WO2012008711A2 - Erlotinib dichloroacetate and anti-cancer agent comprising the same - Google Patents

Erlotinib dichloroacetate and anti-cancer agent comprising the same Download PDF

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WO2012008711A2
WO2012008711A2 PCT/KR2011/004985 KR2011004985W WO2012008711A2 WO 2012008711 A2 WO2012008711 A2 WO 2012008711A2 KR 2011004985 W KR2011004985 W KR 2011004985W WO 2012008711 A2 WO2012008711 A2 WO 2012008711A2
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erlotinib
dichloroacetate
crystalline form
cancer
intensity
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PCT/KR2011/004985
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French (fr)
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WO2012008711A3 (en
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Kyoung Soo Kim
Young Jun Park
Hyun-Nam Song
Dong Youn Kim
Joon Woo Kim
Kyoung-Suk Cho
Keewon Yu
Hyuk Jin Kwon
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Celltrion Chemical Research Institute
Celltrion Pharm, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to erlotinib dichloroacetate and an anti-cancer agent comprising the same. More particularly, the present invention relates to erlotinib dichloroacetate capable of inhibiting epidermal growth factor receptor as well as inducing cancer cells to kill themselves via apoptosis, thereby inhibiting growth of cancer cells and leading to their destruction, and showing significantly enhanced anti-cancer effects by synergy between erlotinib and dichloroacetic acid, and an anti-cancer agent comprising the same.
  • Erlotinib is a generic name of N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine of the following formula (II), which is described in U.S. Patent No. 5,747,498, which is incorporated in its entirety here by reference.
  • Erlotinib is used for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) that has failed at least one prior chemotherapy regimen, or in combination with gemcitabine for the treatment of locally advanced, unresectable or metastatic pancreatic cancer.
  • NSCLC non-small cell lung cancer
  • US Patent No. 6,900,221 which is incorporated in its entirety here by reference, discloses crystalline form A and crystalline form B of erlotinib hydrochloride of the following formula (III), and teaches that crystalline form B is thermodynamically more stable than crystalline form A.
  • erlotinib is manufactured as erlotinib hydrochloride of the following formula (III), and is marketed in many countries under the trade name of TARCEVA.
  • the pharmaceutically acceptable salts of erlotinib are particularly mentioned addition salts with an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, p-toluene sulfonic acid, benzoic acid, citric acid, tartaric acid, succinic acid, oxalic acid, benzene sulfonic acid, methane sulfonic acid, phosphoric acid and mixtures thereof.
  • an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, p-toluene sulfonic acid, benzoic acid, citric acid, tartaric acid, succinic acid, oxalic acid, benzene sulfonic acid, methane sulfonic acid, phosphoric acid and mixtures thereof.
  • erlotinib has been known as a useful anti-cancer drug to prolong a patient's life, it has not been reported that erlotinib alone can treat cancer completely. Further, erlotinib causes toleranace as other anti-cancer agents do, and has the side effects of edema, fatigue, anxiety, headache, depression, vertigo, insomnia, rash, pruritus, xeroderma, erythema, diarrhea, anorexia, nausea, emesis, mucositis, glossalgia, stomatitis, xerostomia, pain, constipation, dyspepsia, dysphagia, weight loss, abdominal pain, dysgensia, hyperbilirubinemia, arthralgia, paresthesia, conjunctivitis, keratoconjunctivitis sicca, dyspnea, cough, infection, etc. with high rate of at least 10%.
  • Dichloroacetic acid is an acetic acid derivative in which two of the three hydrogen atoms of the methyl group have been replaced by chlorine atoms, and has activities of treating lactic acidosis and cancers.
  • dichloroacetic acid is very corrosive and extremely destructive to tissues of the mucous membranes and upper respiratory tract, the salts of dichloroacetic acid such as sodium dichloroacetate and potassium dichloroacetate are therapeutically used.
  • the salts of dichloroacetic acid have been used as drugs for treating lactic acidosis by inhibiting pyruvate dehydrogenase kinase and activating pyruvate dehydrogenase, which is described in Ann Intern Med 108 (1): 58 ⁇ 63 (1988), which is incorporated in its entirety here by reference.
  • erlotinib dichloroacetate prepared from erlotinib and dichloroacetic acid having low toxicity and inducing cancer cells to kill themselves via apoptosis, can show significantly enhanced anti-cancer effects by synergy between erlotinib and dichloroacetic acid. Further, the inventors have found that erlotinib dichloroacetate can eliminate the difficulties in uniformly and completely mixing two ingredients in the same dosage unit in the case of the combination therapy.
  • An object of the present invention is, therefore, to provide erlotinib dichloroacetate showing significantly enhanced anti-cancer effects.
  • Another object of the present invention is to provide an anti-cancer agent comprising erlotinib dichloroacetate.
  • the present invention relates to erlotinib dichloroacetate of the following formula (I).
  • the erlotinib dichloroacetate according to one embodiment of the present invention is particularly a crystalline erlotinib dichloroacetate, more particularly, a crystalline erlotinib dichloroacetate (hereinafter "crystalline form I") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/I o values of at least 10% (I is the intensity of each peak; I o is the intensity of the highest peak) at diffraction angles (2 ⁇ ) of 6.6 ⁇ 0.2, 9.3 ⁇ 0.2, 13.1 ⁇ 0.2, 14.9 ⁇ 0.2, 18.5 ⁇ 0.2, 19.2 ⁇ 0.2, 20.1 ⁇ 0.2, 20.7 ⁇ 0.2, 21.1 ⁇ 0.2, 22.6 ⁇ 0.2, 22.9 ⁇ 0.2, 26.2 ⁇ 0.2, 26.4 ⁇ 0.2, 27.0 ⁇ 0.2, 28.2 ⁇ 0.2, and 28.5 ⁇ 0.2.
  • XRPD X-ray powder diffraction
  • the erlotinib dichloroacetate according to another embodiment of the present invention is a crystalline erlotinib dichloroacetate (hereinafter "crystalline form II") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/I o values of at least 10% (I is the intensity of each peak; I o is the intensity of the highest peak) at diffraction angles (2 ⁇ ) of 6.6 ⁇ 0.2, 8.0 ⁇ 0.2, 13.0 ⁇ 0.2, 16.0 ⁇ 0.2, 16.8 ⁇ 0.2, 20.4 ⁇ 0.2, 20.8 ⁇ 0.2, 21.6 ⁇ 0.2, 22.9 ⁇ 0.2, 23.4 ⁇ 0.2, 26.0 ⁇ 0.2, 26.5 ⁇ 0.2, 27.0 ⁇ 0.2, and 31.1 ⁇ 0.2.
  • XRPD X-ray powder diffraction
  • the erlotinib dichloroacetate according to further embodiment of the present invention is a crystalline erlotinib dichloroacetate (hereinafter "crystalline form III") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/I o values of at least 10% (I is the intensity of each peak; I o is the intensity of the highest peak) at diffraction angles (2 ⁇ ) of 7.6 ⁇ 0.2, 17.7 ⁇ 0.2, 18.2 ⁇ 0.2, 18.5 ⁇ 0.2, 21.7 ⁇ 0.2, 22.7 ⁇ 0.2, 23.2 ⁇ 0.2, 23.8 ⁇ 0.2, 26.6 ⁇ 0.2, and 29.1 ⁇ 0.2.
