WO2014072985A1 - Novel boronic acid derivatives as anti cancer agents - Google Patents

Novel boronic acid derivatives as anti cancer agents Download PDF

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Publication number
WO2014072985A1
WO2014072985A1 PCT/IN2012/000729 IN2012000729W WO2014072985A1 WO 2014072985 A1 WO2014072985 A1 WO 2014072985A1 IN 2012000729 W IN2012000729 W IN 2012000729W WO 2014072985 A1 WO2014072985 A1 WO 2014072985A1
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boronic acid
carbonyl
alkyl
alkoxy
phenylalanine
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PCT/IN2012/000729
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French (fr)
Inventor
Kali Satya Bhujanga Rao Adibhatla
Pulla Reddy Muddasani
Janaki Rama Rao Ravi
Swapna KONDAVEETI
Satyanarayana Kota
Suneel Kumar BATTULA
Venkaiah Chowdary Nannapaneni
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Natco Pharma Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the novel boronic acid derivatives and biological evaluation of them as anti cancer agents.
  • N-terminal peptidyl boronic acid and ester compounds has been described previously (Shenvi et al. U.S. Pat. No. 4,499,082; Shenvi et al. U.S. Pat. No. 4,537,773; Siman et al. WO 91/13904; Kettner et al. J. Biol. Chem. 259 (24), 15106- 15114 (1984)). These compounds have been shown to be inhibitors of certain proteolytic * enzymes.
  • a class of N-terminal tri-peptide boronic ester and acid compounds have been shown to inhibit the growth of cancer cells (Kinder et al. U.S. Pat. No. 5,106,948).
  • a broad class of N-terminal tri-peptide boronic ester and acid compounds and analogues thereof has been shown to inhibit rennin (Kleeeman et. al. U.S. Pat. No. 5,169,841).
  • proteasome component system In the cell, there is a soluble proteolytic pathway that requires ATP and involves covalent conjugation of the cellular proteins with small polypeptide ubiquitin (Ub). Thereafter, the conjugated proteins are hydrolyzed by a 26S proteolytic complex containing a 20S degradative particle called the proteasome component system is known to catalyze the selective degradation of highly abnormal proteins and short-lived regulatory proteins (Goldberg. Eur. J Biochem. 203:9-23 (1992); Goldberg et al.., Nature 357: 375-379 (1992).
  • proteasome inhibitors including apoptosis seem to be mediated through inhibition of NF-kB. They inhibit NF-kB activity in cells by blocking the degradation of 1-kB.
  • Inhibition of NF-kB transcriptional activity plays a beneficial role in cancer by down regulating the expression of various growth, survival and angiogenic factors. It leads to decreased levels of the proapoptotic proteins Bcl-2 AND al/Bfl-1, triggering cytochrome C release, caspase-9 activation, and apoptosis.
  • Dipeptidyl boronic acids are new potent proteasome inhibitors. Boronic acids act as transition state analogues for serine protease because the boron can accept the oxygen lone pair of the active site serine residue. It seems likely that these compounds react similarly with catalytic N-terminal threonine residue of the proteasome catalytic subunits forming tetrahedral adduct. In addition, the boron-threonine bond is much more stable than the carbon-threonine bond found in the hemiacetal formed between peptide aldehydes and the proteasome.
  • Bortezomib (1) (originally code named PS-341 ; Julian Adams et al. ' US Pat No. 5,780,454) is the first therapeutic proteasome inhibitor to be tested in humans. 1 It is approved by FDA for the treatment of multiple myeloma (MM). Bortezomib is marketed as Velcade® by Millennium Pharmaceuticals.
  • the boron atom in the bortezomib binds the catalytic site of the 26S proteasome with high affinity and specificity.
  • R3 is -OBu', benzyloxy, phenyl, dimethylamino phenyl, 3-pyridyl, 2-thiazolyl, 3- thiazolyl, 2-indolyl, and 2-quinolinyl.
  • HDACs histone acetyl transferases and the histone deacetylases
  • the latter family can be divided into two categories; those are Zn 2+ dependent enzymes and NAD + dependent enzymes.
  • Zn 2+ dependent HDACs are closely connected with control of gene expression and cell cycle progression.
  • the inhibition of HDACs causes histone hyperacetylation and leads to transcriptional activation genes such as p2l WAF CIPI 5 Gadd 45, FAS, and caspase-3, which are associated with growth arrest and apoptosis in tumor cells.
  • P 1 is methyl, ter/-butoxy, etc.
  • P 2 is C 1-3 alkyl, phenyl, etc.
  • P 3 is propyl, isopropyl, etc.
  • P 4 is hydrogen and (S)-pinanediol
  • the above compounds have been designed as bortezomib analogues by introduction of various moieties in place of pyrazine, phenyl alanine, and isobutyl at P , P and P respectively.
  • the above analogues have been screened for inhibition of Human 20S proteasome.
  • the analogues obtained from the replacing of P 1 group with 1,2,3,4- tetrahydronaphthyl and 5,6,7,8-tetrahydronaphthyl moieties shown significant activity in which P 2 and P 3 are phenyl and isobutyl respectively and P 4 is hydrogen.
  • R 1 is a pyrazin-2-yl, phenyl, etc.
  • R 2 is a hydrogen, methyl, etc.
  • R is isopropyl, phenyl, etc.
  • the above analogues were synthesized and tested for anticancer activity on ten cancer cell lines including two human hematological cell lines and eight solid tumor cell lines.
  • bortezomib is a highly potent proteasome inhibitor
  • treatment with bortezomib resulted in some severe side effects such as neurologic and cardiovascular adverse effects, fatigue, nausea and vomiting and diarrhea. Therefore, there remains an unmet need to develop new proteasome inhibitors with less toxicity and greater therapeutic index. From the findings based on pharmacological researches and clinical results obtained so far there is still requirement for novel analogues of boronic acid derivatives for better inhibitory action on proteasome.
  • the object of the present invention is to provide a pharmaceutical agent having an anticancer activity.
  • There is another object of the present invention is to design, synthesize novel dipeptidyl boronates and boronic acids.
  • novel dipeptidyl boronates and boronic acids are designated b the general formula (5).
  • X is aromatic or heteroaromatic ring
  • aromatic ring is selected from C 6 -Ci 0 , and is substituted with different functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
  • each alkyl and alkoxy is independently selected from Ci-C 6;
  • trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
  • heteroaromatic ring is selected from 5- and six-membered heterocyclic compounds and is substituted with functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
  • each alkyl and alkoxy is independently selected from Ci-C 6;
  • trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl;
  • Y is an alkyl, hydroxy alkyl, alkoxy alkyl, and thioalkoxy alkyl or aryl or a heteroaryl component and substituents thereof
  • alkyl group is selected from Ci-C 6; aryl moiety is selected from C 6 -Ci 0 ; and is substituted with different functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
  • each alkyl and alkoxy is independently selected from Ci-C ;
  • trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
  • heteroaryl component optionally selected from 5- and six-membered heterocyclic compounds and is substituted with functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
  • each alkyl and alkoxy is independently selected from C
  • trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
  • X is phenyl, 2-methyl-5-nitrophenyl, 4-nitrophenyl, 3,4-dimethylphenyl, 3-nitrophenyl, naphthyl, tetrahydronaphthyl, pyrazinyl, 2-thienyl, 2-tetrahydrofuryl and Isonicotinyl
  • Y is L- alanyl, L-penylalanyl, L-tyrosinyl, L-leucinyl, L-methionyl and D-valinyl
  • the invention most particularly relates to novel boronic acid compounds of the general formula (5) are given below.
