MAMMAL KINASE INHIBITORS TO PROMOTE IN VITRO EMBRYOGENESIS INDUCTION OF PLANTS
20230025843 · 2023-01-26
Inventors
- Pilar SÁNCHEZ TESTILLANO (Madrid, ES)
- Ana MARTINEZ GIL (Madrid, ES)
- Carmen GIL AYUSO-GONTAN (Madrid, ES)
- Eduardo BERENGUER PEINADO (Madrid, ES)
- Elena CARNEROS GARCÍA (Madrid, ES)
- Yolanda PÉREZ PÉREZ (Madrid, ES)
Cpc classification
International classification
Abstract
The present invention relates to the use of mammal kinase inhibitors, preferably human kinase inhibitors, to promote the induction of in vitro embryogenesis, a strategy never used in plants systems before. The results obtained indicated that these inhibitors have beneficial effects in both crop and forest plants in in vitro systems of microspore and somatic embryogenesis.
Claims
1. Use of at least a mammal kinases inhibitor to improve in vitro plant embryogenesis induction, wherein the mammal kinases inhibitor is selected from a compound of Formula (I) or a salt thereof: ##STR00006## wherein: A is —C(R.sup.1).sub.2—, —O— or —NR.sup.1—; E is —NR.sup.1— or —CR.sup.1R.sup.2— and the substituent R.sup.2 is absent if is a second bond between E and G; G is —S—, —NR.sup.1— or —CR.sup.1R.sup.2—and the substituent R.sub.2 is absent if
is a second bond between E and G;
may be a second bond between E and G where the nature of E and G permits and E with G optionally then forms a fused aryl group; R.sup.1 and R.sup.2 are independently selected from hydrogen, (C.sub.1-C.sub.8)alkyl, cycloakyl, haloalkyl, aryl, —(Z).sub.n-aryl, heteroaryl, —OR.sup.3, —C(O)R.sup.3, —C(O)OR.sup.3, —(Z).sub.n—C(O)OR.sup.3— and —S(O).sub.t— or as indicated R.sup.2 can be such that E with G then form a fused aryl group; Z is independently selected from —C(R.sup.3)(R.sup.4)—, —C(O)—, —O—, —C(═NR.sup.3)—, —S(O).sub.t— and —N(R.sup.3)—; n is zero, one or two; t is zero, one or two; R.sup.3 and R.sup.4 are independently selected from hydrogen, (C.sub.1-C.sub.8)alkyl, aryl and heterocyclic; X and Y are independently selected from ═O, ═S, ═N(R.sup.3) and ═C(R.sup.1)(R.sup.2); a compound of Formula (II) or a salt thereof: ##STR00007## wherein: R.sub.1 is selected from H, CN, NO.sub.2, F, Cl, Br, I, or a group X.sub.1—R.sub.1′ wherein X.sub.1 is a single bond or a group selected from C.sub.1-C.sub.6 alkylene, C.sub.2-C.sub.6 alkenylene, C.sub.2-C.sub.6 alkynylene, C.sub.3-C.sub.10 cycloalkylene, C.sub.3-C.sub.10 heterocycloalkylene, arylene and heteroaryl; being X.sub.1 optionally substituted; R.sub.1′ is selected from H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.6 alkoxy, aryl, heteroaryl, C.sub.3-C.sub.10 cycloalkyl or C.sub.3-C.sub.10 heterocycloalkyl; being R.sub.1′ optionally substituted; R.sub.2 is selected from C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, heteroaryl, C.sub.3-Cao cycloalkyl and C.sub.3-C.sub.10 heterocycloalkyl, CN or amino; being R.sub.2 optionally substituted; R.sub.3 is —CH.sub.2— R.sub.3′; R.sub.3′ is selected from heteroaryl, —C(O)OR.sub.12, or R.sub.3′ is selected from —(CH.sub.2).sub.nOR.sub.6e, n being between 1 and 20, with the condition, that R.sub.3′ cannot be —(CH.sub.2).sub.2—OH, R.sub.6e being selected from R.sub.4 and R.sub.5, or R.sub.3′ is selected from —(CH.sub.2).sub.n—(C.sub.3-C.sub.10heterocycloalkyl), with n being 0 to 20; and R.sub.12 is independently selected from H and C.sub.1-C.sub.6 alkyl; R.sub.4 and R.sub.5 are independently selected from: H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7, X.sub.4-cycloalkyl, X.sub.4-cyclobutyl, X.sub.4-cyclopentyl, X.sub.4-cyclohexyl, X.sub.4-cycloheptyl, X.sub.4-benzyl, X.sub.4-pyridinyl, X.sub.4-pirimidinyl, X.sub.4-pyperidinyl, X.sub.4-pyrrolidinyl, X.sub.4-pyrrolyl, X.sub.4-imidazolyl and X.sub.4-pyranyl saturated or unsaturated; X.sub.4 is a single bond or a group selected from C.sub.1-C.sub.6 alkylene, C.sub.2-C.sub.6 alkenylene; being R.sub.4 and R.sub.5 optionally substituted; a compound of Formula (III) or a salt thereof: ##STR00008## wherein: R.sup.1 is selected from H or C.sub.1-C.sub.10 alkyl and R.sup.2 is selected from C.sub.1-C.sub.10 alkyl or C.sub.2-C.sub.10 alkenyl; being optionally substituted by halogen; a compound of Formula (IV) or a salt thereof: ##STR00009## wherein: R.sub.1 is selected from H and C.sub.1-C.sub.5 alkyl, optionally substituted, R.sub.2 is C.sub.5-C.sub.15 alkyl, optionally substituted, R.sub.3 is selected from H, halogen, C.sub.1-C.sub.5 alkyl, optionally substituted, and —(O)— C.sub.1-C.sub.5 alkyl, optionally substituted, n is between 1 and 4, R.sub.4, R.sub.5 y R.sub.6 are each independently selected from H and C.sub.1-C.sub.5 alkyl, optionally substituted; a compound of Formula (V) or a salt thereof ##STR00010## wherein: R.sub.1 is selected from H, C.sub.1-C.sub.6 alkyl, halogen, CF.sub.3, and —O—C.sub.1-C.sub.6.alkyl; and (E,Z)-3-(morpholinoimino)indolin-2-one or a salt thereof.
2. Use according to claim 1 wherein the mammal kinases are human kinases.
3. Use according to claim 2 wherein the human kinases are the kinases GSK3β and/or LRRK2,
4. Use according to any of claims 1 to 3 wherein the kinase GSK3β comprises the sequence selected from the list consisting of SEQ ID NO: 1 to 4, and the kinase LRRK2 comprises the sequence selected from the list consisting of SEQ ID NO: 5 to 7.
