The present invention concerns crystalline salts of cephalotaxine, defined by their solid state analysis patterns, their process of preparation allowing their use for crude cephalotaxine purification including for resolution of racemic or partially racemized cephalotaxine.

In October 2012, the United States Food and Drug Administration (FDA) granted accelerated approval to omacetaxine mepesuccinate (= omacetaxine homoharringtonine = HHT = HO) for the treatment of adult patients with chronic phase or accelerated phase chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs). In 2013, 14 major articles and reviews related to HHT were published in literature. This occurred after a very long and tumultuous period of clinical development (almost 40 years), including early clinical development of HHT and, to a lesser extent, its congeners harringtonine (HA) and deoxyharringtonine (DHA) in China then at the United States National Cancer Institute [Grem JL, Cheson BD, King SA, et al. Cephalotaxine esters: anti-leukemic advance or therapeutic failure? J Natl Cancer Inst 1988; 80:1095-103]. In 1998, the discovering of a new semi-synthetic process by one of us, allowing industrial production of homoharringtonine at the kilo scale using enantiomreically pure cephalotaxine as starting material. [Robin J, Dhal R, Dujardin G, et al. The first semi-synthesis of enantiopure homo-harringtonine via anhydrohomoharnngtonine from a preformed chiral acyl moiety. Tetrahedron Lett 1999: 40:2931-4] [http://www. ascopost. com/issues/

april- 15, -2013/homoharhngtonineomacetaxine-the-little-drug-that-could . aspx] . HHT as protein synthesis inhibitor is the first-in-class cephalotaxane [NEMUMUNAITIS CCP 2013 Pk study of OM administered subcutaneously to patients with advanced solid and hematologic tumors]

Definition see scheme 1

Omacetaxine mepesuccinate

The INN (International Non-proprietary Name) omacetaxine mepesuccinate is a name reserved for homoharringtonine drug substance dedicated for pharmaceutical use regardless of its natural, hemi-synthetic or synthetic origin. Cephalotaxanes are particular alkaloid to date only extracted from the Cephalotaxaceae family which exhibiting the structural formula 1 . Several substituants may be encountered on this core structure: hydroxyl, ether, acyloxy etc. The eventual presence of some additional double bound or intramolecular bridge achieve to definite cephalotaxanes.

Cephalotaxines 2 are cephalotaxanes without acyloxy side-chain. Cephalotaxine 2a and drupacine 2b are example of cephalotaxines.

Harringtonines 5 are particular cephalotaxanes formed by attachment of a branched a-hydroxyacyloxy side-chain at the 3-position of various cephalotaxines moieties.

Cephalotaxines 2 and harringtonines 5 are examples of cephalotaxanes. Several dozen of cephalotaxanes have been isolated from various Cephalotaxus species.

4 is the generic formula of cephalotaxine esters.

Harringtonines 5 (i. e. harringtonine = HA and homoharringtonine = HHT) are particular cephalotaxine esters, alkaloid isolated from rare and endangered conifers belonging to the Cephalotaxus genus. Cephalotaxine and its natural ester are gathered under the generic term of cephalotaxane.

SCHEME 1 : DEFINITION NOMENCLATURE AND NUMBERING OF CEPHALOTAXANES

skeleton

i.e.cephalotaxine, drupacine, = cephalotaxini

1 1 -hydroxycephalotaxine and

so

CEPHALOTAXINE ESTERS HARRINGTONINES HARRINGTONINES CATION

i.e. acetates, itaconates and i.e. = "HARINGTONINIUM" ION so harringtonine.homoharri

ngtonine.desoxyharringt ^{Thls patent salts}

onine.neoharringtonine

and so

Two harringtonines are very promising drugs in the treatment of certain leukemia such as Chronic Myelogenous Leukemia (CML). Both homoharringtonine and harringtonine are used in human chemotherapy of leukemia for 30 years.

The present invention relates to overcome the problems mentioned above. It also demonstrated that the absolute configuration in the deposited homoharringtonine Cambridge Structural Database seems to be the opposite of that commonly retained in the literature. SCHEME 2: ANION MOEITIES OF SOME SALT S CITED IN THIS INVENTION

hydrogen (2S)-malate hydrogen (2R)-malate hydrogen (2S,3S)-tartrate hydrogen (2R,3R)-tartrate

hydrogen succinate hydrogen itaconate hydrogen fumarate hydrogen maleate

hydrogen malonate hydrogen tartronate hydrogen benzoate hyd

Due to the therapeutic benefit of the esters of cephalotaxine described above and now the existence of at least a semisynthetic process available at industrial scale, there are countless syntheses of cephalotaxine described in literature [(a) Auerbach, J.; Weinreb, S. M. J. Am. Chem. Soc. 1972, 94, 7172; (b) Semmelhack, M. F.; Chong, B. P.; Jones, L. D. J. Am.Chem. Soc.1972, 94, 8629; (c) Semmelhack, M. F.; Chong, B. P.; Stauffer, R. D.; Rogerson, T. D. Chong, A.; Jones, L. D. J. Am. Chem. Soc. 1975, 97, 2507; (d) Weinreb, S. M. Auerbach, J. J. Am. Chem. Soc. 1975, 97, 2503; (e) Burkholder, T. P.; Fuchs P. L. J. Am. Chem. Soc. 1988, 1 10, 2341 ; (f) Kuehne, M. E.; Bornmann, W. G. Parsons, W. H.; Spitzer, T. D.; Blount, J. F.; Zubieta, J. J. Org. Chem. 1988, 53 3439; (g) Burkholder, T. P.; Fuchs, P. L. J. Am.Chem. Soc. 1990, 1 12, 9601 (h) Ishibashi, H.; Okano, M.; Tamaki, H.; Maruyama, K.; Yakura, T.; Ikeda, M. J. Chem. Soc., Chem. Commun. 1990, 1436; (i) Ikeda, M.; Okano, M.; Kosaka, K.; Kido, M.; Ishibashi, H. Chem. Pharm. Bull. 1993, 41 , 276; (j) Lin, X.; Kavash, R. W.; Mariano, P. S. J. Am. Chem. Soc. 1994, 1 16, 9791 ; (k) Lin, X.; Kavash, R. W.; Mariano, P. S. J. Org. Chem.1996, 61 , 7335; (I) Tietze, L. F.; Schirok, H. Angew. Chem., Int. Ed. 1997, 36, 1 124; (m) Koseki, Y.; Sato, H.; Watanabe, Y.; Nagasaka, T. Org. Lett. 2002, 4, 885; (n) Suga, S.; Watanabe, M.; Yoshida, J. J. Am. Chem.Soc. 2002, 124, 14824; (o) Li, W.-D. Z.; Wang, Y.- Q. Org. Lett. 2003, 5, 2931 ; (p) Li, W.-D. Z.; Ma, B.-C. J. Org. Chem. 2005, 70, 3277; (q) Ma, B.-C; Wang, Y.-Q.; Li, W.-D. Z. J. Org. Chem. 2005, 70, 4528; (r) Li, W.-D. Z.; Wang, X.-W. Org. Lett. 2007, 9, 121 1 ; (s) Sun, Mo-ran; Lu, Hong- tao; Wang, Yan-zhi; Yang, Hua; Liu, Hong-min J. Org. Chem. (2009), 74(5), 2213; (t) Zhao, Yu-Ming; Gu, Peiming; Zhang, Hai-Jun; Zhang, Qing-Wei; Fan, Chun-An; Tu, Yong-Qiang; Zhang, Fu-Min J. Org. Chem (2009), 74(8), 321 1 ; Li, Wei-Dong Z.; Wang, Yong-Qiang Org. Letters (2009), 1 1 (8), 1865; (u) Li Wei-Dong Z; Duo Wei-Guo; Zhuang Cheng- Han Organic letters (201 1 ), 13(13), 3538; (v) Xiao, Kai-Jiong; Luo, Jie-Min; Xia, Xiao-Er; Wang, Yu; Huang, Pei-Qiang Chemistry - A European Journal (2013), 19(39), 13075; (w) Zhang, Zhi-Wei; Zhang, Xiao-Fang; Feng, Juan; Yang, Yi-Hua; Wang, Cui-Cui; Feng, Jia-Cai; Liu, Shouxin J. Org. Chem. (2013), 78(2), 786; (x) Xing, Ping; Huang, Zuo-gang; Jin, Yun; Jiang, Biao Synthesis (2013), 45(5), 596]. Much of these syntheses, including non- chiral could be industrially applicable, but no method of resolution of racemic cephalotaxine has been described to date. In addition, there are sources of natural cephalotaxine coming from not endangered Cephalotaxus species (eg, Cephalotaxus fortunei), but most of them produce a partially racemized cephalotaxine.

Moreover, to date, the toxicity of the unnatural enantiomer that could contaminate clinically active esters (semi-synthetic harringtonine and homoharringtonine) remains unknown. The resolution of the partially or completely racemized cephalotaxine therefore is of great economic interest. Secondly, the non-chiral purification of cephalotaxine without using the chromatographic techniques also has great interest.

The present invention purpose is to overcome the difficulties mentioned above, namely: - prepare crystalline cephalotaxine salts and use this process for generation of cephalotaxine exhibiting a high level of purity, including enantiomeric.

- definitely remove any doubt about the absolute configuration of the cephalotaxane series.

provide a preparative method of resolution of racemic or partially racemized cephalotaxine.

provide an efficient method of preparation of (3R,4R,5R)-(+)- cephalotaxine as starting material for hemi-synthesis of enantiomer of potentially bioactive (+)-cephalotaxine esters.

The present invention concerns novel water soluble crystalline salts of cephalotaxine and their use to implement new processes for purification including enantiomeric and determine the absolute configuration of the series. The present invention provides water soluble salts of cephalotaxines, definite by their solid state analysis patterns, their process of preparation from cephalotaxines and commercial organic acid by mixing their solution in an organic solvent

An embodiment of the invention is a crystalline rac-cephalotaxine polymorphic form A having substantially the same IR spectrum, in the solid state as set out in figure 3.2, the same single crystal X-ray diffractogram as set out in figure 1 .1 .a, 1 .1 .b and 1 .1 .c and the same DSC curve as set out in figure 2.3.

Another embodiment of the invention is a crystalline rac-cephalotaxine polymorphic form B having substantially the same IR spectrum, in the solid state as set out in figure 3.3 and the same DSC curve as set out in figure 2.4. A preferred embodiment of the invention is a crystalline (3S,4S,5R,2'S)-(-)- cephalotaxine hydrogen malate having substantially the same IR spectrum, in the solid state as set out in figure 3.5, the same single crystal X-ray diffractogram as set out in figure 1 .2. a and 1 .2.b, the same X-ray powder pattern as set out in figure 1 .2.c and the same DSC curve as set out in figure 2.5.a and 2.5.b.

A further preferred embodiment of the invention provides a crystalline (3R,4R,5S,2'R)-(+)-cephalotaxine hydrogen malate having substantially the same IR spectrum, in the solid state as set out in figure 3.5, the same single crystal X-ray diffractogram as set out in figure 1 .3. a and 1 .3.b, the same X-ray powder pattern as set out in figure 1 .3.c and the same DSC curve as set out in figure 2.6. Yet, a preferred embodiment of the invention is a crystalline (3S,4S,5R,2'R)-(-)- cephalotaxine hydrogen malate having substantially the same IR spectrum, in the solid state as set out in figure 3.7, the same single crystal X-ray diffractogrann as set out in figure 1 .4. a and 1 .4.b, the same X-ray powder pattern as set out in figure 1 .4.c and the same DSC curve as set out in figure 2.7.

Yet, a further preferred aspect of the invention is a crystalline (3S,4S,5R,2'S,3'S)-(-)-cephalotaxine hydrogen tartrate having substantially the same IR spectrum, in the solid state as set out in figure 3.9 the same single crystal X-ray diffractogrann as set out in figure 1 .1 1 .a and 1 .1 1 .b, the same X- ray powder pattern as set out in figure 1 .1 1 .c and the same DSC curve as set out in figure 2.8.

Yet, another embodiment of the invention is a crystalline (3S,4S,5R,2'R,3'R)-(- )-cephalotaxine hydrogen tartrate having substantially the same IR spectrum, in the solid state as set out in figure 3.10 the same single crystal X- ray diffractogrann as set out in figure 1 .12. a and 1 .12.b and the same DSC curve as set out in figure 2.9.

Yet, another prefered aspect of this invention provides a crystalline (3S,4S,5R)- (-)-cephalotaxine hydrogen succinate having substantially the same IR spectrum, in the solid state as set out in figure 3.8, the same single crystal X- ray diffractogrann as set out in figure 1 .5 and the same DSC curve as set out in figure 2.10.

Yet, a further prefered aspect of this invention is a crystalline (3S,4S,5R)-(-)- cephalotaxine hydrogen itaconate having substantially the same IR spectrum, in the solid state as set out in figure 3.10, the same single crystal X- ray diffractogrann as set out in figure 1 .6. a and 1 .6.b, the same X-ray powder pattern as set out in figure 1 .6c and the same DSC curve as set out in figure 2.1 1

Yet, a prefered aspect of this invention provides a crystalline (3S,4S,5R)-(-)- cephalotaxine hydrogen fumarate having substantially the same IR spectrum, in the solid state as set out in figure 3.1 1 , the same single crystal X- ray diffractogrann as set out in figure 1 .9. a and 1 .9.b, the same X-ray powder pattern as set out in figure 1 .9.c and the same DSC curve as set out in figure 2.12.

Yet, a another aspect of the invention provides a crystalline (3S,4S,5R)-(-)- cephalotaxine hydrogen maleate having substantially the same IR spectrum, in the solid state as set out in figure 3.12, the same single crystal X-ray diffractogrann as set out in figure 1 .14. a and 1 .14.b, the same X-ray powder pattern as set out in figure 1 .14.c and the same DSC curve as set out in figure 2.13.

In addition, another embodiment provides a crystalline (3S,4S,5R)-(-)- cephalotaxine hydrogen malonate having substantially the same IR spectrum, in the solid state as set out in figure 3.13, the same single crystal X- ray diffractogrann as set out in figure 1 .10. a and 1 .10.b, the same X-ray powder pattern as set out in figure 1 .10.c and the same DSC curve as set out in figure 2.14.

Moreover, a prefered embodiment of this invention provides a crystalline (3S,4S,5R)-(-)-cephalotaxine hydrogen glutarate having substantially the same IR spectrum, in the solid state as set out in figure 3.14, the same single crystal X-ray diffractogrann as set out in figure 1 .7. a and 1 .7.b, the same X-ray powder pattern as set out in figure 1 .7.c and the same DSC curve as set out in figure 2.15.

Also, a prefered aspect of this invention provides a crystalline (3S,4S,5R)-(-)- cephalotaxine hydrogen tartronate having substantially the same IR spectrum, in the solid state as set out in figure 3.15, the same single crystal X- ray diffractogrann as set out in figure 1 .13. a and 1 .13.b, the same X-ray powder pattern as set out in figure 1 .13.c and the same DSC curve as set out in figure 2.13.

Yet, a preferred aspect of this invention provides a crystalline (3S,4S,5R)-(-)- cephalotaxine dihydrogen citrate having substantially the same IR spectrum, in the solid state as set out in figure 3.16, the same X-ray powder pattern as set out in figure 1 .15 and the same DSC curve as set out in figure 2.17.

Yet, a another aspect of this invention provides a crystalline (3S,4S,5R)-(-)- cephalotaxine hydrogen benzoate having substantially the same IR spectrum, in the solid state as set out in figure 3.17, the same single crystal X- ray diffractogrann as set out in figure 1 .8. a and 1 .8.b, the same X-ray powder pattern as set out in figure 1 .8.C and the same DSC curve as set out in figure 2.13.

A preferred embodiment of the invention is a general method for preparing a salt of the present invention by mixing a crude organic solution of cephalotaxine with a pure organic solution of an organic acid followed by isolation of the formed crystals and regeneration of cephalotaxine from its salt by base treatment, and then by extraction with an organic solvent followed by a final recrystallization.

Another preferred embodiment of the invention is a process for resolution of racemic or partially racemized cephalotaxine, whether natural or synthetic origin, as depicted on sheme 2, comprising:

(i) mixing a hot alcoholic solution of pure (2S)-malic acid with a hot alcoholic solution of racemized cephalotaxine,

(ii) crystallizing by cooling and recovering of the formed crystals of (3S, 4S, 5R, 2'R) - (-)-cephalotaxine malate,

(iii) regenerating of the unnatural enantiomer (3S, 4S, 5R) - (-)-cephalotaxine by base treatment of its salt, solvent extraction, concentration and crystallization.

Yet another embodiment of the invention is a process for resolution of racemic or partially racemized cephalotaxine, whether natural or synthetic origin, as depicted on scheme 3, comprising:

(i) mixing a hot alcoholic solution of pure (2R)-malic acid with a hot alcoholic solution of racemized cephalotaxine,

(ii) crystallizing by cooling and recovering of the formed crystals of (3R, 4R, 5S, 2'S) - (+)-cephalotaxine malate,

(iii) regenerating of the unnatural enantiomer (3R, 4R, 5S) - (+)-cephalotaxine by base treatment of its salt, solvent extraction, concentration and crystallization.

Another preferred aspect of the present invention is the use of said (-)- cephalotaxine with a high level of enantiomeric purity for the preparation of hemisynthetic biologically active esters exhibiting a very low diastereomeric impurity content especially cardiotoxic or antimitotic and finally their introduction in a pharmaceutical formulation for human or veterinary use.

Yet another aspect of the present invention is the use of unnatural (+)- cephalotaxine showing a high level of enantiomeric purity for the preparation of potentially biologically active esters exhibiting a very low level of diastereomeric impurity and finally their introduction in a pharmaceutical formulation for human or veterinary use in view of their use for pharmacologic screening.

Figures:

Figure 1 .1 . a Single crystal X-ray diffraction of rac-cephalotaxine exhibiting natural (3S,4S,5R) enantiomer combined with a molecule of methanol (drawn with ORTEP-3 software)

Figure 1 .1 . b Single crystal X-ray diffraction of rac-cephalotaxine exhibiting unnatural (3R,4R,5S)-(+) enantiomer combined with a molecule of methanol (drawn with ORTEP-3 software)

Figure 1 .1 . c Single crystal X-ray diffraction of rac-cephalotaxine exhibiting packing of an enantiomeric pair and two molecule of methanol per unit cell (drawn with Mercury 3.3 software)

Figure 1 .2.a Single crystal X-ray diffraction of natural (3S,4S,5R,2'S)-(-)- cephalotaxine hydrogen malate (drawn with ORTEP-3 software)

Figure 1 .2.b Single crystal X-ray diffraction of (3S,4S,5R,2'S)-(-)-cephalotaxine hydrogen malate exhibiting unit cell content (drawn with ORTEP-3 software) Figure 1 .2.c X-ray powder diffraction of (3S,4S,5R,2'S)-(-)-cephalotaxine hydrogen malate

Figure 1 .3. a Single crystal X-ray diffraction of unatural (3R,4R,5S,2'R)-(+)- cephalotaxine hydrogen malate (drawn with ORTEP-3 software)

Figure 1 .3.b Single crystal X-ray diffraction of (3R,4R,5S,2'R)-(+)-cephalotaxine hydrogen malate exhibiting unit cell content (drawn with ORTEP-3 software) Figure 1 .3.C X-ray powder diffraction of (3R,4R,5S,2'R)-(+)-cephalotaxine hydrogen malate

Figure 1 .4. a Single crystal X-ray diffraction of natural (3S,4S,5R,2'R)- cephalotaxine hydrogen malate (drawn with ORTEP-3 software)

Figure 1 .4.b Single crystal X-ray diffraction of (3S,4S,5R,2'R)-cephalotaxine hydrogen malate exhibiting unit cell content (drawn with ORTEP-3 software) Figure 1 .6.b Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen itaconate (packing drawn with ORTEP- 3 software)

Figure 1 .4.c X-ray powder diffraction of (3S,4S,5R,2'R)-cephalotaxine hydrogen malate

Figure 1 .5 Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen succinate (packing drawn with Mercury 3.3 software)

Figure 1 .6. a Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen itaconate (drawn with Mercury 3.3 software) Figure 1 .6.b Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen itaconate (packing drawn with ORTEP- 3 software)

Figure 1 .6.C X-ray powder diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen itaconate

Figure 1 .7. a Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen glutarate exhibiting three different ion pairs conformation per asymetric unit (drawn with ORTEP-3 software)

Figure 1 .7.b Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen glutarate exhibiting packing in unit cell content (drawn with Mercury 3.3 software).

