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Solution‐ and Solid‐state Structures of the (—)‐n‐Heptylcarbamate of Geneseroline and Its Hydrochloride Salt
Solution- and Solid-state Structures of the (-)-PHeptylcarbamate of Geneseroline and Its Hydrochloride Salt ENRICOREDENTI*,MAURIZIODELCANALE*, GABRIELEAMAH*, PAOLO VENTURA*, ALESSIA BACCHI*',
AND
GIANCARLO PELIZZIS
Received July 6, 1994, from the *Chemical and Biopharmaceutical Department, Chiesi Farmaceutici S.p.A., Via Palermo 26/A, 1-43100 Parma, Italy, and 'Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Centro di Studio per la Strutfuristica Accepted for publication May 30, 1995@. Diffrattometrica del CNR, Wale delle Scienze, L43100 Parma, /tab, Abstract 0 The solid state structures of the (-)-nheptylcarbamate of geneseroline and its hydrochloride salt were determined by single crystal X-ray diffraction analysis. Both compounds gave crystals belonging to the orthorhombic P212121 space group with a = 27.597(7) A, b = 8.899(2) A, c = 9.290(2) A, V = 2281.5(9) A3, Z = 4, and R = 0.06820forthe base and a = 11.300(1) A, b = 8.3485(5) A, c = 24.141(2) A, V = 2277.3(3) A3, Z= 4, and R = 0.0482 for the salt. X-ray and 'H NMR analysis revealed that the base is a 1,2-oxazine derivative. The sixmembered ring adopts a 4Gchair conformation in the solid-state, whereas, in CDC13solution, it exists as a mixture of two possible chair conformers, 4C1 and lC4, with the Nmethyl group in the equatorial position (ratio FX 7525). The salt is an Noxide derivative; the five-membered ring adopts different envelope conformations in the solid-state and in CDCI3 solution, suggesting a certain flexibility. In more polar solvents, the salt partially undergoes fast inversion at the tetrahedral nitrogen, giving rise to the corresponding epimer.
A
0 O,-NHCH,
Introduction The n-heptylcarbamate of geneseroline {( -)-2,3,4,4a,9,9ahexahydro-2,4aa,9a-trimethyl-l,2-oxazino[6,5-b]indol-6-ol nheptylcarbamate, B}' is a higher homologue of geneserine (Nmethyl derivative), an alkaloid which was first isolated in 1915 by Polonovski from the basic extracts of Calabar bean seeds2 and later obtained from physostigmine by oxidation with H z O ~ The . ~ structure for such alkaloids with a cis-fused tetrahydro-1,2-oxazine ring (instead of the N-oxide ring21 was proposed by Hootele5 and independently supported by NOE measurements.6 Since the absolute configuration a t the indoline 4a-carbon was already k n o ~ nthe , ~absolute ~ ~ stereochemistry of the alkaloid was determined to be 4aS,9aS (see Scheme 1). The n-heptyl derivative was obtained according to a synthesis which gave excellent yields for all the n-alkyl derivatives; the base was hence converted quantitatively into the crystalline hydrochloride salt with concentrated HCl. The salt adopts the N-oxide structureg {(-)-1,2,3,3a,8,8ahexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-01 n-heptylcarbamate N-oxide hydrochloride, A}. Like physostigmine and tacrine n-alkyl homologues, n-alkylcarbamates of geneserolinel inhibit acetylcholinesterase activitylo-l3and, even for this set of compounds, the optimal inhibitory profile was found for the n-heptyl derivative. With respect to the deoxy analog heptastigmine, the n-heptylcarbamate of geneseroline also shows a more prolonged action and a better therapeutic index. The therapeutic interest €or these compounds is due to their potential use in the treatment of the Alzeheimer's disease.14J5 Although a large number of other neurotransmitters and neuropeptides systems are also affected, a deficiency of cholinergic activity is suspected to play a major role in the expression of such p~ychopathology.'~The rationale for this therapy is the reversible anticholinesterasic action of the drug through its carbamate function, which possibly improves the activity of cholinergic neurons by slowing the @Abstractpublished in Advance ACS Abstracts, July 1, 1995.
1126 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
C
D
A
0
Scheme 1 degradation of the neurotransmitter acetylcholine. Here the structural study of A and B in the solution- and solid-state carried out by single crystal X-ray analysis, NMR spectroscopy, and mass spectrometry is reported.
Experimental Section Chemistry-The synthesis is reported in the Scheme I. A solution of 1.5 g (4.6 mmol) of geneserine hydrochloride'6 (D, HPLC purity > 98%)in 6 mL of concentrated sulfuric acid was heated and stirred a t 80 "C for 15 min. The solution was cooled to 0 "C and poured in ice water. A 30%solution of ammonia was added dropwise with stirring to pH = 7.5. Methylene chloride (50 mL) was added t o the mixture. The biphasic mixture was stirred for 5 min and partitioned, and the organic portion was dried over NaZS04, filtered, and evaporated under reduced pressure, t o give 1.0 g (4.3 mmol, 93%) of geneseroline (C) intermediate product as a white solid: [ a ]-158" ~ (c = 0.1, CHCl3); mp 142-145 "C; HPLC purity '97%; EI MS 234 mlz (M+). To 0.9 g (3.8 mmol) of C dissolved in 40 mL of toluene was added a catalytic amount (15 mg) of sodium and the mixture was stirred to complete dissolution. A solution of 55 mg (3.8 mmol) of n-heptyl isocyanate in 20 mL of toluene was added dropwise and the resulting solution stirred a t room temperature for 30 min, then a solution of 100 mg of ammonium chloride in 1 mL of water was added. The mixture was dried over NanS04, filtered, and evaporated under
0022-3549/953184-1 126$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
reduced pressure to give a n oil that was crystallized from petroleum ether a t 0 "C, to yield 1.1g (2.9 mmol) of the n-heptylcarbamate of -121" (c = 0.1, geneseroline (B) as a white crystalline solid: [ a l ~ CHC13); mp 81-82 "C; HPLC purity > 98%; IR 1660 cm-' (C = 0 carbamate); IH NMR (CDC13) 6, ppm 0.85 (t, 3H), 1.2 (s, 3H, CH34a), 1.1-1.5 (m, 8H), 1.6 (m, 2H), 1.96 (m, 2J = -14 Hz, l H , H4'), 2.14 (m, = -14 Hz, l H , H4), 2.5 (m, 2J = -12 Hz, l H , H37, 2.55 (s, 3H, CH3-N2), 2.66 (m, 2J = -12 Hz, l H , H3), 2.85 (s, 3H, CH3N9), 3.3 (q, 2H, CHZa-NH), 4.7 (s, l H , H9a), 4.95 (br, l H , N-H), 6.4 (d, J&h,, = 8.2 Hz, l H , H8), 6.7-6.9 (m, 2H, H5,7); 13CNMR (CDC13) d , ppm 14.7, 23.2, 26.1 (C4a-CH3), 27.4, 29.6, 30.6, 32.3 ((241, 32.4, 33.4 (N9-CH3),41.1 (C4a), 42.0,46.8 (NB-CHd 54.2 (C3), 102.7 (Cga), 107.4 (C8), 116.3 (C5), 120.8 (C7), 137.0 (C4b), 144.3 (C6), 148.2 (C8a), 156.1 (C=O); EI MS 375 mlz (M+). Upon dissolving 1.0 g of the base in 5 mL of acetone, a stoichiometric amount of ethereal HC1 and 7 mL of diethyl ether were added. The solution was allowed to stir for 8 h at 0 "C and the white solid which separated was filtered to yield 1.0 g (2.4 mmol) of the n-heptylcarbamate of gkneseroline hydrochloride (A) as a crystalline ~ (c = 1,HzO); mp 146-147 "C; HPLC purity > 99%; solid: [ a ]-136" IR 1720 cm-l (C=O carbamate); lH NMR (CDC13)6, ppm 0.9 (t, 3H), 1.2-1.6 (m, 8H), 1.6 (s, 3H, CH3-3a), 1.75 (m, 2H), 2.3 (dd, *5= -12.5
Table 1-Crystal Data and Summary of Intensity Data Collection and Structure Refinementd
B A Compound Formula CziH34ClN303 CziH33N303 41 1.97 375.51 MVY 11.300(1) 27.597(7) a, A 8.3485(5) 8.899(2) b, A 24.141(2) 9.290(2) c, A 2277.4(3) 2281.5(9) V, A3 4 4 Z 1.202 1.093 D,, g ~ m - ~ 816 888 F(000) 5.9 16.8 p !cm-' 3-70 3-65 6' range, deg 1-1 7 741 Standard reflection 251 1 (+h,+k,+l) 5828 (kh,+k,+l) Number of measured reflections 2485 2206 (& = 0.047) Number of independent reflections 1391 1718 Number of observed reflections 320 253 Parameters refined 0.23 0.31 Max height in final AFrnap, e A-3 0.0482 0.0682 R (observed data) Hz,1H,H3'),2.7(m,~J~-l2.5Hz,1H,H3),2.93(m,~J=-l1H~, 0.1511 0.1759 wR2 (all data) lH,H2'),3,3(q, 2H,CHza-NH),3.32 ( s , ~ H , C H ~ - N3.5 ~ )(s,3H,CH3, a Features common to both analyses include crystal system (orthorhombic), N l ) , 3.92 (dd, 2J = -11 Hz, lH, H2), 5.05 (br, l H , N-H), 5.9 (s, lH, space group (P212121),scan mode (6' - 26'), and criterion for observation (6> = 2.3 Hz, l H , H4), HSa), 6.6 (d, Jartho = 8.2 Hz, lH, H7), 6.9 (d, Jmeta = 2.3 Hz, lH, H6), 13.1 (br, l H , 0-H); 4u(F0)). 7.0 (dd, Jofiho = 8.2 Hz, Jmeta 13CNMR (CDC13) 6, ppm 14.7, 23.2, 26.7 (C3a-CH31,27.3, 29.6, 30.5, Table 2-Atomic Coordinatts (x104)and Equivalent Isotropic 32.4, 37.7 (N8-CHz), 38.1 (C3), 42.0, 48.8 (Nl-CH31, 53.9 (C3a) 64.3 Displacement Parameters (A2 x 104) (One-Third Trace of the Diagonalized (C2), 109.3 (C7), 111.8(C8a), 117.3 (C4), 123.2 (C6), 135.0 (C3b), 146.2 Matrix) for Compound B (esd Values in Parentheses) (C5), 146.5 (C7a), 155.5 (C=O); EI MS: 375 mlz (M+). Apparatus-Melting points (uncorrected) were determined on a Atom Xla Ylb ZlC uw Buchi apparatus. Optical rotations were measured at room temperature on a Perkin-Elmer 241 automatic polarimeter. Infrared spectra 1081(25) 5822(7) 9130(6) 1588(2) were taken either in Nujol or as a KBr disk on a Perkin-Elmer 297 3440(5) 904(20) 6359(5) 435(2) spectrophotometer. HPLC analyses were run on a Hewlett-Packard 916(20) 3614(6) 4512(6) -129(2) 1050 system provided with a W - v i s detector (1 = 245 nm). A 1268(38) 6418(11) 10301(8) 1303(4) p-Bondapack C18 analytical column (Waters, 10 um, 300 x 3.9 mm 920(25) 7542(8) 3907(7) 1650(2) i.d.1 was employed. The mobile phase was 0.1 M phosphate buffer 909(26) 6679(9) -270(3) 2390(8) pH = 3.0:acetonitrile in the ratio 88:12 for C ( t =~8.7 min) and D ( t ~ 1232(50) 9670(10) 842(4) 6891(13) = 4.3 min) and 62:38 for B and A ( t = ~ 9.8 min), respectively. The 1150(41) 5485(13) 9301(9) 577(3) flow rate was 2 mumin. 