with coupling constant 7.78 Hz. The observed dynamic effect can be explained in terms of hydrogen transfer from the environment (inter- or intramolecular) to the quinoline-N1 nitrogen atom, resulting in a broadened resonance line of the neighbouring H8 proton. IH NMR spectra (500 MHz) of buspirone (1) and kaspar (2) dihydrochlorides have shown four broadened resonances corresponding to piperazine protons, thus indicating NMR fast inversion on the piperazine N11 nitrogen atom and the presence of a (not protonated) lone pair on the atom. Thus the secondary protonation site can be assigned to the aromatic N1 (pyrimidine or quinoline) nitrogen atom.
206
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
expect that in the putative receptor binding site, where the protonated N14 nitrogen atom is expected to bind to aspartic acid residue (Asp 116 is located in the third transmembrane domain of the 5-HT tA receptor [29]) (Fig. 7), the piperazine ring adopts a rather stiff conformation with respect to the chair-chair interconversions. The aromatic substituent at Nll nitrogen atom exhibits, however, a relative freedom to change its configuration from axial to equatorial, with a very small preference for equatorial. Thus the active relative configuration of aromatic and piperazine moieties at the receptor binding site may be dependent only on the receptor structural requirements. The problem of the relative configuration of the aromatic and piperazine moieties, and thus the
4. Conclusions
In free bases of buspirone (1), kaspar (2) and mesmar (3) at room temperature the piperazine ring undergoes relatively fast chair-chair interconversions with an estimated energy barrier falling within the range of 40 kJ mo1-1 (38.9 _+ 0.4 kJ mo1-1 for compound 1). In the protonated species the inversion on the N14 nitrogen atom is hindered and the respective energy barrier is considerably higher, ca. 75 kJ mo1-1 (76.5 +__0.6 kJ mo1-1 for compound 1), making the process NMR slow. The protonated molecules, however, still have the possibility of inversion on the Nll nitrogen atom, allowing permanent change of the N14-aromatic ring relative distance. One may thus
I
Cys 1g4 o
"l______2j
i~rtCO2H ~
Ext III
f
~ 2 ~ O
3~ 106
NIt2 Fig. 7. Putative interactions of aspartic acid residues with N1 and N14 nitrogen atoms of compound 2.
Z. Chilmonczyk et al./Journal of Molecular Structure 385 (1996) 195-207
relative distance between the aromatic ring centroid and the N14 nitrogen atom, corresponds to agonisticantagonistic activity of the 5-HTIA receptor ligands since it was suggested [5,6] that for agonists the distance should fall in the range of 5.2 A and for antagonists 5.6 ,~. Another important observation stems from the fact that in kaspar (2) monohydrochloride the hydrogen atom, formerly assumed to be entirely connected to the N14 nitrogen atom (which further is assumed to bind to Asp 116 residue), can be exchanged at room temperature between two protonation sites - - piperazine N14 and aromatic N1 nitrogen atoms. The open question is whether a similar hydrogen exchange can occur at the receptor binding site and the protonated N1 nitrogen atom can bind to Asp 116 or to the other aspartic acid residue - - Asp 82 - - located in the second transmebrane domain. It should be noted that both aspartic acid residues (Asp 82 and Asp 116) were expected to be involved in binding the N14 nitrogen atom (corresponding to dipropylamino moiety in 8hydroxydipropylaminoteralin, a potent 5-HTIA agonist). We believe that all possibilities discussed here can be used for the refinement of interactions for the known homology models of the 5-HT~A receptor with its ligands (for a review see Refs. [30,31]).
Acknowledgements We thank Dr S. Szymariski for valuable discussions. This study was supported by the Polish State Committee for Scientific Research (KBN), Grant No. 6 6378 92 03 (1992-1995).
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