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Conformational analysis of four spiro[cyclohexane-1,3′-indolin]-2′-one derivatives
Journal of Molecular Structure 654 (2003) 187–196 www.elsevier.com/locate/molstruc
Conformational analysis of four spiro[cyclohexane-1,30 -indolin]20 -one derivatives Judit Hala´sz, Benjamin Poda´nyi*, Andrea Sa´nta-Csutor, Zsolt Bo¨cskei, Ka´lma´n Simon, Miklo´s Hanusz, Istva´n Hermecz Chinoin Pharmaceutical and Chemical Works Company Ltd (a member of the Sanofi-Synthelabo Group), To´ u. 1-5, H-1045 Budapest, Hungary Received 16 January 2003; revised 20 March 2003; accepted 20 March 2003
Abstract SR 121463 is a potent and selective, orally active vasopressin V2 receptor antagonist. During the synthesis of SR 121463, the formation of the stereochemistry of the cyclohexyl moiety is one of the most important steps. Conformational analysis (via NMR studies and, for cis-3, also via X-ray structure determination) of the isomers obtained in this step is reported. q 2003 Elsevier Science B.V. All rights reserved. Keywords: X-ray diffraction; NMR; Configuration; Conformation
1. Introduction The importance of arginine vasopressin (AVP) in the regulation of blood pressure and volume and in the control of the fluid and electrolyte balances is well established. AVP plays a major role as an antidiuretic hormone regulating the water and solute excretion by the kidney through specific interaction with the renal V2 receptors [1,2]. Receptor-specific AVP V2 antagonists, known as ‘aquaretic agents’, could be of major therapeutic value for the treatment of a number of waterretaining disorders, such as SIADH (syndrome of inappropriate antidiuretic hormone secretion), liver cirrhosis, certain stages of congestive heart failure * Corresponding author. Tel.: þ 361-369-2500/2538; fax: þ 361370-5597. E-mail address: [email protected] (B. Poda´nyi).
and hypertension, and the nephritic syndrome [3 –6]. SR 121463 (Scheme 1) is the most potent and selective, orally active V2 antagonist described so far. The action of SR 121463 is purely aquaretic [7], with no changes in urine Naþ and Kþ excretion unlike the situation with other well known diuretic agents such as furosemide or hydrochlorothiazide. Total syntheses of SR 121463 were reported recently [8,9]. During the synthesis of SR 121463 [8,9], the important intermediates 1 and 2 are the compounds in which the stereochemistry of the cyclohexyl moiety is formed (Scheme 2). Both the reduction of oxo compound 1 and the ring opening of the cyclic ketal moiety of 2 (Scheme 2) may yield isomers of 3 and 4, respectively, and theirs unambiguous identification is needed. This can be achieved only if the conformation of the spiro[cyclohexane1,30 -indolin]-20 -one ring system is established.
0022-2860/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-2860(03)00226-6
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2. Experimental 2.1. Synthesis of compounds 3-4
Scheme 1. SR 121463.
Accordingly, we decided to perform detailed investigations. The key intermediates in the synthesis appear to be cis-3 or cis-4 [8,9] (Scheme 2). The corresponding trans isomers of 3 and 4, which are potential impurities, were also prepared as model compounds for the NMR investigations. Structural characterisation and conformational analysis of these four compounds are reported.
2.1.1. cis-5 0 -Ethoxy-4-hydroxyspiro[cyclohexane1,3 0 -indolin]-2 0 -one (cis-3) 50 -Ethoxyspiro[cyclohexane-1,30 -indoline]-4,20 dione 1 [10] (38.89 g, 0.15 mol) was suspended in methanol (500 ml). Solid NaBH4 (5.67 g, 0.15 mol) was added in small portions with vigorous stirring and water-cooling. A clear solution was formed, which progressively transformed to a suspension. After stirring for 15 min, the precipitate was removed by filtration, and washed with water (100 ml) and methanol (20 ml) to give the crude product (18.15 g, 46.3%, mp. 227 –228 8C). Recrystallization from 96% ethanol (400 ml) yielded pure cis-3 (14.11 g, mp. 228– 229 8C) (lit. mp. 225 8C [11]). 2.1.2. trans-5 0 -Ethoxy-4-hydroxyspiro[cyclohexane1,3 0 -indolin]-2 0 -one (trans-3) The above methanolic mother liquor of cis-3 was evaporated to dryness, the residue was suspended in
Scheme 2.
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water (200 ml), the suspension was filtered and the solid was washed thoroughly with water and some methanol to give the crude trans isomer (17.25 g, mp. 165 –195 8C). The purification was performed first by crystallization from 96% ethanol (130 ml) and another ‘crop’ of cis-3 was removed (4.8 g, mp. 227– 228 8C). The evaporated filtrate was then further purified by column chromatography on neutral Al2O3 (600 g), with elution with chloroform: i-propanol (9:1). After evaporation, the crude product (6.45 g) was recrystallized from ethyl acetate to afford pure trans-3 (4.65 g, mp. 185– 186 8C) (lit. mp. 170 8C [11]). 2.1.3. cis-5 0 -Ethoxy-4-(2-hydroxyethoxy) spiro[cyclohexane-1,3 0 -indolin]-2 0 -one (cis-4) To a stirred suspension of 50 -ethoxyspiro[cyclohexane-1,30 -indoline]-4,20 -dione cyclic 4-ethylene ketal 2 [11] (40.4 g, 0.133 mol) in dichloromethane (250 ml), a freshly prepared 0.29 M Zn(BH4) 2 solution [12] (275 ml, 0.08 mol) and trimethylchlorosilane (43.5 ml, 0.35 mol) were added dropwise at 0 8C. The mixture was stirred at room temperature for 16 h, and then quenched by the addition of 1N HCl solution (250 ml) and ethyl acetate (250 ml). The organic layer was separated, washed twice with water, and dried over MgSO4. After evaporation, the oily residue was crystallized from diethyl ether, and filtered off. Recrystallization from toluene (260 ml) afforded cis-4 (19.3 g, mp. 125 8C) (lit. mp. 125 8C [12]; 123– 124 8C [13]). 