924(33) 4350( 10) 8389(8) 868(3) Crystallography-All X-ray measurements were made a t room 960(36) 4335(11) 8822(9) 1403(3) temperature on a computer-controlled Siemens AED diffractometer 792(30) 6358(8) 4458(8) 1391(3) using Cu Ka radiation (2 = 1.54 178 8). Automatic peak search, 734(26) 4830(8) 6816(7) 924(3) centering, and indexing procedures established in both cases an 831(28) 5389(8) 5862(7) 592(3) orthorhombic primitive lattice and the systematic extinctions observed 784(28) 4437(7) 5610(8) 742(3) identified unambiguously the space group as P212121. 837(31) 5250(9) 3993(8) 1199(3) The unit cell dimensions and other relevant crystal data are 869(32) 4938(8) 4696(9) 1534(3) presented in Table 1 together with details of data collection and 787(29) 5774(10) 3174(8) 4 3 ) refinement. No crystal decay was observed. The intensity data were 1958(11) 1106(36) -765(4) 6302(10) processed with a peak-profile analysis procedure and corrected for 1699(62) 10724(13) 1592(5) 7764(15) Lorentz and polarization factors. For B a n absorption correction 1328(46) 8472(9) 649(4) 2782( 11) (empirical surface) was also applied following the method of Walker 7478(11) 1241(42) 2180(3) 3831 (12) and Stuart.l7 For both structures the phase problem was solved by 121l(38) 591(10) -804(3) 5480(12) application of direct methods. Refinement was by full-matrix least1506(50) 245(14) -1338(4) 5 174(14) squares methods employing anisotropic thermal parameters for all 1740(59) -1032(15) -1 417(5) 4313(15) non-hydrogen atoms and isotropic ones for the hydrogen atoms, which 2419(96) -1467(21) -1918(6) 4044(25) were in part located from difference maps (those bonded to O(l), N(3), 2977(134) -1 054(24) -2307(7) 3777(32) C(1), C(2), C(4), C(7), C(9), C(lO), C(13), and C(14) for A and to "3) 2385(92) -2768(5) 3223(23) -1 678(19) for B) and in part placed a t calculated riding positions. In the refinement of B the heptyl chain atoms were subject to rigid bond restraints. The absolute configuration was tested by refining both enantiomers for each compound. In both cases the lower R and R, for distortionless enhancement by polarization transfer (DEFT), values corresponded to the previously known absolute configuration steady state nuclear Overhauser effect difference (NOEDIFF), 2D a t the indoline 4a-carbon a t ~ m . Neutral ~,~ atom scattering factors lH,'H correlation (COSY), and 2D lH,13C correlation (XHCORRDC) were used, those for the non-hydrogen atoms being corrected for experiments. The coupling constants values found were confirmed anomalous dispersion. All the calculations were performed on a t 400.1 MHz as the spin systems were not first order a t 200.1 MHz. GOULD POWERNODE 6040 and ENCORE91 computers using the The 13C NMR solid-state spectra were obtained using the crossprogram packages SHELXSS6,l8 SHELXL92,l9 PARST,20 and polarization magic-angle spinning (CP-MAS) accessory on a Bruker ORTEP.21 Final atomic parameters are reported in Tables 2 and 3, CXP 300 instrument. The spectra were measured with a contact time whereas bond distances and angles are given in Tables 4 and 5. of 1 ms, which was not optimized. The side bands were assigned by carrying out the experiments a t different spinning rates (3.05,4.96, N M R Spectroscopy--'H and 13C NMR spectra were obtained at and 5.46 kHz, respectively). 200.1 and 50.3 MHz in different deuterated solvents on a Bruker ACF Mass Spectrometry-The electron impact (EI) mass spectra were 200 instrument. The residual protons for each solvent were used as recorded a t 70 eV on a VG Micromass ZAB-2F spectrometer for direct internal references, Standard Bruker microprograms were employed
Journal of Pharmaceutical Sciences / 1127 Vol. 84, No. 9, September 1995
Table 3-Atomic Coordinates (xIO4) and Equivalent Isotropic Displacement Parameters (A2 x lo4) (One-Third Trace of the Diagonalized Matrix) for Compound A (esd Values in Parentheses) ~
Ma
Ylb
ilC
482.2( 11) -1804(3) -6100(3) 4960(3) -2632(3) -271 5(4) -6737(4) -3769(4) -3500( 4) -2859(4) -2263(4) -3581 (4) -371 O(4) -4517(4) -51 87(4) -5048(4) 4229(5) -5850(4) -6733(6) -2709(5) -1984(4) -21 13(6) -7076( 10) -7967(6) -8260(9) -9296( 14) -1 0069( 11) -1 1020(10)
6218.0(17) 5733(4) -788(4) -2717(5) 5521(4) 3974(4) -3275(5) 5026(7) 3367(6) 2585(5) 3990(5) 2766(5) 1945(5) 742(5) 358(6) 1140(7) 2394(6) -2329(7) -4979(7) 7028(6) 1308(6) 4686(7) -5488( 10) -4959(10) -5728( 12) -5001(19) -5325( 19) -4599( 16)
-10581.7(6) -10014(1) -1 1256(2) -11631(2) -1 0453(2) -1 1327(1) -1 1240(2) -10165(2) -9967(2) -10455(2) -10781(2) -11388(2) -10888(2) -1 0849(2) -1 1309(2) -1 1803(2) -11850(2) -1 1402(2) -1 1360(3) -10777(3) -10259(2) -1 1798(2) -1 1887(4) -12184(3) -12758(4) -1 3000(7) -13237(8) -1 3476(4)
ueq
939(6) 906(16) 1041(19) 1007(18) 734(17) 798( 17) 950(22) 867(25) 808(23) 656( 18) 685(18) 691(19) 638(18) 722(18) 813(23) 860(24) 854(24) 806(23) 1060(31) 1057(28) 791(20) 1lOO(31) 221 l(80) 1455(37) 1899(52) 3166(145) 31 13(171) 2498( 104)
introduction of the sample without heating. The ion source was kept at 60 “C. The collision activation-mass analyzed ion kinetic energy (CA-MIKE) spectrum was recorded operating a t 8 kV accelerating voltage. Molecular Mechanics-The minimized energy geometries of the molecular mechanics calculated model compounds were determined by the PCMODEL-pi program.zz PCMODEL-pi is a n enhanced version of Allinger’s MMX programz3 with the pi-VESCF routinesz4 incorporated for localized n-electron systems.
Results and Discussion X-ray Diffraction Studies-For the sake of clarity, we have chosen to number the atoms as in geneserine hydrochloride,16 which is different from the IUPAC notation in the Scheme 1. A perspective view of compound B is shown in 1
Figure 1, together with the labeling employed. The N(2)1
C(4)-C(3)-C(6)-C(5) ring shows an envelope conformation, with C(4) 0.49 A out of the N(Z)-C(3)-C(6)-C(5) plane, and the ring least-squares plane makes an angle of 5.1(3)” with the adjacent aromatic ring. The oxazine ring shows a chair conformation with the N-bonded C(13) methyl group in the equatorial position. A statistical analysis has been carried out with the Quest93 and Gstat93 packages of the Cambridge Crystallographic Data Centre, to compare the conformations assumed by the oxazine ring in all nineteen 1,2-oxazine compounds present in the crystallographic literature as well as to examine the behavior of the ring embedded in different crystal environments. The conformation of the ring has been described by the six torsion angles: tl,C(3)-C(4)-0(1)-N(l); t 2 , C(2)-C(3)-C(4)-0(1); t3, C(l)-C(Z)-C(3)-C(4); t4,N(1)C(l)-C(Z)-C(:3); t5, O(1)-N( 1)-C( l)-C(2); t6, C(4)-0( 1)-
Table 4-Selected Bond Distances (A) for Compounds A and B Compound B
Compound B
Compound A 1.425(5) 1.412(6) 1.364(7) 1.193(6) 1.518(6) 1.560(6) 1.485(7) 1.415(5) 1.412(6) 1.452(7) 1.334(6) 1.451(7)
1.444(10) 1.423(9) 1.336(10) 1.245(10) 1.461(15) 1.447(11) 1.492(17) 1.422(11) 1.400(10) 1.466(10) 1.313(11) 1.463(12)
Compound A 1.497(7) 1.530(6) 1.565(6) 1.517(6) 1.529(6) 1.396(6) 1.370(6) 1.360(6) 1.382(7) 1.368(7) 1.402(8)
1.489(16) 1.544(13) 1.530(12) 1.530(10) 1.523(13) 1.397(11) 1.394(11) 1.368(10) 1.401(10) 1.365(12) 1.368(12)
Table 5-Selected Bond Angles (deg) for Compounds A and B ~~
~
~
Compound A
CompoundB
C(8)-0(2)-C(11)
Nil )-O(1)-C(4) O(I )-N( 1)-C( 1)
118.4(6) 101.6(8) 107.0(6) 106.1(8)
C(l )-N(l)-C(l3)
109.9(9)
C(5)-N(Z)-C(I 5) C(4)-N(2)-C( 15) C(4)-N(2)-C(5) C(l l)-N(3)-C(12) N(l)-C( 1)-C(2) C( 1)-C(2)-C(3) C(Z)-C(3)-C(14) C(2)-C(3)-C(6) C(2)-C(3)-C(4) C(6)-C(3)-C(14) C(4)-C(3)-C(14) C(4)-C(3)-C(6)
119.6(6) 121.6(7) 108.6(7) 122.4(7) 106.1(9) 114.9(9) 111.4(8) 113.2(6) 111.3(7) 110.1(7) 111.2(7) 99.1(6)
O(1)-N( 1)-C( 13)
C(8)-0(2)-C(ll) O(1)-N( 1)-C( 13) O(1)-N( 1)-C(4) O(1)-N(1)-C(1) C(4)-N( 1)-C(13) C( 1)-N(1)-C( 13) C(1)-N(1)-C(4) C(5)-N(2)-C(15) C(4)-N(2)-C(15) C(4)-N(2)-C(5) C(Il)-N(3)-C(12) N(1)-C(1)-C(2) C(l)-C(Z)-C(3) C(Z)-C(3)-C( 14) C(Z)-C(3)-C(6) C(Z)-C(3)-C(4) C(6)-C(3)-C(14) C(4)-C(3)-C(14) C(4)-C(3)-C(6)
1128 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
~~
~
~~
~~
117.7(4) 109.0(4) 107.7(3) 104.4(3) 116.2(3) 115.0(4) 103.6(3) 122.4(4) 123.9(4) 110.7(3) 121.3(5) 103.0(4) 104.2(4) 111.4(4) 112.4(3) 105.7(3) 112.2(3) 113.6(4) 101.0(3)
N(2)-C(4)-C(3) O(1)-C(4)-C(3) O(l)-C(4)-N(2) N(2)-C(5)-C(lO) N(2)-C(5)-C(6) C(6)-C(5)-C(lO) C(3)-C(6)-C(5) C(5)-C(6)-C(7) C(3)-C(6)-C(7) C(6)-C(7)-C(8) 0(2)-C(8)-C(7) C(7)-C(8)-C(9) 0(2)-C(8)-C(9) C(8)-C(9)-C(lO) C(5)-C( 10)-C(9) 0(3)-C(ll)-N(3) 0(2)-C(ll)-N(3) 0(2)-C(11)-0(3)
~
~
~~
-~ ~~
~
Compound A
Compound B 104.1(7) 112.6(7) 104.0(7) 130.6(7) 108.3(6) 121.0(7) 108.4(6) 120.4(7) 130.8(6) 117.7(6) 120.3(6) 121.8(7) 117.7(7) 121.1(8) 118.0(7) 125.3(8) 111.7(7) 123.0(8)
N(Z)-C(4)-C(3)
N( 1)-C(4)-C(3) N(l)-C(4)-N(2) N(2)-C(5)-C(I 0) N(2)-C(5)-C(6) C(6)-C(5)-C(I 0) C(3)-C(6)-C(5) C(5)-C( 6)-C( 7) C(3)-C(6)-C(7) C(6)-C( 7)-C( 8) 0(2)-C(8)-C(7) C(7)-C( 8)-C( 9) 0(2)-C(8)-C(9) C(8)-C(9)-C(IO) C(5)-C( 1O)-C(9) 0(3)-C(ll)-N(3) 0(2)-C(lI)-N(3) 0(2)-C(11)-0(3)
107.8(3) 104.1(3) 112.6(3) 128.0(4) 109.5(4) 122.4(4) 110.9(4) 119.5(4) 129.6(4) 118.9(4) 119.0(4) 121.8(5) 119.1(4) 120.2(5) 117.2(5) 127.4(4) 109.1(4) 123.4(5)
mation with the C(2) atom at the flap (see Conclusion section). I
I
B
C(I2 (161
Cl17
01
Figure 1-ORTEP view of compound 8 . The thermal ellipsoids are drawn at the 50% probability level.