Figure 1 .7.c X-ray powder diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen glutarate

Figure 1 .8. a Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine benzoate (drawn with Mercury 3.3 software)

Figure 1 .8.b Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine benzoate (packing drawn with ORTEP- 3 software)

Figure 1 .8.c X-ray powder diffraction of (3S,4S,5R)-(-)-cephalotaxine benzoate Figure 1 .9. a Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen fumarate (drawn with Mercury 3.3 software)

Figure 1 .9.b Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen fumarate (packing drawn with Mercury 3.3 software)

Figure 1 .9.C X-ray powder diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen fumarate

Figure 1 .10. a Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen malonate (drawn with Mercury 3.3 software)

Figure 1 .10.b Single crystal X-ray diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen malonate (packing drawn with Mercury 3.3 software)

Figure 1 .10.C X-ray powder diffraction of (3S,4S,5R)-(-)-cephalotaxine hydrogen malonate

Figure 1 .1 1 . a Single crystal X-ray diffraction of (3S,4S,5R,2'S,3'S)-(-)- cephalotaxine hydrogen tartrate drawn with Mercury 3.3 software)

Figure 1 .1 1 .b Single crystal X-ray diffraction of (3S,4S,5R,2'S,3'S)-(-)- cephalotaxine hydrogen tartrate (packing drawn with Mercury 3.3 software) Figure 1 .1 1 .C X-ray powder diffraction of (3S,4S,5R,2'S,3'S)-(-)-cephalotaxine hydrogen tartrate Figure 1 .12.a Single crystal X-ray diffraction of (3S,4S,5R,2'R,3'R)-(-)- cephalotaxine hydrogen tartrate (drawn with Mercury 3.3 software)

Figure 1 .12.b Single crystal X-ray diffraction of (3S,4S,5R,2'R,3'R)-(-)- cephalotaxine hydrogen tartrate (packing drawn with Mercury 3.3 software) Figure 1 .13. a Single crystal X-ray diffraction of (3S,4S,5R)-cephalotaxine hydrogen tartronate (2 different views drawn with Mercury 3.3 software)

Figure 1 .13.b Single crystal X-ray diffraction of (3S,4S,5R)-cephalotaxine hydrogen tartronate (packing drawn with Mercury 3.3 software)

Figure 1 .13.C X-ray powder diffraction of (3S,4S,5R)-cephalotaxine hydrogen tartronate

Figure 1 .14. a Single crystal X-ray diffraction of (3S,4S,5R)-cephalotaxine hydrogen maleate (drawn with Mercury 3.3 software)

Figure 1 .14.b Single crystal X-ray diffraction of (3S,4S,5R)-cephalotaxine hydrogen maleate (packing drawn with Mercury 3.3 software)

Figure 1 .14.C X-ray powder diffraction of (3S,4S,5R)-cephalotaxine hydrogen maleate

Figure 1 .15 X-ray powder diffraction of (3S,4S,5R)-cephalotaxine dihydrogen citrate

Figure 2.1 Differential Scanning Calorimetry of (3S,4S,5R)-(-)-cephalotaxine Figure 2.2 Differential Scanning Calorimetry of crude rac-cephalotaxine form A Figure 2.3 DSC of purified rac-cephalotaxine form A

Figure 2.4 DSC of purified rac-cephalotaxine form B

Figure 2.5.a DSC of (3S,4S,5R,2'S)-(-)-cephalotaxine hydrogen malate

Figure 2.5.b DSC of (3S,4S,5R,2'S)-(-)-cephalotaxine hydrogen malate (alternative preparation)

Figure 2.6 DSC of (3R,4R,5S,2'R)-(+)-cephalotaxine hydrogen malate

Figure 2.7 DSC of (3S,4S,5R,2'R)-cephalotaxine hydrogen malate

Figure 2.8 DSC of (3S,4S,5R,2'S,3'S)-cephalotaxine hydrogen tartrate

Figure 2.9 DSC of (3S,4S,5R,2'S,3'R)-cephalotaxine hydrogen tartrate

Figure 2.10 DSC of (3S,4S,5R,2'R)-cephalotaxine hydrogen succinate

Figure 2.1 1 DSC of (3S,4S,5R,2'R)-(-)-cephalotaxine hydrogen itaconate Figure 2.12 DSC of (3S,4S,5R,2'R)-cephalotaxine hydrogen fumarate

Figure 2.13 DSC of (3S,4S,5R,2'R)-cephalotaxine hydrogen maleate

Figure 2.14 DSC of (3S,4S,5R,2'R)-cephalotaxine hydrogen malonate

Figure 2.15 DSC of (3S,4S,5R,2'R)-(-)-cephalotaxine hydrogen glutarate Figure 2.16 DSC of (3S,4S,5R,2'R)-cephalotaxine hydrogen tartronate Figure 2.17 DSC of (3S,4S,5R,2'R)-cephalotaxine dihydrogen citrate

Figure 2.18 DSC of (3S,4S,5R,2'R)-(-)-cephalotaxine benzoate

Figure 3.1 : IR spectrum purified natural (3S,4S,5R)-(-)-cephalotaxine base Figure 3.2: IR spectrum purified natural rac-(±)-cephalotaxine base form A crystallized in methanol

Figure 3.3: IR spectrum purified natural rac-(±)-cephalotaxine base form B crystallized in ethyl acetate

Figure 3.4 IR spectrum of (3S,4S,5R,2'S)-(-)-cephalotaxine hydrogen malate Figure 3.5 IR spectrum of (3S,4S,5R,2'S)-(-)-cephalotaxine hydrogen malate (alternate preparation)

Figure 3.6 IR spectrum of (3R,4R,5S,2'R)-(+)-cephalotaxine hydrogen malate Figure 3.7 IR spectrum of (3S,4S,5R,2'R)-(-)-cephalotaxine hydrogen malate Figure 3.8 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen succinate Figure 3.9 IR spectrum of (3S,4S,5R,2'S,3'S)-cephalotaxine hydrogen tartrate Figure 3.10 IR spectrum of (3S,4S,5R,2'R,3'R)-cephalotaxine hydrogen tartrate Figure 3.10b IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen itaconate Figure 3.1 1 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen fumarate Figure 3.12 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen maleate Figure 3.13 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen malonate Figure 3.14 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen glutarate Figure 3.15 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen tartronate Figure 3.16 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine dihydrogen citrate Figure 3.17 IR spectrum of (3S,4S,5R)-(-)-cephalotaxine hydrogen benzoate. Example 1 : General procedure for experimental methods

1.1 General procedures for salts preparation

Cation and anion components were dissolved separately in a solvent at a concentration close of saturation and at a temperature close of boiling then both solutions were mixed under stirring then slowly cooled and evaporated. After a period ranging from a few minutes up to several days, crystal salt was collected. A sample of the batch of crystals was kept suspended in its mother liquors for the subsequent X-ray diffraction analysis. The remainder of the batch was dried under vacuum for further solid characterisation, comparative stability studies and drug formulation. 1.2 General procedure for direct isolation of pure cephalotaxine from crude Cephalotaxus extract, involving cephalotaxine salts

Dry leaves of a cultivar of Cephalotaxus selected and cultivated in Europe exhibit very high rate of cephalotaxine (8-12 grams per kilo of dry leaves) analysed. HPLC purity was ranged from 90 to 95 %. No related compound was higher than 2 %. 25 kilograms of these dry leaves were extracted by an hydromethanolic solution of tartaric acid. After usual treatment, crude cephalotaxine base was obtained as a pale yellow powder (174 grams, 0.55 mol) then was dissolved in hot methanol 1 .2 L then solid weak organic acid (0.55 mol) was added in small portions. Warming and stirring was continued until complete dissolution. After 48 hour at + 4°C, very pure crystallin cephalotaxine salts were obtained (yiled: 65-80% from crude cephalotaxine).

1.3 General procedure for large scale reverse phase chromatographic purification et crystallization involving cepgalotaxine salt

A crude cephalotaxine enriched extract (content: 1 mole, 315 g) was dissolved in 2.5 L of methanol containing 1 mole of a weak organic acid, then heated until complete dissolution. Quantity of water was added and adjusted on the rate of mobile phase (ranged 40-50%, according the batch of stationnary phase). The solution was then injected on a preparative column containing 7 kg of stationary phase RP-18 (or equivalent) and eluted using methanol-water mixture as mobile phase (acetonitrile-water may be also suitable). Unwanted fraction was discarded and selected fractions (checked by high speed analytical HPLC and TLC) were collected, combined and concentrated under reduced pressure. Concentrated cephalotaxine salt solution was alkalinized (NaOH 2N) then re- extracted for purification recristalization or formation of suitable salt. The semi- purified cephalotaxine base is then precipitated by alkalinization. The precipitate is then filtered off, titled, and taken up in 1 equivalent of weak organic acid. Methanol addition, warming and mixing are continued until full dissolution of suspension. Cooling the solution deposited an abundant precipitate of crystalin cephalotaxine salt which is drained and dried under vacuum. Depending on the purity of the obtained product including enantiomeric, salt crystallization is repeated as necessary, until constant optical rotation.

1.4 General procedures for solid state characterization Single crystal X-Ray Diffractions (XRD) material and methods

KappaCCD, Nonius diffractometer, Mo - Ko radiation (λ = 0.71073 A). The structure was solved by direct methods using the SHELXS-97 program [Sheldrick G.M., Acta Cryst. A64 (2008), 1 12-122], and then refined with full-matrix least- square methods based on F ² (SHELXL-2013) [Sheldrick G.M., (2013)] with the aid of the WINGX [L. J. Farrugia, J. Appl. Cryst., 2012, 45, 849-854] program. All non-hydrogen atoms were refined with anisotropic atomic displacement parameters. Except nitrogen and oxygen linked hydrogen atoms that were introduced in the structural model through Fourier difference maps analysis, H atoms were finally included in their calculated positions.

Collected information: atomic positions; unit cell composition; crystal packing anisotropic displacement parameters; bond lengths, dihedral and torsion angles, hydrogen bounding.

Original files with all parameters are includes on a CD and may be visualized and handled in using ORTEP-3 software (ORTEP = Oak Ridge Thermal- Ellipsoid Plot Program) available free of charge on the Internet: http://www.chem.gla.ac.uk/~louis/software/ortep3/

X-Ray Powder Diffraction (XRPD)

Diagrams were measured on a Bruker AXS D8 Advance diffractometer, Bragg- Brentano geometry (θ-2 Θ), CuK = 1 .5406 A, 600 ms / pixel, rotation: 0.25 / sec. For each chart, the calculated patern from the single crystal structure, when

available, is upped mentioned.

Differential Scanning Ca!orimetry (DSC)

The DSC analysis was performed using a Perkin Elmer DSC 4000 apparatus. The scan rate was 5 ° C / min and the scanning range of of temperature 40 to 230 ° C. The accurately weighed quantity was ranged from 1 to 3 mg. All operations were performed under nitrogen atmosphere. The measured values were the Onset, the Peak and the value of the free enthalpy variation. The eventual product decomposition and the vaporization of solvent crystallization (methanol and / or water) were recorded. The value of the change in free energy, was given only as a guideline to assess the endothermicity or exotermicity of the transition. Results were average of triplicate experiment. Only one experiment was given as example of termic curve. Positive sign is used as convention for exothermic transition.

Melting Point Checking Melting points were measured manually for visual checking of the one determined with DSC. A Bucchi B-545 melting point apparatus was used and mp are uncorrected.

Infrared Spectra

All vibrational spectra were recorded on a Perkin Elmer IR FT Spectrum 2 apparatus equipped with diamond ATR accessory that is to say using Attenuated Total Reflection technique. The crystalline solids were crushed directly by in situ compression on the diamond window and the amorphous state has been demonstrated by dissolving the product in deuterated methanol then generating the film by in situ evaporation on the diamond window.

1.5 General procedures for liquid state and solution characterizations

Nuclear Magnetic Resonance (NMR)

NMR spectra were recorded automatically on a Bruker Avance III spectrometer NanoBay - 400MHz (9.4 Tesla magnet) with a BBFO + probe and sampler 120 positions, allows for automatic mode NMR experiments one and two dimensions mainly for nuclei: 1 H, 2H, 1 1 B, 13C, 15N, 19F, 27AI, 31 P, 1 19Sn or on Bruker Avance III - 600MHz spectrometer.

Dissolving salts for ¹³ C NMR: 30 mg of compound were dissolved in 600 μΙ_ (5% m/V) of methanol D ₄ or deuterium oxyde (or both if specified)

Water suppression: The irradiation technique known as "watergate" (Selective pulse flanked by gradient pulses) was used for proton NMR in the presence of

D ₂ O and/or MOD ₄ as solvents.

High Performance Liquid Chromatography (HPLC)

Routine experiments were performed on a Waters HPLC-MS-DAD coupled system (3100 pump, DAD 996 detector, 3100 mass detector).

Solubility determination

Solubility in water at 25 ° C was measured semi-quantitatively at a threshold of 5 g per 100 mL

Optical rotation

Specific rotations [a] _D were measured at 25 °C on a Perkin-Elmer 241 polarimeter with sodium (589 nm) lamp. Concentration is given in g / 100 mL

Example 2: Preparation and analysis of (3S,4S,5RM-)-cephalotaxine base for comparison with its salts

O— CH

Preparation of (-)-cephalotaxine was performed by methanol recrystalisation of crude (-)-cephalotaxine coming from a pilot batch extraction and chromatography of Cephalotaxus fortunei var. alpina purchased in province of Shichuan in China and generously gifted by Oncopharm S.A. company.

Analysis of (-)-cephalotaxine base alkaloid

NMR spectra were performed in deuterated methanol for comparison with salt in the same solvent (MeOD4) By methanol recrystallisation of an alkaloid from natural source, it results fine white prisms (mp 135.5-138°C, by DSC (see below and figure 2.1 ) used for all experiences.

Differential Scanning Calorimetry

As seen on figure 2.1 , thermic curve shows the following event:

An endothermic transition (melting point) at 135.5-138 °C (ΔΗ=87 ± 10 J/g) (average of triplicate experiment)

Ή NMR (400 MHz, Benzene-d _fi ) δ ¹ H NMR (400 MHz, Benzene-c/ ₆ ) δ 6.59 (s, 1 H), 6.50 (s, 1 H), 5.41 (s, OH), 5.36 (d, J = 3.2 Hz, 1 H), 4.63 (s, 1 H), 4.51 (d, J = 9.3 Hz, 1 H), 3.57 (ddd, J = 14.3, 1 1 .9, 7.7 Hz, 1 H), 3.37 (d, J = 9.4 Hz, 1 H), 3.27 (d, J = 2.7 Hz, OH), 2.87 (ddd, J = 8.6, 4.4 Hz, 1 H), 2.77 (ddd, J = 12.0, 10.9, 7.1 Hz, 1 H), 2.63 (ddd, J = 10.9, 7.8, 1 .2 Hz, 1 H), 2.49 (ddd, J = 9.1 , 6.9 Hz, 1 H), 2.17 (ddd, J = 14.3, 7.1 , 1 .2 Hz, 1 H), 1 .89 (ddd, J = 12.1 , 9.6, 8.6 Hz, 1 H), 1 .70 (ddd, 1 H), 1 .61 - 1 .47 (m, 2H)..

Ή NMR (400 MHz. Methanol-d..)* δ ¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 6.68 (s, 1 H), 6.66 (s, 1 H), 5.89 (s, 1 H), 5.88 (d, J = 1 .3 Hz, 1 H), 5.03 (d, J = 0.6 Hz, 1 H), 4.89 (s, 3H), 3.72 (s, 3H), 3.67 (d, J = 9.3 Hz, 1 H), 3.43 (ddd, J = 14.4, 12.2, 8.1 Hz, 1 H), 2.93 (td, J = 9.3, 3.8 Hz, 1 H), 2.86 (td, J = 1 1 .7, 7.2 Hz, 1 H), 2.67 - 2.59 (m, 2H), 2.33 (dd, J = 14.5, 6.5 Hz, 1 H), 2.04 - 1 .92 (m, 1 H), 1 .91 - 1 .85 (m, 1 H), 1 .85 - 1 .66 (m, 2H).

*Partial presuppression of water signal using "watergate" irradiation ¹³ C NMR (101 MHz, MeOD) δ 163.18, 147.78, 147.14, 135.31 , 130.75, 1 13.65, 1 10.89, 101 .89, 97.67, 74.32, 72.08, 58.72, 57.52, 54.38, 49.57, 43.93, 32.26, 20.69.

IR (Diamond ATR, solid) cm ¹ 3217, 2931 , 2874, 281 1 , 2740, 2323, 2051 , 1981 , 1647, 1622, 1501 , 1486, 1462, 1452, 1379, 1364, 1439, 1337, 1299, 1275, 1256, 1219, 1 187, 1 159, 1 107, 1089, 1080, 1060, 1035, 988, 970, 940, 933, 926, 901 , 860, 850, 805, 758, 737, 71 1 , 675, 592, 544, 508, 471 , 459. See figure 3.1

Optical rotation [a] ²⁵ _D -186 ± 4 (c 1 .04, CHCI ₃ ); [a] ²⁵ _D -236 ± 4 (c 0.2, MeOH).

Example 3: Preparation and analyses of partially and fully racemized cephalotaxine

Partially racemized cephalotaxine was a by-product of large scale preparation of cephalotaxine from the plant material collected in China. As reported in the literature, this cephalotaxine of natural origin, typically provides a product which can reach at least 10% of racemization. The mother liquors resulting from successive crystallization of this material have thus given a partially racemized mixture containing 40% of unnatural enantiomer having the analytical characteristics described below:

Differential Scanning Calorimetry

As seen on figure 2.2, thermic curve show several events:

A first endothermic transition at 128.5-131 .5 °C (ΔΗ=12 ± 5 J/g) close of melting point of (-)-cephalotaxine.

- A second endothermic transition at 153.5-156.5°C (ΔΗ=65 ± 10 J/g) corresponding to melting point.of rac-cephalotaxine

Optical rotation [a] ²⁵ _D -35 ± 4 (c 0.19, MeOH). Preparation of purified rac-cephalotaxine form A In a flask equipped with a stirring system, above crystalline partially racemized (+)/(-)-cephalotaxine (70 g) was dissolved in dry methanol (160 ml_) at 60°C. then substantially saturated solution is left standing at 25 ° C. After 2h hours, the first large rhombohedral crystals begin to deposit on the walls and the bottom of the flask. After 48 h the crystallization process being completed, the mother liquors were decanted and the crystals were washed quickly with chilled dry methanol. Wet sample is taken for subsequent single crystal X-ray analysis, and the crystals were dried at 50 ° C under vacuum. A sample was taken for recrystallization in ethyl acetate for the purpose of comparison with the literature and other solid state analyses (form B) see below.

Methanol crystallized rac-cephalotaxine exhibited the following analytical data:

Analysis of rac -cephalotaxine base alkaloid form A

NMR spectra were performed in deuterated methanol for comparison with salt in the same solvent (MeOD4) By methanol recrystallisation of an alkaloid from natural source, it results fine white prisms (mp 157-158.5°C, by DSC, see figures 2.2 and 2.3). As seen by single crystal X-ray diffraction (see figures 1 .1 a, 1 .1 b, 1 .1 c), this form A was a methanolate undescribed in literature.

Differential Scanning Calorimetry of crude form A

As seen on figure 2.2, thermic curve show the following events:

A first endothermic transition (melting point) at 128.5-131 .5 °C (ΔΗ=12 ±

2 J/g) (average of triplicate experiment)

A second endothermic transition (melting point) at 153.5-156.5 °C

(ΔΗ=65 ± 10 J/g) (truncated average of triplicate experiments)

Differential Scanning Calorimetry of purified form A

As seen on figure 2.3, thermic curve show the following events:

An endothermic transition (melting point) at 157-158.5 °C (ΔΗ=62 ± 10 J/g) (average of triplicate experiment)

¹ H NMR (400 MHz, Methanol-ck)* δ was strictly superimposble to the one of (-

)-cephalotaxine.