  • XRPD X-ray powder diffraction
  • the erlotinib dichloroacetate according to further embodiment of the present invention is a crystalline erlotinib dichloroacetate hydrate (hereinafter "crystalline form IV") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/I o values of at least 10% (I is the intensity of each peak; I o is the intensity of the highest peak) at diffraction angles (2 ⁇ ) of 7.2 ⁇ 0.2, 9.1 ⁇ 0.2, 14.6 ⁇ 0.2, 17.0 ⁇ 0.2, 18.0 ⁇ 0.2, 20.1 ⁇ 0.2, 20.9 ⁇ 0.2, 23.7 ⁇ 0.2, 25.9 ⁇ 0.2, 27.0 ⁇ 0.2, 28.0 ⁇ 0.2, 29.0 ⁇ 0.2, 32.0 ⁇ 0.2, and 32.9 ⁇ 0.2.
  • XRPD X-ray powder diffraction
  • the erlotinib dichloroacetate of the above formula (I) according to the present invention can be prepared by reacting erlotinib of the following formula (II) and dichloroacetic acid of the following formula (IV).
  • the erlotinib dichloroacetate of the present invention can be prepared by dissolving erlotinib and dichloroacetic acid in an organic solvent, followed by stirring.
  • the erlotinib dichloroacetate can be prepared by suspending erlotinib in an organic solvent and adding dichloroacetic acid to the resulting supspension, followed by stirring, or by dissolving erlotinib and dichloroacetic acid together in an organic solvent, followed by stirring.
  • the dichloroacetic acid can be used in an amount of approximately 1 equivalent based on the amount of erlotinib.
  • the process for preparing the erlotinib dichloroacetate of the present invention can further optionally include the step of:
  • the organic solvent can include one or more selected from alcohols such as methanol, ethanol, isopropanol, 1-butanol and 1-hexanol; ethers such as tetrahydrofuran, dioxane, diethyl ether and diisopropyl ether; nitriles such as acetonitrile; ketones such as acetone and 2-butanone; esters such as ethyl acetate and isopropyl acetate; and chlorinated hydrocarbons such as dichloromethane, chloroform and 1,2-dichloroethane.
  • alcohols such as methanol, ethanol, isopropanol, 1-butanol and 1-hexanol
  • ethers such as tetrahydrofuran, dioxane, diethyl ether and diisopropyl ether
  • nitriles such as acetonitrile
  • ketones such as acetone and 2-butanone
  • the precipitating solvent can include one or more selected from alcohols such as ethanol, isopropanol, 1-butanol and 1-hexanol; ethers such as tetrahydrofuran, dioxane, diethyl ether and diisopropyl ether; nitriles such as acetonitrile; ketones such as acetone and 2-butanone; hydrocarbons such as n-pentane and n-hexane; aromatic hydrocarbons such as benzene, toluene and xylene; esters such as ethyl acetate and isopropyl acetate; and chlorinated hydrocarbons such as dichloromethane, chloroform and 1,2-dichloroethane.
  • alcohols such as ethanol, isopropanol, 1-butanol and 1-hexanol
  • ethers such as tetrahydrofuran, dioxane, diethyl ether and diis
  • the reaction time can be 1 to 5 hours, and the reaction temperature can be 20 to 60 °C.
  • the process for preparing the erlotinib dichloroacetate can further include washing and drying the solid obtained after filtering.
  • the present invention further relates to an anti-cancer agent comprising the erlotinib dichloroacetate together with a pharmaceutically acceptable carrier.
  • the anti-cancer agent of the present invention can be used for treating non-small cell lung cancer (NSCLC) or pancreatic cancer.
  • the anti-cancer agent of the present invention can optionally include biologically active substances, in addition to the erlotinib dichloroacetate.
  • the anti-cancer agent according to the present invention can be formulated as tablets, capsules, granules, powders, emulsions, suspensions, syrups, etc.
  • the above various forms of the anti-cancer agent can be prepared in a manner well known in the art using a pharmaceutically acceptable carrier(s) which are usually used for each form.
  • the pharmaceutically acceptable carriers include excipient, filler, extender, binder, disintegrator, lubricant, preservative, antioxidant, isotonic agent, buffer, coating agent, sweetening agent, dissolvent, base, dispersing agent, wetting agent, suspending agent, stabilizer, colorant, flavoring agent, etc.
  • the anti-cancer agent of the present invention can contain 1 to 90 wt%, particularly 3 to 50 wt% of the inventive erlotinib dichloroacetate depending on the form thereof.
  • the particular dosage of the present anti-cancer agent can be varied with species of mammals including a human-being, administration route, body weight, gender, age, severity of disease, judgment of doctor, etc.
  • administration route 0.5 to 10 mg of the active ingredient is administered per kg of body weight a day for oral use.
  • the total daily dosage can be administered once or over several times depending on the severity of disease, judgment of doctor, etc.
  • the erlotinib dichloroacetate of the present invention can inhibit epidermal growth factor receptor as well as induce cancer cells to kill themselves via apoptosis, thereby inhibit growth of cancer cells and lead to their destruction. Further, the erlotinib dichloroacetate can increase anti-cancer effects by synergy between erlotinib and dichloroacetic acid. Still further, since the erlotinib dichloroacetate can have the same or higher clinical efficacy at a lower dosage due to the synergy effect, the side effects can be decreased.
  • the erlotinib dichloroacetate in accordance with the present invention can eliminate the difficulties in uniformly and completely mixing erlotinib and dichloroacetic acid in the same dosage unit.
  • the crystalline erlotinib dichloroacetate in accordance with the present invention has good moisture and thermal stability, and low hygroscopicity.
  • the crystalline erlotinib dichloroacetate can be effectively used for preparing a pharmaceutical composition.
  • the erlotinib dichloroacetate in accordance with the present invention can be effectively used for preparing an anti-cancer agent for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) that has failed at least one prior chemotherapy regimen, or in combination with gemcitabine for the treatment of locally advanced, unresectable or metastatic pancreatic cancer.
  • NSCLC locally advanced or metastatic non-small cell lung cancer
  • Fig. 1 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate (crystalline form I) obtained in Example 1.
  • Fig. 2 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate (crystalline form I) obtained in Example 1.
  • Fig. 3 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate (crystalline form II) obtained in Example 6.
  • Fig. 4 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate (crystalline form II) obtained in Example 6.
  • Fig. 5 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate (crystalline form III) obtained in Example 7.
  • Fig. 6 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate (crystalline form III) obtained in Example 7.
  • Fig. 7 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate hydrate (crystalline form IV) obtained in Example 8.
  • XRPD X-ray powder diffraction
  • Fig. 8 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate hydrate (crystalline form IV) obtained in Example 8.
  • Fig. 9 is a diagram illustrating the absorbance values (%) for the human lung cancer NCI-H460 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the erlotinib dichloroacetate, erlotinib hydrochloride and dichloroacetic acid, respectively, based on 100% of the absorbance value for the control.