Figure 2-ORTEP view of compound A. The thermal ellipsoids are drawn at the 50% probability level.
N(l)-C(l). The possible random occurrence of different absolute configurations among the compounds has been taken into account.25 A cluster analysis with a single-linkage algorithm and Minkowsky metric has been applied on the space defined by the torsion angles,26 with the aim of characterizing the distribution of the conformations for the 1,2-oxazinerings. One major conformational cluster (chair, seven compounds) and three less populated ones (half-chair, four compounds; skew, three compounds; skew-boat, two compounds)have been identified (Table 6). The three compounds left are outliers in the conformational space spanned by the endocyclic torsion angles. The conformation of the oxazine ring in compound B is comparable with those found in the compounds of the largest cluster. It is noteworthy that compound B is situated at the border of cluster 1, as shown by the values of t2, t3, t5, and t6, which fall just outside the ranges defined by the other seven compounds; on the other hand, the compound corresponding to the refcode FITXOV, which is chemically similar to compound B, falls within cluster 4. A perspective view of compound A is shown in Figure 2. As expected, the protonation occurs on O(1). The compound adopts the N-oxide five-membered ring conformation showing an envelope conformation with C(1) lying 0.63 A out of the lane of the remaining atoms (maximum displacement of 0.06 ). In the previously structurally characterized geneserine hydrochloride,l6 the N-oxide ring adopts an envelope confor-
1
Unlike B, the five-memberedN(2)-C(4)-C(3)-C(6)-C(5) ring is planar within experimental error and almost coplanar with the adjacent six-membered ring, as already found in geneserine hydrochloride. The different conformational behavior of this ring in the two compounds can be attributed to different steric characteristics of the 1,2-oxazine six-membered and the N-oxide five-membered rings. The two compounds differ markedly also in the conformation shown by the heptylic chain, which is extended in B and folded in A, as is evident by the torsion angles N(3)-C(12)-C(16)-C(17), which is -179.1(8)" in B and -42(1)' in A. Accordin ly, in B the atoms N(3) through C(21) are located within 0.13 of their common plane, while in A N(3) is 0.98 A out of the plane defined by the atoms C(12) through C(21), which show a maximum displacement of 0.24 A from the plane of best fit. In both compounds the chain atoms have a large thermal motion which progressively increases toward the end of the chain. The different conformation of the two n-heptylic chains is due to the different role played by the carbonyl O(3) atom in the packing of the two compounds. The two crystal packings are shown in Figures 3 and 4, respectively. In A the oxygen carbonyl atom is not involved in any hydrogen bond, while in B it acts as a hydrogen bond acceptor with the carbamate N(3) donor atom at -x, y 1/2, -z - 1/2 [N(3)...0(3), 2.904(9) A; N(3)-H-- -0(3),141"l. In A, the N(3) and O(1) atoms are hydrogen bonded to the free C1 ion [N(3)..*Cl (X - 1, y - 1, z ) , 3.547(4) A, N(3)-H*.*Cl, 151"; 0(1).*.C1, 2.953(3)A; O(l)-H-.*Cl, 152"). Both of the molecular arrangements are stabilized by hydrogen-bonded chains extending along the [1,1,0]direction in A and along the [0,1,0] direction in B. The packing motif of compound A resembles closely the one found for geneserine hydrochloride,16despite the presence of one water molecule in the asymmetric unit of the latter. The crystallization solvent does not interfere with the formation of the hydrogen-bonded chains which are common to the packing of both hydrochlorides. N M R and Mass Studies-Free Base-The lH NMR data of B (see Scheme 1)are consistent with the structure of a cisfused tetrahydro-1,2-oxazine ring as previously reported for gene~erine.~ , ~ the MS analysis was indicative of this Also, structure: loss of M+ - 16, M i - 17 or Mf - 18, as might be expected for an N-oxide structure, was not observed, whereas the CA-MIKE spectrum of the molecular ion gave rise to m/z 316 (M+ - C2H5NO) and mlz 315 (M+ - Cz&NO) in analogy to the results reported for geneserineZ7and flustrarine B, another 1,2-0xazine derivative.28 The vicinal IH coupling constants were analyzed to determine the conformation in solution of the molecule. Before proceeding with this study, the IH assignments were first unambiguously established by a homonuclear correlation experiment and NOE's measurements. The 1,2-oxazine ring should adopt a chair conformat i ~ nbut , ~the ~ values of the experimental coupling constants are completely out of the expected range for a single conformation. As already reported for g e n e ~ e r i n e ,it~ ~can be supposed that a rapid equilibrium exists in solution between the two possible chair conformations lCq and 4C1with the N-methyl group in the equatorial position (see Scheme 2). These two forms were built up and minimized by molecular mechanics calculations using the Allinger Force Field MMX and the theoretical 3J coupling constants estimated from the Karplus equation modified by Altona et aL31 The results are reported in Table 7. From the experimental 3J,the population of each conformer can be calculated by solving the well-known time average equation:32
+
+
Jay= P(I)J(I) P(II)J(II)+ ..., P(n)J(n) where J ( n )are the predicted values for the pure conformers. Journal of Pharmaceufical Sciences / 1129 Vol. 84, No. 9, September 1995
Table 6-Cluster Analysis Results Compared with Data for Compound B and Data Relative to the Cambridge Crystallographic Database Quest (Oct 93) Sl
Compound 6
-59.9
Cmean maximum minimum
-65.7 -54.2 -73.8
Cmean maximum min imum
-55.0 -50.6 -59.3
Cmean maximum minimum
t2 39.6
t3 (a) Cluster Analysis Resultsa -36.5
t4 51.9
t5
t6
-71.6
77.2
Cluster 1 55.0 -49.2 54.6 -64.5 60.7 -45.5 58.4 -62.4 46.5 -53.1 50.6 -67.6 Compounds, 7; Refcodes,CXBAXR, FETZOT, FEVPIF, FEVPOL, FIBLIL, FUHHEV, VORDEL
71.O 76.6 65.1
61.2 64.2 58.9
Cluster 2 -37.1 6.9 -30.1 12.8 -43.9 1.o Compounds, 4; Refcodes, JAMGEJ, SOSZEF, SOSZEF, YAHBAK
2.0 2.8 0.5
22.7 27.9 16.5
-39.6 -33.5 -50.5
26.4 33.3 22.7
Cluster 3 -2.8 --12.5 -1.9 -8.9 -3.7 --17.5 Compounds, 3; Refcodes, FRGLXB, MNPHOX, MNPHOX
0.3 1.3 -0.8
27.1 33.3 23.4
Cmean maximum minimum
-48.7 -44.9 -52.5
-19.3 -13.8 -24.9
-37.5 -32.7 -42.3
82.7 84.4 81.O
BAZGIS CETHOZ VORDAH
-13.7 69.9 -54.0
9.6 -49.6 22.0
0.7 12.3 -41.7
8.6 -56.4 67.9
Cluster 4 59.2 --2a.7 62.7 --27.6 55.7 -29.8 Compounds, 2; Refcodes, FITXOV, JEBXIX Isolated Fragments -1.5 8.3 1.7
-3.9 12.9 6.7
(b) Data Relative to Questb BAZGIS FUHHEV 6-Chloro-3acyano-2-cyclohexyl-4a-methyl-1 aa,3,4,4a,5,1 O-hexahydronaphth3,4-Bis(methoxycarbonyl)-5-(methylthio)-6-oxo-6K1,2-0xazine CsHdiosSi (2,3-e)(1,2)0xazine H. Gotthardt, 0. M. Huss, C. M. Weisshuhn CzoH25ChNzOi C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Acta Crystallogr., Sect. B, 1982, 38, 680 J. Cryst. Spectrosc. 1987, 17, 515 CETHOZ 2-(Chloroethanoyl)-4-methyl-5-methylene-6-hydro-l,2-oxazine JAMGEJ CsHioCIiNiOz Ethyl 6-methyl-6-phenyl-5,6-dihydro-4H-l,2-oxazine-3-carboxylate B. Tinant, J. P. Declercq, G. Germain, M. van Meerssche Ci4Hi7Ni03 D. J. Chadwick, T. L. Gilchrist, W. Stretch Bull. Soc. Chim. Belg. 1980, 89, 407 Acta Crysfallogr. C 1989, 45, 976 CXBAXR N(pCarboxybenzy1)tetrahydro-1,Poxazine JEBXIX C12H15N103 Ethyl rek(1R,3S,6aff,EaF@bS)-5,5,6,6,8b-pentamethyI-6a, 7,8,8a-tetrahydrocyclopenta(1,2,3-h~)isooxazolo(2,3-b)( 1,2)oxazine-l-carboxylate F. G. Riddell, P.Murray-Rust, J. Murray-Rust Tetrahedron 1974, 30, 1007 Ci6Hz7Ni04 S. E. Denmark, Young-Choon Moon, C. B. W. Senanayake FETZOT 2-Cyclohexyl-4a-methyl-3,4,4aa,5,10,1Oaa-hexahydro-6-nitronaphth- J. Am. Chem. SOC.1990, 112,31 1 (2,3-e)(1,2)oxazine-3P-carbonitrile MNPHOX CzoHz&”03 6-(Dimethylamino)-5-(4-nitrophenylamino)-3-(2,4,6-trimethylphenyl)-6~-1,2-oxazine C. W. Holzapfel, G. J. Kruger, M.S. Van Dyk CziHz4N403 A. C. Villa, A. G. Manfredotti, C. Guastini, P. Trimarco Acta Ctystallogr. C 1987, 43, 514 Cryst. Struct. Commun. 1980, 9,523 FEVPIF 9-Chloro-2-cyclohexyI-4~-methyl-3,4,4a,5,10,1Oa-hexahydronaphth- SOSZEE 4,4’-Bi(5,6-dihydro-3-methyl-4H1,2-oxazine) (2,3-e)(1,2)0xazine-3/?-carbonitrile CzoHz5CliNz0i CIOHI~NZ~Z S. Shatzmiller, R. Lidor, E. Bahar, I. Goldberg C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Liebigs Ann. Chem. 1991,851 Acta Crystallogr. C 1987, 43,598 FEVPOL VORDAH 2-Cyclohexyl-9-iodo-4/?-methyl-3,4,4a,5,10,1Oaa-hexahydronaphth(6R)-2-((R)-2-Hydrox~2-phenylacetyl)-6-((S)-5-methoxycarbonyl-2-oxopyrrolidinyl)3,6-dihydro-2H-l,2-oxazine (2,3-e)(1,2)oxazine-3~-carbonitrile CzoHzsIiNzOt Ci8HzoNz06 A. Defoin, J. Pires, I. Tissot, T. Tschamber, D. Bur, M. Zehnder, J. Streith C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Tetrahedron: Asymmetry 1991,2, 1209 Acta Ctystallogr. C 1987, 43,598 FlBLlL VORDEL 2-(gNitrobenzoyl)tetrahydro-2H1,Poxazine (4R,5R,6S)-6-(t-ButyIdimethylsiloxy)-2-[(2R,5R)-(2,5-dimethylpyrrolidinocarbonyl]Cii HizN204 tetrahydro-2Kl,2-oxazine-4,5-diol J. E.Johnson, R. Hodzi, S. L. Todd, P. de Meester, S. S. C. Chu C17H&205Si3 J. Heterocycl. Chem. 1986, 23, 1423 A. Defoin, J. Pires, I. Tissot, T. Tschamber, D. Bur, M. Zehnder, J. Streith Tetrahedron: Asymmetry 1991,2,1209