¹³ C NMR (101 MHz, MeOD) δ 163.20, 147.80, 147.16, 135.31 , 130.75, 1 13.66, 1 10.90, 101 .90, 97.68, 74.33, 72.1 1 , 58.73, 57.53, 54.39, 49.58, 43.93, 32.27, 20.69. IR (Diamond ATR, solid) cm ¹ 3565, 3289, 2932, 2881 , 2810, 2744, 2051 , 1978, 1648, 1621 , 1498, 1486, 1453, 1414, 1381 , 1363, 1335, 1315, 1280, 1269, 1221 , 1 161 , 1 1 12, 1094, 1057, 1029, 986, 925, 898, 864, 849, 819, 806, 783, 752, 735, 703, 670, 589, 537, 496, 470, 458. See figure 3.2

Optical rotation [a] ²⁵ _D - 2 ± 4 (c 0.4, MeOH/H ₂ O 1/1 )

Single crystal X-ray diffraction

see figures 1 .1 .a, 1 .1 .b and 1 .1 .c (pa ;king):

From a suspension in its above mothei liquor, a suitable small single crystal of size 0.1 8 x 0.1 1 x 0.08 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C ₁₉ ¾ ₅ iV 0 ₅

Extended formula C ₁₈ H ₂ iN0 ₄ , CH ₄ 0

Formula weight 347.4

Temperature 150(2) K

Wavelength 0.71073 A

Crystal system, space group triclinic, P 1

Unit cell dimensions a = 7.5250(3) X, a = 84.165(2) ^° b =

8.2528(3) X, jS = 86.575(2) ^°

c = 15.6938(6) X, γ = 64.2890(10) ^°

Volume 873.45(6) X ³

Z, Calculated density 2 , 1.321 (g. cm ^{' 1} )

Absorption coefficient 0.095 mm ^"1

F(000) 372

Crystal size 0.18 x 0.11 x 0.08 mm

Crystal color colourless

Theta range for data collection 2.94 to 27.48 ^°

h_min, h_max -9 , 9

k_min, k_max -9 , 10

l_min, l_max -20 , 20

Reflections collected / unique 11180 / 3963 [ ^a R(int) = 0.0261] Reflections [I > 2σ] 3212

Completeness to theta_max 0.992 Absorption correction type multi-scan

Max. and min. transmission 0.992 , 0.880

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 3963 / 0 / 228

''Goodness -of-f it 1.04

Final R indices [I > 2σ] ^c Ri = 0.0403, ^d wR ₂ = 0.0975

R indices (all data) ^c Ri = 0.0519, ^d wR ₂ = 0.1047 Larg peak and hole 0.326 and -0.245 e.X ^"3 Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z Occ. U(eq)

CI 0.30249(19) 0.39691(17) 0.37659(8) 1 0.0213(3)

HI 0.2419 0.4556 0.4267 1 0.026

C2 0.35338(18) 0.22367(17) 0.36832(8) 1 0.0193(3)

C3 0.44833(18) 0.16020(16) 0.28335(8) 1 0.0181(3)

H3 0.3633 0.1186 0.2538 1 0.022

C4 0.43905(17) 0.33820(16) 0.23320(8) 1 0.0168(2)

H4 0.3367 0.3719 0.1886 1 0.02

C5 0.35214(18) 0.48958(16) 0.29714(8) 1 0.0194(3)

C6 0.1704(2) 0.65454(18) 0.26044(9) 1 0.0263(3)

H6A 0.1895 0.6802 0.1984 1 0.032

H6B 0.0505 0.6333 0.2689 1 0.032

C7 0.1521(2) 0.81201(19) 0.31048(10) 1 0.0353(4)

H7A 0.1292 0.9207 0.2712 1 0.042

H7B 0.0422 0.842 0.353 1 0.042

C8 0.3493(2) 0.74310(18) 0.35483(9) 1 0.0303(3)

H8A 0.4021 0.8347 0.3475 1 0.036

H8B 0.3351 0.7133 0.4168 1 0.036

N9 0.48020(16) 0.57966(14) 0.31239(7) 1 0.0216(2)

CIO 0.6601(2) 0.47504(18) 0.36098(9) 1 0.0255(3)

H10A 0.6237 0.451 0.421 1 0.031

H10B 0.7358 0.5476 0.3614 1 0.031

Cl l 0.79155(19) 0.29548(18) 0.32437(8) 1 0.0222(3)

H11A 0.9283 0.2556 0.3434 1 0.027 HUB 0.7463 0.2036 0.3481 1 0.027

C12 0.79212(18) 0.30408(16) 0.22789(8) 1 0.0192(3)

C13 0.62549(18) 0.32197(16) 0.18509(8) 1 0.0173(2)

C14 0.62752(18) 0.32610(17) 0.09523(8) 1 0.0211(3)

H14 0.5147 0.34 0.0655 1 0.025

C15 0.7979(2) 0.30933(19) 0.05183(8) 1 0.0251(3)

C16 0.96079(19) 0.29306(19) 0.09367(9) 1 0.0256(3)

C17 0.96175(19) 0.29209(18) 0.18107(9) 1 0.0243(3)

H17 1.0742 0.2835 0.2092 1 0.029

C18 1.0417(2) 0.2605(3) -0.04437(10) 1 0.0400(4)

H18A 1.0696 0.3348 -0.0921 1 0.048

H18B 1.1109 0.1323 -0.0566 1 0.048

04 0.32522(14) 0.09826(12) 0.42300(6) 1 0.0256(2)

C19 0.2295(2) 0.1594(2) 0.50256(9) 1 0.0315(3)

H19A 0.3079 0.203 0.5331 1 0.047

H19B 0.2166 0.0595 0.5375 1 0.047

H19C 0.0982 0.2579 0.4917 1 0.047

01 0.63919(13) 0.01493(12) 0.29328(6) 1 0.0218(2)

HOI 0.642(3) -0.062(3) 0.2591(13) 1 0.05

02 0.83342(15) 0.31397(17) -0.03576(6) 1 0.0384(3)

03 1.10804(15) 0.28622(16) 0.03506(7) 1 0.0379(3)

031 0.60879(15) 0.77830(13) 0.18849(6) 1 0.027

H31 0.568(3) 0.706(3) 0.2186(12) 1 0.05

C32 0.4881(2) 0.8509(2) 0.11549(10) 1 0.0373(4)

H32A 0.3493 0.891 0.1331 1 0.056

H32B 0.5234 0.7581 0.0751 1 0.056

H32C 0.5081 0.9537 0.0878 1 0.056

X-ray data showed that rac-cephalotaxine is a true racemic (as methanolate) (see figures 1 .1 .a, 1 .1 .b and 1 .1 .c)

Example 4 Preparation and analysis of AcOEt crystallized (±)- cephalotaxine form B

In a 50 mL-Erlenmeyer flask, a sample of (3.15 g) of above MeOH crystallized (±)-cephalotaxine form B was dissolved in dry ethyl acetate by stirring at 70°C. After complete disolution, the solution is filtered on a small Buchner funnel then then allowed to cool at room temperature. After 48 h the solution deposited thin translucent needles which were collected and dried under vacuum. This solid exhibited the following analytical data:

Differential Scanning Calorimetry

As seen on figure 2.4, thermic curve show several events:

- A first endothermic melting point at 123-126 °C (ΔΗ=33 ± 5 J/g) litt 122- 124 [Burkholder and fuchs J. Am. Chem. Soc. 9601 (1990); Ikeda et al. Chem. Pharm. Bull. 276 (1993)]

A second peak at 130 °C (ΔΗ=-24 ± 5 J/g) showing an exothermic transition corresponding to recrystallisation.

- A third peak showing an endothermic transition corresponding to a second melting point at 157-159°C (ΔΗ=85 ± 10 J/g). Latter thermal data correspond to above ones found for rac-cephalotaxine form A (see figure 2.3)

¹ H NMR (400 MHz, Methanol-c/ ₄ )* δ was strictly superimposable to the one of the compound of example 3 [(3S,4S,5R)-cephalotaxine form A].

¹³ C NMR (101 MHz, MeOD) δ was strictly superimposable to the one of the compound of example 3 [(3S,4S,5R)-cephalotaxine form A]. IR (Diamond ATR, solid) cm ¹ 3238, 3059, 2998, 2961 , 2931 , 2887, 2802, 2740, 1645, 1622, 1505, 1497, 1484, 1463, 1452, 1442, 1380, 1362, 1336, 1317, 1281 , 1269, 1252, 1218, 1203, 1 185, 1 158, 1 107, 1090, 1059, 1037, 1016, 986, 937, 900, 859, 847, 806, 756, 737, 720, 71 1 , 677, 662, 592, 544, 504, 471 , 459. See figure 3.3

Optical rotation Γα1 ²⁵ η +4 ± 4 (c 0.39, MeOH/H ₂ O 1/1 )

Example 5: Resolution of rac-cephalotaxine. 5.1 Preparation and analyses of (3S,4S,5R,2'S)-(-)-cephalotaxine hydrogen malate

Rac-cephalotaxine (100g) was dissolved in dry methanol (mL) at 60°C under argon. A solution of 2S-(-)-malic acid (natural form) in dry methanol was added under stirring. After standing for 24 hours the solution leaves deposit white prismatic crystals (a small sample was collected with its mother liquor for further single crystal diiffraction-X analysis), which on draining then drying (31 g), exhibited the following analytical features:

Differential Scanning Calorimetrv

DSC curve (see figure 2.5a) indicated two transition points:

(i) one endotermic transition coresponding to melting point 224.5-229.5 (AH=175 ± 20J/g)

(ii) one exotermic transition at 237°C (ΔΗ=-23 ± 5 J/g) corresponding to decomposition. ¹ H NMR (400 MHz, Methanol-ck)* δ 6.83 (s, 1 H), 6.79 (s, 1 H), 5.97 (d, J = 1 .1 Hz, 1 H), 5.95 (d, J = 1 .1 Hz, 1 H), 5.12 (s, 1 H), 4.81 (d, J = 9.0 Hz, 1 H), 4.25 (dd, J = 7.5, 5.5 Hz, 1 H), 3.91 (d, J = 9.1 Hz, 1 H), 3.83 (s, 3H), 3.62 (ddd, J = 14.7, 12.1 , 8.3 Hz, 1 H), 3.55 - 3.45 (m, 1 H), 3.17 (dd, J = 12.4, 8.1 Hz, 1 H), 2.78 (dd, J = 15.9, 5.5 Hz, 1 H), 2.58 - 2.45 (m, 2H), 2.15 (dd, J = 9.3, 3.4 Hz, 3H), 2.01 - 1 .90 (m, 1 H).

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR APT* (101 MHz. MeOD) δ 179.42, 176.30, 169.05, 149.38, 148.67, 131 .59, 127.82, 1 14.35, 1 1 1 .81 , 102.69, 92.97, 78.60, 73.83, 69.44, 58.47, 55.33, 53.87, 41 .92, 40.38, 29.27, 19.77.

*APT = Attached Proton Test

IR (Diamond ATR. solid) cm-1 3397, 2918, 1723, 1653, 1614, 1507, 1490, 1460, 1446, 141 1 , 1367, 1327, 1301 , 1279, 1265, 1222, 1 177, 1 100, 1087, 1070, 1056, 1035, 971 , 930, 901 , 866, 837, 801 , 783, 706, 645, 598, 568, 501 , 475. (see figure 3.4)

Optical rotation [a] ²⁵ _D -196 ± 4 (c 2.06, H ₂ O); [a] ²⁵ _D -175 ± 4 (c 0.23, MeOH).

A. Single crystal X-ray diffraction (see figure 1 .2. a and 1 .2.b) From a suspension in its mother liquor, a suitable single crystal of size 0.32 x 0.24 x 0.12 mm was finally selected and implemented on the diffractometer. Structural data

Empirical formula CziR _ll N O ₉

Extended formula CwHzzNOi, C ₄ HsOs

Formula weight 449.45

Temperature 150(2) K

Wavelength 0.71073 A

Crystal system, space group monoclinic, P

Unit cell dimensions a = 8.1040(6) A, a = 90 ^°

b = 17.7574(9) ^° A, β = 115.838(2) ^° c = ..3193(6) X, γ = 90 ^°

Volume 1077.51(12) λ ³

Z, Calculated density 2 , 1.385 (g. cm.- ¹ )

Absorption coefficient 0.108 mm ^{" 1}

F(000) 476

Crystal size 0.32 x 0.24 x 0.12 mm

Crystal color colourless

Theta range for data collection 2.93 to 27.48 ^°

h_min, h_max - 10 , 10

k_min, k_max -22 , 23

l_min, l_max -10 , 10

Reflections collected / unique 9650 / 2534 [ ^a R(int) = 0.0649]

Reflections [I > 2σ] 2310

Completeness to theta_max 0.992

Absorption correction type multi-scan

Max. and min. transmission 0.987 , 0.875

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 2534 / 1 / 302

f ^t Goodness-of-fit 1.039

Final R indices [I > 2σ] ^c Ri = 0.0406, ^d wR ₂ = 0.091 1

R indices (all data) ^c Ri = 0.0452, ^d wR ₂ = 0.0941 Largest diff. peak and hole 0.249 and -0.223 e.X ^"3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters {A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom x y z Occ. U(eq)

CI 0.8147(3) 0.99947(14) 0.3868(3) 1 0.0172(5)

HI 0.7365 1.0384 0.3158 1 0.021 C2 0.9964(3) 0.99955(13) 0.4512(3) 1 0.0173(5)

C3 1.0909(3) 0.93393(13) 0.5695(3) 1 0.0171(5)

H3 1.1515 0.9028 0.5104 1 0.021

C4 0.9288(3) 0.88771(12) 0.5760(3) 1 0.0156(5)

H4 0.9295 0.8378 0.5205 1 0.019

C5 0.7506(3) 0.92875(13) 0.4425(3) 1 0.0171(5)

C6 0.6296(4) 0.87917(15) 0.2835(3) 1 0.0215(5)

H6A 0.6841 0.8739 0.1982 1 0.026

H6B 0.6139 0.8285 0.3244 1 0.026

C7 0.4460(4) 0.92080(17) 0.1973(4) 1 0.0280(6)

H7A 0.4456 0.9578 0.1081 1 0.034

H7B 0.3435 0.885 0.138 1 0.034

C8 0.4304(4) 0.96044(17) 0.3534(4) 1 0.0277(6)

H8A 0.4108 1.0152 0.33 1 0.033

H8B 0.3264 0.9397 0.3717 1 0.033

N9 0.6107(3) 0.94596(12) 0.5166(3) 1 0.0195(4)

H9 0.587(5) 0.901(2) 0.565(5) 1 0.05

CIO 0.6555(4) 1.00726(14) 0.6523(3) 1 0.0227(6)

H10A 0.5684 1.0048 0.7065 1 0.027

H10B 0.639 1.0566 0.5918 1 0.027

Cl l 0.8517(4) 1.00196(14) 0.8006(4) 1 0.0228(5)

H11A 0.9367 1.0226 0.755 1 0.027

HUB 0.8615 1.034 0.9018 1 0.027

C12 0.9125(3) 0.92294(13) 0.8691(3) 1 0.0190(5)

C13 0.9505(3) 0.87055(13) 0.7629(3) 1 0.0167(5)

C14 1.0175(3) 0.79864(14) 0.8302(3) 1 0.0188(5)

H14 1.0461 0.7632 0.7606 1 0.023

C15 1.0404(3) 0.78095(13) 0.9992(3) 1 0.0197(5)

C16 0.9984(4) 0.83173(15) 1.1019(3) 1 0.0212(5)

C17 0.9363(4) 0.90271(15) 1.0411(3) 1 0.0217(5)

H17 0.9099 0.9375 1.1136 1 0.026

C18 1.1197(5) 0.72862(17) 1.2668(4) 1 0.0321(7)

H18A 1.2513 0.7324 1.3508 1 0.039

H18B 1.0649 0.6872 1.3072 1 0.039

C19 1.0258(4) 1.11320(15) 0.3190(4) 1 0.0302(6)

H19A 0.9819 1.1456 0.3876 1 0.045 H19B 0.9221 1.0982 0.2072 1 0.045

H19C 1.1155 1.1408 0.2921 1 0.045

01 1.2263(2) 0.95964(10) 0.7371(2) 1 0.0203(4)

HOI 1.319(5) 0.928(2) 0.776(5) 1 0.05

02 1.0281(3) 0.79842(11) 1.2631(2) 1 0.0303(5)

03 1.0996(3) 0.71370(11) 1.0904(3) 1 0.0305(5)

04 1.1107(2) 1.04707(9) 0.4219(2) 1 0.0224(4)

C21 0.4814(3) 0.79884(14) 0.7365(3) 1 0.0216(5)

C22 0.4762(4) 0.71555(15) 0.7849(3) 1 0.0223(5)

H22 0.3485 0.7027 0.7648 1 0.027

C23 0.6046(4) 0.70283(15) 0.9811(4) 1 0.0327(7)

H23A 0.7323 0.7118 0.9998 1 0.039

H23B 0.5759 0.74 1.0539 1 0.039

C24 0.5913(4) 0.62472(15) 1.0454(4) 1 0.0248(6)

021 0.5142(3) 0.81085(11) 0.6038(3) 1 0.0319(5)

022 0.4515(3) 0.84621(11) 0.8302(3) 1 0.0345(5)

023 0.5262(4) 0.66798(12) 0.6758(3) 1 0.0409(6)

H23 0.546(5) 0.702(2) 0.600(5) 1 0.05

024 0.7488(3) 0.60313(12) 1.1757(3) 1 0.0425(6)

H24 0.747(5) 0.561(2) 1.213(5) 1 0.05

025 0.4542(3) 0.58678(11) 0.9900(3) 1 0.0342(5)

B. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.108 °(2 Θ) at 19.602 °(2Θ) (for view of diagrams and experimental details, see figure 1 .2.c)

Example 6: Resolution of rac-cephalotaxine. Preparation and analyses of (3R,4R,5S,2'RM+)-cephalotaxine hydrogen malate (enantiomer of example 5)

Rac-cephalotaxine (100g) was dissolved in dry methanol (mL)at 60°C under argon. A solution of 2R-(+)-malic acid (natural form) in dry methanol was added under stirring. After standing for 24 hours the solution leaves deposit white prismatic crystals, which on draining then drying (27g), exhibited below analytical features. Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

Differential Scanning Calorimetrv

DSC curve (see figure 2.6) indicated two transition points:

(i) one endothermic transition coresponding to melting point 224-231 (AH=143 ± 20J/g)

(ii) one exothermic transition at 238°C corresponding to decomposition. ¹ H NMR (400 MHz, Methanol-rf ₄ )* δ was strictly suprimposable to the one of example 5

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR APT* (101 MHz, Methanol-d ₄ ) δ was strictly suprimposable to the one of example 5

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^"1 , was strictly suprimposable to the one of example 5 See figure 3.6.

Optical rotation [a] ²⁵ _D +194 ± 2 (c 0.97, H ₂ O); [a] ²⁵ _D +176 ± 2 (c 0.22, MeOH) A. Single crystal X-ray diffraction (see figures 1 .3.a and 1 .3.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.41 x 0.24 x 0.21 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C iH iN Og

Extended formula C ₁₈ H ₂ 2N0 ₄ , C ₄ /i ₅ 0 ₅

Formula weight 449.45

Temperature 150(2) K

Wavelength 0.71073 ^° A

Crystal system, space group monoclinic, P

Unit cell dimensions a = 8.0931 (4) λ, a = 90 ^°

b = 17.7334(7) ^° A, β = 115.8350(10) ^° c = 8.3060(3) ^° A, γ = 90 ^° Volume 1072.92(8) λ ³

Z, Calculated density 2 , 1.391 (g. cm.- ¹ )

Absorption coefficient 0.108 mm ^"1

F(000) 476

Crystal size 0.41 x 0.24 x 0.21 mm

Crystal color colourless

Theta range for data collection 2.93 to 27.47 ^°

h_min, h_max - 10 , 10

k_min, k_max -23 , 20

l_min, l_max -10 , 7

Reflections collected / unique 9553 / 2530 [ ^a R(int) = 0.042]

Reflections [I > 2σ] 2420

Completeness to theta_max 0.995

Absorption correction type multi-scan

Max. and min. transmission 0.977 , 0.883

Refinement method Full-matrix least-squares

Data / restraints / parameters 2530 / 1 / 302

f ^t Goodness-of-fit 1.041

Final R indices [I > 2σ] ^c Ri = 0.0313, ^d wR ₂ 0.0782

R indices (all data) ^c Ri = 0.0332, ^d wR ₂ = 0.0796 Largest cliff. peak and hole 0.254 and -0.164 e.A

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI 0.6856(2) 0.21150(10) 1.1131(2) 1 0.0173(3)

HI 0.764 0.1724 1.1838 1 0.021

C2 0.5030(2) 0.21156(10) 1.0490(2) 1 0.0171(3)

C3 0.4090(2) 0.27739(10) 0.9304(2) 1 0.0162(3)

H3 0.3483 0.3087 0.9894 1 0.019

C4 0.5710(2) 0.32337(9) 0.9242(2) 1 0.0152(3)

H4 0.5706 0.3733 0.9801 1 0.018

C5 0.7493(2) 0.28223(10) 1.0572(2) 1 0.0164(3)

C6 0.8709(2) 0.33204(12) 1.2170(2) 1 0.0210(4)

H6A 0.8866 0.3828 1.1763 1 0.025

H6B 0.8166 0.3373 1.3027 1 0.025 C7 1.0542(3) 0.29036(13) 1.3026(3) 1 0.0280(4)

H7A 1.1569 0.3262 1.3616 1 0.034

H7B 1.0548 0.2534 1.3922 1 0.034

C8 1.0694(3) 0.25057(13) 1.1467(3) 1 0.0269(4)

H8A 1.1735 0.2711 1.1281 1 0.032

H8B 1.0887 0.1958 1.1702 1 0.032

N9 0.8892(2) 0.26509(9) 0.9836(2) 1 0.0191(3)

H9 0.908(4) 0.308(2) 0.938(4) 1 0.05

CIO 0.8444(3) 0.20376(11) 0.8475(3) 1 0.0234(4)

H10A 0.8605 0.1543 0.9079 1 0.028

H10B 0.9318 0.2062 0.7935 1 0.028

Cl l 0.6481(3) 0.20914(10) 0.6985(3) 1 0.0220(4)

H11A 0.6387 0.1774 0.5968 1 0.026

HUB 0.5627 0.1882 0.7435 1 0.026

C12 0.5873(2) 0.28838(10) 0.6310(2) 1 0.0190(4)

C13 0.5494(2) 0.34075(10) 0.7369(2) 1 0.0163(3)

C14 0.4828(2) 0.41276(10) 0.6700(2) 1 0.0188(3)

H14 0.4548 0.4483 0.7401 1 0.023

C15 0.4592(3) 0.43030(10) 0.5007(3) 1 0.0206(4)

C16 0.5021(3) 0.37954(11) 0.3982(2) 1 0.0210(4)

C17 0.5647(3) 0.30846(11) 0.4593(3) 1 0.0215(4)

H17 0.5921 0.2737 0.3873 1 0.026

C18 0.3809(3) 0.48234(13) 0.2331(3) 1 0.0318(5)

H18A 0.436 0.5236 0.1926 1 0.038

H18B 0.2492 0.4786 0.1488 1 0.038

C19 0.4742(3) 0.09791(12) 1.1812(3) 1 0.0305(5)

H19A 0.5247 0.0669 1.1156 1 0.046

H19B 0.3826 0.0687 1.2021 1 0.046

H19C 0.5734 0.113 1.2963 1 0.046

01 0.38921(18) 0.16410(7) 1.07809(19) 1 0.0223(3)

02 0.27383(18) 0.25146(8) 0.76315(18) 1 0.0206(3)

H2 0.193(4) 0.284(2) 0.727(4) 1 0.05

03 0.4721(2) 0.41260(9) 0.23709(18) 1 0.0301(3)

04 0.4006(2) 0.49771(9) 0.40984(19) 1 0.0298(3)

C21 1.0186(2) 0.41236(11) 0.7638(3) 1 0.0219(4)

C22 1.0229(3) 0.49577(11) 0.7148(3) 1 0.0218(4) H22 1.1507 0.5086 0.7346 1 0.026

C23 0.8955(3) 0.50835(11) 0.5194(3) 1 0.0318(5)

H23A 0.9242 0.471 0.4467 1 0.038

H23B 0.7675 0.4995 0.5005 1 0.038

C24 0.9094(3) 0.58645(11) 0.4547(3) 1 0.0241(4)

021 0.9858(2) 0.40026(9) 0.8964(2) 1 0.0314(3)

022 1.0486(2) 0.36491(9) 0.6698(2) 1 0.0341(4)

023 0.9742(3) 0.54334(9) 0.8250(2) 1 0.0408(4)

H023 0.956(4) 0.5130(19) 0.898(4) 1 0.05

024 1.0457(2) 0.62442(9) 0.5098(2) 1 0.0335(4)

025 0.7511(2) 0.60792(9) 0.3243(3) 1 0.0414(5)

H025 0.757(4) 0.648(2) 0.298(4) 1 0.05

ORTEP diagram representation of single crystal X-ray diffraction of this compound is exhibited on figure 1 .3. a and is the mirror image of the one of example 5 showed on figure 1 .2.a

B. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.106° (2Θ) at 19.627° (2Θ). The sample is without impurity, as shown by the good agreement between calculated (red) and experimental (black) patterns (for view of diagrams and experimental details, see figure 1 .3.c).