  • Fig. 10 is a diagram illustrating the absorbance values (%) for the colon cancer SW620 L6 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the erlotinib dichloroacetate, erlotinib hydrochloride and dichloroacetic acid, respectively, based on 100% of the absorbance value for the control.
  • the light yellow crystalline solid formed was filtered, washed with 10 ml of ethyl acetate and dried under vacuum at 60 °C for 20 hours to give 9.90 g of the target compound. The yield was 94.76%.
  • the obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 1 and 2, respectively.
  • the light yellow crystalline solid formed was filtered, washed with 10 ml of diisopropyl ether and dried under vacuum at 60 °C for 20 hours to give 4.77 g of the target compound. The yield was 91.22%.
  • the light yellow crystalline solid formed was filtered, washed with 10 ml of diisopropyl ether and dried under vacuum at 60 °C for 20 hours to give 4.91 g of the target compound. The yield was 93.90%.
  • the light yellow crystalline solid formed was filtered, washed with 5 ml of tetrahedrofuran and dried under vacuum at 60 °C for 20 hours to give 4.43 g of the target compound. The yield was 84.72%.
  • the light yellow crystalline solid formed was filtered, washed with 5 ml of acetonitrile and dried under vacuum at 60 °C for 20 hours to give 4.76 g of the target compound. The yield was 91.03%.
  • the light yellow crystalline solid formed was filtered, washed with 10 ml of isopropanol and dried under vacuum at 60 °C for 20 hours to give 4.99 g of the target compound.
  • the yield was 95.43%.
  • the obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 3 and 4, respectively.
  • the light yellow crystalline solid formed was filtered, washed with 10 ml of ethanol and dried under vacuum at 60 °C for 20 hours to give 9.21 g of the target compound. The yield was 88.15%.
  • the obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 5 and 6, respectively.
  • the light yellow crystalline solid formed was filtered, washed with 10 ml of methanol and dried under vacuum at 60 °C for 20 hours to give 3.81 g of the target compound. The yield was 72.86%.
  • the obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 7 and 8, respectively.
  • Erlotinib hydrochloride (crystalline form B) was prepared in accordance with the process described in U.S. Patent Publication No. 2009-0131665, which is incorporated in its entirety here by reference.
  • the light yellow crystalline solid formed was filtered and dried under vacuum at 60 °C for 20 hours to give 20.35 g of the target compound. The yield was 93.13%.
  • the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II), erlotinib dichloroacetate (crystalline form III) and erlotinib dichloroacetate hydrate (crystalline form IV) obtained in Examples 1, 6, 7 and 8, respectively, have distinctively characteristic peaks in the X-ray powder diffraction (XRPD) patterns.
  • the observed characteristic peaks shown in the XRPD patterns of Figs. 1, 3, 5 and 7 are listed in Tables 1, 2, 3 and 4, respectively, wherein ‘2 ⁇ ’ is diffraction angle, ‘d’ is interplanar spacing, and 'I/I 0 ' is relative intensity of the peak.
  • the moisture and thermal stability of an active ingredient in a pharmaceutical composition is an important factor on the perspective of the production process and long-term storage of the pharmaceutical composition.
  • the moisture and thermal stabilities of the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II), erlotinib dichloroacetate (crystalline form III) and erlotinib dichloroacetate hydrate (crystalline form IV) respectively obtained in Examples 1, 6, 7 and 8, and the erlotinib hydrochloride (crystalline form B) obtained in Comparative Example 1 were measured.
  • each erlotinib acid addition salt was stored in a sealed state under an accelerated condition (a temperature of 40 °C and a relative humidity of 75 %), and after 0 (zero), 3, 7, 14 and 28 days, the remaining rate of the active ingredient was analyzed with high performance liquid chromatography (HPLC). The results are listed in Table 5.
  • Table 5 Salts Initial 3 days 7 days 14 days 28 days Erlotinib hydrochloride (crystalline form B) 100 100.1 100.1 100.0 100.0 Erlotinib dichloroacetate (crystalline form I) 100 100.1 100.0 100.0 100.1 Erlotinib dichloroacetate (crystalline form II) 100 100.0 100.0 100.0 Erlotinib dichloroacetate (crystalline form III) 100 100.1 100.0 100.0 Erlotinib dichloroacetate (crystalline form IV) 100 100.0 100.0 100.0 99.9
  • the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II), erlotinib dichloroacetate (crystalline form III) and erlotinib dichloroacetate hydrate (crystalline form IV) showed the same level of good stability even when exposed to the accelerated condition for 28 days, as compared with the known erlotinib hydrochloride (crystalline form B). Such a result suggests that the erlotinib dichloroacetate of the present invention has good chemical stability to be effectively used for preparing a pharmaceutical composition.
  • each of the erlotinib dichloroacetate (crystalline form I), erlotinib hydrochloride (crystalline form B) and dichloroacetic acid was dissolved in dimethylsulfoxide (DMSO) to prepare 10 mM solution, and then 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M solutions were respectively prepared using serum free medium. 100 ⁇ l of each prepared solution was seeded to the wells and incubated at 37 °C for 48 hours.
  • DMSO dimethylsulfoxide
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • Control shows the absorbance that was measured for serum free medium containing no drug.
  • the absorbance values for the cells respectively treated with erlotinib dichloroacetate, erlotinib hydrochloride and dichloroacetic acid were measured and the mean of four values was calculated, as the results (%) are represented in Figs. 9 and 10.
  • the absorbance values for the human lung cancer NCI-H460 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the erlotinib dichloroacetate were 68.27%, 71.08%, 79.08% and 82.12%, respectively, based on 100% of the absorbance value for the control.
  • the absorbance values for the NCI-H460 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the erlotinib hydrochloride were 94.04%, 96.76%, 101.72% and 102.49%, respectively.
  • the absorbance values for the NCI-H460 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the dichloroacetic acid were 83.99%, 85.04%, 86.76% and 88.08%, respectively.
  • the absorbance values for the colon cancer SW620 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the erlotinib dichloroacetate were 75.54%, 81.14%, 91.65% and 96.26%, respectively, based on 100% of the absorbance value for the control.
  • the absorbance values for the SW620 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the erlotinib hydrochloride were 97.14%, 97.24%, 98.81% and 103.11%, respectively.
  • the absorbance values for the SW620 cells which were treated with 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M and 0.1 ⁇ M of the dichloroacetic acid were 99.78%, 101.02%, 100.01% and 104.01%, respectively.
  • the erlotinib dichloroacetate of the present invention has stronger anti-cancer effects than the erlotinib hydrochloride and generates synergy by the addition of dichloroacetic acid.

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Abstract

The present invention relates to erlotinib dichloroacetate and an anti-cancer agent comprising the same. The erlotinib dichloroacetate of the present invention can inhibit epidermal growth factor receptor as well as induce cancer cells to kill themselves via apoptosis, thereby inhibit growth of cancer cells and lead to their destruction, and show significantly enhanced anti-cancer effects by synergy between erlotinib and dichloroacetic acid.