1130 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
Table 6 (Continued) FITXOV 2H2-Cyclohexyl-4~-methyl-3,4,4aa,9aa-tetrahydrobenzothieno(3,2~e)( 1,2)oxazine3a-carbonitrile CieHzzNzOiSi C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Acfa Crysta//ogr.C 1987, 43, 1367 FRGLXB 3-(Ethoxycarbonyl)-6-(methoxycarbonyl)-5-phenyl-6~1 ,Poxazine CikMW5 Y. Nakada, T. Hata, C. Tamura, T. Iwaoka, M. Kondo, J. Ide Tetrahedron Left. 1981, 22,473
YAHBAK 4-(pBromophenyl)-5-hydroxy-3-(methoxycarbonyl)-5,6-di hydro-4#1,2oxazine Cl2H1~BrlN1O4 K. Harada, Y. Shimozono, E. Kaji, H. Takayanagi, H. Ogura, S. Zen Chem. Pharm. Bull. 1992, 44 1921
a Cmean = circular mean for torsion angles within a cluster, Refcode = Cambridge Crystallographic Database reference. For compounds FRGLXB and JEBXIX the R value is not reported in the original papers. All other compounds have R c 10%. A connectivity search has been also carried out which did not give any other compound suitable for comparison: t l *comp '1 ,Poxazine'
t2 *comp '1,2)oxazine' quest t l .or.t2
It Scheme 2
The calculated percentages of the two forms 4C1 and lC4 were 75 & 5% and 25 & 5%, respectively. As previously observed, the N-methyl group should prefer the equatorial position; therefore, the complete inversion should involve both ring and nitrogen inversion (see Scheme 2 again). The existence of such conformational equilibria was further confirmed by lowering the temperature; at 244 K the four singlet resonances of the molecule a t room temperature split into doublets. For the bridgehead methine proton, the integrated intensity ratio of the peaks gave approximately the same percentages of 4C1and lC4 reported before, indicating that a t 305 and 244 K the equilibrium constant for the two conformers is nearly identical within the experimental error (Figure 5). HCZ Salt-An analysis of the lH NMR 3Jvalues was used to establish whether the molecule A exists as a N-oxide derivative in solution as observed for geneserolineg and for geneserine hydrochloride.16 For the salt as well, the lH assignments were first unambiguously established by correlation spectroscopy and NOES measurements. In CDCl3 the vicinal coupling constants of the spin system CHz-CHz are typical of a five-membered ring, confirming that the molecule adopts this structure. The experimental values are consistent with the 3Jestimated from the envelope conformation with the 3-carbon a t the flap, indicating that this form is largely prevalent in solution (see Table 8). The spectra of A in more polar solvents like DMSO-d6 or D20 show the signals of another molecule in slow exchange on the NMR time scale. To assign its structure, a steady state NOE experiment was performed. By irradiating the pyrrolic
Figure 3-Packing diagram of compound 6. Table 7-Theoretical and Experimental Coupling Constants (3J in Hz) for Vicinal Proton Pairs in Compound B (SeeScheme 2)
Proton Pair
3Jexp
H3 H4 H3 H4' H3' H4 H3' H4'
4.5 5.1 4.3 9.7
75:25 'Jcaic 4.6 5.0 4.3 9.7
' C4
4C~
12.8 3.1 3.4 2.7
1.9 5.6 4.6 12.0
N-methyl group, a significant enhancement of the 8a-proton was observed, indicating that the second molecule is the epimer arising from inversion a t the tetrahedral nitrogen (see Scheme 3). Its absolute configuration is 3aS,BaS,lS. A careful analysis of the spectrum in CDCl3 pointed out that such epimer is present in this solvent too, but in negligible amounts (3-4%). The percentages of the two epimers in different solvents are reported in Table 9. The ratio between the two epimers does not change, even if the solutions were allowed to stand for 1 week a t room temperature, indicating Journal of Pharmaceutical Sciences / 1131 Vol. 84, No. 9, September 1995
0
& 7 -L&J I
(33
/
HO
I
"'./
C"3
2 :
CH3
\,,,
HO
3aS. 8aS. 1s
3aS. 8aS. 1R
Scheme 3 Table 9-Percentages of the Two Epimers of Compound A in Different Deuterated Solvents at 303 K A
Deuterated Solvent
1R
1S
Deuterated Solvent
1R
1S
Water (pD = 2.0) Water (pD= 5 0) Trifluoroethanol Ethanol
62
38
73
27
80 80
20 20
Dimethyl sulfoxide Acetone Chloroform Dioxane
80 94 96
20 6 4 0
100
E
Figure 4-Packing diagram of compound A.
l
160 rio iio loo io 60 in in i PPM Figure 6-Comparison of the CDC13 solution-state (bottom) and solid-state (top) all-decoupled i3C spectra of compound A. Solid circles denote sidebands, 7
7.0
6.0
5.0
4.b PPti
.
2.0
3.0
L
'
1.0
Figure 5--'H NMR spectrum of compound A in CDC13: at 303 K (bottom) and 244 K (top). Solid circles indicate the splitted signal of the bridgehead methine proton. Table 8-Dihedral Angles ($) and Corresponding Theoretical and Experimental Coupling Constants (Vin Hz) for Vicinal Proton Pairs in Compound A Proton Pair
d, deg
3Jca~c
H2 H3' H2 H3 H2' H3 H2' H3'
76 -43 -1 60 -4 1
0.7 6.2 11.5 6.4
3Jexp
0.8 5.5 13.1
5.7
that the equilibrium in each solvent has been already reached during the time required to run the spectra. Unexpectedly, the vicinal coupling constants of the spin system CHz-CHz in the IS epimer are very different from the corresponding ones in the 1R epimer, and they are completely out of the expected range for a single conformation. The values observed can be explained only supposing a fluxional system with 1132 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
different conformers in rapid equilibrium. To investigate whether the two epimers are present in the solid-state too or they form just in solution, the CP-MAS 13C NMR spectrum was recorded. Figure 6 compares the solid-state and solution spectra: carbons directly bonded to nitrogen atoms can be perceived immediately, as they do not give rise to sharp peaks.33 The other signals were assigned by comparison with those of the solution spectrum (see Experimental Section); in the CP-MAS spectrum no splitting of the signals was observed, indicating that in the solid-state the molecule is present as a single epimer. The presence of a single set of peaks confirms the crystallographic finding, i.e. that a single crystalline form is present and that the molecules in the unit cell are eq~ivalent.~~
Conclusions The n-heptylcarbamate of geneseroline base is a 1,2-oxazine derivative; it exists in solution as a mixture of two possible chair conformers 4C1 and ICq (with the N-methyl group in the equatorial position), whereas in the solid-state only the former is present.
Its hydrochloride salt is instead an N-oxide derivative; the five-membered ring adopts an envelope conformation in both states, but in the solid-state the atom at the flap is the 2-carbon, while in solution it is prevalently the 3-carbon. The latter conformation corresponds to the one observed for geneserine hydrochloride both in the solution- and solidsuggest that such a moiety is state'16 These and the packing forces are predominant in defining the conformation in the crystal. Interestingly, in solution, the salt partially undergoes fast inversion at the tetrahedral nitrogen, giving rise to the corresponding 1sepimer whose N-oxide fivemembered ring adopts a more flexible conformation. The amount of the latter epimer increases with the polarity of the solvent,34but it is always present as a minor species, indicating that it is less stable than the former. The results of force field calculations in vacuum are qualitatively consistent with these experimental findings.
References and Notes 1. Chiesi, P.; Delcanale, M.; Servadio, V.; Ghidini, E., PCT Int. Appl. Patent 93 03041, 1993; Chem. Abstr. 1993, 119, 95904. 2. Polonovski, M. Bull. SOC.Chim. Fr. 1915, 17, 245-289. 3. Polonovski, M. Bull. SOC.Chim. Fr. 1917,21, 191-200. 4. Polonovski, M. Bull. SOC.Chim. Fr. 1925, 37, 744-759. 5. Hootelk, C. Tetrahedron Lett. 1969,32, 2713-2716. 6. Robinson, B.; Moorcroft, D. J . Chem. Soc.(C) 1970,2077-2078. 7. Hill, R. K.; Newkome, G. R. Tetrahedron 1969,25,1249-1260. 8. Pauling, P.; Petcher, T. J. J . Chem. SOC.Perkin Trans 2 1973, 1.142-1.14.5 - - .- - - -. 9. Yu, Q. S.; Yeh, H. C. J.; Brossi, A,; Flippen-Anderson, J . L. J . Nut. Prod. 1989, 52, 332-336. 10. Brufani. M.: Marta., M.:, PomDoni. M. Eur. J . Biochem. 1986. 157, 115-120. 11. Brufani, M.; Castellano, C.; Marta, M.; Oliviero, A,; Pagella, P. G.; Pavone, F.; Pomponi, M.; Rugarli, P. L. Pharmacol. Biochem. Behau. 1987,26, 625-629. 12. De Sarno, P.; Pomponi, M.; Giacobini, E.; Tang, X. C.; Williams, E. Neurochem. Res. 1989,14, 971-977.
13. Marta, M.; Castellano, C.; Oliviero, A.; Pavone, F.; Pagella, P. G.; Brufani, M.; Pomponi, M. Life Sci. 1988,43,1921-1928. 14. Pomponi, M.; Giardina, B.; Gatta, F.; Marta, M. Med. Chem. Res. 1992,2, 306-327 and references cited therein. 15. Brzostowska, M.; x.; ~ ~N. H.;~ Rapoport, i ~s, 1,; ~, ~ A. Med. Chem. Res. 1992,2, 238-246. 16. Bacchi, A.; Pelizzi, G.; Redenti, E.; Delcanale, M.; Amari, G.; Ventura, P. Acta Crystallogr. 1994, '250, 1126-1130. 17. Walker, N.; Stuart, D. Acta Crystallogr, 1983, A39, 158-166, 18. Sheldrick, G. M. S H E U S 8 6 A Program for Structure Determination, University of Gottingen, D, 1986. 19. Sheldrick, G. M. SHELXL92 A Program for Structure Refinement, University of Gttingen, D, 1992.
;::~ 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
I
34.