Example 7: Alternate preparation and analyses of (3S,4S,5R,2'SM-)- cephalotaxine hydrogen malate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial (2R)-(-)-malic acid (unnatural form) according to the above general procedure included in example 1 , then isolated as a white prismatic solid mp 224.5-229.5°C (dec.) measured by DSC, see figure 2.5. b). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

Differential Scanning Calorimetrv

DSC curve (see figure 2.5b) indicated two transition points:

(i) one endothermic transition corresponding to melting point 224.5-229.5

(AH=163 ± 20J/g)

(ii) one exothermic transition at 235.5°C (ΔΗ=-24 ± 5 J/g) corresponding to decomposition.

¹ H NMR (400 MHz, Methanol-ck)* δ was strictly superimposable to those of example 5 and 6.

^1J C NMR APT* (101 MHz, MeOD) δ was strictly superimposable to those of example 5 and 6.

IR (Diamond ATR, solid) cm ¹ was strictly superimposable to those of example 5 and 6. See figure 3.5

Optical rotation [a] ²⁵ _D -204 ± 4 (c 0.89, H ₂ O).

Example 8: Preparation and analyses of (3S,4S,5R,2'R)-cephalotaxine hydrogen malate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial (2R)-(+)-malic acid (unnatural form) according to the above general procedure included in example 1 , then isolated as a white prismatic solid mp 214-218°C measured by DSC, see figure 2.7). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.7)

¹ H NMR (400 MHz, Methanol-ck)* δ 8.38 (s, 1 H), 8.35 (s, 1 H), 7.52 (d, J = 1 .1 Hz, 1 H), 7.51 (d, J = 1 .1 Hz, 1 H), 6.67 (s, 1 H), 6.37 (d, J = 9.0 Hz, 1 H), 5.82 (dd, J = 7.4, 5.6 Hz, 1 H), 5.47 (d, J = 9.1 Hz, 1 H), 5.39 (s, 3H), 5.18 (ddd, J = 14.8, 12.1 , 8.3 Hz, 1 H), 5.10 - 5.00 (m, 1 H), 4.73 (dd, J = 12.3, 8.0 Hz, 1 H), 4.34 (dd, J = 15.9, 5.6 Hz, 1 H), 4.08 (ddd, J = 20.7, 15.4, 7.1 Hz, 2H), 3.77 - 3.64 (m, 3H), 3.58 - 3.47 (m, 1 H)

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR APT* (101 MHz, Methanol-d ₄ ) δ 179.39, 176.29, 168.98, 149.33, 148.62, 131 .59, 127.82, 1 14.35, 1 1 1 .80, 102.67, 93.02, 78.55, 73.81 , 69.43, 58.47, 55.31 , 53.84, 48.71 , 41 .92, 40.36, 29.25, 19.77.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^"1 3392, 2931 , 2839, 2650, 1721 , 1656, 1609, 1506, 1488, 1462, 1446, 1399, 1386, 1370, 1320, 1267, 1219, 1 175, 1098, 1069, 1055, 1035, 984 , 970, 936, 899, 868, 844, 826, 800, 782, 641 , 596, 567, 523, 501 , 472, 453. See figure 3.7

Optical rotation [a] ²⁵ _D -191 ± 4 (c 0.60, H ₂ O).

X-ray crystallographic studies

A, Single crystal X-ray diffraction (see figure 1 .4.a and 1 .4.b)

From a suspension in its mother liquor, a s suitable single crystal of size 0.53 x 0.4 x 0.33 mm was finally selected and implem ented on the diffractometer.

Structural data

Empirical formula CziU _ll N

Extended formula CwHzzNOi, C ₄ HsOs

Formula weight 449.45

Temperature 150(2) K

Wavelength 0.71073 ^° A

Crystal system, space group monoclinic, P

Unit cell dimensions a = 8.0993(4) ^° A, a = 90 ^° b = 17.1887(8) A, β = 117.024(2) ^° c 8.5330(4) A, γ = 90 ^°

Volume 1058.23(9) A ³

Z, Calculated density 2 , 1.411 (g. cm ^{' 1} )

Absorption coefficient 0.110 mm ^"1

F(000) 476

Crystal size 0.53 x 0.4 x 0.33 mm

Crystal color colourless

Theta range for data collection 2.93 to 27.48 ^°

h_min, h_max - 10 , 9

k_min, k_max -20 , 22

l_min, l_max -9 , 11

Reflections collected / unique 7614 / 2501 [ ^a R(int) = 0.0413]

Reflections [I > 2σ] 2350

Completeness to theta_max 0.997

Absorption correction type multi-scan

Max. and min. transmission 0.964 , 0.881

Refinement method Full-matrix least-squares

Data / restraints / parameters 2501 / 1 / 302

f ^t Goodness-of-fit 0.975

Final R indices [I > 2σ] 0.035, ^a wR ₂ -- 0.0878

R indices (all data) : 0.0375, ^d wR ₂ = 0.0897 Largest cliff. peak and hole 0.262 and -0.19 e.A ^"

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI 0.1586(3) 0.00389(12) 0.5857(3) 1 0.0167(4)

HI 0.2365 -0.0353 0.6621 1 0.02

C2 -0.0254(3) 0.00323(12) 0.5135(3) 1 0.0165(4)

C3 -0.1180(3) 0.06976(12) 0.3910(3) 1 0.0150(4)

H3 -0.1822 0.1033 0.4422 1 0.018

C4 0.0487(3) 0.11603(12) 0.3901(3) 1 0.0146(4)

H4 0.0463 0.1686 0.4394 1 0.018

C5 0.2269(3) 0.07496(12) 0.5301(3) 1 0.0147(4)

C6 0.3474(3) 0.12806(14) 0.6850(3) 1 0.0205(5)

H6A 0.3641 0.1797 0.6427 1 0.025

H6B 0.2908 0.1352 0.7653 1 0.025 C7 0.5329(3) 0.08496(16) 0.7777(3) 1 0.0262(5)

H7A 0.6364 0.1221 0.836 1 0.031

H7B 0.5319 0.0484 0.867 1 0.031

C8 0.5514(3) 0.04103(14) 0.631 1(3) 1 0.0216(5)

H8A 0.6569 0.0615 0.6149 1 0.026

H8B 0.5718 -0.0151 0.6593 1 0.026

N9 0.3703(2) 0.05385(1 1) 0.4663(2) 1 0.0160(4)

H9 0.386(5) 0.098(2) 0.418(5) 1 0.05

CIO 0.3259(3) -0.01 165(13) 0.3385(3) 1 0.0199(4)

H10A 0.341 -0.0614 0.4019 1 0.024

H10B 0.4157 -0.01 13 0.2895 1 0.024

Cl l 0.1292(3) -0.00759(13) 0.1868(3) 1 0.0192(4)

H11A 0.1211 -0.0427 0.0915 1 0.023

HUB 0.0412 -0.0269 0.2289 1 0.023

C12 0.0706(3) 0.07312(13) 0.1 121(3) 1 0.0160(4)

C13 0.0336(3) 0.13038(12) 0.2094(3) 1 0.0147(4)

C14 -0.0265(3) 0.20444(12) 0.1360(3) 1 0.0165(4)

H14 -0.0518 0.2438 0.2002 1 0.02

C15 -0.0477(3) 0.21888(13) -0.0300(3) 1 0.0188(4)

C16 -0.0076(3) 0.16289(14) -0.1231(3) 1 0.0190(4)

C17 0.0521(3) 0.08984(14) -0.0564(3) 1 0.0185(4)

H17 0.0799 0.0519 -0.1219 1 0.022

C18 -0.0937(4) 0.27094(17) -0.2853(4) 1 0.0345(6)

H18A -0.0023 0.3065 -0.2943 1 0.041

H18B -0.2155 0.2799 -0.3882 1 0.041

C19 -0.0586(3) -0.1 1 178(15) 0.6468(4) 1 0.0289(5)

H19A 0.0027 -0.1441 0.5942 1 0.043

H19B -0.154 -0.1424 0.6593 1 0.043

H19C 0.0332 -0.0938 0.7629 1 0.043

01 -0.2504(2) 0.04159(10) 0.2239(2) 1 0.0189(3)

HOI -0.338(5) 0.071(2) 0.184(5) 1 0.05

02 -0.0378(3) 0.19238(12) -0.2847(2) 1 0.0313(4)

03 -0.1067(3) 0.28632(1 1) -0.1269(2) 1 0.0303(4)

04 -0.1428(2) -0.04604(9) 0.5359(2) 1 0.0223(4)

C21 0.4721(3) 0.19787(13) 0.2233(3) 1 0.0201(5)

C22 0.4256(3) 0.28151(13) 0.1525(3) 1 0.0202(5) H22 0.2915 0.2839 0.0652 1 0.024

C23 0.5404(3) 0.30577(14) 0.0617(4) 1 0.0256(5)

H23A 0.673 0.3045 0.1485 1 0.031

H23B 0.5218 0.2673 -0.0313 1 0.031

C24 0.4948(3) 0.38547(14) -0.0198(3) 1 0.0215(5)

021 0.4871(3) 0.18783(10) 0.3759(2) 1 0.0280(4)

022 0.4889(2) 0.14813(10) 0.1265(2) 1 0.0270(4)

023 0.4569(3) 0.33372(11) 0.2928(3) 1 0.0389(5)

H23 0.474(5) 0.301(2) 0.380(5) 1 0.05

024 0.3145(3) 0.39496(12) -0.1259(3) 1 0.0386(5)

H24 0.305(5) 0.442(2) -0.158(5) 1 0.05

025 0.6083(2) 0.43567(11) 0.0066(2) 1 0.0278(4) β. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.102°(2Θ) at 15.515°(2Θ). The sample is without impurity, as shown by the good agreement between calculated (red) and experimental (black) patterns (for view of diagrams and experimental details, see figure 1 .4.c).

Example 9: Preparation and analyses of (3S,4S,5R,2'S,3'S)-cephalotaxine hydrogen tartrate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial (2S,3S)-tartaric acid according to the above general procedure included in example 1 , then isolated as large white prisms mp 212.5-218.5°C measured by DSC, see figure 2.8). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below). Differential Scanning Calorimetry

DSC curve (see figure 2.8) indicated two transition points:

(i) one endothermic transition corresponding to melting point 212.5-218.5 (AH=105± 20J/g)

(ii) one exothermic transition at 228°C (ΔΗ=-63± 10J/g) corresponding to decomposition.

¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.83 (s, 1 H), 6.79 (s, 1 H), 5.96 (d, J = 1 .1 Hz, 2H), 5.95 (d, J = 1 .1 Hz, 2H), 5.12 (s, 1 H), 4.81 (d, J = 9.1 Hz, 2H), 4.37 (s, 2H), 3.91 (d, J = 9.1 Hz, 1 H), 3.83 (s, 3H), 3.62 (ddd, J = 14.7, 12.1 , 8.3 Hz, 1 H), 3.56 - 3.45 (m, 1 H), 3.18 (dd, J = 12.3, 8.3 Hz, 1 H), 2.55 (dd, J = 14.8, 6.8 Hz, 1 H), 2.25 - 2.07 (m, 3H), 2.06 - 1 .89 (m, 1 H).

Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR APT* (101 MHz, Methanol-d4) δ 176.85, 169.05, 149.39, 148.67, 131 .56, 127.78, 1 14.36, 1 1 1 .82, 102.69, 92.97, 78.65, 74.10, 73.83, 58.48, 55.31 , 53.88, 48.75, 40.35, 29.24, 19.77.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^-1 3386, 2935, 1751 , 1650, 1607, 1502, 1486, 1454, 1421 , 1393, 1371 , 1344, 1324, 1278, 1236, 1216, 1 163, 1089, 1069, 1032, 972, 946, 929, 892, 860, 842, 812, 799, 785, 752, 718, 663, 622, 593, 563, 498, 457. See figure 3.9

Optical rotation [a] ²⁵ _D -197 ± 4 (c 0.81 , H ₂ O).

X-ray crystallographic studies

A. Single crystal X-ray diffraction (see figure 1 .1 1 .a and 1 .1 1 .b)

From a suspension in its mother liquor, a suitable single crystal of size 0.48 x 0.44 x 0.21 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula -?V Oio

Extended formula

Formula weight 465.45

Temperature 150(2) K

Wavelength 0.71073 ^° A Crystal system, space group monoclinic, P

Unit cell dimensions a = 8.0461 (4) A, a = 90 ^°

b = 15.9064(8) X, β = 115.223(2) ^° c = 8.89: 5(5) X, γ = 90 ^°

Volume 1030.29(9) X ³

Z, Calculated density 2 , 1.5 (g. cm.- ¹ )

Absorption coefficient 0.119 mm ^"1

F(000) 492

Crystal size 0.48 x 0.44 x 0.21 mm

Crystal color colourless

Theta range for data collection 3.08 to 27.48 ^°

h_min, h_max - 10 , 9

k_min, k_max -20 , 17

l_min, l_max -11 , 11

Reflections collected / unique 8870 / 2443 [ ^a R(int) = 0.0307] Reflections [I > 2σ] 2320

Completeness to theta_max 0.997

Absorption correction type multi-scan

Max. and min. transmission 0.975 , 0.899

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 2443 / 1 / 314

f ^t Goodness-of-fit 1.069

Final R indices [I > 2σ] ^c Ri = 0.0293, ^d wR ₂ = 0.0733

R indices (all data) ^c Ri = 0.0313, ^d wR ₂ = 0.0745 Largest cliff. peak and hole 0.21 and -0.178 e.X ^-3

para meters (A ² x 10 ³ ). U(eq) is defined as one of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI 0.5882(3) 0.00953(12) 0.0451(2) 1 0.0166(4)

HI 0.5912 -0.0324 -0.0298 1 0.02

C2 0.4766(3) 0.00863(12) 0.1213(2) 1 0.0166(4)

C3 0.5046(2) 0.07986(12) 0.2395(2) 1 0.0152(4)

H3 0.3947 0.1176 0.1934 1 0.018

C4 0.6742(2) 0.12805(12) 0.2385(2) 1 0.0140(3)

H4 0.6323 0.1868 0.2022 1 0.017

C5 0.7087(2) 0.08653(11) 0.0951(2) 1 0.0139(4)

C6 0.6727(3) 0.14624(13) -0.0514(2) 1 0.0200(4) H6A 0.5393 0.1545 -0.1 187 1 0.024

H6B 0.7311 0.2016 -0.01 17 1 0.024

C7 0.7601(3) 0.10137(15) -0.1518(3) 1 0.0231(4)

H7A 0.6777 0.0571 -0.2233 1 0.028

H7B 0.7889 0.1416 -0.2223 1 0.028

C8 0.9354(3) 0.06310(14) -0.0191(2) 1 0.0194(4)

H8A 0.9508 0.0044 -0.0479 1 0.023

H8B 1.0443 0.096 -0.0083 1 0.023

N9 0.9121(2) 0.06605(10) 0.14237(19) 1 0.0144(3)

H9 0.979(5) 0.1 1 1(2) 0.201(4) 1 0.05

CIO 0.9884(3) -0.01042(13) 0.2463(2) 1 0.0185(4)

H10A 1.1235 -0.01 1 0.2857 1 0.022

H10B 0.9384 -0.061 1 0.177 1 0.022

Cl l 0.9427(3) -0.01392(12) 0.3955(2) 1 0.0177(4)

H11A 0.8152 -0.0346 0.3581 1 0.021

HUB 1.0252 -0.0552 0.4761 1 0.021

C12 0.9601(2) 0.06880(12) 0.4831(2) 1 0.0165(4)

C13 0.8351(2) 0.13463(12) 0.4085(2) 1 0.0149(4)

C14 0.8500(3) 0.21078(13) 0.4943(2) 1 0.0167(4)

H14 0.7661 0.2556 0.4455 1 0.02

C15 0.9904(3) 0.21794(13) 0.6514(2) 1 0.0180(4)

C16 1.1 147(2) 0.15401(13) 0.7225(2) 1 0.0185(4)

C17 1.1042(3) 0.07941(13) 0.6428(2) 1 0.0190(4)

H17 1.1913 0.036 0.6936 1 0.023

C18 1.2023(3) 0.26475(16) 0.8912(3) 1 0.0304(5)

H18A 1.2974 0.2987 0.8765 1 0.036

H18B 1.2055 0.2782 1.001 1 0.036

C19 0.3124(3) -0.1 1380(14) -0.0028(3) 1 0.0257(4)

H19A 0.2797 -0.0937 -0.1 16 1 0.039

H19B 0.2134 -0.1493 -0.0018 1 0.039

H19C 0.4263 -0.1465 0.0356 1 0.039

01 0.33858(18) -0.04353(9) 0.10499(19) 1 0.0215(3)

02 0.52678(19) 0.04971(10) 0.39762(16) 1 0.0189(3)

H02 0.495(5) 0.088(2) 0.437(4) 1 0.05

03 1.23641(19) 0.17734(10) 0.88165(17) 1 0.0246(3)

04 1.0245(2) 0.28351(10) 0.76200(18) 1 0.0263(3) C21 1.2684(3) 0.22316(13) 0.4100(2) 1 0.0192(4)

C22 1.2562(3) 0.31136(12) 0.4758(2) 1 0.0184(4)

H22 1.1347 0.3164 0.4808 1 0.022

C23 1.2674(2) 0.38055(13) 0.3593(2) 1 0.0161(4)

H23 1.2526 0.4364 0.4037 1 0.019

C24 1.4560(3) 0.37806(13) 0.3557(2) 1 0.0172(4)

021 1.3994(2) 0.17909(10) 0.50324(19) 1 0.0281(3)

022 1.1463(2) 0.20408(9) 0.26781(18) 1 0.0256(3)

023 1.3955(2) 0.32283(11) 0.63745(18) 1 0.0285(4)

H023 1.454(4) 0.280(3) 0.648(4) 1 0.05

024 1.1258(2) 0.37101(11) 0.19783(19) 1 0.0266(3)

H024 1.111(4) 0.316(3) 0.191(4) 1 0.05

025 1.4864(2) 0.33636(10) 0.25670(19) 1 0.0250(3)

026 1.5882(2) 0.42363(11) 0.4688(2) 1 0.0270(4)

H026 1.556(4) 0.447(2) 0.526(5) 1 0.05

X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.089° (2Θ) at 19.928 °(2 Θ). There are additional diffraction peaks, for example at 14.770° (2 Θ), 17.970° (2 Θ), 19.420° (2 Θ), which can be readily attributed to impurities, without change of diffraction lines corresponding to these of calculated from single-crystal data (red) (for view of diagrams and experimental details, see figure 1 .1 1 .c).

Example 10: Preparation and analyses of (3S,4S,5R,2'R,3'R)- cephalotaxine hydrogen tartrate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial (2R,3R)-tartaric acid according to the above general procedure included in example 1 , then isolated as large white prisms mp -°C measured by DSC, see figure 2.). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.9)

DSC curve (see figure 2.9) indicated two transition points:

(i) one endothermic transition corresponding to melting point 199 (ΔΗ=24± 5J/g)

(ii) one exothermic transition at 223°C (ΔΗ=-50± 10J/g) corresponding to decomposition. ¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.82 (s, 1 H), 6.78 (s, 1 H), 5.98 - 5.95 (m, 1 H), 5.94 (s, 1 H), 5.1 1 (s, 1 H), 4.80 (d, J = 9.0 Hz, 1 H), 4.38 (s, 2H), 3.91 (d, J = 9.0 Hz, 1 H), 3.82 (s, 3H), 3.68 - 3.55 (m, 1 H), 3.50 (d, J = 9.6 Hz, 1 H), 3.22 - 3.10 (m, 1 H), 2.54 (dd, J = 14.8, 6.9 Hz, 1 H), 2.16 (s, 3H), 2.03 - 1 .88 (m, 1 H).

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR APT* (101 MHz, Methanol-d4) δ 176.50, 168.95, 149.32, 148.61 , 131 .56, 127.78, 1 14.34, 1 1 1 .81 , 102.67, 93.05, 78.60, 74.33, 73.95, 73.85, 73.80, 58.49, 55.28, 53.85, 52.60, 48.70, 40.34, 29.23, 19.77.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ¹ 3378, 2884, 2589, 1721 , 1654, 1616, 1507, 1489, 1460, 1442, 1368, 1329, 1275, 1219, 1 1 14, 1095, 1080, 1064, 1055, 1033, 969, 938, 929, 894, 856, 836, 800, 677, 600, 565, 533, 501 , 471 .See figure 3.10 Optical rotation [a] ²⁵ _D -141 ± 4 (c 0.74, H ₂ O). X-ray crystallographic studies

Single crystal X-ray diffraction (see figure 1 .12. a and 1 .12.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.57 x 0.48 x 0.39 mm was finally selected and implemented on the diffractometer.