5. Use according to any of claims 1 to 4 wherein the embryogenesis is somatic and/or by microspores.
6. Use according to any of claims 1 to 5 wherein the plants are crops plants, preferably Brassica spp. and/or Hordeum spp, or wherein the plants are forest plants, preferably Quercus spp.
7. Use according to any of claims 1 to 6, wherein the mammal kinases inhibitor is selecting from a list consisting of: 4-benzyl-2-methyl-1,2,4-thiadozilidine-3,5-dione (TDZD8), 5-(2-Morpholinethylimino)-2,3-diphenyl-2,5-dihydro-1,2,4-thiadiazole (VP3.15), 3-acetyl-4-(1-methyl-1H-indol-3-yl)-1H-pirrol-2,5-dione (VP3.36), 4-hydroxy-1-ethyl-N′-palmitoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (VP0.7), N-(6-methylbenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.3), N-(6-fluorobenzothiazole-2-yl)-4-morpholinobenzamide, (JZ1.6), N-(6-bromobenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.24), and (E,Z)-3-(morpholinoimino)indolin-2-one (IGS4.75).
8. Method to induce in vitro plant embryogenesis, comprising: a. culturing the microspores and/or explants in a culture medium suitable for embryo development; and b. adding mammal kinase inhibitors to the culture medium of a); and c. culturing for a period sufficient to obtain embryos.
9. Method according to claim 8 wherein the mammal kinases are human kinases, preferably GSK3β and/or LRRK2.
10. Method according to any of claims 8 to 9 wherein the kinase GSK3β comprises the sequence selected from the list consisting of SEQ ID NO: 1 to 4, and the kinase LRRK2 comprises the sequence selected from the list consisting of SEQ ID NO: 5 to 7.
11. Method according to any of claims 8 to 10 wherein the embryogenesis is somatic and/or by microspores.
12. Method according to any of claims 8 to 11 wherein the plants are crops plants, preferably Brassica spp. and/or Hordeum spp, or wherein the plants are forest plants, preferably Quercus spp.
13. Method according to any of claims 8 to 12, wherein the mammal kinases inhibitor is selecting from a compound of Formula (I) or a salt thereof: ##STR00011## wherein: A is —C(R.sup.1).sub.2—, —O— or —NR.sup.1—; E is —NR.sup.1— or —CR.sup.1R.sup.2— and the substituent R.sup.2 is absent if is a second bond between E and G; G is —S—, —NR.sup.1— or —CR.sup.1R.sup.2— and the substituent R.sup.2 is absent if
is a second bond between E and G;
may be a second bond between E and G where the nature of E and G permits and E with G optionally then forms a fused aryl group; R.sup.1 and R.sup.2 are independently selected from hydrogen, (C.sub.1-C.sub.8)alkyl, cycloakyl, haloalkyl, aryl, —(Z).sub.n-aryl, heteroaryl, —OR.sup.3, —C(O)R.sup.3, —C(O)OR.sup.3, —(Z).sub.n—C(O)OR.sup.3— and —S(O).sub.t— or as indicated R.sup.2 can be such that E with G then form a fused aryl group; Z is independently selected from —C(R.sup.3)(R.sup.4)—, —C(O)—, —O—, —C(═NR.sup.3)—, —S(O).sub.t— and —N(R.sub.3)—; n is zero, one or two; t is zero, one or two; R.sup.3 and R.sup.4 are independently selected from hydrogen, (C.sub.1-C.sub.8)alkyl, aryl and heterocyclic; X and Y are independently selected from ═O, ═S, ═N(R.sup.3) and ═C(R.sup.1)(R.sup.2); a compound of Formula (II) or a salt thereof: ##STR00012## wherein: R.sub.1 is selected from H, CN, NO.sub.2, F, Cl, Br, I, or a group X.sub.1—R.sub.1′ wherein X.sub.1 is a single bond or a group selected from C.sub.1-C.sub.6 alkylene, C.sub.2-C.sub.6 alkenylene, C.sub.2-C.sub.6 alkynylene, C.sub.3-C.sub.10 cycloalkylene, C.sub.3-C.sub.10 heterocycloalkylene, arylene and heteroaryl; being X.sub.1 optionally substituted; R.sub.1′ is selected from H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.6 alkoxy, aryl, heteroaryl, C.sub.3-C.sub.10 cycloalkyl or C.sub.3-C.sub.10 heterocycloalkyl; being R.sub.1′ optionally substituted; R.sub.2 is selected from C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, heteroaryl, C.sub.3-C.sub.10 cycloalkyl and C.sub.3-C.sub.10 heterocycloalkyl, CN or amino; being R.sub.2 optionally substituted; R.sub.3 is —CH.sub.2— R.sub.3′; R.sub.3′ is selected from heteroaryl, —C(O)OR.sub.12, or R.sub.3′ is selected from —(CH.sub.2).sub.nOR.sub.6e, n being between 1 and 20, with the condition, that R.sub.3′ cannot be —(CH.sub.2).sub.2—OH, R.sub.6e being selected from R.sub.4 and R.sub.5, or R.sub.3′ is selected from —(CH.sub.2).sub.n—(C.sub.3-C.sub.10heterocycloalkyl), with n being 0 to 20; and R.sub.12 is independently selected from H and C.sub.1-C.sub.6 alkyl; R.sub.4 and R.sub.5 are independently selected from: H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7 X.sub.4-cycloalkyl, X.sub.4-cyclobutyl, X.sub.4-cyclopentyl, X.sub.4-cyclohexyl, X.sub.4-cycloheptyl, X.sub.4-benzyl, X.sub.4-pyridinyl, X.sub.4-pirimidinyl, X.sub.4-pyperidinyl, X.sub.4-pyrrolidinyl, X.sub.4-pyrrolyl, X.sub.4-imidazolyl and X.sub.4-pyranyl saturated or unsaturated; X.sub.4 is a single bond or a group selected from C.sub.1-C.sub.6 alkylene, C.sub.2-C.sub.6 alkenylene; being R.sub.4 and R.sub.5 optionally substituted; a compound of Formula (III) or a salt thereof: ##STR00013## wherein: R.sup.1 is selected from H or C.sub.1-C.sub.10 alkyl and R.sup.2 is selected from C.sub.1-C.sub.10 alkyl or C.sub.2-C.sub.10 alkenyl; being optionally substituted by halogen; a compound of Formula (IV) or a salt thereof: ##STR00014## wherein: R.sub.1 is selected from H and C.sub.1-C.sub.5 alkyl, optionally substituted, R.sub.2 is C.sub.5-C.sub.15 alkyl, optionally substituted, R.sub.3 is selected from H, halogen, C.sub.1-C.sub.5 alkyl, optionally substituted, and —(O)— C.sub.1-C.sub.5 alkyl, optionally substituted, n is between 1 and 4, R.sub.4, R.sub.5 y R.sub.6 are each independently selected from H and C.sub.1-C.sub.5 alkyl, optionally substituted; a compound of Formula (V) or a salt thereof: ##STR00015## wherein: R.sub.1 is selected from H, C.sub.1-C.sub.6 alkyl, halogen, CF.sub.3, and —O—C.sub.1-C.sub.6.alkyl; and (E,Z)-3-(morpholinoimino)indolin-2-one or a salt thereof.