Description

ERLOTINIB DICHLOROACETATE AND ANTI-CANCER AGENT COMPRISING THE SAME
The present invention relates to erlotinib dichloroacetate and an anti-cancer agent comprising the same. More particularly, the present invention relates to erlotinib dichloroacetate capable of inhibiting epidermal growth factor receptor as well as inducing cancer cells to kill themselves via apoptosis, thereby inhibiting growth of cancer cells and leading to their destruction, and showing significantly enhanced anti-cancer effects by synergy between erlotinib and dichloroacetic acid, and an anti-cancer agent comprising the same.
Erlotinib is a generic name of N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine of the following formula (II), which is described in U.S. Patent No. 5,747,498, which is incorporated in its entirety here by reference.
Figure PCTKR2011004985-appb-I000001
Erlotinib is used for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) that has failed at least one prior chemotherapy regimen, or in combination with gemcitabine for the treatment of locally advanced, unresectable or metastatic pancreatic cancer.
US Patent No. 6,900,221, which is incorporated in its entirety here by reference, discloses crystalline form A and crystalline form B of erlotinib hydrochloride of the following formula (III), and teaches that crystalline form B is thermodynamically more stable than crystalline form A. Particularly, erlotinib is manufactured as erlotinib hydrochloride of the following formula (III), and is marketed in many countries under the trade name of TARCEVA.
Figure PCTKR2011004985-appb-I000002
US Patent No. 6,706,721, which is incorporated in its entirety here by reference, discloses anhydrous and hydrate forms of erlotinib mesylate. Also, WO 2008/122776, which is incorporated in its entirety here by reference, describes a process for the preparation of erlotinib and its pharmaceutically acceptable salts. As the pharmaceutically acceptable salts of erlotinib are particularly mentioned addition salts with an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, p-toluene sulfonic acid, benzoic acid, citric acid, tartaric acid, succinic acid, oxalic acid, benzene sulfonic acid, methane sulfonic acid, phosphoric acid and mixtures thereof. However, physiochemical properties such as moisture and thermal stability and hygroscopicity of the acid addition salts of erlotinib have never been reported in the above patent documents.
Although erlotinib has been known as a useful anti-cancer drug to prolong a patient's life, it has not been reported that erlotinib alone can treat cancer completely. Further, erlotinib causes toleranace as other anti-cancer agents do, and has the side effects of edema, fatigue, anxiety, headache, depression, vertigo, insomnia, rash, pruritus, xeroderma, erythema, diarrhea, anorexia, nausea, emesis, mucositis, glossalgia, stomatitis, xerostomia, pain, constipation, dyspepsia, dysphagia, weight loss, abdominal pain, dysgensia, hyperbilirubinemia, arthralgia, paresthesia, conjunctivitis, keratoconjunctivitis sicca, dyspnea, cough, infection, etc. with high rate of at least 10%.
Therefore, a combined modality therapy administering erlotinib together with a complementary drug has been suggested to overcome the problems described above. However, the combined modality therapy also cannot destruct cancer cells completely, and has difficulties in completely and uniformly mixing two active ingredients in the same dosage unit.
Dichloroacetic acid is an acetic acid derivative in which two of the three hydrogen atoms of the methyl group have been replaced by chlorine atoms, and has activities of treating lactic acidosis and cancers. However, since dichloroacetic acid is very corrosive and extremely destructive to tissues of the mucous membranes and upper respiratory tract, the salts of dichloroacetic acid such as sodium dichloroacetate and potassium dichloroacetate are therapeutically used.
Particularly, the salts of dichloroacetic acid have been used as drugs for treating lactic acidosis by inhibiting pyruvate dehydrogenase kinase and activating pyruvate dehydrogenase, which is described in Ann Intern Med 108 (1): 58~63 (1988), which is incorporated in its entirety here by reference.
A recent study published in Cancer Cell 11 (1): 37~51 (2007), which is incorporated in its entirety here by reference, testing dichloroacetic acid on in vitro cancer cell lines and a rat model, found that dichloroacetic acid restored mitochondrial function, thus restoring apoptosis, killing cancer cells in vitro, and shrinking the tumors in the rats.
The present inventors have researched on erlotinib in order to improve the anti-cancer effects and to reduce the side effects described above, and found that erlotinib dichloroacetate, prepared from erlotinib and dichloroacetic acid having low toxicity and inducing cancer cells to kill themselves via apoptosis, can show significantly enhanced anti-cancer effects by synergy between erlotinib and dichloroacetic acid. Further, the inventors have found that erlotinib dichloroacetate can eliminate the difficulties in uniformly and completely mixing two ingredients in the same dosage unit in the case of the combination therapy.
An object of the present invention is, therefore, to provide erlotinib dichloroacetate showing significantly enhanced anti-cancer effects.
Another object of the present invention is to provide an anti-cancer agent comprising erlotinib dichloroacetate.
The present invention relates to erlotinib dichloroacetate of the following formula (I).
Figure PCTKR2011004985-appb-I000003
The erlotinib dichloroacetate according to one embodiment of the present invention is particularly a crystalline erlotinib dichloroacetate, more particularly, a crystalline erlotinib dichloroacetate (hereinafter "crystalline form I") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 6.6±0.2, 9.3±0.2, 13.1±0.2, 14.9±0.2, 18.5±0.2, 19.2±0.2, 20.1±0.2, 20.7±0.2, 21.1±0.2, 22.6±0.2, 22.9±0.2, 26.2±0.2, 26.4±0.2, 27.0±0.2, 28.2±0.2, and 28.5±0.2.
The erlotinib dichloroacetate according to another embodiment of the present invention is a crystalline erlotinib dichloroacetate (hereinafter "crystalline form II") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 6.6±0.2, 8.0±0.2, 13.0±0.2, 16.0±0.2, 16.8±0.2, 20.4±0.2, 20.8±0.2, 21.6±0.2, 22.9±0.2, 23.4±0.2, 26.0±0.2, 26.5±0.2, 27.0±0.2, and 31.1±0.2.
The erlotinib dichloroacetate according to further embodiment of the present invention is a crystalline erlotinib dichloroacetate (hereinafter "crystalline form III") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 7.6±0.2, 17.7±0.2, 18.2±0.2, 18.5±0.2, 21.7±0.2, 22.7±0.2, 23.2±0.2, 23.8±0.2, 26.6±0.2, and 29.1±0.2.
The erlotinib dichloroacetate according to further embodiment of the present invention is a crystalline erlotinib dichloroacetate hydrate (hereinafter "crystalline form IV") showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 7.2±0.2, 9.1±0.2, 14.6±0.2, 17.0±0.2, 18.0±0.2, 20.1±0.2, 20.9±0.2, 23.7±0.2, 25.9±0.2, 27.0±0.2, 28.0±0.2, 29.0±0.2, 32.0±0.2, and 32.9±0.2.
The erlotinib dichloroacetate of the above formula (I) according to the present invention can be prepared by reacting erlotinib of the following formula (II) and dichloroacetic acid of the following formula (IV).