2.1.4. trans-5 0 -Ethoxy-4-(2-hydroxyethoxy) spiro[cyclohexane-1,3 0 -indolin]-2 0 -one (trans-4) A solution of 50 -ethoxyspiro[cyclohexane-1,30 indoline]-4,20 -dione cyclic 4-ethylene ketal 2 [11] (15.15 g, 0.05 mol) in dichloromethane (100 ml) was added dropwise to a stirred solution formed from LiAlH4 (1.9 g, 0.05 mol) in diethyl ether (100 ml) and AlCl3 (26.6 g, 0.02 mol) in diethyl ether (150 ml) at 5 8C. The mixture was left to warm to room temperature and stirred for 2.5 h. The pH was set to 1 with 2N H2SO4 (150 ml) and extracted with ethyl acetate (2 £ 250 ml). The organic extract was washed with water, dried over Na2SO4 and evaporated. The residue contained the cis and trans isomers in a ratio of 3:2. These were separated by column chromatography on neutral Al2O3 (600 g), with elution with chloroform: i-propanol (9:1). After evaporation, the crude
189
product (4.1 g) was crystallized twice from ethyl acetate to afford pure trans-4 (2.6 g, mp. 178-179 8C) (lit. mp. 180 8C [11]). 2.2. NMR measurements, spectrum simulation and AM1 calculations The NMR spectra were recorded in pyridine-d5 and acetone-d6 on Bruker AVANCE DRX-400 and DMX-800 NMR spectrometers, using standard Bruker software. Spectrum simulation was carried out with the gNMR program [14]. AM1 calculations were performed with HyperChem software [15]. Starting geometries were constructed by the Model Builder function of the program setting the torsion angles for the theoretical values of the two chair conformations Table 1 Crystal data and experimental parameters for the X-ray diffraction studies on cis-3 cis-3 Empirical formula Formula weight Crystal system Space group Colour of crystal Unit cell dimensions ˚) a (A ˚) b (A ˚) c (A a (8) b (8) g (8) ˚ 3) Volume (A Z Density (calculated) Crystal size (mm) m (Cu Ka) (mm21) Scan type Scan width u range Reflections collected Independent reflections Absorption correction Data/restraints/parameters Final R indices ½I . 2sigmaðIÞ R1 WR2 Largest diff. peak Largest diff. hole
C15H19NO3 261.31 Monoclinic P21=a Colourless 8.611(2) 13.802(1) 11.504(2) 90 99.15(1) 90 1349.8(3) 4 1.286 g cm23 0.5 £ 0.1 £ 0.1 0.725 v=2u ð1:57 – 0:30tan uÞ8 3.89–75.17 2881 2687 ½RðintÞ ¼ 0:0501 None 2674/0/179 0.0551 0.1315 0.238 ˚ 23 20.217 eA
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of the cyclohexyl ring. No restrains were used during the calculations. 2.3. X-ray crystallography Data on cis-3 were collected on an MSC Rigaku AFC6s diffractometer. The crystal data and refinement parameters are summarized in Table 1. Data reduction, structure solution and refinement were performed with teXsan [16], SHELXS-86 [17] and SHELXL-93 [18], respectively. All hydrogen atoms but H6 were generated.
3. Results and discussion 3.1. X-ray crystallography and theoretical calculations A single-crystal could be grown from one of the two isomers of the hydroxy derivative 3 (mp. 228-229 8C) and its X-ray structure demonstrated that
it was the cis isomer. In the cis isomer, the 20 -oxo group of the indolinone ring and the OH group of the cyclohexyl moiety are on the same side of the sixmembered ring, whereas they are on opposite sides in the trans isomer. The ORTEP plot of cis-3 is presented in Fig. 1. The cyclohexane ring has the expected chair conformation, with the OH oxygen atom in the equatorial, and the carbon of the amide group in the axial position. Two stable conformers of both isomers may exist (Scheme 3), denoted eq and ax according to the equatorial or axial steric position of the C-1 substituent respectively. The upper structure (cis-eq) corresponds to the conformation found in the solid state by X-ray crystallography for cis-3. The recently reported N-benzyl derivative of cis-3 was found to exhibit the axial conformation in the crystal structure [8]. The axial position of the OH in the solid state was explained by the pairwise hydrogen-bonds formed by the hydroxy groups and the amide carbonyls [8]. It is worthy of mention that we observed a very similar dimer formation for our cis-3 compound (Fig. 1). In cis-3,
Fig. 1. ORTEP diagram with intermolecular hydrogen-bonds of cis-3.
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Scheme 3. Conformational equilibria of 3 and 4.
the N – H forms a second intermolecular hydrogen˚; bond with the OH group [O2_a· · ·O1_b ¼ 2.791(4) A O2_a – H6_a…O1_b ¼ 168(1)8; N1_d· · ·O2_a ¼ ˚ ; N1_d – H7_d…O2_a ¼ 173(1)8. Sym2.874(4) A metry transformation used to generate equivalent molecules (a, b, c, d): a ðx; y; zÞ; b ð2x þ 2; 2y þ 1; -z þ 2Þ; c ð2x þ 1; 2y þ 1; 2z þ 2Þ; d ðx þ 1; y; zÞ]. AM1 semiempirical energy calculations [15] resulted in that the energy difference between the two conformers is small; in solution, therefore, the two conformers may exist in equilibrium. This question was studied by NMR. 3.2. Assignment of the NMR spectra The NMR spectra were measured in different solvents in order to achieve the best 1H signal