14. Method according to any of claims 8 to 13, wherein the mammal kinase inhibitor is selecting from a list consisting of: 4-benzyl-2-methyl-1,2,4-thiadozilidine-3,5-dione (TDZD8), 5-(2-Morpholinethylimino)-2,3-diphenyl-2,5-dihydro-1,2,4-thiadiazole (VP3.15), 3-acetyl-4-(1-methyl-1H-indol-3-yl)-1H-pirrol-2,5-dione (VP3.36), 4-hydroxy-1-ethyl-N′-palmitoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (VP0.7), N-(6-methylbenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.3), N-(6-fluorobenzothiazole-2-yl)-4-morpholinobenzamide, (JZ1.6), N-(6-bromobenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.24) and (E,Z)-3-(morpholinoimino)indolin-2-one (IGS4.75).
15. Method according to any of claims 8 to 14 wherein the mammal kinase inhibitor concentration ranges from 0.5 μM to 100 μM.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0145] Methodology
[0146] 1.1. Microspore Embryogenesis of B. napus, Through Isolated Microspore Culture (Protocol without Inhibitors)
[0147] B. napus L. (rapeseed) cv. ‘Topas’ line DH407 plants were used as donor plants. Rapeseed seeds were germinated and grew under controlled conditions (relative humidity 60%, 15° C. under long-day photoperiod 16 h light and 8 h dark at 10° C.) in a growth chamber in pots containing a mixture of organic substrate and vermiculite (2/1, v/v).
[0148] Flower buds containing vacuolated microspores, the most responsive stage for microspore induction, were isolated for microspore culture as previously described (Prem et al., 2012 BMC Plant Biology 12, 127). The selected buds were surface-sterilized in 5.0% (v/v) commercial bleach (5% active chlorine) for 20 min and then rinsed 6-7 times with sterile distilled water. Ten to 15 buds were crushed using a cold mortar and pestle in 5 ml of cold NLN-13 medium containing 13% sucrose (w/v). The suspension was filtered through 48 μm nylon mesh and the filtrate collected in 15-ml falcon centrifuge tubes. The crushed buds were rinsed with 5 ml NLN-13 to make up the volume to 10 ml and the filtrate was then centrifuged at 1100 rpm for 5 min at 4° C. The pellet was re-suspended in 10 ml of cold NLN-13 and centrifuged as mentioned above. This process was repeated three times for washing of the microspores. The final pellet was suspended in the NLN-13, and the cell density was adjusted to 10,000 cells per ml. The cell suspension was then poured into 90-mm Petri dishes (10 ml per Petri dish) and cultured in darkness. For embryogenesis induction, microspore cultures were subjected to an in vitro stress treatment of 32° C. for 15 days. In response to the inductive treatment, responsive microspores divide and produce multicellular structures or proembryos, still confined within the microspore wall (exine). Such structures are considered to be the first sign of embryogenesis initiation; they can be found after 4-6 days in culture. When globular/heart shaped embryos were observed (around 20 days), cultures were shifted to 25° C. on a gyratory shaker at 60 rpm until complete development and maturation of the embryos was observed (
[0149] 1.2. Microspore Embryogenesis of H. vulgare, Through Isolated Microspore Culture (Protocol without Inhibitors)
[0150] H. vulgare L. cv. Igri plants were used as donor plants. Seeds were vernalized in soil for one month at 4° C., and then transferred for one month in a plant growth chamber at 18° C. for germination and growth. Finally, plants were transferred to a greenhouse under 18° C. temperature.
[0151] Spikes containing microspores at the stage of vacuolated microspore, the most responsive stage for embryogenesis induction, were collected and surface sterilized by immersion in 5% bleach for 20 min, followed by 4 washes with sterile distilled water. Isolated microspore culture was settled as previously described (Rodŕiguez-Serrano et al., 2012, Journal of Experimental Botany 63(5), 2007-2024). The sterilized spikes were pre-treated at 4° C. for 21-24 days as stress treatment to induce microspore embryogenesis. Microspore were isolated blending spikes in 20 ml of pre-cooled 0.4 M mannitol at 4° C., using a Waring Blender pre-cooled in a refrigerator at −20° C., and the extract was filtered through a 100 μm nylon mesh into a beaker pre-cooled at −20° C. The collected microspore suspension was transferred into a 50 ml tube and centrifuged at 800 rpm for 10 min at 4° C. After removing the supernatant, the pellet was resuspended in 4 ml of pre-cooled 0.55 M maltose and transferred in 15 ml falcon tube. 1.5 ml of 0.4 M mannitol solution were cautiously added unmixed. After gradient centrifugation at 800 rpm for 10 min at 4° C., the interphase band consisting of an almost pure population of vacuolated microspores was resuspended in 0.4M mannitol solution giving a final volume of 10 ml. After counting cells in the Neubauer chamber, the pelleted microspores were diluted in an appropriate volume of KBP medium to obtain a cell density of 1.1×10.sup.5 cells per ml, and plated in 30 mm Petri dishes, at a volume of 1 ml per plate. Then, microspore cultures were incubated at 25° C. in the dark, and microspores reprogrammed and produced multicellular structures/proembryos that can be found after 4-6 days in culture, as the first sign of embryogenesis initiation. Proembryos further developed and produced coleoptylar and mature embryos (
[0152] 1.3. Microspore Embryogenesis of Q. Suber, Through Anther Culture (Protocol without Inhibitors)
[0153] Branches with several catkins were cut and collected from G. suber trees in the countryside (El Pardo region, Madrid, Spain), during the flowering period (from early May to early-mid June). Cut tips of branches were immediately covered with moist cotton and aluminium foil, and transferred to the laboratory, where they were kept in the dark at 4° C. for several days, until use for in vitro culture. Selected catkins were separated from branches and sterilized by immersion in 70% ethanol for 30-60 s, under vacuum, to aid penetration of the solvent. They were then immersed in 2% sodium hypochlorite with 1% Tween-20 for 20 min, with magnetic stirring. After three washes in sterile distilled water, catkins were prepared for dissection and anther excision.