Structural data Empirical formula C22¾7-?V Oio

Extended formula Ci8¾2-? On, C^HsOe

Formula weight 465.45

Temperature 150(2) K

Wavelength 0.71073 A

Crystal system, space group monoclinic, P

Unit cell dimensions a = 8.3302(3) A, a 90

b = 17.2827(7) ^° A, β = 118.537(2) 8.6775(4) X, γ = 90 ^°

Volume 1097.51(8) X ³

Z, Calculated density 2 , 1.408 (g. cm ^{' 1} )

Absorption coefficient 0.112 mm ^"1

F(000) 492

Crystal size 0.57 x 0.48 x 0.39 mm

Crystal color colourless

Theta range for data collection 2.92 to 27.48 ^°

h_min, h_max -8 , 10

k_min, k_max -20 , 22

l_min, l_max -11 , 11

Reflections collected / unique 9621 / 2595 [ ^a R(int) = 0.0397]

Reflections [I > 2σ] 2410

Completeness to theta_max 0.998

Absorption correction type multi-scan

Max. and min. transmission 0.957 , 0.900

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 2595 / 2 / 314

f ^t Goodness-of-fit 1.035

Final R indices [I > 2σ] ^c Ri = 0.0367, ^d wR ₂ = 0.0871

R indices (all data) ^c Ri = 0.040 L ^d wR ₂ = 0.0892 Larg peak and hole 0.198 and -0.214 e.X ^"3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom y occ. U(eq)

CI 0.1140(3) 0.42383(14) 0.5508(3) 1 0.0186(4) HI 0.1932 0.4604 0.6342 1 0.022

C2 -0.0675(3) 0.42485(13) 0.4788(3) 1 0.0185(4) C3 -0.1631(3) 0.36277(13) 0.3447(3) 1 0.0172(4)

H3 -0.2282 0.3274 0.3877 1 0.021

C4 -0.0028(3) 0.31783(13) 0.3402(3) 1 0.0164(4)

H4 -0.0042 0.265 0.3864 1 0.02

C5 0.1761(3) 0.35731(14) 0.4826(3) 1 0.0182(4)

C6 0.3016(3) 0.30123(16) 0.6274(3) 1 0.0250(5)

H6A 0.2546 0.2915 0.711 1 0.03

H6B 0.311 0.2513 0.5764 1 0.03

C7 0.4875(3) 0.3416(2) 0.7189(4) 1 0.0372(7)

H7A 0.5013 0.3707 0.8229 1 0.045

H7B 0.5878 0.3033 0.7572 1 0.045

C8 0.4906(3) 0.39631(18) 0.5834(3) 1 0.0294(6)

H8A 0.5019 0.4506 0.6235 1 0.035

H8B 0.5943 0.3842 0.5619 1 0.035

N9 0.3102(2) 0.38368(12) 0.4187(2) 1 0.0202(4)

H9 0.331(5) 0.345(2) 0.369(5) 1 0.05

CIO 0.2587(3) 0.45094(14) 0.2969(3) 1 0.0253(5)

H10A 0.345 0.4545 0.2489 1 0.03

H10B 0.2703 0.499 0.3635 1 0.03

Cl l 0.0631(4) 0.44499(14) 0.1444(3) 1 0.0244(5)

H11A -0.0233 0.4611 0.1864 1 0.029

HUB 0.0495 0.4814 0.0509 1 0.029

C12 0.0123(3) 0.36496(13) 0.0674(3) 1 0.0193(4)

C13 -0.0189(3) 0.30534(13) 0.1600(3) 1 0.0172(4)

C14 -0.0671(3) 0.23105(13) 0.0874(3) 1 0.0188(4)

H14 -0.0875 0.1902 0.1494 1 0.023

C15 -0.0839(3) 0.21917(14) -0.0762(3) 1 0.0206(5)

C16 -0.0497(3) 0.27718(15) -0.1647(3) 1 0.0219(5)

C17 -0.0006(3) 0.34992(15) -0.0984(3) 1 0.0225(5)

H17 0.024 0.3891 -0.1611 1 0.027

C18 -0.1419(5) 0.17298(18) -0.3374(3) 1 0.0402(7)

H18A -0.0707 0.1361 -0.3682 1 0.048

H18B -0.2708 0.1712 -0.4311 1 0.048

C19 -0.0953(4) 0.53615(15) 0.6216(4) 1 0.0317(6)

H19A 0.0093 0.519 0.7312 1 0.048

H19B -0.1843 0.562 0.6477 1 0.048 H19C -0.0536 0.5724 0.5613 1 0.048

01 -0.1790(2) 0.47070(11) 0.5115(2) 1 0.0240(4)

H02 -0.394(5) 0.370(2) 0.143(5) 1 0.05

02 -0.2930(2) 0.39713(10) 0.1831(2) 1 0.0215(3)

03 -0.0714(3) 0.24888(11) -0.3240(2) 1 0.0329(4)

04 -0.1306(3) 0.15178(10) -0.1742(2) 1 0.0325(4)

C21 0.4178(3) 0.24908(14) 0.1684(3) 1 0.0232(5)

C22 0.3770(3) 0.16558(14) 0.0991(3) 1 0.0249(5)

H22 0.2624 0.165 -0.0163 1 0.03

C23 0.5353(3) 0.13674(14) 0.0731(3) 1 0.0198(4)

H23 0.5479 0.171 -0.0134 1 0.024

C24 0.5020(4) 0.05427(15) 0.0042(3) 1 0.0246(5)

021 0.4259(3) 0.25941(12) 0.3162(2) 1 0.0350(5)

022 0.4396(2) 0.29748(10) 0.0751(2) 1 0.0300(4)

023 0.3547(4) 0.11656(12) 0.2172(3) 1 0.0476(6)

H023 0.368(5) 0.145(2) 0.290(5) 1 0.05

024 0.7005(2) 0.13932(11) 0.2352(2) 1 0.0258(4)

H024 0.716(5) 0.0948(13) 0.272(5) 1 0.05

025 0.3535(3) 0.04586(12) -0.1453(2) 1 0.0353(5)

H025 0.335(5) -0.002(2) -0.172(5) 1 0.05

026 0.6071(3) 0.00273(12) 0.0829(3) 1 0.0472(6)

Example 11 : Preparation and analyses of (3S,4S,5RM-)-cephalotaxine hydrogen succinate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial succinic acid according to the above general procedure included in example 1 , then isolated as large white prisms mp 226-229°C measured by DSC, see figure 2.8). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.10)

DSC curve (see figure 2.10) indicated one endothermic transition corresponding to melting point 226-229°C (ΔΗ=135± 20J/g)

¹ H NMR (400 MHz, Methanol-ck)* δ 6.82 (s, 1 H), 6.78 (s, 1 H), 5.96 (d, J = 1 .1 Hz, 1 H), 5.94 (d, J = 1 .2 Hz, 1 H), 4.81 (d, J = 9.0 Hz, 1 H), 3.90 (d, J = 9.1 Hz, 1 H), 3.61 (ddd, J = 14.7, 12.2, 8.3 Hz, 1 H), 3.52 - 3.43 (m, 1 H), 3.30 - 3.23 (m, 2H), 3.15 (dd, J = 12.4, 7.6 Hz, 1 H), 2.54 (m, 1 H), 2.50 (s,4H), 2.20 - 2.06 (m, 3H), 2.01 - 1 .88 (m, 1 H).

Partial presuppression of water signal using "watergate" irradiation

NMR APT* (101 MHz, D,Q) δ 179.20, 168.81 , 149.28, 148.57, 131 .72, 127.93, 1 14.35, 1 1 1 .79, 102.65, 93.17, 78.36, 73.84, 58.43, 55.43, 53.86, 48.74, 40.48, 32.84, 29.36, 19.80.

*APT = Attached Proton Test

IR (KBr, solid), cm ¹ 3229, 2958, 1988, 1691 , 1650, 1566, 1502, 1487, 1463, 1436, 1373, 1350, 1257, 1216, 1 151 , 1 121 , 1 101 , 1077, 1065, 1054, 1032, 970, 927, 903, 890, 835, 801 , 740, 710, 673, 640, 591 , 563, 506, 476, 458. See figure 3.8

Optical rotation [a] ²⁵ _D -205 ± 4 (c 1 .15, H ₂ O).

X-ray crystallographic studies

Single crystal X-ray diffraction (see figure 1 .5)

From a suspension in its mother liquor, a suitable single crystal of size 0.58 x 0.53 x 0.27 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C22H2iN OB

Extended formula C18H22N O4, CnH^On

Formula weight 433.45

Temperature 150(2) K

Wavelength 0.71073 A Crystal system, space group monoclinic, P 2

Unit cell dimensions a = 9.8102(8) A, a = 90

b = 8.6029(5) A, jS = 97.603(3) c = 12.1177(11) A, γ = 90 °

Volume 1013.70(14) A ³

Z, Calculated density 2 , 1.42 (g.cm ^{" 1} )

Absorption coefficient 0.108 mm ^"1

F(000) 460

Crystal size 0.58 x 0.53 x 0.27 mm

Crystal color colourless

Theta range for data collection 2.91 to 27.48 °

h_min, h_max -12 , 12

k_min, k_max -10 , 11

l_min, l_max -15 , 12

Reflections collected / unique 8818 / 2444 [ ^a R(int) = 0.0307]

Reflections [7 > 2σ] 2189

Completeness to theta_max 0.985

Absorption correction type multi-scan

Max. and min. transmission 0.971 , 0.881

Refinement method Full-matrix least-squares on F ²

Data / restraints / parameters 2444 / 1 / 291

^Goodness-of-fit 1.04

Final R indices [I > 2σ] ^c Ri = 0.0486, ^d wR ₂ = 0.1052

R indices (all data) ^c Ri = 0.0552, ^d wR ₂ = 0.1092 Largest diff. peak and hole 0.561 and -0.42 e.A ^-3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI -0.0814(4) 0.6206(4) 0.8127(3) 1 0.0314(7)

HI -0.1663 0.6639 0.8272 1 0.038

C2 -0.0619(3) 0.4708(4) 0.7935(2) 1 0.0246(6)

C3 0.0816(3) 0.4292(3) 0.7768(2) 1 0.0233(6)

H3 0.1237 0.3658 0.8417 1 0.028

C4 0.1560(3) 0.5909(4) 0.7798(2) 1 0.0260(6)

H4 0.2295 0.5882 0.8453 1 0.031

C5 0.0465(4) 0.7119(4) 0.8086(3) 1 0.0328(7) C6 0.0916(6) 0.8047(5) 0.9165(3) 1 0.0585(13)

H6A 0.0639 0.7489 0.9815 1 0.07

H6B 0.1927 0.8175 0.928 1 0.07

C7 0.0217(5) 0.9622(5) 0.9038(3) 1 0.0519(11)

H7A 0.0899 1.0466 0.9206 1 0.062

H7B -0.0485 0.9714 0.9551 1 0.062

C8 -0.0455(5) 0.9719(4) 0.7829(3) 1 0.0482(10)

H8A -0.146 0.9547 0.7775 1 0.058

H8B -0.0288 1.0747 0.7506 1 0.058

N9 0.0216(3) 0.8450(3) 0.7239(2) 1 0.0324(6)

H9 0.103(5) 0.879(5) 0.713(4) 1 0.05

CIO -0.0581(3) 0.8056(4) 0.6142(3) 1 0.0304(7)

H10A -0.0572 0.896 0.5637 1 0.037

H10B -0.1549 0.7851 0.6245 1 0.037

Cl l -0.0010(3) 0.6645(4) 0.5601(2) 1 0.0259(6)

H11A -0.0345 0.5689 0.5933 1 0.031

HUB -0.036 0.6641 0.4796 1 0.031

C12 0.1541(3) 0.6628(4) 0.5747(2) 1 0.0259(6)

C13 0.2277(3) 0.6270(4) 0.6788(3) 1 0.0260(6)

C14 0.3719(3) 0.6260(4) 0.6926(3) 1 0.0348(8)

H14 0.4231 0.6006 0.7623 1 0.042

C15 0.4369(3) 0.6627(5) 0.6027(3) 1 0.0375(8)

C16 0.3636(4) 0.6982(5) 0.5004(3) 1 0.0393(9)

C17 0.2229(3) 0.7005(4) 0.4836(3) 1 0.0314(7)

H17 0.1736 0.7266 0.4132 1 0.038

C18 0.5862(4) 0.7088(8) 0.4820(3) 1 0.0602(14)

H18A 0.6413 0.8044 0.4771 1 0.072

H18B 0.6327 0.6225 0.4478 1 0.072

C19 -0.2903(3) 0.3952(4) 0.8025(3) 1 0.0358(8)

H19A -0.3228 0.4708 0.7445 1 0.054

H19B -0.2935 0.4418 0.8759 1 0.054

H19C -0.3493 0.3029 0.7944 1 0.054

01 -0.1510(2) 0.3512(3) 0.79193(18) 1 0.0277(5)

02 0.0832(2) 0.3417(3) 0.67846(17) 1 0.0249(4)

H02 0.165(4) 0.296(6) 0.695(4) 1 0.05

03 0.4526(3) 0.7303(5) 0.4243(2) 1 0.0626(10) 04 0.5763(3) 0.6736(4) 0.5965(2) 1 0.0559(9)

C21 0.3306(4) 1.0933(4) 0.7768(3) 1 0.0395(9)

C22A 0.3912(5) 0.9841(7) 0.8622(5) 0.656(6) 0.0349(8)

H22A 0.3611 0.8771 0.8412 0.656(6) 0.042

H22B 0.3592 1.0094 0.9342 0.656(6) 0.042

C23A 0.5496(6) 0.9936(8) 0.8745(5) 0.656(6) 0.0349(8)

H23A 0.5798 1.1012 0.8938 0.656(6) 0.042

H23B 0.582 0.9656 0.8031 0.656(6) 0.042

C22B 0.4560(11) 1.0517(14) 0.8880(9) 0.344(6) 0.0349(8)

H22C 0.5207 1.1403 0.8998 0.344(6) 0.042

H22D 0.4118 1.039 0.9563 0.344(6) 0.042

C23B 0.5330(11) 0.9100(15) 0.8693(9) 0.344(6) 0.0349(8)

H23C 0.4692 0.823 0.8471 0.344(6) 0.042

H23D 0.5915 0.927 0.8098 0.344(6) 0.042

C24 0.6142(4) 0.8767(6) 0.9703(3) 1 0.0502(12)

021 0.3473(3) 1.2353(3) 0.7722(2) 1 0.0386(6)

022 0.2440(5) 1.0258(5) 0.7071(3) 1 0.0865(13)

023 0.5534(3) 0.8895(5) 1.0607(2) 1 0.0722(12)

H023 0.586(4) 0.823(6) 1.117(4) 1 0.05

024 0.7171(3) 0.8031(4) 0.9674(2) 1 0.0527(8)

Example 12: Preparation and analyses of (3S,4S,5R)-cephalotaxine hydrogen itaconate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial itaconic acid according to the above general procedure included in example 1 , then isolated as large white prisms mp 222-229°C measured by DSC, see figure 2.9). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.11)

DSC curve indicated endotermic transition coresponding to melting point 222- 228.5 (AH=127 ± 20J/g) ¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.82 (s, 1 H), 6.78 (s, 1 H), 5.99 (d, J = 1 .7 Hz, 1 H), 5.96 (d, J = 1 .1 Hz, 1 H), 5.94 (d, J = 1 .1 Hz, 1 H), 5.49 (q, J = 1 .2 Hz, 1 H), 5.1 1 (s, 1 H), 4.81 (d, J = 9.0 Hz, 1 H), 3.90 (d, J = 9.1 Hz, 1 H), 3.82 (s, 3H), 3.61 (ddd, J = 14.8, 12.1 , 8.3 Hz, 1 H), 3.53 - 3.42 (m, 1 H), 3.29 - 3.24 (m, 3H), 3.15 (dd, J = 12.5, 8.1 Hz, 1 H), 2.54 (dd, J = 14.8, 6.8 Hz, 1 H), 2.20 - 2.06 (m, 3H), 2.02 - 1 .87 (m, 1 H).

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR (101 MHz, MeOD) ^* δ 176.97, 173.90, 168.90, 149.31 , 148.60, 139.97, 131 .65, 127.87, 125.00, 1 14.34, 1 1 1 .79, 102.65, 93.08, 78.45, 73.83, 58.45, 55.38, 53.86, 48.75, 42.33, 40.44, 29.31 , 19.79.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^"1 3350, 2969, 2870, 1645, 1585, 1508, 1488, 1463, 1446, 1416, 1387, 1363, 1276, 1253, 1216, 1 162, 1 1 17, 1 100, 1081 , 1059, 1031 , 969, 938, 924, 891 , 848, 835, 825, 803, 773, 708, 673, 601 , 565, 537, 504, 465. See figure 3.10b

Optical rotation Γα1 ²⁵ η -178 ± 4 (c 1 .14, H _? O).

X-ray crystallographic studies

A. Single crystal X-ray diffraction (see figure 1 .6. a and 1 .6.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.5 x 0.45 x 0.32 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula

Formula weight 445.46

Temperature 150(2) K

Crystal system, space group orthorhombic, P 2i 2i 2i Unit cell dimensions a = 7.9590(6) X, a = 90 ^° b =

1 1.8849(9) X, jS = 90 ^° c = 22.2676(14) X, γ 90

Volume 2106.3(3) X ³

Z, Calculated density 4 , 1.405 {g. cmT ¹ )

Absorption coefficient 0.107 mm ^"1

F(000) 944

Crystal size 0.5 x 0.45 x 0.32 mm

Crystal color colourless

Theta range for data collection 3.08 to 27.46 ^°

h_min, h_max -7 , 10

k_min, k_max -15 , 14

l_min, l_max -23 , 28

Reflections collected / unique 10261 / 2750 [ ^a R(int) = 0.044]

Reflections [I > 2σ] 2494

Completeness to theta_max 0.995

Absorption correction type multi-scan

Max. and min. transmission 0.966 , 0.792

Refinement method Full-matrix least-squares on F ²

Data / restraints / parameters 2750 / 0 / 299

"Goodness-of-fit 1.087

Final R indices [I > 2σ] ^c Ri = 0.0444, ^d wR ₂ = 0.1072

R indices (all data) ^c Ri = 0.0498, ^d wR ₂ = 0.1098 Largest diff. peak and hole 0.273 and -0.249 e.X ^"3 Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI 1.0487(3) 0.8806(2) 0.93357(11) 1 0.0207(5)

HI 1.1505 0.9114 0.9486 1 0.025

C2 0.8982(4) 0.8924(2) 0.95977(11) 1 0.0213(5)

C3 0.7538(3) 0.8392(2) 0.92631(11) 1 0.0196(5)

H3 0.7 0.7807 0.9523 1 0.023

C4 0.8438(3) 0.7807(2) 0.87232(11) 1 0.0181(5)

H4 0.8375 0.6982 0.8808 1 0.022

C5 1.0340(3) 0.8121(2) 0.87735(11) 1 0.0178(5) C6 1.1485(3) 0.7083(2) 0.87513(12) 1 0.0227(6)

H6A 1.1485 0.6688 0.9143 1 0.027

H6B 1.1106 0.6553 0.8436 1 0.027

C7 1.3239(4) 0.7540(3) 0.86060(18) 1 0.0388(8)

H7A 1.3899 0.765 0.8978 1 0.047

H7B 1.3854 0.7015 0.834 1 0.047

C8 1.2941(4) 0.8662(3) 0.82904(14) 1 0.0273(6)

H8A 1.3389 0.9293 0.8533 1 0.033

H8B 1.3488 0.8671 0.7891 1 0.033

N9 1.1050(3) 0.87514(18) 0.82288(9) 1 0.0184(5)

H9 1.077(5) 0.833(3) 0.7854(16) 1 0.05

CIO 1.0469(4) 0.9926(2) 0.81197(12) 1 0.0208(5)

H10A 1.0958 1.0202 0.7738 1 0.025

H10B 1.0881 1.0416 0.8448 1 0.025

Cl l 0.8545(4) 1.0010(2) 0.80856(12) 1 0.0209(5)

H11A 0.8096 1.0109 0.8497 1 0.025

HUB 0.8242 1.0688 0.7851 1 0.025

C12 0.7708(3) 0.8995(2) 0.78021(11) 1 0.0189(5)

C13 0.7673(3) 0.7960(2) 0.81069(11) 1 0.0169(5)

C14 0.6975(3) 0.6998(2) 0.78326(11) 1 0.0175(5)

H14 0.6973 0.629 0.803 1 0.021

C15 0.6298(3) 0.7122(2) 0.72697(11) 1 0.0188(5)

C16 0.6300(3) 0.8140(2) 0.69740(11) 1 0.0203(5)

C17 0.6996(4) 0.9087(2) 0.72263(12) 1 0.0225(6)

H17 0.6996 0.9784 0.7017 1 0.027

C18 0.4869(4) 0.6918(2) 0.64159(12) 1 0.0268(6)

H18A 0.364 0.6983 0.6474 1 0.032

H18B 0.5082 0.6525 0.6031 1 0.032

C19 0.9923(4) 0.9883(3) 1.04567(13) 1 0.0350(7)

H19A 1.0469 1.0477 1.0222 1 0.053

H19B 1.0738 0.9287 1.0545 1 0.053

H19C 0.95 1.0199 1.0834 1 0.053

01 0.8551(3) 0.94231(17) 1.01196(8) 1 0.0278(5)

02 0.6303(3) 0.91864(18) 0.90782(9) 1 0.0280(5)

H02 0.579(5) 0.933(4) 0.9390(17) 1 0.05

03 0.5632(3) 0.80206(17) 0.64085(8) 1 0.0303(5) 04 0.5612(3) 0.63074(16) 0.69053(8) 1 0.0262(4)

C21 1.0799(4) 0.8329(2) 0.66808(12) 1 0.0247(6)

C22 1.0093(4) 0.7890(3) 0.60870(12) 1 0.0287(6)

H22A 1.0045 0.7059 0.6111 1 0.034

H22B 0.8924 0.8165 0.6048 1 0.034

C23 1.1031(3) 0.8205(2) 0.55185(11) 1 0.0201(5)

C24 1.0934(3) 0.9415(2) 0.52980(12) 1 0.0212(5)

C25 1.1874(4) 0.7455(3) 0.51974(13) 1 0.0295(6)

H25A 1.1905 0.669 0.5321 1 0.035

H25B 1.2448 0.7681 0.4843 1 0.035

021 1.1264(3) 0.93677(18) 0.66962(9) 1 0.0333(5)

022 1.0860(3) 0.77060(17) 0.71190(8) 1 0.0326(5)

023 1.0830(3) 1.01705(16) 0.57062(9) 1 0.0340(5)

H23 1.101(5) 0.987(3) 0.6182(16) 1 0.051

024 1.0976(3) 0.96199(17) 0.47602(8) 1 0.0306(5)

B. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.077° (2Θ) at 16.795° (2 Θ). The sample is without impurity, as shown by the good agreement between calculated (red) and experimental (black) patterns (for view of diagrams and experimental details, see figure 1 .6.c).