separation. Pyridine-d5 appeared to be the most appropriate solvent for the cis isomer of both derivatives and trans-3, whereas acetone-d6 was so for trans-4. The 1H and 13C signal assignments (Table 2) were based on the coupling patterns, and the NOESY [19], HMQC [20] and HMBC [21] spectra. The protons of the cyclohexyl ring are denoted a or b, indicating their positions below or above the reference plane of the cyclohexyl ring. The bhydrogens of the methylene groups, 2-H2 and 3-H2, were identified on the basis of their cross-peaks with 40 -H in the NOESY spectra (Table 3). The vicinal proton-proton coupling constants were determined by first-order approximation from the 400 MHz proton spectrum of cis-4, and by first-order analysis of the 1H NMR spectrum of trans-4,
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Table 2 1 H and 13C chemical shifts [ppm] and some proton-proton coupling constants (J [Hz]) of 3 and 4 in pyridine-d5 1
1 2
a b a b
3 20 30 3a0 40 50 60 70 7a0 80 90 OCH2 CH2OH OH NH
13
H
C
cis-3a
trans-3a
cis-4a
trans-4a,b
cis-3
trans-3
cis-4
trans-4c
4.20 2.68 2.18 2.31 1.87 – – – 7.20 – 6.89 6.98 – 3.97 1.33 – – 6.25 11.43
4.20 2.46 2.18 2.18 2.04 – – – 7.41 – 6.91 7.03 – 3.86 1.29 – – 6.36 11.53
3.66 2.49 2.04 2.21 1.74 – – – 7.18 – 6.90 6.99 – 3.98 1.34 3.81 4.05 6.32 11.40
3.60 2.23 1.77 1.69 1.82 – – – 7.00 – 6.76 6.83 – 4.00 1.34 3.60 3.69 3.60 9.13
67.9 30.1
67.7 30.6
76.1 26.4
76.5 27.7
31.4
31.0
30.9
31.4
182.3 46.9 137.5 111.5 154.7 112.6 109.5 135e 63.9 14.7 – – – –
182.3 47.8 137.2 112.9 154.5 112.6 109.7 135.2 63.7 14.7 – – – –
182.0 46.9 137.2 111.6 154.7 112.6 109.5 135.2 63.8 14.7 70.4 61.8 – –
182.7 48.5 138.5 113.8d 155.1 113.7d 111.0 135.9 65.3 15.9 71.1 63.1 – –
a
Coupling constants [Hz] of cis-3: J1,OH ¼ 4.2; J2a,2b ¼ 12.7; J3a,3b ¼ 13.8; J40 ,60 ¼ 2.6; J60 ,70 ¼ 8.5; J80 ,90 ¼ 6.9; coupling constants [Hz] of trans-3: J1,OH ¼ 4.2; J40 ,60 ¼ 2.6; J60 ,70 ¼ 8.5; J80 ,90 ¼ 6.9; coupling constants [Hz] of cis-4: J2a,2b ¼ 13.1; J3a,3b ¼ 13.8; J40 ,60 ¼ 2.6; J60 ,70 ¼ 8.5; J80 ,90 ¼ 6.9; JOCH2,CH2OH¼ JCH2OH,OH ¼ 5.4; coupling constants [Hz] of trans-4 b: J2a,2b ¼ 13.5; J3a,3b ¼ 13.0; J40 ,60 ¼ 2.3; J60 ,70 ¼ 8.8; J80 ,90 ¼ 6.9. b Measured in acetone-d6 at 800 MHz. c Measured in acetone-d6. d Tentative assignment. e The correct chemical shift could not be determined because of overlapping with the solvent signal.
Table 3 Characteristic spatial proximities on the basis of the phase-sensitive NOESY spectra Cross-peaks
1-H 2-Ha 2-Hb 3-Ha 3-Hb 40 -H 60 -H 70 -H 80 -H2 OCH2 NH
cis-3
trans-3
cis-4
2-Ha; 2-Hb; 3-Hb 1-H; 2-Hb; 3-Ha 1-H; 2-Ha; 3-Hb; 40 -H 2-Ha; 3-Hb 1-H; 2-Hb; 3-Ha; 40 -H 2-Hb; 3-Hb; 80 -H2 80 -H2
2-Ha; 2-Hb þ 3-Ha 1-H; 2-Hb þ 3-Ha; 3-Hb 1-H; 2-Ha; 3-Hb; 40 -H 2-Ha; 3-Hb 2-Ha; 2-Hb þ 3-Ha; 40 -H 2-Hb þ 3-Ha; 3-Hb; 80 -H2 80 -H2
40 -H; 60 -H –
40 -H; 60 -H –
2-Ha; 2-Hb; 3-Hb; OCH2 1-H; 2-Hb; 3-Ha; OCH2 1-H; 2-Ha; 3-Hb; OCH2; 40 -H 2-Ha; 3-Hb 1-H; 2-Hb; 3-Ha; 40 -H 2-Hb; 3-Hb; 80 -H2 80 -H2 NH 40 -H; 60 -H 1-H; 2-Ha; 2-Hb 70 -H
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measured at 800 MHz. For cis-3, the coupling constants were determined by spectrum simulation, performed with the gNMR program. 3.3. Identification of the isomers and qualitative conformational analysis based on NOE measurements NOE cross-peaks of the NOESY spectra are listed in Table 3. The cross-peaks between 1-H and 3-Hb in cis-3 and cis-4 prove that these compounds are cis isomers, since these protons are not in steric proximity in the conformers of the trans isomer. Further crosspeaks of the 2D NOESY spectra, however, i.e. the interactions between aromatic proton 40 -H and 2-Hb, and 3-Hb, prove the existence of both conformers in solution. The former cross-peak relates to the cis-eq, and the latter to the cis-ax conformer. In the conformers of trans-3 obtained in the calculations, 40 -H showed spatial proximity to 3-Hb in the trans-ax conformer, and to 2-Hb in the trans-eq form. Cross-peaks due to these interactions were observed in the NOESY spectrum, but the signals of
193
2-Hb and 3-Ha overlapped. The AM1 calculation revealed that there can not be spatial proximity between 3-Ha and 40 -H in either conformer, and consequently there can not be a NOESY cross-peak between these protons, so the obtained cross-peak is an indication of the 40 -H,2-Hb spatial proximity. According to the models obtained in the AM1 calculations, the distances between 40 -H and 2-Hb in the trans-eq, and between 40 -H and 3-Hb in the trans-ax conformers are nearly identical. The signal corresponding to the trans-eq conformer is more intense than the cross-peak of the trans-ax form, indicating that the amount of the trans-eq form is larger than that of the trans-ax form in the conformational equilibrium. 3.4. Quantitative determination of the conformer ratio from the coupling constants The vicinal proton – proton coupling constants were first determined from the 1H NMR spectrum, and the coupling constants for both conformers were
Table 4 Determination of the ratio of the conformers from the coupling constant data (J [Hz]) Dihedral anglesa
cis-3c
cis-4c
trans24d
a b c d
3-Hb,2-Ha 3-Hb,2-Hb 3-Ha,2-Ha 3-Ha,2-Hb 2-Hb,1-H 2-Ha,1-H 3-Hb,2-Ha 3-Hb,2-Hb 3-Ha,2-Ha 3-Ha,2-Hb 2-Hb,1-H 2-Ha,1-H 3-Hb,2-Ha 3-Hb,2-Hb 3-Ha,2-Ha 3-Ha,2-Hb 2-Hb,1-H 2-Ha,1-H
Jcalculatedb
‘eq’
‘ax’
‘eq’
‘ax’
174.8 55.9 57.0 261.9 258.4 2176.3 175.6 56.7 57.4 261.5 257.4 2175.5 62.1 256.4 255.3 2173.8 177.8 59.6
63.5 255.2 254.6 2173.3 52.3 264.8 63.6 254.8 254.1 2172.5 54.7 263.3 176.6 58.0 58.2 260.4 61.3 257.3
13.59 3.77 3.62 2.63 4.34 11.33 13.61 3.64 3.48 2.72 4.50 11.30 2.61 3.73 3.91 13.54 11.44 4.11
2.38 4.01 4.10 13.50 2.86 3.22 2.36 4.05 4.15 13.47 2.55 3.41 13.64 3.39 3.38 2.92 3.73 2.27
Determined from the results of the AM1 calculations. Calculated via the modified Karplus equation [15]. Measured in pyridine-d5. Measured in acetone-d6.