[0154] Anther culture and microspore embryogenesis induction were performed as previously described (Testillano et al. 2018, Forestry Sciences Vol. 84. Springer International Publishing AG. pp. 93-105). Anthers containing vacuolated microspores, the most responsive stage for embryogenesis induction, were carefully excised from sterilized catkins under aseptic conditions and plated in Petri dishes of 90 mm diameter on solid induction medium which contained Sommer medium macronutrients, Murashige and Skoog (MS) micronutrients and vitamins, as well as 30 g/L sacarose and activated charcoal. Anthers were placed in linear arrays of 10-12 anthers each, with a gap of around 5 mm between each anther, and up to 100 anthers per Petri dish. Embryogenesis was induced by stress treatment at 33° C. in darkness for 5 days. After this inductive treatment, the anther cultures were transferred to 25° C. in darkness. In the following 20-30 days, responsive anthers become swollen and proembryos and small proembryogenic masses were visible as very small white structures emerging from the anther interior, breaking the tissues of the anther wall. After some more days, proembryos and proembryogenic masses grew and formed globular embryos by direct and indirect embryogenesis from individual microspores.
[0155] Microspore-derived embryogenic masses and embryos were transferred to new plates with proliferation medium which has a similar composition to induction medium except that it does not contain activated charcoal and is supplemented with 0.5 g/L glutamine. They were kept at 25° C. in darkness and sub-cultured every month in the same medium, where embryogenic masses can proliferate and spontaneously originate new globular embryos, which further developed heart-shaped, torpedo and cotyledonary embryos. In proliferation medium, some of these embryos produced new embryos by secondary and recurrent embryogenesis.
[0156] 1.4. Somatic Embryogenesis of G. suber, Through Immature Zygotic Embryos Culture (Protocol without Inhibitors)
[0157] Immature pollinated acorns were collected from G. suber L. (cork oak) trees in the countryside (El Pardo region, Madrid, Spain) during fruit development period (late August and September), transferred to the laboratory and kept at 4° C. for one week before in vitro culture initiation. Immature acorns were selected at the most responsive stage to somatic embryogenesis induction; they are those with small size, around 1 cm diameter, and green colour; they contain immature zygotic embryos at the early cotyledonary stage.
[0158] Immature zygotic embryos were carefully excised from the acorns by dissecting the surrounding tissues with the help of scalpel and forceps. After dissection, explants (immature zygotic embryos) were sterilized by immersion in 70% ethanol for 30 s and in 2% sodium hypochlorite for 20 min, followed by three rinses in sterile distilled water of 10 min each. Five explants were placed per plate.
[0159] Somatic embryogenesis was induced as previously described (Testillano et al. 2018, Plant Cell Culture Protocols, eds. V. M. Loyola-Vargas & N. Ochoa-Alejo. Springer and Bussines Media. pp. 247-256). Explants were first cultured in solid induction medium, which contains Sommer macronutrients, MS micronutrients and vitamins, 0.5 mg/l Glutamine, 30 g/l Sucrose, and 0.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D), for one month at 25° C. and 16/8h light/darkness. During this induction period, cell reprogramming occurs in some responsive cells which initiated the embryogenesis pathway, producing small proembryogenic masses. Then, the explants were transferred to solid proliferation medium, with the same composition but growth regulator-free (without 2,4-D). During the next weeks of culture in the proliferation medium, proembryogenic masses proliferated and protruded from different parts of the explants; they produce new embryogenic masses and embryos, which in turn give rise to new embryos, that developed to fully developed cotyledonary embryos, by recurrent and secondary embryogenesis (
[0160] 1.5. Treatment with Mammal Kinases Inhibitors on Microspore Embryogenesis Cultures of B. napus and H. vulgare in Liquid Media
[0161] The compounds were added to the microspore liquid culture media by using stock solutions of 10 mM in DMSO. Appropriate volumes of stock solutions of the drugs were added to the culture media to get the selected working concentrations of the inhibitors, keeping DMSO concentration below 0.2%. [0162] In B. napus microspore cultures: 4-benzyl-2-methyl-1,2,4-thiadozilidine-3,5-dione (TDZD8), 5-(2-Morpholinethylimino)-2,3-diphenyl-2,5-dihydro-1,2,4-thiadiazole (VP3.15), 3-acetyl-4-(1-methyl-1H-indol-3-yl)-1H-pirrol-2,5-dione (VP3.36), 4-hydroxy-1-ethyl-N′-palmitoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (VP0.7), N-(6-methylbenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.3), N-(6-fluorobenzothiazole-2-yl)-4-morpholinobenzamide, (JZ1.6) and N-(6-bromobenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.24). and (E,Z)-3-(morpholinoimino)indolin-2-one (IGS4.75) were tested at 3-4 different concentrations, ranging from 0.5 to 5 μM. [0163] In H. vulgare microspore cultures: 4-benzyl-2-methyl-1,2,4-thiadozilidine-3,5-dione (TDZD8) and N-(6-bromobenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.24) inhibitors at the selected concentrations (2.5 μM and 5p, respectively) were tested.
[0164] The compounds were added from culture initiation and their effect on embryogenesis efficiency was assessed. Several plates of the same cultures were kept without the inhibitors, as controls.
[0165] 1.6. Evaluation of the Effect of Mammal Kinases Inhibitors Over In Vitro Embryogenesis Induction in Isolated Microspore Cultures of B. napus and H. vulgare
[0166] Embryogenesis induction was quantified in control and treated-cultures by the number of proembryos formed (considered the first sign of embryogenesis initiation), as previously described, and by the number of embryos produced after 40 days. Proembryos were easily identified under inverted microscope in 4 day-culture plates as rounded multicellular structures with higher size and density than microspores, still surrounded by the exine (special microspore wall). Embryos produced after 40 days in culture were quantified through images captured under a stereo microscope. Randomly obtained micrographs from inverted and stereo microscopes were collected from untreated and treated microspore culture plates. Mean percentage of proembryos and mean number of embryos per plate were obtained from three independent experiments per each in vitro system and treatment. A minimum of 1000 proembryos were counted for each treatment and plant species. Results on proembryos were expressed as percentages (percent change) and referred to the mean percentage of proembryos in control cultures, which has been normalized to 100%.
