Patent Grant
8318731
This application is a national stage application of PCT Application No. PCT/EP2008/059833, filed Jul. 25, 2008, which application claims priority from EP Patent Application No. 07113297.1, filed Jul. 27, 2007, all of which are hereby incorporated by reference in their entirety.
The present invention relates to pyrrolopyrimidines or pharmaceutically acceptable salts thereof, processes for preparing them, pharmaceutical compositions containing them and their use in therapy. The invention particularly relates to compounds that are polo-like kinase (PLKs) inhibitors useful for the treatment of disease states mediated by PLK, in particular such compounds that are useful in the treatment of pathological processes which involve an aberrant cellular proliferation, such as tumour growth, rheumatoid arthritis, restenosis and atherosclerosis.
The majorities of human cancers show no obvious inheritance pattern, but appear to develop through a multistep process in which genetic changes alter the normal control over cell division. A multitude of factors, including viruses, exposure to ultraviolet radiation and carcinogens, may contribute to a weakening of checkpoint controls in cell division. Also the aging process involves cumulative oxidative damage to gene promoters and a selective reduction in expression of mitotic regulators and centrosome proteins including cyclins A, B, F and PLKs. In combination, an incremental loss of checkpoint controls increases the probability of mitotic errors over multiple cell divisions that in combination with activating mutations in proto-oncogenes and loss of tumour suppressors lead to the progression of benign lesions into malignant tumours.
Polo-like kinases (PLKs) are key enzymes that control mitotic entry of proliferating cells and regulate many aspects of mitosis necessary for successful cytokinesis, including centrosome duplication and maturation; DNA damage checkpoint activation; bipolar spindle formation; Golgi fragmentation and assembly; and chromosome segregation (Barr, F. A. et al., Nat. Rev. Mol. Cell Biol. 2004, 5, 429-441). PLKs are found in organisms as diverse as yeast and human and contain two conserved domains, the N-terminal catalytic kinase domain and a C-terminal region composed of the so-called polo-boxes. In yeasts a single PLK exist, whereas four distinct PLKs have been identified to date in mammals. Whereas PLK1, PLK2 and PLK3 are expressed in all tissues and structurally homologous in that they comprise the N-terminal catalytic kinase domain and two polo-boxes, PLK 4 differs not only in structure, compared to the other PLKs it has only one polo-box, but also in the distribution of PLK4 mRNA in adults that is restricted to certain tissues such as testes and thymus (Karn, T. et al., Oncol. Rep. 1997, 4, 505-510; Fode, C. et al., Proc. Natl. Acad. Sci. USA 1994, 91, 6388-6392). It is still under investigation whether these differences also result in a unique physiological role for PLK4.
Given the established role of PLKs as mitotic regulators, they have been regarded as validated mitotic cancer targets for a number of years. In addition, recent studies demonstrate that changes of intracellular levels of PLKs are involved in the control of cell growth. For example, PLK1 when fused to an antennapedia peptide and efficiently internalized into cells caused an inhibition of cancer cell proliferation (Yuan, J., et al., Cancer Res. 62, 2002, 4186-4190), whereas downregulation of PLK1 by antisense induced the growth inhibition of cancer cells (Spankuch-Schmitt, B., et al., Oncogene 21, 2002, 3162-3171). PLK2 was recently found to be a novel p53 target gene and RNAi silencing of PLK2 leads to mitotic catastrophe in taxol-exposed cells (Burns, T F., et al., Mol Cell Biol. 23, 2003, 5556-5571). For PLK3 it was found that it induces cell cycle arrest and apoptosis through perturbation of microtubule integrity (Wang, Q., et al., Mol Cell Biol. 22, 2002, 3450-3459) and PLK4 was shown to be transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Li, J., et al., Neoplasia 7, 2005, 312-323). PLK4 was also found to be required for centriole duplication and flagella development. The absence of centrioles, and hence basal bodies, compromises the meiotic divisions and the formation of sperm axonemes (Bettencourt-Dias M., et al., Current Biology 15, 2005, 2199-2207).
All of this confirms that targeting PLKs with conventional small-molecule agents may be a valid and effective anticancer strategy with potential to synergize with established DNA-damage and antimitotic chemotherapies.
Relatively few reports of selective small-molecule PLK inhibitors have appeared to date.
Kyowa Hakko Kogyu has disclosed trisubstituted diamino-pyrimidine compounds as PLK1 inhibitors (PCT Int. Pat. Appl. Publ. WO 2004043936). Active compounds contain either a tetrazole group or a nitrile function at the pyrimidine C-5 position. A variety of (hetero)arylalkylamino groups are tolerated at C-2, whereas the C-4 substituent does not appear to be critical for PLK1 inhibitory activity.
A closely related pharmacophore, 5-nitro-N2,N4-diarylpyrimidine-2,4-diamines, was disclosed by GlaxoSmithKline (PCT Int. Pat. Appl. Publ. WO 2004074244). Again a variety of pyrimidine C-2 and C4 arylamine substituents were tolerated and the C-5 nitro group was a determinant of activity.
GlaxoSmithKline has also reported on a different pharmacophore, the 5-benzimidazol-1-yl-3-aryloxy-thiophene-2-carboxylic acid amides (PCT Int. Pat. Appl. Publ. WO 2004014899). Different substituents on the benzimidazole benzene ring were tolerated and a range of aryl ethers are present in the active compounds.
Onconova are developing amino-substituted 1(E)-2,6-dialkoxystyryl 4-substituted benzyl sulfones as cell-cycle agents with CDK inhibitory properties (PCT Int. Pat. Appl. Publ. WO 2003072062). Although reported as ATP-non-competitive kinase inhibitors, the relevant tumour cell target appears to be PLK1.
Certain macrocyclic compounds have been disclosed by the Applicants as inhibitors of tyrosine kinases (PCT Int. Pat. Appl. Publ. WO2004105765, WO2005058318, WO2005058913, WO2006061415 and WO2006061417) and as glycogen synthase kinase inhibitors (PCT Int. Pat. Appl. Publ. WO 2007003525).
It is accordingly an object of the present invention to provide selective small-molecule PLK inhibitors useful in the treatment of cell proliferative disorders.
The present invention concerns macrocyclic pyrrolopyrimidines having PLK inhibitory activity, particularly against PLK4. The invention further relates to methods for their preparation and pharmaceutical compositions comprising them. The invention also relates to the use of the macrocyclic pyrrolopyrimidines for the manufacture of a medicament for the treatment of cell proliferative disorders, including cancer, rheumatoid arthritis, restenosis and atherosclerosis. In the treatment of cancers, said cancers include lung cancer (especially non small-cell lung cancer), breast cancer, liver cancer, ovarian cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastrointestinal cancer such as colon, rectal or stomach cancer and papillary carcinomas (such as papillary thyroid cancer) as well as in squamous cell cancers of the head and neck and in oesophageal cancers including oropharyngeal cancer.
The compounds of the present invention are distinguishable from the above cited prior art because of their structure and their pharmacological activity as PLK inhibitors, in particular their activity against PLK4.
In addition to their activity against PLK4 certain of the compounds according to the invention have been found to have activity against Aurora B kinase. Aurora B is required for chromosome alignment, kinetochore-microtubule biorientation, activation of the spindle assembly checkpoint and cytokinesis. Aurora B is upregulated in various cancers such as colorectal cancer and high-grade gliomas, and Aurora B overexpression in CHO cells results in an increased invasiveness, suggesting a role for Aurora B in tumourigenesis (Carvajal, R. D. et al., Clin. Cancer Res. (2006) 12(23), 6869-6875).
The present invention relates to compounds of formula:
the N-oxide forms, the pharmaceutically acceptable addition salts, the quaternary amines and the stereochemically isomeric forms thereof, wherein
In an alternative embodiment, the invention relates to compounds of formula:
the N-oxide forms, the pharmaceutically acceptable addition salts, the quaternary amines and the stereochemically isomeric forms thereof, wherein
In an embodiment the present invention provides compounds of formula (I) and the N-oxide forms, the pharmaceutically acceptable addition salts, the quaternary amines and the stereochemically isomeric forms thereof, wherein one or more of the following restrictions apply:
In a further embodiment the present invention provides compounds of formula (I) and the N-oxide forms, the pharmaceutically acceptable addition salts, the quaternary amines and the stereochemically isomeric forms thereof, wherein one or more of the following restrictions apply:
In a further embodiment the present invention provides compounds of formula (I) and the N-oxide forms, the pharmaceutically acceptable addition salts, the quaternary amines and the stereochemically isomeric forms thereof, wherein one or more of the following restrictions apply:
In a further embodiment the present invention provides compounds of formula (I) and the N-oxide forms, the pharmaceutically acceptable addition salts, the quaternary amines and the stereochemically isomeric forms thereof, wherein one or more of the following restrictions apply:
In the compounds of formula (I) the —X2—Y—X1— linkage is advantageously attached to the 5-position of the pyrrolopyrimidine ring system and to the 3′-position of the phenyl ring.
Specific compounds according to the invention include:
Other specific compounds according to the invention include:
A more specific group of compounds according to the invention includes:
Two particular compounds according to the invention comprise:
As used herein;
When any variable occurs more than one time in any constituent, each definition is independent.
It will be appreciated that some of the compounds of formula (I) and their N-oxides, addition salts, quaternary amines and stereochemically isomeric forms may contain one or more centers of chirality and exist as stereochemically isomeric forms.
The term “stereochemically isomeric forms” as used hereinbefore or hereinafter defines all the possible stereoisomeric forms which the compounds of formula (I) and their N-oxides, addition salts, quaternary amines or physiologically functional derivatives may possess. Unless otherwise mentioned or indicated, the chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms, said mixtures containing all diastereomers and enantiomers of the basic molecular structure as well as each of the individual isomeric forms of formula (I) and their N-oxides, salts, solvates, quaternary amines substantially free, i.e. associated with less than 10%, preferably less than 5%, in particular less than 2% and most preferably less than 1% of the other isomers. Stereochemically isomeric forms of the compounds of formula (I) are obviously intended to be embraced within the scope of this invention.
For therapeutic use, salts of the compounds of formula (I) are those wherein the counter ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not are included within the ambit of the present invention.
The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove or hereinafter are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds of formula (I) are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.
The compounds of formula (I) containing an acidic proton may also be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. primary, secondary and tertiary aliphatic and aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, tromethamine, quinuclidine, pyridine, quinoline and isoquinoline; the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. Conversely the salt form can be converted by treatment with acid into the free acid form.
The term addition salt as used hereinabove also comprises the solvates which the compounds of formula (I), as well as the salts thereof, are able to form. Such solvates are for example hydrates, alcoholates and the like.
The term “quaternary amine” as used hereinbefore defines the quaternary ammonium salts which the compounds of formula (I) are able to form by reaction between a basic nitrogen of a compound of formula (I) and an appropriate quaternizing agent, such as, for example, an optionally substituted alkyl halide, aryl halide or arylalkyl halide, e.g. methyl iodide or benzyl iodide. Other reactants with good leaving groups may also be used, such as alkyl trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl p-toluenesulfonates. A quaternary amine has a positively charged nitrogen. Pharmaceutically acceptable counterions include for example chloro, bromo, iodo, trifluoroacetate and acetate. The counterion of choice can be made using ion exchange resin columns.
The N-oxide forms of the present compounds are meant to comprise the compounds of formula (I) wherein one or several tertiary nitrogen atoms are oxidized to the so-called N-oxide.
Some of the compounds of formula (I) may also exist in their tautomeric form. Such forms although not explicitly indicated in the above formula are intended to be included within the scope of the present invention.
The compounds of this invention can be prepared by any of several standard synthetic processes commonly used by those skilled in the art of organic chemistry and described for instance in the following references; “Heterocyclic Compounds”—Vol. 24 (part 4), p 261-304, Fused Pyrimidines, Wiley-Interscience; Chem. Pharm. Bull., Vol 41(2), 362-368 (1993); and J. Chem. Soc., Perkin Trans. 1, 2001, 130-137. The compounds are generally prepared from starting materials which are either commercially available or prepared by standard means obvious to those skilled in the art.
In general the compounds of formula (I) can be prepared as shown in Scheme 1 below wherein a pyrrolopyrimidine of formula (II) is converted, directly (for example by reaction with a compound of formula X4—Y2—X2—H (III)) or indirectly, into a compound of formula (IV) which is then reacted with an aniline of formula (V) to form a compound of formula (VI). The compound of formula (VI) can then be optionally deprotected if desired before cyclisation to form a compound of formula (I).
In the above Scheme:
In the above reaction of the compound of formula (II) with the compound of formula (III) the leaving group Z2 is advantageously a halo group such as a chlorine group and the group Z1 is advantageously an aldehyde (—CHO) group. The reaction can be effected by reductive amination for example by treating the compound of formula (II) in an organic solvent such as THF/DMF with an appropriate amine using a reducing agent such as for example sodium triacetoxyborohydride in an organic solvent for example dichloromethane, at r.t.
The reaction of the resulting compound of formula (IV) with the aniline compound of formula (V) is advantageously effected under acidic conditions for example with 4N hydrochloric acid in dioxane, the reaction being effected in an organic solvent such as acetonitrile or tert-butanol at an elevated temperature for example under reflux.
The resulting compound of formula (VI) can optionally be treated to remove any desired protecting groups for example ester groups such as tert-butyloxy-carbonyl ester groups can be converted to the parent free carboxy group, or an amide group such as a tert-butyloxycarbonylamino group can be converted to the free amino group. Such deprotection can be effected in conventional manner for example by treatment under acidic conditions for example using aqueous hydrochloric acid in dioxane at an elevated temperature for example about 60° C. Alternatively deprotection of a carboxy group can be effected under basic conditions for example using a base such as lithium hydroxide in THF/H2O 1/1 at r.t., followed by deprotection of an amino group by addition of aqueous hydrochloric acid in an appropriate solvent such as for example dioxane, or by addition of trifluoroacetic acid in an appropriate solvent such as dichloromethane.
The cyclisation of the compound of formula (VI) can be effected by treatment with a cyclising agent such as 1-benzotriazolyloxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP) in the presence of a base such as triethylamine and in an organic solvent such as dimethylformamide at r.t.
Other cyclisation agents which may be employed include 1,3-dicyclohexyl-carbodiimide (DCC), N,N′-carbonyldiimidazole (CDI), 1-[bis(dimethylamino)-methylene]-1H-benzotriazoliumhexafluorophophate-(1-)3-oxide (HBTU) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) in the presence or absence of hydroxybenzotriazole (HOBt).
The above general process is illustrated by the following more specific processes which describe the preparation of various classes of compounds of formula (I) above.
In these processes, illustrated by reaction Schemes 2-6, the various reaction stages, namely reductive amination, reaction of the pyrrolopyrimidine and the aniline compound, deprotection and cyclisation are all conducted in conventional manner for example using the reactions conditions and reagents described above for the general process illustrated in Scheme 1.
The compounds of formula (I) in which the —X2—Y—X1 grouping is —CH2—NR7—C1-6alkyl-NR3—CO—C1-6alkyl-X1, represented by formula (Ia), can be prepared as described below in Scheme 2 in which Y2a is —C1-6alkyl-NR3— and Y1a is —CO—C1-6alkyl-such that —Y2a—Y1a— is —C1-6alkyl-NR3—CO—C1-6alkyl-, P1 is an amino protecting group and P2 is a carboxy protecting group:
In the above scheme P1 is an amino protecting group such as tert-butoxycarbonyl (Boc) and P2 is a carboxy protecting group such as a C1-6alkyl group for example an ethyl group. Both protecting groups can be removed in one step by hydrolysis under acidic conditions for example using aqueous hydrochloric acid in an organic solvent such as dioxane. Alternatively the groups can be removed separately for example as described above using basic conditions to deprotect the carboxy group and then acidic conditions to deprotect the amino group.
The compounds of formula (I) in which —X2—Y—X1— grouping is —CH2—NR7—C1-6alkyl-CO—NR3—C1-6alkyl-X1—, represented by formula (Ib), can be prepared as described below in Scheme 3 in which Y1b is —NR3—C1-6alkyl- and Y2b is —C1-6alkyl-CO— such that —Y2b—Y1b— is —C1-6alkyl-CO—NR3—C1-6alkyl-:
The compounds of formula (I) containing a —X2—Y— grouping in which a C3 or C4alkyl group is directly attached to the pyrrolopyrimidine ring system may be prepared using an acetylenic intermediate as described in Scheme 4 below in which Ry is a R3, R4 or R7 group, Rz is a —CH2OH, —OCH2-phenyl or —OCH2COOP2 group and P3 is an N-protecting group:
In the above Scheme the Sonogashira reaction of the compound of formula (IVc) and the acetylenic compound of formula (Vc) is generally carried out in the presence of Pd(Ph3P)2Cl2 and cuprous iodide (CuI) and a base such as triethylamine or diethylamine, in an organic solvent such as dimethylacetamide or dimethylformamide, for example at r.t. The subsequent reduction may be effected for example by hydrogenation in the presence of a nickel catalyst in an organic solvent such as tetrahydrofuran.
The compounds of formula (I) in which the —X2—Y—X1— grouping is —(CH2)3 or 4—NR3—CO—C1-6alkyl-NR6—CH2—, represented by formula (Ic), can be prepared as described in Scheme 4a below using a compound of formula (VIIc) in which Rz is —CH2OH, Ry is R3, Y2c is —(CH2)3 or 4—NR3— and Y1c is —CO—C1-6alkyl-such that —Y2c—Y1c— is —(CH2)3 or 4—NR3—CO—C1-6alkyl-:
In the above Scheme the oxidation of the compound of formula (VIIc1) may be effected for example by treatment with manganese dioxide in an organic solvent such as tetrahydrofuran at r.t. The deprotection of the compound of formula (IXc) can be effected for example by treatment with a base such as aqueous sodium hydroxide in an appropriate solvent such as tetrahydrofuran or methanol, at r.t.
The procedure described in Scheme 4a can also be used to prepare compounds of formula (I) in which —X2—Y—X1— is —(CH2)3 or 4—NR4—CO-Het2-CH2—, represented by formula (Id), as described in Scheme 4b below in which Y2d is —(CH2)3 or 4—NR4— and Y1d is —CO-Het2- such that —Y2d—Y1d— is —(CH2)3 or 4—NR4—CO-Het2-:
Compounds of formula I in which the —X2—Y—X1— grouping is —(CH2)3 or 4—NR7—C1-6alkyl-NR3CO—CH2—O—, represented by formula (Ie), can be prepared as described in Scheme 4c below using a compound of formula (VIIc) in which Rz is a protected —OCH2COOP2 group, Ry is R7, Y2e is —C1-6alkyl-NR3— and Y1e is —CO—CH2— such that —Y2e—Y1e— is —C1-6alkyl-NR3—CO—CH2—:
The procedure described in Scheme 4c can be used to prepare compounds of formula (I) in which —X2—Y—X1— is —(CH2)3 or 4—NR7—C1-6alkyl-Het1-CO—CH2—O—, represented by formula (If), as described in Scheme 4d below in which Y2f is —CH2-Het1- and Y1f is —CO—CH2— such that —Y2f—Y1f— is —CH2-Het1-CO—CH2—:
Compounds of formula (I) in which the —X2—Y—X1— grouping is —(CH2)2—NR7—C1-6alkyl-NR3—CO—CH2O—, represented by formula (Ig) below, may be prepared as described in Scheme 5 below in which in which Y2g is —C1-6alkyl-NR3— and Y1g is —CO—CH2— such that —Y2g—Y1g— is —C1-6alkyl-NR3—CO—CH2—:
In the above Scheme the compound of formula (IIg) in which Z1 is an iodine atom may be subjected to a Stille reaction using (CH3(CH2)3)3SnCH2CHCH2 and Pd(Ph3P)4, in an organic solvent such as toluene at an elevated temperature for example at reflux. The resulting compound of formula (IIIg) can then be oxidised for example using osmium tetroxide, 4-methylmorpholine 4-oxide (NMO) and NaIO4 in an aqueous system, or by ozonolysis.
Compounds of formula (I) in which the X2—Y—X1— grouping is —CH2—NR7—C1-6alkyl-NR3—CO—C1-6alkyl-NR4—CO—C1-6alkyl-, represented by formula (Ih), may be prepared as described in Scheme 6 below in which Y1h is —CO—C1-6alkyl-, Y2h is —C1-6alkyl-NR3— and Y3h is —CO—C1-6alkylNR4— such that —Y2h—Y3h—Y1h is —C1-6alkyl-NR3—CO—C1-6alkyl-NR4—CO—C1-6alkyl-:
In the above Scheme the initial deprotection of the amino function in the compound of formula (IIh) can be effected by treatment with anhydrous 4N hydrochloric acid in dioxane in an organic solvent such as ethanol. The reaction of the compound of formula (IIIb) with the compound of formula P1Y3hOH can be effected using the same reagents and reaction conditions described above for the cyclisation reaction for example using PyBOP in the presence of a base such as triethylamine for example at 25° C.
In the above scheme compounds of formula (I) can be effected by for example alkylation reactions with for example alkylhalides in an organic solvent as for example dimethylformamide in the presence of a base such as cesium carbonate.
The starting materials used in the above syntheses may be prepared in conventional manner or are available commercially.
For the preparation of the starting materials of formula (IIa) above the following process can be used:
In the above Scheme the Z2 leaving group is preferably a halogen atom such as a chlorine atom and Z1 is generally a leaving group such as a halogen atom for example a bromine atom which is introduced by treating a compound of formula (1) with N-bromosuccinimide in an organic solvent such as dichloromethane and then reacting the resulting compound of formula (2) with butyllithium in an organic solvent such as tetrahydrofuran at −78° C. and subsequently quenching the resulting reaction product with dimethylformamide in an organic solvent such as tetrahydrofuran, also at −78° C.
For the preparation of the N-protected pyrrolopyrimidine starting materials of formula (IIc), used for example in Scheme 4, these can prepared in accordance with the following reaction Scheme:
In the above reaction scheme the Z2 leaving group is preferably a halogen atom such as a chlorine atom and the leaving group Z1 is generally an iodine atom which is introduced by treating the compound of formula (3) with N-iodosuccinimide in an organic solvent for example dichloromethane at r.t. for about one hour, and then reacting the resulting compound of formula (4) with sodium hydride and introduction of an appropriate P3 protecting group which is preferably an organosulphonyl group such as benzenesulphonyl for example by treatment with benzenesulphonyl chloride at a temperature between r.t. and 0° C.
The aniline starting materials can be prepared as described in the literature for example by the procedures described in the above-mentioned patent specifications relating to macrocyclic tyrosine kinase inhibitors, namely PCT Int. Pat. Appl. Publ. WO 2004105765, WO 2005058318, WO 2005058913, WO2006061415, WO2006061417 and WO 2007003525.
Compounds of formula (V) in which —X1— is —O— can be prepared from the corresponding nitrophenol in accordance with the following scheme in which Z is a leaving group for example a halogen atom such as chlorine:
The reaction of the compounds of formulae (5) and (6) can be effected under alkaline conditions for example in the presence of a base such cesium carbonate in an organic solvent for example dimethylformamide. The resulting compound of formula (6) can then be reduced in conventional manner to the aniline compound of formula (V), for example by hydrogenation with palladium/carbon in the presence of a 4% thiophene solution in diisopropyl ether and in an organic solvent such as tetrahydrofuran.
For the preparation of such compounds in which X1 is —NR5— the corresponding nitro compound can be obtained for example in accordance with the procedure described in Tetrahedron Vol. 45, No. 24, pp 7817-7826, 1989.
Compounds of formula (V) in which —Y1—X1— is —C1-6alkyl-NR3—CH2— may be prepared for example by reductive amination of a corresponding commercially available 2-nitrobenzaldehyde in accordance with the following scheme:
The compound of formula (7) is subjected to reductive amination in conventional manner for example as described above to form a nitro compound of formula (8) which is then reduced under conventional conditions for example as described above to form the desired aniline of formula (V).
Compounds of formula (V) in which —Y1—X1— is —CO—C1-6alkyl-may be prepared for example by subjecting a 2-nitrobenzaldehyde of formula (9) to a Wittig reaction to obtain a compound of formula (10) which is then reduced in accordance with the following scheme in which n is 0 to 4:
The compound of formula (9) is reacted with a (triphenylphosphorylidene)alkanoic acid ester in an organic solvent such as tetrahydrofuran and the resulting compound of formula (10) is then reduced under conventional conditions for example as described above to form the desired aniline of formula (V).
Where necessary or desired, any one or more of the following further steps in any order may be performed:
Compounds of formula (I), N-oxides, addition salts, quaternary amines and stereochemical isomeric forms thereof can be converted into further compounds according to the invention using procedures known in the art.
It will be appreciated by those skilled in the art that in the processes described above the functional groups of intermediate compounds may need to be blocked by protecting groups.