Jmeasuredc,d
‘eq’/’ax’ ratio
10.4 3.8 3.8 6.1 3.5 8.9 10.2 3.9 4.1 6.1 3.8 8.7 ,7 4.0 4.3 9.3 8.0c 3.8c
72:28
68:32 70:30 70:30
69:31 67:33 60:40
61:39 55:45
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then calculated from the molecular geometry obtained in the AM1 calculation by using the modified Karplus equation [22]. In consequence of the fast interconversion of the conformers, the measured coupling constants are weighted averages. The ratio of the two conformers can be calculated from the comparison of the measured and calculated coupling constants. From the coupling constants, the ratio of the two conformers was calculated to be about , 7:3 for both cis-3 and cis-4 (Table 4). The ratio of the conformers was determined from the coupling constants between protons with significantly different dihedral angles in the two conformers. For both compounds, the 3-Hb, 2-Ha, 3-Ha,2-Hb and 2-Ha,1-H coupling constants were significant, since these protons are antiperiplanar in one conformer (dihedral angle , 1808) and gauche
oriented in the other conformer (dihedral angle , 608) (Table 4). Strong coupling appeared between 2-Hb and 3-Ha in the spectrum of trans-3, which prevented determination of the coupling constants, but the splitting of the 1-H signal for the trans-3 isomer is very similar to that observed for the 1-H signal for the cis-3 isomer, where the 1-H signal relates to the axial situation. If the dominant conformer were trans-ax, where 1-H is equatorial, the 1-H signals would differ significantly. This suggests that in the dominant conformer of the trans isomer this proton is in the axial position (trans-eq conformer). For trans-4, comparison of the measured and the calculated coupling constants (Table 4) indicated that the ratio of the trans-eq and trans-ax conformers was about 55– 60:45 – 40.
Fig. 2. 1H NMR spectra of cis-4 (a) and trans-4 (b) in acetone-d6 at 285 8C.
J. Hala´sz et al. / Journal of Molecular Structure 654 (2003) 187–196
3.5. Low-temperature NMR measurements The conformational equilibria of 3 and 4 were also investigated in detail by means of dynamic NMR measurements. At room temperature, the ring inversion is fast and this yields only one signal set. At low temperature, where the ring inversion is sufficiently slow, two signal sets appear and the intensities of the signals correspond to the populations of the conformers. The measurements were carried out in acetone-d6. The proton spectra of both isomers of 3 and 4 at about 2 85 8C showed two NH and OH signals (two separated OH signals also appeared for 4) and two aromatic signal sets. Since the coupling constants and the intensities of the NOESY cross-peaks demonstrate that the dominant conformer of both isomers is the eq. form, the integral values of the NH, OH or aromatic signals lead to a cis-eq: cis-ax value of 79:21 in cis-3, and of 68:32 in cis-4 (Fig. 2), while trans-eq: trans-ax is 86:14 in trans-3, and 62:38 in trans-4 (Fig. 2). The positions of the amide group in the two conformers differ, which can be reflected in the NH chemical shift; in the cis isomers, the CyO is quasi-axial in the ‘equatorial’, and quasi-equatorial in the ‘axial’ conformer. In the trans isomer, the position of the amide group is opposite to that in the cis isomer. CyO is quasi-axial in the ‘axial’, and quasi-equatorial in the “equatorial” conformer. In cis-3 and cis-4, the NH chemical shift relating to the cis-eq form is lower than that in the corresponding ‘axial’ form [dðNHÞcis-eq ¼ 10:19 ppm (cis-3) and dðNHÞcis-eq ¼ 9:84 ppm (cis-4); dðNHÞcis-ax ¼ 10:42 ppm (cis-3) and dðNHÞcis-ax ¼ 10:09 ppm (cis-4)]. In trans-3 and trans-4, the chemical shift of the NH signal of the major conformer is higher than that of the minor conformer [dðNHÞtrans-eq ¼ 10:34 ppm (trans-3) and dðNHÞtrans-eq ¼ 10:13 ppm (trans-4); dðNHÞtrans-ax ¼ 10:04 ppm (trans-3) and dðNHÞtrans-ax ¼ 9:87 ppm (trans-4)], which suggests that the CyO group is quasi-equatorial in the major conformer. These data further support that the dominant conformer of both trans-3 and trans-4 is the trans-eq form. The conformational ratios obtained via the NMR methods are summarized in Table 5.
195
Table 5 Conformational ratio of 3 and 4 determined from coupling constants values and by dynamic NMR measurements cis-3
Coupling constants Dynamic NMR
trans-3
‘eq’
‘ax’
,7
,3
79
21
‘eq’
86
cis-4 ‘ax’
14
trans-4
‘eq’
‘ax’
‘eq’
‘ax’
,7
,3
,6
,4
68
32
62
38
4. Conclusions The cis and trans isomers of 3 and 4, containing a spiro[cyclohexane-1,30 -indolin]-20 -one ring system, were identified by means of 2D NOESY measurements. The conformational equilibria of the cis and trans isomers of 3 and 4 in solution were investigated by NMR spectroscopy. The dominant forms were identified as the two chair conformers of the cyclohexane ring, and the ratios of the conformers were determined from the coupling constant data and from low-temperature NMR measurements. In both isomers, the conformer containing an equatorial OR group in the cyclohexane ring is the dominant, but the other conformer also exists in significant amounts.