Figure PCTKR2011004985-appb-I000004
Figure PCTKR2011004985-appb-I000005
The process for preparing the erlotinib dichloroacetate of the present invention is described in more detail below.
The erlotinib dichloroacetate of the present invention can be prepared by dissolving erlotinib and dichloroacetic acid in an organic solvent, followed by stirring. Particularly, the erlotinib dichloroacetate can be prepared by suspending erlotinib in an organic solvent and adding dichloroacetic acid to the resulting supspension, followed by stirring, or by dissolving erlotinib and dichloroacetic acid together in an organic solvent, followed by stirring.
In one embodiment, the dichloroacetic acid can be used in an amount of approximately 1 equivalent based on the amount of erlotinib.
In one embodiment, the process for preparing the erlotinib dichloroacetate of the present invention can further optionally include the step of:
(i) stirring the reaction solution and filtering the solid formed;
(ii) lowering the temperature of the reaction solution with stirring and filtering the solid formed;
(iii) adding a precipitating solvent to the reaction solution with stirring and filtering the solid formed; or
(iv) evaporating the reaction solution under reduced pressure, adding a precipitating solvent to the obtained residue with stirring, and filtering the solid formed.
In one embodiment, the organic solvent can include one or more selected from alcohols such as methanol, ethanol, isopropanol, 1-butanol and 1-hexanol; ethers such as tetrahydrofuran, dioxane, diethyl ether and diisopropyl ether; nitriles such as acetonitrile; ketones such as acetone and 2-butanone; esters such as ethyl acetate and isopropyl acetate; and chlorinated hydrocarbons such as dichloromethane, chloroform and 1,2-dichloroethane.
In one embodiment, the precipitating solvent can include one or more selected from alcohols such as ethanol, isopropanol, 1-butanol and 1-hexanol; ethers such as tetrahydrofuran, dioxane, diethyl ether and diisopropyl ether; nitriles such as acetonitrile; ketones such as acetone and 2-butanone; hydrocarbons such as n-pentane and n-hexane; aromatic hydrocarbons such as benzene, toluene and xylene; esters such as ethyl acetate and isopropyl acetate; and chlorinated hydrocarbons such as dichloromethane, chloroform and 1,2-dichloroethane.
In one embodiment, the reaction time can be 1 to 5 hours, and the reaction temperature can be 20 to 60 ℃.
In one embodiment, the process for preparing the erlotinib dichloroacetate can further include washing and drying the solid obtained after filtering.
The present invention further relates to an anti-cancer agent comprising the erlotinib dichloroacetate together with a pharmaceutically acceptable carrier. In particular, the anti-cancer agent of the present invention can be used for treating non-small cell lung cancer (NSCLC) or pancreatic cancer.
In one embodiment, the anti-cancer agent of the present invention can optionally include biologically active substances, in addition to the erlotinib dichloroacetate.
The anti-cancer agent according to the present invention can be formulated as tablets, capsules, granules, powders, emulsions, suspensions, syrups, etc. The above various forms of the anti-cancer agent can be prepared in a manner well known in the art using a pharmaceutically acceptable carrier(s) which are usually used for each form. Examples of the pharmaceutically acceptable carriers include excipient, filler, extender, binder, disintegrator, lubricant, preservative, antioxidant, isotonic agent, buffer, coating agent, sweetening agent, dissolvent, base, dispersing agent, wetting agent, suspending agent, stabilizer, colorant, flavoring agent, etc.
In one embodiment, the anti-cancer agent of the present invention can contain 1 to 90 wt%, particularly 3 to 50 wt% of the inventive erlotinib dichloroacetate depending on the form thereof.
The particular dosage of the present anti-cancer agent can be varied with species of mammals including a human-being, administration route, body weight, gender, age, severity of disease, judgment of doctor, etc. By way of example, 0.5 to 10 mg of the active ingredient is administered per kg of body weight a day for oral use. The total daily dosage can be administered once or over several times depending on the severity of disease, judgment of doctor, etc.
The erlotinib dichloroacetate of the present invention can inhibit epidermal growth factor receptor as well as induce cancer cells to kill themselves via apoptosis, thereby inhibit growth of cancer cells and lead to their destruction. Further, the erlotinib dichloroacetate can increase anti-cancer effects by synergy between erlotinib and dichloroacetic acid. Still further, since the erlotinib dichloroacetate can have the same or higher clinical efficacy at a lower dosage due to the synergy effect, the side effects can be decreased.
Also, the erlotinib dichloroacetate in accordance with the present invention can eliminate the difficulties in uniformly and completely mixing erlotinib and dichloroacetic acid in the same dosage unit.
Also, the crystalline erlotinib dichloroacetate in accordance with the present invention has good moisture and thermal stability, and low hygroscopicity. Thus, the crystalline erlotinib dichloroacetate can be effectively used for preparing a pharmaceutical composition.
Accordingly, the erlotinib dichloroacetate in accordance with the present invention can be effectively used for preparing an anti-cancer agent for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) that has failed at least one prior chemotherapy regimen, or in combination with gemcitabine for the treatment of locally advanced, unresectable or metastatic pancreatic cancer.
Fig. 1 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate (crystalline form I) obtained in Example 1.
Fig. 2 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate (crystalline form I) obtained in Example 1.
Fig. 3 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate (crystalline form II) obtained in Example 6.
Fig. 4 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate (crystalline form II) obtained in Example 6.
Fig. 5 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate (crystalline form III) obtained in Example 7.
Fig. 6 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate (crystalline form III) obtained in Example 7.
Fig. 7 is an X-ray powder diffraction (XRPD) pattern of the erlotinib dichloroacetate hydrate (crystalline form IV) obtained in Example 8.
Fig. 8 is a differential scanning calorimeter (DSC) thermogram of the erlotinib dichloroacetate hydrate (crystalline form IV) obtained in Example 8.
Fig. 9 is a diagram illustrating the absorbance values (%) for the human lung cancer NCI-H460 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the erlotinib dichloroacetate, erlotinib hydrochloride and dichloroacetic acid, respectively, based on 100% of the absorbance value for the control.
Fig. 10 is a diagram illustrating the absorbance values (%) for the colon cancer SW620 L6 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the erlotinib dichloroacetate, erlotinib hydrochloride and dichloroacetic acid, respectively, based on 100% of the absorbance value for the control.
The present invention is further illustrated by the following examples, which are not to be construed to limit the scope of the invention.
Example 1: Preparation of erlotinib dichloroacetate (crystalline form I)
7.87 g (20.00 mmol) of erlotinib was suspended in 40 ㎖ of ethyl acetate, and 1.66 ㎖ (20.00 mmol) of dichloroacetic acid was added dropwise. Then, the reaction solution was stirred for 1 hour at 20 to 25 ℃. After the compounds were completely dissolved in the reaction solution and a solid began to be formed, the reaction solution was further stirred for 2 hours at 20 to 25 ℃.
The light yellow crystalline solid formed was filtered, washed with 10 ㎖ of ethyl acetate and dried under vacuum at 60 ℃ for 20 hours to give 9.90 g of the target compound. The yield was 94.76%. The obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 1 and 2, respectively.