Example 13: Preparation and analyses of (3S,4S,5R)-cephalotaxine hydrogen fumarate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial fumaric acid according to the above general procedure included in example 1, then isolated as large white prisms mp -°C measured by DSC, see figure 2.12). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.12)

DSC curve indicated two transition points:

(i) one endothermic transition coresponding to melting point 244.5-249.5 (ΔΗ=33± 5J/g)

(ii) one exothermic transition at 55.5°C (ΔΗ=-10± 5J/g)

corresponding to decomposition.

¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.81 (s, 1H), 6.77 (s, 1H), 6.66 (s, 2H), 5.95 (d, J = 1.1 Hz, 1H), 5.93 (d, J = 1.1 Hz, 1H), 5.10 (s, 1H), 4.79 (d, J = 9.0 Hz, 1H), 3.90 (d, J = 9.0 Hz, 1H), 3.81 (s, 3H), 3.66 - 3.54 (m, 1H), 3.47 (s, 1H), 3.21 -3.13(m, 1H), 2.53 (dd, J = 14.9,6.8 Hz, 1H), 2.15 (s, 3H), 1.93 (d, J= 6.8 Hz, 1H).

*Partial presuppression of water signal using "watergate" irradiation ¹³ C NMR APT* (101 MHz, Methanol-d4) δ 171.39, 169.09, 149.39, 136.21, 131.54, 127.77, 114.37, 111.83, 102.69, 92.90, 78.63, 73.82, 58.47, 55.30, 53.87, 40.36, 29.23, 19.75.

*APT = Attached Proton Test IR (Diamond ATR, solid) cm ¹ 3224, 2984, 2844, 2724, 2352, 1926, 1688, 1646, 1566, 1503, 1485, 1464, 1381, 1370, 1351, 1300, 1249, 1216, 1191, 1166, 1120, 1107, 1067, 1054, 1032, 973, 934, 924, 889, 833, 795, 739, 678, 643, 592, 564, 505, 475. See figure 3.11

Optical rotation [a] ²⁵ _D -192 ± 4 (c 0.97, H ₂ O).

X-ray crystallographic studies

Single crystal X-ray diffraction (see figure 1.9. a and 1.9.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.52 x 0.12 x 0.06 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C iHsN Oa Extended formula C18H22N O4, C4H3 O4

Formula weight 431.43

Temperature 150(2) K

Wavelength 0.71073 A

Crystal system, space group monoclinic, P

Unit cell dimensions a = 9.7627(3) A, a 90

b = 8.6149(3) X, jS = 98.3340(10) 12.0869(5) X, γ = 90 ^°

Volume 1005.83(6) X ³

Z, Calculated density 2 , 1.425 (g. cm ^{' 1} )

Absorption coefficient 0.109 mm ^"1

F(000) 456

Crystal size 0.52 x 0.12 x 0.06 mm

Crystal color colourless

Theta range for data collection 2.91 to 27.48 ^°

h_min, h_max - 12 , 10

k_min, k_max -11 , 10

l_min, l_max -15 , 15

Reflections collected / unique 8945 / 2454 [ ^a R(int) = 0.0356]

Reflections [I > 2σ] 2212

Completeness to theta_max 0.998

Absorption correction type multi-scan

Max. and min. transmission 0.993 , 0.888

Refinement method Full- matrix least-squares on F'

Data / restraints / parameters 2454 / 1 / 290

f ^t Goodness-of-fit 1.059

Final R indices [I > 2σ] ^c Ri = 0.0353, ^d wR ₂ = 0.079

R indices (all data) ^c Ri = 0.0411 , ^d wR ₂ = 0.0827

Largest diff. peak and hole 0.219 and ^" 0.2 e.X ^"3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI 0.5813(2) 1.0051(3) 0.68559(18) 1 0.0193(5)

HI 0.6659 1.0492 0.6705 1 0.023

C2 0.5622(2) 0.8548(3) 0.70331(17) 1 0.0185(5)

C3 0.4177(2) 0.8124(3) 0.72127(18) 1 0.0174(5) H3 0.3735 0.7499 0.656 1 0.021

C4 0.3430(2) 0.9732(3) 0.72019(18) 1 0.0169(4)

H4 0.2685 0.9715 0.6541 1 0.02

C5 0.4521(2) 1.0954(3) 0.69251(17) 1 0.0177(4)

C6 0.3994(2) 1.1934(3) 0.58888(18) 1 0.0229(5)

H6A 0.3 1.2179 0.5868 1 0.028

H6B 0.4114 1.1365 0.5198 1 0.028

C7 0.4860(3) 1.3429(3) 0.5988(2) 1 0.0310(6)

H7A 0.562 1.3359 0.5529 1 0.037

H7B 0.4276 1.4338 0.5737 1 0.037

C8 0.5440(3) 1.3566(3) 0.7230(2) 1 0.0266(5)

H8A 0.646 1.3463 0.7345 1 0.032

H8B 0.5193 1.4582 0.753 1 0.032

N9 0.47869(19) 1.2258(2) 0.78001(15) 1 0.0191(4)

H9 0.387(3) 1.264(4) 0.793(3) 1 0.05

CIO 0.5622(2) 1.1826(3) 0.88930(19) 1 0.0211(5)

H10A 0.5643 1.2715 0.9414 1 0.025

H10B 0.6585 1.1607 0.8775 1 0.025

Cl l 0.5041(2) 1.0414(3) 0.94188(17) 1 0.0183(5)

H11A 0.5365 0.9462 0.9078 1 0.022

HUB 0.5399 1.0392 1.0228 1 0.022

C12 0.3470(2) 1.0416(3) 0.92637(18) 1 0.0180(5)

C13 0.2715(2) 1.0087(3) 0.82109(18) 1 0.0176(5)

C14 0.1261(2) 1.0142(3) 0.80547(18) 1 0.0197(5)

H14 0.0736 0.9917 0.7348 1 0.024

C15 0.0626(2) 1.0529(3) 0.89548(19) 1 0.0236(5)

C16 0.1375(2) 1.0843(4) 0.99912(19) 1 0.0277(6)

C17 0.2794(2) 1.0813(3) 1.01715(18) 1 0.0226(5)

H17 0.33 1.1051 1.0883 1 0.027

C18 -0.0867(3) 1.1054(5) 1.0147(2) 1 0.0430(8)

H18A -0.1388 1.2031 1.0196 1 0.052

H18B -0.1365 1.021 1.0478 1 0.052

C19 0.7897(2) 0.7744(3) 0.6881(2) 1 0.0287(6)

H19A 0.8299 0.841 0.7502 1 0.043

H19B 0.7892 0.8301 0.6173 1 0.043

H19C 0.8452 0.6797 0.6875 1 0.043 01 0.64988(16) 0.7340(2) 0.70190(13) 1 0.0229(4)

02 0.41993(17) 0.7229(2) 0.81997(13) 1 0.0204(3)

H02 0.332(3) 0.682(5) 0.814(3) 1 0.05

03 -0.07737(17) 1.0706(3) 0.89987(14) 1 0.0404(6)

04 0.04902(18) 1.1208(4) 1.07429(15) 1 0.0496(7)

C21 0.1692(2) 1.4786(3) 0.7295(2) 1 0.0216(5)

C22 0.0849(2) 1.4046(3) 0.6288(2) 1 0.0234(5)

H22 0.1122 1.4242 0.5578 1 0.028

C23 -0.0235(3) 1.3151(3) 0.6317(2) 1 0.0264(5)

H23 -0.0515 1.2935 0.7022 1 0.032

C24 -0.1037(2) 1.2464(3) 0.5305(2) 1 0.0236(5)

021 0.16097(17) 1.6248(2) 0.73457(14) 1 0.0234(4)

022 0.24330(18) 1.3968(2) 0.79899(14) 1 0.0292(4)

023 -0.05277(17) 1.2763(2) 0.43690(14) 1 0.0259(4)

H023 -0.098(3) 1.219(5) 0.376(3) 1 0.05

024 -0.20603(19) 1.1669(3) 0.53407(15) 1 0.0366(5)

X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.105° (2Θ) at 19.372° (2Θ) The sample slightly differs from the structure obtained from single-crystal (calculated in red). There are visible variations in the angular position of some diffraction lines, together with the presence of additional peaks (for view of diagrams and experimental details, see figure 1 .9.c).

Example 14: Preparation and analyses of (3S,4S,5R)-cephalotaxine hydrogen maleate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial maleic acid according to the above general procedure included in example 1 , then isolated as large white prisms mp -°C measured by DSC, see figure 2.13). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.13)

DSC curve indicated endothermic transition corresponding to melting point 216- 219 (AH= 85 ± 20J/g)

¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.83 (s, 1 H), 6.79 (s, 1 H), 6.24 (s, 2H), 5.96 (d, J = 1 .1 Hz, 1 H), 5.95 (d, J = 1 .1 Hz, 1 H), 5.12 (s, 1 H), 4.82 (d, J = 9.0 Hz, 1 H), 3.92 (d, J = 9.1 Hz, 1 H), 3.83 (s, 3H), 3.69 - 3.56 (m, 1 H), 3.50 (d, J = 9.1 Hz, 1 H), 3.22 - 3.13 (m, 1 H), 2.54 (dd, J = 14.7, 7.2 Hz, 1 H), 2.17 (s, 3H), 1 .95 (s, 1 H).

Partial presuppression of water signal using "watergate" irradiation

^1j C NMR APT* (101 MHz, Methanol-d4) δ 170.72, 169.10, 149.37, 148.66, 136.71 , 131 .41 , 127.74, 1 14.40, 1 1 1 .87, 102.68 , 92.70, 78.59, 73.70, 58.48, 55.20, 53.85, 48.73, 40.45, 29.13, 19.73.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^"1 3393, 2973, 2867, 1645, 1586, 1505, 1485, 1369, 1349, 1303, 1280, 1255, 1215, 1 161 , 1 102, 1080, 1058, 1032, 997, 972, 934, 926, 861 , 849, 836, 803, 775, 701 , 682, 599, 566, 551 , 505, 468. See figure 3.12

Optical rotation [a] ²⁵ _D -192 ± 4 (c 1 .16, H ₂ O).

X-ray crystallographic studies

Single crystal X-ray diffraction (see figure 1 .14. a and 1 .14.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.58 x 0.4 x 0.29 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C ₂₂ ¾ ₅ i 08

Extended formula C18H22N O4, C4H3O4

Formula weight 431.43

Temperature 150(2) K Wavelength 0.71073 A

Crystal system, space group orthorhombic, P i 2i 2i

Unit cell dimensions a = 7.9879(4) X, a = 90 ^° b = 1 1.7505(4)

X, β = 90 ^° c = 22.0488(12) X, γ 90

Volume 2069.54(17) X ³

Z, Calculated density 4 , 1.385 (g. cm.- ¹ )

Absorption coefficient 0.106 mm ^"1

F(000) 912

Crystal size 0.58 x 0.4 x 0.29 mm

Crystal color colourless

Theta range for data collection 3.08 to 27.47 ^°

h_min, h_max - 10 , 10

k_min, k_max -8 , 15

l_min, l_max -23 , 28

Reflections collected / unique 9525 / 2691 [ ^a R(int) = 0.0375]

Reflections [I > 2σ] 2377

Completeness to theta_max 0.991

Absorption correction type multi-scan

Max. and min. transmission 0.970 , 0.883

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 2691 / 0 / 290

f ^t Goodness-of-fit 1.048

Final R indices [I > 2σ] ^c Ri = 0.042, ^d wR ₂ = 0.0976

R indices (all data) ^c Ri = 0.0493, ^d wR ₂ = 0.1017 Largest diff. peak and hole 0.267 and -0.281 e.X ^-3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI 0.4898(3) 0.1270(2) 0.93035(12) 1 0.0265(5)

HI 0.3893 0.0951 0.9459 1 0.032

C2 0.6411(3) 0.1121(2) 0.95451(11) 1 0.0254(5)

C3 0.7818(3) 0.1710(2) 0.92182(11) 1 0.0243(5)

H3 0.8324 0.2294 0.9492 1 0.029

C4 0.6906(3) 0.23201(19) 0.86875(11) 1 0.0219(5)

H4 0.6986 0.3152 0.8777 1 0.026 C5 0.5002(3) 0.2010(2) 0.87526(11) 1 0.0229(5)

C6 0.3870(3) 0.3062(2) 0.87602(13) 1 0.0293(5)

H6A 0.3901 0.3437 0.9162 1 0.035

H6B 0.4225 0.3616 0.8448 1 0.035

C7 0.2117(3) 0.2612(2) 0.86240(18) 1 0.0448(8)

H7A 0.1472 0.3173 0.8384 1 0.054

H7B 0.1504 0.2453 0.9005 1 0.054

C8 0.2377(3) 0.1517(2) 0.82615(14) 1 0.0338(6)

H8A 0.1908 0.0854 0.8481 1 0.041

H8B 0.1835 0.1571 0.7859 1 0.041

N9 0.4259(2) 0.14088(17) 0.81970(10) 1 0.0229(4)

H9 0.455(4) 0.182(3) 0.7815(15) 1 0.05

CIO 0.4803(3) 0.02146(19) 0.80756(12) 1 0.0250(5)

H10A 0.43 -0.0048 0.769 1 0.03

H10B 0.4387 -0.0285 0.8404 1 0.03

Cl l 0.6712(3) 0.01092(19) 0.80350(12) 1 0.0231(5)

H11A 0.7167 0.0006 0.8449 1 0.028

HUB 0.6992 -0.058 0.7798 1 0.028

C12 0.7555(3) 0.11232(19) 0.77457(11) 1 0.0214(5)

C13 0.7625(3) 0.21690(18) 0.80557(11) 1 0.0196(5)

C14 0.8324(3) 0.31381(19) 0.77780(11) 1 0.0217(5)

H14 0.8357 0.3851 0.7981 1 0.026

C15 0.8951(3) 0.30142(19) 0.72070(11) 1 0.0229(5)

C16 0.8918(3) 0.1988(2) 0.69058(11) 1 0.0266(5)

C17 0.8229(3) 0.1032(2) 0.71614(11) 1 0.0252(5)

H17 0.8209 0.0329 0.6948 1 0.03

C18 1.0234(4) 0.3245(2) 0.63190(13) 1 0.0393(7)

H18A 1.1473 0.3224 0.6324 1 0.047

H18B 0.987 0.3637 0.5944 1 0.047

C19 0.5611(5) -0.0040(3) 1.03595(15) 1 0.0533(9)

H19A 0.4792 0.0514 1.051 1 0.08

H19B 0.6092 -0.0458 1.0703 1 0.08

H19C 0.5055 -0.0576 1.0085 1 0.08

01 0.6916(3) 0.05439(18) 1.00407(8) 1 0.0381(5)

02 0.9087(2) 0.09505(16) 0.90211(8) 1 0.0303(4)

H02 0.955(5) 0.066(3) 0.9315(16) 1 0.05 03 0.9583(3) 0.21166(16) 0.63306(8) 1 0.0367(5)

04 0.9631(2) 0.38412(14) 0.68369(8) 1 0.0332(4)

C21 0.4312(4) 0.1862(2) 0.66587(13) 1 0.0335(6)

C22 0.4970(6) 0.2112(3) 0.60444(17) 1 0.0676(13)

H22 0.5632 0.2782 0.602 1 0.081

C23 0.4797(6) 0.1566(3) 0.55258(17) 1 0.0715(13)

H23 0.5359 0.191 0.5193 1 0.086

C24 0.3868(4) 0.0500(3) 0.53703(15) 1 0.0381(7)

021 0.4598(3) 0.25441(16) 0.70676(9) 1 0.0409(5)

022 0.3513(3) 0.0935(2) 0.67422(11) 1 0.0534(6)

023 0.3842(3) 0.0187(2) 0.48333(11) 1 0.0563(7)

024 0.3156(3) -0.00357(17) 0.57942(10) 1 0.0499(6)

H024 0.336(4) 0.049(3) 0.6325(17) 1 0.05

X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.143° (2Θ) at 15.590° (2Θ) (for view of diagrams and experimental details, see figure 1 .14.c).

Example 15: Preparation and analyses of (3S,4S,5R)-cephalotaxine hydrogen malonate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial malonic acid according to the above general procedure included in example 1 , then isolated as large white prisms mp -°C measured by DSC, see figure 2.14). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below). DSC analysis (see figure 2.14)

DSC curve indicated endotermic transition coresponding to melting point 194- 198°C (ΔΗ= 321 ± 20J/g)

¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.85 (s, 1 H), 6.81 (s, 1 H), 5.98 (d, J = 1 .1 Hz, 1 H), 5.97 (d, J = 1 .1 Hz, 1 H), 5.14 (s, 1 H), 4.91 (s, 7H), 4.83 (d, J = 9.1 Hz, 1 H), 3.93 (d, J = 9.1 Hz, 1 H), 3.85 (s, 3H), 3.64 (ddd, J = 14.7, 12.1 , 8.2 Hz, 1 H), 3.51 (t, J = 8.7 Hz, 1 H), 3.19 (dd, J = 12.2, 8.4 Hz, 1 H), 2.56 (dd, J = 14.9, 6.8 Hz, 1 H), 2.25 - 2.09 (m, 3H), 2.05 - 1 .90 (m, 1 H).

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR APT* (101 MHz, Methanol-d4) δ 175.03, 169.06, 149.36, 148.65, 131 .56, 127.80, 1 14.36, 1 1 1 .81 , 102.69, 92.92, 78.61 , 73.80, 58.47, 55.30, 53.86, 48.75, 40.37, 29.23, 19.76.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^"1 2893, 1716, 1653, 1581 , 1501 , 1487, 1465, 1425, 1389, 1372, 1354, 1330, 1314, 1282, 1260, 1243, 1220, 1 150, 1 119, 1096, 1082, 1061 , 1032, 971 , 933, 926, 891 , 845, 805, 790, 774, 740, 688, 654, 591 , 576, 564, 505, 474, 459 ee figure 3.13

Optical rotation [a] ²⁵ _D -195 ± 4 (c 0.79, H ₂ O).