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Conformational analysis of four spiro[cyclohexane-1,30 -indolin]20 -one derivatives Judit Hala´sz, Benjamin Poda´nyi*, Andrea Sa´nta-Csutor, Zsolt Bo¨cskei, Ka´lma´n Simon, Miklo´s Hanusz, Istva´n Hermecz Chinoin Pharmaceutical and Chemical Works Company Ltd (a member of the Sanofi-Synthelabo Group), To´ u. 1-5, H-1045 Budapest, Hungary Received 16 January 2003; revised 20 March 2003; accepted 20 March 2003
Abstract SR 121463 is a potent and selective, orally active vasopressin V2 receptor antagonist. During the synthesis of SR 121463, the formation of the stereochemistry of the cyclohexyl moiety is one of the most important steps. Conformational analysis (via NMR studies and, for cis-3, also via X-ray structure determination) of the isomers obtained in this step is reported. q 2003 Elsevier Science B.V. All rights reserved. Keywords: X-ray diffraction; NMR; Configuration; Conformation
1. Introduction The importance of arginine vasopressin (AVP) in the regulation of blood pressure and volume and in the control of the fluid and electrolyte balances is well established. AVP plays a major role as an antidiuretic hormone regulating the water and solute excretion by the kidney through specific interaction with the renal V2 receptors [1,2]. Receptor-specific AVP V2 antagonists, known as ‘aquaretic agents’, could be of major therapeutic value for the treatment of a number of waterretaining disorders, such as SIADH (syndrome of inappropriate antidiuretic hormone secretion), liver cirrhosis, certain stages of congestive heart failure * Corresponding author. Tel.: þ 361-369-2500/2538; fax: þ 361370-5597. E-mail address: [email protected] (B. Poda´nyi).
and hypertension, and the nephritic syndrome [3 –6]. SR 121463 (Scheme 1) is the most potent and selective, orally active V2 antagonist described so far. The action of SR 121463 is purely aquaretic [7], with no changes in urine Naþ and Kþ excretion unlike the situation with other well known diuretic agents such as furosemide or hydrochlorothiazide. Total syntheses of SR 121463 were reported recently [8,9]. During the synthesis of SR 121463 [8,9], the important intermediates 1 and 2 are the compounds in which the stereochemistry of the cyclohexyl moiety is formed (Scheme 2). Both the reduction of oxo compound 1 and the ring opening of the cyclic ketal moiety of 2 (Scheme 2) may yield isomers of 3 and 4, respectively, and theirs unambiguous identification is needed. This can be achieved only if the conformation of the spiro[cyclohexane1,30 -indolin]-20 -one ring system is established.
0022-2860/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-2860(03)00226-6
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2. Experimental 2.1. Synthesis of compounds 3-4
Scheme 1. SR 121463.
Accordingly, we decided to perform detailed investigations. The key intermediates in the synthesis appear to be cis-3 or cis-4 [8,9] (Scheme 2). The corresponding trans isomers of 3 and 4, which are potential impurities, were also prepared as model compounds for the NMR investigations. Structural characterisation and conformational analysis of these four compounds are reported.
2.1.1. cis-5 0 -Ethoxy-4-hydroxyspiro[cyclohexane1,3 0 -indolin]-2 0 -one (cis-3) 50 -Ethoxyspiro[cyclohexane-1,30 -indoline]-4,20 dione 1 [10] (38.89 g, 0.15 mol) was suspended in methanol (500 ml). Solid NaBH4 (5.67 g, 0.15 mol) was added in small portions with vigorous stirring and water-cooling. A clear solution was formed, which progressively transformed to a suspension. After stirring for 15 min, the precipitate was removed by filtration, and washed with water (100 ml) and methanol (20 ml) to give the crude product (18.15 g, 46.3%, mp. 227 –228 8C). Recrystallization from 96% ethanol (400 ml) yielded pure cis-3 (14.11 g, mp. 228– 229 8C) (lit. mp. 225 8C [11]). 2.1.2. trans-5 0 -Ethoxy-4-hydroxyspiro[cyclohexane1,3 0 -indolin]-2 0 -one (trans-3) The above methanolic mother liquor of cis-3 was evaporated to dryness, the residue was suspended in
Scheme 2.
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water (200 ml), the suspension was filtered and the solid was washed thoroughly with water and some methanol to give the crude trans isomer (17.25 g, mp. 165 –195 8C). The purification was performed first by crystallization from 96% ethanol (130 ml) and another ‘crop’ of cis-3 was removed (4.8 g, mp. 227– 228 8C). The evaporated filtrate was then further purified by column chromatography on neutral Al2O3 (600 g), with elution with chloroform: i-propanol (9:1). After evaporation, the crude product (6.45 g) was recrystallized from ethyl acetate to afford pure trans-3 (4.65 g, mp. 185– 186 8C) (lit. mp. 170 8C [11]). 2.1.3. cis-5 0 -Ethoxy-4-(2-hydroxyethoxy) spiro[cyclohexane-1,3 0 -indolin]-2 0 -one (cis-4) To a stirred suspension of 50 -ethoxyspiro[cyclohexane-1,30 -indoline]-4,20 -dione cyclic 4-ethylene ketal 2 [11] (40.4 g, 0.133 mol) in dichloromethane (250 ml), a freshly prepared 0.29 M Zn(BH4) 2 solution [12] (275 ml, 0.08 mol) and trimethylchlorosilane (43.5 ml, 0.35 mol) were added dropwise at 0 8C. The mixture was stirred at room temperature for 16 h, and then quenched by the addition of 1N HCl solution (250 ml) and ethyl acetate (250 ml). The organic layer was separated, washed twice with water, and dried over MgSO4. After evaporation, the oily residue was crystallized from diethyl ether, and filtered off. Recrystallization from toluene (260 ml) afforded cis-4 (19.3 g, mp. 125 8C) (lit. mp. 125 8C [12]; 123– 124 8C [13]). 