Patent Application
20160016929
The present invention relates to neuronal gap junction blocking compounds having improved pharmacokinetic properties, the compounds being useful for the treatment or prevention of a range of conditions including migraine, epilepsy, non-epileptic seizures, brain injury (including stroke, intracranial haemorrhage and trauma induced) or cardiovascular disease including myocardial infarction, coronary revascularization or angina.
Cortical spreading depolarization (CSD) is a wave of depolarisation with consequent depressed electrical activity which spreads across the surface of the cerebral cortex (at a rate of 2-6 mm/min) usually followed by hyperaemia and neuronal hyperpolarisation. The reduction in electrical activity is a consequence of neuron depolarisation and swelling, with K+ efflux, Na and Ca influx and electrical silence. This abnormal neuronal activity is associated with delayed neuronal damage in a number of pathological states including cerebral ischaemia (arising from e.g. stroke, haemorrhage and traumatic brain injury Strong et al., 2002 Fabricius et al., 2006; Dreier et al., 2006 Dohmen et al., 2008), epilepsy and the aura associated with migraine (Lauritzen 1994; Goadsby 2007). As the CSD wave moves across the cortex it is associated with a reactive increase in local blood flow which may serve to help restore the more normal ionic balance of the neurons affected. After the CSD induced hyperaemia the local increase in blood flow attenuates (oligaemia) potentially resulting in imbalances in energy supply and demand. Under certain conditions, the reactive hyperaemia is not observed, but instead the local vasculature constricts resulting in ischaemia which in turn can lead to neuronal death. The conditions triggering this abnormal response in experimental models are high extracellular levels of K+ and low NO availability. These conditions are typically seen in ischaemic areas of the brain, and clusters of CSD waves in these circumstances result in spreading ischaemia (see Dreier 2011). Of particular importance is the spreading ischaemia seen after sub-arachnoid haemorrhage (SAH), in the penumbra of an infarct and after traumatic brain injury where delayed neuronal damage can have a significant effect on clinical outcomes (Dreier et al., 2006, 2012; Hartings et al., 2011a, 2011b; Fabricius et al., 2006).
Given the detrimental effect of clusters of CSDs in humans and experimental animals, and the poor prognosis associated with CSDs, there is an unmet medical need for new compounds useful for inhibiting CSDs for patients with and without brain injuries. Without wishing to be bound by theory, the spread of CSD is believed to be mediated by gap junctions rather than by neuronal synaptic communication (Nedergard et al., 1995; Rawanduzy et al., 1997, Saito et al., 1997), the gap junctions providing a means of spreading the depolarisation in the absence of normal synaptic communication. Gap junctions are comprised of connexin proteins of which there are 21 in the human genome. Each Gap junction is made of two hemichannels, each comprising six connexin monomers.
Gap junctions are also implicated in a number of other disease states including hereditary diseases of the skin and ear (e.g. keratitis-ichthyosis deafness syndrome, erythrokeratoderma variabilis, Vohwinkel's syndrome, and hypotrichosis-deafness syndrome). Blockade of gap junction proteins has been shown to beneficial in some preclinical models of pain (e.g. Spataro et al., 2004 J Pain 5, 392-405, Wu et al., 2012 J Neurosci Res. 90, 337-45). This is believed to be a consequence of gap junction blockade in the spinal cord resulting in a reduction in the hypersensitivity of the dorsal horn to sensory nerve input. In addition gap junctions and their associated hemichannels have been implicated in neurodegenerative diseases including Alzheimer's disease, Parkinson's Disease, Huntington's Disease and amyotrophic lateral sclerosis (Takeuchi et al 2011 PLoS One.; 6, e21108).
Tonabersat (SB-220453/PRX201145) is a gap junction blocker (Silberstein, 2009; Durham and Garrett, 2009) which binds selectively and with high affinity to a unique stereo-selective site in rat and human brains. Consistent with its action on gap junctions Tonabersat also inhibits high K+ evoked CSD in cats (Smith et al., 2000; Read et al., 2000; Bradley et al., 2001) and rats (Read et al., 2001).
However, known gap junction blockers, including Tonabersat and Carabersat, suffer from undesirable physiochemical properties. Tonabersat is a crystalline solid with a high melting point (152-153C) and with a relatively high lipophilicity (log P 3.32). The compound has no readily ionisable groups and consequently has a low aqueous solubility of 0.025 mg/ml over a range of pH values including pH of 7.4. The low aqueous solubility of Tonabersat makes both intravenous (IV) and oral (PO) modes of administration problematic. The poor aqueous solubility prevents rapid injection of the required dose of Tonabersat which is required for the treatment of head injuries and stroke or for emergency treatment of epileptic seizures where the patient may be unconscious and unable to swallow an oral drug. At present the effective plasma concentrations needed to reduce the cortical spreading depression caused by head injury or stroke can only be reached by slow IV infusion given over a period of hours. With respect to the PO administration of Tonabersat for the treatment of other indications, solubility limited dissolution of the tablet form of Tonabersat given PO leads to a significant “food effect” with differences in the maximum blood concentration of Tonabersat (Cmax) seen depending on whether the drug is given with or without food. These differences make it difficult to accurately predict the plasma exposure of Tonabersat when given orally, thus increasing the risk of under or over dosing the patient.
Therefore it is an object of the present invention to provide gap junction blocker compounds having improved physiochemical properties thus improving the utility of these agents in treating a range of disease states.
The present invention makes available three classes of compounds, each class having one or more solubilising pro-drug groups.
