WO2015128463A1 - Water soluble salts of harringtonine and their pharmaceutical applications - Google Patents

Abstract

A isolated harringtonines solid salt having formula 1, made by reacting a cephalotaxine ester having formula 2 in which R is, but not limited to, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl or heterocycloalkyi, with an acid having general formula AH in a crystallization solvent, wherein the salt has a water or methanol solubility of approximately 5 mg/mL to approximately 100 mg/mL, and its pharmaceutical applications.

Description

WATER SOLUBLE SALTS OF HARRINGTONINE AND THEIR

PHARMACEUTICAL APPLICATIONS

The present invention concerns solid state salts of harringtonine, definite by their analysis patterns, their process of preparation allowing their use as drug substance for blending alone or in combination in pharmaceutical composition particularly useful for treatment of cancers, leukemias and lymphoma including in using subcutaneous or oral routes of administration

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) [H. Kantarjian et al Homoharringtonine/ Omacetaxine Mepesuccinate: The Long and Winding Road to Food and Drug Administration Approval, Clinical Lymphoma, Myeloma & Leukemia, Vol. 13(5) 530 (2013)]. 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. In 1998, the discovering of a new semi-synthetic process by one of us, allowing industrial production of homoharringtonine at the kilo scale.

The historical comparative study of harringtonine and its natural counterpart, homoharringtonine shows that the activities and toxicity in animals and humans are strictly equivalent to the point that a number of clinical trials made in China used indistinctly both similar, or even sometime as a mixture. The development literature of the late Matthew Suffness has identified several dozen clinical trials carried out in China involving harringtonine alone or in combination [M. Suffness et al, Cephalotaxine esters: antileukemic advance or therapeutic failure?, J Natl Cancer Inst. 1095 (1988)]

The historical reasons for the clinical development homoharringtonine instead of harringtonine are very simple. At the time when these substances were extracted from nature, indeed to the detriment of biodiversity, homoharringtonine was more abundant and easier to purify by chromatography that harringtonine. The invention of a process for the industrial synthesis of semi Homoharringtonine by one of us (JPR) came in 1998 reinforced this phenomenon and trigger a revival of clinical development of HHT in Western countries [JP Robin et al, The first semi-synthesis of enantiopure homoharringtonine via anhydrohomoharringtonine from a preformed chiral acyl moiety, Tetrahedron Letters, 2931 (1999)].

The recent editorial of Kanrarjian et al. perfectly summarizes current status of HHT /http://www. ascopost.com/issues/april-15,- 2013/ homoharringtonine omacetaxine-the-little-drug-that-could.aspx].

However, despite all these progresses, several drawbacks remain:

(i) The cost of treatment for omacetaxine (Synribo) is prohibitive: $28,000 for induction, $14,000 for monthly treatments), this give about 180.000

$ per year, per patient [H. Kantarjian et al., Cancer Drugs in the United States: Justum Pretium— The Just Price, Journal of Clinical Oncology, 2013, p3600; H. Kantarjian, personal communication. "The price of drugs for chronic myeloid leukemia (CML) is a reflection of the unsustainable prices of cancer drugs: from the perspective of a large group of CML experts" blood 2013 p4439]

(ii) The use of parenteral route of administration exclusively in inpatient while the competitors products are administered orally on an outpatient basis, considerably retards its development.

(iii) There is some local intolerance to this product when administered subcutaneously [Chronic myeloid leukemia: overview of new agents and comparative analysis., H. Kantarjian et al, Curr Treat Options Oncol. 2013 Jun;14(2):127 (2013)]

(iv) On the other hand, although it has been known for almost 40 years, there is still a slight doubt regarding the absolute configuration of this series of natural product [see Steitz above]

Definition see scheme 1A

CEPHALOTAXINE ESTERS HARRINGTONINES HARRINGTON INE CATION

i.e. acetates, itaconates... = "HARRINGTON INIUM" ION

Cephalotaxanes are particular alkaloid to date only extracted exclusively the Cephalotaxaceae family which exhibiting the structural formula 1_. Several substituants may be encountered on this core structure: hydroxyl, ether, acyloxy side chain... The eventual presence of some additional double bound or intramolecular bridge achieve to definite cephalotaxanes.

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

Harringtonines 5 (sometime called cephalotaxine esters) are particular cephalotaxanes formed by attachment of a branched a-hydroxyacyloxy side- chain at the 3-position of various cephalotaxines moieties (including drupacine). Another definition of harringtonines may be mixte 1 -O-cephalotaxyl 4-O-methyl esters of 2-alkyl malic acid.

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.

Harringtoids are semi-synthetic derivatives of harringtonines. Harringtonic acids are the free version of side-chain of harringtonines

^*ln cancer area, for definition of term see [Suffness et al in Journal of Natural Products 1982 p 1 Current Status of the NCI Plant and Animal Product Program] CYTOTOXICITY is toxicity to tumor cells in culture; ANTITUMOR is in vivo activity in experimental systems; ANTINEOPLASTIC or ANTICANCER are the reserved terms for reported clinical trials data.

(1 ) The first cephatotaxine ester isolated from cephalotaxus harringtonia

(2) "Homo" means one more carbone than harringtonine; Named omacetaxine (DCI = USAN) as active pharmaceutical ingredient.

(3) "nor" means one more less carbon

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.

harringtonine itself has been the subject of many studies in vitro, in vivo and clinical:

-In myeloid leukemia [M. Suffness et al, Cephalotaxine esters: antileukemic advance or therapeutic failure?, J Natl Cancer Inst. 1095 (1988,); Kantarjian et a\]Effectiveness of Homoharringtonine(Omacetaxine Mepesuccinate) for Treatmentof Acute Myeloid Leukemia: A Meta-Analysis of Chinese Studies; Clinical Lymphoma Myeloma and Leukemia

Some mixture of acid and alkaloid have been in situ prepared and administrated but, surprisingly, never salts including crystalline have been isolated and described in literature.

The natural purification of HA and HO has caused significant damage to the environment. In 1999, the first hemi-synthesis of HHT was reported. This synthesis involves the direct esterification of cephalotaxine extracted from leaves of Cephalotaxus, not from the bark. Only one 70th of the amount of Cephalotaxus is required compared with its natural counterpart, and this semisynthetic HA is also purer (99.7%). The following editorial perfectly summarizes the status of this drug: http://www.ascoDost.com/issues/april -15.- 2013/homoharringtonine. Omacetaxine-the-little-drug-that-could.aspx Recent scientific discovering regarding mechanism of activity of harringtonines

The team of Steitz [Steitz et al, U2504 determines the species specificity of the A-site cleft antibiotics: the structures of tiamulin, homoharringtonine, and bruceantin bound to the ribosome, J Mol Biol. 389(1 ), 146 (2009)] recently demonstrated that homoharringtonine when in place on its active site was protonated in a neutral media, implying that protonation is imperative condition for the manifestation of the activity of this anticancer agent.

In addition, the team of Takano et al [J. Org. Chem. p 8251 (1997)] demonstrated experimentally that when the nitrogen lone pair of homoharringtonine was occupied by an oxygen atom, the cytotoxic activity was divided by a factor of at least 50. The authors concluded that "the nitrogen lone pair on the cephalotaxine skeleton appears to be essential for its activity".

The above mentioned team of Steitz showed that the absolute configuration of homoharringtonine deposited in the Cambridge Structural Database seems to be the opposite of that commonly adopted in the literature.