[0167] In order to evaluate whether proembryo structures of treated cultures, identified under the inverted microscope for quantification, were actually dividing microspores, similar to the same structures in control cultures, a simply staining technique was performed to visualize nuclei inside proembryos. Samples from control and treated-cultures of 4 days, containing proembryos, were stained with 10 μg/mL 4′,6-diamidine-2-phenyl indole dihydrochloride (DAPI). Squash preparations were analysed under fluorescence microscopy using UV excitation for observing nuclei.
[0168] 1.7. Evaluation of Quality/Germination Capacity of Embryos Produced after Treatment with Mammal Kinases Inhibitors
[0169] To evaluate the quality of embryos produced in microspore embryogenesis cultures in the presence of the mammal kinases inhibitors, embryo germination assays were performed. B. napus microspore cotyledonary embryos originated from control and treated-cultures were used for in vitro embryo germination and conversion to plantlets as previously described (Prem et al., 2012, BMC Plant Biology 12, 127). The 34-40 old dicotyledonous embryos, after air desiccation on sterile filter paper were germinated in MS medium containing sucrose 2% (w/v) and gelled with 7 g/L bacteriological agar (w/v). Microspore derived-embryos were incubated for 15-20 days at 18° C. in darkness conditions till activation of radicle and plumule, and quantified in terms of percentage of embryos showing normal growth, similar to zygotic embryo germination.
[0170] 1.8. Treatments with Kinases Inhibitors on Microspore and Somatic Embryogenesis Cultures of G. suber in Solid Media
[0171] Since the in vitro systems of G. suber were two-step processes in solid culture media, a different strategy than in liquid microspore cultures was applied for the treatments with the mammal kinases inhibitors. During in vitro embryogenesis of Q. suber, after incubation in induction medium, the transfer of explants to proliferating medium involves the multiplication of proembryogenic masses, embryogenesis initiation, by recurrent and secondary embryogenesis, and embryo development. Therefore, treatments with the mammal kinases inhibitors were performed during the first 15-30 days in proliferating media, and afterwards, explants with emerging embryos were transferred to fresh proliferating media without the inhibitor.
[0172] Since solid media involve much less diffusion and availability of compounds to cells in comparison with liquid media, as referred in other in vitro systems, the concentration of the mammal kinases inhibitors used in solid media was around 10× higher than in liquid media, in the range of 25 to 100 μM. Appropriate volumes of stock solutions of 10 mM in DMSO of the selected compounds were added to cooled media, before its gelling, keeping DMSO concentration below 0.2%. Mock parallel plates of the same cultures were kept as controls.
[0173] 1.9. Evaluation of the Effect of Inhibitors Over In Vitro Embryogenesis Induction in Microspore and Somatic Embryogenesis Cultures of Q. Suber
[0174] Embryogenesis induction efficiency was quantified in control and treated-cultures by the number of cotyledonary embryos produced by 15-30 days of treatment (culture medium containing the inhibitor) followed by 30 days of recovery (culture medium without inhibitor). Embryo production was estimated as the number of cotyledonary embryos originated per gram of embryogenic masses at culture initiation.
[0175] Results
[0176] 1.1. Effect of Kinases Inhibitors Over Microscope Embryogenesis Cultures B. napus
[0177] To evaluate the effect of the kinase inhibitors over in vitro embryogenesis induction, we first tested them in B. napus microspore embryogenesis, as a model platform to check the mammal kinases inhibitors and different concentrations. After these analyses, one selected mammal kinases inhibitor of each category was tested in other two plant species, H. vulgare and Q. suber, with different in vitro systems. The efficiency of embryogenesis induction was evaluated in control cultures and cultures treated with the mammal kinases inhibitors, at different concentrations. The results for the GSK3β inhibitors and LRRK2 inhibitors tested are shown as the percentage of proembryos (first sign of embryogenesis initiation) in
[0178] The results showed that the increase of embryogenesis induction efficiency provided by the use of the inhibitors was in the range of 20-25% for GSK3β inhibitors and 23-30% for LRRK2 inhibitors.
[0179] To confirm that proembryos quantified in treated cultures were multicellular microspores that have initiated embryogenesis, squash preparations from control and treated cultures at 4 days were stained with DAPI and observed under fluorescence microscopy. Results showed that proembryos from treated cultures contained several nuclei (
[0180] Taking into account these results in B. napus, the compounds that were selected for testing in other in vitro embryogenesis systems were: [0181] 4-benzyl-2-methyl-1,2,4-thiadozilidine-3,5-dione (TDZD.8) as GSK3β inhibitor and [0182] N-(6-bromobenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.24) as LRRK2 inhibitor.
[0183] As it is showed in
[0184] The quality of the embryos produced in microspore cultures treated with the mentioned inhibitors was evaluated by germination assays. Fully developed cotyledonary embryos from control and treated cultures, produced after 30 days were desiccated and cultured under germination conditions. Results showed that embryos from treated cultures germinated very well, producing roots and hypocotyl, similarly and in the same proportion than embryos from control cultures (
[0185] 1.2. Effect of Kinase Inhibitors Over Microscope Embryogenesis Cultures of H. vulgare
[0186] The selected inhibitors, TDZ.8 and JZ1.24 were tested in microspore embryogenesis cultures of a different crop, H. vulgare. The inhibitors were firstly applied at the same concentrations that provided the best results in B. napus, 0.5 μM TDZD8 and 2.5 μM JZ1.24, but the results obtained (percentage of proembryos) in H. vulgare treated cultures using these concentrations were similar to control cultures. Therefore, two slightly higher concentrations were tested for both inhibitors (1 μM and 2.5 μM for TDZD8; and 2.5 μM and 5 μM for JZ1.24). The results showed that the two inhibitors lead to an increase in the embryogenesis initiation in H. vulgare, when used at slightly different concentrations than in B. napus, 2.5 μM TDZD8 and 5 μM JZ1.24. This indicates that optimal concentrations of these inhibitors could differ among species, probably due to differences in cell wall and permeability properties, and the specific features of each plant and in vitro system. The quantification of the proembryos formed at 4 days showed that treatments with the two inhibitors at the selected concentrations enhanced embryogenesis induction efficiency in H. vulgare, being the increase in proembryo formation of 27% in the case of 2.5 μM TDZD8, and 47% in the case of 5 μM JZ1.24-treated cultures (
[0187] Untreated and treated cultures further developed and total number of embryos produced per plate at 40 days was quantified. Microspore cultures treated with these inhibitors produced more embryos than control cultures, being the increment of 22% for JZ1.24 and 15% for TDZD8 (
[0188] The results indicated that small molecule inhibitors of mammalian GSK3β and LRRK2 produced a similar promoting effect in H. vulgare than in B. napus microspore cultures, an increase of in vitro embryogenesis induction efficiency. 1.3. Effect of Kinase Inhibitors Over Microspore and Somatic Embryogenesis Cultures of G. suber
[0189] In order to evaluate the possibility to extend the findings from B. napus and H. vulgare to more distant species and processes, the selected inhibitors, TDZD.8 and JZ1.24, were applied to a forest woody species G. suber, in which two different embryogenesis in vitro systems were established, somatic embryogenesis from immature zygotic embryos and microspore embryogenesis, two culture systems that consisted in two-step cultures in solid media.