Functional groups, which are desirable to protect, include hydroxy, amino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl groups (e.g. tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), benzyl and tetrahydropyranyl. Suitable protecting groups for amino include tert-butyloxycarbonyl or benzyloxycarbonyl. Suitable protecting groups for carboxylic acid include C1-6alkyl or benzyl esters.
The protection and deprotection of functional groups may take place before or after a reaction step.
Additionally, the N-atoms in compounds of formula (I) can be methylated by art-known methods using CH3—I in a suitable solvent such as, for example 2-propanone, tetrahydrofuran or dimethylformamide. Alternatively N-atoms can be alkylated by treatment with an appropriate aldehyde and a reducing agent such as NaBH(OAc)3.
Compounds of formula (I) in which R8 is hydrogen, obtained in certain of the synthetic routes described above, can be converted into compounds of formula (I) in which R8 is other than hydrogen in conventional manner for example by reaction with sodium hydride in dimethylformamide and treatment with an appropriate R8 derivative for example an organosulphonyl derivative such as benzenesulfonyl chloride, or a halo alkyl derivative such as methyl iodide. Alternatively the conversion can be effected using an appropriate R8 derivative such as R8OH in a Mitsunobu reaction with for example Ph3P and DEAD in an appropriate organic solvent such as tetrahydrofuran. Such conversions are further described by D. J. Calderwood et al, Bioorg. Med. Chem. Lett. 12, (2002), 1683-1686.
The compounds of formula (I) can also be converted into each other following art-known procedures of functional group transformation of which some examples are mentioned hereinafter.
The compounds of formula (I) may also be converted to the corresponding N-oxide forms following art-known procedures for converting a trivalent nitrogen into its N-oxide form. Said N-oxidation reaction may generally be carried out by reacting the starting material of formula (I) with 3-phenyl-2-(phenylsulfonyl)oxaziridine or with an appropriate organic or inorganic peroxide. Appropriate inorganic peroxides comprise, for example, hydrogen peroxide, alkali metal or earth alkaline metal peroxides, e.g. sodium peroxide, potassium peroxide; appropriate organic peroxides may comprise peroxy acids such as, for example, benzenecarboperoxoic acid or halo substituted benzenecarboperoxoic acid, e.g. 3-chlorobenzenecarboperoxoic acid, peroxoalkanoic acids, e.g. peroxoacetic acid, alkylhydroperoxides, e.g. t-butyl hydroperoxide. Suitable solvents are, for example, water, lower alkanols, e.g. ethanol and the like, hydro-carbons, e.g. toluene, ketones, e.g. 2-butanone, halogenated hydrocarbons, e.g. dichloromethane, and mixtures of such solvents.
Pure stereochemically isomeric forms of the compounds of formula (I) may be obtained by the application of art-known procedures. Diastereomers may be separated by physical methods such as fractional crystallization and chromatographic techniques, e.g. counter-current distribution, liquid chromatography and the like.
Some of the compounds of formula (I) and some of the intermediates in the present invention may contain an asymmetric carbon atom. Pure stereochemically isomeric forms of said compounds and said intermediates can be obtained by the application of art-known procedures. For example, diastereoisomers can be separated by physical methods such as fractional crystallization or chromatographic techniques, e.g. counter current distribution, liquid chromatography and the like methods. Enantiomers can be obtained from racemic mixtures by first converting said racemic mixtures with suitable resolving agents such as, for example, chiral acids, to mixtures of diastereomeric salts or compounds; then physically separating said mixtures of diastereomeric salts or compounds by, for example, fractional rystallization or chromatographic techniques, e.g. liquid chromatography and the like methods; and finally converting said separated diastereomeric salts or compounds into the corresponding enantiomers. Pure stereochemically isomeric forms may also be obtained from the pure stereochemically isomeric forms of the appropriate intermediates and starting materials, provided that the intervening reactions occur stereospecifically.
An alternative manner of separating the enantiomeric forms of the compounds of formula (I) and intermediates involves liquid chromatography, in particular liquid chromatography using a chiral stationary phase.
Some of the intermediates and starting materials as used in the reaction procedures mentioned hereinabove are known compounds and may be commercially available or may be prepared according to art-known procedures.
We have now surprisingly found that, the pyrrolopyrimidines defined hereinbefore possess potent anti-tumour activity. Without wishing to imply that the compounds disclosed in the present invention possess pharmacological activity only by virtue of an effect on a single biological process, it is believed that the compounds provide an anti-tumour effect by way of inhibition of one or more of protein kinases that are involved in the regulation of cellular mitosis and which lead to cytogenetic catastrophe in case of abberant activity.
It is thus an object of the present invention to provide the compounds of the present invention for use as a medicine. As used herein the compounds of the present invention includes the compounds of formula (I) as defined hereinbefore, including all subgroups and combinations thereof
In one aspect, the compounds of the present invention may be useful for the treatment or prevention of cell proliferative disorders, including cancer, rheumatoid arthritis, restenosis and atherosclerosis. In the treatment of cancers said cancers include lung cancer (especially non small-cell lung cancer), breast cancer, liver cancer, ovarian cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastrointestinal cancer such as colon, rectal or stomach cancer and papillary carcinomas (such as papillary thyroid cancer), squamous cell cancers of the head and neck, oesophageal cancers including oropharyngeal cancer, and fast-dividing leukaemias such as acute myelogenous leukaemia (AML).
The compounds of the present invention may be administered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains a compound of formula (I) and one or more additional therapeutic agents, as well as administration of the compound of formula (I) and each additional therapeutic agents in its own separate pharmaceutical dosage formulation. For example, a compound of formula (I) and a therapeutic agent may be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations.
Where separate dosage formulations are used, the compounds of the present invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).
For example the compounds of the present invention could be used in combination with other anti-cancer agents. Examples of anti-cancer agents are:
The term “platinum coordination compound” is used herein to denote any tumour cell growth inhibiting platinum coordination compound which provides platinum in the form of an ion.
The term “taxane compounds” indicates a class of compounds having the taxane ring system and related to or derived from extracts from certain species of yew (Taxus) trees.
The term “topisomerase inhibitors” is used to indicate enzymes that are capable of altering DNA topology in eukaryotic cells. They are critical for important cellular functions and cell proliferation. There are two classes of topoisomerases in eukaryotic cells, namely type I and type II. Topoisomerase I is a monomeric enzyme of approximately 100,000 molecular weight. The enzyme binds to DNA and introduces a transient single-strand break, unwinds the double helix (or allows it to unwind) and subsequently reseals the break before dissociating from the DNA strand. Topisomerase II has a similar mechanism of action which involves the induction of DNA strand breaks or the formation of free radicals.
The term “camptothecin compounds” is used to indicate compounds that are related to or derived from the parent camptothecin compound which is a water-insoluble alkaloid derived from the Chinese tree Camptothecin acuminata and the Indian tree Nothapodytes foetida.
The term “podophyllotoxin compounds” is used to indicate compounds that are related to or derived from the parent podophyllotoxin, which is extracted from the mandrake plant.
The term “anti-tumour vinca alkaloids” is used to indicate compounds that are related to or derived from extracts of the periwinkle plant (Vinca rosea).
The term “alkylating agents” encompass a diverse group of chemicals that have the common feature that they have the capacity to contribute, under physiological conditions, alkyl groups to biologically vital macromolecules such as DNA. With most of the more important agents such as the nitrogen mustards and the nitrosoureas, the active alkylating moieties are generated in vivo after complex degradative reactions, some of which are enzymatic. The most important pharmacological actions of the alkylating agents are those that disturb the fundamental mechanisms concerned with cell proliferation in particular DNA synthesis and cell division. The capacity of alkylating agents to interfere with DNA function and integrity in rapidly proliferating tissues provides the basis for their therapeutic applications and for many of their toxic properties.
The term “anti-tumour anthracycline derivatives” comprise antibiotics obtained from the fungus Strep. peuticus var. caesius and their derivatives, characterised by having a tetracycline ring structure with an unusual sugar, daunosamine, attached by a glycosidic linkage.
Amplification of the human epidermal growth factor receptor 2 protein (HER 2) in primary breast carcinomas has been shown to correlate with a poor clinical prognosis for certain patients. Trastuzumab is a highly purified recombinant DNA-derived humanized monoclonal IgG1 kappa antibody that binds with high affiniity and specificity to the extracellular domain of the HER2 receptor.
Many breast cancers have estrogen receptors and growth of these tumours can be stimulated by estrogen. The terms “estrogen receptor antagonists” and “selective estrogen receptor modulators” are used to indicate competitive inhibitors of estradiol binding to the estrogen receptor (ER). Selective estrogen receptor modulators, when bound to the ER, induces a change in the three-dimensional shape of the receptor, modulating its binding to the estrogen responsive element (ERE) on DNA.
In postmenopausal women, the principal source of circulating estrogen is from conversion of adrenal and ovarian androgens (androstenedione and testosterone) to estrogens (estrone and estradiol) by the aromatase enzyme in peripheral tissues. Estrogen deprivation through aromatase inhibition or inactivation is an effective and selective treatment for some postmenopausal patients with hormone-dependent breast cancer.
The term “antiestrogen agent” is used herein to include not only estrogen receptor antagonists and selective estrogen receptor modulators but also aromatase inhibitors as discussed above.
The term “differentiating agents” encompass compounds that can, in various ways, inhibit cell proliferation and induce differentiation. Vitamin D and retinoids are known to play a major role in regulating growth and differentiation of a wide variety of normal and malignant cell types. Retinoic acid metabolism blocking agents (RAMBA's) increase the levels of endogenous retinoic acids by inhibiting the cytochrome P450-mediated catabolism of retinoic acids.
DNA methylation changes are among the most common abnormalities in human neoplasia. Hypermethylation within the promotors of selected genes is usually associated with inactivation of the involved genes. The term “DNA methyl transferase inhibitors” is used to indicate compounds that act through pharmacological inhibition of DNA methyl transferase and reactivation of tumour suppressor gene expression.
The term “kinase inhibitors” comprises potent inhibitors of kinases that are involved in cell cycle progression and programmed cell death (apoptosis).
The term “farnesyltransferase inhibitors” is used to indicate compounds that were designed to prevent farnesylation of Ras and other intracellular proteins. They have been shown to have effect on malignant cell proliferation and survival.
The term “histone deacetylase inhibitor” or “inhibitor of histone deacetylase” is used to identify a compound, which is capable of interacting with a histone deacetylase and inhibiting its activity, more particularly its enzymatic activity. Inhibiting histone deacetylase enzymatic activity means reducing the ability of a histone deacetylase to remove an acetyl group from a histone.
The term “other inhibitors of the ubiquitin-proteasome pathway” is used to indentify compounds that inhibit the targeted destruction of cellular proteins in the proteasome, including cell cycle regulatory proteins.
The term “telomerase inhibitor” refers to compounds which target, decrease or inhibit the activity of telomerase, especially compounds which inhibit the telomerase receptor.
The term “matrix metalloproteinase inhibitor” includes but is not limited to, collagen peptidomimetic and non-peptidomimetic inhibitors.
The compounds of the present invention can be used as “radiosensitizer” and/or “chemosensitizer”.
Radiosensitizers are known to increase the sensitivity of cancerous cells to the toxic effects of ionizing radiation. Several mechanisms for the mode of action of radiosensitizers have been suggested in the literature including: hypoxic cell radiosensitizers (e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds) mimicking oxygen or alternatively behave like bioreductive agents under hypoxia; non-hypoxic cell radiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNA bases and preferentially incorporate into the DNA of cancer cells and thereby promote the radiation-induced breaking of DNA molecules and/or prevent the normal DNA repair mechanisms; and various other potential mechanisms of action have been hypothesized for radiosensitizers in the treatment of disease. Many cancer treatment protocols currently employ radiosensitizers in conjunction with radiation of x-rays. Examples of x-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.
Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives, tin etioporphyrin, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.
Radiosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds which promote the incorporation of radiosensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and/or oxygen to the target cells; chemotherapeutic agents which act on the tumour with or without additional radiation; or other therapeutically effective compounds for treating cancer or other disease. Examples of additional therapeutic agents that may be used in conjunction with radiosensitizers include, but are not limited to: 5-fluorouracil, leucovorin, 5′-amino-5′deoxythymidine, oxygen, carbogen, red cell transfusions, perfluorocarbons (e.g., Fluosol 10 DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, and LBSO. Examples of chemotherapeutic agents that may be used in conjunction with radiosensitizers include, but are not limited to: adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of the same.
Chemosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds which promote the incorporation of chemosensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and/or oxygen to the target cells; chemotherapeutic agents which act on the tumour or other therapeutically effective compounds for treating cancer or other disease.
In view of the above described pharmacological properties, the compounds of formula (I) or any subgroup thereof, their N-oxides, pharmaceutically acceptable addition salts, quaternary amines and stereochemically isomeric forms, may be used as a medicine. In particular, the present compounds can be used for the manufacture of a medicament for treatment of any one of the disease conditions mentioned hereinbefore, in particular for the manufacture of a medicament for the treatment of cancer including lung cancer (especially non small-cell lung cancer), breast cancer, liver cancer, ovarian cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastrointestinal cancer such as colon, bladder, rectal or stomach cancer and papillary carcinomas (such as papillary thyroid cancer), squamous cell cancers of the head and neck, oesophageal cancers including oropharyngeal cancer, and fast-dividing leukaemias such as acute myelogenous leukaemia (AML).
In view of the utility of the compounds of formula (I) there is provided a method of treating warm-blooded animals, including humans, suffering from or a method of preventing warm-blooded animals, including humans, to suffer from any one of the diseases mentioned hereinbefore, such as cancer including lung cancer (especially non small-cell lung cancer), breast cancer, liver cancer, ovarian cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastrointestinal cancer such as colon, bladder, rectal or stomach cancer and papillary carcinomas (such as papillary thyroid cancer) as well as in squamous cell cancers of the head and neck and in oesophageal cancers including oropharyngeal cancer. Said methods comprise the administration, i.e. the systemic or topical administration, preferably oral administration, of an effective amount of a compound of formula (I), a N-oxide form, a pharmaceutically acceptable addition salt, a quaternary amine or a possible stereoisomeric form thereof, to warm-blooded animals, including humans.
One skilled in the art will recognize that a therapeutically effective amount of the compounds of the present invention is the amount sufficient to have anti-tumour activity and that this amount varies inter alias, depending on the type of disease, the concentration of the compound in the therapeutic formulation, and the condition of the patient. Generally, the amount of a compound of the present invention to be administered as a therapeutic agent for treating cell proliferative disorders such as cancer, rheumatoid arthritis, restenosis and atherosclerosis will be determined on a case by case by an attending physician.
Generally, a suitable dose is one that results in a concentration of the compounds of the present invention at the treatment site in the range of 0.5 nM to 200 μM, and more usually 5 nM to 50 μM. To obtain these treatment concentrations, a patient in need of treatment likely will be administered between 0.01 mg/kg to 250 mg/kg body weight, in particular from 0.1 mg/kg to 50 mg/kg body weight. The amount of a compound according to the present invention, also referred to here as the active ingredient, which is required to achieve a therapeutically effect will be, of course vary on case-by-case basis, for example with the particular compound, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated. A method of treatment may also include administering the active ingredient on a regimen of between one and four intakes per day. In these methods of treatment the compounds according to the invention are preferably formulated prior to admission. As described herein below, suitable pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients.
The present invention also provides compositions for preventing or treating cell proliferative diseases, such as cancer, rheumatoid arthritis, restenosis and atherosclerosis. Said compositions comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier or diluent.
While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical composition. Accordingly, the present invention further provides a pharmaceutical composition comprising a compound according to the present invention, together with a pharmaceutically acceptable carrier or diluent. The carrier or diluent must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof
The pharmaceutical compositions of this invention may be prepared by any methods well known in the art of pharmacy, for example, using methods such as those described in Gennaro et al. Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical preparations and their Manufacture). A therapeutically effective amount of the particular compound, in base form or addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for systemic administration such as oral, percutaneous or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions: or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wettable agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause any significant deleterious effects on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on or as an ointment.
It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
The present compounds can be used for systemic administration such as oral, percutaneous or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like. The compounds are preferably orally administered. The exact dosage and frequency of administration depends on the particular compound of formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, extent of disorder and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
The following examples illustrate the present invention.
In obtaining the compounds described in the examples below, the following experimental protocols were followed unless otherwise indicated.
Unless otherwise stated, reaction mixtures were magnetically stirred at r.t. Where solutions were “dried,” they were generally dried over a drying agent such as Na2SO4 or MgSO4. Where mixtures, solutions, and extracts were “concentrated”, they were typically concentrated on a rotary evaporator under reduced pressure.
Hereinafter, the term “DMA” means N,N-dimethylacetamide, “DIPEA” means N-ethyl-N-(1-methylethyl)-2-propanamine, “DCM” means dichloromethane, “MeOH” means methanol, “EtOAc” means ethyl acetate, “HBTU” means 1-[bis(dimethylamino)methylene]-1H-benzotriazoliumhexafluorophosphate(1-)3-oxide, “DMF” means N,N-dimethylformamide, “TFA” means trifluoroacetic acid, “PyBOP” means 1-benzotriazolyloxytripyrrolidinylphosphonium hexafluorophosphate, “EtOH” means ethanol, “DIPE” means diisopropyl ether, “THF” means tetrahydrofuran, “LCMS” means Liquid Chromatography/Mass spectrometry, “eq.” means equivalent, “t-BuOH” means t-butanol, “HPLC” means high-performance liquid chromatography, “HOAc” acetic acid, “BOC-anhydride” means di-tert-butyl dicarbonate, “DMAP” means N,N-Dimethyl-4-pyridinamine, “DIAD” means diisopropyl diazodicarboxylate, “DMSO” means dimethyl sulfoxide, “CH3CN” means acetonitrile, “r.t.” means room temperature, “p.a.” means pro analyse, “aq.” means aqueous, “(Ph)3P” means triphenylphosphine and “Et2O” means diethyl ether.
For some compounds that were purified by reversed phase high-performance liquid chromatography (HPLC) the used method is described below (indicated in the compound procedure with HPLC method A, HPLC method B, HPLC method C). When necessary, these methods can be slightly adjusted by a person skilled in the art to obtain a more optimal result for the separation.
HPLC Method A
The product was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). Three mobile phases were used (phase A: a 0.25% NH4HCO3 solution in H2O; phase B: CH3OH; phase C: CH3CN). First, 75% A and 25% B with a flow rate of 40 ml/min was hold for 0.5 minutes. Then a gradient was applied to 100% B in 41 minutes with a flow rate of 80 ml/min. Then a gradient was applied to 100% C in 20 minutes with a flow rate of 80 ml/min and hold for 4 minutes.
HPLC Method B
The product was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). Two mobile phases were used (phase A: a 0.25% NH4HCO3 solution in H2O; phase B: CH3CN). First, 85% A and 15% B with a flow rate of 40 ml/min was hold for 0.5 minutes. Then a gradient was applied to 10% A and 90% B in 41 minutes with a flow rate of 80 ml/min. Then a gradient was applied to 100% C in 20 minutes with a flow rate of 80 ml/min and hold for 4 minutes.
HPLC Method C
The product was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). Three mobile phases were used (phase A: a 0.25% NH4HCO3 solution in H2O; phase B: CH3OH; phase C: CH3CN). First, 75% A and 25% B with a flow rate of 40 ml/min was hold for 0.5 minutes. Then a gradient was applied to 50% B and 50% C in 41 minutes with a flow rate of 80 ml/min. Then a gradient was applied to 100% C in 20 minutes with a flow rate of 80 ml/min and hold for 4 minutes.
Reaction under N2 atmosphere. A mixture of 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (0.010 mol) in THF (65 ml; dry) was stirred at −78° C. BuLi (0.022 mol; 2.5 M) was added dropwise at −78° C. Then the mixture was stirred for 30 minutes at −78° C. DMF (2 ml; dry) was added dropwise at −78° C. and the reaction mixture was stirred at r.t. for 1 hour. An aq. NH4Cl solution was added (decomposition). This mixture was extracted with EtOAc. The separated organic layer was dried, filtered and the solvent was evaporated. The residue (1.9 g) was stirred in DIPE, filtered off and dried. Yield: 1.420 g of intermediate 1.
A mixture of N-(2-aminoethyl)-N-methylcarbamic acid 1,1-dimethylethyl ester (1.044 g, 0.0060 mol) and NaBH(OAc)3 (3.2 g, 0.015 mol) in DCM (50 ml) was stirred at r.t. A solution of intermediate 1 (0.905 g, 0.0050 mol) in THF/DMF 1/1 (70 ml) was added dropwise. Stirring was continued for 16 hours. Then more NaBH(OAc)3 (3.2 g, 0.015 mol) was added. Formaldehyde (2 ml; 40%) dissolved in THF (10 ml) was added dropwise. Stirring was continued for 16 hours The reaction mixture was poured into H2O, basified with K2CO3 and extracted with DCM. The organic layer was dried (MgSO4), filtered and the solvent was evaporated. The residue was purified on silica (eluent: DCM/(MeOH/NH3) 90/10. Yield: 1.6 g of intermediate 2 (90.6%).
A mixture of 2-methoxy-5-nitrophenol (0.001 mol), 4-chlorobutanoic acid methyl ester (0.0013 mol) and Cs2CO3 (1.63 g) in DMF (30 ml) was stirred at r.t. When the reaction was finished, the solvent was evaporated. The residue was partitioned between H2O and toluene (3×). The organic layers were combined, dried (MgSO4), filtered and the solvent was evaporated. Yield: 0.250 g of intermediate 3.
A mixture of intermediate 3 (0.0008 mol) in MeOH (40 ml) was hydrogenated with Pd/C 10% (1 g) as a catalyst in the presence of a thiophene solution (0.1 ml; 4% in DIPE). After uptake of H2 (3 eq.), the catalyst was filtered off and the filtrate was evaporated. Yield: 0.158 g of intermediate 4.
3-Nitrophenol (5 g, 0.036 mol) was dissolved in DMA (70 ml). 5-Bromopentanoic acid methyl ester (7.7 g, 0.0395 mol) was added. K2CO3 (5.5 g, 0.0395 mol) was added to the stirred solution and the mixture was stirred overnigth at 60° C. After completion of the reaction (TLC monitoring), the mixture was partitioned between EtOAc and brine. The layers were separated and the organic layer dried (MgSO4), filtered and concentrated. The resulting product was dried under high vacuum. Yield: 9.3 g of intermediate 5 (used as such in next reaction step).
Intermediate 5 (9.3 g., 0.036 mol) was dissolved in EtOH (175 ml). A thiophene solution (4 ml; 2% in DIPE) was added, followed by addition of Pd/C 10% as catalyst. After a N2 purge, H2 was introduced through a gas-bag. The reaction mixture was hydrogenated for 40 hours at r.t. The catalyst was filtered off over a Celite-pad. HCl in dioxane (13 ml; 4 N) was added to the filtrate and the mixture was concentrated under reduced pressure. THF was added to the residue and the resulting solid was filtered off, washed with DIPE and dried. Yield: 7.0 g of intermediate 6 (.HCl).
First intermediate 4 was converted into its HCl-salt.
A mixture of intermediate 2 (0.00042 mol) and the HCl-salt of intermediate 4 (1.1 eq.; 0.00046 mol) in CH3CN/2-propanol 3/1 (2 ml) and HCl/2-propanol (q.s.) was heated for 4 hours at 80° C. The solvent was evaporated and the crude residue was purified by flash column chromatography over silica gel (eluent: DCM/MeOH gradient). The product fractions were collected and the solvent was evaporated, yielding intermediate 7.
A mixture of intermediate 7 (max. 0.00042 mol) and LiOH (3 eq.) was stirred in THF/H2O 1/1 (5 ml) at r.t. for 6 hours. HOAc (q.s.) was added until pH=6-7. The reaction mixture was extracted with DCM (3×5 ml). The organic layers were combined, dried, filtered and the solvent was evaporated. The residue was stirred in TFA/DCM 1/1 (5 ml) for 6 hours at r.t. The solvent was evaporated. Yield: intermediate 8 (used in next reaction step, without further purification).
A mixture of intermediate 2 (0.00042 mol) and intermediate 6 (1.1 eq.; 0.00046 mol) in CH3CN/2-propanol (2 ml) and HCl/2-propanol (q.s.) was heated for 4 hours at 80° C. The solvent was evaporated and the crude residue was purified by flash column chromatography over silica gel (eluent: DCM/MeOH gradient). The product fractions were collected and the solvent was evaporated. Yield: intermediate 9.