In brief, the fact that the real active form of harringtonines would be their nitrogen-protonated version was recently supported by the work of Seitz et al. and, indirectly by the work of Takano et al. The present invention relates to overcome the problems mentioned above regarding harringtonine. 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 MOIETIES OF SOME SALT S CITED IN THIS INVENTION

h drogen (2S)-malate hydrogen (2R)-malate hydrogen (2S)-tartrate hydrogen (2R)-tartrate h

hydrogen succinate hydrogen tartronate dihydrogen citrate

The example of single crystal X-ray diffraction of harringtonine hydrogen (2S)-malate exhibited in figure 7a clearly indicates that the alkaloid moeity was efficiently protonated by the processes described in the present invention (proton numbered Hn9 on ORTEP diagram on figure 7a). The same finding was made on homoharringtonines salts in a previous invention. An example of diagram of (2S)-malate of homoharringtonine is represented in Figure 7d (proton H9). Moreover as shown in Figures 7a and 7d, the conformation in the crystalline state are so close together on the one hand, and so close to the configuration of homoharringtonine in its active site on the other hand (see above cited reference of Seitz), that we can expect the same activity for both molecules. In addition, the shortest distance between said proton carried by the nitrogen is close to two angstroms, showing the reality of the formation of a salt and not a mere co-crystal (for detail see figure 7b) Final determination of absolute configuration of harringtonine, homo-harringtonine and cephalotaxine: based upon the well known absolute configuration of 2S-malate and the relative configuration of the salt seen on figure 7a and 7d the absolute configuration of harringtonine or homoharringtonine and cephalotaxine are 3S,4S,5R,2'R and 3S,4S,5R respectively. This confirm definitely the predictive observation of Seitz et al (see above reference) based on biological observation of omacetaxine in its ribosome site at the crystalin state.

The purpose of this invention is to overcome the above mentioned difficulties by the following actions:

-generate a hydrosoluble form of the molecule of harringtonine exhibiting a protonated nitrogen and which can be formulated as is it.

-provide the medical community with a cheaper drug, easier to use including orally, with an improved local tolerance when administrated subcutaneously and exhibiting a good long-term stability

-assess the new chemical entities object of this invention by means of preclinical study conducted adequately.

-incidentally definitively confirm the absolute configuration of the harringtonine itself from one of its chiral organic salts

-improve purification of harringtonine prepared from natural source In brief:

The present invention concerns novel water soluble solid salts of harringtonine and their use as new chemical entities for the formulation of new cancer chemotherapeutic agents, or immunosuppressive or antiparasitic and to implement new processes for purification including enantiomeric and determine the absolute configuration of the series.

The present invention describes the preparation of crystalline salts of harringtonine as nitrogen-protonated form, stable and soluble in water and their use for the manufacture of pharmaceutical composition useful in the treatment of cancers, leukemias, lymphpmas, immune disease and as reversal agents.

The present invention provides solid salts of harringtonine, definite by their analysis patterns, their process of preparation from harringtonine free base and commercial organic acid allowing their use as drug substance for blending alone or in combination in pharmaceutical composition particularly useful for treatment of cancer in using oral mode of administration.

A preferred embodiment of the invention is a crystalline harringtonine hydrogen (2S)-malate having substantially the same IR spectrum, in the solid state as set out in figure 2, the same single crystal X-ray diffractogram as set out in figure 7a and 7b, the same X-ray powder pattern as set out in figure 7c and the same DSC curve as set out in figure 6 and the same 1 H NMR spectra as set out in figure 4b.

A further preferred embodiment of the invention provides a harringtonine hydrogen 2R-malate having substantially the same IR spectrum, in the solid state as set out in figure 3a

A further preferred aspect of the invention is a glassy solid harringtonine hydrogen (2S,3S)-tartrate having substantially the same IR spectrum, in the solid state as set out in figure 3b

Yet, a further embodiment of the invention is a glassy solid harringtonine hydrogen (2R,3R)-tartrate having substantially the same IR spectrum, in the solid state as set out in figure 3c

Yet, another embodiment of the invention provides a glassy solid harringtonine hydrogen (2S)-citramalate having substantially the same IR spectrum, in the solid state as set out in figure 3d

Yet, a prefered aspect of this invention is a solid harringtonine hydrogen (2R)-citramalate having substantially the same IR spectrum, in the solid state as set out in figure 3e

Yet, a further prefered aspect of this invention is a solid harringtonine hydrogen itaconate having substantially the same IR spectrum, in the solid state as set out in figure 3f

Yet, another prefered aspect of this invention provides a solid harringtonine hydrogen succinate having substantially the same IR spectrum, in the solid state as set out in figure 3g

Yet, a another aspect of the invention provides a solid harringtonine hydrogen tartronate having substantially the same IR spectrum, in the solid state as set out in figure 3h

Moreover, a prefered embodiment of this invention provides a crystalline harringtonine dihydrogen citrate having substantially the same IR spectrum, in the solid state as set out in figure 3i and the same DSC curve as set out in figure 6.

Another aspect of the invention provides a process of purification, including enantiomeric, which use formation of any of the salt of this invention to perform fractional crystallization.

Yet, a preferred aspect of this invention provides a pharmaceutical composition comprising an effective amount of one of the salts of this invention, together with one or more pharmaceutical acceptable inactive components such as carriers, excipients, adjuvants or diluents.

Yet, a preferred aspect of this invention provides a pharmaceutical dosage form comprising an effective amount of one of the harringtonine salts, dedicated to an oral mode of administration selected among, for example, capsules, dragees, emulsions, granules, pills, powders, solutions, suspensions, tablets, microemulsions, elixirs, syrups, tea or powders for reconstitution

Yet, a another aspect of this invention provides a pharmaceutical dosage form comprising an effective amount of one of the harringtonine salts of this invention dedicated to a subcutaneous mode of administration in non-acidic condition allowing a good locale tolerance and no reaction at the injection site Another aspect of the invention is the use of at least the solid form comprising an effective amount of one of the harringtonine salts described in the invention for preparing the above pharmaceutical composition as (i) chemotherapeutic agent, (ii) enhancer of other chemotherapeutic agents (iii) new efficacy after failure of other agents (iv) for inhibiting tumors growth in animal, (v) for inhibiting mammalian parasites, (vi) as immunosuppressive agent, or (vii) as reversal agent.

A preferred embodiment of the invention describes a method for treating mammalian tumors including leukemia and lymphoma which comprises oral administering to a mammal an antitumor effective amount of the solid form of one salt described in this invention.

A further preferred embodiment of the invention describes a method for treating mammalian tumors which comprises implantable pharmaceutical preparation administering to a mammal an antitumor effective amount of the solid form of at least one salt described in this invention.

Yet, invention is also concerned with the use of solid form as defined above, for the preparation of pharmaceutical compositions for the treatment of cancer particularly, ovarian serous high-grade carcinoma including those resistant to existing therapy, breast cancer including triple negative breast carcinoma and eventually their metastasis, pancreatic cancer including ductal adenocarcinoma.

Yet, a preferred embodiment of the invention describes the use of solid form as defined above, for the preparation of pharmaceutical compositions for the treatment of lymphocytic leukemia and lymphoma

Finally, the invention is also concerned with the use of solid form as defined above, for the preparation of pharmaceutical compositions for the treatment of leukemias particularly acute myelod leukemia (AML), myelodysplastic syndroma (MDS) and myeloproliferative disorders including chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, myelosclerosis and myelomonocytic leukemia.