2.1.4. trans-5 0 -Ethoxy-4-(2-hydroxyethoxy) spiro[cyclohexane-1,3 0 -indolin]-2 0 -one (trans-4) A solution of 50 -ethoxyspiro[cyclohexane-1,30 indoline]-4,20 -dione cyclic 4-ethylene ketal 2 [11] (15.15 g, 0.05 mol) in dichloromethane (100 ml) was added dropwise to a stirred solution formed from LiAlH4 (1.9 g, 0.05 mol) in diethyl ether (100 ml) and AlCl3 (26.6 g, 0.02 mol) in diethyl ether (150 ml) at 5 8C. The mixture was left to warm to room temperature and stirred for 2.5 h. The pH was set to 1 with 2N H2SO4 (150 ml) and extracted with ethyl acetate (2 £ 250 ml). The organic extract was washed with water, dried over Na2SO4 and evaporated. The residue contained the cis and trans isomers in a ratio of 3:2. These were separated by column chromatography on neutral Al2O3 (600 g), with elution with chloroform: i-propanol (9:1). After evaporation, the crude
189
product (4.1 g) was crystallized twice from ethyl acetate to afford pure trans-4 (2.6 g, mp. 178-179 8C) (lit. mp. 180 8C [11]). 2.2. NMR measurements, spectrum simulation and AM1 calculations The NMR spectra were recorded in pyridine-d5 and acetone-d6 on Bruker AVANCE DRX-400 and DMX-800 NMR spectrometers, using standard Bruker software. Spectrum simulation was carried out with the gNMR program [14]. AM1 calculations were performed with HyperChem software [15]. Starting geometries were constructed by the Model Builder function of the program setting the torsion angles for the theoretical values of the two chair conformations Table 1 Crystal data and experimental parameters for the X-ray diffraction studies on cis-3 cis-3 Empirical formula Formula weight Crystal system Space group Colour of crystal Unit cell dimensions ˚) a (A ˚) b (A ˚) c (A a (8) b (8) g (8) ˚ 3) Volume (A Z Density (calculated) Crystal size (mm) m (Cu Ka) (mm21) Scan type Scan width u range Reflections collected Independent reflections Absorption correction Data/restraints/parameters Final R indices ½I . 2sigmaðIÞ R1 WR2 Largest diff. peak Largest diff. hole
C15H19NO3 261.31 Monoclinic P21=a Colourless 8.611(2) 13.802(1) 11.504(2) 90 99.15(1) 90 1349.8(3) 4 1.286 g cm23 0.5 £ 0.1 £ 0.1 0.725 v=2u ð1:57 – 0:30tan uÞ8 3.89–75.17 2881 2687 ½RðintÞ ¼ 0:0501 None 2674/0/179 0.0551 0.1315 0.238 ˚ 23 20.217 eA
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of the cyclohexyl ring. No restrains were used during the calculations. 2.3. X-ray crystallography Data on cis-3 were collected on an MSC Rigaku AFC6s diffractometer. The crystal data and refinement parameters are summarized in Table 1. Data reduction, structure solution and refinement were performed with teXsan [16], SHELXS-86 [17] and SHELXL-93 [18], respectively. All hydrogen atoms but H6 were generated.
3. Results and discussion 3.1. X-ray crystallography and theoretical calculations A single-crystal could be grown from one of the two isomers of the hydroxy derivative 3 (mp. 228-229 8C) and its X-ray structure demonstrated that
it was the cis isomer. In the cis isomer, the 20 -oxo group of the indolinone ring and the OH group of the cyclohexyl moiety are on the same side of the sixmembered ring, whereas they are on opposite sides in the trans isomer. The ORTEP plot of cis-3 is presented in Fig. 1. The cyclohexane ring has the expected chair conformation, with the OH oxygen atom in the equatorial, and the carbon of the amide group in the axial position. Two stable conformers of both isomers may exist (Scheme 3), denoted eq and ax according to the equatorial or axial steric position of the C-1 substituent respectively. The upper structure (cis-eq) corresponds to the conformation found in the solid state by X-ray crystallography for cis-3. The recently reported N-benzyl derivative of cis-3 was found to exhibit the axial conformation in the crystal structure [8]. The axial position of the OH in the solid state was explained by the pairwise hydrogen-bonds formed by the hydroxy groups and the amide carbonyls [8]. It is worthy of mention that we observed a very similar dimer formation for our cis-3 compound (Fig. 1). In cis-3,
Fig. 1. ORTEP diagram with intermolecular hydrogen-bonds of cis-3.
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191
Scheme 3. Conformational equilibria of 3 and 4.
the N – H forms a second intermolecular hydrogen˚; bond with the OH group [O2_a· · ·O1_b ¼ 2.791(4) A O2_a – H6_a…O1_b ¼ 168(1)8; N1_d· · ·O2_a ¼ ˚ ; N1_d – H7_d…O2_a ¼ 173(1)8. Sym2.874(4) A metry transformation used to generate equivalent molecules (a, b, c, d): a ðx; y; zÞ; b ð2x þ 2; 2y þ 1; -z þ 2Þ; c ð2x þ 1; 2y þ 1; 2z þ 2Þ; d ðx þ 1; y; zÞ]. AM1 semiempirical energy calculations [15] resulted in that the energy difference between the two conformers is small; in solution, therefore, the two conformers may exist in equilibrium. This question was studied by NMR. 3.2. Assignment of the NMR spectra The NMR spectra were measured in different solvents in order to achieve the best 1H signal
separation. Pyridine-d5 appeared to be the most appropriate solvent for the cis isomer of both derivatives and trans-3, whereas acetone-d6 was so for trans-4. The 1H and 13C signal assignments (Table 2) were based on the coupling patterns, and the NOESY [19], HMQC [20] and HMBC [21] spectra. The protons of the cyclohexyl ring are denoted a or b, indicating their positions below or above the reference plane of the cyclohexyl ring. The bhydrogens of the methylene groups, 2-H2 and 3-H2, were identified on the basis of their cross-peaks with 40 -H in the NOESY spectra (Table 3). The vicinal proton-proton coupling constants were determined by first-order approximation from the 400 MHz proton spectrum of cis-4, and by first-order analysis of the 1H NMR spectrum of trans-4,