  • Bortezomib is meant for the treatment of multiple myeloma and mantle cell lymphoma.
  • In vitro studies of bortezomib and dipeptidyl boronates and boronic acids of the present invention have been carried out on different cancer cell lines like prostate cancer, lung cancer, head and neck cancer and breast cancer. Results of in vitro study were compared with standard compounds of those cell lines. Surprisingly, bortezomib and its analogues of the present invention showed better activity than the standard compounds.
  • MTT proliferation assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay first described by Mosmann in 1983, is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. The number of surviving cells is directly proportional to the level of the formazan product created. The colour then is quantified using simple colorimetric assay.
  • formulation of boronic acid according to the methods of invention results in formation of a chemical derivative of the boronic acid compound preferably formation of a boronate ester compound.
  • the composition according to the present invention comprises a boronic acid compound and a compound having at least two hydroxyl groups in lyophilized powder.
  • a compound having at least two hydroxyl group derived from sugar.
  • the moiety derived from a compound having at least two hydroxyl groups may be attached to boron by the oxygen atoms of any two of its hydroxyl groups.
  • the dihydroxy compound is sugar preferably a monosaccharide or disaccharide, more preferably a reduced sugar and most preferably sorbitol or mannitol.
  • composition according to the present invention is in the form of a lyophilized powder.
  • method of formulating a boronic acid compound comprising preparing a mixture of boronic acid, dihydroxy compound, water and water miscible solvent and then lyophilizing the mixture.
  • the water miscible solvent is selected from alcohol and more preferably ethanol or tert-butanol.
  • composition according to the present invention can be readily reconstituted by adding an aqueous solvent.
  • the reconstitution solvent is suitable for pharmaceutical administration.
  • reconstitution solvents include, without limitation, water, saline, and phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the compositions according to the present invention are preferably reconstituted with sterile saline (0.9%w/v). Upon reconstitution in aqueous medium, equilibrium is established between boronate ester present in the composition and the boronic acid.
  • the compounds are administered by injection, more preferably intravenous injection, but also by subcutaneous or intraperitoneal injection, and the like.
  • the compounds may be formulated into tablets, capsules, syrups, powders, or other suitable forms for administration orally.
  • a solution of (+) pinanediol(l S)-l -chloro-3-methyIbutane- 1-boronate (12,70g, 0.25 moles) in cyclohexane (150 mL) was added to the above reaction mass drop wise under nitrogen atmosphere during 60 min. The resulting yellow coloured solution was stirred for 65 min at -25 to -20°C.
  • Trifluoroacetic acid (77g, 0.67 moles) and diisopropyl ether (500 mL) were charged into 2 L three necked round bottomed flask reaction mass was cooled to -10°C (ice-salt bath). The solution of (li?)-(S)-pinanediol-l-bis(trimethylsilyl)amino-3-methylbutane-l- boronate (13) obtained above was added drop wise to the reaction mixture. After completion of addition the reaction mass was stirred for 6-8 h at below 0°C.
  • N,N-diisopropylethylamine (52.45g, 0.41 moles) was added drop wise to the above reaction mass, during 20-30 min. The reaction mass was then stirred for 35 min at -2 to 0°C for 35 min. TLC was Checked for reaction completion, using mobile phase: Toluene: ethyl acetate (1 : 1), detection: UV at 254 nm; and 0.75% aq. potassium permanganate solution.
  • the solvent was distilled off at 40-45°C under reduced pressure to obtain an oily product.
  • the resulting crude product was dissolved in ethyl acetate washed with water (2x300 mL), 1% aq. H 3 P0 4 solution (390 mL), 2%aq. K 2 C0 3 solution (390 mL) followed by 10% aq. NaCl solution (350 mL).
  • Cell lines DU-145, A-549, NCI-H292, Cal-27, MDA-MB-231 and PC3 Receiveived from ATCC
  • Cell Titer 96 Non-Radioactive Cell Proliferation MTT Assay kit Promega
  • DMSO cell biology grade
  • Trypan blue Cell growth medium as prescribed by ATCC.

Abstract

The invention relates to synthesis and anticancer activity of novel boronic acid derivatives of formula 5 or pharmaceutical acceptable salts and esters thereof. Anti cancer activity of the compounds is evaluated by in vitro study on cancer cell lines like prostate cancer, lung cancer, head and neck cancer or breast cancer.

Description

NOVEL BORONIC ACID DERIVATIVES AS ANTI CANCER AGENTS
FIELD OF THE INVENTION
The present invention relates to the novel boronic acid derivatives and biological evaluation of them as anti cancer agents.
BACK GROUND OF THE INVENTION
The synthesis of N-terminal peptidyl boronic acid and ester compounds has been described previously (Shenvi et al. U.S. Pat. No. 4,499,082; Shenvi et al. U.S. Pat. No. 4,537,773; Siman et al. WO 91/13904; Kettner et al. J. Biol. Chem. 259 (24), 15106- 15114 (1984)). These compounds have been shown to be inhibitors of certain proteolytic * enzymes. A class of N-terminal tri-peptide boronic ester and acid compounds have been shown to inhibit the growth of cancer cells (Kinder et al. U.S. Pat. No. 5,106,948). A broad class of N-terminal tri-peptide boronic ester and acid compounds and analogues thereof has been shown to inhibit rennin (Kleeeman et. al. U.S. Pat. No. 5,169,841).
In the cell, there is a soluble proteolytic pathway that requires ATP and involves covalent conjugation of the cellular proteins with small polypeptide ubiquitin (Ub). Thereafter, the conjugated proteins are hydrolyzed by a 26S proteolytic complex containing a 20S degradative particle called the proteasome component system is known to catalyze the selective degradation of highly abnormal proteins and short-lived regulatory proteins (Goldberg. Eur. J Biochem. 203:9-23 (1992); Goldberg et al.., Nature 357: 375-379 (1992). Several effects of proteasome inhibitors, including apoptosis seem to be mediated through inhibition of NF-kB. They inhibit NF-kB activity in cells by blocking the degradation of 1-kB. Inhibition of NF-kB transcriptional activity plays a beneficial role in cancer by down regulating the expression of various growth, survival and angiogenic factors. It leads to decreased levels of the proapoptotic proteins Bcl-2 AND al/Bfl-1, triggering cytochrome C release, caspase-9 activation, and apoptosis.
Dipeptidyl boronic acids are new potent proteasome inhibitors. Boronic acids act as transition state analogues for serine protease because the boron can accept the oxygen lone pair of the active site serine residue. It seems likely that these compounds react similarly with catalytic N-terminal threonine residue of the proteasome catalytic subunits forming tetrahedral adduct. In addition, the boron-threonine bond is much more stable than the carbon-threonine bond found in the hemiacetal formed between peptide aldehydes and the proteasome.