Example 1 : General procedure for experimental methods

1.1 General procedures for salts preparation General procedure 1A:

Alkaloid base and acid components are dissolved separately in equimolecular concentration in a solvent at a concentration close of saturation and at a temperature close of boiling then both solutions are mixed under stirring then slowly cooled and evaporated. After a period ranging from a few minutes up to several days, crystal salt is collected. A sample of the batch of crystals is 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.

General procedure 1 B:

Alkaloid base and acid components are intimately mixed by grinding in a mortar. A small amount (0.01 mmol) of the reaction mixture is loaded into an aluminium capsule and placed under nirogen atmosphere, in the furnace of a differential scanning calorimetry (DSC) apparatus for thermodynamic studies. Condition were reproduced at 0.1 mmol. Scales. Crystals were collected, then analyzed in the solid state or dissolved in minimum of a suitable solvent for recrystallization. General procedure 1 C:

Alkaloid base and acid components are intimately mixed by grinding in a mortar in the presence of a suitable solvent. The mixture is abandoned for several days.

1.2 General procedures for solid state characterization

Single crystal X-Ray diffractions material and methods

Kappa CCD, Nonius diffractometer, M o - Ka 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 Diffraction Powder

Diagrams were measured on a Bruker AXS D8 Advance diffractometer, Bragg-Brentano geometry (θ-2 Θ), CuK a = 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 Calorimetry (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 temperature was 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.

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.

1.3 General procedures for liquid state and solution characterizations

Nuclear Magnetic Resonance

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 B, 13C, 15N, 19F, 27AI, 31 P, 1 19Sn or on Bruker Avance III - 600MHz spectrometer.

Dissolving salts for ¹³C NMR: 30 mg of compound were dissolved in 600 μΐ_ (5% m/V) of methanol D₄ 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 D₂O and/or MOD₄ as solvents.

Most of 13C spectra were recorded according to Attached Proton Test mode (APT: includes quaternary carbons) or DEPT135: Distortionless Enhancement by Polarization Transfer (non-quaternary carbons only).

High Performance Liquid Chromatography

Routine experiments were performed on a Waters HPLC-MS-DAD coupled system (3100 pump, DAD 996 detector, 3100 mass detector). Solubility determination: Solubility in sterile water for injection at 25 ° C was measured semi-quantitatively at a threshold of 50 mg per mL

Example 2: Analysis of alkaloid base for comparison with their salts

2.2 Analysis of harringtonine base alkaloid for comparison

Batch #: 62H0301 from SIGMA

NMR spectra were performed in deuterated methanol for comparison with salt in the same solvent.

¹H NMR (400 MHz, Benzene-d₆) δ 6.54 (s, 1 H), 6.46 (s, 1 H), 6.13 (d, J = 9.8 Hz, 1 H), 5.46 (d, J = 1 .4 Hz, 1 H), 5.34 (d, J = 1 .4 Hz, 1 H), 4.67 (s, 1 H), 3.98 (s, 1 H), 3.44 (d, J = 9.8 Hz, 1 H), 3.35 (s, 3H), 3.22 (s, 3H), 3.21 - 3.14 (m, 1 H), 2.84 (m, 1 H), 2.75 (m, 1 H), 2.54 (dd, J = 10.8, 7.5 Hz, 1 H), 2.47 - 2.37 (m, 2H), 2.19 - 2.08 (m, 2H), 1 .83 - 1 .57 (m, 5H), 1 .47 (m, 3H), 1 .09 (s, 3H), 1 .06 (s, 3H).

1H NMR (400 MHz, Chloroform-d) δ 6.61 (s, 1 H), 6.54 (s, 1 H), 5.99 (d,

J = 9.8 Hz, 1 H), 5.87 (d, J = 1 .4 Hz, 1 H), 5.86 (s, 1 H), 5.07 (s, 1 H), 3.78 (d, J = 9.8 Hz, 1 H), 3.69 (s, 3H), 3.57 (s, 3H), 3.16 - 3.04 (m, 2H), 2.93 (td, J = 12.0, 1 1 .6, 7.0 Hz, 1 H), 2.62 - 2.53 (m, 2H), 2.38 (dd, J = 14.1 , 6.8 Hz, 1 H), 2.28 (d, J = 16.5 Hz, 1 H), 2.03 (dt, J = 12.1 , 9.6 Hz, 1 H), 1 .94 - 1 .86 (m, 2H), 1 .80 - 1 .66 (m, 5H), 1 .66 - 1 .54 (m, 3H), 1 .32 - 1 .18 (m, 1 H), 1 .16 (s, 3H), 1 .14 (s, 3H). ¹H NMR (400 MHz, Methanol-d₄) δ 6.67 (s, 1 H), 6.59 (s, 1 H), 5.98 (dd, J = 9.8, 0.8 Hz, 1 H), 5.89 (d, J = 1 .2 Hz, 1 H), 5.84 (d, J = 1 .2 Hz, 1 H), 5.22 (d, J = 0.6 Hz, 1 H), 3.89 (d, J = 9.8 Hz, 1 H), 3.70 (s, 3H), 3.55 (s, 3H), 3.26 - 3.15 (m, 1 H), 3.02 - 2.83 (m, 2H), 2.65 (d, J = 8.1 Hz, 2H), 2.44 (dd, J = 14.2, 6.8 Hz, 1 H), 2.18 (d, J = 16.1 Hz, 1 H), 2.08 - 1 .91 (m, 2H), 1 .86 (d, J = 16.1 Hz, 2H), 1 .62 - 1 .49 (m, 3H), 1 .38 - 1 .27 (m, 1 H), 1 .15 (s, 3H), 1 .12 (s, 3H).

¹³C NMR APT* (101 MHz, Methanol-d4) δ 174.61 , 171 .78, 159.95, 148.23, 147.35, 134.51 , 129.95, 1 14.02, 1 10.91 , 102.14, 100.86, 75.86, 75.48, 72.19, 70.76, 57.86, 56.54, 54.65, 52.04, 44.41 , 43.92, 37.57, 35.10, 32.20, 29.91 , 28.56, 20.88.

^*APT = Attached Proton Test

IR (Diamond ATR, solid) cm ¹ 3338.6, 2973.0, 1743.9, 1723.7, 1655.4, 1623.9, 1502.8, 1485.9, 1438.7, 1364.3, 1339.5, 1306.4, 1277.4, 1261 .2, 1223.0, 1203.8, 1 161 .7, 1 1 10.3, 1082.4, 1061 .6, 1032.9, 1021 .1 , 989.9, 927.4, 850.1 , 805.6, 722.8, 709.3, 690.1 , 670.3, 614.8, 548.8, 498.9, 479.6, 472.6, 460.7, 452.9 (see figure 1 b).

Solubility in sterile water for injection: lower than 10 img/mL

This ionic compound was obtained from commercial harringtonine (batch # 52H00301 ) mixed with commercial (2S)-malic acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as an off-white crystalline solid mp 159-167°C (DSC) from Methanol. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media ¹H NMR (400 MHz, Methanol-d₄) δ 6.80 (s, 1H), 6.74 (s, 1H), 6.08 (d, J = 9.5 Hz, 1 H), 5.96 (d, J = 1.0 Hz, 1 H), 5.91 (d, J = 1.0 Hz, 1 H), 5.33 (s, 1 H), 4.31 (dd, J = 7.5, 5.1 Hz, 1H), 4.17 (d, J = 9.6 Hz, 1H), 3.81 (s, 3H), 3.53 (s, 3H), 3.45-3.30 (m, 2H), 3.19 (dd, J = 11.2, 7.1 Hz, 1H), 2.75 (dd, J = 15.9, 5.1 Hz, 1H), 2.67 (dd, J = 13.9, 6.1 Hz, 1H), 2.57 - 2.46 (m, 1H), 2.29 - 2.09 (m, 4H), 1.98 (dd, J = 9.1, 3.9 Hz, 1 H), 1.89 (d, J = 16.1 Hz, 1 H), 1.59 - 1.46 (m, 3H), 1.28 (d, J= 12.3 Hz, 1H), 1.13 (s, 3H), 1.10 (s, 3H).