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192
Table 2 1 H and 13C chemical shifts [ppm] and some proton-proton coupling constants (J [Hz]) of 3 and 4 in pyridine-d5 1
1 2
a b a b
3 20 30 3a0 40 50 60 70 7a0 80 90 OCH2 CH2OH OH NH
13
H
C
cis-3a
trans-3a
cis-4a
trans-4a,b
cis-3
trans-3
cis-4
trans-4c
4.20 2.68 2.18 2.31 1.87 – – – 7.20 – 6.89 6.98 – 3.97 1.33 – – 6.25 11.43
4.20 2.46 2.18 2.18 2.04 – – – 7.41 – 6.91 7.03 – 3.86 1.29 – – 6.36 11.53
3.66 2.49 2.04 2.21 1.74 – – – 7.18 – 6.90 6.99 – 3.98 1.34 3.81 4.05 6.32 11.40
3.60 2.23 1.77 1.69 1.82 – – – 7.00 – 6.76 6.83 – 4.00 1.34 3.60 3.69 3.60 9.13
67.9 30.1
67.7 30.6
76.1 26.4
76.5 27.7
31.4
31.0
30.9
31.4
182.3 46.9 137.5 111.5 154.7 112.6 109.5 135e 63.9 14.7 – – – –
182.3 47.8 137.2 112.9 154.5 112.6 109.7 135.2 63.7 14.7 – – – –
182.0 46.9 137.2 111.6 154.7 112.6 109.5 135.2 63.8 14.7 70.4 61.8 – –
182.7 48.5 138.5 113.8d 155.1 113.7d 111.0 135.9 65.3 15.9 71.1 63.1 – –
a
Coupling constants [Hz] of cis-3: J1,OH ¼ 4.2; J2a,2b ¼ 12.7; J3a,3b ¼ 13.8; J40 ,60 ¼ 2.6; J60 ,70 ¼ 8.5; J80 ,90 ¼ 6.9; coupling constants [Hz] of trans-3: J1,OH ¼ 4.2; J40 ,60 ¼ 2.6; J60 ,70 ¼ 8.5; J80 ,90 ¼ 6.9; coupling constants [Hz] of cis-4: J2a,2b ¼ 13.1; J3a,3b ¼ 13.8; J40 ,60 ¼ 2.6; J60 ,70 ¼ 8.5; J80 ,90 ¼ 6.9; JOCH2,CH2OH¼ JCH2OH,OH ¼ 5.4; coupling constants [Hz] of trans-4 b: J2a,2b ¼ 13.5; J3a,3b ¼ 13.0; J40 ,60 ¼ 2.3; J60 ,70 ¼ 8.8; J80 ,90 ¼ 6.9. b Measured in acetone-d6 at 800 MHz. c Measured in acetone-d6. d Tentative assignment. e The correct chemical shift could not be determined because of overlapping with the solvent signal.
Table 3 Characteristic spatial proximities on the basis of the phase-sensitive NOESY spectra Cross-peaks
1-H 2-Ha 2-Hb 3-Ha 3-Hb 40 -H 60 -H 70 -H 80 -H2 OCH2 NH
cis-3
trans-3
cis-4
2-Ha; 2-Hb; 3-Hb 1-H; 2-Hb; 3-Ha 1-H; 2-Ha; 3-Hb; 40 -H 2-Ha; 3-Hb 1-H; 2-Hb; 3-Ha; 40 -H 2-Hb; 3-Hb; 80 -H2 80 -H2
2-Ha; 2-Hb þ 3-Ha 1-H; 2-Hb þ 3-Ha; 3-Hb 1-H; 2-Ha; 3-Hb; 40 -H 2-Ha; 3-Hb 2-Ha; 2-Hb þ 3-Ha; 40 -H 2-Hb þ 3-Ha; 3-Hb; 80 -H2 80 -H2
40 -H; 60 -H –
40 -H; 60 -H –
2-Ha; 2-Hb; 3-Hb; OCH2 1-H; 2-Hb; 3-Ha; OCH2 1-H; 2-Ha; 3-Hb; OCH2; 40 -H 2-Ha; 3-Hb 1-H; 2-Hb; 3-Ha; 40 -H 2-Hb; 3-Hb; 80 -H2 80 -H2 NH 40 -H; 60 -H 1-H; 2-Ha; 2-Hb 70 -H
J. Hala´sz et al. / Journal of Molecular Structure 654 (2003) 187–196
measured at 800 MHz. For cis-3, the coupling constants were determined by spectrum simulation, performed with the gNMR program. 3.3. Identification of the isomers and qualitative conformational analysis based on NOE measurements NOE cross-peaks of the NOESY spectra are listed in Table 3. The cross-peaks between 1-H and 3-Hb in cis-3 and cis-4 prove that these compounds are cis isomers, since these protons are not in steric proximity in the conformers of the trans isomer. Further crosspeaks of the 2D NOESY spectra, however, i.e. the interactions between aromatic proton 40 -H and 2-Hb, and 3-Hb, prove the existence of both conformers in solution. The former cross-peak relates to the cis-eq, and the latter to the cis-ax conformer. In the conformers of trans-3 obtained in the calculations, 40 -H showed spatial proximity to 3-Hb in the trans-ax conformer, and to 2-Hb in the trans-eq form. Cross-peaks due to these interactions were observed in the NOESY spectrum, but the signals of
193
2-Hb and 3-Ha overlapped. The AM1 calculation revealed that there can not be spatial proximity between 3-Ha and 40 -H in either conformer, and consequently there can not be a NOESY cross-peak between these protons, so the obtained cross-peak is an indication of the 40 -H,2-Hb spatial proximity. According to the models obtained in the AM1 calculations, the distances between 40 -H and 2-Hb in the trans-eq, and between 40 -H and 3-Hb in the trans-ax conformers are nearly identical. The signal corresponding to the trans-eq conformer is more intense than the cross-peak of the trans-ax form, indicating that the amount of the trans-eq form is larger than that of the trans-ax form in the conformational equilibrium. 3.4. Quantitative determination of the conformer ratio from the coupling constants The vicinal proton – proton coupling constants were first determined from the 1H NMR spectrum, and the coupling constants for both conformers were
Table 4 Determination of the ratio of the conformers from the coupling constant data (J [Hz]) Dihedral anglesa
cis-3c
cis-4c
trans24d
a b c d
3-Hb,2-Ha 3-Hb,2-Hb 3-Ha,2-Ha 3-Ha,2-Hb 2-Hb,1-H 2-Ha,1-H 3-Hb,2-Ha 3-Hb,2-Hb 3-Ha,2-Ha 3-Ha,2-Hb 2-Hb,1-H 2-Ha,1-H 3-Hb,2-Ha 3-Hb,2-Hb 3-Ha,2-Ha 3-Ha,2-Hb 2-Hb,1-H 2-Ha,1-H
Jcalculatedb
‘eq’
‘ax’
‘eq’
‘ax’
174.8 55.9 57.0 261.9 258.4 2176.3 175.6 56.7 57.4 261.5 257.4 2175.5 62.1 256.4 255.3 2173.8 177.8 59.6
63.5 255.2 254.6 2173.3 52.3 264.8 63.6 254.8 254.1 2172.5 54.7 263.3 176.6 58.0 58.2 260.4 61.3 257.3
13.59 3.77 3.62 2.63 4.34 11.33 13.61 3.64 3.48 2.72 4.50 11.30 2.61 3.73 3.91 13.54 11.44 4.11
2.38 4.01 4.10 13.50 2.86 3.22 2.36 4.05 4.15 13.47 2.55 3.41 13.64 3.39 3.38 2.92 3.73 2.27
Determined from the results of the AM1 calculations. Calculated via the modified Karplus equation [15]. Measured in pyridine-d5. Measured in acetone-d6.