A mixture of intermediate 9 (max. 0.00042 mol) and LiOH (3 eq.) was stirred in THF/H2O 1/1 (5 ml) at r.t. for 6 hours. HOAc (q.s.) was added until pH 6-7. The reaction mixture was extracted with DCM (3×5 ml). The organic layers were combined, dried, filtered and the solvent was evaporated. The residue was stirred in TFA/DCM 1/1 (5 ml) for 6 hours at r.t. The solvent was evaporated. Yield: intermediate 10 (used in next reaction step, without further purification).
A mixture of 4-chloro-5-iodo-7-(phenylsulfonyl)-7H-Pyrrolo[2,3-d]pyrimidine (0.005 mol), (5-amino-2-methoxyphenoxy)acetic acid ethyl ester (0.0055 mol) and HCl/dioxane (0.5 ml) in
t-BuOH (100 ml) was stirred for 40 hours at 80° C. The mixture was purified by HPLC. Three product fraction groups were collected and the solvent was evaporated. The main product fraction group yielded 1.5 g of intermediate 11.
A mixture of intermediate 11 (0.0246 mol), dichlorobis(triphenylphosphine)palladium (0.00246 mol) and CuI (0.00246 mol) in Et3N (250 ml) and DMA (250 ml) was stirred at r.t. A solution of N-methyl-N-2-propyn-1-yl-carbamic acid 1,1-dimethylethyl ester (0.0492 mol) in DMA (q.s.) was added dropwise and the resultant reaction mixture was stirred for 2 hours at r.t. The mixture was poured out into H2O and was extracted 3× with EtOAc. The combined organic layers were washed with H2O (2×), dried, filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (eluent: EtOAc/hexane from 30/70 to 70/30). The desired product fractions were collected and the solvent was evaporated. The residue was stirred in DIPE, filtered off and dried. Yield: 11.8 g of intermediate 12 (79.9%).
A mixture of intermediate 12 (0.019 mol) in THF (250 ml) was hydrogenated with Raney Nickel (1 g) as a catalyst. After uptake of H2 (2 eq.), the catalyst was filtered off and the filtrate was evaporated. Yield: 12.8 g of intermediate 13 (quantitative yield; used in next reaction step, without further purification).
A mixture of intermediate 13 (0.019 mol) in EtOH (700 ml; p.a.) and HCl/dioxane (60 ml; 4 N) was stirred for 4 days at r.t. and then for 16 hours at 50° C. The solvent was evaporated. The residue was taken up in DIPE. The precipitate was filtered off and dried. Yield: 10.6 g of intermediate 14 (0.2 HCl; 89.5%).
A mixture of intermediate 14 (0.001 mol) and Et3N (0.0003 mol) in DCM (50 ml) was stirred at r.t. NaBH(OAc)3 (0.003 mol) was added portionwise. 4R-4-formyl-2,2-dimethyl-3-oxazolidinecarboxylic acid 1,1-dimethylethyl ester (0.0015 mol) was added portionwise. The resultant reaction mixture was stirred for 16 hours at r.t. The mixture was poured out into H2O and was then alkalized with K2CO3. This mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. Yield: intermediate 15 (quantitative yield; used in next reaction step, without further purification).
A mixture of intermediate 15 (0.001 mol) in dioxane (30 ml), H2O (30 ml) and HCl (15 ml; 36%) was stirred for 2 hours at 60° C. The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. CH3CN was added and was then evaporated again (2×). Yield: 0.458 g of intermediate 16 (76.6%; S-enantiomer).
A mixture of PyBOP (2.600 g) and Et3N (0.300 g) in DMF (50 ml) was stirred at r.t. under N2 atmosphere. A solution of intermediate 16 (0.00076 mol) in DMF (50 ml) was added dropwise. The resultant reaction mixture was stirred for one hour at r.t. H2O was added (decomposition). The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated, yielding intermediate 17 (S-enantiomer).
A mixture of 4-chloro-5-iodo-7-(phenylsulfonyl)-7H-Pyrrolo[2,3-d]pyrimidine (0.005 mol), 5-amino-2-methoxy-benzenemethanol (0.0051 mol) and HCl/dioxane (0.5 ml; 1 N) in t-BuOH (100 ml) was stirred for 3 days at 80° C. The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. The residue was taken up into H2O. This mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. Yield: 1.85 g of intermediate 18 (69%).
Reaction mixture (I): A mixture of intermediate 18 (0.0003 mol), dichlorobis(triphenylphosphine)palladium (0.00006 mol) and CuI (0.00006 mol) in Et3N (5 ml) and DMA (5 ml) was stirred at r.t. A solution of N-methyl-N-2-propyn-1-yl-carbamic acid, 1,1-dimethylethyl ester (0.00075 mol) in DMA (2 ml) was added dropwise. The reaction mixture was stirred for one hour at r.t. Reaction mixture (II): A mixture of A (0.003 mol), dichlorobis(triphenylphosphine)palladium (0.0006 mol) and CuI (0.0006 mol) in Et3N (50 ml) and DMA (50 ml) was stirred at r.t. A solution of N-methyl-N-2-propyn-1-yl-carbamic acid, 1,1-dimethylethyl ester (0.0075 mol) in DMA (20 ml) was added dropwise. The reaction mixture was stirred for one hour at r.t. Reaction mixtures (I) and (II) were combined and combined mixture was poured out into H2O. This mixture was extracted with EtOAc (3×). The separated organic layer was washed with H2O (3×), dried, filtered and the solvent evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. The residue was taken up into H2O, then extracted with EtOAc. The separated organic layer was dried, filtered and the solvent evaporated. Yield: 0.7 g of intermediate 19 (35.7%).
A mixture of intermediate 19 (0.0012 mol) in THF (50 ml) was hydrogenated with Raney Nickel (catalytic quantity) as a catalyst. After uptake of H2 (2 eq.), the catalyst was filtered off and the filtrate was evaporated. Yield: 0.7 g of intermediate 20 (quantitative yield; used in next reaction step, without further purification).
A mixture of intermediate 20 (0.0012 mol) and MnO2 (7 g; activated) in THF (100 ml) was stirred for 2 hours at r.t. The catalyst was filtered off and the filtrate was evaporated. Yield: 0.550 g of intermediate 21 (78.6%).
A mixture of intermediate 21 (0.001 mol), N-methyl-β-alanine 1,1-dimethylethyl ester hydrochloride (0.001 mol) and NaBH(OAc)3 (0.003 mol) in DCM (50 ml) and Et3N (0.2 g) was stirred overnight at r.t. More N-methyl-β-alanine 1,1-dimethylethyl ester hydrochloride (0.0005 mol) was added and the reaction mixture was stirred overnight at r.t. The mixture was purified by column chromatography over silica gel (eluent: DCM/(MeOH/NH3) 95/5). The product fractions were collected and the solvent was evaporated. Yield: 0.950 g of intermediate 22 (oil).
A mixture of intermediate 22 (0.001 mol) in dioxane (20 ml), H2O (20 ml) and HCl (20 ml; 36%) was stirred for one hour at 60° C. The solvent was evaporated. CH3CN was added, then evaporated again (3×), yielding intermediate 23 (quantitative yield; used in next reaction step, without further purification).
A mixture of PyBOP (0.005 mol) and Et3N (0.010 mol) in DMF (50 ml) was stirred at r.t. under N2 atmosphere. A solution of intermediate 23 (0.001 mol) in DMF (50 ml) was added dropwise. The resultant reaction mixture was stirred for 1 hour at r.t. H2O was added. The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.106 g of intermediate 24.
A mixture of intermediate 21 (0.00024 mol), 4-(methylamino)-butanoic acid 1,1-dimethylethyl ester hydrochloric acid (0.00036 mol) and NaBH(OAc)3 (0.00072 mol) in DCM (10 ml) and Et3N (0.036 g) was stirred for 16 hours at r.t. The reaction mixture was poured out into H2O and this mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.075 g of intermediate 25.
A mixture of intermediate 25 (0.0001 mol) in dioxane (20 ml), H2O (20 ml) and HCl (10 ml; 36%) was stirred for one hour at 60° C. The solvent was evaporated. CH3CN was added and was evaporated again (3×). Yield: intermediate 26 as a hydrochloric acid salt form (0.3 HCl) (quantitative yield; used in next reaction step, without further purification).
A mixture of PyBOP (0.0005 mol) and Et3N (0.0010 mol) in DMF (20 ml) was stirred at r.t. under N2 atmosphere. A solution of intermediate 26 (0.0001 mol) in DMF (20 ml) was added dropwise. The resultant reaction mixture was stirred for one hour at r.t. H2O was added (decomposition). The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.044 g of intermediate 27.
A mixture of glycine 1,1-dimethylethyl ester hydrochloride (0.0056 mol) and NaBH(OAc)3 (3.535 g) in DCM (55 ml) and Et3N (0.84 g) was stirred at r.t. A solution of intermediate 1 (0.0056 mol) in THF (22 ml) and DMF (22 ml) was added dropwise. The reaction mixture was stirred for 5 hours. Formaldehyde (2 ml) and NaBH(OAc)3 were added. The reaction mixture was stirred overnight. The mixture was poured out into H2O, alkalized with K2CO3 and then extracted with DCM. The organic layer was separated, dried, filtered and the solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.857 g of intermediate 29 (49%).
A mixture of intermediate 29 (0.00032 mol), [3-(5-amino-2-methoxyphenoxy)propyl]-carbamic acid 1,1-dimethylethyl ester (0.000358 mol) and HCl/dioxane (5 drops; 4 N) in CH3CN (2 ml) was stirred for 3 hours at 75° C. The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.097 g of intermediate 30 (53.2%).
A mixture of intermediate 30 (0.00017 mol) in dioxane (10 ml), H2O (10 ml) and HCl (5 ml; 36%) was stirred for one hour at 60° C. The solvent was evaporated. CH3CN was added, then evaporated again (3×). The residue was dried (vacuum, 50° C., 16 hours). Yield: intermediate 31 as a HCl-salt (0.3 HCl) (quantitative yield; used in next reaction step, without further purification).
A mixture of intermediate 1 (0.008 mol) in DCM (100 ml) and THF (100 ml) was stirred at r.t. NaBH(OAc)3 (0.024 mol) was added portionwise. A solution of methyl[3-(methylamino)propyl]carbamic acid 1,1-dimethylethyl ester (0.008 mol) in DCM (q.s.) was added dropwise. The reaction mixture was stirred overnight at r.t. The mixture was purified by HPLC. The product fractions were collected and the solvent was evaporated. The residue was taken up into H2O, then extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. Yield: 0.800 g of intermediate 32.
A mixture of intermediate 32 (0.00027 mol), (5-amino-2-methoxyphenoxy)acetic acid ethyl ester (0.0003 mol) and HCl/2-propanol (3 drops) in CH3CN (2 ml) was stirred for 3 hours at 80° C. The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.059 g of intermediate 33 (39.3%).
A mixture of intermediate 33 (0.00010 mol) in dioxane (10 ml), H2O (10 ml) and HCl (5 ml; 36%) was stirred for 2 hours at 60° C. The solvent was evaporated. CH3CN was added, then evaporated again (3×). Yield: intermediate 34 as a HCl-salt (0.3 HCl)(quantitative yield; used in next reaction step, without further purification).
(Reaction mixture I): A mixture of intermediate 2 (0.00026 mol) and 3-amino-benzenepropanoic acid ethyl ester hydrochloride (0.00026 mol) in CH3CN (5 ml) was stirred for 3 hours at 80° C. (Reaction mixture II): A mixture of intermediate 2 (0.00052 mol) and 3-amino-benzenepropanoic acid ethyl ester hydrochloride (0.00052 mol) in CH3CN (10 ml) was stirred for 5 hours at 80° C. Reaction mixture (I) and (II) were combined. The solvent was evaporated. The residue was purified by HPLC. Yield: 0.217 g of intermediate 35 (54.6%).
A mixture of intermediate 35 (0.00025 mol) in EtOH (20 ml; p.a.) and HCl/2-propanol (5 ml) was stirred for 32 hours at 25° C. The solvent was evaporated. The residue was stirred in DIPE. The resulting precipitate was filtered off and dried. Yield: 0.099 g of intermediate 36 as a HCl-salt (0.3 HCl) (96.6%).
Reaction under N2 atmosphere. A mixture of intermediate 36 (0.00019 mol) and PyBOP (0.00038 mol) and Et3N (0.0019 mol) in DMF (10 ml) was stirred at r.t. under N2 flow. A solution of N-[(1,1-dimethylethoxy)carbonyl]-N-methylglycine (0.00019 mol) in Et3N (10 ml) was added dropwise and the resultant reaction mixture was stirred for 30 minutes at r.t. The mixture was decomposed with H2O. The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.052 g of intermediate 37 (47.1%).
A mixture of intermediate 37 (0.00009 mol) in dioxane (10 ml), H2O (10 ml) and HCl/2-propanol (5 ml) was stirred for 2 hours at 60° C. The solvent was evaporated. CH3CN was added, then evaporated again (3×). The residue was dried (vacuum, 50° C., 3 hours). Yield: intermediate 38 as a HCl-salt (0.3 HCl) (quantitative yield; used in next reaction step, without further purification).
A mixture of intermediate 21 (0.001 mol), O-(1,1-dimethylethyl)-L-Serine 1,1-dimethylethyl ester hydrochloride (0.0015 mol), Et3N (0.002 mol) and NaBH(OAc)3 (0.003 mol) in DCM (50 ml) was stirred for 16 hours at r.t. Extra O-(1,1-dimethylethyl)-L-Serine 1,1-dimethylethyl ester hydrochloride (0.00075 mol) and NaBH(OAc)3 (0.0015 mol) were added and the mixture was stirred overnight at r.t. Then the mixture was poured out into H2O. This mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. Yield: intermediate 39 (S-enantiomer) (crude; used in next reaction step, without further purification).
A mixture of intermediate 39 (max. 0.001 mol), formaldehyde (5 ml; 40%) and NaBH(OAc)3 (0.003 mol) in THF (50 ml) was stirred for 16 hours at r.t. The mixture was poured out into H2O. This mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. Yield: intermediate 40 (S-enantiomer) (crude, used in next reaction step, without further purification).
A mixture of intermediate 40 (max. 0.001 mol) in H2O (30 ml), dioxane (30 ml) and HCl (15 ml; 36%) was stirred for 6 hours at 60° C. The solvent was evaporated. The residue was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). The mentioned mobile phases were used to apply a gradient (phase A: 0.25% NH4HCO3 solution in H2O; phase B: CH3OH (optional); phase C: CH3CN). The product fractions were collected and the solvent was evaporated. Yield: 0.185 g of intermediate 41 (S-enantiomer).
Reaction under N2 atmosphere. A mixture of PyBOP (0.00160 mol) and Et3N (0.00160 mol) in DMF (25 ml; p.a.) was stirred at r.t. A solution of intermediate 41 (0.000317 mol) in DMF (25 ml; p.a.) was added dropwise. The resultant reaction mixture was stirred for one hour. The solvent was evaporated. Yield: intermediate 42 (S-enantiomer) (quantitative yield; used in next reaction step, without further purification).
A mixture of intermediate 2 (1.6 g, 0.0045 mol) and (5-amino-2-methoxyphenoxy)acetic acid ethyl ester (1.237 g, 0.0055 mol) in t-BuOH (35 ml) and HCl/dioxane (2 ml; 4 N) was stirred at 75° C. for 5 hours. The reaction mixture with intermediate 43 was used as such in the next step.
A mixture of intermediate 43 (0.0045 mol; crude mixture from previous reaction step), t-BuOH (35 ml), H2O (35 ml) and HCl/dioxane (17.5 ml; 4 N) was stirred at 60° C. for 2 hours. The reaction mixture was evaporated to dryness. The residue was purified by HPLC method B. Yield: 0.69 g of intermediate 44 (37%).
2-Bromoacetic acid ethyl ester (15 g, 0.0897 mol) and then K2CO3 (25 g, 0.0179 mol) were added to a solution of 2-(benzyloxy)-5-nitro-phenol (22 g, 0.0897 mol) in DMF (300 ml). The reaction mixture was stirred at r.t. for 12 hours. H2O and ether were added. The separated organic layer was washed with a K2CO3 aq. solution (10%) and with brine, and was then dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica (eluent: petroleum ether/DCM 15:1). The product fractions were collected and the solvent was evaporated. Yield: 15 g of intermediate 45 (51%).
NH4Cl (2.1 g, 0.0398 mol) in H2O (100 ml) was added to a solution of intermediate 45 (11 g, 0.0332 mol) and Fe (9.3 g, 0.1660 mol) in THF (200 ml) and stirred for 25 hours at 85° C. Then, the reaction mixture was cooled to r.t. and filtered through Celite. The solvent was evaporated. The residue was partitioned between EtOAc and a Na2CO3 aq. solution (2 M). The separated organic layer was washed with brine, dried (MgSO4), filtered and the solvent was evaporated. The residue was acidified with HCl in dioxane and the precipitate was filtered off. The product was dissolved in H2O (200 ml) and then extracted with ether. The aq. layer was lyophilized to obtain the residue. Yield: 10 g of intermediate 46 (.HCl; 91%).
A mixture of intermediate 32 (0.400 g, 0.0011 mol), intermediate 46 (0.432 g, 0.0013 mol), t-BuOH (8 ml) and HCl/1,4-dioxane (0.4 ml; 4 N) was stirred for 5 hours at 75° C. The reaction mixture with intermediate 47 was used as such in the next reaction step.
A mixture of intermediate 47 (0.0011 mol; crude mixture from precious reaction step), t-BuOH (8 ml), H2O (8 ml) and HCl (4 ml; 36%) was stirred for 2 hours at 60° C. The reaction mixture was evaporated to dryness and purified by HPLC method B. Yield: 0.045 g of intermediate 48 (8.2%).
Pd/C 10% (2 g) was suspended in MeOH (125 ml) under N2 flow. A thiophene solution (1 ml; 4% in DIPE) was added. N,N-bis(phenylmethyl)-1,3-propanediamine (20 g, 0.0790 mol) and acetone (13.8 g, 0.2370 mol) in MeOH (125 ml) was added. The reaction mixture was stirred under H2 atmosphere until 1 eq. of H2 was absorbed. The catalyst was removed by filtration over Dicalite and the solvent was evaporated. Yield: intermediate 49 (97.0%).
Intermediate 49 (22.6 g, 0.0760 mol) was dissolved in DCM (225 ml) and stirred. DMAP (catalytic amount) was added. BOC-anhydride (17.5 g, 0.0800 mol) was dissolved in DCM (75 ml) and added dropwise. The reaction mixture was stirred at r.t. (CO2 gas development). When the reaction was finished, the solvent was evaporated and the residue was purified by column chromatography (eluent: DCM/MeOH from 100/0 till 92/8. The product fractions were collected and the solvent was evaporated and co-evaporated with toluene. Yield: 27.9 g of intermediate 50 (92.6%).
Pd/C 10% (2.5 g) was suspended in MeOH (125 ml). Intermediate 50 (27.8 g, 0.0700 mol) in MeOH (125 ml) was added (under N2 flow). The reaction mixture was stirred under H2 atmosphere until 2 eq. of H2 were absorbed. The catalyst was removed by filtration over Dicalite and the solvent was evaporated. Yield: 14.3 g of intermediate 51 (94.3%).
A mixture of intermediate 51 (1.30 g, 0.0060 mol), NaBH(OAc)3 (3.2 g, 0.0150 mol) and DCM was stirred at r.t. Intermediate 1 (0.905 g, 0.0050 mol) dissolved in THF/DMF 1/1 (70 ml) was added dropwise. Stirring was continued for 16 hours. Another portion of NaBH(OAc)3 (3.2 g, 0.0150 mol) was added. Formaldehyde (2 ml; 40%) dissolved in THF (10 ml) was added dropwise. Stirring was continued for 16 hours The reaction mixture was poured into H2O, basified with K2CO3 and extracted with DCM. The organic layer was dried with MgSO4, filtered off and the solvent was evaporated. The residue was purified over silica gel (eluent: DCM/(MeOH/NH3) 90/10) Yield: 1.8 g of intermediate 52 (91%).
A mixture of intermediate 52 (1.8 g, 0.0045 mol), (5-amino-2-methoxyphenoxy)acetic acid ethyl ester, t-BuOH (35 ml) and HCl/dioxane (2 ml; 4 N) was stirred for 5 hours at 75° C. The reaction mixture with intermediate 53 was used as such in the next reaction step.
A mixture of intermediate 53 (0.0045 mol; crude reaction mixture from previous reaction step), t-BuOH (35 ml), H2O (35 ml) and HCl (17.5 ml; 36%) was stirred for 2 hours at 60° C. The reaction mixture was evaporated to dryness and the residue was purified on HPLC. Yield: 0.714 g of intermediate 54 (34.8%).
A mixture of 2-amino-5-nitrophenol (7.7 g, 0.05 mol), isocyanatocyclohexane (6.3 g, 0.05 mol) and THF (100 ml) was stirred for 16 hours at r.t. The solvent was evaporated. The residue was stirred in DIPE, filtered off and dried. Yield: 13.44 g of intermediate 55 (96.2%).
A mixture of intermediate 55 (13.4 g, 0.0480 mol), 2-bromo acetic acid 1,1-dimethylethyl ester (10.3 g, 0.0530 mol), K2CO3 (8 g, 0.0580 mol) and DMF (130 ml) was stirred at r.t. for 5 hours. The reaction mixture was poured into H2O and extracted with EtOAc (3×). The organic layer was washed with H2O (2×), filtered over Dicalite, dried (MgSO4), filtered and the solvent was evaporated. The residue was stirred in DIPE and the solid was filtered off and dried. Yield: 9.2 g of intermediate 56 (LCMS 99%). (More but impure intermediate 56 can be obtained by evaporation of the solvent (DIPE) of the filtrate and by stirring the residue in DIPE again. After filtration and drying of the solid, 6.3 g of less pure intermediate 56 (LCMS 91%) was obtained. Total yield: 82.1%)
Pd/C 10% (1.5 g) was suspended in THF (75 ml). Under N2 flow, a thiophene solution (1 ml; 4% in DIPE) was added. Then intermediate 56 (5 g, 0.0127 mol) in THF (75 ml) was added. The reaction mixture was stirred under H2 atmosphere until 3 eq. of H2 were absorbed (3 eq.). The catalyst was removed by filtration over Dicalite and the solvent was evaporated, yielding 5.2 g of the crude product. The crude product was purified by HPLC. Yield: 3.3 g of intermediate 57 (71.5%; LC-MS 98%).
A mixture of intermediate 32 (0.700 g, 0.0019 mol) and intermediate 57 (0.889 g, 0.0025 mol) in t-BuOH (14 ml) and HCl/1,4-dioxane (0.7 ml; 4 N) was stirred for 5 hours at 75° C. The reaction mixture with intermediate 58 was used as such in the next reaction step.
A mixture of intermediate 58 (0.0019 mol; crude mixture from previous reaction step) in H2O (14 ml) and HCl (7 ml; 36%) was stirred for 2 hours at 60° C. The mixture was evaporated to dryness and the residue was purified by HPLC method B. The pure fractions were collected, evaporated to dryness and 3 times co-evaporated with CH3CN. Yield: 0.380 g of intermediate 59 (38%).
2-Chloro-5-nitrophenol (10.0 g, 0.058 mol), bromoacetic acid ethyl ester (9.7 g, 0.058 mol) and K2CO3 (16.0 g, 0.116 mol) were suspended in DMF (150 ml). The reaction mixture was stirred overnight at r.t. H2O (200 ml) and Et2O (200 ml) were added. The organic layer was washed with 10% K2CO3 and saturated brine, and was then dried (Na2SO4). The mixture was filtered. The filtrate was concentrated in vacuo. The residue was suspended in petroleum ether (100 ml). The precipitate was filtered off and dried in vacuo to give 11.0 g of intermediate 67 (73%).
Intermediate 67 (11.0 g, 0.042 mol) and Raney Nickel (2.2 g) were suspended in THF (350 ml) and hydrogenated (under H2 atmosphere) for 2 hours. Then the mixture was filtered. The filtrate was concentrated in vacuo. The residue was dissolved in 4 N HCl/dioxane (35 ml). Then Et2O (100 ml) was added. The precipitate was filtered off and dried in vacuo to give 7.0 g of intermediate 60 (.HCl; 62%).
A mixture of intermediate 32 (3 g, 0.0080 mol), intermediate 60 (2.1 g, 0.0080 mol), t-BuOH (60 ml) and HCl/dioxane (3 ml; 4 N) was stirred for 6 hours at 75° C. The reaction mixture with intermediate 61 was used as such in the next reaction step.
A mixture of intermediate 61 (0.0080 mol; crude reaction mixture from previous reaction step), H2O (60 ml) and HCl (30 ml; 36%) was stirred for 2 hours at 60° C. The reaction mixture was evaporated to dryness and the residue was purified by HPLC method B. The pure fractions were evaporated to dryness and evaporated 2 times with CH3CN Yield: 0.340 g of intermediate 62. The residue was dried in vacuum at 50° C. for 16 hours.