^1jC NMR APT* (101 MHz Methanol-d₄) δ 178.24, 175.34, 174.12, 171.60, 165.23, 149.81, 148.81, 130.72, 126.73, 114.85, 111.85, 102.90, 95.94, 78.37, 75.87, 74.27, 70.67, 68.97, 58.87, 54.22, 53.12, 52.09, 44.30, 41.12, 40.38, 37.45, 35.09, 30.28, 29.65, 29.15, 28.83, 19.91.

*APT = Attached Proton Test

IR (Diamond ATR, solid) cm¹ 3463, 3408.2, 2967, 1756.2, 1741.2, 1703.5, 1657.6, 1505.5, 1489.5, 1447.7, 1374.5, 1359.1, 1335.6, 1289.7, 1266.3, 1226.4, 1207.9, 1150.8, 1108.5, 1081.3, 1031.1, 987.2, 945, 923.4, 902.8, 865.4, 817.6, 771.2, 718.1 , 615.1 , 564.4.

IR (Diamond ATR, film) cm¹ 3442.4, 2966.4, 2502.5, 2156.3, 2005, 1958.5, 1738, 1654.6, 1592, 1504.9, 1488.6, 1463.7, 1439.5, 1371.5, 1265, 1221.6, 1167.3, 1110.3, 1081, 1032.7, 986.6, 928.2 , 855, 805, 709.5, 647, 614.3, 565.

See figure 2

A. Single crystal X-ray diffraction (see figure 7a and 7b) From a suspension in its mother liquor, a suitable single crystal of size

0.24 x 0.18 x 0.16 mm was finally selected and implemented on the diffracto meter.

Structural data

Empirical formula C32H44.14/V 014.57

Extended formula C28H38A/ O9, C4H5O5, 0.57(H2O)

Formula weight 675.9

Temperature 150(2) K

Wavelength 0.71073 A

Crystal system, space group orthorhombic, P 21 21 21 Unit cell dimensions a = 10.9975(2) A, o =

90 ^° b = 15.9436(4) A, β = 90 ^° c = 18.8372(3) A, γ = 90 ^°

Volume 3302.91 (12) A³

Z, Calculated density 4 , 1 .359 (g.cm- )

Absorption coefficient 0.108 mm^~^

F(000) 1438.7

Crystal size 0.24 x 0.18 x 0.16 mm

Crystal color colourless

Theta range for data collection 3.12 to 27.48 ^° h min, h_max -13 , 14

k_min, k max -15 , 20

l_min, l_max -24 , 18

Reflections collected / unique 1 6541 / 4191 [^aR(int)

= 0.0308] Reflections [/ > 2σ] 3635

Completeness to theta_max 0.993

Absorption correction type multi-scan

Max. and min. transmission 0.983 , 0.856

Refinement method Full-matrix least-squares on Data / restraints / parameters 41 91 / 2 / 459

^Goodness-of-fit 1 .042

Final R indices [/ > 2σ] ^cf?1 = 0.0454, ^dwR2 0.1 1 18

R indices (all data) ^cf?1 = CL055, ^dwR2 = 0.1 19 Largest diff. peak and hole 0.298 and -0.303 e.A^-3

Atomic coordinates, site occupancy (%) and equivalent isotropic displacement parameters (A2 x 103). U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

Atom numbering of figure 7a corresponds to below table.

Atom x y z occ. U(eq)

C1 0.6092(3) 0.92767(1 0.731 1 1 (1 1 0.0395(7) H1 0.6281 0.9464 0.7777 1 0.047 C2 0.6067(3) 0.97694(1 0.67446(1 1 0 0333(6)

C3 0.5732(2) 0.93274(1 0.60722(1 1 0 0283(5)

H3 0.4941 0.9547 0.5888 1 0 034

C4 0.5590(3) 0.83921 (1 0.62951 (1 1 0 0267(5)

H4 0.4714 0.8251 0.6218 1 0 032

C5 0.5785(3) 0.83870(1 0.71241 (1 1 0 0342(6)

C6 0.471 1 (3) 0.8016(2) 0.75229(1 1 0 0428(8)

H6A 0.4418 0.75 0.7285 1 0 051

H6B 0.4034 0.8424 0.7548 1 0 051

C7 0.5187(4) 0.7814(2) 0.82656(1 1 0 0590(1 1 )

H7A 0.5046 0.829 0.8593 1 0 071

H7B 0.4783 0.7309 0.846 1 0 071

C8 0.6546(4) 0.7660(2) 0.81631 (1 1 0 0564(1 1 )

H8A 0.7029 0.8062 0.8448 1 0 068

H8B 0.6765 0.7083 0.8308 1 0 068

N9 0.6778(3) 0.77872(1 0.73741 (1 1 0 0370(6)

HN9 0.661 (3) 0.732(2) 0.716(2) 1 0 05

C10 0.8064(3) 0.8010(2) 0.72275(1 1 0 0466(8)

H10A 0.8593 0.7533 0.736 1 0 056

H10B 0.8298 0.8497 0.7524 1 0 056

C11 0.8266(3) 0.82246(1 0.64466(1 1 0 0366(7)

H11A 0.8016 0.8813 0.6364 1 0 044

H11 B 0.9145 0.8181 0.6339 1 0 044

C12 0.7572(2) 0.76631 (1 0.59452(1 1 0 0293(6)

C13 0.6303(2) 0.77493(1 0.58784(1 1 0 0251 (5)

C14 0.5648(2) 0.72070(1 0.5431 1 (1 1 0 0278(5)

H14 0.4791 0.7256 0.5381 1 0 033

C15 0.6284(3) 0.66071 (1 0.50702(1 1 0 0322(6)

C16 0.7526(3) 0.65208(1 0.51376(1 1 0 0355(6)

C17 0.8189(3) 0.70265(1 0.55758(1 1 0 0346(6)

H17 0.9041 0.695 0.5629 1 0 042

C18 0.6841 (4) 0.5463(2) 0.4477(2) 1 0 0540(9)

H18A 0.6731 0.4943 0.4757 1 0 065 H18B 0.6895 0.5311 0.3968 1 0.065

C19 0.6735(3) 1.09847(1 0.73109(1 1 0.0415(7)

H19A 0.7421 1.0664 0.7503 1 0.062

H19B 0.6077 1.1 0.7661 1 0.062

H19C 0.7 1.1558 0.7205 1 0.062

C21 0.6476(3) 1.00078(1 0.50246(1 1 0.0289(5)

C22 0.7610(3) 1.00712(1 0.45519(1 1 0.0302(6)

C23 0.8041(3) 0.91949(1 0.43315(1 1 0.0334(6)

H23A 0.8731 0.9252 0.3997 1 0.04

H23B 0.834 0.8894 0.4757 1 0.04

C24 0.7058(3) 0.8678(2) 0.39846(1 1 0.0361(6)