[0190] Inhibitor treatments were applied at concentrations 10× higher than in liquid media, because of the lower diffusion and availability of compounds in gelled medium. The evaluation of the effects of the compounds over embryogenesis efficiency in the two systems were assessed by the quantification of the embryos produced in control and treated cultures. Results showed that treatments with the two types of inhibitors increased embryogenesis induction efficiency and lead to higher embryo production, in somatic embryogenesis from immature zygotic embryos cultures (
[0191] The results indicated that also in a woody species and in different in vitro embryogenesis systems, involving solid culture media, the small molecule inhibitors of mammalian GSK3β and LRRK2 produced the same effect than in rapeseed and barley systems in liquid media, an increase of in vitro embryogenesis induction efficiency.
CONCLUSIONS
[0192] The present invention deals with a major challenge of in vitro plant propagation techniques, that is to improve the efficiency of embryogenesis induction for rapid production of high numbers of high quality, disease-free and uniform planting material for agroindustry companies, reducing time and costs, in many species of economic interest. The new strategy reported in the present invention uses for the first time in plant in vitro systems inhibitors of mammalian protein kinases, specifically inhibitors of GSK3β and LRRK2 families, which have demonstrated capacity to increase embryogenesis induction and embryo production yield in three different crop and forest species. Moreover, treatments with these inhibitors have been successfully applied to different in vitro protocols, in liquid or solid media, and with direct, indirect and secondary/recurrent embryogenesis, providing similar promoting effects over embryogenesis. Several inhibitors of each group, with different molecular structure, have shown to be able to enhance embryogenesis efficiency, giving additional support to the use of these type of small molecules as new tools to optimize in vitro plant embryogenesis protocols.
[0193] 2. Synthesis and characterisation of the inibitors of the present invention.
[0194] 2.1. Inhibitors of Formula (I)
[0195] 4-benzyl-2-methyl-1,2,4-thiadozilidine-3,5-dione (TDZD8) is disclosed in Martinez A et al. (Martinez A et al. J Med Chem. 2002; 45(6):1292-9).
[0196] 2.2. Inhibitors of Formula (11)
[0197] All of inhibotors of Formula (II), including 5-(2-Morpholinethylimino)-2,3-diphenyl-2,5-dihydro-1,2,4-thiadiazole (VP3.15), are disclosed in EP2484670A1.
[0198] 2.3. Inhibitors of Formula (111)
[0199] 3-acetyl-4-(1-methyl-1H-indol-3-yl)-1H-pirrol-2,5-dione (VP3.36) is disclosed in Perez D I et al. (Perez D I et al. J Med Chem. 2011; 54(12):4042-56).
[0200] 2.4. Inhibitors of Formula (IV)
[0201] 4-hydroxy-1-ethyl-N′-palmitoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (VP0.7) is disclosed in Palomo V et al. (Palomo V et al. J Med Chem. 201; 60(12):4983-5001).
[0202] 2.5. Inhibitors of Formula (V)
[0203] N-(benzothiazole-2-yl)-4-morpholinobenzamide: 276.0 mg of 4-morpholinobenzoic acid (1.3 mmol), 331.00 mg of EDCI (1.4 mmol), 24.4 mg of DMAP (0.3 mmol) and 335 μL (2.4 mmol) of triethylamine were dissolved in dichloromethane. After stirring for 1 hour at room temperature, 200 mg of 2-aminobenzothiazole (1.3 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with saturated solutions of NaHCO.sub.3 and NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by chromatography in a flash column using a mixture of eluents CH.sub.2Cl.sub.2/MeOH (20:1) to obtain a yellow solid (72 mg, 16%). HPLC Purity >95%. MS: m/z 340 [M+1].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 10.21 (s, 1H, NH), 7.90 (d, J=9.0 Hz, 2H), 7.84 (dd, J=8.5, 1.5 Hz, 1H), 7.62 (dd, J=8.3, 1.2 Hz, 1H), 7.44-7.35 (m, 1H), 7.35-7.27 (m, 1H), 4.01-3.71 (m, 4H), 3.49-3.16 (m, 4H). .sup.13C NMR (75 MHz, DMSO-d.sub.6) δ 164.6, 159.1, 154.3, 148.2, 132.2, 129.4, 126.0, 123.7, 121.3, 121.1, 120.7, 113.8, 66.5, 47.4.
[0204] N-(6-methoxybenzothiazole-2-yl)-4-morpholinobenzamide: 230.0 mg of 4-morpholinobenzoic acid (1.1 mmol), 276.6 mg of EDCI (1.4 mmol), 24.43 mg of DMAP (0.2 mmol) and 248 μL (1.7 mmol) of triethylamine were dissolved in dichloromethane. After stirring for 1 hour at room temperature, 200 mg of 2-amino-6-methoxybenzothiazole (1.1 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with saturated solutions of NaHCO.sub.3 and NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by flash column chromatography using a mixture of eluents CH.sub.2Cl.sub.2/MeOH (50:1) to obtain a yellow solid (36 mg, 9%). P.f.: 237.6-240.0° C. HPLC Purity: 95%. MS: m/z 370 [M+H].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 9.47 (s, 1H), 7.88 (d, J=9.0 Hz, 2H), 7.64 (d, J=8.8 Hz, 1H), 7.33 (d, J=2.6 Hz, 1H), 7.04 (dd, J=8.8, 2.6 Hz, 1H), 6.94 (d, J=9.0 Hz, 2H), 3.93-3.83 (m, 7H), 3.36-3.31 (m, 4H). .sup.13C NMR (75 MHz, DMSO-d.sub.6) δ 164.8, 156.9, 156.0, 153.8, 142.7, 132.8, 129.8, 120.8, 120.5, 114.80, 113.1, 104.6, 65.8, 55.6, 46.8.