A mixture of N-(2-hydroxy-4-nitrophenyl)-N-(4-methoxyphenyl)urea (3.85 g, 0.0127 mol), 2-bromo acetic acid 1,1-dimethylethyl ester (2.73 g, 0.0140 mol) and K2CO3 (2.11 g, 0.0152 mol) in DMF (40 ml) was stirred at r.t. The reaction mixture was poured into H2O and extracted with EtOAc (3×). The organic layer was washed with H2O (1×). The EtOAc-layer was dried (MgSO4) and filtered. The solvent was evaporated. The crude product (6.18 g) was purified over silica gel (eluent: 25% EtOAc/hexane). The product fractions were collected and the solvent was evaporated. Yield: 4.99 g of intermediate 63 (94.1%).
Pd/C 10% (1 g) was suspended in THF (q.s.) under N2 flow. A thiophene solution (1 ml; 4% in DIPE) was added. Intermediate 63 (5.0 g, 0.0120 mol) in THF (q.s.) was added. The reaction mixture was stirred under H2 atmosphere until 3 eq. of H2 were absorbed. The catalyst was removed by filtration over Dicalite and the solvent was evaporated. The crude product (5.5 g) was purified by HPLC. The desired fractions were collected and the solvent was evaporated, yielding 2.6 g residue. This residue was stirred in DIPE and filtered off. Yield: 2.32 g of intermediate 64 (49.9%).
Intermediate 32 (0.700 g, 0.0019 mol) and intermediate 64 (0.950 g, 0.0025 mol) in t-BuOH (14 ml) and HCl/1,4-dioxane (0.7 ml; 4 N) was stirred at 75° C. for 5 hours. The reaction mixture with intermediate 65 was used as such in the next step.
A mixture of intermediate 65 (0.0019 mol; crude reaction mixture from previous reaction step), t-BuOH (14 ml), H2O (14 ml) and HCl (7 ml; 36%) was stirred at 60° C. for 2 hours. The reaction mixture was evaporated to dryness and the residue was purified by HPLC method B. The product fractions were collected and the solvent was evaporated. Yield: 0.285 g of intermediate 66.
A mixture of 2-methoxy-5-nitrobenzaldehyde (0.058 mol) in DCM (200 ml) was stirred at r.t. 2-(triphenylphosphoranylidene)acetic acid, methyl ester (0.058 mol) was added portionwise. The reaction mixture was stirred at r.t. More 2-(triphenylphosphoranylidene)acetic acid, methyl ester (6 g) was added and the reaction mixture was stirred overnight at r.t. The solvent was evaporated. The residue was purified by column chromatography over silica gel (eluent: DCM/MeOH 99/1). The product fractions were collected and the solvent was evaporated. Yield: 9.7 g of intermediate 68.
A mixture of intermediate 68 (0.041 mol) in THF (250 ml) was hydrogenated with Raney Nickel as a catalyst. After uptake of H2 (4 eq.), the catalyst was filtered off and the filtrate was evaporated. The residue was taken up into 2-propanone and treated with 4 N HCl/dioxane. The solvent was evaporated. The residue was stirred in DIPE, filtered off and dried, and was then taken up into MeOH (150 ml). This mixture was treated with 4 N HCl/dioxane. The mixture was stirred for 3 hours at 65° C. The mixture was cooled and the solvent was evaporated. The residue was stirred in DIPE. The precipitate was filtered off and dried. Yield: 7.6 g of intermediate 69 (.HCl).
A mixture of intermediate 32 (0.001 mol) and intermediate 69 (0.001 mol) in CH3CN (3 ml) with HCl/dioxane (0.1 ml) was stirred for 4 hours at 80° C. The mixture was cooled. The solvent was evaporated to give residue (I). The reaction was repeated, without addition of the acid. The reaction mixture was stirred for 4 hours at 80° C. The mixture was cooled. The solvent was evaporated to give residue (II). Residues (I) and (II) were combined to give intermediate 70 (.2HCl) (used in next reaction step without further purification).
A mixture of crude intermediate 70 (max. 0.002 mol) in MeOH (60 ml) and HCl/dioxane (20 ml) was stirred for one hour at 60° C. The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and worked-up. Yield: intermediate 71 (used in the next reaction step without further purification).
A mixture of crude intermediate 71 (max. 0.002 mol) in dioxane (20 ml), H2O (20 ml) and HCl (10 ml;
36%) was stirred for 2 hours at 60° C. The solvent was evaporated. CH3CN was added (3×) and was evaporated each time. Yield: 0.184 g of intermediate 72 (.3HCl) (used in the next reaction step without further purification).
K2CO3 (32 g, 0.2292 mol) was added to a solution of 2-ethoxy-5-nitrophenol (21 g, 0.1146 mol) and 2-bromoacetic acid ethyl ester (19 g, 0.1146 mol) in DMF (400 ml) and the reaction mixture was stirred at r.t. for 12 hours. The reaction mixture was extracted with H2O and ether. The separated organic layer was washed with an aq. K2CO3 solution (10%), washed with brine, dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (eluent: petroleum ether/EtOAc 8/1). The desired fractions were collected and the solvent was evaporated. Yield: 13 g of intermediate 73 (42%).
A solution of NH4Cl (2.9 g, 0.0535 mol) in H2O (100 ml) was added to a solution of intermediate 73 (12 g, 0.0446 mol) and Fe (12.5 g, 0.2230 mol) in THF (200 ml) and stirred at 85° C. for 150 minutes. The reaction mixture was cooled to r.t., filtered over Celite and the filtrate's solvent was evaporated. The residue was partitioned between EtOAc and an aq. solution of Na2CO3 (2 M). The separated organic layer was washed with brine, dried (MgSO4), filtered and the filtrate's solvent was evaporated. The residue was acidified with HCl in dioxane. Yield: 10 g of intermediate 74 (81%; .HCl).
A mixture of intermediate 32 (0.40 g, 0.0011 mol) and intermediate 74 (0.352 g, 0.0013 mol) in t-BuOH (8 ml) and 4 N HCl/1,4-dioxane (0.4 ml) was stirred at 75° C. for 5 hours. The reaction mixture which contained intermediate 75 (0.2HCl) was used as such in the next step.
A mixture of intermediate 75 (crude; approximately 0.0011 mol) in t-BuOH (8 ml), H2O (8 ml) and HCl (4 ml; 36%) was stirred at 60° C. for 2 hours. The reaction mixture was evaporated to dryness and the residue was purified by HPLC method B. The product fractions were collected and the solvent was evaporated. Yield: 0.210 g of intermediate 76 (44%).
2,3-Dimethoxy-5-nitrophenol (0.0182 mol) was dissolved in CH3CN (55 ml). Then 4-bromobutanoic acid ethyl ester (0.0219 mol) was added followed by the addition of K2CO3 (0.0273 mol). The reaction mixture was stirred overnight at 80° C. The solid was filtered off and washed with CH3CN. EtOAc (50 ml) was added and the mixture was washed with brine, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography on silica gel, using as eluents a mixture of hexanes:EtOAc (ratios: 30:1-25:1-20:1-15:1-10:1). The product fractions were collected and the solvent was evaporated. The product was dried (vacuum, r.t.) yielding a pale yellow solid. Yield: 2.50 g of intermediate 77 (44%).
Intermediate 77 (0.0080 mol) was dissolved in THF (30 ml). Then, Pt/C 5% (0.50 g) was added and the mixture was stirred at r.t. under H2 atmosphere for 15 hours. The reaction mixture was filtered through a Celite pad and the Celite was washed with THF. The filtrate was evaporated under reduced pressure. The product was dried (vacuum, r.t.) yielding 2.25 g a brown oil. The hydrochloric salt was obtained by purging HCl gas into a solution of the aniline in Et2O. Yield: 2.20 g of intermediate 78 (.HCl).
Intermediate 32 (0.0019 mol) was dissolved in a mixture of CH3CN (4 ml) and 2-propanol (2 ml). Then, intermediate 78 (0.0023 mol) was added followed by the addition of HCl in 1,4-dioxane (1 ml; 4 N). The reaction mixture was heated at 80° C. for 2 hours. The solvent was evaporated and the residue was dried (vacuum, r.t.) yielding intermediate 79 (0.2 HCl) as a brown foam that was used a such in the next reaction step.
Na (5.2 g, 0.226 mol) was added portionwise to 2-ethoxyethanol (90 ml). This mixture was stirred for 1 hour under N2 atmosphere at r.t. Subsequently, 1,2-(methylenedioxy)-4-nitrobenzene (15 g, 0.090 mol) and DMSO (180 ml) were added and the reaction mixture was stirred overnight at r.t. First H2O (250 ml) was added and then a NaOH solution (150 ml; 10%) was added dropwise. The mixture was washed 2 times with DIPE (200 ml). The aq. layer was acidified (to pH 5). The precipitate was filtered off and washed with H2O to yield 17.8 g of intermediate 80 (87%).
A mixture of intermediate 80 (17.8 g, 0.078 mol), ethyl 2-bromoacetate (14.4 g, 0.086 mol), K2CO3 (13.0 g, 0.094 mol) and DMF (300 ml) was stirred overnight at r.t. H2O was added and the product precipitated. The solid was filtered off, washed with H2O and dried. Yield: 22.0 g of intermediate 81 (90%).
Raney Nickel (catalytical amount) was suspended in THF (350 ml) under N2 flow. Intermediate 81 (22.0 g, 0.0702 mol) was added and the reaction mixture was stirred under H2 atmosphere until 3 eq. of H2 were absorbed. Then the catalyst was filtered over Dicalite and the solvent was evaporated. THF and HCl/dioxane (20 ml; 4 N) were added to the residue. The solvent was evaporated. The residue was stirred in DIPE, filtered off and dried. Yield: 19.5 g of intermediate 82 (86.9%; .HCl).
A mixture of intermediate 32 (1.84 g; crude, max 0.005 mol), intermediate 82 (1.60 g, 0.005 mol), HCl/dioxane (2 ml; 4 N) and t-BuOH (40 ml) was stirred for 5 hours at 75° C. Then the solvent was evaporated and the crude intermediate 83 was used as such in the next reaction step.
A mixture of intermediate 83 (crude, max. 0.005 mol), HCl (0.5 ml) and H2O (30 ml) was stirred for 5 hours at 60° C. The solvent was evaporated and the residue (3.8 g) was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). A gradient with 2 mobile phases was applied. Phase A: a 0.25% NH4HCO3 solution in H2O; phase B: CH3CN). The desired fractions were collected. After removal of the solvent, 0.51 g of intermediate 84 was obtained.
A mixture of (2-hydroxy-5-nitrophenoxy)acetic acid ethyl ester (4.82 g, 0.020 mol) and K2CO3 (3.04 g, 0.022 mol) was stirred in DMF (75 ml) at r.t. 2-Bromo-N-(3,4,5-trimethoxyphenyl)acetamide (6.08 g, 0.020 mol) in DMF (75 ml) was added dropwise and the reaction mixture was stirred for 6 hours. Then H2O (300 ml) was added. The compound was filtered off, washed (H2O) and dried. Yield: 7.3 g of intermediate 85 (78.6%).
Raney Nickel (0.5 g) was suspended in THF (300 ml) under N2 flow. Intermediate 85 (7.3 g, 0.0157 mol) was added and the reaction mixture was stirred under H2 atmosphere until 3 eq. of H2 were absorbed. The catalyst was filtered off over Dicalite and the filtrate was evaporated. EtOH and HCl/dioxane (20 ml; 4 N) were added and this mixture was stirred. The solvent was evaporated and the residue was stirred in DIPE. The compound was filtered off and dried. Yield: 6.6 g of intermediate 86 (89.3%; 0.2HCl).
A mixture of intermediate 32 (1.84 g, 0.005 mol), intermediate 86 (3.53 g, 0.0075 mol), dioxane/HCl (2.5 ml; 4 N) and t-BuOH was stirred for 5 hours at 75° C. The solvent was evaporated and the crude residue (with intermediate 87) was used as such in the next reaction step.
A mixture of intermediate 87 (crude; max. 0.005 mol), dioxane/HCl (2 ml; 4 N) and H2O (40 ml) was stirred for 5 hours at 60° C. The solvent was evaporated and the residue (7.1 g) was purified by HPLC. After workup, 0.30 g of intermediate 88 was obtained.
A mixture of 2,3-dimethoxy-5-nitro-benzenemethanol (0.0375 mol) in MeOH (150 ml) was hydrogenated at r.t. for 20 hours with Pt/C 5% (2 g) as a catalyst in the presence of a thiophene solution (1 ml; 4% in DIPE). After uptake of H2 (3 eq.), the catalyst was filtered off and the filtrate was evaporated. The residue was suspended in DIPE. The precipitate was filtered off, washed with DIPE and dried (vacuo). Yield: 5.5 g of intermediate 89 (80%).
A mixture of 4-chloro-5-iodo-7-(phenylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine (0.014 mol), intermediate 89 (0.015 mol) and HCl (1.40 ml; 4 N in 1,4-dioxane) in t-BuOH (278 ml) was stirred for 72 hours at 80° C. The solvent was evaporated. H2O was added and the mixture was alkalized with Et3N. The mixture was extracted with DCM (3×). The organic layers were combined, dried (MgSO4), filtered and the solvent was evaporated. Yield: 6.45 g of intermediate 90 (81%).
Intermediate 90 (0.0104 mol) was dissolved in Et3N (100 ml) and DMA (100 ml), and the solution was purged with N2. Then, bis(triphenylphosphine)dichloropalladium (0.001 mol) and CuI (0.001 mol) were added. This mixture was purged again with N2 and a solution of N-methyl-N-2-propyn-1-yl-carbamic acid, 1,1-dimethylethyl ester (0.0209 mol) in DMA (15 ml) was added dropwise over 30 minutes. The reaction mixture was stirred under N2 over the weekend. The reaction mixture was poured into H2O and was extracted with EtOAc (3×100 ml). The organic layer was washed with H2O (2×100 ml), dried and concentrated to dryness. The residue was purified by column chromatography on silica gel eluting with EtOAc/hexane 3/1, 2/1, 3/2 and 1/1. The product fractions were collected and the solvent was evaporated. Yield: 6.9 g of intermediate 91 (100%).
Intermediate 91 (0.011 mol) was dissolved in THF (120 ml), and Raney Nickel (2 g) was added to the solution. The reaction mixture was purged with H2 and it was stirred at r.t. overnight under H2 atmosphere. The reaction mixture was filtered through Celite and the filtrate was concentrated to dryness. The crude intermediate 92 (6.9 g; 99% yield) was used as such in the next reaction step.
Intermediate 92 (0.0112 mol) was dissolved in THF (250 ml) and the solution was purged with N2. Then, MnO2 (0.245 mol) was added and the reaction mixture was purged with N2. The reaction mixture was stirred overnight at r.t. under N2 atmosphere. Then the mixture was filtered through Celite and the Celite was washed with THF, then with THF/MeOH and with MeOH/DCM. The filtrate was concentrated to dryness. The product was purified by column chromatography on silica eluting with DCM/MeOH (95/5). The product fractions were collected and the solvent was evaporated. Yield: 3.8 g of intermediate 93 (96%).
A mixture of intermediate 93 (0.00205 mol), N-methylglycine 1,1-dimethylethyl ester hydrochloride (0.003075 mol) and Et3N (0.0041 mol) in DCM (102.5 ml) was stirred at r.t. NaBH(OAc)3 (0.00615 mol) was added portionwise. The reaction mixture was stirred for 16 hours at r.t. The reaction mixture was poured out into H2O and this mixture was extracted 2 times with DCM. The organic layers were combined, dried (MgSO4), filtered and concentrated to dryness. The residue was dried (vacuum, r.t.) yielding 1.37 g of intermediate 94 as a yellow solid (90% yield).
A mixture of intermediate 94 (0.0024 mol) in 1,4-dioxane (48 ml), H2O (48 ml) and HCl (24 ml; 37%) was stirred for 2 hours at 60° C. Then the solvent was evaporated, and co-evaporated 3 times with CH3CN. After drying at high vacuum, the obtained residue (with intermediate 95 as a HCl-salt) was used as such in the next reaction step.
A solution of intermediate 95 (0.0024 mol) in DMF (190 ml) was added very slowly (over 1 hour, using a Marlow peristaltic pump) to a solution of HBTU (0.00576 mol) and DIPEA (0.060 mol) in DMF (190 ml). After the addition was completed, the reaction mixture was stirred for an additional hour. Then the mixture was quenched by addition of 7 N NH3 in MeOH (5 ml) and the solvents were evaporated. The reaction mixture was concentrated to dryness and the residue was partitioned between DCM and a saturated aq. K2CO3 solution. The aq. layer was extracted with more DCM. The organic extracts were washed with a saturated aq. K2CO3 solution, then dried, filtered and the filtrate was concentrated to dryness. Yield: Intermediate 96 (quantitative yield; used as a crude in the next reaction step without further purification).
A mixture of [[1-(diphenylmethyl)-3-azetidinyl]methyl]-carbamic acid 1,1-dimethylethyl ester (8 g, 0.0227 mol) and THF (100 ml) was stirred under N2 atmosphere. NaH (1 g, 0.025 mol; 60%) was added and the reaction mixture was stirred for 30 minutes. Dimethylsulphate (3.16 g, 0.025 mol) was added dropwise and the mixture was stirred for 16 hours. More NaH (1 g, 0.025 mol; 60%) and dimethylsulphate (3.16 g, 0.025 mol) were added and stirring was continued for 16 hours. Then, the reaction mixture was decomposed with H2O and the mixture was extracted with DCM. The separated organic layer was dried and the solvent was evaporated. The residue was purified by HPLC. Yield: 4.9 g of intermediate 97. A mixture of intermediate 97 (4.9 g, 0.0135 mol) in THF (50 ml) was hydrogenated at r.t. with Pd/C 10% (1 g) as a catalyst. After uptake of H2 (1 eq.), the catalyst was filtered off and the filtrate containing intermediate 98 was used as such in the next reaction step.
A mixture of the filtrate from the previous reaction step containing intermediate 98 (0.0135 mol) and NaBH(OAc)3 (8.6 g, 0.040 mol) was stirred at r.t. A mixture of intermediate 1 (2 g, 0.011 mol) and THF/DMF (50 ml/50 ml) was added dropwise to the first mixture and the reaction mixture was stirred for 3 hours. Then, the mixture was poured out into H2O and the aq. mixture was extracted with DCM. The organic layer was dried and evaporated to dryness. The residue was purified over silica gel (eluent: DCM/MeOH 90/10). The desired fractions were collected and the solvent was evaporated. Yield: 1.5 g of intermediate 99.
A mixture of 2-methoxy-5-nitrophenol (10 g, 0.0590 mol), K2CO3 (9 g, 0.0650 mol) and 4-chlorobutanoic acid ethyl ester (10 g, 0.0660 mol) in DMF (120 ml) was stirred at 60° C. until the reaction was finished. Then, the mixture was poured out into H2O. The precipitate was filtered off and dried (vacuum). Yield: 8.05 g of intermediate 100.
A mixture of intermediate 100 (14 g, 0.0494 mol) in EtOH (250 ml) was hydrogenated at r.t. with Pd/C 10% (2 g) as a catalyst in the presence of a thiophene solution (2 ml; 4% in DIPE). After uptake of H2 (3 eq.), the catalyst was filtered off and the filtrate was acidified with HCl/2-propanol. The mixture was evaporated and the residue was stirred in DIPE. The precipitate was filtered off and dried. Yield: 13.3 g of intermediate 101 (.HCl).
A mixture of intermediate 99 (0.750 g, 0.002 mol) and intermediate 101 (0.575 g, 0.002 mol) in HCl/dioxane (0.750 ml; 4 N) and t-BuOH (15 ml) was stirred for 5 hours at 75° C. This reaction mixture (containing intermediate 102) was used as such in the next reaction step.
The reaction mixture of the previous reaction step containing crude intermediate 102 (max. 0.002 mol) was heated together with H2O (20 ml) and HO/dioxane (4 ml; 4 N) for 8 hours at 50° C. Then the reaction mixture was cooled, neutralized with Et3N and evaporated to dryness. The residue was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). A gradient with 2 mobile phases was applied. Phase A: a 0.25% NH4HCO3 solution in H2O; phase B: CH3CN). The desired fractions were collected. After workup, 0.180 g of intermediate 103 was obtained.
A solution of (2-hydroxy-5-nitrophenoxy)acetic acid ethyl ester (2 g, 0.0083 mol), tetrahydro-2H-pyran-4-ol (1.13 g, 0.0111 mol) and (Ph)3P (4.35 g, 0.0166 mol) in THF (80 ml) was stirred at r.t. DIAD (3.3 ml, 0.0166 mol) was added dropwise and the reaction mixture was stirred for 3 hours. The solvent was evaporated and the residue was purified over silica gel (eluent: DCM/MeOH 100/0-98/2). The desired fractions were collected and the solvent was evaporated. The crude intermediate 104 was used as such in the next reaction step (quantitative yield).
A mixture of intermediate 104 (2.6 g, 0.008 mol) in THF (120 ml) was hydrogenated at r.t. with Raney Nickel (1 g) as a catalyst. After uptake of H2 (3 eq.), the catalyst was filtered off and the filtrate was evaporated. The residue was treated with DIPE and a few drops of HCl/2-propanol. The mixture was stirred for 1 hour. The solvent was evaporated and the crude residue was used as such in the next reaction step. Yield: 3.2 g of intermediate 105 (crude HCl-salt).
A solution of intermediate 105 (1.6 g, 0.0048 mol), intermediate 32 (1.7 g, 0.0046 mol) and HCl/dioxane (2 ml; 4 N) in t-BuOH (38 ml) was stirred for 6 hours at 75° C. The reaction mixture (with intermediate 106) was used as such in the next reaction step.
The crude reaction mixture from the previous reaction step containing intermediate 106 (approx. 0.0046 mol) was treated with concentrated HCl (1 ml) and H2O (2 ml). The solution was heated for 2 hours to 60° C. Then the solvent was evaporated and the residue was purified by HPLC (RP-18; eluent: (0.25% NH4HCO3 in H2O)/CH3CN v/v 100/0-0/100). The desired fractions were collected and the solvent was evaporated. The residue was repurified under the same HPLC conditions. The desired fractions were collected and the solvent was evaporated and co-evaporated with toluene. The residue was used as such in the next reaction step. Yield: 0.26 g of intermediate 107.
A solution of intermediate 1 (0.91 g, 0.005 mol) in THF/DMF (40 ml/40 ml) was added dropwise at r.t. to a stirred solution of N-(3-Aminopropyl)-N-(methyl)carbamic acid tert-butyl ester (1.13 g, 0.006 mol) and NaBH(OAc)3 (3.2 g, 0.015 mol) in DCM (40 ml). The reaction mixture was stirred for 19 hours and was then poured out into ice-water. The mixture was basified with K2CO3, extracted with DCM, dried (MgSO4), filtered and the solvent was evaporated to yield crude intermediate 108 as an oil that was used as such in the next reaction step.
A solution of intermediate 108 (crude; max. 0.005 mol) and intermediate 82 (0.006 mol) in HCl/dioxane (2 ml; 4 N) and t-BuOH (40 ml) was stirred for 6 hours at 75° C. The reaction mixture (containing intermediate 109) was used as such in the next reaction step.
A mixture of intermediate 109 (crude; max. 0.005 mol), HCl (20 ml; 36%) and H2O (40 ml) was stirred for 2 hours at 60° C. Then the solvent was evaporated and the residue was purified by HPLC (Shandon Hyperprep® C18 HS BDS; 8 μm, 50 mm by 16.5 cm). A gradient with 2 mobile phases was applied: (0.25% NH4HCO3 in H2O)/CH3CN from 100/0 to 80/20 in 35 minutes; then 0/100 for 10 minutes and finally 100/0 for 12 minutes. The desired fractions were collected and the solvent was evaporated. Yield: 0.750 g of intermediate 110 (32% yield).
2-(Difluoromethoxy)-5-nitrophenol (1.68 g, 0.0082 mol) and K2CO3 (2.26 g, 0.0164 mol) were stirred in DMA (20 ml). Then, 2-bromoacetic acid tert-butyl ester (1.32 ml, 0.0090 mol) was added and the resulting reaction mixture was stirred overnight at 60° C. The reaction mixture was cooled and H2O was added. This mixture was extracted with EtOAc (3×300 ml). The combined organic layers were washed with H2O (2×200 ml), dried (MgSO4), filtered and concentrated to dryness. Yield: 3 g of crude intermediate 111 (used as such in the next reaction step).