C25 0.6661(4) 0.7485(3) 0.3298(2) 1 0.0639(11)

H25A 0.6124 0.7818 0.2993 1 0.096

H25B 0.6177 0.7198 0.3661 1 0.096

H25C 0.7091 0.7068 0.301 1 0.096

C31 0.8632(3) 1.05194(1 0.49690(1 1 0.0338(6)

H31A 0.8878 1.0162 0.5374 1 0.041

H31B 0.9347 1.0587 0.4655 1 0.041

C32 0.8259(3) 1.1374(2) 0.52484(1 1 0.0438(8)

H32A 0.7545 1.1301 0.5562 1 0.053

H32B 0.8001 1.1724 0.4842 1 0.053

C33 0.9244(3) 1.1852(2) 0.56618(1 1 0.0450(8)

C34 0.9712(4) 1.1358(3) 0.62879(1 1 0.0536(9)

H34A 0.9024 1.1132 0.6557 1 0.08

H34B 1.0195 1.1726 0.6595 1 0.08

H34C 1.0222 1.0895 0.6118 1 0.08

09 0.8630(3) 1.25847(190.5920(2) 1 0.0901(13)

H09 0.917(4) 1.291(3) 0.607(2) 1 0.05

C35 1.0305(5) 1.2120(3) 0.5197(2) 1 0.0824(16)

H35A 1.093 1.2386 0.5492 1 0.124

H35B 1.002 1.2521 0.4839 1 0.124

H35C 1.065 1.1627 0.496 1 0.124

01 0.6310(2) 1.05932(120.66745(1 1 0.0380(5) 02 0.5839(2) 0.60228(140.45926(1 1 0.0443(5)

03 0.7924(2) 0.58901(140.46960(1 1 0.0499(6)

04 0.66747(160.94408(120.55453(91 0.0275(4)

05 0.55816(191.04265(140.49545(1 1 0.0382(5)

06 0.7328(2) 1.05391(150.39365(1 1 0.0389(5)

H06 0.658(4) 1.069(2) 0.391(2) 1 0.05

07 0.5987(2) 0.88175(180.40232(1 1 0.0499(6)

08 0.7526(2) 0.80258(140.36350(1 1 0.0469(6)

C51 0.7090(3) 0.5732(2) 0.70843(1 1 0.0431(7)

C52 0.7177(3) 0.4893(2) 0.67006(1 1 0.0392(7)

H52 0.7018 0.4437 0.7053 1 0.047

C53 0.8400(3) 0.4730(2) 0.63572(1 1 0.0415(7)

H53A 0.8285 0.4319 0.5969 1 0.05

H53B 0.869 0.5259 0.614 1 0.05

C54 0.9381(3) 0.4404(2) 0.68470(1 1 0.0430(7)

051 0.7969(3) 0.59209(190.74955(1 1 0.0817(10)

052 0.6201(2) 0.61842(140.69489(1 1 0.0412(5)

053 0.6250(2) 0.48657(180.61778(1 1 0.0688(9)

H053 0.577(3) 0.536(2) 0.6376(191 0.05

054 0.9511(3) 0.4777(2) 0.74438(1 1 0.0854(11)

H054 0.895(3) 0.524(2) 0.7592(191 0.05

055 1.0033(2) 0.38077(170.66726(1 1 0.0581(7)

0W1 0.6613(5) 0.6304(3) 0.9546(3) 0.568( 0.0559(18)

HW1A 0.671(7) 0.659(4) 0.994(3) 0.57 0.05

HW1B 0.600(5) 0.607(4) 0.939(4) 0.57 0.05

A. X-ray powder diffraction

The powder sample is well crystallised, with a peak width of 0.119 °(2Θ) at 17.627 °(2Θ). The sample is without impurity, as shown by the good agreement between calculated and experimental patterns (for view of diagrams and experimental details, see figure 7c) Determination of absolute configuration: based upon the well known absolute configuration of 2S-malate and the relative configuration of the salt seen on figure 7a and 7d the absolute configuration of harringtonine or homoharringtonine and cephalotaxine are 3S,4S,5R,2'R and 3S,4S,5R respectively

Solubility in sterile water for injection: higher than 50 img/mL

Example 3: Preparation and analyses of harringtonine hydrogen "'R)-malate

This ionic compound was obtained from commercial harringtonine (batch # 52H00301 ) mixed with commercial (2R)-malic acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media

¹H NMR (400 MHz, Methanol-d₄) δ 6.80 (s, 1 H), 6.74 (s, 1 H), 6.08 (d, J = 9.6 Hz, 1 H), 5.96 (d, J = 1 .0 Hz, 1 H), 5.92 (d, J = 1 .0 Hz, 1 H), 5.33 (s, 1 H), 4.27 (dd, J = 7.4, 5.4 Hz, 1 H), 4.17 (d, J = 9.6 Hz, 1 H), 3.81 (s, 3H), 3.54 (s, 3H), 3.45 - 3.31 (m, 2H), 3.22 - 3.15 (m, 1 H), 2.76 (dd, J = 15.9, 5.4 Hz, 1 H), 2.68 (dd, J = 13.2, 5.3 Hz, 1 H), 2.49 (dd, J = 15.9, 7.5 Hz, 1 H), 2.30 - 2.07 (m, 4H), 1 .97 (d, J = 3.6 Hz, 1 H), 1 .90 (d, J = 16.1 Hz, 1 H), 1 .61 - 1 .48 (m, 3H), 1 .34 - 1 .24 (m, 1 H), 1 .14 (s, 3H), 1 .1 1 (s, 3H).

¹³C NMR APT* (101 MHz, Methanol-d₄) δ 178.99, 175.89, 174.17, 171 .63, 165.17, 149.81 , 148.82, 130.85, 126.85, 1 14.88, 1 1 1 .88, 102.93, 96.11, 78.27, 75.91, 74.34, 70.70, 69.25, 58.89, 54.25, 53.23, 52.13, 44.35, 41.63, 40.50, 37.51, 35.14, 29.71, 29.26, 28.88, 19.97.

*APT = Attached Proton Test

IR (Diamond ATR, film) cm¹ 1732.1, 1654.8, 1505.2, 1488.9, 1439.4, 1371.2, 1333.6, 1264.7, 1221.4, 1167.3, 1109.3, 1080.6, 1029.6, 987, 927.1. See figure 3a.

Solubility in sterile water for injection: higher than 50 img/mL

Example 4: Preparation and analyses of harringtonine hydrogen (2"'S,3"'S)-tartrate

This ionic compound was obtained from commercial harringtonine (batch # 52H00301) mixed with commercial (2S,3S)-tartaric acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a pale yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media

¹H NMR (400 MHz, Methanol^) δ 6.81 (s, 1H), 6.75 (s, 1H), 6.09 (d, J = 9.6 Hz, 1 H), 5.97 (d, J = 1.0 Hz, 1 H), 5.93 (d, J = 1.0 Hz, 1 H), 5.34 (s, 1 H), 4.37 (s, 2H), 4.18 (d, J= 9.6 Hz, 1H), 3.82 (s, 3H), 3.55 (s, 3H), 3.49-3.31 (m, 2H), 3.28 - 3.17 (m, 1H), 2.69 (dd, J = 13.8, 6.0 Hz, 1H), 2.30 - 2.11 (m, 4H), 2.05 - 1.94 (m, 1 H), 1.91 (d, J = 16.1 Hz, 1 H), 1.62 - 1.46 (m, 3H), 1.35 - 1.24 (m, 1H), 1.14 (s, 3H), 1.12 (s, 3H).