M.P.: 144-145 ℃
1H NMR (400MHz, DMSO-d6) : δ= 9.82 (brs, 1 H), 8.54 (s, 1 H), 7.92 (s, 1 H), 7.87 (s, 1 H), 7.83 (d, 1 H, J = 7.6 Hz), 7.39 (t, 1 H, J = 8.0 Hz), 7.23 (d, 1 H, J = 7.2 Hz), 7.22 (s, 1 H), 6.51 (s, 1 H), 4.27 ~ 4.24 (m, 4 H), 4.18(s, 1 H), 3.76 ~ 3.71 (m, 4 H), 3.34 (s, 3 H), 3.32(s, 3 H).
Example 2: Preparation of erlotinib dichloroacetate (crystalline form I)
3.94 g (10.01 mmol) of erlotinib was suspended in 50 ㎖ of chloroform, and 0.83 ㎖ (10.01 mmol) of dichloroacetic acid was added dropwise. Then, the reaction solution was stirred for 1 hour at 20 to 25 ℃. After the compounds were completely dissolved in the reaction solution, 65 ㎖ of diisopropyl ether was added dropwise and the reaction solution was further stirred for 1 hour at 20 to 25 ℃.
The light yellow crystalline solid formed was filtered, washed with 10 ㎖ of diisopropyl ether and dried under vacuum at 60 ℃ for 20 hours to give 4.77 g of the target compound. The yield was 91.22%.
Example 3: Preparation of erlotinib dichloroacetate (crystalline form I)
3.94 g (10.01 mmol) of erlotinib was suspended in 50 ㎖ of methanol, and 0.83 ml (10.01 mmol) of dichloroacetic acid was added dropwise. Then, the reaction solution was stirred for 1 hour at 20 to 25 ℃, and distilled under reduced pressure to be completely concentrated. 50 ㎖ of acetone was added to the residue, and 100 ㎖ of diisopropyl ether was added dropwise to the resulting solution. After a solid began to be formed, the reaction solution was cooled to 0 to 5 ℃ and further stirred for 2 hours.
The light yellow crystalline solid formed was filtered, washed with 10 ㎖ of diisopropyl ether and dried under vacuum at 60 ℃ for 20 hours to give 4.91 g of the target compound. The yield was 93.90%.
Example 4: Preparation of erlotinib dichloroacetate (crystalline form I)
3.94 g (10.01 mmol) of erlotinib was suspended in 10 ㎖ of tetrahydrofuran, and 0.83 ml (10.01 mmol) of dichloroacetic acid was added dropwise. Then, the reaction solution was stirred for 3 hours at 20 to 25 ℃.
The light yellow crystalline solid formed was filtered, washed with 5 ㎖ of tetrahedrofuran and dried under vacuum at 60 ℃ for 20 hours to give 4.43 g of the target compound. The yield was 84.72%.
Example 5: Preparation of erlotinib dichloroacetate (crystalline form I)
3.94 g (10.01 mmol) of erlotinib was suspended in 10 ㎖ of acetonitrile, and 0.83 ml (10.01 mmol) of dichloroacetic acid was added dropwise. Then, the reaction solution was stirred for 3 hours at 20 to 25 ℃.
The light yellow crystalline solid formed was filtered, washed with 5 ㎖ of acetonitrile and dried under vacuum at 60 ℃ for 20 hours to give 4.76 g of the target compound. The yield was 91.03%.
Example 6: Preparation of erlotinib dichloroacetate (crystalline form II)
3.94 g (10.01 mmol) of erlotinib was suspended in 10 ㎖ of isopropanol, and 0.83 ml (10.01 mmol) of dichloroacetic acid was added dropwise. Then, the reaction solution was stirred for 30 minutes at 20 to 25 ℃. After the compounds were completely dissolved in the reaction solution and a solid began to be formed, 65 ㎖ of isopropanol was added and the reaction solution was further stirred for 2 hours at 20 to 25 ℃.
The light yellow crystalline solid formed was filtered, washed with 10 ㎖ of isopropanol and dried under vacuum at 60 ℃ for 20 hours to give 4.99 g of the target compound. The yield was 95.43%. The obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 3 and 4, respectively.
M.P.: 140-142 ℃
Example 7: Preparation of erlotinib dichloroacetate (crystalline form III)
7.87 g (20.00 mmol) of erlotinib was suspended in 60 ㎖ of ethanol, and 1.66 ㎖ (20.00 mmol) of dichloroacetic acid was added dropwise. Then, the reaction solution was stirred for 30 minutes at 20 to 25 ℃. After the compounds were completely dissolved in the reaction solution and a solid began to be formed, 20 ㎖ of ethanol was added and the reaction solution was further stirred for 3 hours at 20 to 25 ℃.
The light yellow crystalline solid formed was filtered, washed with 10 ㎖ of ethanol and dried under vacuum at 60 ℃ for 20 hours to give 9.21 g of the target compound. The yield was 88.15%. The obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 5 and 6, respectively.
M.P.: 142-144 ℃
Example 8: Preparation of erlotinib dichloroacetate hydrate (crystalline form IV)
3.94 g (10.01 mmol) of erlotinib was suspended in 20 ㎖ of distilled water and 0.83 ml (10.01 mmol) of dichloroacetic acid was added dropwise. To the reaction solution was added 50 ㎖ of methanol. Then, the reaction solution was heated to 50 to 55 ℃ and stirred for 1 to 2 hours. After the compounds were completely dissolved in the reaction solution, the reaction solution was slowly cooled to 20 to 25 ℃ and further stirred for 2 hours.
The light yellow crystalline solid formed was filtered, washed with 10 ㎖ of methanol and dried under vacuum at 60 ℃ for 20 hours to give 3.81 g of the target compound. The yield was 72.86%. The obtained crystalline erlotinib dichloroacetate was subjected to X-ray powder diffraction (XRPD) and differential scanning calorimeter (DSC) analyses, and the results are shown in Figs. 7 and 8, respectively.
M.P.: 126-128 ℃
Comparative Example 1: Preparation of erlotinib hydrochloride (crystalline form B)
Erlotinib hydrochloride (crystalline form B) was prepared in accordance with the process described in U.S. Patent Publication No. 2009-0131665, which is incorporated in its entirety here by reference.
20.00 g (50.83 mmol) of erlotinib was added to 440 ㎖ of a mixture of methyl ethyl ketone and distilled water (10:1, v/v) and heated to 30 to 35 ℃ to give a complete solution. 5.15 ㎖ (61.00 mmol) of conc. HCl was added dropwise, and the reaction solution was stirred strongly.
The light yellow crystalline solid formed was filtered and dried under vacuum at 60 ℃ for 20 hours to give 20.35 g of the target compound. The yield was 93.13%.