~ { . ~~ ~~ ~ ~~ ~ ~ Program p :~ for ~ Crystal Structure Illustrations. Report ORNL 3794 (Oak Ridge National Laboratory, Oak Ridge, Tenn.), 1965. PCMODEL-pi program, Version 4.0, Serena Software Inc. P.O. Box 3076, Bloomington, IN, 47402-3076. Allinger, N. L. J . Am. Chem. SOC.1977,99, 8127-8134. Allinser. N. L.; Sprame. J. T. J . A m . Chem. SOC.1973,95,3893. Alle
Supplementary Material Available-Lists of structure factors, atomic coordinates of hydrogen atoms, thermal anisotropic parameters are available from the author. JS9404981
Journal of Pharmaceutical Sciences / 1133 Vol. 84, No. 9, September 1995
~
AND
GIANCARLO PELIZZIS
Received July 6, 1994, from the *Chemical and Biopharmaceutical Department, Chiesi Farmaceutici S.p.A., Via Palermo 26/A, 1-43100 Parma, Italy, and 'Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Centro di Studio per la Strutfuristica Accepted for publication May 30, 1995@. Diffrattometrica del CNR, Wale delle Scienze, L43100 Parma, /tab, Abstract 0 The solid state structures of the (-)-nheptylcarbamate of geneseroline and its hydrochloride salt were determined by single crystal X-ray diffraction analysis. Both compounds gave crystals belonging to the orthorhombic P212121 space group with a = 27.597(7) A, b = 8.899(2) A, c = 9.290(2) A, V = 2281.5(9) A3, Z = 4, and R = 0.06820forthe base and a = 11.300(1) A, b = 8.3485(5) A, c = 24.141(2) A, V = 2277.3(3) A3, Z= 4, and R = 0.0482 for the salt. X-ray and 'H NMR analysis revealed that the base is a 1,2-oxazine derivative. The sixmembered ring adopts a 4Gchair conformation in the solid-state, whereas, in CDC13solution, it exists as a mixture of two possible chair conformers, 4C1 and lC4, with the Nmethyl group in the equatorial position (ratio FX 7525). The salt is an Noxide derivative; the five-membered ring adopts different envelope conformations in the solid-state and in CDCI3 solution, suggesting a certain flexibility. In more polar solvents, the salt partially undergoes fast inversion at the tetrahedral nitrogen, giving rise to the corresponding epimer.
A
0 O,-NHCH,
Introduction The n-heptylcarbamate of geneseroline {( -)-2,3,4,4a,9,9ahexahydro-2,4aa,9a-trimethyl-l,2-oxazino[6,5-b]indol-6-ol nheptylcarbamate, B}' is a higher homologue of geneserine (Nmethyl derivative), an alkaloid which was first isolated in 1915 by Polonovski from the basic extracts of Calabar bean seeds2 and later obtained from physostigmine by oxidation with H z O ~ The . ~ structure for such alkaloids with a cis-fused tetrahydro-1,2-oxazine ring (instead of the N-oxide ring21 was proposed by Hootele5 and independently supported by NOE measurements.6 Since the absolute configuration a t the indoline 4a-carbon was already k n o ~ nthe , ~absolute ~ ~ stereochemistry of the alkaloid was determined to be 4aS,9aS (see Scheme 1). The n-heptyl derivative was obtained according to a synthesis which gave excellent yields for all the n-alkyl derivatives; the base was hence converted quantitatively into the crystalline hydrochloride salt with concentrated HCl. The salt adopts the N-oxide structureg {(-)-1,2,3,3a,8,8ahexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-01 n-heptylcarbamate N-oxide hydrochloride, A}. Like physostigmine and tacrine n-alkyl homologues, n-alkylcarbamates of geneserolinel inhibit acetylcholinesterase activitylo-l3and, even for this set of compounds, the optimal inhibitory profile was found for the n-heptyl derivative. With respect to the deoxy analog heptastigmine, the n-heptylcarbamate of geneseroline also shows a more prolonged action and a better therapeutic index. The therapeutic interest €or these compounds is due to their potential use in the treatment of the Alzeheimer's disease.14J5 Although a large number of other neurotransmitters and neuropeptides systems are also affected, a deficiency of cholinergic activity is suspected to play a major role in the expression of such p~ychopathology.'~The rationale for this therapy is the reversible anticholinesterasic action of the drug through its carbamate function, which possibly improves the activity of cholinergic neurons by slowing the @Abstractpublished in Advance ACS Abstracts, July 1, 1995.
1126 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
C
D
A
0
Scheme 1 degradation of the neurotransmitter acetylcholine. Here the structural study of A and B in the solution- and solid-state carried out by single crystal X-ray analysis, NMR spectroscopy, and mass spectrometry is reported.
Experimental Section Chemistry-The synthesis is reported in the Scheme I. A solution of 1.5 g (4.6 mmol) of geneserine hydrochloride'6 (D, HPLC purity > 98%)in 6 mL of concentrated sulfuric acid was heated and stirred a t 80 "C for 15 min. The solution was cooled to 0 "C and poured in ice water. A 30%solution of ammonia was added dropwise with stirring to pH = 7.5. Methylene chloride (50 mL) was added t o the mixture. The biphasic mixture was stirred for 5 min and partitioned, and the organic portion was dried over NaZS04, filtered, and evaporated under reduced pressure, t o give 1.0 g (4.3 mmol, 93%) of geneseroline (C) intermediate product as a white solid: [ a ]-158" ~ (c = 0.1, CHCl3); mp 142-145 "C; HPLC purity '97%; EI MS 234 mlz (M+). To 0.9 g (3.8 mmol) of C dissolved in 40 mL of toluene was added a catalytic amount (15 mg) of sodium and the mixture was stirred to complete dissolution. A solution of 55 mg (3.8 mmol) of n-heptyl isocyanate in 20 mL of toluene was added dropwise and the resulting solution stirred a t room temperature for 30 min, then a solution of 100 mg of ammonium chloride in 1 mL of water was added. The mixture was dried over NanS04, filtered, and evaporated under
0022-3549/953184-1 126$09.00/0
0 1995, American Chemical Society and American Pharmaceutical Association
reduced pressure to give a n oil that was crystallized from petroleum ether a t 0 "C, to yield 1.1g (2.9 mmol) of the n-heptylcarbamate of -121" (c = 0.1, geneseroline (B) as a white crystalline solid: [ a l ~ CHC13); mp 81-82 "C; HPLC purity > 98%; IR 1660 cm-' (C = 0 carbamate); IH NMR (CDC13) 6, ppm 0.85 (t, 3H), 1.2 (s, 3H, CH34a), 1.1-1.5 (m, 8H), 1.6 (m, 2H), 1.96 (m, 2J = -14 Hz, l H , H4'), 2.14 (m, = -14 Hz, l H , H4), 2.5 (m, 2J = -12 Hz, l H , H37, 2.55 (s, 3H, CH3-N2), 2.66 (m, 2J = -12 Hz, l H , H3), 2.85 (s, 3H, CH3N9), 3.3 (q, 2H, CHZa-NH), 4.7 (s, l H , H9a), 4.95 (br, l H , N-H), 6.4 (d, J&h,, = 8.2 Hz, l H , H8), 6.7-6.9 (m, 2H, H5,7); 13CNMR (CDC13) d , ppm 14.7, 23.2, 26.1 (C4a-CH3), 27.4, 29.6, 30.6, 32.3 ((241, 32.4, 33.4 (N9-CH3),41.1 (C4a), 42.0,46.8 (NB-CHd 54.2 (C3), 102.7 (Cga), 107.4 (C8), 116.3 (C5), 120.8 (C7), 137.0 (C4b), 144.3 (C6), 148.2 (C8a), 156.1 (C=O); EI MS 375 mlz (M+). Upon dissolving 1.0 g of the base in 5 mL of acetone, a stoichiometric amount of ethereal HC1 and 7 mL of diethyl ether were added. The solution was allowed to stir for 8 h at 0 "C and the white solid which separated was filtered to yield 1.0 g (2.4 mmol) of the n-heptylcarbamate of gkneseroline hydrochloride (A) as a crystalline ~ (c = 1,HzO); mp 146-147 "C; HPLC purity > 99%; solid: [ a ]-136" IR 1720 cm-l (C=O carbamate); lH NMR (CDC13)6, ppm 0.9 (t, 3H), 1.2-1.6 (m, 8H), 1.6 (s, 3H, CH3-3a), 1.75 (m, 2H), 2.3 (dd, *5= -12.5
Table 1-Crystal Data and Summary of Intensity Data Collection and Structure Refinementd
B A Compound Formula CziH34ClN303 CziH33N303 41 1.97 375.51 MVY 11.300(1) 27.597(7) a, A 8.3485(5) 8.899(2) b, A 24.141(2) 9.290(2) c, A 2277.4(3) 2281.5(9) V, A3 4 4 Z 1.202 1.093 D,, g ~ m - ~ 816 888 F(000) 5.9 16.8 p !cm-' 3-70 3-65 6' range, deg 1-1 7 741 Standard reflection 251 1 (+h,+k,+l) 5828 (kh,+k,+l) Number of measured reflections 2485 2206 (& = 0.047) Number of independent reflections 1391 1718 Number of observed reflections 320 253 Parameters refined 0.23 0.31 Max height in final AFrnap, e A-3 0.0482 0.0682 R (observed data) Hz,1H,H3'),2.7(m,~J~-l2.5Hz,1H,H3),2.93(m,~J=-l1H~, 0.1511 0.1759 wR2 (all data) lH,H2'),3,3(q, 2H,CHza-NH),3.32 ( s , ~ H , C H ~ - N3.5 ~ )(s,3H,CH3, a Features common to both analyses include crystal system (orthorhombic), N l ) , 3.92 (dd, 2J = -11 Hz, lH, H2), 5.05 (br, l H , N-H), 5.9 (s, lH, space group (P212121),scan mode (6' - 26'), and criterion for observation (6> = 2.3 Hz, l H , H4), HSa), 6.6 (d, Jartho = 8.2 Hz, lH, H7), 6.9 (d, Jmeta = 2.3 Hz, lH, H6), 13.1 (br, l H , 0-H); 4u(F0)). 7.0 (dd, Jofiho = 8.2 Hz, Jmeta 13CNMR (CDC13) 6, ppm 14.7, 23.2, 26.7 (C3a-CH31,27.3, 29.6, 30.5, Table 2-Atomic Coordinatts (x104)and Equivalent Isotropic 32.4, 37.7 (N8-CHz), 38.1 (C3), 42.0, 48.8 (Nl-CH31, 53.9 (C3a) 64.3 Displacement Parameters (A2 x 104) (One-Third Trace of the Diagonalized (C2), 109.3 (C7), 111.8(C8a), 117.3 (C4), 123.2 (C6), 135.0 (C3b), 146.2 Matrix) for Compound B (esd Values in Parentheses) (C5), 146.5 (C7a), 155.5 (C=O); EI MS: 375 mlz (M+). Apparatus-Melting points (uncorrected) were determined on a Atom Xla Ylb ZlC uw Buchi apparatus. Optical rotations were measured at room temperature on a Perkin-Elmer 241 automatic polarimeter. Infrared spectra 1081(25) 5822(7) 9130(6) 1588(2) were taken either in Nujol or as a KBr disk on a Perkin-Elmer 297 3440(5) 904(20) 6359(5) 435(2) spectrophotometer. HPLC analyses were run on a Hewlett-Packard 916(20) 3614(6) 4512(6) -129(2) 1050 system provided with a W - v i s detector (1 = 245 nm). A 1268(38) 6418(11) 10301(8) 1303(4) p-Bondapack C18 analytical column (Waters, 10 um, 300 x 3.9 mm 920(25) 7542(8) 3907(7) 1650(2) i.d.1 was employed. The mobile phase was 0.1 M phosphate buffer 909(26) 6679(9) -270(3) 2390(8) pH = 3.0:acetonitrile in the ratio 88:12 for C ( t =~8.7 min) and D ( t ~ 1232(50) 9670(10) 842(4) 6891(13) = 4.3 min) and 62:38 for B and A ( t = ~ 9.8 min), respectively. The 1150(41) 5485(13) 9301(9) 577(3) flow rate was 2 mumin. 