¹³C NMR APT* (101 MHz, Methanol-d4) δ 176.47, 174.12, 171.59, 165.19, 149.81, 148.81, 130.73, 126.72, 114.85, 111.87, 102.90, 96.01, 78.36, 75.87, 74.29, 73.95, 70.66, 58.88, 54.26, 53.14, 52.09, 48.95, 44.30, 40.40, 37.46, 35.09, 29.66, 29.16, 28.83, 19.92.

*APT = Attached Proton Test

IR (Diamond ATR, film) cm^-1 2968.3, 1737.1 , 1655.1 , 1613.7, 1505.5, 1489.1 , 1439.4, 1372.3, 1264.6, 1222.9, 1 1 14.5, 1081 .4, 1033.5, 928. See figure 3b.

Solubility in sterile water for injection: higher than 50 img/mL

Example 5: Preparation and analyses of harringtonine hydrogen

(2"'R,3"'R)-tartrate

This ionic compound was obtained from commercial harringtonine (batch # 52H00301 ) mixed with commercial (2R,3R)-tartaric acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media

¹H NMR (400 MHz, Methanol-d₄) δ 6.81 (s, 1 H), 6.75 (s, 1 H), 6.09 (d, J = 9.6 Hz, 1 H), 5.97 (d, J = 1 .0 Hz, 1 H), 5.93 (d, J = 1 .0 Hz, 1 H), 5.34 (s, 1 H), 4.37 (s, 2H), 4.19 (d, J = 9.6 Hz, 1 H), 3.82 (s, 3H), 3.55 (s, 3H), 3.47 - 3.32 (m, 2H), 3.21 (dd, J = 1 1 .2, 7.1 Hz, 1 H), 2.69 (dd, J = 13.8, 6.0 Hz, 1 H), 2.30 - 2.1 1 (m, 4H), 1 .91 (d, J = 16.1 Hz, 1 H), 1 .62 - 1 .48 (m, 3H), 1 .36 - 1 .24 (m, 1 H), 1 .14 (s, 3H), 1 .12 (s, 3H).

¹³C NMR APT* (101 MHz, Methanol-d₄) δ 176.52, 174.13, 171 .59, 165.19, 149.82, 148.83, 130.74, 126.72, 1 14.83, 1 1 1 .86, 102.90, 96.02, 78.35, 75.87, 74.30, 73.96, 70.66, 58.88, 54.24, 53.14, 52.09, 48.94, 44.30, 40.41 , 37.46, 35.09, 29.66, 29.17, 28.84, 19.91 . ^*APT = Attached Proton Test

IR (Diamond ATR, film) cm¹ 3445, 2968.1, 1737.4, 1655.1, 1610.9,

1505.6, 1489.1, 1439.7, 1372.3, 1264.8, 1223.1, 1114.4, 1081.3, 1033.2, 928.2. See figure 3c.

Solubility in sterile water for injection: higher than 50 mg/mL.

Example 6: Preparation and analyses of harringtonine hydrogen (2"'S)-citramalate

This ionic compound was obtained from commercial harringtonine (batch # 52H00301) mixed with commercial (2S)-citramalic acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media

¹H NMR (400 MHz, Methanol-d₄) δ 6.80 (s, 1H), 6.74 (s, 1H), 6.09 (d, J = 9.6 Hz, 1H), 5.96 (d, J = 0.9 Hz, 1H), 5.92 (d, J = 0.8 Hz, 1H), 5.33 (s, 1H), 4.17 (d, J = 9.6 Hz, 1 H), 3.82 (s, 3H), 3.54 (s, 4H), 3.44 - 3.31 (m, 2H), 3.24 - 3.14 (m, 1 H), 2.73 - 2.59 (m, 3H), 2.30 - 2.09 (m, 4H), 1.97 (s, 1 H), 1.90 (d, J = 16.1 Hz, 1H), 1.61 -1.46 (m, 3H), 1.37 (s, 3H), 1.34- 1.23 (m, 1H), 1.14 (s, 3H), 1.11 (s, 3H).

¹³C NMR APT* (101 MHz, Methanol-d4) δ 181.23, 176.12, 174.12, 171.59, 165.12, 149.78, 148.79, 130.81, 126.81, 114.83, 111.83, 102.87, 96.06, 78.23, 75.87, 74.30, 73.19, 70.65, 58.84, 54.22, 53.20, 52.08, 48.94, 46.63, 44.30, 40.47, 37.46, 35.10, 29.67, 29.22, 28.84, 26.49, 19.92.

^*APT = Attached Proton Test IR (KBr), cm^"1 3609.7, 3427, 2970.8, 2938.4, 1757.9, 1652.4, 1551.4, 1504.9, 1489.4, 1469.4, 1449.5, 1404.1, 1374.4, 1352.8, 1309.1, 1293.3, 1268.2, 1248.4, 1228.4, 1194.6, 1166.9, 1151, 1128.2, 1111.9, 1085.7, 1064, 1031.5, 1000.8, 921.2. See figure 3d.

Solubility in sterile water for injection: higher than 50 img/mL

Example 7: Preparation and analyses of harringtonine hydrogen

(2"'R)-citramalate

This ionic compound was obtained from commercial harringtonine (batch

# 52H00301) mixed with commercial (2S)-citramalic acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a pale yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media

¹H NMR (400 MHz, Methanol^) δ 6.79 (s, 1H), 6.73 (s, 1H), 6.08 (d, J = 9.6 Hz, 1 H), 5.95 (d, J = 1.0 Hz, 1 H), 5.91 (d, J = 1.0 Hz, 1 H), 5.32 (s, 1 H), 4.15 (d, J= 9.6 Hz, 1H), 3.80 (s, 3H), 3.54 (s, 3H), 3.15 (q, J= 6.7, 6.2 Hz, 1H), 2.65 (d, J = 4.0 Hz, 3H), 2.24 (d, J = 16.2 Hz, 2H), 2.21 - 2.02 (m, 3H), 2.02 - 1.93 (m, 1 H), 1.89 (d, J = 16.1 Hz, 1 H), 1.60 - 1.46 (m, 3H), 1.36 (s, 3H), 1.34 - 1.23 (m, 1H), 1.13 (s, 3H), 1.11 (s, 3H).

¹³C NMR (101 MHz, Methanol-d4)* δ 181.52, 176.35, 174.14, 171.59, 164.90, 149.71, 148.72, 130.98, 126.95, 114.80, 111.79, 102.83, 96.27, 77.97, 75.86, 74.35, 73.19, 70.66, 58.80, 54.25, 53.35, 52.08, 46.80, 44.31, 40.62, 37.47, 35.10, 29.68, 29.36, 28.82, 26.47, 19.97.

^*APT = Attached Proton Test IR (ATR) cm^"1 3412, 2968, 1738, 1655, 1587, 1505, 1489, 1439, 1372, 1341, 1265, 1221, 1163, 1111, 1081, 1030, 969, 927, 831, 803, 784, 708, 614, 561, 502, 473. See figure 3e.