Intermediate 111 (crude; max. 0.0082 mol) was dissolved in EtOAc (q.s.) and the solution was purged using vacuum and N2. Then Pd/C ((catalytic quantity) was added and the reaction mixture was purged with N2. The reaction mixture was stirred overnight at r.t. under H2 atmosphere. The reaction mixture was filtered through Celite and the Celite was washed with more EtOAc, THF/MeOH and with MeOH/DCM. The filtrate was concentrated to dryness. Yield: 2.4 g of intermediate 112 (99%).
A mixture of intermediate 32 (1.26 g, 0.0034 mol), intermediate 112 (1.2 g, 0.0041 mol) and HCl (1 ml; 4 N in 1,4-dioxane) in CH3CN (25 ml) was stirred for 2 hours at 80° C. The reaction mixture was concentrated to dryness and the residue was re-dissolved in DCM and filtered. After washing with H2O, the organic phase was filtered and concentrated to dryness. Yield: 1.4 g of intermediate 113.
Intermediate 113 (0.00226 mol) was dissolved in HCl (25 ml; 36%) and 1,4-dioxane (3 ml), and the reaction mixture was stirred at r.t. for 3 hours. The solvent was evaporated. The residue was stirred in DIPE, filtered off and dried (vacuum, r.t.). Intermediate 114 was used as such in the next reaction step (quantitative yield).
A mixture of (2-hydroxy-5-nitrophenoxy)acetic acid ethyl ester (5 g, 0.0207 mol) and K2CO3 (4.3 g, 0.03105 mol) in DMF (150 ml) was stirred for 20 minutes at r.t. Then 1,2-dibromoethane (17.9 ml) was added and the mixture was heated to 60° C. for 18 hours. The mixture was quenched with ice-water and the product was extracted with toluene. The organic layer was dried (MgSO4), filtered and the solvent was evaporated. Yield: 6.7 g of intermediate 115.
A solution of intermediate 115 (1.4 g, 0.0042 mol) and N-ethyl-2-methoxyethanamine (1 g, 0.010 mol) in DMF (50 ml) was heated up to 100° C. for 18 hours. The solvent was evaporated and the residue was dissolved in DCM. This organic solution was washed with H2O and was then dried (MgSO4), filtered and the solvent was evaporated. The crude intermediate 116 (1.6 g) was used as such in the next reaction step.
A mixture of intermediate 116 (1.6 g; crude) in THF (120 ml) was hydrogenated at r.t. with Raney Nickel as a catalyst. After uptake of H2 (3 eq.), the catalyst was filtered off and the filtrate was evaporated. The residue was treated with HCl (2 ml; 4 N in dioxane) and this mixture was stirred for 15 minutes. The solvent was evaporated and the crude intermediate 117 (1.6 g; 0.2 HCl) was used as such in the next reaction step.
A solution of intermediate 32 (0.96 g, 0.0026 mol) and intermediate 117 (1.6 g, 0.0037 mol) in HCl/dioxane (2 ml; 4 N), t-BuOH (20.8 ml) and DMF (20 ml) was stirred for 6 hours at 75° C. This reaction mixture (containing intermediate 118) was used as such in the next reaction step.
The reaction mixture from the previous reaction step containing intermediate 118 (max. 0.0026 mol) was treated with concentrated HCl (1 ml) and H2O (2 ml). The solution was heated up to 60° C. for 2 hours. Then the solvent was evaporated and the residue was purified by HPLC (RP-18; eluent: (0.25% NH4HCO3 in H2O)/CH3CN v/v 100/0-0/100). The desired fractions were collected and the solvent was evaporates and co-evaporated with toluene. The crude intermediate 119 (0.1 g) was used as such in the next reaction step.
A mixture of (2-hydroxy-5-nitrophenoxy)acetic acid ethyl ester (10 g, 0.0414 mol) and K2CO3 (8.6 g, 0.0621 mol) in DMF (300 ml) was stirred for 1 hour at r.t. Then 1,3-dibromopropane (42 ml, 0.414 mol) was added and the mixture was heated to 60° C. for 18 hours. The mixture was quenched with ice-water and the product was extracted with toluene (3×). The organic layer was dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by flash column chromatography over silica (eluent: DCM). The desired fractions were collected and the solvent was evaporated. Yield: 8.40 g of intermediate 120 (55% yield).
A solution of 4-methoxyphenol (1.56 g, 0.0126 mol) and K2CO3 (1.73 g, 0.0126 mol) in DMF (70 ml) was heated at 60° C. for 1 hour till the appearance of a strong violet pigmentation. Then intermediate 120 (3.5 g, 0.0097 mol) was added and the mixture was heated for 24 hours. The reaction mixture was poured in ice-water and the product was extracted with toluene (3×). The organic layer was dried (MgSO4), filtered and the solvent was evaporated to yield an oil. This oil was purified by flash column chromatography over silica gel (eluent: heptane/EtOAc 4/1). The desired fractions were collected and the solvent was evaporated. Yield: 2.28 g of intermediate 121 (58%).
A mixture of intermediate 121 (2.28 g, 0.0056 mol) in EtOH (60 ml) was hydrogenated at r.t. with Pd/C 10% (0.5 g) as a catalyst in the presence of a thiophene solution (0.3 ml; 4% in DIPE). After uptake of H2 (3 eq.), the catalyst was filtered off and the filtrate was evaporated. The residue was dissolved in DCM and treated with HCl gas for 5 minutes. The solvent was evaporated to obtain crude intermediate 122 (2.12 g; .HCl) as a purple solid that was used a such in the next reaction step.
A solution of intermediate 122 (1 g, 0.002664 mol), intermediate 32 (1.176 g, 0.003196 mol) and HCl/dioxane (1.1 ml; 4 N) in 1-butanol (22 ml) was heated for 30 minutes at 100° C. This mixture was added to a solution of HCl (13 ml; 36%) in H2O (26 ml). The mixture was stirred for 2 hours at 60° C. and was then heated to 80° C. for 20 hours. The mixture was concentrated under reduced pressure and a mixture of LiOH.H2O (0.559 g, 0.01332 mol), THF (20 ml) and H2O (5 ml) was added. The reaction mixture was heated for 30 minutes at 50° C. The solvent was evaporated and the residue was purified by HPLC method C. The desired fractions were collected and the solvent was evaporated and co-evaporated with MeOH. Yield: 0.287 g of intermediate 123 (18.6%).
A solution of intermediate 115 (1.25 g, 0.0036 mol) and 4-chlorobenzenamine (1.37 g, 0.0108 mol) in DMF (30 ml) was heated in the microwave to 125° C. for 90 minutes. After cooling, the reaction mixture was poured into ice-water and the product was extracted with EtOAc (3×). The organic layer was washed with H2O, dried (MgSO4), filtered and the solvent was evaporated to yield an oil. This oil was purified by flash column chromatography over silica gel (eluent: heptane/EtOAc 4/1). The desired fractions were collected and the solvent was evaporated. Yield: 0.7 g of intermediate 124 (50% yield).
A mixture of intermediate 124 (0.66 g, 0.0017 mol) in THF (50 ml) was hydrogenated at r.t. with Pd/C 5% (0.2 g) as a catalyst in the presence of a thiophene solution (4% in DIPE). After uptake of H2 (3 eq), the catalyst was filtered off and the filtrate was evaporated. The residue was dissolved in DCM and treated with HCl gas for 5 minutes. Then the solvent was evaporated to yield 0.749 g of intermediate 125 (.HCl) as a brown solid that was used as such in the next reaction step.
A solution of intermediate 125 (0.749 g, 0.002053 mol), intermediate 32 (0.906 g, 0.002464 mol) and HCl/dioxane (0.8 ml; 4 N) in 1-butanol (18 ml) was heated for 30 minutes at 100° C. This mixture was added to a solution of HCl (0.8 ml, 36%) in H2O (16 ml) and the reaction mixture was stirred for 24 hours at 80° C. Then the solvent was evaporated. A mixture of LiOH.H2O (0.43 g, 0.01025 mol) and THF (15 ml) in H2O (3 ml) was added to the residue and the mixture was heated overnight at 50° C. The solvent was evaporated and the residue was purified by HPLC method C. The desired fractions were collected and the solvent was evaporated and co-evaporated with MeOH. Yield: 0.1 g of intermediate 126 (7.3% yield).
NaH (1.5 g, 0.0375 mol) was stirred in DMF (80 ml; dry) under N2 at r.t. 2-[(Tetrahydro-2H-pyran-2-yl)oxy]ethanol (5.09 ml, 0.0375 mol) was added dropwise and the mixture was stirred for 1 hour. 5-Nitro-1,3-benzodioxole (4.178 g, 0.025 mol) was added portionwise and the reaction mixture was stirred for 5 hours. Ethyl 2-bromoacetate (4.158 ml, 0.0375 mol) in DMF (20 ml; dry) was added dropwise and the mixture was stirred overnight. Subsequently, H2O was added and the product was extracted with EtOAc (3×). The organic layer was washed with H2O, dried and the solvent was evaporated. Yield: 9.5 g of intermediate 127.
Pd/C (2 g, 0.001879 mol) was suspended in EtOH (300 ml) under N2 flow. Intermediate 127 (9.234 g, 0.025 mol) and a thiophene solution (0.025 mol; 4% in DIPE) was added and the reaction mixture was stirred under H2 atmosphere until 3 eq. of H2 were absorbed. Then the catalyst was filtered off over Dicalite and the solvent was evaporated. The residue was stirred in DIPE and the precipitate was filtered off and dried, yielding crude intermediate 128 (8.8 g).
A mixture of intermediate 32 (1.839 g, 0.005 mol), intermediate 128 (3.636 g, 0.006 mol), HCl/dioxane (2.5 ml; 4 M) and t-BuOH (40 ml) was stirred overnight at 75° C. The solvent was evaporated the crude intermediate 129 was used as such in the next reaction step.
A mixture of intermediate 129 (2.933 g, 0.005 mol), HCl/dioxane (2.5 ml; 4 N) and H2O (40 ml) was stirred for 5 hours at 60° C. The solvent was evaporated and the residue was purified by HPLC. The solvent was evaporated, yielding 0.16 g of intermediate 130 (6.7%).
A mixture of 2-chloro-5-nitrophenoxyacetic acid (2 g, 0.0086 mol), pyrrolidine (0.92 ml, 0.011 mol), Cu powder (catalytic amount; dendritic, 3 micron) and K2CO3 (1.5 g, 0.011 mol) in DMF (5 ml) was heated for 2 hours at 150° C. The solvent was evaporated and the residue was stirred in a mixture of MeOH/DCM 5/95. The precipitate was filtered off, washed and dried. The product was dissolved in H2O and treated with HCl (1 N). The formed precipitate was filtered off and washed. Yield: 1.6 g of crude intermediate 131 that was used as such in the next reaction step
A mixture of intermediate 131 (1.6 g, 0.006 mol) in THF (120 ml) was hydrogenated at r.t. with Raney Nickel (1 g) as a catalyst. After uptake of H2 (3 eq.), the catalyst was filtered off and the filtrate was evaporated. The residue was treated with HCl (4 N) in dioxane (2 ml) and this mixture was stirred for 15 minutes. The solvent was evaporated and the crude intermediate 132 (.HCl) was used as such in the next reaction step.
A solution of intermediate 32 (1.58 g, 0.0043 mol), intermediate 132 (1.6 g, 0.0043 mol) and HCl/dioxane (2 ml; 4 N) in t-BuOH (20 ml) was stirred for 18 hours at 75° C. This reaction mixture containing intermediate 133 was used as such in the next reaction step.
The crude reaction mixture from the previous reaction step containing intermediate 133 (max. 0.0043 mol) was treated with concentrated HCl (3 ml) and H2O (5 ml). The solution was heated up to 60° C. for 2 hours. The solvent was evaporated and the residue was purified by HPLC (RP-18; eluent: (0.25% NH4HCO3 in H2O)/CH3CN v/v 100/0-0/100). The desired fractions were collected and the solvent was evaporated. The crude intermediate 134 (0.030 g) was used as such in the next reaction step.
3-Nitro-5-trifluoromethylphenol (3.20 g, 0.0154 mol) was dissolved in CH3CN (46 ml). 4-Bromobutanoic acid ethyl ester (2.6 ml, 0.0185 mol) was added to the solution, followed by the addition of K2CO3 (3.20 g, 0.0232 mol). The reaction mixture was heated at 80° C. overnight. The solid was filtered off and washed with CH3CN. EtOAc (20 ml) was added and the mixture was washed with brine, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography on silica gel, using as eluent a mixture of hexanes:EtOAc (ratio 40/1). The product fractions were collected and the solvent was evaporated. The product was dried (vacuum, r.t.) yielding a pale yellow solid. Yield: 4.40 g of intermediate 135 (89%).
Intermediate 135 (4.40 g, 0.0137 mol) was dissolved in THF (48 ml) at r.t. Pt/C 5% (0.88 g) was added and the mixture was stirred at r.t. under H2 atmosphere for 15 hours. The mixture was filtered through a Celite pad. The Celite was washed with THF. The solvent was evaporated. The product was dried (vacuum, r.t.), yielding 3.96 g of a brown oil. The hydrochloric salt was obtained purging HCl gas into a solution of the aniline in Et2O. Yield: 3.86 g of intermediate 136 (86%; .HCl).
Intermediate 136 (3.86 g, 0.0118 mol) was dissolved in HCl/1,4-dioxane (30 ml; 4 N). The solution was heated at 60° C. overnight. The solvent was evaporated and the residue was stirred in Et2O and filtered off. The product was dried (vacuum, r.t.) yielding 3.40 g of intermediate 137 (96%; .HCl) as a grey solid.
Intermediate 32 (0.70 g, 0.0019 mol) was dissolved in a mixture of CH3CN (4 ml) and 2-propanol (2 ml). Then intermediate 137 (0.69 g, 0.0023 mol) was added, followed by the addition of HCl (1 ml; 4 N in 1,4-dioxane). The reaction mixture was heated at 80° C. for 2 hours. The solvent was evaporated and the residue was dried (vacuum, r.t.), yielding crude intermediate 138 (0.2HCl) as a brown foam. This compounds was used as such in the next reaction step.
A mixture of 1-chloro-4-nitro-2-(phenylmethoxy)benzene (26.29 g, 0.0997 mol) in 1-methyl-1,4-piperazine (50 ml) was heated at 100° C. overnight. The mixture was partitioned between EtOAc and a saturated aq. NaHCO3 solution. The aq. phase was extracted with EtOAc several times. The combined organic layers were washed first with brine, then with 200 ml of HOAc (1%), again with brine and finally with a saturated aq. NaHCO3 solution. Subsequently, the organic layer was dried (MgSO4), filtered and concentrated to dryness. The product was dried (vacuum, r.t.) yielding 23.90 g of intermediate 139 (73%) as a brown syrup.
Intermediate 139 (1.5 g, 0.0045 mol) was suspended in 4 N HCl in 1,4-dioxane (15 ml) and the reaction mixture was heated at 100° C. in a sealed tube overnight. The reaction mixture was concentrated to dryness and the residue was triturated with DIPE, filtered off and dried, giving 1 g of a yellow solid. Yield: 1 g of intermediate 140 (0.2 HCl; 91%).
Intermediate 140 (4.23 g, 0.0136 mol) was dissolved in a mixture of toluene (70 ml) and H2O (30 ml). NaHCO3 (4.0 g, 0.0477 mol) and N,N,N-tributyl-1-butanaminium bromide (0.22 g, 0.0007 mol) were added, followed by the addition of ethyl 2-bromoacetate (1.8 ml, 0.0164 mol). The reaction mixture was heated at 80° C. overnight. The phases were separated and the aq. phase was extracted with EtOAc (3×20 ml). The organic layers were combined, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography on silica gel, eluent: a mixture of DCM/MeOH (ratios: 80/1-70/1-60/1-50/1). The product fractions were collected and the solvent was evaporated. The product was dried (vacuum, r.t.) yielding a pale yellow solid. Yield: 0.75 g of intermediate 141 (17%).
Intermediate 141 (0.75 g, 0.0023 mol) was dissolved in THF (10 ml) at r.t. Then, Pt/C 5% (0.15 g) was added and the mixture was stirred at r.t. under H2 atmosphere for 15 hours. The mixture was filtered through a Celite pad. The Celite was washed with THF. The solvent was evaporated. The residue was dried (vacuum, r.t.), yielding 0.72 g of a brown oil. The hydrochloric acid salt was obtained by purging HCl gas into a solution of the aniline in Et2O. Yield: 0.86 g of intermediate 142 (0.3 HCl; 92%).
Intermediate 32 (0.70 g, 0.0019 mol) was dissolved in a mixture of CH3CN (4 ml) and 2-propanol (2 ml). Then, HCl (1 ml; 4 N in 1,4-dioxane) was added followed by the addition of intermediate 142 (0.53 g, 0.0023 mol). The reaction mixture was heated at 80° C. for 2 hours. The solvent was evaporated and the crude intermediate 143 (0.4 HCl) was used as such in the next reaction step. The product was dried (vacuum, r.t.), yielding a brown foam.
A mixture of 1-chloro-4-nitro-2-(phenylmethoxy)benzene (26.29 g, 0.0997 mol) in morpholine (50 ml) was heated at 100° C. overnight. The mixture was partitioned between EtOAc and a saturated aq. NaHCO3 solution. The aq. phase was extracted with EtOAc several times. The combined organic layers were washed with brine, with 200 ml of HOAc (1%), with brine and finally with a saturated aq. NaHCO3 solution. The organic layer was dried (MgSO4), filtered and concentrated to dryness. The residue was dried (vacuum, r.t.) yielding a brown syrup. Yield: 26.47 g of intermediate 144 (85%).
Intermediate 144 (26.47 g, 0.0842 mol) was dissolved in HCl (185 ml; 4 N in 1,4-dioxane). The solution was stirred at 100° C. overnight in a sealed tube. The solvent was evaporated and the residue was dried (vacuum, r.t.), yielding a brown oil. The product was used as such in the next reaction step. Yield: 21.95 g of intermediate 145 (.HCl).
Intermediate 145 (6.0 g, 0.0230 mol) was dissolved in CH3CN (70 ml) and then ethyl 2-bromoacetate (3.1 ml, 0.0276 mol) was added, followed by the addition of K2CO3 (4.77 g, 0.0345 mol). The reaction mixture was heated at 80° C. overnight. The solid was filtered off and washed with CH3CN. EtOAc (50 ml) was added and the mixture was washed with brine, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography over silica gel (eluent: a mixture of hexanes/EtOAc (ratios: 10/1-8/1-5/1-3/1). The product fractions were collected and the solvent was evaporated. The product was dried (vacuum, r.t.) yielding a pale yellow solid. Yield: 5.88 g of intermediate 146 (82%).
Intermediate 146 (5.88 g, 0.0189 mol) was dissolved in THF (66 ml) and the solution was stirred at r.t. Then, Pt/C 5% (1.18 g) was added and the mixture was stirred at r.t. under H2 atmosphere for 15 hours. The mixture was filtered through a Celite pad. The Celite was washed with THF. The solvent was evaporated. The residue was dried (vacuum, r.t.) yielding 4.67 g of a brown oil. The hydrochloric salt was obtained by purging HCl gas into a solution of the aniline in Et2O. Yield: 3.71 g of compound 147 (55%).
Intermediate 32 (0.70 g, 0.0019 mol) was dissolved in a mixture of CH3CN (4 ml) and 2-propanol (2 ml). Then, HCl (1 ml; 4 N in 1,4-dioxane) was added followed by the addition of intermediate 147 (0.53 g, 0.0023 mol). The reaction mixture was heated at 80° C. for 2 hours. The solvent was evaporated and the residue was dried (vacuum, r.t.), yielding crude intermediate 148 (0.3 HCl) as a brown foam.
Pd/C 10% (4 g) was suspended in MeOH (200 ml) under N2 atmosphere. A 4% thiophene solution (4 ml) was added. The mixture was stirred at 25° C. under H2 atmosphere for pre-hydrogenation. First N-(3-aminopropyl)-N-methylcarbamic acid 1,1-dimethylethyl ester (0.106 mol) and then benzaldehyde (0.106 mol) was added. The reaction mixture was stirred and hydrogenated at 25° C. under H2 atmosphere. After uptake of H2 (1 equiv), formaldehyde (0.106 mol) was added and the reaction mixture was hydrogenated further. After uptake of H2 (1 equiv), the catalyst was filtered off over dicalite and the filtrate was evaporated. The residue was purified over silica gel (eluent: DCM/CH3OH 95/5). The product fractions were collected and the solvent was evaporated to give an oil, yielding 26.5 g (71.7%) of intermediate 149.
Pd/C 10% (2 g, catalyst) was suspended in MeOH (150 ml) under N2 flow. intermediate 149 (0.092 mol) was added. The reaction mixture was stirred under H2 atmosphere until 1 equivalent of H2 was taken up. The catalyst was removed by filtration over dicalite. The filtrate's solvent was evaporated under reduced pressure, yielding 18.4 g (98.9%) of intermediate 150.
A mixture of 4-chloro-2-methyl-1H-pyrrolo[2,3-d]pyrimidine (0.0298 mol) in DCM (150 ml) was stirred at r.t. 1-bromo-2,5-pyrrolidinedione (0.0471 mol) was added portionwise. The reaction mixture was stirred for one hour. The precipitate was filtered off, washed with DIPE, then dried in vacuo at 50° C., yielding 2.5 g (34%) of intermediate 151.
A solution of intermediate 151 (0.01014 mol) in THF (50 ml) was stirred under N2— flow at −78° C. and a solution of n-BuLi 2.5M (0.0305 mol) in THF (10 ml) was added dropwise. After addition, the reaction was continued for one hour at −78° C. A solution of DMF (0.02029 mol) in THF (10 ml) was added dropwise. The reaction mixture was stirred for 30 min at −78° C. The reaction mixture was allowed to reach −30° C., and H2O (10 ml) was added dropwise. The reaction mixture was stirred for 30 min. The organic layer was separated and filtered over silica gel (eluent: THF). The collected fractions were concentrated under reduced pressure and the residue was stirred in a mixture of 2% CH3OH and 98% DCM. The precipitate was filtered off, washed and dried under vacuo, yielding 0.85 g (42.8%) of intermediate 152.
A mixture of intermediate 150 (0.00515 mol) and Na(OAc)3BH (0.01288 mol) in DCM (q.s.) was stirred at r.t. A mixture of intermediate 152 (0.00429 mol) in THF (q.s.) was added dropwise. After addition, stirring was continued for 18 hours. The reaction mixture was treated with H2O. The layers were separated and the water layer was further extracted with DCM. The combined organic layers were concentrated under reduced pressure. The crude residual fraction was purified by high-performance liquid chromatography over an RP-18 column (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/v/v 50/50-0/100). The product fractions were collected, evaporated to dryness and the residue was dried in vacuo at 50° C., yielding 0.87 g (49.3%) of intermediate 153. A mixture of intermediate 153 (0.000655 mol) and the free base of intermediate 82 (0.000655 mol) in HCl/Dioxane (4N) (1 ml) and n-Butanol (20 ml) was heated under microwave conditions for 2 hours at 100° C. The reaction solution was concentrated under reduced pressure. The crude residual fraction (0.43 g; 66.1%) was used as intermediate 154 (0.2HCl) in the next step.
A solution of intermediate 154 (0.00079 mol) and LiOH (0.002371 mol) in THF (20 ml) and H2O (2 ml) was heated to 50° C. and stirred for 1.5 hours. The solvent was removed under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/CH3OH/v/v 100/0/0-60/40/0-0/50/50). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.130 g (32.8%) of intermediate 155.
A mixture of intermediate 153 (0.000655 mol) and the free base of intermediate 60 (0.000655 mol) in HCl/Dioxane (4N) (1 ml) and n-butanol (20 ml) was heated under microwave conditions for 2 hours at 100° C. The solution was concentrated under reduced pressure. The crude residual fraction (0.660 g; 60.1%) was used as intermediate 156(0.2HCl) in the next step.
A solution of intermediate 156 (0.000497 mol) and LiOH (0.001491 mol) in THF (20 ml) and H2O (2 ml) was heated to 50° C. and stirred for 90 min. The solvent was removed under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/v/v 100/0-50/50-0/100). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.030 g (13.5%) of intermediate 157.
Reaction under N2 atmosphere. NaH (0.004488 mol) was added to DMF (12 ml), and the mixture was stirred at r.t. A solution of 1-(phenylmethyl)-4-piperidinemethanol (0.004488 mol) in DMF (4 ml) was added dropwise. The mixture was stirred for one hour. Then a solution of 5-nitro-1,3-benzodioxole (0.002992 mol) in DMF (4 ml) was added dropwise to the reaction mixture and stirring was continued for one hour. A solution of 2-bromoacetic acid 1,1-dimethylethyl ester (0.004488 mol) in DMF (4 ml) was added dropwise and the reaction mixture was stirred for one hour. The reaction mixture was poured out into ice water. The product was extracted with EtOAc (×2). The separated organic layer was dried, filtered and the solvent evaporated, yielding 1.78 g (96.4%) of intermediate 158 used as such in the next step
The catalyst Pt/C 5% (0.5 g) was suspended in THF (100 ml), under N2 flow. A 4% thiophene solution (1 ml) was added. Intermediate 158 (0.002885 mol) was added. The reaction mixture was stirred at 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The filtrate was evaporated under reduced pressure. The residue was dissolved in DCM and treated with HCl gas for 5 minutes. The solvent was removed in vacuo to give a powder, yielding 2.11 g of intermediate 159(.HCl) used in the next reaction step.