X-ray crystallographic studies

Single crystal X-ray diffraction (see figure 1 .10. a and 1 .10.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.51 x 0.23 x 0.1 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C21H25N Oa

Extended formula C18H22NO4, C3H3O4

Formula weight 419.42

Temperature 150(2) K

Wavelength 0.71073 X

Crystal system, space group monoclinic, P

Unit cell dimensions a = 9.4080(2) λ, a = 90 ^°

b = 8.3275(2) X, β = 97.2120(10) ^° c = 12.2817(3) X, γ = 90 ^°

Volume 954.60(4) X ³ Z, Calculated density 2 , 1.459 (g. cm.- ¹ )

Absorption coefficient 0.112 mm ^"1

F(000) 444

Crystal size 0.51 x 0.23 x 0.1 mm

Crystal color colourless

Theta range for data collection 2.96 to 27.48 ^°

h_min, h_max - 12 , 11

k_min, k_max -10 , 10

l_min, l_max -13 , 15

Reflections collected / unique 6539 / 2308 [ ^a R(int) = 0.0342]

Reflections [I > 2σ] 2154

Completeness to theta_max 0.985

Absorption correction type multi-scan

Max. and min. transmission 0.989 , 0.851

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 2308 / 1 / 281

f ^t Goodness-of-fit 1.047

Final R indices [I > 2σ] ^c Ri = 0.0365, ^d wR ₂ = 0.0892

R indices (all data) ^c Ri = 0.0392, ^d wR ₂ = 0.0912 Largest cliff. peak and hole 0.241 and -0.181 e.X ^"3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI 0.4191(2) 0.0398(3) 0.32130(18) 1 0.0163(5)

HI 0.3321 0.0867 0.3374 1 0.02

C2 0.4380(2) -0.1163(3) 0.30537(17) 1 0.0159(4)

C3 0.5869(2) -0.1621(3) 0.28324(18) 1 0.0157(4)

H3 0.6341 -0.225 0.3471 1 0.019

C4 0.6642(2) 0.0040(3) 0.27995(17) 1 0.0146(4)

H4 0.7434 0.0028 0.3424 1 0.018

C5 0.5538(2) 0.1318(3) 0.31051(16) 1 0.0144(4)

C6 0.6097(2) 0.2317(3) 0.41192(17) 1 0.0176(5)

H6A 0.7121 0.2587 0.4111 1 0.021

H6B 0.5997 0.1712 0.4799 1 0.021

C7 0.5183(2) 0.3850(3) 0.40610(19) 1 0.0230(5) H7A 0.4443 0.3777 0.4565 1 0.028

H7B 0.5788 0.4804 0.4261 1 0.028

C8 0.4488(2) 0.3959(3) 0.28666(19) 1 0.0197(5)

H8A 0.3445 0.3753 0.2813 1 0.024

H8B 0.4642 0.5035 0.2559 1 0.024

N9 0.52169(18) 0.2683(2) 0.22655(15) 1 0.0148(4)

H9 0.614(4) 0.307(5) 0.212(3) 1 0.05

CIO 0.4364(2) 0.2259(3) 0.11955(18) 1 0.0172(5)

H10A 0.4361 0.3187 0.0692 1 0.021

H10B 0.3361 0.2049 0.1319 1 0.021

Cl l 0.4938(2) 0.0798(3) 0.06520(17) 1 0.0158(4)

H11A 0.4593 -0.0186 0.0987 1 0.019

HUB 0.4556 0.0794 -0.0137 1 0.019

C12 0.6558(2) 0.0767(3) 0.07647(17) 1 0.0162(4)

C13 0.7345(2) 0.0378(3) 0.17770(17) 1 0.0145(4)

C14 0.8845(2) 0.0324(3) 0.18820(18) 1 0.0181(5)

H14 0.9391 0.0054 0.2561 1 0.022

C15 0.9500(2) 0.0672(3) 0.09741(19) 1 0.0213(5)

C16 0.8722(2) 0.1084(3) -0.00170(18) 1 0.0211(5)

C17 0.7251(2) 0.1145(3) -0.01522(18) 1 0.0185(5)

H17 0.6724 0.143 -0.0836 1 0.022

C18 1.1032(2) 0.1008(5) -0.0252(2) 1 0.0421(8)

H18A 1.1351 0 -0.0574 1 0.051

H18B 1.1734 0.1861 -0.0359 1 0.051

C19 0.2045(2) -0.1950(3) 0.3336(2) 1 0.0273(6)

H19A 0.2108 -0.1225 0.3969 1 0.041

H19B 0.1508 -0.2914 0.349 1 0.041

H19C 0.1553 -0.1404 0.2689 1 0.041

01 0.34688(16) -0.2399(2) 0.31318(14) 1 0.0215(4)

02 0.58456(17) -0.2567(2) 0.18698(13) 1 0.0199(4)

H02 0.645(4) -0.340(5) 0.199(3) 1 0.05

03 0.96437(17) 0.1449(3) -0.07772(14) 1 0.0309(5)

04 1.09483(16) 0.0790(3) 0.08922(14) 1 0.0320(5)

C21 1.0680(2) 0.3467(3) 0.4054(2) 1 0.0212(5)

C22 0.9936(2) 0.4066(3) 0.29686(19) 1 0.0211(5)

H22A 0.9709 0.3134 0.2477 1 0.025 H22B 1.0605 0.4767 0.2626 1 0.025

C23 0.8553(2) 0.5002(3) 0.30541(18) 1 0.0201(5)

021 1.17294(19) 0.4093(3) 0.45441(16) 1 0.0382(5)

022 1.0048(2) 0.2182(3) 0.43948(17) 1 0.0356(5)

H022 1.056(3) 0.180(5) 0.510(3) 1 0.05

023 0.84982(19) 0.5914(3) 0.38471(16) 1 0.0387(5)

024 0.75459(16) 0.4803(2) 0.22799(13) 1 0.0205(4)

X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.070° (2Θ) at 18.880° (2Θ). The sample is without impurity, as shown by the good agreement between calculated (red) and experimental (black) patterns (for view of diagrams and experimental details, see figure 1 .10).

Example 16: Preparation and analyses of (3S,4S,5R)-cephalotaxine hydrogen glutarate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial glutaric acid according to the above general procedure included in example 1 , then isolated as large white prisms mp 204-207°C measured by DSC, see figure 2.10). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.15)

DSC curve indicated endothermic transition coresponding to melting point 204- 206.5°C (AH= 95 ± 10J/g) ¹ H NMR (400 MHz, Methanol-d ₄ )* δ ¹ H NMR (400 MHz, Methanol-c/ ₄ ) δ 6.80 (s, 1 H), 6.77 (s, 1 H), 5.94 (d, J = 0.8 Hz, 1 H), 5.92 (d, J = 1 .1 Hz, 1 H), 5.09 (s, 1 H), 4.79 (d, J = 9.1 Hz, 1 H), 3.88 (d, J = 9.1 Hz, 1 H), 3.80 (s, 3H), 3.62 - 3.52 (m, 1 H), 3.44 (d, J = 9.5 Hz, 1 H), 3.13 (dd, J = 12.3, 8.1 Hz, 1 H), 2.52 (dd, J = 14.7, 6.9 Hz, 1 H), 2.35 (t, J = 7.5 Hz, OH), 2.27 (t, J = 7.5 Hz, 5H), 2.13 (s, 3H), 1 .92 (s, 1 H), 1 .85 (p, J = 7.4 Hz, 3H).

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR (101 MHz, MeOD)** δ 178.93, 168.87, 149.24, 148.54, 131 .65, 127.89, 1 14.36, 1 1 1 .80, 102.63, 93.14, 78.43, 73.82, 58.44, 55.35, 53.82, 51 .97, 48.67, 40.39, 36.1 1 , 35.99, 34.24, 29.30, 22.70, 22.20, 19.79.

**DEPT135: Distortionless Enhancement by Polarization Transfer (non-quaternary carbons only)

IR (ATR, solid), cm ^"1 . 2948, 1697, 1650, 1561 , 1503, 1486, 1463, 1367, 1351 , 1279, 1250, 1218, 1 164, 1 103, 1054, 1030, 972, 936, 925, 889, 844, 797, 738, 709, 676, 593, 562, 504, 472, 455, 670, 589, 537, 470, 458. See figure 3.14 Optical rotation [a] ²⁵ _D -169 ± 4 (c 0.73, H ₂ O).

X-ray crystallographic studies

A. Single crystal X-ray diffraction (see figure 1 .7. a and 1 .7.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.37 x 0.27 x 0.21 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula CmHsiN-i Oi^

Extended formula 3(C _W H ₂ 2N0 ₄ ), 3(C _S H ₇ 0 ₄ )

Formula weight 1342.42

Temperature 150(2) K

Wavelength 0.71073 A

Crystal system, space group monoclinic, P 2

Unit cell dimensions a = 10.0976(2) A, a = 90 ° b = 25.7683(7) A, β = 96.3460(10) ° c = 12.3288(3) A, γ = 90 °

Volume 3188.27(13) A ³

Z, Calculated density 2 , 1.398 (g.cm ^{- 1} )

Absorption coefficient 0.106 mnT ¹

F(000) 1428

Crystal size 0.37 x 0.27 x 0.21 mm Crystal color colourless

Theta range for data collection 1.58 to 27.5

h_min, h_max - 11 , 13

k_min, k_max -32 , 33

l_min, l_max -15 , 16

Reflections collected / unique 28870 / 7474 [ ^a R(int) =

0.0465] Reflections [7 > 2σ] 6431

Completeness to theta_max 0.999

Absorption correction type multi-scan

Max. and min. transmission 0.978 , 0.910

Refinement method Full-matrix least-squares on F ²

Data / restraints / parameters 7474 / 7 / 895

f ^t Goodness-of-fit 1.123

Final R indices [I > 2σ] ^c Ri = 0.0423, ^d wR ₂ = 0.0993

R indices (all data) ^c Ri = 0.0533, ^d wR ₂ = 0.1194 Largest diff. peak and hole 0.617 and -0.281 e.A ^-3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor.

Atom X y z occ. U(eq)

CI -0.0886(3) 0.34837(12) 0.3215(2) 1 0.0191(6)

HI -0.1703 0.3627 0.3387 1 0.023

C2 -0.0684(3) 0.29818(12) 0.3040(2) 1 0.0179(6)

C3 0.0705(3) 0.28452(11) 0.2814(2) 1 0.0169(6)

H3 0.1134 0.2631 0.3432 1 0.02

C4 0.1420(3) 0.33861(11) 0.2825(2) 1 0.0159(6)

H4 0.2115 0.338 0.3469 1 0.019

C5 0.0352(3) 0.37924(12) 0.3104(2) 1 0.0169(6)

C6 0.0820(3) 0.41281(12) 0.4113(2) 1 0.0205(6)

H6A 0.0706 0.3937 0.4794 1 0.025

H6B 0.1771 0.4222 0.4117 1 0.025

C7 -0.0059(3) 0.46180(13) 0.4020(3) 1 0.0261(7)

H7A -0.0779 0.4591 0.45 1 0.031

H7B 0.048 0.4931 0.4228 1 0.031

C8 -0.0639(3) 0.46455(12) 0.2821(3) 1 0.0245(7)

H8A -0.1612 0.4583 0.2745 1 0.029 H8B -0.0468 0.499 0.2508 1 0.029

N9 0.0065(3) 0.42234(10) 0.2253(2) 1 0.0186(5)

H9 0.096(3) 0.4368(17) 0.221(3) 1 0.05

CIO -0.0685(3) 0.40676(12) 0.1 193(2) 1 0.0212(6)

H10A -0.0735 0.4367 0.0687 1 0.025

H10B -0.1607 0.3973 0.1316 1 0.025

Cl l -0.0044(3) 0.36124(12) 0.0665(2) 1 0.0197(6)

H11A -0.0346 0.3285 0.0979 1 0.024

HUB -0.0349 0.361 -0.0126 1 0.024

C12 0.1469(3) 0.36310(12) 0.0823(2) 1 0.0185(6)

C13 0.2151(3) 0.35108(1 1) 0.1844(2) 1 0.0172(6)

C14 0.3554(3) 0.35169(12) 0.1990(2) 1 0.0185(6)

H14 0.4032 0.3427 0.2671 1 0.022

C15 0.4208(3) 0.36573(13) 0.1 1 18(3) 1 0.0230(7)

C16 0.3533(3) 0.37899(13) 0.0122(3) 1 0.0239(7)

C17 0.2166(3) 0.37791(12) -0.0059(2) 1 0.0215(6)

H17 0.1709 0.3868 -0.0749 1 0.026

C18 0.5712(3) 0.38466(18) -0.0023(3) 1 0.0369(9)

H18A 0.6243 0.4169 -0.0038 1 0.044

H18B 0.6186 0.3567 -0.0373 1 0.044

C19 -0.2842(3) 0.27175(14) 0.3338(3) 1 0.0323(8)

H19A -0.2778 0.2912 0.4025 1 0.048

H19B -0.3371 0.2403 0.3406 1 0.048

H19C -0.3273 0.2934 0.2747 1 0.048

01 -0.1528(2) 0.25771(9) 0.30960(18) 1 0.0245(5)

02 0.0682(2) 0.25553(8) 0.18347(17) 1 0.0213(5)

H02 0.153(4) 0.2398(19) 0.190(4) 1 0.05

03 0.4422(2) 0.39231(1 1) -0.06025(19) 1 0.0339(6)

04 0.5564(2) 0.37085(1 1) 0.10798(19) 1 0.0330(6)

C21 0.1039(3) 0.18489(12) -0.31 19(2) 1 0.0189(6)

H21 0.1849 0.2001 -0.3287 1 0.023

C22 0.0883(3) 0.13467(12) -0.2894(2) 1 0.0192(6)

C23 -0.0503(3) 0.1 1962(1 1) -0.2694(2) 1 0.0183(6)

H23 -0.089 0.0969 -0.3306 1 0.022

C24 -0.1272(3) 0.17261(1 1) -0.2761(2) 1 0.0165(6)

H24 -0.1953 0.1698 -0.3412 1 0.02 C25 -0.0241(3) 0.21399(12) -0.3070(2) 1 0.0177(6)

C26 -0.0692(3) 0.24407(13) -0.4115(3) 1 0.0251(7)

H26A -0.164 0.2543 -0.4136 1 0.03

H26B -0.058 0.2228 -0.4768 1 0.03

C27 0.0209(4) 0.29228(14) -0.4075(3) 1 0.0311(8)

H27A 0.0978 0.2863 -0.4493 1 0.037

H27B -0.0295 0.3229 -0.4383 1 0.037

C28 0.0678(4) 0.30050(12) -0.2861(3) 1 0.0274(7)

H28A 0.1656 0.2964 -0.2719 1 0.033

H28B 0.0434 0.3356 -0.2626 1 0.033

N29 -0.0026(2) 0.25951(10) -0.2266(2) 1 0.0187(5)

H29 -0.092(2) 0.2726(17) -0.219(4) 1 0.05

C30 0.0711(3) 0.24762(12) -0.1173(2) 1 0.0227(7)

H30A 0.0691 0.2785 -0.0698 1 0.027

H30B 0.1655 0.2402 -0.1264 1 0.027

C31 0.0121(3) 0.20123(12) -0.0613(2) 1 0.0211(7)

H31A 0.0463 0.1686 -0.0904 1 0.025

H31B 0.0415 0.2025 0.018 1 0.025

C32 -0.1386(3) 0.20094(12) -0.0789(2) 1 0.0187(6)

C33 -0.2036(3) 0.18690(11) -0.1812(2) 1 0.0175(6)

C34 -0.3427(3) 0.18840(12) -0.1998(2) 1 0.0199(6)

H34 -0.3884 0.1779 -0.2678 1 0.024

C35 -0.4108(3) 0.20555(13) -0.1163(3) 1 0.0230(7)

C36 -0.3469(3) 0.21964(13) -0.0161(2) 1 0.0240(7)

C37 -0.2109(3) 0.21754(13) 0.0059(2) 1 0.0230(7)

H37 -0.1674 0.2269 0.0754 1 0.028

C38 -0.5648(4) 0.2296(2) -0.0102(3) 1 0.0455(11)

H38A -0.6168 0.2033 0.0254 1 0.055

H38B -0.615 0.2627 -0.0134 1 0.055

C39 0.3110(3) 0.11381(14) -0.3051(3) 1 0.0334(8)

H39A 0.3101 0.1274 -0.3794 1 0.05

H39B 0.3738 0.0848 -0.2948 1 0.05

H39C 0.3385 0.1413 -0.2524 1 0.05

021 0.1792(2) 0.09602(9) -0.28849(19) 1 0.0263(5)

022 -0.0503(2) 0.09192(8) -0.16968(17) 1 0.0235(5)

H022 -0.140(4) 0.0779(18) -0.174(3) 1 0.05 023 -0.4387(2) 0.23663(11) 0.05147(19) 1 0.0359(6)

024 -0.5470(2) 0.21324(11) -0.11719(19) 1 0.0338(6)

C41 0.0617(3) 0.51567(12) -0.3102(2) 1 0.0189(6)

H41 0.1403 0.5322 -0.3289 1 0.023

C42 0.0504(3) 0.46489(12) -0.2941(2) 1 0.0200(6)

C43 -0.0857(3) 0.44765(12) -0.2695(2) 1 0.0201(6)

H43 -0.1269 0.4263 -0.332 1 0.024

C44 -0.1644(3) 0.50032(11) -0.2677(2) 1 0.0173(6)

H44 -0.2344 0.4989 -0.3316 1 0.021

C45 -0.0652(3) 0.54354(11) -0.2953(2) 1 0.0180(6)

C46 -0.1185(3) 0.57783(12) -0.3926(3) 1 0.0234(7)

H46A -0.1098 0.5598 -0.4623 1 0.028

H46B -0.2135 0.5863 -0.3892 1 0.028

C47 -0.0335(4) 0.62731(13) -0.3837(3) 1 0.0291(8)

H47A 0.0396 0.6249 -0.4308 1 0.035

H47B -0.0887 0.6581 -0.4058 1 0.035

C48 0.0221(3) 0.63088(12) -0.2639(3) 1 0.0240(7)

H48A 0.1204 0.6279 -0.2557 1 0.029

H48B -0.0027 0.6644 -0.2323 1 0.029

N49 -0.0396(2) 0.58613(9) -0.2084(2) 1 0.0183(5)

H49 -0.125(3) 0.6012(16) -0.198(3) 1 0.05

C50 0.0383(3) 0.57164(12) -0.1033(2) 1 0.0216(6)

H50A 0.038 0.6011 -0.0517 1 0.026

H50B 0.1318 0.5648 -0.116 1 0.026

C51 -0.0186(3) 0.52357(12) -0.0525(2) 1 0.0201(6)

H51A 0.015 0.4921 -0.0865 1 0.024

H51B 0.0131 0.5226 0.0264 1 0.024

C52 -0.1696(3) 0.52294(12) -0.0671(2) 1 0.0201(6)

C53 -0.2383(3) 0.51051(11) -0.1691(2) 1 0.0179(6)

C54 -0.3781(3) 0.51107(12) -0.1849(2) 1 0.0225(7)

H54 -0.4251 0.502 -0.2532 1 0.027

C55 -0.4443(3) 0.52516(13) -0.0985(3) 1 0.0235(7)

C56 -0.3770(3) 0.53834(13) 0.0019(2) 1 0.0251(7)

C57 -0.2406(3) 0.53736(13) 0.0207(2) 1 0.0228(7)

H57 -0.1956 0.5461 0.09 1 0.027

C58 -0.5954(4) 0.5456(2) 0.0143(3) 1 0.0529(13) H58A -0.6431 0.5177 0.0493 1 0.064

H58B -0.6484 0.5779 0.0155 1 0.064

C59 0.2713(3) 0.44481(14) -0.3200(3) 1 0.0328(8)

H59A 0.2654 0.4634 -0.3897 1 0.049

H59B 0.3314 0.4151 -0.3225 1 0.049

H59C 0.3059 0.4682 -0.2608 1 0.049

041 0.1404(2) 0.42671(9) -0.3008(2) 1 0.0271(5)

042 -0.0800(2) 0.41752(9) -0.17325(18) 1 0.0245(5)

H042 -0.128(4) 0.3903(19) -0.186(4) 1 0.05

043 -0.4668(2) 0.55323(11) 0.07277(18) 1 0.0360(6)

044 -0.5803(2) 0.53167(11) -0.09563(19) 1 0.0346(6)

ClOl -0.4766(3) 0.65082(13) -0.2385(3) 1 0.0284(7)

H10C -0.5381 0.6806 -0.2358 1 0.034

H10D -0.4995 0.6248 -0.1845 1 0.034

C102 -0.4947(3) 0.62652(14) -0.3543(3) 1 0.0305(8)

H10E -0.4661 0.6516 -0.408 1 0.037

H10F -0.4389 0.595 -0.3558 1 0.037

C103 -0.3352(3) 0.66888(13) -0.2093(2) 1 0.0230(7)

C104 -0.6391(4) 0.61269(16) -0.3838(3) 1 0.0386(9)

H10G -0.6924 0.6449 -0.3822 1 0.046

H10H -0.6665 0.5895 -0.3263 1 0.046

C105 -0.6754(3) 0.58666(14) -0.4933(3) 1 0.0284(7)

O106 -0.2531(3) 0.63972(11) -0.1581(2) 1 0.0373(6)

O107 -0.3074(2) 0.71499(10) -0.2358(2) 1 0.0372(6)

O108 -0.5996(2) 0.60033(10) -0.5707(2) 1 0.0328(6)

H108 -0.625(4) 0.5867(16) -0.628(2) 1 0.049

O109 -0.7663(3) 0.55740(11) -0.5077(2) 1 0.0416(7)

C121 -0.6178(3) 0.47396(12) -0.6732(3) 1 0.0233(7)

H12A -0.6445 0.4372 -0.685 1 0.028

H12B -0.6304 0.4831 -0.597 1 0.028

C122 -0.4689(3) 0.47965(13) -0.6889(3) 1 0.0235(7)

H12C -0.4369 0.447 -0.7194 1 0.028

H12D -0.4583 0.5078 -0.7421 1 0.028

C123 -0.7072(3) 0.50788(12) -0.7501(2) 1 0.0206(6)

C124 -0.3840(3) 0.49197(13) -0.5815(3) 1 0.0240(7)

H12E -0.2909 0.4986 -0.596 1 0.029 H12F -0.4181 0.5238 -0.5494 1 0.029

C125 -0.3865(3) 0.44795(13) -0.5018(3) 1 0.0229(7)

0126 -0.6848(2) 0.55641(9) -0.74868(18) 1 0.0294(5)

0127 -0.7983(2) 0.48784(11) -0.8127(2) 1 0.0392(7)

0128 -0.3182(3) 0.40697(10) -0.5289(2) 1 0.0358(6)

H128 -0.311(4) 0.3850(14) -0.477(3) 1 0.05

0129 -0.4453(2) 0.44909(10) -0.42162(19) 1 0.0330(6)

C141 -0.4524(3) 0.33564(14) -0.2802(3) 1 0.0265(7)

H14A -0.4746 0.3716 -0.2609 1 0.032

H14B -0.4732 0.3129 -0.2197 1 0.032

C142 -0.5417(3) 0.32018(13) -0.3830(3) 1 0.0253(7)

H14C -0.517 0.3407 -0.4456 1 0.03

H14D -0.6355 0.3283 -0.3733 1 0.03

C143 -0.3040(3) 0.33265(11) -0.2888(3) 1 0.0205(6)

C144 -0.5293(3) 0.26207(13) -0.4079(3) 1 0.0254(7)

H14E -0.4352 0.2541 -0.4166 1 0.03

H14F -0.5543 0.2417 -0.3451 1 0.03

C145 -0.6149(3) 0.24578(12) -0.5085(3) 1 0.0222(7)

0146 -0.2281(2) 0.32411(9) -0.20303(19) 1 0.0273(5)

0147 -0.2635(2) 0.34037(9) -0.38072(19) 1 0.0289(5)

0148 -0.5675(2) 0.25902(10) -0.59964(18) 1 0.0282(5)

H148 -0.604(4) 0.2443(17) -0.654(3) 1 0.05

0149 -0.7192(2) 0.22260(12) -0.5068(2) 1 0.0395(7) β. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.073 °(2Θ) at 19.048 °(2 Θ). The sample is without impurity, as shown by the good agreement between calculated (red) and experimental (black) patterns (for view of diagrams and experimental details, see figure 1 .7.c).