Bortezomib (1) (originally code named PS-341 ; Julian Adams et al. ' US Pat No. 5,780,454) is the first therapeutic proteasome inhibitor to be tested in humans.1 It is approved by FDA for the treatment of multiple myeloma (MM). Bortezomib is marketed as Velcade® by Millennium Pharmaceuticals.
Figure imgf000003_0001
The boron atom in the bortezomib binds the catalytic site of the 26S proteasome with high affinity and specificity.
Nobuaki Suzuki et al. J. Med. Chem. 2009, 52, 2909-2922 described the design, synthesis and biological activity of the following boronic acid derivatives (2a) and (2b) as histone deacetylase inhibitors.
Figure imgf000003_0002
Wherein Rl is
Figure imgf000004_0001
R2 is
Figure imgf000004_0002
R3 is -OBu', benzyloxy, phenyl, dimethylamino phenyl, 3-pyridyl, 2-thiazolyl, 3- thiazolyl, 2-indolyl, and 2-quinolinyl.
The acetylation status of lysine residues in nucleosomal histones is tightly controlled by two counteracting enzyme families, the histone acetyl transferases and the histone deacetylases (HDACs). The latter family can be divided into two categories; those are Zn2+ dependent enzymes and NAD+ dependent enzymes. The Zn2+ dependent HDACs are closely connected with control of gene expression and cell cycle progression. The inhibition of HDACs causes histone hyperacetylation and leads to transcriptional activation genes such as p2lWAF CIPI 5 Gadd 45, FAS, and caspase-3, which are associated with growth arrest and apoptosis in tumor cells.
Zhu, Y.Q. et al. described the 3D-QSAR studies on tripeptide aldehyde derivatives as proteasome inhibitors (Bioorg. Med. Chem. 2006, 14, 1483-1496). Design, synthesis and biological evaluation of tripeptide boronic acids as proteasome inhibitors also has been discussed in the literature (Yongqiang Zhu et. al Bioorg. Med. Chem. letters 2009, 14, 6851-6861). Furthermore, Yongqiang Zhu et. al J. Med. Chem. 2009, 52, 4192-4199 describes the design, synthesis, biological evaluation and structure activity relationship (SAR) of the following dipeptidyl boronates and boronic acids (3) as proteasome inhibitors. This study is particularly focused on structure activity relationship of a-amino acid boronates.
Figure imgf000005_0001
Wherein P1 is methyl, ter/-butoxy, etc.
P2 is C 1-3 alkyl, phenyl, etc.
P3 is propyl, isopropyl, etc.
P4 is hydrogen and (S)-pinanediol
The above compounds have been designed as bortezomib analogues by introduction of various moieties in place of pyrazine, phenyl alanine, and isobutyl at P , P and P respectively. Initially the above analogues have been screened for inhibition of Human 20S proteasome. The analogues obtained from the replacing of P1 group with 1,2,3,4- tetrahydronaphthyl and 5,6,7,8-tetrahydronaphthyl moieties shown significant activity in which P2 and P3 are phenyl and isobutyl respectively and P4 is hydrogen.
Also Yongqiang Zhu et. al J. Med. Chem. 2010, 53, 1990-1999 describes the synthesis, in vitro, in vivo biological evaluation, docking studies and structure activity relationship (SAR) of dipeptidyl boronic acids (4) composed of β-amino acids.
Figure imgf000006_0001
4
Wherein R1 is a pyrazin-2-yl, phenyl, etc.
R2 is a hydrogen, methyl, etc.
R is isopropyl, phenyl, etc.
The above analogues were synthesized and tested for anticancer activity on ten cancer cell lines including two human hematological cell lines and eight solid tumor cell lines.
SUMMARY OF THE INVENTION
Nowadays more structurally diverse compounds are being designed and synthesized to clearly and extensively elucidate the SAR so that much better candidates are obtained and developed.
Although bortezomib is a highly potent proteasome inhibitor, treatment with bortezomib resulted in some severe side effects such as neurologic and cardiovascular adverse effects, fatigue, nausea and vomiting and diarrhea. Therefore, there remains an unmet need to develop new proteasome inhibitors with less toxicity and greater therapeutic index. From the findings based on pharmacological researches and clinical results obtained so far there is still requirement for novel analogues of boronic acid derivatives for better inhibitory action on proteasome.
The object of the present invention is to provide a pharmaceutical agent having an anticancer activity. There is another object of the present invention is to design, synthesize novel dipeptidyl boronates and boronic acids.
According to the object of the present invention the novel dipeptidyl boronates and boronic acids are designated b the general formula (5).
Figure imgf000007_0001
5
Wherein,
X is aromatic or heteroaromatic ring;
Where aromatic ring is selected from C6-Ci0, and is substituted with different functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from Ci-C6;
trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
heteroaromatic ring is selected from 5- and six-membered heterocyclic compounds and is substituted with functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from Ci-C6;
trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl;
Y is an alkyl, hydroxy alkyl, alkoxy alkyl, and thioalkoxy alkyl or aryl or a heteroaryl component and substituents thereof
alkyl group is selected from Ci-C6; aryl moiety is selected from C6-Ci0; and is substituted with different functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from Ci-C ;
trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
heteroaryl component optionally selected from 5- and six-membered heterocyclic compounds and is substituted with functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from C|-C6>
trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
The compound of general formula (5) wherein:
X is phenyl, 2-methyl-5-nitrophenyl, 4-nitrophenyl, 3,4-dimethylphenyl, 3-nitrophenyl, naphthyl, tetrahydronaphthyl, pyrazinyl, 2-thienyl, 2-tetrahydrofuryl and Isonicotinyl
The compound of general formula (5) wherein:
Y is L- alanyl, L-penylalanyl, L-tyrosinyl, L-leucinyl, L-methionyl and D-valinyl
The invention most particularly relates to novel boronic acid compounds of the general formula (5) are given below.
N-(Phenyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5a)
N-(Tetrahydronaphthyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5b)
N-(2-Thiophene)carbonyl-L-phenylalanine-L-leucine boronic acid (5c)
N-(2-Tetrahydrofuran)carbonyl-L-phenylalanine-L-leucine boronic acid (5d)
N-(4-Nicotincarbonyl-L-phenylalanine-L-leucine boronic acid (5e)
N-(2-Methyl-5-nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid(5f) N-(4-Nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5g)
N-(2-Naphtyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5h)
N-(3,4-Dimethylphenyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5i)
N-(3-Nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5j)
N-(2-Pyrazine)carbonyl-L-alanine-L-leucine boronic acid (5k)
N-(2-Pyrazine)carbonyl)-L-tyrosine-L-leucine boronic acid (51)
N-(2-Pyrazine) carbonyl-L-leucine-L-leucine boronic acid (5m)
N-(2-Pyrazine) carbonyl-L-methionine-L-leucine boronic acid (5n)
N-(2-Pyrazine) carbonyl-D-valine-L-leucine boronic acid (5o)
IN VITRO STUDIES
Bortezomib is meant for the treatment of multiple myeloma and mantle cell lymphoma. In vitro studies of bortezomib and dipeptidyl boronates and boronic acids of the present invention have been carried out on different cancer cell lines like prostate cancer, lung cancer, head and neck cancer and breast cancer. Results of in vitro study were compared with standard compounds of those cell lines. Surprisingly, bortezomib and its analogues of the present invention showed better activity than the standard compounds.