[0205] N-(6-trifluoromethylbenzothiazole-2-yl)-4-morpholinobenzamide: 189.9 mg of 4-morpholinobenzoic acid (0.9 mmol), 228.53 mg of EDCI (1.2 mmol), 22.41 mg of DMAP (0.2 mmol) and 223 μL (1.5 mmol) of triethylamine were dissolved in dichloromethane. After stirring for 1 hour at room temperature, 200 mg of 2-amino-6-trifluorobenzothiazole (0.9 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with saturated solutions of NaHCO.sub.3 and NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by automatic flash column chromatography (Biotage®Isolera One) using a mixture of eluents hexane/AcOEt to obtain a yellow solid (79 mg, 26%). P.f.: 218.5-218.5° C. HPLC Purity: 95%. MS: m/z 408 [M+H].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 10.85 (s, 1H), 8.13 (s, 1H), 7.89 (d, J=9.0 Hz, 1H), 7.57-7.53 (m, 2H), 6.84 (d, J=9.0 Hz, 2H), 3.87-3.83 (m, 4H), 3.31-3.26 (m, 4H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 163.8, 160.8, 153.4, 149.4, 131.2, 128.5, 124.9 (d, J=32.5 Hz), 124.3, 122.1 (d, J=3.4 Hz), 119.6 (d, J=32.2 Hz), 118.0 (d, J=4.2 Hz), 112.7, 65.4, 46.3, 28.6. C.sub.19H.sub.16F.sub.3N.sub.3O.sub.2S: Theoretical (%) C, 56.01; H, 3.96; N, 10.31; S, 7.87. Found (%) C, 56.13; H, 3.98; N, 10.38; S, 7.59.
[0206] N-(6-methylbenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.3): 252.4 mg of 4-morpholinobenzoic acid (1.2 mmol), 303.5 mg of EDCI (1.58 mmol), 20.06 mg of DMAP (0.2 mmol) and 272 μL (1.9 mmol) of triethylamine were dissolved in dichloromethane. After stirring for 1 hour at room temperature, 200 mg of 2-amino-6-methylbenzothiazole (1.2 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with solutions of HCl (0.1M), saturated NaHCO.sub.3 and saturated NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by automatic flash column chromatography (Biotage®Isolera One) using a mixture of eluents hexane/AcOEt to obtain a yellow solid (43 mg, 10%). P.f.: 287.7-288.8° C. MS (ESI+): m/z 354 [M+H].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 10.56 (s, 1H), 7.89 (d, J=8.9 Hz, 2H), 7.63 (s, 1H), 7.42 (d, J=8.3 Hz, 1H), 7.16 (dd, J=8.3, 1.7 Hz, 1H), 6.85 (d, J=8.9 Hz, 1H), 3.87-3.83 (m, 4H), 3.29-3.26 (m, 4H), 2.46 (s, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 164.7, 158.5, 154.2, 146.1, 133.7, 132.3, 129.4, 127.5, 121.3, 121.1, 120.3, 113.8, 66.5, 47.5, 21.4. C.sub.19H.sub.19N.sub.3O.sub.2S: Theoretical (%) C, 64.57; H, 5.42; N, 11.89; S, 9.07. Found (%) C, 64.33; H, 5.38; N, 11.85; S, 8.96.
[0207] N-(6-chlorobenzothiazole-2-yl)-4-morpholinobenzamide: 224.4 mg of 4-morpholinobenzoic acid (1.1 mmol), 269.89 mg of EDCI (1.4 mmol), 26.4 mg of DMAP (0.2 mmol) and 242 μL (1.7 mmol) of triethylamine were dissolved in dichloromethane. After stirring for 1 hour at room temperature, 200 mg of 2-amino-6-chlorobenzothiazole (1.1 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with solutions of HCl (0.1M), saturated NaHCO.sub.3 and saturated NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by automatic flash column chromatography (Biotage®Isolera One) using a mixture of eluents hexane/AcOEt to obtain a white solid (96 mg, 24%). P.f.: 245.4-246.4° C. HPLC Purity: 97%. MS: m/z 374 [M+H].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 10.25 (s, 1H), 7.89 (d, J=8.9 Hz, 2H), 7.81 (d, J=2.1 Hz, 1H), 7.52 (d, J=8.7 Hz, 1H), 7.33 (dd, J=8.7, 2.1 Hz, 1H), 6.89 (d, J=9.0 Hz, 2H), 3.92-3.82 (m, 4H), 3.33-3.30 (m, 4H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 164.5, 159.3, 154.4, 146.8, 139.7, 133.5, 129.4, 126.7, 121.5, 121.0, 120.8, 113.7, 66.5, 47.4. C.sub.18H.sub.16ClN.sub.3O.sub.2S: Theoretical (%) C, 57.83; H, 4.31; N, 11.24; S, 8.58. Found (%) C, 57.56; H, 4.09; N, 11.43; S, 8.40.
[0208] N-(6-fluorobenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.6): 168.20 mg of 4-morpholinobenzoic acid (1.2 mmol), 296.3 mg of EDCI (1.5 mmol), 29.05 mg of DMAP (0.2 mmol) and 265 μL (1.9 mmol) of triethylamine were dissolved in dichloromethane. After stirring for 1 hour at room temperature, 200 mg of 2-amino-6-fluorobenzothiazole (1.2 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with solutions of HCl (0.1M), saturated NaHCO.sub.3 and saturated NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by automatic flash column chromatography (Biotage®Isolera One) using a mixture of eluents hexane/AcOEt to obtain a white solid (79 mg, 19%). P.f.: 228.3-229.3° C. HPLC Purity: 98%. MS: m/z 358 [M+H].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 9.96 (s, 1H), 7.81 (d, J=8.9 Hz, 2H), 7.74 (d, J=2.1 Hz, 1H), 7.49 (d, J=8.6 Hz, 1H), 7.28 (dd, J=8.7, 2.1 Hz, 1H), 6.84 (d, J=8.7 Hz, 2H), 3.87-3.85 (m, 4H), 3.34-3.30 (m, 4H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 164.4, 159.2, 154.4, 147.0, 138.7, 133.6, 129.3, 126.8, 121.6, 121.0, 120.8, 113.8, 66.5, 47.4. C.sub.18H.sub.16FN.sub.3O.sub.2S: Theoretical (%) C, 60.49; H, 4.51; N, 11.76; S, 8.97. Found (%) C, 60.68; H, 4.50; N, 11.55; S, 8.72.