Example 17: Preparation and analyses of (3S,4S,5R)-cephalotaxine hydrogen tartronate This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial tartronic acid according to the above general procedure included in example 1, then isolated as white prisms mp 192-196°C measured by DSC, see figure 2.16). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis. (see below).

DSC analysis (see figure 2.16)

DSC curve indicated endotermic transition coresponding to melting point 192- 195-5°C (ΔΗ= 144 ±20J/g)

¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.83 (s, 1 H), 6.79 (s, 1 H), 5.96 (d, J = 1.0 Hz, 1H), 5.95 (d, J= 1.1 Hz, 1H), 5.12 (s, 1H), 4.81 (d, J= 9.0 Hz, 1H), 4.43 (s, 1H), 3.91 (d, J = 9.1 Hz, 1H), 3.83 (s, 3H), 3.68 - 3.55 (m, 1H), 3.50 (s, 1H), 3.23-3.14 (m, 1H), 2.55 (dd, J= 14.8, 6.8 Hz, 1H), 2.22-2.10 (m, 3H), 1.94 (m, 1H).

Partial presuppression of water signal using "watergate" irradiation

^1j C NMR APT* (101 MHz, Methanol-d4) δ 174.47, 169.05, 149.38, 148.66, 131.53, 127.77, 114.35, 111.81, 102.69, 92.94, 78.66, 73.81, 58.48, 55.29, 53.87, 48.76, 40.34, 29.23, 19.77.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^"1 3468, 2876, 1981, 1718, 1655, 1598, 1504, 1489, 1375, 1354, 1262, 1241, 1215, 1095, 1080, 1055, 1032, 1022, 968, 937, 923, 885, 841 , 823, 795, 780, 708, 670, 570, 530, 499, 478, 458. See figure 3.15

Optical rotation [a] ²⁵ _D -183 ± 4 (c 0.08, H ₂ O). X-ray crystallographic studies

A. Single crystal X-ray diffraction (see figure 1 .13.a and 1 .13.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.4 x 0.35 x 0.2 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C21H25N O9

Extended formula C ₁₈ H ₂ 2N0 ₄ , C ₃ ¾0 ₅

Formula weight 435.42

Temperature 150(2) K

Wavelength 0.71073 X

Crystal system, space group monoclinic, P

Unit cell dimensions a = 9.3310(9) λ, a = 90 ^°

b = 8.5210(8) X, jS = 99.047(4) ^° c = 12.3828(10) X, γ = 90 ^°

Volume 972.30(15) X ^J

Z, Calculated density 2 , 1.487 (g. cm.- ¹ )

Absorption coefficient 0.117 mm ^"1

F(000) 460

Crystal size 0.45 x 0.14 x 0.09 mm

Crystal color colourless

Theta range for data collection 2.91 to 27.46 ^°

h_min, h_max - 12 , 11

k_min, k_max -9 , 11

l_min, l_max -13 , 16

Reflections collected / unique 8243 / 2344 [ ^a R(int) = 0.0468]

Reflections [I > 2σ] 1950

Completeness to theta_max 0.987

Absorption correction type multi-scan

Max. and min. transmission 0.990 , 0.886

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 2344 / 1 / 293

f ^t Goodness-of-fit 1.049

Final R indices [I > 2σ] ^c Ri = 0.049, ^d wR ₂ = 0.1233

R indices (all data) ^c Ri = 0.0624, ^d wR ₂ = 0.1332 Largest diff. peak and hole 0.574 and -0.288 e.X ^"3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A ² x 10 ³ ). U(eq) is defined as one third of the trace of the orthogonalized L¾ tensor. Atom X y z occ. U(eq)

CI -0.0603(3) 0.2543(5) 0.8083(3) 1 0.0206(7)

HI -0.1523 0.2959 0.8169 1 0.025

C2 -0.0331(3) 0.1021(4) 0.7924(3) 1 0.0194(7)

C3 0.1228(4) 0.0667(4) 0.7835(3) 1 0.0219(7)

H3 0.1682 0.0112 0.8515 1 0.026

C4 0.1933(3) 0.2319(4) 0.7811(3) 1 0.0200(7)

H4 0.2751 0.2348 0.8439 1 0.024

C5 0.0752(3) 0.3491(4) 0.8106(2) 1 0.0199(7)

C6 0.1209(4) 0.4336(5) 0.9197(3) 1 0.0278(8)

H6A 0.1136 0.3627 0.9819 1 0.033

H6B 0.2219 0.4724 0.926 1 0.033

C7 0.0142(5) 0.5703(6) 0.9174(3) 1 0.0404(11)

H7A 0.0598 0.66 0.9607 1 0.049

H7B -0.0736 0.5379 0.9473 1 0.049

C8 -0.0239(4) 0.6143(5) 0.7965(3) 1 0.0307(8)

H8A -0.1304 0.6142 0.7733 1 0.037

H8B 0.014 0.7199 0.7832 1 0.037

N9 0.0475(3) 0.4902(4) 0.7342(2) 1 0.0215(6)

H9 0.139(5) 0.526(7) 0.723(4) 1 0.05

CIO -0.0405(3) 0.4578(5) 0.6239(3) 1 0.0248(8)

H10A -0.0374 0.5514 0.577 1 0.03

H10B -0.1429 0.4406 0.633 1 0.03

Cll 0.0117(3) 0.3164(5) 0.5664(3) 1 0.0224(7)

H11A -0.0244 0.2196 0.5972 1 0.027

HUB -0.0297 0.3208 0.4878 1 0.027

C12 0.1755(3) 0.3086(5) 0.5780(3) 1 0.0215(7)

C13 0.2594(3) 0.2702(5) 0.6785(3) 1 0.0223(7)

C14 0.4112(3) 0.2688(5) 0.6907(3) 1 0.0265(8)

H14 0.4693 0.2441 0.7586 1 0.032

C15 0.4729(3) 0.3050(6) 0.5988(3) 1 0.0313(10)

C16 0.3901(4) 0.3424(6) 0.5001(3) 1 0.0326(10)

C17 0.2417(3) 0.3442(6) 0.4865(3) 1 0.0291(9)

H17 0.1855 0.3685 0.4177 1 0.035

C18 0.6229(4) 0.3522(8) 0.4781(3) 1 0.0493(14) H18A 0.6809 0.4484 0.4729 1 0.059

H18B 0.669 0.265 0.4434 1 0.059

C19 -0.2720(4) 0.0166(5) 0.7920(3) 1 0.0322(9)

H19A -0.28 0.0665 0.8622 1 0.048

H19B -0.3308 -0.0792 0.7839 1 0.048

H19C -0.307 0.0892 0.7322 1 0.048

01 -0.1232(2) -0.0222(3) 0.7888(2) 1 0.0264(6)

02 0.1346(3) -0.0303(4) 0.6921(2) 1 0.0305(6)

H02 0.187(5) -0.103(8) 0.708(4) 1 0.05

03 0.4794(3) 0.3753(6) 0.4235(2) 1 0.0551(12)

04 0.6190(2) 0.3160(5) 0.5907(2) 1 0.0466(10)

C21 0.3694(4) 0.6959(5) 0.8255(3) 1 0.0350(9)

C22 0.5338(4) 0.6628(5) 0.8633(3) 1 0.0342(9)

H22 0.5785 0.7628 0.8948 1 0.041

C23 0.5554(4) 0.5413(5) 0.9539(3) 1 0.0336(9)

021 0.2983(3) 0.7589(4) 0.8939(3) 1 0.0499(9)

022 0.3173(3) 0.6489(5) 0.7318(2) 1 0.0530(11)

023 0.6011(3) 0.6250(5) 0.7744(2) 1 0.0459(8)

H023 0.621(5) 0.523(8) 0.744(4) 1 0.05

024 0.6185(4) 0.4131(4) 0.9298(3) 1 0.0478(9)

H024 0.641(5) 0.357(8) 0.982(4) 1 0.05

025 0.5189(4) 0.5694(5) 1.0420(3) 1 0.0533(10) β. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.101 ° (2Θ) at 18.586° (2 Θ). The sample is without impurity, as shown by the good agreement between calculated (red) and experimental (black) patterns (for view of diagrams and experimental details, see figure 1 .13.c).

Example 18: Preparation and analyses of (3S,4S,5R)-cephalotaxine dihydrogen citrate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial citric acid according to the above general procedure included in example 1 , then isolated as fine needle mp -°C measured by DSC, see figure 2.17). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.17)

DSC curve indicated endotermic transition coresponding to melting point 210.5- 219.5°C(AH= 223±20J/g)

¹ H NMR (400 MHz, Methanol-d ₄ )* δ 6.85 (s, 1H), 6.81 (s, 1H), 5.98 (s, 1H), 5.97 (s, 1H), 5.14 (s, 1H), 4.83 (d, J = 9.1 Hz, 1H), 3.93 (d, J = 9.1 Hz, 1H), 3.85 (s, 3H), 3.64 (ddd, J = 14.7, 12.1, 7.8 Hz, 1H), 3.52 (d, J = 9.7 Hz, 1H), 2.82 (d, J = 15.4 Hz, 2H), 2.74 (d, J = 15.4 Hz, 2H), 2.56 (dd, J = 14.8, 6.8 Hz, 1H), 2.19 (s, 3H), 1.97 (s, 1H).

*Partial presuppression of water signal using "watergate" irradiation

¹³ C NMR APT* (101 MHz, Methanol-d4) δ 179.20, 174.91, 168.95, 149.31, 148.60, 131.57, 127.80, 114.37, 111.82, 102.66, 93.06, 78.60, 73.98, 73.81, 58.50, 55.31 , 53.85, 48.70, 44.76, 40.35, 29.24, 19.79.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ^"1 3445, 2971, 2712, 1981, 1758, 1688, 1649, 1529, 1500, 1462, 1375, 1345, 1314, 1295, 1261, 1218, 1182, 1145, 1121, 1085, 1054, 1043, 1018, 970, 939, 925, 898, 879, 847, 831, 802, 785, 753, 702, 675, 644, 599, 565, 544, 489, 456. See figure 3.16

Optical rotation [a] ²⁵ _D -175 ± 4 (c 0.88, H ₂ O). X-ray crystallographic studies

Single crystal X-ray diffraction not available (no suitable crystals)

X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.088° (2Θ) at 17 597° (2Θ). Calculated (red) pattern is not available (for view of diagrams and experimental details, see figure 1 .15)

Example 19: Preparation and analyses of (3S,4S,5R)-cephalotaxine benzoate

This ionic compound was obtained from natural (3S,4S,5R)-(-)-cephalotaxine with commercial benzoic acid according to the above general procedure included in example 1 , then isolated as large white prisms mp 184-186.5°C measured by DSC, see figure 2.18). Several potentially acceptable crystals were kept suspended in their mother liquors for the subsequent X-ray diffraction analysis, (see below).

DSC analysis (see figure 2.18)

DSC curve indicated endotermic transition coresponding to melting point 184- 186.5°C (ΔΗ= 102 ± 20J/g)

¹ H NMR (400 MHz, Methanol-d ₄ )* δ. 7.96 - 7.90 (m, 2H), 7.39 (dt, J = 27.4, 7.1 Hz, 4H), 6.80 (s, 1 H), 6.76 (s, 1 H), 5.92 (s, 1 H), 5.91 (s, 1 H), 5.09 (s, 1 H), 4.79 (d, J = 9.0 Hz, 1 H), 3.88 (d, J = 9.1 Hz, 1 H), 3.80 (s, 3H), 3.66 - 3.53 (m, 1 H), 3.45 (s, 1 H), 3.13 (dd, J = 12.2, 8.1 Hz, 1 H), 2.51 (dd, J = 14.8, 6.9 Hz, 1 H), 2.13 (d, J = 3.0 Hz, 3H), 1 .93 (s, 1 H). Partial presuppression of water signal using "watergate" irradiation

^1j C NMR (101 MHz, MeOD) ^** δ 174.19, 168.80, 149.25, 148.55, 137.67, 131 .74, 131 .67, 130.34, 128.82, 127.89, 1 14.31 , 1 1 1 .76, 102.57, 93.16, 78.32, 73.83, 58.42, 55.44, 53.85, 48.75, 40.48, 29.37, 19.80.

**DEPT135: Distortionless Enhancement by Polarization Transfer (non-quaternary carbons only)

IR (Diamond ATR, solid) cm ^"1 3244, 3064, 2944, 2893, 2834, 1648, 1603, 1569, 1504, 1486, 1459, 1449, 1359, 1323, 1269, 1214, 1 162, 1 1 1 1 , 1082, 1061 , 1034, 969, 936, 861 , 838, 815, 801 , 779, 724, 690, 669, 658, 599, 563, 496, 470, 454 See figure 3.17

Optical rotation [a] ²⁵ _D -175 ± 4 (c 0.87, H ₂ O).

X-ray crystallographic studies

A. Single crystal X-ray diffraction (see figure 1 .8. a and 1 .8.b)

From a suspension in its mother liquor, a suitable single crystal of size 0.4 x 0.35 x 0.2 mm was finally selected and implemented on the diffractometer.

Structural data

Empirical formula C32H33N OB

Extended formula C18H22N O4, C14H11O4

Formula weight 559.59

Temperature 150(2) K

Wavelength 0.71073 A

Crystal system, space group orthorhombic, P i 2i

Unit cell dimensions a = 8.0462(2) X, a = 15.3628(3)

X, β = 90 ^° c = 21.9444(6) X, γ 90

Volume 2712.59(11) X ³

Z, Calculated density 4 , 1.37 (g. cm.- ¹ )

Absorption coefficient 0.099 mm ^{" 1}

F(000) 1184

Crystal size 0.4 x 0.35 x 0.2 mm

Crystal color colourless

Theta range for data collection 3 to 27.48 ⁰

h_min, h_max - 10 , 9

k_min, k_max -19 , 17

1 min, 1 max -28 . 13 Reflections collected / unique 13404 / 3499 [ ^a R(int) = 0.0407]

Reflections [I > 2σ] 3109

Completeness to theta_max 0.993

Absorption correction type multi-scan

Max. and min. transmission 0.980 , 0.898

Refinement method Full- matrix least-squares on F ²

Data / restraints / parameters 3499 / 0 / 380

f ^t Goodness-of-fit 1.017

Final R indices [I > 2σ] ^c Ri = 0.0361 , ^d wR ₂ = 0.081 1

R indices (all data) ^c Ri = 0.0427, ^d wR ₂ = 0.0844 Largest cliff. peak and hole 0.175 and -0.248 e.X ^{" 3}

Atomic coordinates, site occupancy (%) and equiva isotropic displacement parameters (A ² x lO ³ ). U(eq) is defined as one third of the trace of the orthogonal Lin tensor.

Atom X y z occ. U(eq)

CI 0.6431(3) 1.03138(12) 0.82499(9) 1 0.0186(4)

HI 0.5457 1.0636 0.8346 1 0.022

C2 0.7952(3) 1.06437(12) 0.82281(9) 1 0.0197(4)

C3 0.9313(3) 0.99972(12) 0.81 134(9) 1 0.0207(4)

H3 0.9921 1.0156 0.7732 1 0.025

C4 0.8344(2) 0.91256(12) 0.80139(9) 1 0.0171(4)

H4 0.8482 0.8974 0.7574 1 0.021

C5 0.6461(3) 0.93569(12) 0.81017(8) 1 0.0173(4)

C6 0.5366(3) 0.91 173(12) 0.75532(9) 1 0.0213(4)

H6A 0.5488 0.9552 0.7223 1 0.026

H6B 0.5671 0.8537 0.7392 1 0.026

C7 0.3588(3) 0.91 131(14) 0.77997(10) 1 0.0245(5)

H7A 0.2905 0.8674 0.7585 1 0.029

H7B 0.3065 0.9692 0.7749 1 0.029

C8 0.3763(3) 0.88836(14) 0.84756(10) 1 0.0260(5)

H8A 0.3276 0.9347 0.8734 1 0.031

H8B 0.3194 0.8327 0.8567 1 0.031

N9 0.5604(2) 0.88047(10) 0.85897(7) 1 0.0176(3)

H9 0.591(4) 0.8241(18) 0.8525(12) 1 0.05

CIO 0.6019(3) 0.89693(13) 0.92416(9) 1 0.0206(4)

H10A 0.5503 0.8511 0.9496 1 0.025 H10B 0.5542 0.9536 0.9366 1 0.025

Cl l 0.7888(3) 0.89802(12) 0.93576(9) 1 0.0206(4)

H11A 0.8329 0.956 0.9246 1 0.025

HUB 0.8088 0.8894 0.9799 1 0.025

C12 0.8834(3) 0.82933(12) 0.90072(9) 1 0.0189(4)

C13 0.9014(2) 0.83553(11) 0.83720(9) 1 0.0166(4)

C14 0.9852(3) 0.77006(12) 0.80475(9) 1 0.0195(4)

H14 0.995 0.7725 0.7616 1 0.023

C15 1.0525(3) 0.70231(12) 0.83745(9) 1 0.0213(4)

C16 1.0369(3) 0.69717(12) 0.89987(9) 1 0.0219(4)

C17 0.9511(3) 0.75864(12) 0.93242(9) 1 0.0220(4)

H17 0.9379 0.7534 0.9753 1 0.026

C18 1.1881(3) 0.58400(14) 0.86858(11) 1 0.0311(5)

H18A 1.3105 0.5869 0.8724 1 0.037

H18B 1.1554 0.5221 0.8653 1 0.037

C19 0.7167(3) 1.21105(13) 0.83243(12) 1 0.0331(6)

H19A 0.6467 1.1991 0.868 1 0.05

H19B 0.649 1.2079 0.7953 1 0.05

H19C 0.7649 1.2694 0.8361 1 0.05

01 0.84758(19) 1.14764(8) 0.82925(7) 1 0.0255(3)

02 1.04503(19) 0.99388(9) 0.86046(7) 1 0.0261(3)

H02 1.107(4) 1.0393(18) 0.8608(13) 1 0.05

03 1.1116(2) 0.62236(9) 0.92110(7) 1 0.0338(4)

04 1.1359(2) 0.63015(9) 0.81573(7) 1 0.0321(4)

C21 0.6434(3) 0.63750(12) 0.83265(9) 1 0.0219(4)

C22 0.6839(3) 0.57568(12) 0.88376(9) 1 0.0214(4)

C23 0.6376(3) 0.59702(14) 0.94281(10) 1 0.0266(5)

H23 0.5775 0.6492 0.9502 1 0.032

C24 0.6784(3) 0.54284(16) 0.99121(10) 1 0.0328(5)

H24 0.6463 0.558 1.0315 1 0.039

C25 0.7658(3) 0.46679(15) 0.98071(10) 1 0.0336(6)

H25 0.7941 0.4297 1.0138 1 0.04

C26 0.8122(4) 0.44469(14) 0.92172(11) 1 0.0347(6)

H26 0.8721 0.3924 0.9144 1 0.042

C27 0.7710(3) 0.49885(13) 0.87349(10) 1 0.0286(5)

H27 0.8025 0.4834 0.8332 1 0.034 021 0.6994(2) 0.61466(9) 0.78001(7) 1 0.0281(4)

H21 0.697(4) 0.6690(15) 0.7433(13) 1 0.05

022 0.5625(2) 0.70420(8) 0.84265(7) 1 0.0278(4)

C31 0.7277(3) 0.69683(12) 0.64758(9) 1 0.0204(4)

C32 0.7599(3) 0.76150(12) 0.59754(9) 1 0.0194(4)

C33 0.8707(3) 0.74026(13) 0.55122(10) 1 0.0253(5)

H33 0.9305 0.687 0.5528 1 0.03

C34 0.8935(3) 0.79672(15) 0.50296(10) 1 0.0335(5)

H34 0.9722 0.7831 0.4721 1 0.04

C35 0.8025(3) 0.87297(15) 0.49930(10) 1 0.0323(5)

H35 0.8157 0.9105 0.4652 1 0.039

C36 0.6924(3) 0.89464(13) 0.54521(10) 1 0.0296(5)

H36 0.6298 0.947 0.5427 1 0.036

C37 0.6734(3) 0.83966(12) 0.59508(9) 1 0.0242(5)

H37 0.6013 0.8556 0.6275 1 0.029

031 0.6975(2) 0.72777(9) 0.70057(6) 1 0.0292(4)

032 0.72676(19) 0.61814(8) 0.63451(7) 1 0.0246(3) β. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.076° (2Θ) at 17.018° (2 Θ). The sample is without impurity, as shown by the good agreement between calculated (red) and experimental (black) patterns (for view of diagrams and experimental details, see figure 1 .8.c).