MTT proliferation assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay first described by Mosmann in 1983, is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. The number of surviving cells is directly proportional to the level of the formazan product created. The colour then is quantified using simple colorimetric assay. Cell lines, DU-145 (prostate cancer), A-549 (lung cancer), NCI-H292 (lung cancer), Cal- 27 (Head and neck cancer), MDA-MB-231 (breast cancer) and PC3 (prostate cancer) received from ATCC and the protocols and methods are as per manufacturers instruction. Novel dipeptidyl boronates and boronic acids of the present invention have been
Figure imgf000010_0001
Scheme-1
The key intermediate (li?)-(15,2S',3 ?,5S)-pinanediol leucine boronate trifluoroacetate salt (6) has been prepared by the following synthetic scheme (I. Fraser Pickersgill et. al US 20100174072A1
Figure imgf000010_0002
Scheme-2 PHARMACEUTICAL COMPOSITION
The synthesized compounds were formulated into pharmaceutical dosage form for administration to animal subject using commonly understood formulation techniques well known in the art. In some preferred embodiments, formulation of boronic acid according to the methods of invention results in formation of a chemical derivative of the boronic acid compound preferably formation of a boronate ester compound.
In these embodiments, the composition according to the present invention comprises a boronic acid compound and a compound having at least two hydroxyl groups in lyophilized powder. The term "a compound having at least two hydroxyl group" derived from sugar. The moiety derived from a compound having at least two hydroxyl groups may be attached to boron by the oxygen atoms of any two of its hydroxyl groups.
Another aspect of the present invention, the dihydroxy compound is sugar preferably a monosaccharide or disaccharide, more preferably a reduced sugar and most preferably sorbitol or mannitol.
The composition according to the present invention is in the form of a lyophilized powder. And the method of formulating a boronic acid compound comprising preparing a mixture of boronic acid, dihydroxy compound, water and water miscible solvent and then lyophilizing the mixture. The water miscible solvent is selected from alcohol and more preferably ethanol or tert-butanol.
The composition according to the present invention can be readily reconstituted by adding an aqueous solvent. Preferably, the reconstitution solvent is suitable for pharmaceutical administration. Examples of reconstitution solvents include, without limitation, water, saline, and phosphate buffered saline (PBS). For clinical use, the compositions according to the present invention are preferably reconstituted with sterile saline (0.9%w/v). Upon reconstitution in aqueous medium, equilibrium is established between boronate ester present in the composition and the boronic acid.
Preferably the compounds are administered by injection, more preferably intravenous injection, but also by subcutaneous or intraperitoneal injection, and the like.
Besides, injection, other routes of administration may also be used. The compounds may be formulated into tablets, capsules, syrups, powders, or other suitable forms for administration orally.
The invention will be fully described in conjunction with the following specific examples, which are not to be construed as limiting the scope of invention.
Example 1
Preparation of (lR)-(lS,2S,3R,5S)-pinanediol leucine boronate trifluoroacetate salt (6)
Figure imgf000012_0001
(+) Pinanedio l-2-methy I propane- 1 -boronate (11, 60 g, 0.25 moles), /ert-butyl methyl ether (300 mL), and methylene dichloride (1 12.5 g (84.9 mL), 1.32 moles) were charged into 2 L four necked round bottomed flask. The reaction mass was cooled to -65 to -60° C using liquid nitrogen-acetone bath. LDA (lithium diisopropylamide) solution prepared from diisopropyl- amine (33.3 g, 0.33 moles), rc-BuLi 1.6 M solution in hexane (196.8 mL, 0.32 moles) in tetrahydrofuran was added drop wise to the above reaction mass during 50 min under nitrogen atmosphere. The resulting pale yellow coloured cloudy solution was stirred for 20 min at -60 to -55° C. Then ZnCl2 solution (64 g, 0.47 moles) in tetrahydrofuran was added drop wise during 50 min. under nitrogen atmosphere. The reaction mass was allowed to stir for 35 min at -50 to -45°C.
The reaction mass was warmed to -10°C and 10% sulphuric acid (prepared from 36 g of cone. Sulphuric acid and 324 mL of water) was added below 0°C during 40 min. Layers were separated and the aq. layer was extracted with tert- butyl methyl ether (120 mL), and combined organic layer was washed with water followed by brine solution. The organic layer was separated, dried over sodium sulphate and solvent was evaporated under reduced pressure at 45°C, to yield (+) pinanediol(l.ST)-l-chloro-3-methylbutane -1 - boronate (12) as yellow coloured oil. [70g, 96.7%, purity: 87% (GC).]
The above compound was taken to next step without further purification.
Lithium bis(trimethylsilyl)amide (20% in tetrahydrofuran), 226 mL (46g, 0.27 moles) was charged into 1L four necked round bottomed flask and cooled to -25 to -20°C using liquid nitrogen-acetone bath. A solution of (+) pinanediol(l S)-l -chloro-3-methyIbutane- 1-boronate (12,70g, 0.25 moles) in cyclohexane (150 mL) was added to the above reaction mass drop wise under nitrogen atmosphere during 60 min. The resulting yellow coloured solution was stirred for 65 min at -25 to -20°C. Solvent was distilled off at 45°C under reduced pressure to attain a cream to yellow coloured suspension. The resulting suspension was triturated with cyclohexane (120 mL) and filtered, and the residue was washed with diisopropyl ether (150mL). The filtrate obtained contains ( 1 i?)-(iS)-pinanediol- 1 -bis(trimethylsilyl)amino-3 -methylbutane- 1 - boronate (13), which was taken to next step without product isolation. Trifluoroacetic acid (77g, 0.67 moles) and diisopropyl ether (500 mL) were charged into 2 L three necked round bottomed flask reaction mass was cooled to -10°C (ice-salt bath). The solution of (li?)-(S)-pinanediol-l-bis(trimethylsilyl)amino-3-methylbutane-l- boronate (13) obtained above was added drop wise to the reaction mixture. After completion of addition the reaction mass was stirred for 6-8 h at below 0°C. The product was filtered from the reaction mass and washed with diisopropyl ether to afford the required product (l/?)-(lS,2S,3i?,55)-pinanediol leucine boronate trifluoroacetate salt (6) as off white coloured solid. (50g, % yield: 57.6). (a)D 25 + 9.8° (c=l in methanol); mp: 125-140 °C; GC: 98.8%; Ή NMR (400 MHz CDC13): 7.79 (s, 1H), 4.33 (dd, 1H), 2.91 (t, 1H), 2.25 (m, 2H), 2.03 (t, 1H), 1.86-1.9 (m, 3H), 1.77 (m, 2H), 1.6 (m, 2H), 1.38 (s, 3H), 1.27 (s, 3H), 1.05 (m, 1H), 0.92 (dd, 6H), 0.8 (s, 3H); MS (ESI), m/z = 266.28 (M+l - CF3COOH).