A solution of intermediate 32 (0.002556 mol), intermediate 159 (0.005112 mol) and HCl/Dioxane (4N) (2 ml) in 1-butanol (40 ml) was stirred and heated at 100° C. for one hour, then the resulting mixture was used as intermediate 160(0.3HCl) in the next reaction step.
The crude intermediate 160 (max. 0.0025 mol) was added to a solution of 36% HCl solution (11 ml) in H2O (22 ml). The reaction mixture was stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH.H2O (0.0125 mol) in THF (22 ml) and 5 ml of H2O were added and the reaction mixture was heated at 50° C. for 10 hours. An extra 5 equivalent of LiOH.H2O (0.0125 mol), dissolved in 5 ml of H2O was added and the heating was continued for an additional one hour. The reaction mixture was concentrated under reduced pressure. The concentrate was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN starting with 100/0 for 5 min, then from 100/0 to 70/30 over 25 min, then 0/100 for 10 min, and 100/0 for 10 min; Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 16.5 cm). The desired fractions were concentrated under reduced pressure to give a brown powder, yielding 0.2 g (10.5%) of intermediate 161.
A solution of (2-hydroxy-5-nitrophenoxy)acetic acid ethyl ester (0.004146 mol), Ph3P (0.008292 mol) and 2-[(4-chlorophenyl)thio]ethanol (0.006219 mol) in anhydrous THF (30 ml) was stirred under N2 flow at r.t. A solution of DIAD (0.008292 mol) in anhydrous THF (10 ml) was added dropwise. The resultant reaction mixture was stirred for 6 hours at r.t. The solvent was evaporated under reduced pressure. The residue was dissolved in DCM. The organic solution was washed with H2O (×2). The organic layer was separated, dried (MgSO4), filtered and the solvent was evaporated. The crude residue was stirred in diethyl ether and the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated, yielding 0.841 g (49.25%) of intermediate 162.
Pt/C 10% (0.500 g) was suspended in THF (50 ml), under N2 flow. A 4% thiophene solution (0.5 ml) was added. Intermediate 162 (0.002042 mol) was added and the reaction mixture was stirred under H2 atmosphere until 3 equivalents of H2 were taken up. The catalyst was removed by filtration over dicalite. The filtrate was evaporated under reduced pressure. The residue was dissolved in DCM and treated with HCl gas for 5 minutes. The solvent was removed in vacuo, yielding 0.663 g (77.6%) of intermediate 163(.HCl)
A solution of intermediate 163 (0.001133 mol), intermediate 32 (0.001359 mol) and HCl/Dioxane (4N) (0.5 ml) in 1-butanol (10 ml) was stirred and heated at 100° C. for 30 min. The reaction mixture was used as intermediate 164(0.2HCl) in the next step.
Crude intermediate 164 (0.001133 mol) was added to a solution of 36% HCl solution (5 ml) in H2O (10 ml). The reaction mixture was stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH (0.005665 mol) and THF (10 ml) in 5 ml of H2O was added and the reaction mixture was heated at 50° C. for 10 hours. An extra 5 equivalents of LiOH (0.005665 mol), dissolved in 5 ml of H2O, was added and the heating was continued for an additional one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.25% ammonium carbonate in H2O/CH3CN from 100/0 to 70/30 aver 60 min, then 0/100 for 20 min; Shandon 8 nm, Hyperprep C18 HS DBS 50 mm by 16.5 cm). The product fractions were collected and the solvent was evaporated under reduced pressure, yielding 0.240 g (32.6%) of intermediate 165.
DMF (12 ml) was treated with NaH (0.0038 mol), under N2-flow at r.t. A solution of 2-ethoxy-ethanol (0.0038 mol) in DMF (6 ml) was added dropwise. The resultant mixture was stirred for one hour. Then a solution of 5-methoxy-6-nitro-1,3-benzodioxole (0.00254 mol) in DMF (6 ml) was added dropwise and the reaction mixture was stirred for one hour. A solution of 2-bromoacetic acid 1,1-dimethylethyl ester (0.0038 mol) in DMF (6 ml) was added dropwise and the reaction mixture was stirred for 2 hours at r.t. The reaction was poured out into ice water and stirred for 15 minutes. The desired product was filtered off, washed with H2O and dried in vacuo at 50° C., yielding 0.42 g (44.6%) of intermediate 166
The catalyst Pd/C 10% (0.200 g) was suspended in THF (100 ml), under N2 flow. A 4% thiophene solution (1 ml) was added. Intermediate 166 (0.00113 mol) was added and the reaction mixture was stirred at 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The solvent was removed under reduced pressure, yielding 0.39 g (53.53%) of crude residue was used as intermediate 167 in the next step.
A mixture of intermediate 32 (0.00106 mol) and intermediate 167 (0.00106 mol) in HCl/Dioxane (4N) (1 ml) and n-Butanol (10 ml) was heated under microwave conditions for 2 hours at 100° C. The solution was concentrated under reduced pressure. The crude residual fraction was used as intermediate 168(0.2HCl) in the next step.
A mixture of intermediate 168 (0.000703 mol) in concentrated HCl (2 ml) and dioxane (20 ml) was heated to 85° C. for 4 hours. The solvent was removed under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/v/v 100/0-75/25-0/100). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.145 g (39.9%) of intermediate 169.
DMF (6 ml) was treated with NaH (0.0019 mol) under N2-flow at r.t. Then, a solution of MeOH (0.0019 mol) in DMF (3 ml) was added dropwise. The resultant reaction mixture was stirred for one hour. Then, a solution of 5-methoxy-6-nitro-1,3-benzodioxole (0.00127 mol) in DMF (3 ml) was added dropwise and the reaction mixture was stirred for one hour. A solution of 2-bromoacetic acid 1,1-dimethylethyl ester (0.0019 mol) in DMF (3 ml) was added dropwise and the reaction mixture was stirred for 2 hours. The reaction was poured out into ice water and stirred for 15 minutes. The product was filtered off, washed with H2O and dried in vacuo at 50° C., yielding 0.22 g (55.4%) of intermediate 170.
The catalyst Pd/C 10% (0.100 g) was suspended in THF (40 ml) under N2 flow. A 4% thiophene solution (0.1 ml) was added. Intermediate 170 (0.000702 mol) was added and the reaction mixture was hydrogenated at a temperature of 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The filtrate was concentrated under reduced pressure, yielding 0.2 g (54.3%) of residue was used as intermediate 171 in the next step.
A mixture of intermediate 32 (0.000706 mol) and intermediate 171 (0.000706 mol) in HCl/Dioxane (4N) (1 ml) and n-Butanol (10 ml) was heated under microwave conditions for 2 hours at 100° C. The solution was concentrated under reduced pressure, yielding 0.362 g (30.9%) of crude residual fraction, used as intermediate 172 (2HCl) in the next step.
A solution of intermediate 172 (0.000703 mol) in concentrated HCl (1 ml) and dioxane (10 ml) was heated to 95° C. and stirred for 90 minutes. The solvent was removed under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.50% NH4HCO3 in H2O)/CH3CN/v/v 80/20-20/80-0/100). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.078 g (24.2%) of intermediate 173.
A solution of 2-methoxy-5-nitrobenzenamine (0.0119 mol) and DIPEA (0.0238 mol) in DMF (65 ml) was stirred at r.t. A solution of 2-bromoacetic acid 1,1-dimethylethyl ester (0.0238 mol) in DMF (10 ml) was added dropwise. The reaction mixture was heated to 85° C. for 18 hours. The reaction mixture was poured out into ice water. The product was extracted with EtOAc. The separated organic layer was dried (MgSO4), filtered and the solvent evaporated, yielding 2.1 g (45.03%) of intermediate 174.
A solution of intermediate 174 (0.00354 mol) and K2CO3 (0.00531 mol) in CH3CN (60 ml) was stirred at ambient temperature for 20 minutes. CH3I (2 equiv) was added. The reaction mixture was heated to 80° C. and stirred for 18 hours. An extra amount of CH3I (2 equiv) was added and the reaction mixture was heated for an additional 18 hours. Another 2 equivalents of CH3I were added and the reaction mixture was stirred for 18 hours at 80° C. The solvent was removed under reduced pressure. The residual fraction was dissolved in EtOAc and washed with H2O twice. The organic layer was dried (MgSO4), filtered and evaporated to dryness, yielding 0.8 g (76.2%) of intermediate 175.
The catalyst Pd/C 10% (0.100 g) was suspended in THF (100 ml), under N2 flow. a 4% Thiophene solution (1 ml) was added, then intermediate 175 (0.0027 mol) was added and the reaction mixture was stirred at a 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The solvent was removed under reduced pressure. The residue was dissolved in DCM and treated with HCl gas for 5 min. The solvent was removed under reduced pressure, yielding 0.91 g (44.7%) of intermediate 176 (0.2HCl) used as such in the next step.
A mixture of intermediate 176 (0.00265 mol) and intermediate 32 (0.00265 mol) in HCl/Dioxane (4N) (2 ml) and n-butanol (50 ml) was heated to 100° C. for one hour under microwave conditions. The solvent was removed under reduced pressure, yielding 1.3 g (54.16%) of crude intermediate 177(0.2HCl) used as such in the next step.
A solution of intermediate 177 (0.00201 mol), Dioxane (50 ml), concentrated HCl (2 ml) and H2O (5 ml) was heated to 85° C. for 4 hours. The solvent was removed under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/CH3OH/v/v 75/25/0-0/100/0-0/0/100). The desired fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.020 g (2.26%) of intermediate 178.
A solution of DMF (25 ml) was treated with NaH (0.0018 mol), under N2-flow at r.t. A solution of 2-[[4-(trifluoromethyl)-2-pyridinyl]oxy]ethanol (0.0018 mol) in DMF (5 ml) was added dropwise. After addition, the mixture was stirred for one hour. Then a solution of 5-nitro-1,3-benzodioxole (0.0012 mol) in DMF (5 ml) was added dropwise and the resultant reaction mixture was stirred for 4 hours at r.t. A solution of 2-bromoacetic acid 1,1-dimethylethyl ester (0.0018 mol) in DMF (5 ml) was added dropwise and the entire mixture was stirred for 18 hours. The reaction mixture was poured out into ice water. The product was extracted with EtOAc (3 times) and the organic layer was dried (MgSO4), filtered and evaporated to dryness. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN//v/v 90/10-10/90-0/100). The product fractions were collected and evaporated to dryness yielding 0.26 g (47.4%) used as intermediate 179 in the next step.
Catalyst Pd/C 10% (0.100 g) was suspended in THF (100 ml) under N2 flow. A 4% thiophene solution (1 ml) was added. Intermediate 179 (0.000567 mol) was added. The reaction mixture was stirred at a temperature of 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The filtrate was evaporated under reduced pressure. The residual fraction was dissolved in DCM and treated with HCl gas for 2 min. The reaction mixture was concentrated and the residue (0.3 g; 61.4%) was used as intermediate 180 in the next step.
A solution of intermediate 180 (0.000645 mol), intermediate 32 (0.000774 mol) and HCl/Dioxane (4N) (0.3 ml) in 1-butanol (6 ml) was heated at 100° C. for 30 min. The obtained mixture was used as intermediate 181(0.2HCl) in the next step
Intermediate 181 (max. 0.000645 mol) was added to a solution of 36% HCl (3 ml) in H2O (6 ml). The reaction mixture was stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH (0.003225 mol) and THF (6 ml) in 5 ml of H2O was added and the reaction mixture was heated at 50° C. for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by HPLC (0.5% ammonium carbonate in H2O/MeOH/CH3CN from 75/25/0 to 0/50/50 over 44 min, then from 0/50/50 to 0/0/100 over 13 min; Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 16.5 cm) The desired fractions were collected and concentrated under reduced pressure, yielding 0.025 g (5.20%) of intermediate 182.
A solution of 4-methoxyphenol (0.01494 mol) and K2CO3 (0.01494 mol) in anhydrous DMF (80 ml) was heated at 60° C. for 30 minutes (strong purple pigmentation appeared). Intermediate 115 (0.01149 mol) was added and the reaction mixture was heated at 60° C. for 22 hours. The mixture was poured out into ice water. The product was extracted with toluene (3×). The organic fraction was dried (MgSO4), filtered and evaporated to dryness. The oil was purified by HPLC (0.5% ammonium carbonate in H2O/CH3CN from 80/20 to 0/100 over 60 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 16.5 cm). The product fractions were concentrated under reduced pressure to give a yellow oil, yielding 0.380 g (8.45%) of intermediate 183.
Pd/C 10% (0.100 g) was suspended in THF (50 ml) under N2 flow. A 4% thiophene solution (0.1 ml) was added and then intermediate 183 (0.000971 mol) was added. The reaction mixture was stirred under H2 atmosphere until 3 equivalents of H2 were taken up. The catalyst was removed by filtration over dicalite. The solution was concentrated under reduced pressure. The residue was dissolved in DCM and treated with HCl gas for 5 minutes. The solvent was removed in vacuo to give 0.5025 g (93.66%) of a solid used as intermediate 184(.HCl) in the next step.
A solution of intermediate 184 (0.001262 mol), intermediate 32 (0.001514 mol) and HCl/Dioxane (4N) (0.5 ml) in 1-Butanol (10 ml) was heated at 100° C. for 30 minutes, yielding intermediate 185(0.2HCl) used as such in the next step.
Crude intermediate 185 (max. 0.001262 mol) was added to a solution of 36% HCl solution (6 ml) in H2O (12 ml). The reaction mixture was stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH (0.00631 mol) and THF (12 ml) in 5 ml of H2O was added and the reaction mixture was heated at 50° C. for 10 hours. An extra 5 equivalent of LiOH (0.00631 mol), dissolved in 5 ml of H2O, was added and the heating was continued for an additional one hour. The reaction mixture was concentrated under reduced pressure and purified by HPLC (0.5% ammonium carbonate in H2O/MeOH/CH3CN from 75/25/0 to 0/0/100 over 57 min, then 0/0/100 for 4 min; Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure, yielding 0.040 g (4.9%) of intermediate 186.
A solution of (2-hydroxy-5-nitrophenoxy)acetic acid ethyl ester (0.009246 mol), (Ph)3P (0.018493 mol) and 2-(2-thiazolylamino)ethanol (0.01387 mol) in anhydrous THF (80 ml) was stirred under N2 flow at r.t. while a solution of DIAD (0.018493 mol) in anhydrous THF (10 ml) was added dropwise. After addition the reaction mixture was stirred for 6 hours.
The reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 and washed with H2O (×2). The organic layer was dried (MgSO4), filtered and evaporated to dryness. The residue was purified by HPLC (eluent: CH2Cl2/ethanol starting with 100/0 for 15 min, then from 100/0 to 95/5 over 15 min, then 95/5 for 5 min, and from 90/10 to 0/100 over 15 min; Shandon 25-40 μm, silica gel 60, 50 mm by 20 cm). The product fractions were concentrated under reduced pressure, yielding 3.2 g (92%) of intermediate 187.
The catalyst Pd/C 10% (0.300 g) was suspended in THF (100 ml), under N2 flow. A 4% thiophene solution (1 ml) was added. Intermediate 187 (0.002722 mol) was added. The reaction mixture was stirred at 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The solvent was removed under pressure, yielding 1.44 g (112.9%) crude residue, used as intermediate 188 in the next step.
A solution of intermediate 188 (0.002265 mol), intermediate 32 (0.002718 mol) and HCl/Dioxane (4N) (1 ml) in 1-butanol (20 ml) was heated at 100° C. for 30 min. The resulting mixture was used as intermediate 189(0.2HCl) in the next step.
Intermediate 189 (max. 0.002265 mol) was added to a solution of 36% HCl (10 ml) in H2O (20 ml). The reaction mixture was stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH (0.011325 mol) and THF (20 ml) in 5 ml of H2O was added and the reaction mixture was heated at 50° C. for 4 hours. The reaction mixture was concentrated under reduced pressure and the concentrate was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 100/0 to 50/50 over 40 min, then 0/100 for 10 min and 100/0 for 10 min; Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure, yielding 0.120 g (9.60%) of intermediate 190.
A solution of DMF (42 ml) was treated with NaH (0.0158 mol) under N2-flow at r.t. A solution of 2-(methyl-2-thiazolylamino)ethanol (0.0158 mol) in DMF (12 ml) was added dropwise and then the reaction mixture was stirred for one hour. A solution of 5-nitro-1,3-benzodioxole (0.0105 mol) in DMF (12 ml) was added dropwise and stirring was continued for one hour. A solution of 2-bromoacetic acid 1,1-dimethylethyl ester (0.0158 mol) in DMF (12 ml) was added dropwise and the mixture was stirred for one hour. The reaction mixture was poured out into ice water. The product was extracted with EtOAc (twice) and the organic layer was dried, filtered and evaporated to dryness. The residue was purified by HPLC (0.5% ammonium bicarbonate in H2O/MeOH/CH3CN from 85/15/0 to 46/25/29 over 24 min, then 0/50/50 for 4 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å, 50 mm by 20 cm). The product fractions were collected and evaporated under reduced pressure, yielding 3 g (69.55%) of intermediate 191.
Catalyst Pd/C 10% (0.300 g) was suspended in THF (100 ml), under N2 flow. A 4% thiophene solution (1 ml) was added, then intermediate 191 (0.00244 mol) was added. The reaction mixture was stirred at a temperature of 25° C. under H2 atmosphere until 3 equivalent of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The solvent was removed under reduced pressure, yielding 1.09 g (70.6% (60% P) of intermediate 192.
A solution of intermediate 192 (0.002265 mol), intermediate 32 (0.002718 mol) and HCl/Dioxane (4N) (1 ml) in 1-butanol (20 ml) was heated at 100° C. for 30 min. The mixture was used as intermediate 193(0.2HCl) in the next step
Intermediate 193 (2.265 mmol) was added to a solution of 36% HCl (10 ml) in H2O (20 ml). The reaction mixture was stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH (0.011325 mol) and THF (20 ml) in 5 ml of H2O was added and the reaction mixture was heated at 50° C. for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 100/0 to 50/50 over 40 min, then 0/100 for 10 min and 100/0 for 10 min; Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure, yielding 0.440 g (25.9%) of intermediate 194.
A solution of DMF (35 ml) was treated with NaH (0.00898 mol), under N2-flow, at r.t. A solution of 2-(methyl-2-pyridinylamino)ethanol (0.00898 mol) in DMF (15 ml) was added dropwise. The reaction mixture was stirred for one hour. Then a solution of 5-nitro-1,3-benzodioxole (0.00598 mol) in DMF (15 ml) was added dropwise and stirring was continued for 4 hours. A solution of 2-bromoacetic acid 1,1-dimethylethyl ester (0.00898 mol) in DMF (15 ml) was added dropwise and the entire mixture was stirred for 18 hours. The reaction was completed and the mixture was poured out into ice water. The product was extracted with EtOAc (3 times) and the organic layer was dried (MgSO4), filtered and evaporated to dryness. The crude residue (3.1 g; 74.5%) was used as intermediate 195 in the next step.
Catalyst Pd/C 10% (0.5 g) was suspended in THF (100 ml), under N2 flow. A 4% thiophene solution (1 ml) was added. Intermediate 195 (0.00595 mol) was added. The reaction mixture was stirred at 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2 and treated with HCl gas for 5 min. The solvent was removed under reduced pressure. The crude dark brown solid (2.3 g; 79.3%) was used as intermediate 196(0.2HCl) in the next step.
A solution of intermediate 196 (0.001133 mol), intermediate 32 (0.001359 mol) and HCl/Dioxane (4N) (0.5 ml) in 1-butanol (10 ml) was heated at 100° C. for 30 minutes. The mixture was used as intermediate 197(0.3HCl) in the next step.
Intermediate 197 (0.001133 mol) was added to a solution of 36% HCl (5 ml) in H2O (10 ml). The reaction mixture was stirred at 60° C. for 4 hours. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH (0.005665 mol) and THF (10 ml) in 5 ml of H2O was added and the reaction mixture was heated at 50° C. for 10 hours. An extra 5 equivalent of LiOH (0.005665 mol), dissolved in 5 ml of H2O, was added and the heating was continued for an additional one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 100/0 to 60/40 over 60 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure, yielding 0.200 g (84%) of intermediate 198.
A solution of (2-hydroxy-5-nitrophenoxy)acetic acid ethyl ester (0.009582 mol), (Ph)3P (0.019163 mol) and 2-(2-pyrimidinylamino)ethanol (0.014372 mol) in anhydrous THF (83 ml) was stirred under N2 flow at r.t., while a solution of DIAD (0.019163 mol) in anhydrous THF (10 ml) was added dropwise. After addition, the reaction mixture was stirred for 24 hours. An additional 0.5 eq. of (Ph)3P and DIAD and 0.38 eq. of 2-(2-pyrimidinylamino)ethanol was added and the reaction mixture was stirred under N2 flow for 24 hours. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in DIPE. The precipitate was filtered off. The filtrate was concentrated under reduced pressure and the residue was purified by HPLC (0.5% ammonium carbonate in H2O/MeOH from 70/30 to 30/70 over 24 min; then 0/100 for 8 min, Shandon 8 μm, Hyperprep C18 HS DBS 50 mm by 21 cm). The product fractions were collected and reduced under reduced pressure, yielding 1 g of intermediate 199.(mixture of methyl and ethyl ester)
Catalyst Pd/C 10% (0.100 g) was suspended in THF (100 ml) under N2 atmosphere. A 4% Thiophene solution (1 ml) was added. Intermediate 199 was added and the reaction mixture was stirred at a temperature of 25° C. under H2 atmosphere until 3 equivalents of hydrogen were taken up. The catalyst was removed by filtration over dicalite. The solvent was removed under reduced pressure, yielding 0.169 g of intermediate 200.
A mixture of intermediate 32 (0.00061 mol) and HCl/Dioxane (4N) (0.000265 mol) in Butanol (4.5 ml) was added to intermediate 200 (0.254 mmol) and the reaction mixture was heated at 100° C. for 30 min. The precipitate was filtered off, yielding intermediate 201(0.3HCl).
Crude intermediate 201 (0.000338 mol) was added to a solution of 36% HCl solution (1.4 ml) in H2O (2.8 ml). The reaction mixture was stirred at 60° C. for one hour. The reaction mixture was concentrated under reduced pressure. A mixture of LiOH (0.00169 mol) and THF (1.4 ml) in 0.5 ml of H2O was added and the reaction mixture was heated at 50° C. for one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.2% ammonium bicarbonate in H2O/CH3CN starting with 100/0 for 10 min, then from 100/0 to 0/100 over 60 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å 50 mm by 21 cm). The product fractions were collected and concentrated under vacuum, yielding 0.030 g (15.6%) of intermediate 202.
60% NaH in mineral oil (0.03804 mol) was suspended in DMF (60 ml). The suspension was stirred for 30 min. A solution of 2-ethoxy-ethanol (0.03804 mol) in DMF (40 ml) was added dropwise over a 10-min period. The mixture was stirred for 15 min. 4-methoxy-6-nitro-1,3-benzodioxole (0.02536 mol) was added portionwise. The reaction mixture was stirred for 16 hours at r.t. 2-bromoacetic acid ethyl ester (0.03804 mol) was added and the resulting reaction solution was stirred for an additional 4 hours. The solvent was evaporated. The residue was partitioned between brine and EtOAc (3×100 ml). The organic layers were dried (MgSO4), filtered and concentrated to dryness, yielding intermediate 203 used in the next reaction step without further purification.
Intermediate 203 (0.02536 mol) was dissolved in EtOH (100 ml) and this solution was hydrogenated under atmospheric pressure at r.t. with Pd/C 10% (1.59 g) as a catalyst After uptake of H2 (3 equiv), the catalyst was filtered off over a pad of Celite and the filtrate was evaporated. A brown oil was obtained which was partitioned between an aqueous NaHCO3 solution and EtOAc (3×100 ml). The organic layers were combined, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash chromatography using hexane/EtOAc mixtures as eluents, yielding 72% of intermediate 204.