Jmeasuredc,d
‘eq’/’ax’ ratio
10.4 3.8 3.8 6.1 3.5 8.9 10.2 3.9 4.1 6.1 3.8 8.7 ,7 4.0 4.3 9.3 8.0c 3.8c
72:28
68:32 70:30 70:30
69:31 67:33 60:40
61:39 55:45
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then calculated from the molecular geometry obtained in the AM1 calculation by using the modified Karplus equation [22]. In consequence of the fast interconversion of the conformers, the measured coupling constants are weighted averages. The ratio of the two conformers can be calculated from the comparison of the measured and calculated coupling constants. From the coupling constants, the ratio of the two conformers was calculated to be about , 7:3 for both cis-3 and cis-4 (Table 4). The ratio of the conformers was determined from the coupling constants between protons with significantly different dihedral angles in the two conformers. For both compounds, the 3-Hb, 2-Ha, 3-Ha,2-Hb and 2-Ha,1-H coupling constants were significant, since these protons are antiperiplanar in one conformer (dihedral angle , 1808) and gauche
oriented in the other conformer (dihedral angle , 608) (Table 4). Strong coupling appeared between 2-Hb and 3-Ha in the spectrum of trans-3, which prevented determination of the coupling constants, but the splitting of the 1-H signal for the trans-3 isomer is very similar to that observed for the 1-H signal for the cis-3 isomer, where the 1-H signal relates to the axial situation. If the dominant conformer were trans-ax, where 1-H is equatorial, the 1-H signals would differ significantly. This suggests that in the dominant conformer of the trans isomer this proton is in the axial position (trans-eq conformer). For trans-4, comparison of the measured and the calculated coupling constants (Table 4) indicated that the ratio of the trans-eq and trans-ax conformers was about 55– 60:45 – 40.
Fig. 2. 1H NMR spectra of cis-4 (a) and trans-4 (b) in acetone-d6 at 285 8C.
J. Hala´sz et al. / Journal of Molecular Structure 654 (2003) 187–196
3.5. Low-temperature NMR measurements The conformational equilibria of 3 and 4 were also investigated in detail by means of dynamic NMR measurements. At room temperature, the ring inversion is fast and this yields only one signal set. At low temperature, where the ring inversion is sufficiently slow, two signal sets appear and the intensities of the signals correspond to the populations of the conformers. The measurements were carried out in acetone-d6. The proton spectra of both isomers of 3 and 4 at about 2 85 8C showed two NH and OH signals (two separated OH signals also appeared for 4) and two aromatic signal sets. Since the coupling constants and the intensities of the NOESY cross-peaks demonstrate that the dominant conformer of both isomers is the eq. form, the integral values of the NH, OH or aromatic signals lead to a cis-eq: cis-ax value of 79:21 in cis-3, and of 68:32 in cis-4 (Fig. 2), while trans-eq: trans-ax is 86:14 in trans-3, and 62:38 in trans-4 (Fig. 2). The positions of the amide group in the two conformers differ, which can be reflected in the NH chemical shift; in the cis isomers, the CyO is quasi-axial in the ‘equatorial’, and quasi-equatorial in the ‘axial’ conformer. In the trans isomer, the position of the amide group is opposite to that in the cis isomer. CyO is quasi-axial in the ‘axial’, and quasi-equatorial in the “equatorial” conformer. In cis-3 and cis-4, the NH chemical shift relating to the cis-eq form is lower than that in the corresponding ‘axial’ form [dðNHÞcis-eq ¼ 10:19 ppm (cis-3) and dðNHÞcis-eq ¼ 9:84 ppm (cis-4); dðNHÞcis-ax ¼ 10:42 ppm (cis-3) and dðNHÞcis-ax ¼ 10:09 ppm (cis-4)]. In trans-3 and trans-4, the chemical shift of the NH signal of the major conformer is higher than that of the minor conformer [dðNHÞtrans-eq ¼ 10:34 ppm (trans-3) and dðNHÞtrans-eq ¼ 10:13 ppm (trans-4); dðNHÞtrans-ax ¼ 10:04 ppm (trans-3) and dðNHÞtrans-ax ¼ 9:87 ppm (trans-4)], which suggests that the CyO group is quasi-equatorial in the major conformer. These data further support that the dominant conformer of both trans-3 and trans-4 is the trans-eq form. The conformational ratios obtained via the NMR methods are summarized in Table 5.
195
Table 5 Conformational ratio of 3 and 4 determined from coupling constants values and by dynamic NMR measurements cis-3
Coupling constants Dynamic NMR
trans-3
‘eq’
‘ax’
,7
,3
79
21
‘eq’
86
cis-4 ‘ax’
14
trans-4
‘eq’
‘ax’
‘eq’
‘ax’
,7
,3
,6
,4
68
32
62
38
4. Conclusions The cis and trans isomers of 3 and 4, containing a spiro[cyclohexane-1,30 -indolin]-20 -one ring system, were identified by means of 2D NOESY measurements. The conformational equilibria of the cis and trans isomers of 3 and 4 in solution were investigated by NMR spectroscopy. The dominant forms were identified as the two chair conformers of the cyclohexane ring, and the ratios of the conformers were determined from the coupling constant data and from low-temperature NMR measurements. In both isomers, the conformer containing an equatorial OR group in the cyclohexane ring is the dominant, but the other conformer also exists in significant amounts.
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