924(33) 4350( 10) 8389(8) 868(3) Crystallography-All X-ray measurements were made a t room 960(36) 4335(11) 8822(9) 1403(3) temperature on a computer-controlled Siemens AED diffractometer 792(30) 6358(8) 4458(8) 1391(3) using Cu Ka radiation (2 = 1.54 178 8). Automatic peak search, 734(26) 4830(8) 6816(7) 924(3) centering, and indexing procedures established in both cases an 831(28) 5389(8) 5862(7) 592(3) orthorhombic primitive lattice and the systematic extinctions observed 784(28) 4437(7) 5610(8) 742(3) identified unambiguously the space group as P212121. 837(31) 5250(9) 3993(8) 1199(3) The unit cell dimensions and other relevant crystal data are 869(32) 4938(8) 4696(9) 1534(3) presented in Table 1 together with details of data collection and 787(29) 5774(10) 3174(8) 4 3 ) refinement. No crystal decay was observed. The intensity data were 1958(11) 1106(36) -765(4) 6302(10) processed with a peak-profile analysis procedure and corrected for 1699(62) 10724(13) 1592(5) 7764(15) Lorentz and polarization factors. For B a n absorption correction 1328(46) 8472(9) 649(4) 2782( 11) (empirical surface) was also applied following the method of Walker 7478(11) 1241(42) 2180(3) 3831 (12) and Stuart.l7 For both structures the phase problem was solved by 121l(38) 591(10) -804(3) 5480(12) application of direct methods. Refinement was by full-matrix least1506(50) 245(14) -1338(4) 5 174(14) squares methods employing anisotropic thermal parameters for all 1740(59) -1032(15) -1 417(5) 4313(15) non-hydrogen atoms and isotropic ones for the hydrogen atoms, which 2419(96) -1467(21) -1918(6) 4044(25) were in part located from difference maps (those bonded to O(l), N(3), 2977(134) -1 054(24) -2307(7) 3777(32) C(1), C(2), C(4), C(7), C(9), C(lO), C(13), and C(14) for A and to "3) 2385(92) -2768(5) 3223(23) -1 678(19) for B) and in part placed a t calculated riding positions. In the refinement of B the heptyl chain atoms were subject to rigid bond restraints. The absolute configuration was tested by refining both enantiomers for each compound. In both cases the lower R and R, for distortionless enhancement by polarization transfer (DEFT), values corresponded to the previously known absolute configuration steady state nuclear Overhauser effect difference (NOEDIFF), 2D a t the indoline 4a-carbon a t ~ m . Neutral ~,~ atom scattering factors lH,'H correlation (COSY), and 2D lH,13C correlation (XHCORRDC) were used, those for the non-hydrogen atoms being corrected for experiments. The coupling constants values found were confirmed anomalous dispersion. All the calculations were performed on a t 400.1 MHz as the spin systems were not first order a t 200.1 MHz. GOULD POWERNODE 6040 and ENCORE91 computers using the The 13C NMR solid-state spectra were obtained using the crossprogram packages SHELXSS6,l8 SHELXL92,l9 PARST,20 and polarization magic-angle spinning (CP-MAS) accessory on a Bruker ORTEP.21 Final atomic parameters are reported in Tables 2 and 3, CXP 300 instrument. The spectra were measured with a contact time whereas bond distances and angles are given in Tables 4 and 5. of 1 ms, which was not optimized. The side bands were assigned by carrying out the experiments a t different spinning rates (3.05,4.96, N M R Spectroscopy--'H and 13C NMR spectra were obtained at and 5.46 kHz, respectively). 200.1 and 50.3 MHz in different deuterated solvents on a Bruker ACF Mass Spectrometry-The electron impact (EI) mass spectra were 200 instrument. The residual protons for each solvent were used as recorded a t 70 eV on a VG Micromass ZAB-2F spectrometer for direct internal references, Standard Bruker microprograms were employed
Journal of Pharmaceutical Sciences / 1127 Vol. 84, No. 9, September 1995
Table 3-Atomic Coordinates (xIO4) and Equivalent Isotropic Displacement Parameters (A2 x lo4) (One-Third Trace of the Diagonalized Matrix) for Compound A (esd Values in Parentheses) ~
Ma
Ylb
ilC
482.2( 11) -1804(3) -6100(3) 4960(3) -2632(3) -271 5(4) -6737(4) -3769(4) -3500( 4) -2859(4) -2263(4) -3581 (4) -371 O(4) -4517(4) -51 87(4) -5048(4) 4229(5) -5850(4) -6733(6) -2709(5) -1984(4) -21 13(6) -7076( 10) -7967(6) -8260(9) -9296( 14) -1 0069( 11) -1 1020(10)
6218.0(17) 5733(4) -788(4) -2717(5) 5521(4) 3974(4) -3275(5) 5026(7) 3367(6) 2585(5) 3990(5) 2766(5) 1945(5) 742(5) 358(6) 1140(7) 2394(6) -2329(7) -4979(7) 7028(6) 1308(6) 4686(7) -5488( 10) -4959(10) -5728( 12) -5001(19) -5325( 19) -4599( 16)
-10581.7(6) -10014(1) -1 1256(2) -11631(2) -1 0453(2) -1 1327(1) -1 1240(2) -10165(2) -9967(2) -10455(2) -10781(2) -11388(2) -10888(2) -1 0849(2) -1 1309(2) -1 1803(2) -11850(2) -1 1402(2) -1 1360(3) -10777(3) -10259(2) -1 1798(2) -1 1887(4) -12184(3) -12758(4) -1 3000(7) -13237(8) -1 3476(4)
ueq
939(6) 906(16) 1041(19) 1007(18) 734(17) 798( 17) 950(22) 867(25) 808(23) 656( 18) 685(18) 691(19) 638(18) 722(18) 813(23) 860(24) 854(24) 806(23) 1060(31) 1057(28) 791(20) 1lOO(31) 221 l(80) 1455(37) 1899(52) 3166(145) 31 13(171) 2498( 104)
introduction of the sample without heating. The ion source was kept at 60 “C. The collision activation-mass analyzed ion kinetic energy (CA-MIKE) spectrum was recorded operating a t 8 kV accelerating voltage. Molecular Mechanics-The minimized energy geometries of the molecular mechanics calculated model compounds were determined by the PCMODEL-pi program.zz PCMODEL-pi is a n enhanced version of Allinger’s MMX programz3 with the pi-VESCF routinesz4 incorporated for localized n-electron systems.
Results and Discussion X-ray Diffraction Studies-For the sake of clarity, we have chosen to number the atoms as in geneserine hydrochloride,16 which is different from the IUPAC notation in the Scheme 1. A perspective view of compound B is shown in 1
Figure 1, together with the labeling employed. The N(2)1
C(4)-C(3)-C(6)-C(5) ring shows an envelope conformation, with C(4) 0.49 A out of the N(Z)-C(3)-C(6)-C(5) plane, and the ring least-squares plane makes an angle of 5.1(3)” with the adjacent aromatic ring. The oxazine ring shows a chair conformation with the N-bonded C(13) methyl group in the equatorial position. A statistical analysis has been carried out with the Quest93 and Gstat93 packages of the Cambridge Crystallographic Data Centre, to compare the conformations assumed by the oxazine ring in all nineteen 1,2-oxazine compounds present in the crystallographic literature as well as to examine the behavior of the ring embedded in different crystal environments. The conformation of the ring has been described by the six torsion angles: tl,C(3)-C(4)-0(1)-N(l); t 2 , C(2)-C(3)-C(4)-0(1); t3, C(l)-C(Z)-C(3)-C(4); t4,N(1)C(l)-C(Z)-C(:3); t5, O(1)-N( 1)-C( l)-C(2); t6, C(4)-0( 1)-
Table 4-Selected Bond Distances (A) for Compounds A and B Compound B
Compound B
Compound A 1.425(5) 1.412(6) 1.364(7) 1.193(6) 1.518(6) 1.560(6) 1.485(7) 1.415(5) 1.412(6) 1.452(7) 1.334(6) 1.451(7)
1.444(10) 1.423(9) 1.336(10) 1.245(10) 1.461(15) 1.447(11) 1.492(17) 1.422(11) 1.400(10) 1.466(10) 1.313(11) 1.463(12)
Compound A 1.497(7) 1.530(6) 1.565(6) 1.517(6) 1.529(6) 1.396(6) 1.370(6) 1.360(6) 1.382(7) 1.368(7) 1.402(8)
1.489(16) 1.544(13) 1.530(12) 1.530(10) 1.523(13) 1.397(11) 1.394(11) 1.368(10) 1.401(10) 1.365(12) 1.368(12)
Table 5-Selected Bond Angles (deg) for Compounds A and B ~~
~
~
Compound A
CompoundB
C(8)-0(2)-C(11)
Nil )-O(1)-C(4) O(I )-N( 1)-C( 1)
118.4(6) 101.6(8) 107.0(6) 106.1(8)
C(l )-N(l)-C(l3)
109.9(9)
C(5)-N(Z)-C(I 5) C(4)-N(2)-C( 15) C(4)-N(2)-C(5) C(l l)-N(3)-C(12) N(l)-C( 1)-C(2) C( 1)-C(2)-C(3) C(Z)-C(3)-C(14) C(2)-C(3)-C(6) C(2)-C(3)-C(4) C(6)-C(3)-C(14) C(4)-C(3)-C(14) C(4)-C(3)-C(6)
119.6(6) 121.6(7) 108.6(7) 122.4(7) 106.1(9) 114.9(9) 111.4(8) 113.2(6) 111.3(7) 110.1(7) 111.2(7) 99.1(6)
O(1)-N( 1)-C( 13)
C(8)-0(2)-C(ll) O(1)-N( 1)-C( 13) O(1)-N( 1)-C(4) O(1)-N(1)-C(1) C(4)-N( 1)-C(13) C( 1)-N(1)-C( 13) C(1)-N(1)-C(4) C(5)-N(2)-C(15) C(4)-N(2)-C(15) C(4)-N(2)-C(5) C(Il)-N(3)-C(12) N(1)-C(1)-C(2) C(l)-C(Z)-C(3) C(Z)-C(3)-C( 14) C(Z)-C(3)-C(6) C(Z)-C(3)-C(4) C(6)-C(3)-C(14) C(4)-C(3)-C(14) C(4)-C(3)-C(6)
1128 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
~~
~
~~
~~
117.7(4) 109.0(4) 107.7(3) 104.4(3) 116.2(3) 115.0(4) 103.6(3) 122.4(4) 123.9(4) 110.7(3) 121.3(5) 103.0(4) 104.2(4) 111.4(4) 112.4(3) 105.7(3) 112.2(3) 113.6(4) 101.0(3)
N(2)-C(4)-C(3) O(1)-C(4)-C(3) O(l)-C(4)-N(2) N(2)-C(5)-C(lO) N(2)-C(5)-C(6) C(6)-C(5)-C(lO) C(3)-C(6)-C(5) C(5)-C(6)-C(7) C(3)-C(6)-C(7) C(6)-C(7)-C(8) 0(2)-C(8)-C(7) C(7)-C(8)-C(9) 0(2)-C(8)-C(9) C(8)-C(9)-C(lO) C(5)-C( 10)-C(9) 0(3)-C(ll)-N(3) 0(2)-C(ll)-N(3) 0(2)-C(11)-0(3)
~
~
~~
-~ ~~
~
Compound A
Compound B 104.1(7) 112.6(7) 104.0(7) 130.6(7) 108.3(6) 121.0(7) 108.4(6) 120.4(7) 130.8(6) 117.7(6) 120.3(6) 121.8(7) 117.7(7) 121.1(8) 118.0(7) 125.3(8) 111.7(7) 123.0(8)
N(Z)-C(4)-C(3)
N( 1)-C(4)-C(3) N(l)-C(4)-N(2) N(2)-C(5)-C(I 0) N(2)-C(5)-C(6) C(6)-C(5)-C(I 0) C(3)-C(6)-C(5) C(5)-C( 6)-C( 7) C(3)-C(6)-C(7) C(6)-C( 7)-C( 8) 0(2)-C(8)-C(7) C(7)-C( 8)-C( 9) 0(2)-C(8)-C(9) C(8)-C(9)-C(IO) C(5)-C( 1O)-C(9) 0(3)-C(ll)-N(3) 0(2)-C(lI)-N(3) 0(2)-C(11)-0(3)
107.8(3) 104.1(3) 112.6(3) 128.0(4) 109.5(4) 122.4(4) 110.9(4) 119.5(4) 129.6(4) 118.9(4) 119.0(4) 121.8(5) 119.1(4) 120.2(5) 117.2(5) 127.4(4) 109.1(4) 123.4(5)
mation with the C(2) atom at the flap (see Conclusion section). I
I
B
C(I2 (161
Cl17
01
Figure 1-ORTEP view of compound 8 . The thermal ellipsoids are drawn at the 50% probability level.