Solubility in sterile water for injection: higher than 50 img/mL

Example 8: Preparation and analyses of harringtonine hydrogen itaconate o

This ionic compound was obtained from commercial harringtonine (batch

# 52H00301) mixed with commercial itaconic acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media.

¹H NMR (400 MHz, Methanol-d₄) δ 6.79 (s, 1H), 6.73 (s, 1H), 6.08 (d, J = 9.6 Hz, 1 H), 6.02 (d, J = 1.6 Hz, 1 H), 5.95 (d, J = 0.9 Hz, 1 H), 5.91 (d, J = 0.9 Hz, 1 H), 5.52 (d, J = 1.3 Hz, 1 H), 5.32 (s, 1 H), 4.15 (d, J = 9.6 Hz, 1 H), 3.81 (s, 3H), 3.54 (s, 3H), 3.48 (m, 1H), 3.19 - 3.08 (m, 1H), 2.73 - 2.60 (m, 1H), 2.25 (m, 1H), 2.22 - 2.07 (m, 3H), 1.90 (d, J = 16.1 Hz, 1H), 1.61 - 1.46 (m, 3H), 1.36 - 1.25 (m, 1 H), 1.14 (s, 3H), 1.12 (s, 3H).

¹³C NMR (101 MHz, MeOD)^** δ 125.29, 114.51, 111.50, 102.54, 96.02, 74.06, 58.50, 53.96, 53.09, 51.79, 48.70, 44.03, 41.50, 40.37, 37.18, 34.82, 29.39, 29.10, 28.53, 19.69.

*^*DEPT135: Distortionless Enhancement by Polarization Transfer (non- quaternary carbons only) IR (Diamond ATR, solid) cm ¹ 3418, 2967, 1736, 1655, 1568, 1505, 1489, 1440, 1371 , 1265, 1220, 1 164, 1 1 12, 1081 , 1032, 928, 856, 827, 804, 729, 690, 674, 614, 557, 504, 473. See figure 3f.

IR (Diamond ATR, film) cm ¹ 3437, 2969, 2868, 2845, 1742, 1655, 1572, 1505, 1489, 1464, 1440, 1373, 1267, 1223, 1 1 13, 1082, 1057, 1033, 931 , 828, 736, 615, 562, 506, 475

Solubility in sterile water for injection: higher than 50 img/mL

Example 9: Preparation and analyses of harringtonine hydrogen succinate

This ionic compound was obtained from commercial harringtonine (batch # 52H00301 ) mixed with commercial itaconic acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media. 1H NMR (400 MHz, Methanol-d₄) δ 6.77 (s, 1 H), 6.71 (s, 1 H), 6.06 (d, J

= 9.6 Hz, 1 H), 5.94 (d, J = 1 .1 Hz, 1 H), 5.90 (d, J = 1 .1 Hz, 1 H), 5.31 (s, 1 H), 4.1 1 (d, J = 9.7 Hz, 1 H), 3.79 (s, 3H), 3.54 (s, 3H), 3.45 - 3.36 (m, 1 H), 3.22 - 3.10 (m, 1 H), 3.07 (q, J = 5.5 Hz, 1 H), 2.67 - 2.56 (m, 1 H), 2.49 (s, 4H), 2.23 (d, J = 16.1 Hz, 1 H), 2.20 - 2.02 (m, 3H), 1 .89 (d, J = 16.1 Hz, 1 H), 1 .61 - 1 .46 (m, 3H), 1 .35 - 1 .24 (m, 1 H), 1 .14 (s, 3H), 1 .1 1 (s, 3H). ^1jC NMR (101 MHz, Methanol)^** δ 114.45, 111.43, 102.48, 96.43, 74.15, 58.41, 53.98, 53.35, 51.79, 48.72, 44.04, 40.63, 37.19, 34.82, 32.06, 29.41, 29.33, 28.50, 19.76.

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

IR (Diamond ATR, solid) cm¹ 3398, 2967, 2845, 2077, 1736, 1655, 1570, 1505, 1489, 1464, 1440, 1371, 1332, 1265, 1220, 1165, 1112, 1081, 1059, 1033, 927, 832, 804, 768, 752, 709, 690, 658, 614, 562, 503, 475. See figure 3g.

IR (Diamond ATR, film) cm¹ 3704, 3681, 3377, 2969, 2923, 2868, 2845, 2069, 1737, 1655, 1571, 1505, 1489, 1463, 1439, 1371, 1332, 1266, 1221, 1166, 1114, 1081, 1058, 1033, 929, 833, 805, 768, 752, 736, 710, 690, 661, 614, 563, 504, 475.

Solubility in sterile water for injection: higher than 50 mg/mL.

Example 10: Preparation and analyses of harringtonine hydrogen tartronate

commercial tartronic acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as a yellow glassy solid. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media.

¹H NMR (400 MHz, Methanol-d₄) δ 6.81 (s, 1H), 6.75 (s, 1H), 6.09 (d, J = 9.6 Hz, 1 H), 5.97 (s, 1 H), 5.93 (s, 1 H), 5.34 (s, 1 H), 4.18 (d, J = 9.6 Hz, 1 H), 3.82 (s, 3H), 3.55 (s, 3H), 2.69 (m, 1H), 2.26 (d, J= 16.1 Hz, H), 1.97 (m, 1H), 1 .91 (d, J = 16.1 Hz, 1 H), 1 .62 - 1 .45 (m, 3H), 1 .31 (m, 1 H), 1 .14 (s, 3H), 1 .12 (s, 3H).

¹³C NMR (101 MHz, MeOD)^** δ 1 14.56, 1 1 1 .55, 102.61 , 95.74, 73.99, 58.58, 53.97, 52.87, 51 .80, 48.69, 44.02, 40.13, 37.17, 34.82, 29.38, 28.89, 28.54, 19.64.

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

IR (ATR), cm^"1 3425.4, 2968.5, 1738.3, 1654.7, 1505.0, 1488.8, 1439.9, 1372.4, 1222.6, 1 162.9, 1082.9, 1034.3, 928.2, 804.3, 615.0, 472.4, 452.6. See figure 3h.

Solubility in sterile water for injection: higher than 50 mg/mL.

Example 11 : Preparation and analyses of harringtonine dihydrogen citrate

This ionic compound was obtained from commercial harringtonine (batch # 52H00301 ) mixed with commercial citric acid according to the general procedure A in which the solvent was methanol-d₄, then isolated as uncolored prismatic solid solid mp 129-136.5 °C (DSC) from MeOH. Kinetic of the reaction was checked by ¹H NMR directly on the reaction media.

¹H NMR (400 MHz, Methanol-d₄) δ 6.79 (s, 1 H), 6.73 (s, 1 H), 6.08 (d, J = 9.7 Hz, 1 H), 5.96 (s, 1 H), 5.91 (s, 1 H), 5.33 (s, 1 H), 4.17 (d, J = 9.6 Hz, 1 H), 3.81 (s, 3H), 3.53 (s, 3H), 2.77 (d, J = 15.4 Hz, 2H), 2.72 - 2.63 (m, 3H), 2.31 - 2.07 (m, 4H), 1.96 (s, 1H), 1.89 (d, J= 16.1 Hz, 1H), 1.62 - 1.44 (m, 3H), 1.32 - 1.23 (m, 1H), 1.13 (s, 3H), 1.11 (s, 3H).

¹³C NMR(101 MHz, CD₃OD)* δ 179.16, 174.86, 174.12, 171.58, 165.14, 149.77, 148.78, 130.77, 126.76, 114.86, 111.86, 102.87, 96.08, 78.31, 75.86, 74.30, 73.99, 70.66, 58.88, 54.23, 53.16, 52.09, 48.92, 44.74, 44.30, 40.42, 37.46, 35.10, 29.67, 29.18, 28.83, 19.94.