[0209] N-(6-ethoxybenzothiazole-2-yl)-4-morpholinobenzamide: 213.1 mg of 4-morpholinobenzoic acid (1.0 mmol), 256.2 mg of EDCI (1.3 mmol), 25.12 mg of DMAP (0.2 mmol) were dissolved in dichloromethane. After stirring for 6 hours at room temperature, 200 mg of 2-amino-6-ethoxybenzothiazole (1.0 mmol) and 229 μL of triethylamine (1.9 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with solutions of HCl (0.1M), saturated NaHCO.sub.3 and saturated NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by automatic flash column chromatography (Biotage®Isolera One) using a mixture of eluents hexane/AcOEt to obtain a yellow solid (20 mg, 5%). P.f.: 222.8-223.8° C. HPLC Purity: 95%. MS: m/z 384 [M+H].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 8.07 (d, J=8.6 Hz, 2H), 7.54 (d, J=8.9 Hz, 1H), 7.35-7.18 (m, 1H), 7.04 (dd, J=8.9, 2.4 Hz, 1H), 6.89 (d, J=8.6 Hz, 2H), 4.07 (q, J=6.9 Hz, 2H), 3.89-3.69 (m, 4H), 3.38-3.25 (m, 4H), 1.43 (t, J=6.9 Hz, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 164.4, 157.1, 156.0, 154.2, 142.3, 133.3, 129.3, 121.3, 121.2, 119.7, 115.5, 114.2, 113.8, 106.0, 104.9, 99.5, 66.5, 64.1, 64.1, 47.5, 14.8.
[0210] N-(6-bromobenzothiazole-2-yl)-4-morpholinobenzamide (JZ1.24): 180.9 mg of 4-morpholinobenzoic acid (0.9 mmol), 217.6 mg of EDCI (1.1 mmol), 21.33 mg of DMAP (0.2 mmol) were dissolved in dichloromethane. After stirring for 6 hours at room temperature, 200 mg of 2-amino-6-bromobenzothiazole (0.9 mmol) and 195 μl of triethylamine (1.4 mmol) were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with solutions of HCl (0.1M), saturated NaHCO.sub.3 and saturated NaCl, respectively. Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by automatic flash column chromatography (Biotage®Isolera One) using a mixture of eluents hexane/AcOEt to obtain a yellow solid (41 mg, 11%). P.f.: 237.5-238.5° C. HPLC Purity: 98%. MS: m/z 418 [M+H]+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 10.51 (s, 1H), 7.96 (s, 1H), 7.87 (d, J=9.0 Hz, 3H), 7.44 (dd, J=8.6, 1.9 Hz, 2H), 7.38 (d, J=8.6 Hz, 2H), 6.86 (d, J=9.0 Hz, 3H), 3.89-3.83 (m, 11H), 3.33-3.26 (m, 11H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 165.2, 160.4, 154.8, 147.1, 134.1, 130.0, 129.9, 124.3, 122.1, 121.1, 117.2, 114.1, 66.9, 47.8.
[0211] N-(6-propoxybenzothiazole-2-yl)-4-morpholinobenzamide: 248.8 mg of 4-morpholinobenzoic acid (1.2 mmol), 299.00 mg of EDCI (1.6 mmol), 29.3 mg of DMAP (0.2 mmol) were dissolved in dichloromethane. After stirring for 6 hours at room temperature, 250 mg of 2-amino-6-propoxybenzothiazole (1.2 mmol) and 267.6 μL (1.9 mmol) of triethylamine were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with a HCl solution (0.1M). Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by flash column chromatography using a mixture of eluents CH.sub.2Cl.sub.2/MeOH (50:1) to obtain a yellow solid (127 mg, 27%). HPLC Purity>95%. MS: m/z 398 [M+H]+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 7.88 (d, J=9.0 Hz, 2H), 7.46 (d, J=8.9 Hz, 1H), 7.31 (d, J=2.5 Hz, 1H), 6.96 (dd, J=8.9, 2.5 Hz, 1H), 6.88 (d, J=9.0 Hz, 2H), 3.98 (t, J=6.6 Hz, 2H), 3.89-3.83 (m, 4H), 3.32-3.27 (m, 4H), 1.85 (h, J=7.3 Hz, 2H), 1.06 (t, J=7.4 Hz, 2H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 163.6, 156.3, 155.2, 153.3, 141.27, 132.2, 128.4, 120.4, 120.3, 114.5, 112.8, 103.9, 69.2, 65.5, 46.5, 21.6, 9.5. C.sub.21H.sub.23N.sub.3O.sub.3S: Theoretical (%) C, 63.46; H, 5.83; N, 10.57; S, 8.07. Found (%) C, 63.73; H, 5.74, N, 10.09; S, 7.71.
[0212] N-(6-isopropylbenzothiazole-2-yl)-4-morpholinobenzamide: 269.4 mg of 4-morpholinobenzoic acid (1.3 mmol), 324.00 mg of EDCI (1.7 mmol), 32.00 mg of DMAP (0.3 mmol) were dissolved in dichloromethane. After stirring for 6 hours at room temperature, 250 mg of 2-amino-6-isopropylbenzothiazole (1.3 mmol) and 290.0 μL (2.1 mmol) of triethylamine were added. The reaction is left under stirring at room temperature during the night. After this time period the crude was washed with a HCl solution (0.1M). Next, the organic phase is dried on anhydrous magnesium sulfate, the solvent is evaporated at low pressure and is purified by flash column chromatography using a mixture of eluents CH.sub.2Cl.sub.2/MeOH (50:1) to obtain a yellow solid (218.4 mg, 44%). HPLC Purity>95%. MS: m/z 382 [M+H].sup.+. .sup.1H NMR (300 MHz, CDCl.sub.3) δ 10.35 (s, 1H), 7.89 (d, J=9.0 Hz, 2H), 7.68 (d, J=1.7 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.26-7.22 (m, 1H), 6.88 (d, J=9.0 Hz, 2H), 3.89-3.81 (m, 4H), 3.33-3.25 (m, 4H), 3.03 (p, J=6.9 Hz, 1H), 1.31 (d, J=6.9 Hz, 6H). .sup.13C NMR (75 MHz, CDCl.sub.3) δ 163.7, 157.7, 153.2, 145.3, 143.9, 131.3, 128.4, 124.0, 120.4, 119.4, 119.1, 117.5, 112.8, 65.5, 46.5, 33.2, 23.3. C.sub.21H.sub.23N.sub.3O.sub.2S: Theoretical (%) C, 66.12; H, 6.08; N, 11.00; S, 8.40. Found (%) C, 66.09; H, 6.13; N, 10.69; S, 8.54.
[0213] 2.6. Inhibitor of formula (E,Z)-3-(morpholinoimino)indolin-2-one (IGS4.75): is disclosed in Salado I G. et al., (Salado I G. et al., Eur J Med Chem. 2017 Sep. 29; 138:328-342).