7 EXAMPLE 2
Preparation of N-tert-butoxycarbonyl-L-phenylalanine (7a)
Figure imgf000014_0001
Di-tert-butyldicarbonate (26.96g) in acetone (90.4mL) was added to a solution of L- phenylalanine (20g) in aqueous IN NaOH (128 mL) by maintaining pH 7.5-8.0. After stirring at room temperature for 18h, reaction mass was diluted with DM water (100 mL) and washed with of hexane (60 mL). Aqueous layer was cooled to 10°C then pH was adjusted to 2-2.5. The resulting suspension was extracted with ethyl acetate (240 mLx2). Combined organic phases were dried over sodium sulphate and solvent was evaporated under reduced pressure to give of Boc-L-phenylalanine as pale yellow colored viscous oil. The above oil was crystallized from hexane (150 mL) at 5-10°C for lh and the resulting white colured solid was filtered and dried under reduced pressure at RT for 3h to afford Boc-L-Phenylalanine, yield: 22.85g (71.4%). Ή NMR values (400 MHz, CDC13) δ 7.21 (5H, m), 7.013 (1H, d) ,4.13 (1H, m), 3.0(lH,m),2.841(lH,d),1.3(9H,s); MS (ESI): m/z 264.3 (M-l)
Similarly the following Boc-protecting amino acids were prepared.
N-tert-butoxycarbonyl-L-alanine (7b)
N-tert-butoxycarbonyl-L-tyrosine (7c)
N-tert-butoxycarbonyl-L-leucine (7d)
N-tert-butoxycarbonyl-L-methionine (7e)
N-tert-butoxycarbonyl-D-valine (7f)
EXAMPLE 3
Preparation of (lSjlS^jS^-pinanediol-N-Boc-yff-il-pheny -L-alanine-L- leucineboronate 8a)
Figure imgf000015_0001
(li?)-(lS,2S,3i?,5S)-Pinanediol leucine boronate trifluoroacetate salt (50g, 0.13 moles), BOC-L-phenylalanine (7a, 35g, 0.13 moles), TBTU (48.16g, 0.150 moles) and methylene chloride (760 mL) were charged into in 3 L 4 necked round bottomed flask at room temperature under nitrogen atmosphere and stirred for 5 min. Resulting off white coloured suspension was cooled to -2°C using ice-salt bath. N,N-diisopropylethylamine (52.45g, 0.41 moles) was added drop wise to the above reaction mass, during 20-30 min. The reaction mass was then stirred for 35 min at -2 to 0°C for 35 min. TLC was Checked for reaction completion, using mobile phase: Toluene: ethyl acetate (1 : 1), detection: UV at 254 nm; and 0.75% aq. potassium permanganate solution.
The solvent was distilled off at 40-45°C under reduced pressure to obtain an oily product. The resulting crude product was dissolved in ethyl acetate washed with water (2x300 mL), 1% aq. H3P04 solution (390 mL), 2%aq. K2C03 solution (390 mL) followed by 10% aq. NaCl solution (350 mL). Organic layer was separated, dried over sodium sulphate and the solvent was evaporated under reduced pressure at 45 °C to afford ( 1 S,2S,3/?,5S)-pinanediol-N-BOC- β-( 1 -pheny l)-L-alanine-L-leucineboronate (8a) as white coloured foamy solid (67. Og (Yield: 98.5%). The compound was taken to next step without further purification.
Similarly the following compounds were prepared.
(lS,2S,3R,5S)-Pinanediol-N-Boc-L-alanine-L-leucine boronate (8b)
(lS,2S,3R,5S)-Pinanediol-N-Boc-L-tyrosine-L-leucineboronate (8c)
(lS,2S,3R,5S)-Pinanediol-N-Boc-L-leucine-L-leucineboronate (8d)
(1 S,2S,3R,5S)-Pinanediol-N-Boc-L-methionine-L-leucine boronate (8e)
(l S,2S,3R,5S)-Pinanediol-N-Boc-D-valine-L-leucine boronate (8f) EXAMPLE 4
Preparation of (lS,2S,3^»5S)-pinanediol-^-(l-phenyl)-L-alanine-L-leucine boronate HC1 (9a
Figure imgf000016_0001
(lS'^.S^ii^^-Pinanediol-N-BOC-^-il-pheny -L-alanine-L-leucineboronate (8a, 67g, 0.13 moles) was dissolved in ethyl acetate (200mL) and cooled to 10 to 15°C. Then EtOAc-HCl solution (830 mL, assay: 11.5% w/v; 2.61 moles) was added to the reaction mass and stirred for 4 h. TLC was checked for completion of reaction. The reaction mass was concentrated under reduced pressure at 40-45 °C to yield white coloured suspension. To the resulting suspension was added 450 mL of fresh ethyl acetate, stirred for 2h at room temperature and filtered to afford (lS^S^^SSypinanediol-P-O-pheny -L-alanine- L-leucineboronate HC1 as white crystalline solid (70g; % yield: 79.84);
(a)D 25+8.3° (c=l in methanol); mp: 200-205 °C; purity 95.6 % (HPLC); Ή NMR (400 MHz CDC13): δ 8.23 (br s, 2H), 7.71 (brs, IH), 7.24- 7.35 (m, 5H), 4.67 (m, IH), 4.22 (dd, IH), 3.29-3.42 (m, 2H), 2.90 (t, IH), 2.27 (m, 2H), 2.1 1 (m, 2H), 2.02 (t, IH), 1.77- 1.82 (m, 2H), 1.46 (m, 2H), 1.33 .(s, 3H), 1.26 (s, 3H), 0.84 (m, 6H), 0.8 (s, 3H); MS (ESI), m/z = 447.1 1 (M-l). Similarly, the following compounds were synthesized
(l S,2S,3R,5S)-Pinanediol-L-alanine-L-leucine boronate HC1 salt (9b)
(lS,2S,3R,5S)-Pinanediol-L-tyrosine-L-leucine boronate HC1 salt (9c)
(l S,2S,3R,5S)-Pinanediol-L-leucine-L-leucineboronate HC1 salt (9d)
(lS,2S,3R,5S)-Pinanediol-L-methionine-L-leucine boronate HC1 salt (9e)
(lS,2S,3R,5S)-Pinanediol-D-valine-L-leucine boronate HC1 salt (9f)
EXAMPLE 5
Preparation of (lS,2S,3R,5S)-Pinanediol-N-(phenyl)carbonyl p-(l-phenyI)-L- alanine-L-Ieucine boronate 10a)
Figure imgf000017_0001
Charged 9a (5.7g, 0.013 moles), Benzoic acid (1.7g, 0.014 moles), TBTU (4.48g, 0.014 moles) and MDC (40 mL) in a 4N RBF and stirring was given for 20 min under N2 atmosphere. To the RM, DIPEA (5.7g, 0.044 moles) was added drop wise at 0°C. RM turned to light brownish colored solution. The solution was stirred at 15-20°C for 2 h and Concentrated in rotavapor to get brown colured oil. The resulting oil is dissolved in ethyl acetate (54 mL) and was washed with water, 1% H3P04 solution, 2% K2C03 solution and 10% NaCl. Organic layer separated and dried over Na2S04. Solvent was reduced under reduced pressure to afford the title compound as white colored foamy solid (6.5g; 99. l%yield). This compound is taken to next step without purification.
Similarly, the following compound were synthesized
(l S,2S,3R,5S)-Pinanediol-N-(tetrahydronaphthyl)carbonyl-L-phenylalanine-L-leucine boronate (10b) using 5,6,7, 8-tetrahydronaphthoic acid.