Figure 2-ORTEP view of compound A. The thermal ellipsoids are drawn at the 50% probability level.
N(l)-C(l). The possible random occurrence of different absolute configurations among the compounds has been taken into account.25 A cluster analysis with a single-linkage algorithm and Minkowsky metric has been applied on the space defined by the torsion angles,26 with the aim of characterizing the distribution of the conformations for the 1,2-oxazinerings. One major conformational cluster (chair, seven compounds) and three less populated ones (half-chair, four compounds; skew, three compounds; skew-boat, two compounds)have been identified (Table 6). The three compounds left are outliers in the conformational space spanned by the endocyclic torsion angles. The conformation of the oxazine ring in compound B is comparable with those found in the compounds of the largest cluster. It is noteworthy that compound B is situated at the border of cluster 1, as shown by the values of t2, t3, t5, and t6, which fall just outside the ranges defined by the other seven compounds; on the other hand, the compound corresponding to the refcode FITXOV, which is chemically similar to compound B, falls within cluster 4. A perspective view of compound A is shown in Figure 2. As expected, the protonation occurs on O(1). The compound adopts the N-oxide five-membered ring conformation showing an envelope conformation with C(1) lying 0.63 A out of the lane of the remaining atoms (maximum displacement of 0.06 ). In the previously structurally characterized geneserine hydrochloride,l6 the N-oxide ring adopts an envelope confor-
1
Unlike B, the five-memberedN(2)-C(4)-C(3)-C(6)-C(5) ring is planar within experimental error and almost coplanar with the adjacent six-membered ring, as already found in geneserine hydrochloride. The different conformational behavior of this ring in the two compounds can be attributed to different steric characteristics of the 1,2-oxazine six-membered and the N-oxide five-membered rings. The two compounds differ markedly also in the conformation shown by the heptylic chain, which is extended in B and folded in A, as is evident by the torsion angles N(3)-C(12)-C(16)-C(17), which is -179.1(8)" in B and -42(1)' in A. Accordin ly, in B the atoms N(3) through C(21) are located within 0.13 of their common plane, while in A N(3) is 0.98 A out of the plane defined by the atoms C(12) through C(21), which show a maximum displacement of 0.24 A from the plane of best fit. In both compounds the chain atoms have a large thermal motion which progressively increases toward the end of the chain. The different conformation of the two n-heptylic chains is due to the different role played by the carbonyl O(3) atom in the packing of the two compounds. The two crystal packings are shown in Figures 3 and 4, respectively. In A the oxygen carbonyl atom is not involved in any hydrogen bond, while in B it acts as a hydrogen bond acceptor with the carbamate N(3) donor atom at -x, y 1/2, -z - 1/2 [N(3)...0(3), 2.904(9) A; N(3)-H-- -0(3),141"l. In A, the N(3) and O(1) atoms are hydrogen bonded to the free C1 ion [N(3)..*Cl (X - 1, y - 1, z ) , 3.547(4) A, N(3)-H*.*Cl, 151"; 0(1).*.C1, 2.953(3)A; O(l)-H-.*Cl, 152"). Both of the molecular arrangements are stabilized by hydrogen-bonded chains extending along the [1,1,0]direction in A and along the [0,1,0] direction in B. The packing motif of compound A resembles closely the one found for geneserine hydrochloride,16despite the presence of one water molecule in the asymmetric unit of the latter. The crystallization solvent does not interfere with the formation of the hydrogen-bonded chains which are common to the packing of both hydrochlorides. N M R and Mass Studies-Free Base-The lH NMR data of B (see Scheme 1)are consistent with the structure of a cisfused tetrahydro-1,2-oxazine ring as previously reported for gene~erine.~ , ~ the MS analysis was indicative of this Also, structure: loss of M+ - 16, M i - 17 or Mf - 18, as might be expected for an N-oxide structure, was not observed, whereas the CA-MIKE spectrum of the molecular ion gave rise to m/z 316 (M+ - C2H5NO) and mlz 315 (M+ - Cz&NO) in analogy to the results reported for geneserineZ7and flustrarine B, another 1,2-0xazine derivative.28 The vicinal IH coupling constants were analyzed to determine the conformation in solution of the molecule. Before proceeding with this study, the IH assignments were first unambiguously established by a homonuclear correlation experiment and NOE's measurements. The 1,2-oxazine ring should adopt a chair conformat i ~ nbut , ~the ~ values of the experimental coupling constants are completely out of the expected range for a single conformation. As already reported for g e n e ~ e r i n e ,it~ ~can be supposed that a rapid equilibrium exists in solution between the two possible chair conformations lCq and 4C1with the N-methyl group in the equatorial position (see Scheme 2). These two forms were built up and minimized by molecular mechanics calculations using the Allinger Force Field MMX and the theoretical 3J coupling constants estimated from the Karplus equation modified by Altona et aL31 The results are reported in Table 7. From the experimental 3J,the population of each conformer can be calculated by solving the well-known time average equation:32
+
+
Jay= P(I)J(I) P(II)J(II)+ ..., P(n)J(n) where J ( n )are the predicted values for the pure conformers. Journal of Pharmaceufical Sciences / 1129 Vol. 84, No. 9, September 1995
Table 6-Cluster Analysis Results Compared with Data for Compound B and Data Relative to the Cambridge Crystallographic Database Quest (Oct 93) Sl
Compound 6
-59.9
Cmean maximum minimum
-65.7 -54.2 -73.8
Cmean maximum min imum
-55.0 -50.6 -59.3
Cmean maximum minimum
t2 39.6
t3 (a) Cluster Analysis Resultsa -36.5
t4 51.9
t5
t6
-71.6
77.2
Cluster 1 55.0 -49.2 54.6 -64.5 60.7 -45.5 58.4 -62.4 46.5 -53.1 50.6 -67.6 Compounds, 7; Refcodes,CXBAXR, FETZOT, FEVPIF, FEVPOL, FIBLIL, FUHHEV, VORDEL
71.O 76.6 65.1
61.2 64.2 58.9
Cluster 2 -37.1 6.9 -30.1 12.8 -43.9 1.o Compounds, 4; Refcodes, JAMGEJ, SOSZEF, SOSZEF, YAHBAK
2.0 2.8 0.5
22.7 27.9 16.5
-39.6 -33.5 -50.5
26.4 33.3 22.7
Cluster 3 -2.8 --12.5 -1.9 -8.9 -3.7 --17.5 Compounds, 3; Refcodes, FRGLXB, MNPHOX, MNPHOX
0.3 1.3 -0.8
27.1 33.3 23.4
Cmean maximum minimum
-48.7 -44.9 -52.5
-19.3 -13.8 -24.9
-37.5 -32.7 -42.3
82.7 84.4 81.O
BAZGIS CETHOZ VORDAH
-13.7 69.9 -54.0
9.6 -49.6 22.0
0.7 12.3 -41.7
8.6 -56.4 67.9
Cluster 4 59.2 --2a.7 62.7 --27.6 55.7 -29.8 Compounds, 2; Refcodes, FITXOV, JEBXIX Isolated Fragments -1.5 8.3 1.7
-3.9 12.9 6.7
(b) Data Relative to Questb BAZGIS FUHHEV 6-Chloro-3acyano-2-cyclohexyl-4a-methyl-1 aa,3,4,4a,5,1 O-hexahydronaphth3,4-Bis(methoxycarbonyl)-5-(methylthio)-6-oxo-6K1,2-0xazine CsHdiosSi (2,3-e)(1,2)0xazine H. Gotthardt, 0. M. Huss, C. M. Weisshuhn CzoH25ChNzOi C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Acta Crystallogr., Sect. B, 1982, 38, 680 J. Cryst. Spectrosc. 1987, 17, 515 CETHOZ 2-(Chloroethanoyl)-4-methyl-5-methylene-6-hydro-l,2-oxazine JAMGEJ CsHioCIiNiOz Ethyl 6-methyl-6-phenyl-5,6-dihydro-4H-l,2-oxazine-3-carboxylate B. Tinant, J. P. Declercq, G. Germain, M. van Meerssche Ci4Hi7Ni03 D. J. Chadwick, T. L. Gilchrist, W. Stretch Bull. Soc. Chim. Belg. 1980, 89, 407 Acta Crysfallogr. C 1989, 45, 976 CXBAXR N(pCarboxybenzy1)tetrahydro-1,Poxazine JEBXIX C12H15N103 Ethyl rek(1R,3S,6aff,EaF@bS)-5,5,6,6,8b-pentamethyI-6a, 7,8,8a-tetrahydrocyclopenta(1,2,3-h~)isooxazolo(2,3-b)( 1,2)oxazine-l-carboxylate F. G. Riddell, P.Murray-Rust, J. Murray-Rust Tetrahedron 1974, 30, 1007 Ci6Hz7Ni04 S. E. Denmark, Young-Choon Moon, C. B. W. Senanayake FETZOT 2-Cyclohexyl-4a-methyl-3,4,4aa,5,10,1Oaa-hexahydro-6-nitronaphth- J. Am. Chem. SOC.1990, 112,31 1 (2,3-e)(1,2)oxazine-3P-carbonitrile MNPHOX CzoHz&”03 6-(Dimethylamino)-5-(4-nitrophenylamino)-3-(2,4,6-trimethylphenyl)-6~-1,2-oxazine C. W. Holzapfel, G. J. Kruger, M.S. Van Dyk CziHz4N403 A. C. Villa, A. G. Manfredotti, C. Guastini, P. Trimarco Acta Ctystallogr. C 1987, 43, 514 Cryst. Struct. Commun. 1980, 9,523 FEVPIF 9-Chloro-2-cyclohexyI-4~-methyl-3,4,4a,5,10,1Oa-hexahydronaphth- SOSZEE 4,4’-Bi(5,6-dihydro-3-methyl-4H1,2-oxazine) (2,3-e)(1,2)0xazine-3/?-carbonitrile CzoHz5CliNz0i CIOHI~NZ~Z S. Shatzmiller, R. Lidor, E. Bahar, I. Goldberg C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Liebigs Ann. Chem. 1991,851 Acta Crystallogr. C 1987, 43,598 FEVPOL VORDAH 2-Cyclohexyl-9-iodo-4/?-methyl-3,4,4a,5,10,1Oaa-hexahydronaphth(6R)-2-((R)-2-Hydrox~2-phenylacetyl)-6-((S)-5-methoxycarbonyl-2-oxopyrrolidinyl)3,6-dihydro-2H-l,2-oxazine (2,3-e)(1,2)oxazine-3~-carbonitrile CzoHzsIiNzOt Ci8HzoNz06 A. Defoin, J. Pires, I. Tissot, T. Tschamber, D. Bur, M. Zehnder, J. Streith C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Tetrahedron: Asymmetry 1991,2, 1209 Acta Ctystallogr. C 1987, 43,598 FlBLlL VORDEL 2-(gNitrobenzoyl)tetrahydro-2H1,Poxazine (4R,5R,6S)-6-(t-ButyIdimethylsiloxy)-2-[(2R,5R)-(2,5-dimethylpyrrolidinocarbonyl]Cii HizN204 tetrahydro-2Kl,2-oxazine-4,5-diol J. E.Johnson, R. Hodzi, S. L. Todd, P. de Meester, S. S. C. Chu C17H&205Si3 J. Heterocycl. Chem. 1986, 23, 1423 A. Defoin, J. Pires, I. Tissot, T. Tschamber, D. Bur, M. Zehnder, J. Streith Tetrahedron: Asymmetry 1991,2,1209
1130 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