^*APT = Attached Proton Test (includes quaternary carbons)

IR (Diamond ATR, solid) cm¹ 3396, 2970, 1750, 1746, 1732, 1651, 1505, 1488, 1402, 1368, 1269, 1228, 1171 , 1149, 1113, 1086, 1029, 930, 908, 884, 830, 751 , 706, 615, 566, 513, 469. See figure 3i.

IR (Diamond ATR, film) cm^-1 3433, 2969, 1735, 1656, 1587, 1506, 1490, 1441 , 1373, 1265, 1223, 1113, 1083, 1033, 929, 615, 479.

Solubility in sterile water for injection: higher than 50 img/mL

Claims

What is claimed is:

1. A isolated harringtonines solid salt having formula 1 ,

FORMULA 1

made by reacting a cephalotaxine ester having formula 2,

FORMULA 2

in which R is, but not limited to, alkyl, aryl, cycloalkyi, heteroalkyi, heteroaryl or heterocycloalkyi, with an acid having general formula AH in a crystallization solvent, wherein the salt has a water or methanol solubility of approximately 5 mg/mL to approximately 100 mg/mL.

2. The salt of claim 1 , in which the cephalotaxine ester reactant is harringtonine having formula 3

3. The salts of claim 1 and 2 having the formula 4

FORMULA 4

in which the acid is an organic acid having the formula ACOOH of which the anion formula is below developed as

4. The salts of claim 3, in which the organic acid is an organic diacid having formula

In which n comprise between 0 and 6 and the interchain moiety may be alkyliden, aryliden including those branched and/or with heterosubstituted variations.

5. The salts of claim 4, in crystalline form, in which the organic diacid is a malic acid having formula

6. The salts of claim 4, in crystalline form, in which the organic diacidinic acid having formula

7. The salts of claim 4, in crystalline form, in which the organic diacidtaric acid having formula

9. The salts of claim 4, in crystalline form, in which the malic acid is of configuration 2R having formula

10. The salts of claim 4, in crystalline form, in which the malic acid is a racemic mixture having formula

H

The salts of claim 4, in which the malate is as an hemimalate

14. The solid salt named harringtonine hydrogen (R)-malate diastereomer of the one of claiml 3 exhibiting the below formula:

17. The solid salt named harringtonine hydrogen (2R,3R)-tartrate diastereomer of the salt of the claim 16 exhibiting the below formula

18. The solid salt named harnngtonine hydrogen (2S)-citramalate exhibiting the below formula:

19. The solid salt named harringtonine hydrogen (2R)-citramalate diastereomer of the salt of the claim 18 exhibiting the below formula:

20. The solid salt named harnngtonine hydrogen itaconate exhibiting the below formula:

21. The solid salt named harringtonine hydrogen tartronate exhibiting the below formula:

23. A method of analysis of the salts of claims 1 to 22 by proton and/or carbon 13, nuclear magnetic resonance in solution in deuterated solvent able to dissolve both the said salts and the corresponding free base, comprising deuterated methanol.

24. A method of determination of absolute configuration of harringtonine based on the use of single crystal X-ray diffraction of a crystalline salt prepared by combination of the said harringtonine with a chiral acid having a known absolute configuration.

25. The method of determination of absolute configuration of the claim 24 in which the crystalline salt is the one mentioned in claims 13 namely harringtonine hydrogen (2S)-malate

26. The cation of harringtonine or harringtoninium which is the nitrogen-protonated form of harringtonine in the solid state and exhibiing the absolute configuration as described in figure 7a and 7b as well as in the below formula

27. The cation of claim 26 in solution in a suitable solvent exhibting specific proton and carbon 13 NMR features

28. The cation of claim 27 in which the suitable solvent is water or deuterated water.

29. The cation of claim 27 in which the suitable solvent is an organic polar solvent or a deuterated organic polar solvent

30. The cation of claim 27 in which the suitable solvent is an alcohol or a deuterated alcohol.

31. The cation of claim 27 in which the suitable solvent is an methanol or deuterated methanol.

32. The process of preparation of salts of claims 1 to 21 comprising contacting a hamngtonine with an organic acid in suspension or in solution in a suitable solvent, preferably an alcohol or in the solid state either at the amorphous state or at the crystalline state then recrystallized said salt in a suitable solvent, preferably an alcohol.

33. The process of claim 7 in which the harringtonine is an hamngtonine analog natural, hemisynthetic or synthetic having general formula

In which R, R3 are independently alkyl, aryl, aralkyl, and their equivalent branched and heterosubstituted.

34. The process of preparation of claims 26 to 31 in which the organic acid is a diacid

35. The process of preparation of claim 32 in which the diacid is a malic acid

36. The process of preparation of claim 32 in which the diacid is a tartaric acid

37. The process of preparation of claim 32 in which the diacid is succinic acid

38. The process of preparation of claim 32 in which the diacid is itaconic acid

39. The process of preparation of claims 26 to 31 in which the organic acid belongs but are not limited to the following list: acetic, propanoic, butanoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, palmitic, oleic, benzoic, p-toluene sulfonic, succinic, itaconic, malic, tartaric, citramalic, tartronic, maleic, fumaric, citric

40. A pharmaceutical dosage form comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a harringtonine salt and/or solvate of any one of claims 1 to 21 .

41. The pharmaceutical dosage form of claim 40 dedicated to an oral mode of administration selected among, for example, capsules, dragees, emulsions, granules, pills, powders, solutions, suspensions, tablets, microemulsions, elixirs, syrups or powders for reconstitution

42. The pharmaceutical dosage form of claim 40 may be osmotically and topically administered dosage forms for example, gels, creams, gels, inhalants, lotions, ointments, powders or pastes.

43. The pharmaceutical dosage form of claim 40 may be parenterally, including intrathecally, administered dosage forms, as, for example, aqueous or oleaginous solution, pseudosolution or suspensions.

44. A method of treatment comprising administering a therapeutically effective amount of a pharmaceutical dosage form of claim 40 to 43 to a patient or an animal suffering from cancer including their metastasis, leukemia, lymphoma, parasitic disease, ocular proliferation and/or immune disorder.

45. The method of treatment of claim 44 in which the leukemia is a myeloid leukemia.

46. The method of treatment of claim 44 and 45 in which the leukemia is any stages of chronic myeloid leukemia including when resisting, relapsing or refractory existing targeted or not therapies.

47. The method of treatment of claim 44 in which the leukemia is a myeloproliferative disorder.

48. The method of treatment of claim 47 in which the myeloproliferative disorder is polycytemia vera.

49. The method of treatment of claim 47 in which the myeloproliferative disorder is myeloslerosis or myelofibrosis.

50. The method of treatment of claim 47 in which the myeloproliferative disorder is essential thrombocytemia.

51. The method of treatment of claim 44 in which the leukemia is any form of myelodysplastic syndroma.

52. The method of treatment of claim 44 in which the cancer is an ovarian cancer

53. The method of treatment of claim 52 in which the ovarian cancer is ovarian serous high-grade carcinoma including those resistant to existing therapy

54. The method of treatment of claim 44 in which the cancer is a breast cancer and eventually its metastasis

55. The method of treatment of claim 54 in which the breast cancer is triple negative breast carcinoma

56. The method of treatment of claim 44 in which the cancer is a pancreatic cancer.

57. The method of treatment of claim 56 in which the pancreatic cancer is pancreatic ductal adenocarcinoma.