(l S,2S,3R,5S)-Pinanediol-N-(2-thienylcarbonyl)-L-phenylalanine-L-leucine boronate (10c) using 2-thiophenecarboxylic acid.
(l S,2S,3R,5S)-Pinanediol-N-(tetrahydrofurancarbonyl)-L-phenylalanine-L-leucine boronate (lOd) using tetrahydrofuran-2-carboxylic acid.
(lS,2S,3R,5S)-Pinanediol-N-(4-nicotincarbonyl)-L-phenylalanine-L-leucine boronate (lOe) using nicotinic acid.
(l S,2S,3R,5S)-Pinanediol-N-(2-methyl-5-nitrophenyl)carbonyl-L-phenylalanine-L- leucine boronate (lOf) using 2-methyl-5-nitrobenzoic acid.
(lS,2S,3R,5S)-Pinanediol-N-(4-nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronate (lOg) using 4-nitrophenyl benzoic acid
(l S,2S,3R,5S)-Pinanediol-N-(2-naphthyl)carbonyl-L-phenylalanine-L-leucine boronate (10h) using 2-naphthoic acid
(lS,2S,3R,5S)-Pinanediol-N-(3,4-dimethylphenyl)carbonyl-L-phenylalanine-L-leucine boronate (lOi) using 3,4-dimethyl benzoic acid
(lS,2S,3R,5S)-Pinanediol-N-(3-nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronate (lOj) using 3-nitrobenzoic acid
EXAMPLE 6
Preparation of (lS,2S,3R»5S)-Pinanediol-N-(2-pyrazinecarbonyl)-L-alanine-L- leucine boronate 10k)
Figure imgf000019_0001
Charged (9b, 3.65g, 0.0098 moles), 2-pyrazine carboxylic acid (1.3g, 0.0105 moles), TBTU (3.46g, 0.0107 moles) and MDC (43.8mL) in a 4N RBF and stirring was given for 20 min under N2 atmosphere. To the reaction mixture, DIPEA (5.2mL, 3.86g, 0.030 moles) was added drop wise at 0°C. The resulting solution was stirred at 15-20°C for 2 h and concentrated in Rota apparatus then subjected to high vacuum, the resulting pale yellow colored oily crude was dissolved in EtOAc (20 mL) and washed with water (2x25 mL) followed by l%H3PO4(0.29g in 24.2mL water) solution, 2%K2C03 solution(0.46g in 24.2mL water) and 10% NaCl solution (2.4g in 29.2mL water). Layers were separated and organic layer was dried over Na2S04 and solvent was evaporated to give white colored foamy solid. Wt. of the compound: 3.62g (83.6%), the compound was taken to next step without further purification.
Similarly, the following compounds have been synthesized
(1 S,2S,3R,5S)-Pinanediol-N-(pyrazinecarbonyl)-L-tyrosine-L-leucine boronate (101) using 9c as starting material
(1 S,2S,3R,5S)-Pinanediol-N-(pyrazinecarbonyl)-L-leucine-L-leucineboronate (10m) using 9d as starting material
(lS,2S,3R,5S)-Pinanediol-N-(pyrazinecarbonyl)-L-methionine-L-leucineboronate (10η) using 9e as starting material
(1 S,2S,3R,5S)-Pinanediol-N-(pyrazinecarbonyl)-D-valine-L-leucineboronate (10ο) using 9f as starting material EXAMPLE 7
Figure imgf000020_0001
10a (6g, 0.01 16 moles), methanol (48 mL), n- heptane (48 mL), and isobutyl boronic acid (2.14g, 0.0210 moles) were charged in a 4N RBF and the RM was cooled to below 25°C. IN aq.HCl solution (36 mL) was added drop wise and the reaction mixture was maintained for 5 h at rt. layers were separated aq. layer was washed with heptane and layers were separated, aq. layer was concentrated under vacuum at 40-45°C to give pale yellow coloured suspension. Acetone was added to the suspension and stirred for lh at rt and product was filtered off. Wt. of the compound: 1.8g (40.5%)
Infrared Data(KBr) : cin 1 331 1.3; 2952.2; 1954.5; 1623.9; 1528.8; 1079.7; 951.0; 744.6; 692.7 EXAMPLE 8
Preparation of N-(tetrahydronaphthyI)carbonyI-L-pheny.alanine-L-Ieucine boronic acid (5b)
Figure imgf000020_0002
10b (3.8g, 0.0067 moles), methanol (30 mL), n-heptane (30 mL), and isobutyl boronic acid (1.24g, 0.0122 moles), were charged in a 4N RBF and the RM was cooled to below 25 C. IN HC1 solution (23mL) was added drop wise and the reaction mixture was maintained for 5 h at rt. After completion of reaction, layers were separated. Aqueous layer was concentrated to give brownish white coloured solid and acetone (25 ml) was added to the reaction mass stirred for 45min at rt. product was filtered off and dried to give the title compound. Wt. of the product: 2.3g (79.2%).
Ή NMR (400 MHz,CDC13): δ 7.2(8H, t), 7.0 (2H, d), 6.9(1H, d), 6.8(1H, d), 6.3(lH,d), 4.9 (1H, t), 3.2(H, d), 3.1(2H, d), 2.9(1H, s), 2.6(2H, t), 2.5(2H,s), 1.63-1.70(6H, m), 1.43-1.47(2H, m),1.2(2H, s), 0.80-0.85(7H, m) Similarly the following compounds were prepared.
N-(2-Thiophene)carbonyl-L-phenylalanine-L-Ieucine boronic acid (5c): Crystallized from EtOAc: heptane (1 :9); Yield: 35%; mp: 130.4°C; purity (HPLC): 98.64%; Ή NMR (DMSO-flk): δ 8.82 (brs, 1H), 8.72 (d, 1H, J= 8.4Hz), 7.82 (d, 1H, J= 2.8Hz), 7.7 (d, IH, J= 4.8Hz), 7.54 (m, IH), 7.1-7.31 (m, 6H), 4.8 (m, IH), 2.9-3.1 (m, 2H), 2.6 (brs, IH), 1.55 (t, IH, J= 6.4Hz), 1.25(m, 2H), 0.75-0.83 (m, 7H); 13C NMR (OMSO-d6): S 173.5, 170.6, 161, 150.0, 139.1, 137.5, 131.0, 129.2, 128.7, 127.8, 126.4, 54.6, 43.0, 37.1 , 25.0, 23.0, 22.5; MS (ESI), m/z = 388.21 (M+ ion), 387.21 (M-lpeak), 369.20 (M-1-H20); IR (KBr): cm-1 3295.4, 3089.3, 2952.4, 1628.2, 1537.7, 1384.1, 1281.5, 1200.6. N-(2-Tetrahydrofuran)carbonyl-L-phenyIalanine-L-leucine boronic acid (5d):
Crystallized from EtOAc: heptane (1 :9); Yield: 43%; mp: 69.9 °C; purity (HPLC): 98.89%;1H NMR (DMSO-efe): δ 7.19-7.28 (m, 5H), 6.9 (m, IH), 4.83 (m, IH), 4.7 (m, IH), 4.2 (brs, IH), 3.6-3.8 (m, 2H), 3.0-3.23 (m, 2H), 2.2 (m, IH), 1.80 (m, 4H), 1.5 (m, 2H), 1.25(m, '2H), 0.86 (m, 7H); 13C NMR (DMSO-i¾): δ 174.7 173.5, 136.0, 129.2, 128.5, 127.0, 78.1, 69.3, 52.2, 39.9, 37.6, 31.8, 30.0, 28.9, 25.8, 22.6, 14.0; MS (ESI), m/z = 376.23 (M+ ion), 375.22 (M-lpeak), 357.22 (M-1-H20); IR (KBr): cm'1 3404.1, 3065.2, 2953.4, 1654.3, 1521.0, 1394.4, 1238.6, 1201.1, 1072.4.