A 37% HCl solution (0.230 ml) was added to a solution of intermediate 1 (0.01116 mol) and intermediate 204 (0.02233 mol) in CH3CN (50 ml). The reaction mixture was stirred and refluxed for 5 hours, then heated at 45° C. overnight. The solvent was evaporated and the residue was partitioned between an aqueous Na2CO3 solution and EtOAc and extracted. The organic layers were combined, dried (MgSO4), filtered and the solvent was evaporated. The residue was purified by flash chromatography (eluent:n-Hexane/EtOAc 1:1). The product fractions were collected and the solvent was evaporated, yielding 3.0 g (60%) of intermediate 205.
A mixture of intermediate 205 (0.00218 mol) and intermediate 98 (0.00262 mol) in THF (q.s.) was stirred for one hour at r.t. NaBH(OAc)3 (0.00654 mol) was added. The resultant reaction mixture was stirred at r.t. for 16 hours. The mixture was concentrated, and the concentrate was partitioned between an aqueous NaHCO3 solution and EtOAc, and extracted. The organic layers were combined, dried (MgSO4), filtered and the solvent was evaporated to dryness. Intermediate 206 was used in the next reaction step without further purification.
Intermediate 206 (0.00218 mol) was dissolved in CH3CN (30 ml). 37% HCl solution (3 ml) and H2O (9 ml) were added. The reaction mixture was stirred at 60° C. for 2 hours. The crude reaction mixture was concentrated to dryness (under oil pump vacuum). The crude residue was used as intermediate 207(0.2HCl) in the next reaction step.
60% NaH in mineral oil (0.03804 mol) was suspended in DMF (60 ml). The suspension was stirred for 30 min. A solution of 2-methoxy-ethanol (0.03804 mol) in DMF (40 ml) was added dropwise over a 10-min period. The mixture was stirred for 15 min. 4-methoxy-6-nitro-1,3-benzodioxole (0.02536 mol) was added portionwise. The resultant reaction mixture was stirred for 16 hours at r.t. 2-bromoacetic acid ethyl ester (0.03804 mol) was added and the resulting reaction solution was stirred for an additional 4 hours. The solvent was evaporated. The residue was partitioned between brine and EtOAc (3×100 mL). The organic layers were combined, dried (MgSO4), filtered and the solvent was evaporated, yielding crude intermediate 208 used as such in next reaction step.
A solution of intermediate 208 (0.02536 mol) in EtOH (100 ml) was hydrogenated under atmospheric H2 pressure at r.t. with Pd/C 10% (1.67 g) as a catalyst. After uptake of H2 (3 equiv), the catalyst was filtered off over a pad of Celite and the filtrate was evaporated. A brown oil was obtained which was partitioned between an aqueous NaHCO3 solution and EtOAc (3×100 ml). The organic layers were combined, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography (eluent: hexane/EtOAc mixtures). The product fractions were collected and the solvent was evaporated, yielding 81% of intermediate 209.
To a solution of intermediate 32 (0.0037 mol) and intermediate 209 (1.2 equiv; 0.0045 mol) in CH3CN (45 ml), HCl/Dioxane (4N) (0.00555 mol) was added and the reaction mixture was stirred and for 5 hours at 80° C. Then a 12% HCl solution (45 ml) was added. The mixture was heated for 2 hours at 60° C. The mixture was concentrated to dryness and the residue was dried under high vacuum, yielding intermediate 210(0.2HCl).
Intermediate 8 (0.00025 mol) was dissolved in DMF (10 ml). This solution was added dropwise to a mixture of HBTU (2.2 eq., 0.00055 mol) and DIPEA (30 eq., 0.0075 mol) in DMF, using a Watson-Marlow peristaltic pump (0.50 rpm). The reaction mixture was stirred for one hour at r.t. and was quenched by addition of NH3/MeOH (1 ml). The solvent was evaporated. The residue was washed with a saturated aq. Na2CO3 solution and was then purified by flash column chromatography over silica gel (eluent:DCM/MeOH mixture). The products were precipitated in a mixture of EtOAc/Et2O, filtered off and dried. Yield: 0.016 g of compound 1.
Intermediate 10 (0.00025 mol) was dissolved in DMF (10 ml). This solution was added dropwise to a mixture of HBTU (2.2 eq., 0.00055 mol) and DIPEA (30 eq., 0.0075 mol) in DMF, using a Watson-Marlow peristaltic pump (0.50 rpm). The reaction mixture was stirred for one hour at r.t. and was then quenched by addition of NH3/MeOH (1 ml). The solvent was evaporated. The residue was washed with a saturated aq. Na2CO3 solution and was then purified by flash column chromatography over silica gel (eluent: DCM/MeOH mixture). The products were precipitated in a mixture of EtOAc/Et2O, filtered off and dried. Yield: 0.015 g of compound 2.
A mixture of intermediate 17 (0.00076 mol) in MeOH (30 ml) and NaOH (15 ml; 1 N) was stirred for 16 hours at r.t. More NaOH (15 ml; 1 N) was added and the reaction mixture was stirred for 16 hours at r.t. The resulting precipitate was filtered off, washed with CH3CN and was then dried. Yield:0.236 g of compound 3 (70.6%; S-enantiomer).
A mixture of intermediate 24 (0.00019 mol) in MeOH (30 ml) and NaOH (15 ml; 1 M) was stirred for 2 hours at r.t. This mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. The residue was purified over silica gel on a glass filter (eluent: DCM/(MeOH/NH3) 90/10). The product fractions were collected and the solvent was evaporated. Yield: 0.105 g of compound 4.
A mixture of intermediate 27 (0.00007 mol) in MeOH (10 ml) and NaOH (3 ml; 1 M) was stirred for 1 hour at r.t. This mixture was poured out into H2O. The mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. The residue (0.033 g) was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.016 g of compound 5 (15.8%).
Reaction under N2 atmosphere. A mixture of PyBOP (0.00085 mol) and Et3N (0.0017 mol) in DMF (25 ml) was stirred at r.t. under N2 flow. A solution of intermediate 31 (0.00017 mol) in DMF (25 ml) was added dropwise and the resultant reaction mixture was stirred for one hour at r.t. The mixture was treated with H2O (decomposition). The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.026 g of compound 6 (38.6%).
A mixture of PyBOP (0.0005 mol) and Et3N (0.0010 mol) in DMF (20 ml) was stirred at r.t. under N2 atmosphere. A solution of intermediate 34 (0.0001 mol) in DMF (20 ml) was added dropwise. The resultant reaction mixture was stirred for 1 hour at r.t. H2O was added (decomposition). The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.022 g of compound 7 (53.7%).
Reaction under N2 atmosphere. A mixture of PyBOP (0.00045 mol) and Et3N (0.0009 mol) in DMF (20 ml; dry) was stirred under N2 flow. A solution of intermediate 38 (0.00009 mol) in DMF (20 ml; dry) was added dropwise. The reaction mixture was stirred for 1 hour at r.t. H2O was added (decomposition). The solvent was evaporated. The residue was purified by HPLC. The product fractions were collected and the solvent was evaporated. Yield: 0.032 g of compound 8.
A mixture of intermediate 42 (max. 0.000317 mol) in MeOH (25 ml) and NaOH (25 ml; 1 N) was stirred for 2 hours at r.t. This mixture was extracted with DCM. The separated organic layer was dried, filtered and the solvent evaporated. The residue was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). The mentioned mobile phases were used to apply a gradient (phase A: 0.25% NH4HCO3 solution in H2O; phase B: CH3OH (optional); phase C: CH3CN). The product fractions were collected and the solvent was evaporated. Yield: 0.120 g of compound 9 (S-enantiomer). Yield over 5 reaction steps: 28.3%.
A mixture of PyBOP (4.28 g, 0.0082 mol), Et3N (0.82 g, 0.0082 mol) and DMF (100 ml) was stirred at r.t. under N2 flow. Intermediate 44 (0.680 g, 0.0017 mol) in DMF (100 ml) was added dropwise to the mixture and stirring was continued for 1 hour. The reaction mixture was cooled on a cold waterbath and H2O (20 ml) was added dropwise to the reaction mixture. The mixture was continued stirring for 1 hour. Then the solvent was evaporated to dryness and the residue was purified by HPLC method C. The pure fractions were evaporated to dryness and evaporated with MeOH at 60° C. The residue was stirred in DIPE, filtered off and dried. Yield: 0.275 g of compound 10 (42%).
A mixture of PyBOP (0.26 g, 0.0005 mol) and Et3N (0.050 g, 0.0005 mol) in DMF (20 ml) was stirred at r.t. under N2 flow. A solution of intermediate 48 (0.045 g, 0.0001 mol) in DMF (20 ml) was added dropwise to the mixture. Stirring was continued for 1 hour. Then, the reaction mixture was cooled on a cold waterbath, and H2O (20 ml) was added dropwise. Stirring was continued for 1 hour. The reaction mixture was evaporated to dryness and the residue was purified by HPLC method A. The pure fractions were evaporated to dryness. The residue was taken up into H2O and was extracted with DCM. The organic layer was separated, dried, filtered and the solvent was evaporated. The residue was stirred in DIPE, filtered off and dried. Yield: 0.026 g of compound 11 (59.4%).
A mixture of PyBOP (4.20 g, 0.0080 mol), Et3N (0.8 g, 0.0080 mol) and DMF (100 ml) was stirred at r.t. under N2 flow. Intermediate 54 (0.696 g, 0.0016 mol) in DMF (100 ml) was added dropwise. Stirring was continued for 1 hour. The reaction mixture was cooled on a cold waterbath and H2O (20 ml) was added dropwise. Stirring was continued for 1 hour. Then the solvent was evaporated to dryness and the residue was purified by HPLC method A. The pure fractions were evaporated to dryness and evaporated with MeOH at 60° C. The residue was stirred in DIPE, filtered off and dried. Yield: 0.338 g of compound 12 (48.2%).
A mixture of PyBOP (1.836 g, 0.0035 mol) and Et3N (0.353 g, 0.0035 mol) in DMF (50 ml) was stirred at r.t. under N2 flow. A solution of intermediate 59 (0.380 g, 0.0007 mol) in DMF (50 ml) was added dropwise. Stirring was continued for 1 hour. The reaction mixture was cooled on a cold waterbath and H2O (20 ml) was added dropwise. Stirring was continued for 1 hour. The reaction mixture was evaporated to dryness and purified by HPLC method B. The pure fractions were collected, evaporated to dryness, stirred in dipe, filtered off and dried. Yield: 0.048 g of compound 13.
A mixture of PyBOP (2.080 g, 0.0040 mol), Et3N (0.400 g, 0.0040 mol) and DMF (50 ml) was stirred at r.t. under N2 flow. Intermediate 62 (0.340 g, 0.0008 mol) in DMF (50 ml) was added dropwise. The reaction mixture was cooled on a cold waterbath and H2O (20 ml) was added dropwise. Stirring was continued for 1 hour. The reaction mixture was evaporated to dryness and purified by HPLC. The pure fractions were evaporated to dryness. Yield: 0.184 g of compound 14 (56%).
A mixture of PyBOP (1.300 g, 0.0025 mol), Et3N (0.253 g, 0.0025 mol) and DMF (50 ml) was stirred at r.t. under N2 flow. Then intermediate 66 (0.270 g, 0.0005 mol) in DMF (50 ml) was added dropwise. Stirring was continued for 1 hour. The reaction mixture was cooled on a cold waterbath and H2O (20 ml) was added dropwise. Stirring was continued for 1 hour. The reaction mixture was evaporated to dryness and purified by HPLC method C. The pure fractions were collected and evaporated to dryness. The residue was stirred in dipe, filtered off and dried. Yield: 0.073 g of compound 15.
A mixture of intermediate 72 (0.0011 mol) in DMF (75 ml) was added dropwise to a mixture of PyBOP (0.0055 mol) and Et3N (0.011 mol) in DMF (75 ml). The reaction mixture was stirred at r.t. under N2 atmosphere until the reaction was finished. The solvent was evaporated. The residue (0.037 g) was stirred in DIPE, filtered off and dried. Yield: 0.015 g of compound 30.
A mixture of PyBOP (1.450 g, 0.0028 mol) and Et3N (0.28 g, 0.0028 mol) in DMF (30 ml) was stirred at r.t. under N2 flow. Intermediate 76 (0.280 g, 0.0006 mol) in DMF (30 ml) was added dropwise. The resultant reaction mixture was stirred for one hour. The reaction mixture was cooled on a cold water-bath. H2O (20 ml) was added dropwise and stirring was continued for 1 hour. The solvent was evaporated. The residue was purified by HPLC (HPLC method A). The product fractions were collected and the solvent was evaporated. Yield: 0.040 g of compound 63 (17%).
A mixture of PyBOP (1.040 g, 0.002 mol) and Et3N (0.2 g, 0.002 mol) in DMF (25 ml) was stirred at r.t. under N2 flow. A solution of intermediate 103 (0.180 g, 0.00040 mol) in DMF (25 ml) was added dropwise and the mixture was stirred for 1 hour. Then the reaction mixture was cooled on a cold water-bath, H2O (20 ml) was added dropwise and the mixture was stirred for 1 hour. Subsequently, the solvent was evaporated to dryness and the residue was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). A gradient with 3 mobile phases was applied. Phase A: a 0.25% NH4HCO3 solution in H2O; phase B: CH3OH; phase C: CH3CN). The desired fractions were collected and the solvent was evaporated. The residue was stirred in DIPE, filtered off and dried. Yield: 0.070 g of compound 73.
Intermediate 79 (0.0019 mol) in DMF (10 ml) was added over about 2 hours to a solution of HBTU (0.0042 mol) and DIPEA (0.0571 mol) in DMF (350 ml), using a Watson Marlow pump (0.75 rpm). After the addition was completed, the reaction mixture was stirred for 30 minutes. Then, 2 ml of ammonia (7 N solution in MeOH) was added and the mixture was stirred for 30 additional minutes. The solvent was evaporated and the crude was dissolved in DCM, washed with a 1 M Na2CO3 solution, dried (MgSO4), filtered and concentrated to dryness. The product was purified by flash column chromatography on silica gel, using as eluent a mixture of DCM/MeOH (+1% NH3/MeOH) (ratios: 100/0-70/1-60/1-50/1). The product fractions were collected and the solvent was evaporated. The residue was dried (vacuum, r.t.) yielding a white solid. Yield: 0.015 g of compound 69.
A solution of intermediate 143 (crude; approx. 0.0009 mol) in DMF (10 ml) was added over a 3 hour period to a solution of HBTU (0.73 g, 0.0019 mol) and DIPEA (4.4 ml, 0.0262 mol) in DMF (155 ml), using a Watson Marlow pump (0.75 rpm). After the addition was completed, the reaction mixture was stirred for 30 minutes. Then, 1 ml of NH3 (7 N solution in MeOH) was added and the mixture was stirred for 30 minutes. The solvent was evaporated and the crude was dissolved in DCM. The organic solution was washed with an aq. 1 M Na2CO3 solution, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography over silica gel (eluent: a mixture of DCM/MeOH (ratios: 100/0-70/1-60/1-50/1-40/1-30/1). The product fractions were collected and the solvent was evaporated. The residue was dried (vacuum, r.t.) yielding a white solid. Yield: 0.106 g of compound 84.
A mixture of PyBOP (2.73 g, 0.00524 mol), Et3N (0.53 g, 0.00524 mol) and DMF (50 ml) was stirred at r.t. under N2 flow. A mixture of intermediate 84 (0.51 g, 0.00105 mol) in DMF (80 ml) was added dropwise over 2 hours. The reaction mixture was stirred for 1 hour and was then cooled on a cold waterbath. H2O (20 ml) was added dropwise and the mixture was stirred again for 1 hour. Then the solvent was evaporated and the residue (4.3 g) was purified by HPLC (a gradient with NH4OAc buffer/CH3CN and MeOH was applied). After workup, 0.75 g of residue was obtained. H2O and K2CO3 were added to the residue and the product was extracted with DCM. The solvent of the organic layer was evaporated to yield 0.31 g of compound 70 (63%).
Intermediate 138 (crude, max. 0.0019 mol) in DMF (10 ml) was added over about 2 hours to a solution of HBTU (1.58 g, 0.0042 mol) and DIPEA (9.7 ml, 0.0571 mol) in DMF (350 ml), using a Watson Marlow pump (0.75 rpm). After the addition was completed, the reaction mixture was stirred for 30 minutes. Then, 2 ml of NH3 (7 N solution in MeOH) was added and the mixture was stirred for 30 minutes. The solvent was evaporated and the crude was dissolved in DCM. The organic solution was washed with an aq. 1 M Na2CO3 solution, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (eluent:DCM/MeOH (+1% NH3/CH3OH) (ratios: 100/0-70/1-60/1-50/1-40/1-30/1). The product fractions were collected and the solvent was evaporated. The products were dried (vacuum, r.t.), yielding 0.054 g of compound 83 (6%) as a white solid.
A solution of intermediate 148 (crude; approx. 0.0009 mol) in DMF (10 ml) was added over a 3-hour period to a solution of HBTU (0.73 g, 0.0019 mol) and DIPEA (4.4 ml, 0.0262 mol) in DMF (155 ml), using a Watson Marlow pump (0.75 rpm). After the addition was completed, the reaction mixture was stirred for 30 minutes. Then, 1 ml of NH3 (7 N solution in MeOH) was added and the mixture was stirred for 30 minutes. The solvent was evaporated and the residue was dissolved in DCM. The organic solution was washed with an aq. 1 M Na2CO3 solution, dried (MgSO4), filtered and concentrated to dryness. The residue was purified by flash column chromatography over silica gel (eluent: a mixture of DCM/MeOH (ratio: 100/0-70/1-60/1-50/1-40/1-30/1). The product fractions were collected and the solvent was evaporated. The product was dried (vacuum, r.t.) yielding 0.089 g of compound 85 as a white solid.
A mixture of PyBOP (1.22 g, 0.00235 mol), Et3N (0.24 g, 0.00235 mol) and DMF (30 ml) was stirred under N2 flow at r.t. A mixture of intermediate 88 (0.30 g, 0.00047 mol) in DMF (40 ml) was added dropwise over 2 hours. The reaction mixture was stirred for 1 hour. Subsequently, the mixture was cooled on a cold waterbath, H2O (20 ml) was added dropwise and the mixture was stirred for another hour. Then the solvent was evaporated and the residue was purified by reversed-phase HPLC (Shandon Hyperprep® C18 BDS (Base Deactivated Silica) 8 μm, 250 g, I.D. 5 cm). A gradient with 3 mobile phases was applied. Phase A: a 0.25% NH4HCO3 solution in water; phase B: CH3OH; phase C: CH3CN). The desired fractions were collected and the solvent was evaporated. Yield: 0.115 g of compound 71 (39.5%).
A mixture of intermediate 96 (0.0024 mol) in NaOH (58 ml; 1 M solution) and MeOH (100 ml) was stirred for 2 hours at r.t. The reaction mixture was extracted with DCM. The aq. layer was extracted with more DCM. The organic extracts were washed with a saturated aq. K2CO3 solution, dried, filtered and the filtrate was concentrated to dryness. The residue was purified by column chromatography over silica gel eluting with DCM/MeOH. The product fractions were collected and the solvent was evaporated. The residue was purified by reversed-phase column chromatography. The product fractions were collected and the solvent was evaporated. The residue was recrystallized from CH3CN, filtered off and dried. Yield: 0.099 g of compound 72 (10%).
A solution of intermediate 107 (0.26 g, 0.00052 mol) in DMF (40 ml) was added at r.t. under N2 flow to a stirred mixture of PyBOP (1.3 g, 0.0025 mol) and Et3N (0.34 ml, 0.0025 mol) in DMF (40 ml). The reaction mixture was stirred for 1 hour and H2O (15 ml) was added. The mixture was stirred for 1 hour. Subsequently, the solvent was evaporated and the residue was purified by HPLC (RP-18; eluent: (0.25% NH4HCO3 in H2O)/CH3CN v/v 100/0-0/100). The desired fractions were collected and the solvent was evaporated. Yield: 0.17 g of compound 74.
A solution of intermediate 110 (0.750 g, 0.0016 mol) in DMF (117 ml) was added dropwise at r.t. under N2 flow to a stirred mixture of PyBOP (4.11 g, 0.0079 mol) and Et3N (1.10 ml, 0.0079 mol) in DMF (117 ml). The mixture was stirred for 1 hour and then H2O (15 ml) was added and stirring was continued for 1 hour. Then the mixture was evaporated and the residue was purified by HPLC (Shandon Hyperprep® C18 HS BDS; 8 μm, 50 mm by 16.5 cm). A gradient with 2 mobile phases was applied: (0.25% NH4HCO3 in H2O)/CH3CN from 100/0 to 80/20 in 10 minutes; then 0/100 for 8 minutes and finally 100/0 for 12 minutes. After workup, the powder was dissolved in MeOH and concentrated under reduced pressure. The solid was dried (vacuum, 50° C.). Yield: 0.230 g of compound 75 (31% yield).
A solution of intermediate 114 (crude; max. 0.00226 mol) in DMF (115 ml) was added very slowly (over 1 one hour using a Marlow peristaltic pump) to a solution of HBTU (1.896 g, 0.005 mol) and DIPEA (0.0565 mol) in DMF (55 ml). The reaction mixture was stirred for an additional hour and was then quenched with 4 ml of 7 N NH3 in MeOH. The reaction mixture was concentrated to dryness and the residue was partitioned between DCM and a saturated aq. K2CO3 solution. The aq. layer was extracted with more DCM. The organic extracts were washed with a saturated aq. K2CO3 solution, dried, filtered and the filtrate was concentrated to dryness. The residue was purified by column chromatography (eluent: DCM/MeOH gradient). The product fractions were collected and the solvent was evaporated. The residue was recrystallized from CH3CN, filtered off and dried. Yield: 0.043 g of compound 76 (4% yield).
A solution of intermediate 119 (crude; 0.1 g, max. 0.00018 mol) in DMF (25 ml) was added dropwise at r.t. under N2 flow to a stirred mixture of PyBOP (0.468 g, 0.0009 mol) and Et3N (0.126 ml, 0.0009 mol) in DMF (25 ml). The mixture was stirred for 1 hour and then H2O (20 ml) was added and stirring was continued for 1 hour. The solvent was evaporated and the residue was purified by HPLC (RP-18; eluent: (0.25% NH4HCO3 in H2O)/CH3CN v/v 100/0-0/100). The desired fractions were collected and the solvent was evaporated. Yield: 0.004 g of compound 77.
A solution of intermediate 123 (0.287 g, 0.000496 mol) in DMF (35 ml) was dropwise added at r.t. under N2 flow to a stirred mixture of PyBOP (1.29 g, 0.00248 mol) and Et3N (0.35 ml) in DMF (70 ml). The mixture was stirred for 1 hour and then H2O (4.6 ml) was added and stirring was continued for 1 hour. The solvent was evaporated. The residue was purified by HPLC method B. The desired fractions were collected and the solvent was evaporated. The solid was dried (vacuum, 50° C.). Yield: 0.1828 g of compound 78 (65.7%).
A solution of intermediate 126 (0.1 g, 0.000176 mol) in DMF (13 ml) was dropwise added at r.t. under N2 flow to a stirred mixture of PyBOP (0.458 g, 0.00088 mol) and Et3N (0.13 ml) in DMF (13 ml). The mixture was stirred for 1 hour and then H2O (1.6 ml) was added and stirring was continued for 1 hour. The solvent was evaporated. The residue was purified by HPLC method C. The desired fractions were collected and the solvent was evaporated. The solid was dried (vacuum, 50° C.). Yield: 0.0657 g of compound 79 (67.8%).
A solution of intermediate 130 (0.160 g, 0.000349 mol) in DMF (25 ml) was added dropwise at r.t. under N2 flow to a stirred mixture of PyBOP (0.907 g, 0.001745 mol) and Et3N (0.174 g) in DMF (25 ml). The mixture was stirred for 1 hour and then H2O was added. The solvent was evaporated and the residue was purified by HPLC method B. After workup, the residue was stirred in DIPE. The precipitate was filtered off and dried. Yield: 0.055 g of compound 80 (35.7%).
A mixture of compound 70 (0.1 g, 0.000213 mol) and Cs2CO3 (0.069537 g, 0.000213 mol) in DMF (10 ml) was stirred at r.t. for 15 minutes. CH3I (0.013286 ml, 0.000213 mol) was added and the reaction mixture was stirred for 18 hours. Then the solvent was evaporated. The residue was purified by HPLC (RP-18; eluent: (0.25% NH4HCO3 in H2O)/CH3CN v/v 100/0-0/100). The desired fractions were collected and the solvent was evaporated and co-evaporated with toluene. The residue was dried (vacuum, 50° C.). Yield: 0.042 g of compound 81 (40.7%).