Experimental Example 1: X-ray structure analysis of crystalline erlotinib dichloroacetate
As shown in Figs. 1, 3, 5 and 7, the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II), erlotinib dichloroacetate (crystalline form III) and erlotinib dichloroacetate hydrate (crystalline form IV) obtained in Examples 1, 6, 7 and 8, respectively, have distinctively characteristic peaks in the X-ray powder diffraction (XRPD) patterns. The observed characteristic peaks shown in the XRPD patterns of Figs. 1, 3, 5 and 7 are listed in Tables 1, 2, 3 and 4, respectively, wherein ‘2θ’ is diffraction angle, ‘d’ is interplanar spacing, and 'I/I0' is relative intensity of the peak.
Table 1
2θ d I/I0 2θ d I/I0
6.6120 13.36837 100 23.8402 3.73249 8.94
9.2866 9.52336 11.50 25.0289 3.55786 6.55
13.1025 6.75718 13.70 26.1716 3.40505 60.18
13.5029 6.55766 6.94 26.4245 3.37303 42.80
14.9108 5.94152 12.62 26.9752 3.30541 20.37
16.6186 5.33459 8.38 28.1519 3.16987 15.92
17.6684 5.01991 7.92 28.5382 3.12784 10.93
18.5228 4.79025 29.90 29.3902 3.03907 5.72
19.1765 4.62841 16.39 29.8875 2.98963 6.84
20.0659 4.42521 14.91 31.3903 2.84984 4.44
20.6721 4.29680 25.87 32.6335 2.74406 5.58
21.0734 4.21587 23.26 34.4562 2.60297 4.18
22.5931 3.93563 37.41 35.5362 2.52629 8.14
22.9363 3.87750 12.02 36.6405 2.45265 3.40
Table 2
2θ d I/I0 2θ d I/I0
6.5723 13.44905 100 21.6049 4.11334 20.44
7.9780 11.08229 22.71 22.8776 3.88732 19.63
12.9565 6.83295 22.60 23.3969 3.80220 16.84
14.7426 6.00891 4.80 25.9535 3.43316 35.75
15.9316 5.56305 20.73 26.4644 3.36804 19.30
16.7804 5.28352 32.21 27.0396 3.29768 18.11
20.4133 4.35069 14.88 31.0649 2.87895 12.50
20.8314 4.26430 15.54
Table 3
2θ d I/I0 2θ d I/I0
7.6210 11.60061 100 26.6368 3.34663 17.19
8.9437 9.88775 9.05 27.8625 3.20213 6.21
11.8985 7.43805 2.95 28.3160 3.15187 7.79
15.0768 5.87648 6.93 29.0865 3.07011 12.44
16.7493 5.29325 8.94 29.6563 3.01241 4.61
17.7167 5.00633 15.77 31.3090 2.85706 3.08
18.1769 4.88061 19.45 32.0511 2.79258 3.81
18.5364 4.78676 22.28 33.1538 2.70218 4.45
21.7141 4.09291 32.39 35.8578 2.50437 3.21
22.6546 3.92507 39.94 36.5906 2.45588 1.50
23.1850 3.83647 31.59 37.2175 2.41594 2.34
23.7956 3.73938 12.70 38.4084 2.34373 2.21
25.0373 3.55668 8.74 39.0321 2.30770 1.73
25.6646 3.47115 4.98
Table 4
2θ d I/I0 2θ d I/I0
7.1663 12.33567 100 25.8875 3.44177 11.6
9.0533 9.76826 30.49 26.2819 3.39101 7.38
11.0797 7.98584 4.46 27.0126 3.30092 25.58
11.7158 7.55364 4.38 27.9333 3.19417 13.27
14.5691 6.08006 21.25 29.0344 3.07549 17.48
15.7063 5.64232 8.19 30.4149 2.93898 6.71
16.2238 5.46351 9.28 31.1529 2.87102 8.95
16.9713 5.2245 11.35 31.5744 2.83365 5.58
17.9656 4.93754 31.02 32.0424 2.79332 12.11
18.4106 4.81918 4.88 32.8977 2.72262 12.58
20.0913 4.41967 19.03 34.1307 2.62704 5.99
20.9354 4.24335 28.81 34.8104 2.57729 7.06
21.72 4.09181 8.09 35.608 2.52136 5.94
23.6511 3.7619 28.27 36.2294 2.47953 5.75
24.6313 3.61438 5.65 38.571 2.33422 0.25
Experimental Example 2: Moisture and thermal stability test
The moisture and thermal stability of an active ingredient in a pharmaceutical composition is an important factor on the perspective of the production process and long-term storage of the pharmaceutical composition. Thus, the moisture and thermal stabilities of the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II), erlotinib dichloroacetate (crystalline form III) and erlotinib dichloroacetate hydrate (crystalline form IV) respectively obtained in Examples 1, 6, 7 and 8, and the erlotinib hydrochloride (crystalline form B) obtained in Comparative Example 1 were measured.
Particularly, each erlotinib acid addition salt was stored in a sealed state under an accelerated condition (a temperature of 40 ℃ and a relative humidity of 75 %), and after 0 (zero), 3, 7, 14 and 28 days, the remaining rate of the active ingredient was analyzed with high performance liquid chromatography (HPLC). The results are listed in Table 5.
Table 5
Salts Initial 3 days 7 days 14 days 28 days
Erlotinib hydrochloride (crystalline form B) 100 100.1 100.1 100.0 100.0
Erlotinib dichloroacetate (crystalline form I) 100 100.1 100.0 100.0 100.1
Erlotinib dichloroacetate (crystalline form II) 100 100.0 100.0 100.0 100.0
Erlotinib dichloroacetate (crystalline form III) 100 100.1 100.0 100.0 100.0
Erlotinib dichloroacetate (crystalline form IV) 100 100.0 100.0 100.0 99.9
As shown in Table 5, the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II), erlotinib dichloroacetate (crystalline form III) and erlotinib dichloroacetate hydrate (crystalline form IV) showed the same level of good stability even when exposed to the accelerated condition for 28 days, as compared with the known erlotinib hydrochloride (crystalline form B). Such a result suggests that the erlotinib dichloroacetate of the present invention has good chemical stability to be effectively used for preparing a pharmaceutical composition.
Experimental Example 3: Hygroscopicity test
The hygroscopicities of the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II), erlotinib dichloroacetate (crystalline form III) and erlotinib dichloroacetate hydrate (crystalline form IV) respectively obtained in Examples 1, 6, 7 and 8 were compared with that of the known erlotinib hydrochloride (crystalline form B) obtained in Comparative Example 1.
Each compound was exposed to the condition of a temperature of 40 ℃ and a relative humidity of 75 % for 2 hours, 8 hours, 24 hours and 3 days, and then the amount of moisture contained in the compound was measured with Karl-Fischer titrator. The percentage amounts (weight %) of moisture contained in the active ingredients are listed in Table 6.