N-(4-Nicotincarbonyl-L-phenylalanine-L-Ieucine boronic acid (5e): Crystallized from EtOAc; Yield: 56%; mp: 124.7 °C; purity (HPLC): 99.07%; Ή NMR (DMSO-<¾: δ 9.06 (d, 1H, J= 8.4 Hz), 8.9 (d, 2H, J= 8.4 Hz), 8.7- 8.66 (dd, 2H, J= 10.4 Hz, J=4.8Hz), 7.3-7.55 (m, 5H), 4.83 (m, 1H), 3.3-3.5 (m, 2H), 2.6 (brs, 1H), 1.54 (m, 2H), 1.35(m, 2H), 1.15 (m, 1H), 0.75-0.8 (m, 6H); 13C NMR (DMSO-<¾): <5 173.3; 170.4, 164.6, 150.0, 141.0, 140.7, 138.4, 137.4, 129.2, 128.0, 126.2, 121.3, 54.8, 52.5, 36.9, 24.8, 23.2, 22.5, 22.1 ; MS (ESI), m/z = 383.23 (M+ ion), 382.22 (M-lpeak), 364.22(M-1-H20); IR (KBr): cm"1 3430.3, 3307.5, 3066.2, 2950.3, 1633.1 , 1532.8, 1398.8, 1284.4, 1209.8.
Similarly the following compounds were synthesized
N-(2-Methyl-5-nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid(5f)
N-(4-Nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5g)
N-(2-Naphtyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5h)
N-(3,4-Dimethylphenyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5i)
N-(3-Nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid (5j) EXAMPLE 9
Preparation of N-(2-pyrazine)carbonyl-L-alanine-L-Ieucine boronic acid (5k)
Figure imgf000022_0001
10k (3.5g, 0.008 moles), methanol (28 mL), n-heptane (28 mL), and isobutyl boronic acid (1.5g, 0.0147 moles), were charged in a 4N RBF and the reaction mixture was cooled to below 25°C. IN HCI solution (21mL) was added drop wise and the reaction mixture was maintained for 5h at rt. The completion of reaction was determined by TLC. Reaction mixture contained aqueous and organic layers. Product lied in aqueous layer which was confirmed by TLC. Solvent was evaporated from aqueous layer to give brownish yellow colored crude. The crude was dissolved in THF (50mL) and distilled off the solvent. The compound was purified by crystallization using ethyl acetate (20mL).The solution was filtered and dried under vacuum for 4 h at rt to afford the title compound. Wt. of the compound: 1.6g (65.5%).Melting point: 178-180°C
ESIMS: m/z : 308 (M+) EXAMPLE 10
Preparation N-(2-pyrazine)carbonyl)-L-tyrosine-L-Ieucine boronic acid (51)
Figure imgf000023_0001
101 (2.29g, 0.0043 moles), methanol (18.3 mL), n-heptane (18.3 mL), and isobutyl boronic acid (0.80g, 0.0078 moles), were charged in a 4N RBF and the reaction mixture was cooled to below 25°C. IN HCl solution (13.8 mL) was added drop wise and the reaction mixture was maintained for 5h at rt. The completion of reaction was determined by TLC. Reaction mixture contained aqueous and organic layers. Product lied in aqueous layer which was confirmed by TLC. Solvent was evaporated from aqueous layer to give brownish yellow colored crude. The compound was purified by crystallization using heptane(20 mL).The solution was filtered and dried under vacuum for 4 h at rt. wt. of the title compound: 0.7g (41%). Melting point: 150-156°C Ή NMR (400 MHz,CDC13): δ 9.1(1H, s), 8.8(1H, d), 8.6(1H, d), 7.0(1H, d), 6.5 (1H, d), 4.67-4.72(lH, m), 3.0(3H, t), 2.9 (1H, d), 1.5(1H, d), 1.27-1.39(3H, m), 0.81-0.85(9H, m) EXAMPLE 11
Preparation of N-(2-Pyrazine) carbonyl-L-leucine-L-leucine boronic acid (5m)
Figure imgf000024_0001
10m (2.2g, 0.0045 moles), methanol (17.5mL), n-heptane (17.5mL), and isobutyl boronic acid (0.83g, 0.0082), were charged in a 4N RBF and the reaction mixture was cooled to below 25°C. IN HCl solution (13.2mL) was added drop wise and the reaction mixture was maintained for 5 h at rt. The completion of reaction was determined by TLC. Reaction mixture contained aqueous and organic layers. Product lied in aqueous layer which was confirmed by TLC. Solvent was evaporated from aqueous layer to give brownish yellow colored crude. The compound was purified by crystallization using heptane(20mL).The solution was filtered and dried under vacuum for 4 h at rt. Wt. of the compound: lg (62.9%) Melting point: 102- 104°C
Ή NMR (400 MHz,CDCl3): δ 9.1 (1H, s), 8.8 (1H, d) , 8.7(1H, s), 4.55-4.58(lH, m), 3.01-3.04 (1H, m), 1.5(4H, t), 1.2(1H, d), 0.8-0.9(12H, m) ' EXAMPLE 12
Pre aration of N-(2-Pyrazine) carbonyl-L-methionine-L-leucine boronic acid (5n)
Figure imgf000024_0002
10η (3g, 0.006 moles), methanol (24mL), n-heptane (24mL), and isobutyl boronic acid (l.lg, 0.0108 moles), were charged in a 4N RBF and the reaction mixture was cooled to below 25°C. IN aq. HCl solution (18.1mL) was added drop wise and the reaction mixture was maintained for 5 h at rt. The completion of reaction was determined by TLC. Reaction mixture contained aqueous and organic layers. Product lied in aqueous layer which was confirmed by TLC. Solvent was evaporated from aqueous layer to give brownish yellow colored crude. The compound was purified by crystallization using heptane(20mL).The solution was filtered and dried under vacuum for 4 h at rt to give the title compound 1.7g (77.3%).