Table 6 (Continued) FITXOV 2H2-Cyclohexyl-4~-methyl-3,4,4aa,9aa-tetrahydrobenzothieno(3,2~e)( 1,2)oxazine3a-carbonitrile CieHzzNzOiSi C. W. Holzapfel, G. J. Kruger, M. S. Van Dyk Acfa Crysta//ogr.C 1987, 43, 1367 FRGLXB 3-(Ethoxycarbonyl)-6-(methoxycarbonyl)-5-phenyl-6~1 ,Poxazine CikMW5 Y. Nakada, T. Hata, C. Tamura, T. Iwaoka, M. Kondo, J. Ide Tetrahedron Left. 1981, 22,473
YAHBAK 4-(pBromophenyl)-5-hydroxy-3-(methoxycarbonyl)-5,6-di hydro-4#1,2oxazine Cl2H1~BrlN1O4 K. Harada, Y. Shimozono, E. Kaji, H. Takayanagi, H. Ogura, S. Zen Chem. Pharm. Bull. 1992, 44 1921
a Cmean = circular mean for torsion angles within a cluster, Refcode = Cambridge Crystallographic Database reference. For compounds FRGLXB and JEBXIX the R value is not reported in the original papers. All other compounds have R c 10%. A connectivity search has been also carried out which did not give any other compound suitable for comparison: t l *comp '1 ,Poxazine'
t2 *comp '1,2)oxazine' quest t l .or.t2
It Scheme 2
The calculated percentages of the two forms 4C1 and lC4 were 75 & 5% and 25 & 5%, respectively. As previously observed, the N-methyl group should prefer the equatorial position; therefore, the complete inversion should involve both ring and nitrogen inversion (see Scheme 2 again). The existence of such conformational equilibria was further confirmed by lowering the temperature; at 244 K the four singlet resonances of the molecule a t room temperature split into doublets. For the bridgehead methine proton, the integrated intensity ratio of the peaks gave approximately the same percentages of 4C1and lC4 reported before, indicating that a t 305 and 244 K the equilibrium constant for the two conformers is nearly identical within the experimental error (Figure 5). HCZ Salt-An analysis of the lH NMR 3Jvalues was used to establish whether the molecule A exists as a N-oxide derivative in solution as observed for geneserolineg and for geneserine hydrochloride.16 For the salt as well, the lH assignments were first unambiguously established by correlation spectroscopy and NOES measurements. In CDCl3 the vicinal coupling constants of the spin system CHz-CHz are typical of a five-membered ring, confirming that the molecule adopts this structure. The experimental values are consistent with the 3Jestimated from the envelope conformation with the 3-carbon a t the flap, indicating that this form is largely prevalent in solution (see Table 8). The spectra of A in more polar solvents like DMSO-d6 or D20 show the signals of another molecule in slow exchange on the NMR time scale. To assign its structure, a steady state NOE experiment was performed. By irradiating the pyrrolic
Figure 3-Packing diagram of compound 6. Table 7-Theoretical and Experimental Coupling Constants (3J in Hz) for Vicinal Proton Pairs in Compound B (SeeScheme 2)
Proton Pair
3Jexp
H3 H4 H3 H4' H3' H4 H3' H4'
4.5 5.1 4.3 9.7
75:25 'Jcaic 4.6 5.0 4.3 9.7
' C4
4C~
12.8 3.1 3.4 2.7
1.9 5.6 4.6 12.0
N-methyl group, a significant enhancement of the 8a-proton was observed, indicating that the second molecule is the epimer arising from inversion a t the tetrahedral nitrogen (see Scheme 3). Its absolute configuration is 3aS,BaS,lS. A careful analysis of the spectrum in CDCl3 pointed out that such epimer is present in this solvent too, but in negligible amounts (3-4%). The percentages of the two epimers in different solvents are reported in Table 9. The ratio between the two epimers does not change, even if the solutions were allowed to stand for 1 week a t room temperature, indicating Journal of Pharmaceutical Sciences / 1131 Vol. 84, No. 9, September 1995
0
& 7 -L&J I
(33
/
HO
I
"'./
C"3
2 :
CH3
\,,,
HO
3aS. 8aS. 1s
3aS. 8aS. 1R
Scheme 3 Table 9-Percentages of the Two Epimers of Compound A in Different Deuterated Solvents at 303 K A
Deuterated Solvent
1R
1S
Deuterated Solvent
1R
1S
Water (pD = 2.0) Water (pD= 5 0) Trifluoroethanol Ethanol
62
38
73
27
80 80
20 20
Dimethyl sulfoxide Acetone Chloroform Dioxane
80 94 96
20 6 4 0
100
E
Figure 4-Packing diagram of compound A.
l
160 rio iio loo io 60 in in i PPM Figure 6-Comparison of the CDC13 solution-state (bottom) and solid-state (top) all-decoupled i3C spectra of compound A. Solid circles denote sidebands, 7
7.0
6.0
5.0
4.b PPti
.
2.0
3.0
L
'
1.0
Figure 5--'H NMR spectrum of compound A in CDC13: at 303 K (bottom) and 244 K (top). Solid circles indicate the splitted signal of the bridgehead methine proton. Table 8-Dihedral Angles ($) and Corresponding Theoretical and Experimental Coupling Constants (Vin Hz) for Vicinal Proton Pairs in Compound A Proton Pair
d, deg
3Jca~c
H2 H3' H2 H3 H2' H3 H2' H3'
76 -43 -1 60 -4 1
0.7 6.2 11.5 6.4
3Jexp
0.8 5.5 13.1
5.7
that the equilibrium in each solvent has been already reached during the time required to run the spectra. Unexpectedly, the vicinal coupling constants of the spin system CHz-CHz in the IS epimer are very different from the corresponding ones in the 1R epimer, and they are completely out of the expected range for a single conformation. The values observed can be explained only supposing a fluxional system with 1132 / Journal of Pharmaceutical Sciences Vol. 84, No. 9, September 1995
different conformers in rapid equilibrium. To investigate whether the two epimers are present in the solid-state too or they form just in solution, the CP-MAS 13C NMR spectrum was recorded. Figure 6 compares the solid-state and solution spectra: carbons directly bonded to nitrogen atoms can be perceived immediately, as they do not give rise to sharp peaks.33 The other signals were assigned by comparison with those of the solution spectrum (see Experimental Section); in the CP-MAS spectrum no splitting of the signals was observed, indicating that in the solid-state the molecule is present as a single epimer. The presence of a single set of peaks confirms the crystallographic finding, i.e. that a single crystalline form is present and that the molecules in the unit cell are eq~ivalent.~~
Conclusions The n-heptylcarbamate of geneseroline base is a 1,2-oxazine derivative; it exists in solution as a mixture of two possible chair conformers 4C1 and ICq (with the N-methyl group in the equatorial position), whereas in the solid-state only the former is present.
Its hydrochloride salt is instead an N-oxide derivative; the five-membered ring adopts an envelope conformation in both states, but in the solid-state the atom at the flap is the 2-carbon, while in solution it is prevalently the 3-carbon. The latter conformation corresponds to the one observed for geneserine hydrochloride both in the solution- and solidsuggest that such a moiety is state'16 These and the packing forces are predominant in defining the conformation in the crystal. Interestingly, in solution, the salt partially undergoes fast inversion at the tetrahedral nitrogen, giving rise to the corresponding 1sepimer whose N-oxide fivemembered ring adopts a more flexible conformation. The amount of the latter epimer increases with the polarity of the solvent,34but it is always present as a minor species, indicating that it is less stable than the former. The results of force field calculations in vacuum are qualitatively consistent with these experimental findings.
References and Notes 1. Chiesi, P.; Delcanale, M.; Servadio, V.; Ghidini, E., PCT Int. Appl. Patent 93 03041, 1993; Chem. Abstr. 1993, 119, 95904. 2. Polonovski, M. Bull. SOC.Chim. Fr. 1915, 17, 245-289. 3. Polonovski, M. Bull. SOC.Chim. Fr. 1917,21, 191-200. 4. Polonovski, M. Bull. SOC.Chim. Fr. 1925, 37, 744-759. 5. Hootelk, C. Tetrahedron Lett. 1969,32, 2713-2716. 6. Robinson, B.; Moorcroft, D. J . Chem. Soc.(C) 1970,2077-2078. 7. Hill, R. K.; Newkome, G. R. Tetrahedron 1969,25,1249-1260. 8. Pauling, P.; Petcher, T. J. J . Chem. SOC.Perkin Trans 2 1973, 1.142-1.14.5 - - .- - - -. 9. Yu, Q. S.; Yeh, H. C. J.; Brossi, A,; Flippen-Anderson, J . L. J . Nut. Prod. 1989, 52, 332-336. 10. Brufani. M.: Marta., M.:, PomDoni. M. Eur. J . Biochem. 1986. 157, 115-120. 11. Brufani, M.; Castellano, C.; Marta, M.; Oliviero, A,; Pagella, P. G.; Pavone, F.; Pomponi, M.; Rugarli, P. L. Pharmacol. Biochem. Behau. 1987,26, 625-629. 12. De Sarno, P.; Pomponi, M.; Giacobini, E.; Tang, X. C.; Williams, E. Neurochem. Res. 1989,14, 971-977.
13. Marta, M.; Castellano, C.; Oliviero, A.; Pavone, F.; Pagella, P. G.; Brufani, M.; Pomponi, M. Life Sci. 1988,43,1921-1928. 14. Pomponi, M.; Giardina, B.; Gatta, F.; Marta, M. Med. Chem. Res. 1992,2, 306-327 and references cited therein. 15. Brzostowska, M.; x.; ~ ~N. H.;~ Rapoport, i ~s, 1,; ~, ~ A. Med. Chem. Res. 1992,2, 238-246. 16. Bacchi, A.; Pelizzi, G.; Redenti, E.; Delcanale, M.; Amari, G.; Ventura, P. Acta Crystallogr. 1994, '250, 1126-1130. 17. Walker, N.; Stuart, D. Acta Crystallogr, 1983, A39, 158-166, 18. Sheldrick, G. M. S H E U S 8 6 A Program for Structure Determination, University of Gottingen, D, 1986. 19. Sheldrick, G. M. SHELXL92 A Program for Structure Refinement, University of Gttingen, D, 1992.
;::~ 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
I
34.
~ { . ~~ ~~ ~ ~~ ~ ~ Program p :~ for ~ Crystal Structure Illustrations. Report ORNL 3794 (Oak Ridge National Laboratory, Oak Ridge, Tenn.), 1965. PCMODEL-pi program, Version 4.0, Serena Software Inc. P.O. Box 3076, Bloomington, IN, 47402-3076. Allinger, N. L. J . Am. Chem. SOC.1977,99, 8127-8134. Allinser. N. L.; Sprame. J. T. J . A m . Chem. SOC.1973,95,3893. Alle
Supplementary Material Available-Lists of structure factors, atomic coordinates of hydrogen atoms, thermal anisotropic parameters are available from the author. JS9404981
Journal of Pharmaceutical Sciences / 1133 Vol. 84, No. 9, September 1995
~