A solution of intermediate 134 (0.030 g, 0.0000642 mol) in DMF (30 ml) was added dropwise at r.t. under N2 flow to a stirred mixture of PyBOP (0.1669 g, 0.000321 mol) and Et3N (0.0447 ml, 0.000321 mol) in DMF (30 ml). The mixture was stirred for 1 hour and then H2O (20 ml) was added. The mixture was stirred for 1 hour and then the solvent was evaporated. The residue was purified by HPLC method C. The product fractions were collected and the solvent was evaporated. The residue was dried (vacuum, 50° C.). Yield: 0.008 g of compound 82 (27.7%).
Reaction under N2 atmosphere. A solution of intermediate 155 (0.00026 mol) in DMF (40 ml) was added dropwise to a mixture of PyBOP (0.000779 mol) and Et3N (0.001298 mol) in DMF (40 ml), stirred at r.t. Stirring was continued for one hour. Then H2O (10 ml) was added and the resultant mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/CH3OH/v/v 100/0/0-70/30/0-0/50/50). The desired fractions were collected and evaporated to dryness. Then, the product was repurified over silica gel (eluent: DCM/DCM-CH3OH 9/1//EtOH v/v 100/0/0-0/100/0-0/0/100). The product fractions were collected and evaporated to dryness. The residual fraction was dried in vacuo at 50° C., yielding 0.025 g (19.9%) of compound 147.
A solution of intermediate 157 (0.0000671 mol) in DMF (15 ml) was added dropwise to a mixture of PyBOP (0.000201 mol) and Et3N (0.00036 mol) in DMF (15 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour. Then H2O (5 ml) was added and the entire mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure. The crude residual fraction was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/v/v 100/0-50/50-0/100). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.021 g (72.9%) of compound 148.
A solution of intermediate 161 (0.000332 mol) in DMF (25 ml) was added dropwise to a mixture of PyBOP (0.001662 mol) and Et3N (0.25 ml) in DMF (25 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour, then 2 ml of H2O was added and the reaction mixture was stirred for one hour. The solvent was evaporated under reduced pressure and the residue was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 100/0 to 70/30 over 60 minutes, then 0/100 for 10 minutes; Shandon 8 μm, Hyperprep C18 BDS 100 Å 50 mm by 16.5 cm).
The product fractions were collected and the solvent was evaporated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.047 g (23.7%) of compound 149.
A solution of intermediate 165 (0.00041 mol) in DMF (30 ml) was added dropwise to a mixture of PyBOP (0.002051 mol) and Et3N (0.3 ml) in DMF (30 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour, then 2 ml of H2O was added and the resultant reaction mixture was stirred for one hour at r.t. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 100/0 to 50/50 over 40 minutes, then 0/100 for 10 minutes and 100/0 for 10 minutes; Shandon 8 μm, Hyperprep C18 BDS 100 Å 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.110 g (46.8%) of compound 150.
A solution of intermediate 169 (0.000281 mol) in DMF (50 ml) was added dropwise to a mixture of PyBOP (0.000982 mol) and Et3N (0.001403 mol) in DMF (50 ml), stirred at r.t. under N2-flow. Stirring was continued for one hour, then 5 ml H2O was added and the entire mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure. The crude residual fraction was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/v/v 100/0-50/50-0/100). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.035 g (25.0%) of compound 151.
A solution of intermediate 173 (0.00017 mol) in DMF (20 ml) was added dropwise to a mixture of PyBOP (0.000851 mol) and Et3N (0.000851 mol) in DMF (20 ml), stirred at r.t. under N2-flow. Stirring was continued for one hour, then 10 ml H2O was added and the entire mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3OH/CH3CN/v/v 75/25/0-0/50/50-0/100). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.005 g (3.74%) of compound 152.
A solution of intermediate 178 (0.0000453 mol) in DMF (10 ml) was added dropwise to a mixture of PyBOP (0.000136 mol) and Et3N (0.000226 mol) in DMF (10 ml), the reaction mixture was stirred at r.t. under N2-flow. Stirring was continued for one hour, then 5 ml of H2O was added and the mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure. The crude residue was purified by high-performance liquid chromatography over RP-18 (eluent:(0.25% NH4HCO3 in H2O)/CH3CN/CH3OH/v/v 100/0/0-70/30/0-0/50/50). The product fractions were collected and evaporated to dryness. The residue was dried in vacuo at 50° C., yielding 0.005 g (26.1%) of compound 153.
A solution of intermediate 182 (0.0000414 mol) in DMF (3 ml) was added dropwise to a mixture of PyBOP (0.000207 mol) and Et3N (0.3 ml) in DMF (3 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour. Then 0.5 ml of H2O was added and the mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.2% ammonium bicarbonate in H2O/CH3CN from 80/20 to 0/100 over 60 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å, 50 mm by 21 cm). The product fractions were collected and concentrated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.0038 g (15.7%) of compound 154.
A solution of intermediate 186 (0.0000708 mol) in DMF (5 ml) was added dropwise to a mixture of PyBOP (0.000354 mol) and Et3N (0.5 ml) in DMF (5 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour. H2O (0.5 ml) was added and the mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 100/0 to 0/100 over 40 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å, 50 mm by 16.5 cm), then (0.25% ammonium bicarbonate in H2O/CH3CN from 80/20 to 30/70 over 50 min, then from 30/70 to 0/100 over 10 min and 0/100 for 8 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å, 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.0141 g (33.5%) of compound 155.
A solution of intermediate 190 (0.000222 mol) in DMF (15 ml) was added dropwise to a mixture of PyBOP (0.00111 mol) and Et3N (1.5 ml) in DMF (15 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour, then 0.5 ml of H2O was added and the entire mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 100/0 to 50/50 over 40 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å, 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.0621 g (48.7%) of compound 156.
A solution of intermediate 194 (0.000793 mol) in DMF (50 ml) was added dropwise to a mixture of PyBOP (0.00397 mol) and Et3N (5 ml) in DMF (50 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour. H2O (0.5 ml) was added. The resultant reaction mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.25% ammonium bicarbonate in H2O/CH3CN from 90/10 to 30/70 over 40 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å, 50 mm by 16.5 cm). The product fractions were collected and concentrated under reduced pressure. The residue was further purified (eluent: DCM/MeOH/ethanol from 100/0/0 to 95/5/0 over 40 min, then 95/5/0 for 10 min; and 0/0/100 for 10 min, Shandon 5 μm, Kromasil silica gel 100 Å 50 mm by 20 cm). The product fractions were collected and concentrated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.088 g (20.25%) of compound 157.
A solution of intermediate 198 (0.000365 mol) in DMF (27 ml) was added dropwise to a mixture of PyBOP (0.001823 mol) and Et3N (2.7 ml) in DMF (27 ml) stirred at r.t. under N2 flow. Stirring was continued for one hour, then 2 ml of H2O was added and the entire mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (0.2% ammonium bicarbonate in H2O/CH3CN from 80/20 to 0/100 over 60 min, then 0/100 for 10 min; Shandon 8 μm, Hyperprep C18 BDS 100 Å, 50 mm by 21 cm). The product fractions were collected and concentrated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.129 g (66%) of compound 158.
A solution of intermediate 202 (0.0000653 mol) in DMF (4 ml) was added dropwise to a mixture of PyBOP (0.000327 mol) and Et3N (0.4 ml) in DMF (4 ml), stirred at r.t. under N2 flow. Stirring was continued for one hour, then 1 ml of H2O was added and the entire mixture was stirred for one hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by HPLC (DCM/CH3OH/EtOH from 100/0/0 to 90/10/0 40 min, then 90/10/0 for 10 min and 0/0/100 for 10 min; Shandon 5 μm, 200 g Kromasil silica gel 100 Å 50 mm by 20 cm). The product fractions were collected and concentrated under reduced pressure. The solid was dried under vacuum at 50° C., yielding 0.0188 g (51.7%) of compound 159.
A solution of intermediate 207 (0.00218 mol) in DMF (109 ml) was added very slowly (over a 60-min period, using a Marlow peristaltic pump) to a solution of PyBOP (0.00479 mol) and DIPEA (0.0545 mol) in DMF (54 ml). After the addition was completed, the reaction mixture was stirred for an additional 1 hour before being quenched by adding 4 ml of 7 N ammonia in MeOH. The reaction mixture was concentrated to dryness and the residue was partitioned between EtOAc and a saturated aqueous sodium bicarbonate solution. The organic extracts were washed with brine, dried and concentrated to dryness. The residue was purified by column chromatography over silica gel (eluent: DCM-CH3OH gradient). The pure fractions were combined and concentrated. The residue was recrystallized from CH3CN, filtered off and dried, yielding 0.250 g (23%) of compound 160.
A solution of intermediate 210 (0.0037 mol) in DMF (190 ml) was added very slowly (over a 60 min period, using a Marlow peristaltic pump), to a solution of PyBOP (0.00814 mol) and DIPEA (0.0925 mol) in DMF (95 ml). After the addition was completed, the reaction mixture was stirred for an additional 1 hour before being quenched by adding 4 ml of 7 N ammonia in MeOH. The reaction mixture was concentrated to dryness. The residue was partitioned between EtOAc and a saturated aqueous sodium bicarbonate solution. The organic extracts were washed with brine, dried, filtered and the filtrate was concentrated to dryness. The residue was purified by column chromatography over silica gel (eluent: DCM-CH3OH gradient). The product fractions were collected and the solvent was evaporated. The residue was recrystallized from CH3CN, filtered off and dried, yielding 0.148 g (8.2%) of compound 161.
The compounds in Table 1 were prepared by analogy to one of the procedures described above, indicated by Ex. No. The exemplified procedures are indicated by a ‘*’. All the compounds are free bases.
Compound Identification
Melting Points
For a number of compounds, melting points (m.p.) were determined with a DSC823e (Mettler-Toledo). Melting points were measured with a temperature gradient of 30° C./minute. Maximum temperature was 400° C.
Values are peak values and are obtained with experimental uncertainties that are commonly associated with this analytical method.
LCMS
For LCMS-characterization of the compounds of the present invention, the following methods were used.
General Procedure A
The HPLC measurement was performed using an Alliance HT 2790 (Waters) system comprising a quaternary pump with degasser, an autosampler, a column oven (set at 40° C. unless otherwise indicated), a diode-array detector (DAD) and a column as specified in the respective methods below. Flow from the column was split to a MS detector. The MS detector was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 1 second using a dwell time of 0.1 second. The capillary needle voltage was 3 kV and the source temperature was maintained at 140° C. (DSC). N2 was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data system.
General Procedure B
The LC measurement was performed using an Acquity UPLC (Waters) (Ultra Performance Liquid Chromatography) system comprising a binary pump, a sample organizer, a column heater (set at 55° C.), a diode-array detector (DAD) and a column as specified in the respective methods below. Flow from the column was split to a MS detector. The MS detector was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 0.18 seconds using a dwell time of 0.02 seconds. The capillary needle voltage was 3.5 kV and the source temperature was maintained at 140° C. (DSC). N2 was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data system.
General Procedure C
The HPLC measurement was performed using an Agilent 1100 series liquid chromatography system comprising a binary pump with degasser, an autosampler, a column oven, a UV detector and a column as specified in the respective methods below. Flow from the column was split to a MS spectrometer. The MS detector was configured with an electrospray ionization source. The capillary voltage was 3 kV, the quadrupole temperature was maintained at 100° C. and the desolvation temperature was 300° C. N2 was used as the nebulizer gas. Data acquisition was performed with an Agilent Chemstation data system.
LCMS Method 1
In addition to general procedure B: Reversed phase UPLC (Ultra Performance Liquid Chromatography) was carried out on a bridged ethylsiloxane/silica (BEH) C18 column (1.7 μm, 2.1×50 mm) with a flow rate of 0.8 ml/min. Two mobile phases (mobile phase A: 0.1% formic acid in H2O/MeOH 95/5; mobile phase B: MeOH) were used to run a gradient condition from 95% A and 5% B to 5% A and 95% B in 1.3 minutes and hold for 0.2 minutes. An injection volume of 0.5 μl was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.
LCMS Method 2
In addition to general procedure A: Column heater was set at 60° C. Reversed phase HPLC was carried out on an Xterra MS C18 column (3.5 μm, 4.6×100 mm) with a flow rate of 1.6 ml/min. Three mobile phases (mobile phase A: 95% 25 mM ammoniumacetate+5% CH3CN; mobile phase B: CH3CN; mobile phase C: MeOH) were employed to run a gradient condition from 100% A to 50% B and 50% C in 6.5 minutes, to 100% B in 0.5 minute and hold these conditions for 1 minute and reequilibrate with 100% A for 1.5 minutes. An injection volume of 10 μl was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.
LCMS Method 3
In addition to general procedure A: Reversed phase HPLC was carried out on an Xterra MS C18 column (3.5 μm, 4.6×100 mm) with a flow rate of 1.6 ml/min. Three mobile phases (mobile phase A: 95% 25 mM ammoniumacetate+5% CH3CN; mobile phase B: CH3CN; mobile phase C: MeOH) were employed to run a gradient condition from 100% A to 1% A, 49% B and 50% C in 6.5 minutes, to 1% A and 99% B in 1 minute and hold these conditions for 1 minute and reequilibrate with 100% A for 1.5 minutes. An injection volume of 10 μl was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.
LCMS Method 4
In addition to general procedure A: Column heater was set at 45° C. Reversed phase HPLC was carried out on an Xterra MS C18 column (3.5 μm, 4.6×100 mm) with a flow rate of 1.6 ml/min. Three mobile phases (mobile phase A: 0.1% formic acid in H2O/MeOH 95/5; mobile phase B: CH3CN; mobile phase C: MeOH) were employed to run a gradient condition from 100% A to 1% A, 49% B and 50% C in 7 minutes and hold these conditions for 1 minute. An injection volume of 10 μl was used. Cone voltage was 10 V for positive ionization mode.
LCMS Method 5
In addition to general procedure A: Column heater was set at 45° C. Reversed phase HPLC was carried out on an Atlantis C18 column (3.5 μm, 4.6×100 mm) with a flow rate of 1.6 ml/min. Two mobile phases (mobile phase A: 70% MeOH+30% H2O; mobile phase B: 0.1% formic acid in H2O/MeOH 95/5) were employed to run a gradient condition from 100% B to 5% B+95% A in 9 minutes and hold these conditions for 3 minutes. An injection volume of 10 μl was used.
LCMS Method 6
In addition to general procedure C: Reversed phase HPLC was carried out on a YMC-Pack ODS-AQ C18 column (4.6×50 mm) with a flow rate of 2.6 ml/min. A gradient run was used from 95% water and 5% CH3CN to 95% CH3CN in 4.80 minutes and was hold for 1.20 minutes. Mass spectra were acquired by scanning from 100 to 1400. Injection volume was 10 μl Column temperature was 35° C.
LCMS Method 7
In addition to general procedure C: Reversed phase HPLC was carried out on a YMC-Pack ODS-AQ C18 column (4.6×50 mm) with a flow rate of 2.6 ml/min. A gradient run was used from 95% H2O and 5% CH3CN to 95% CH3CN in 7.30 minutes and was hold for 1.20 minutes. Mass spectra were acquired by scanning from 100 to 1000. Injection volume was 10 μl Column temperature was 35° C.
LCMS Method 8
In addition to general procedure C: Reversed phase HPLC was carried out on a YMC-Pack ODS-AQ C18 column (4.6×50 mm) with a flow rate of 2.6 ml/min. A gradient run was used from 88% H2O and 12% CH3CN to 88% CH3CN in 3.40 minutes and was hold for 1.20 minutes. Mass spectra were acquired by scanning from 110 to 1000. Injection volume was 10 μl Column temperature was 35° C.
LCMS Method 9
In addition to the general procedure A: Reversed phase HPLC was carried out on an Xterra MS C18 column (3.5 μm, 4.6×100 mm) with a flow rate of 1.6 ml/min. Three mobile phases (mobile phase A: 95% 25 mM ammoniumacetate+5% acetonitrile; mobile phase B: acetonitrile; mobile phase C: methanol) were employed to run a gradient condition from 100% A to 1% A, 49% B and 50% C in 6.5 minutes, to 1% A and 99% B in 1 minute and hold these conditions for 1 minute and reequilibrate with 100% A for 1.5 minutes. An injection volume of 10 μl was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.
LCMS Method 10
In addition to the general procedure A: Column heater was set at 60° C. Reversed phase HPLC was carried out on an Xterra MS C18 column (3.5 μm, 4.6×100 mm) with a flow rate of 1.6 ml/min. Three mobile phases (mobile phase A: 95% 25 mM ammoniumacetate+5% acetonitrile; mobile phase B: acetonitrile; mobile phase C: methanol) were employed to run a gradient condition from 100% A to 50% B and 50% C in 6.5 minutes, to 100% B in 0.5 minute and hold these conditions for 1 minute and reequilibrate with 100% A for 1.5 minutes. An injection volume of 10 μl was used. Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.
C1. Kinase Profiling
The in vitro inhibition of a panel of kinases was assessed using either the scintillation proximity assay (SPA) as described by Cook, N. D. et al., Advances in Experimental Medicine and Biology (1991), 36; p. 525-528.
In the SPA technology the activity of the kinase of interest is measured using an appropriate biotinylated substrate that is incubated with the aforementioned kinase protein in the presence of (33P) radiolabeled ATP. (33P) Phosphorylation of the substrate is subsequently measured through binding of the phosphorylated substrate to streptavidine coated beads that are based on the scintillant poly(vinyl toluene) (PVT-Beads). The scintillation intensity is detected by imaging on Leadseeker.
All kinases are pre-diluted to a 10× working concentration prior to addition into the assay. The composition of the dilution buffer for each kinase is detailed below.
C1.1 PLK-4 Human
In a final reaction volume of 30 μl, PLK4 (h) (19 μg/ml) is incubated with 50 mM Hepes pH 8.0, 10 mM MgCl2, 50 mM NaCl, 1 mM NaF, 1 mM DTT, 10 μM of peptide Biotin-RPRGQRDSSYYWE-OH, 1 μM ATP and 2 nM [γ-33P-ATP] (6.0 μCi/ml). After incubation of 60 minutes at r.t., the reaction is stopped by addition of 40 μL of stop solution containing 8.7 mM EDTA, BSA 0.17%, 0.17% Triton X-100, 1.7 mg/ml SPA beads (GE-healthcare). The plate is centrifuged and read for Scintillation imaging on Leadseeker.
Table 3 provides the pIC50 values of the compounds according to the invention, obtained using the above mentioned kinase assay.
C.2. Cellular Proliferation Assay
Cellular proliferation of these compounds was tested on a panel of different cell lines in the presence of 10% FCS serum (37° C. and 5% (v/v) CO2). In a first step these cells were seeded and incubated for 24 hours in the absence of compound. In the second step the cells were incubated for 72 hours with the compounds to be tested for 72 hours. The viable cell number was finally assessed in a standard Alamar blue cell viability assay.
The viable cell number was assessed by incubation for either 4 h (HCT-116, H1299) 6 h (U87-MG) or 24 h (A2780, PC3, MDA-MB-231) with Alamar blue (Resazurin 9 μg/ml, K-Ferrocyanide 90 μM, K-Ferricyanide 90 μM) and the converted fluorescent product was quantified on a fluorescent plate readed (544 nm/590 nm). Effect of the compounds is calculated as of on control cells.
pIC50 values obtained for compounds tested are presented in Table 4.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07113297 | Jul 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/059833 | 7/25/2008 | WO | 00 | 1/26/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/016132 | 2/5/2009 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4067726 | Sasse et al. | Jan 1978 | A |
| 4160836 | Vandenberk et al. | Jul 1979 | A |
| 4442278 | Giants | Apr 1984 | A |
| 5679683 | Bridges et al. | Oct 1997 | A |
| 5721237 | Myers et al. | Feb 1998 | A |
| 5770599 | Gibson | Jun 1998 | A |
| 5821240 | Himmelsbach et al. | Oct 1998 | A |
| 6002008 | Wissner et al. | Dec 1999 | A |
| 6265410 | Bridges et al. | Jul 2001 | B1 |
| 6288082 | Wissner et al. | Sep 2001 | B1 |
| 6344459 | Bridges et al. | Feb 2002 | B1 |
| 6414148 | Thomas et al. | Jul 2002 | B1 |
| 6521620 | Bridges et al. | Feb 2003 | B1 |
| 6602863 | Bridges et al. | Aug 2003 | B1 |
| 6794395 | Roth et al. | Sep 2004 | B1 |
| 7067507 | Pulley et al. | Jun 2006 | B2 |
| 7312225 | Luecking et al. | Dec 2007 | B2 |
| 7648975 | Freyne et al. | Jan 2010 | B2 |
| 7655642 | Freyne et al. | Feb 2010 | B2 |
| 7799772 | Freyne et al. | Sep 2010 | B2 |
| RE42353 | Thomas et al. | May 2011 | E |
| 20020173646 | Thomas et al. | Nov 2002 | A1 |
| 20030087908 | Geuns-Meyer et al. | May 2003 | A1 |
| 20030186987 | Bridges et al. | Oct 2003 | A1 |
| 20040048880 | Himmelsbach et al. | Mar 2004 | A1 |
| 20040116388 | Armistead et al. | Jun 2004 | A1 |
| 20070078132 | Freyne et al. | Apr 2007 | A1 |
| 20080219975 | Perera et al. | Sep 2008 | A1 |
| 20090075999 | Blanchard et al. | Mar 2009 | A1 |
| 20100029627 | Papanikos et al. | Feb 2010 | A1 |
| 20100069424 | Freyne et al. | Mar 2010 | A1 |
| 20100105668 | Freyne et al. | Apr 2010 | A1 |
| 20100152174 | Freyne et al. | Jun 2010 | A1 |
| 20100160310 | Freyne et al. | Jun 2010 | A1 |
| 20100173913 | Freyne | Jul 2010 | A1 |
| 20100190786 | Diels et al. | Jul 2010 | A1 |
| 20100204197 | Diels et al. | Aug 2010 | A1 |
| 20100222574 | Rombouts et al. | Sep 2010 | A1 |
| 20110009404 | Buijnsters et al. | Jan 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 807899 | Jan 1959 | GB |
| 1465451 | Feb 1977 | GB |
| 1542514 | Mar 1979 | GB |
| WO-9519774 | Jul 1995 | WO |
| WO-9607657 | Mar 1996 | WO |
| WO-9609294 | Mar 1996 | WO |
| WO-9633980 | Oct 1996 | WO |
| WO-9639145 | Dec 1996 | WO |
| WO-9732880 | Sep 1997 | WO |
| WO-9738983 | Oct 1997 | WO |
| WO-9813354 | Apr 1998 | WO |
| WO-9843960 | Oct 1998 | WO |
| WO-9933792 | Jul 1999 | WO |
| WO-9933793 | Jul 1999 | WO |
| WO-9933795 | Jul 1999 | WO |
| WO-9933815 | Jul 1999 | WO |
| WO-0018761 | Apr 2000 | WO |
| WO-0055159 | Sep 2000 | WO |
| WO-0116130 | Mar 2001 | WO |
| WO-0220479 | Mar 2002 | WO |
| WO-02083654 | Oct 2002 | WO |
| WO 03072062 | Sep 2003 | WO |
| WO-03072062 | Sep 2003 | WO |
| WO-03082290 | Oct 2003 | WO |
| WO-2004009562 | Jan 2004 | WO |
| WO-2004014899 | Feb 2004 | WO |
| WO 2004014899 | Feb 2004 | WO |
| WO-2004026829 | Apr 2004 | WO |
| WO-2004026881 | Apr 2004 | WO |
| WO-2004037814 | May 2004 | WO |
| WO-2004043936 | May 2004 | WO |
| WO 2004043936 | Jun 2004 | WO |
| WO-2004074224 | Sep 2004 | WO |
| WO 2004074224 | Sep 2004 | WO |
| WO-2004078682 | Sep 2004 | WO |
| WO-2004105765 | Dec 2004 | WO |
| WO 2004105765 | Dec 2004 | WO |
| WO 2005058318 | Jun 2005 | WO |
| WO-2005058318 | Jun 2005 | WO |
| WO-2005058913 | Jun 2005 | WO |
| WO 2005058913 | Jun 2005 | WO |
| WO 2006061415 | Jun 2006 | WO |
| WO-2006061415 | Jun 2006 | WO |
| WO-2006061417 | Jun 2006 | WO |
| WO 2006061417 | Jun 2006 | WO |
| WO-2007003525 | Jan 2007 | WO |
| WO 2007003525 | Jan 2007 | WO |
| WO-2007058267 | May 2007 | WO |
| WO-2007058627 | May 2007 | WO |
| WO-2007058628 | May 2007 | WO |
| WO-2008006884 | Jan 2008 | WO |
| WO-2008049902 | May 2008 | WO |
| WO-2008155421 | Dec 2008 | WO |
| WO-2009016132 | Feb 2009 | WO |
| WO-2009112439 | Sep 2009 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20100204197 A1 | Aug 2010 | US |