Melting point:81-83uC
Ή NMR (400 MHz,CDC13): δ 9.1(1H, d), 8.87-8.89 (1H, m), 8.76-8.80(2H, m), 7.7(1H, d), 4.7 (1H, d), 4.5(1H, t), 3.01-3.05(1H, m), 2.45-2.48(4H, m), 2.01-2.08(7H, m), 1.5(1H, t), 1.3(1H, t), 1.2(1H, m), 1.1(1H, t), 0.8(9H, s)
EXAMPLE 13
Preparation of N-(2-P razine) carbon l-D-valine-L-leucine boronic acid 5o
Figure imgf000025_0001
lOo (2.36g, 0.0050 moles), methanol (18.8mL), n-heptane (18.8mL), and isobutyl boronic acid (0.93g, 0.0091 moles), were charged in a 4N RBF and the reaction mixture was cooled to below 25°C. IN HCl solution (14.2mL) was added drop wise and the reaction mixture was maintained for 5h at rt. The completion of reaction was determined by TLC. Reaction mixture contained aqueous and organic layers. Product lied in aqueous layer which was confirmed by TLC. Solvent was evaporated from aqueous layer to give brownish yellow colored crude. The compound was piirified by crystallization using heptane(20mL).The solution was filtered and dried under vacuum for 4 h at rt to afford the title compound lg (59.5%). Melting point:83-85°C
Ή NMR (400 MHz,CDC13): δ 9.3(1H, s), 8.7(1H, d ), 8.5(1H, s), 8.3(1H, d ), 8.0(1H, s), 4.7(1H, t), 3.2(1H, d), 2.2(1H, t), 1.55-1.60(1H, m), 1.4(1H, t),1.2(2H, s), 1.0(6H, t), 0.73-0.92(2H, m) EXAMPLE 14
In vitro evaluation of compounds
In vitro evaluation of novel analogues of bortezomib has been carried out by using MTT proliferation assay on different cancer cell lines. DU-145 (prostate cancer), A-549 (lung cancer), NCI-H292 (lung cancer), Cal-27. (Head and neck cancer), MDA-MB-231 (breast cancer) and PC3 (prostate cancer) were employed for the biological activity of the compounds. All cell lines mentioned above were received from ATCC and the protocols based on ATCC and as per manufacturers instruction.
Using the USFDA approved drugs vorinostat, sorafenib, erlotinib, lapatinib as positive controls, biological activity of the compounds of the present invention were tested.
Materials used:
Cell lines DU-145, A-549, NCI-H292, Cal-27, MDA-MB-231 and PC3 (Received from ATCC), Cell Titer 96 Non-Radioactive Cell Proliferation MTT Assay kit (Promega), DMSO (cell biology grade), Trypan blue, Cell growth medium as prescribed by ATCC.
Instruments used:
Bio-safety cabinet (Esco), refrigerated centrifuge (eppendorf), water bath (Thermo), C02 incubator (Binder), Hemo-cytometer (bright lite), multi well plate reader (Biotek). Assay procedure:
Experiments were carried out as per the instructions provided with the assay kit.
Briefly assay procedure, cells were plated at a density of 1,000 to 10,000 cells per well (depending on cell line growth properties) in 96-well plates and allowed to grow overnight incubating at 37°C in 5% C02. The following day, cells were treated with the test compounds and proto drugs allowed to incubate up to 96 hours. MTT reagent was then added to the treated cells for an additional 4 hours. After 4hrs, solubilizing agent was added to all the wells and incubated over night. Readings were measured at 570riM by using multi well plate reader. The IC50 values were calculated using Gen 5 software. All experiments carried out by taking nano molar quantities of the compounds.
Results
All compounds showed better activity compared to thet positive controls except the compound 5o which contains d-isomer of valine as one of the fragments of the structural formula. Results are summarized in the following table.
Figure imgf000027_0001
Figure imgf000027_0002
Figure imgf000028_0001
control)
Erlotinib
(positive - - 428 - - - control)
Lapatinib
1
(positive - - - 1024 - - control)
Sorafenib
(positive - - - 8158 - - control)
L-Phe= L-phenylalanine; L-Ala= L-Alanine; L-Tyr= L-tyrosine; L-Leu= L-Leucine; L- Met=L-Methionine; D-Val= D-V aline EXAMPLE 15
Preparation of lyophilized formulation of boronic acid
Boronic acid: 40mg
D-mannitol: 400mg
Water for injection: 24 mL
rt-Butanol: 16 mL
Boronic acid, rt-butanol, water for injection were charged into a container and stir to dissolve at 40°C. Then D-mannitol was added to the solution and stir to dissolve at the same temperature. The solution was cooled to ambient temperature and filtered through a 0.45μηι membrane. One milliliter aliquots were placed in a 5mL serum bottles, split rubber tubes were partially inserted into the bottles. The filled bottles were subjected to lyophilization process at about -50°C for about 45h to get formulated lyophilized powder of boronic acid.

Claims

We claim
1. A boronic acid derivative of formula 5
Figure imgf000030_0001
5
Wherein,
X is aromatic or heteroaromatic ring;
Where aromatic ring is selected from C6-Ci0, and is substituted with different functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from C]-C6;
trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl r tribromomethyl
heteroaromatic ring is selected from 5- and six membered heterocycliic compounds and is substituted with functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from Ci-C6;
trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl;
Y is an alkyl, hydroxy alkyl, alkoxy alkyl, and thioalkoxy alkyl or aryl or a heteroaryl component and substituents thereof
alkyl group is selected from Ci-C6;
aryl moiety is selected from C -Ci0; and is substituted with different functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from Ci-C6;
1 trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
heteroaryl component optionally selected from 5- and six membered heterocyclic compounds and is substituted with functional groups like hydrogen, hydroxy, alkyl, alkoxy, alkoxy carbonyl, halo, nitro, amino, amido, cyano, carboxylic, trihaloalkyl, sulfonyl;
each alkyl and alkoxy is independently selected from Ci-C6;
trihaloalkyl is independently selected from trifluoromethyl, trichloromethyl or tribromomethyl
2. The compound of claim 1 wherein:
X is phenyl, 2-methyl-5-nitrophenyl, 4-nitrophenyl, 3,4-dimethylphenyl, 3-nitrophenyl, naphthyl, tetrahydronaphthyl, pyrazinyl, 2-thenyl, 2-tetrahydrofuryl and Isonicotinyl
3. The compound of claim 1 wherein:
Y is L- alanyl, L-penylalanyl, L-tyrosinyl, L-leucinyl, L-methionyl and D-valinyl
4. A boronic acid derivative selected from
N-(Phenyl)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(Tetrahydronaphthyl)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(2-Thiophene)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(2-Tetrahydrofuran)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(4-Nicotincarbonyl-L-phenylalanine-L-leucine boronic acid
N-(2-Methyl-5-nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(4-Nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(2-Naphtyl)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(3,4-Dimethylphenyl)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(3-Nitrophenyl)carbonyl-L-phenylalanine-L-leucine boronic acid
N-(2-Pyrazine)carbonyl-L-alanine-L-leucine boronic acid
2 N-(2-Pyrazine)carbonyl)-L-tyrosine-L-leucine boronic acid
N-(2-Pyrazine) carboriyl-L-leucine-L-leucine boronic acid
N-(2-Pyrazine) carbonyl-L-methionine-L-leucine boronic acid
N-(2-Pyrazine) carbonyl-D-valine-L-leucine boronic acid
5. A pharmaceutical composition, comprising a compound of claim 1, or a pharmaceutically accept salt thereof, and pharmaceutically acceptable carrier or diluent.
6. A pharmaceutical composition, comprising a compound of claim 4, or a pharmaceutically accept salt thereof, and pharmaceutically acceptable carrier or diluent.
7. A method of treating cancer by administering therapeutically effective amount of compound of claim 1 to a subject in need thereof.
3
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