Table 6
Salts Initial 2 hours 8 hours 24 hours 3 days
Erlotinib hydrochloride (crystalline form B) 0.27 0.38 0.27 0.33 0.33
Erlotinib dichloroacetate (crystalline form I) 0.50 0.35 0.63 0.81 0.87
Erlotinib dichloroacetate (crystalline form II) 2.13 1.37 1.24 0.95 0.70
Erlotinib dichloroacetate (crystalline form III) 0.42 0.32 0.26 0.28 0.39
Erlotinib dichloroacetate (crystalline form IV) 3.09 7.64 7.55 7.73 7.61
As shown in Table 6, the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II) and erlotinib dichloroacetate (crystalline form III) showed similar non-hygroscopicity even when exposed to the high humidity condition, as compared with the known erlotinib hydrochloride (crystalline form B). Such a result suggests that the erlotinib dichloroacetate (crystalline form I), erlotinib dichloroacetate (crystalline form II) and erlotinib dichloroacetate (crystalline form III) have good moisture stability to be effectively used for preparing a pharmaceutical composition.
Experimental Example 4: Cytotoxicity test (MTT assay)
Human lung cancer NCI-H460 cells and colon cancer SW620 cells were seeded to a 94-well plate in 2.5 x 104 cells/㎖ and cultured for 24 hours, respectively. Each sample was seeded to 4 wells and cultured (n=4).
After 24 hours each of the erlotinib dichloroacetate (crystalline form I), erlotinib hydrochloride (crystalline form B) and dichloroacetic acid was dissolved in dimethylsulfoxide (DMSO) to prepare 10 mM solution, and then 3 μM, 1 μM, 0.3 μM and 0.1 μM solutions were respectively prepared using serum free medium. 100 ㎕ of each prepared solution was seeded to the wells and incubated at 37 ℃ for 48 hours.
20 ㎕ of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each sample and cultured for 2 hours. The absorbance was measured at 510 nm using enzyme-linked immunosorbent assay (ELISA). The MTT was used after dissolving it in phosphate buffered saline (PBS) in 5 mg/ml and filtering the resulting solution.
Control shows the absorbance that was measured for serum free medium containing no drug. The absorbance values for the cells respectively treated with erlotinib dichloroacetate, erlotinib hydrochloride and dichloroacetic acid were measured and the mean of four values was calculated, as the results (%) are represented in Figs. 9 and 10.
The absorbance values for the human lung cancer NCI-H460 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the erlotinib dichloroacetate were 68.27%, 71.08%, 79.08% and 82.12%, respectively, based on 100% of the absorbance value for the control.
Meanwhile, the absorbance values for the NCI-H460 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the erlotinib hydrochloride were 94.04%, 96.76%, 101.72% and 102.49%, respectively. Moreover, the absorbance values for the NCI-H460 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the dichloroacetic acid were 83.99%, 85.04%, 86.76% and 88.08%, respectively.
The above experimental results show that the erlotinib dichloroacetate of the present invention has more potent anti-cancer effects, as compared with erlotinib (erlotinib hydrochloride) or dichloroacetic acid, and generates strong synergy between erlotinib and dichloroacetic acid.
The absorbance values for the colon cancer SW620 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the erlotinib dichloroacetate were 75.54%, 81.14%, 91.65% and 96.26%, respectively, based on 100% of the absorbance value for the control.
Meanwhile, the absorbance values for the SW620 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the erlotinib hydrochloride were 97.14%, 97.24%, 98.81% and 103.11%, respectively. Moreover, the absorbance values for the SW620 cells which were treated with 3 μM, 1 μM, 0.3 μM and 0.1 μM of the dichloroacetic acid were 99.78%, 101.02%, 100.01% and 104.01%, respectively.
The above experimental results show that the erlotinib dichloroacetate of the present invention has more potent anti-cancer effects, as compared with erlotinib (erlotinib hydrochloride) or dichloroacetic acid, and generates strong synergy between erlotinib and dichloroacetic acid.
Consequently, the erlotinib dichloroacetate of the present invention has stronger anti-cancer effects than the erlotinib hydrochloride and generates synergy by the addition of dichloroacetic acid.

Claims (8)

  1. Erlotinib dichloroacetate of the following formula (I):
    Figure PCTKR2011004985-appb-I000006
  2. The erlotinib dichloroacetate of Claim 1, wherein the erlotinib dichloroacetate is a crystalline form.
  3. The erlotinib dichloroacetate of Claim 1, wherein the erlotinib dichloroacetate is a crystalline form showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 6.6±0.2, 9.3±0.2, 13.1±0.2, 14.9±0.2, 18.5±0.2, 19.2±0.2, 20.1±0.2, 20.7±0.2, 21.1±0.2, 22.6±0.2, 22.9±0.2, 26.2±0.2, 26.4±0.2, 27.0±0.2, 28.2±0.2, and 28.5±0.2.
  4. The erlotinib dichloroacetate of Claim 1, wherein the erlotinib dichloroacetate is a crystalline form showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 6.6±0.2, 8.0±0.2, 13.0±0.2, 16.0±0.2, 16.8±0.2, 20.4±0.2, 20.8±0.2, 21.6±0.2, 22.9±0.2, 23.4±0.2, 26.0±0.2, 26.5±0.2, 27.0±0.2, and 31.1±0.2.
  5. The erlotinib dichloroacetate of Claim 1, wherein the erlotinib dichloroacetate is a crystalline form showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 7.6±0.2, 17.7±0.2, 18.2±0.2, 18.5±0.2, 21.7±0.2, 22.7±0.2, 23.2±0.2, 23.8±0.2, 26.6±0.2, and 29.1±0.2.
  6. The erlotinib dichloroacetate of Claim 1, wherein the erlotinib dichloroacetate is a crystalline erlotinib dichloroacetate hydrate showing an X-ray powder diffraction (XRPD) pattern characterized by peaks having I/Io values of at least 10% (I is the intensity of each peak; Io is the intensity of the highest peak) at diffraction angles (2θ) of 7.2±0.2, 9.1±0.2, 14.6±0.2, 17.0±0.2, 18.0±0.2, 20.1±0.2, 20.9±0.2, 23.7±0.2, 25.9±0.2, 27.0±0.2, 28.0±0.2, 29.0±0.2, 32.0±0.2, and 32.9±0.2.
  7. An anti-cancer agent comprising the erlotinib dichloroacetate of any one of Claims 1 to 6 with a pharmaceutically acceptable carrier.
  8. The anti-cancer agent of Claim 7, wherein the anti-cancer agent is used for treating non-small cell lung cancer (NSCLC) or pancreatic cancer.
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WO2013109142A1 (en) * 2012-01-16 2013-07-25 Stichting Het Nederlands Kanker Instituut Combined pdk and mapk/erk pathway inhibition in neoplasia
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WO2013109142A1 (en) * 2012-01-16 2013-07-25 Stichting Het Nederlands Kanker Instituut Combined pdk and mapk/erk pathway inhibition in neoplasia
CN102584719A (en) * 2012-02-02 2012-07-18 瑞阳制药有限公司 Preparing technology of erlotinib hydrochloride
WO2014118737A1 (en) 2013-01-31 2014-08-07 Ranbaxy Laboratories Limited Erlotinib salts
EP3842042A4 (en) * 2018-08-22 2022-08-17 Metafines.Co.Ltd. Pharmaceutical composition for treating cancer comprising metal ion-bound ionic compound
RU2780016C1 (en) * 2018-08-22 2022-09-19 Метафинес.Ко.Лтд. Pharmaceutical composition for cancer treatment, containing ionic compound related to metal ion
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