In a first aspect, the present invention makes available a class of compounds of formula (I) or a hydrate, solvate, or pharmaceutically acceptable salt thereof:
wherein
Z1, Z2, and Z3 are each independently selected from H, F, or Cl,
A is a direct bond, —C(O)O*—, C(R3)(R4)O*—, —C(O)NH* wherein the atom marked * is directly connected to R1,
R3 and R4 are selected independently from H, fluoro, C1-4 alkyl, or C1-4 fluoroalkyl, or R3 and R4 together with the atom to which they are attached form a cyclopropyl group,
R1 is selected from groups [1], [2], [3], [4], [5], [6], [7], [8], [9] or [10] wherein the atom marked ** is directly connected to A:
n is 0, 1, 2, or 3,
R5 is hydrogen,
R6 is selected from —CH2CH(OH)CH2OH, or —CH2CH2R9;
R7 and R7b are independently selected from H, C1-4 alkyl, or C1-4 fluoroalkyl;
R8 and R8b are selected from:
(i) H, C1-4 alkyl, or C1-4 fluoroalkyl, or
(ii) the side chain of a natural or unnatural alpha-amino acid;
or R7 and R8 together with the atom to which they are attached form a C3-7 carbocyclic ring;
R9 is selected from —N(R11)(R12), or —N+(R11)(R12)(R13)X−, N(R11)C(O)R14, —SO3H or —OP(O)(OH)2;
wherein R11, R12, and R13 are independently selected from H, C1-4 alkyl, or C1-4 fluoroalkyl, or
R11 and R12 together with the nitrogen atom to which they are attached form a 4-7 membered heterocyclic ring optionally substituted with one or more groups selected from H, fluoro, C1-4 alkyl, C1-4 fluoroalkyl, C1-4 alkoxy, or —C(O)R3;
or in the case where R1 is group [7], R9 is —NR11R12 wherein R11 is hydrogen C1-4 alkyl, or C1-4 fluoroalkyl, and R12 is C1-4 alkyl, or C1-4 fluoroalkyl, and wherein R12 joins together with R8b such that R12 and R8b together with the nitrogen to which R12 is attached form a 5 or 6 membered cyclic amine group;
R14 is H, C1-4 alkyl, or C1-4 fluoroalkyl;
X− is a pharmaceutically acceptable anion;
R15 is 3-pyridyl or 1,4-dihydro-1-methyl-pyridin-3-yl;
R27 is individually selected from H, C1-4 alkyl, or C1-4 fluoroalkyl; and
R28 is individually selected from H, C1-4 alkyl, or C1-4 fluoroalkyl.
In a second aspect, the present invention makes available a class of compounds of formula (II) or a hydrate, solvate, or pharmaceutically acceptable salt thereof:
wherein
Z1, Z2, and Z3 are each independently selected from H, F, or Cl,
A is a direct bond and R1 is H,
R2 is B—R21 wherein,
B is a direct bond, —C(O)O*—, C(R23)(R24)O*—, —C(O)NH* wherein the atom marked * is directly connected to R21,
R23 and R24 are selected independently from hydrogen, fluoro, C1-4 alkyl, or C1-4 fluoroalkyl, or
R23 and R24 together with the atom to which they are attached form a cyclopropyl group,
R21 is selected from groups [1], [2], [3], [4], [5], [6], [7], [8], [9] or [10] wherein the atom marked ** is directly connected to B:
n is 0, 1, 2, or 3,
R5 is hydrogen,
R6 is selected from —CH2CH(OH)CH2OH, or —CH2CH2R9;
R7 and R7b are independently selected from H, C1-4 alkyl, or C1-4 fluoroalkyl;
R8 and R8b are selected from:
(i) H, C1-4 alkyl, or C1-4 fluoroalkyl, or
(ii) the side chain of a natural or unnatural alpha-amino acid;
or R7 and R8 together with the atom to which they are attached form a C3-7 carbocyclic ring;
R9 is selected from —N(R11)(R12), or —N+(R11)(R12)(R13)X−, N(R11)C(O)R14, —SO3H or —OP(O)(OH)2;
wherein R11, R12, and R13 are independently selected from H, C1-4 alkyl, or C1-4 fluoroalkyl, or
R11 and R12 together with the nitrogen atom to which they are attached form a 4-7 membered heterocyclic ring optionally substituted with one or more groups selected from H, fluoro, C1-4 alkyl, C1-4 fluoroalkyl, C1-4 alkoxy, or —C(O)R3;
or in the case where R1 is group [7], R9 is —NR11R12 wherein R11 is hydrogen, C1-4 alkyl, or C1-4 fluoroalkyl, and R12 is C1-4 alkyl, or C1-4 fluoroalkyl, and wherein R12 joins together with R8b such that R12 and R8b together with the nitrogen to which R12 is attached form a 5 or 6 membered cyclic amine group;
R14 is H, C1-4 alkyl, or C1-4 fluoroalkyl;
X− is a pharmaceutically acceptable anion,
R15 is 3-pyridyl or 1,4-dihydro-1-methyl-pyridin-3-yl;
In an embodiment of the second aspect of the invention, the group R2 is any solubilising group, including but not limited to the group B—R21 as defined above.
In a third aspect, the present invention makes available a class of compounds of formula (IIIa) or (IIIb), or a hydrate, solvate, or pharmaceutically acceptable salt thereof:
wherein Z1, Z2, and Z3 are each independently selected from H, F, or Cl; and
R2 and -A-R1 are both H; and
In the case of formula (IIIa):
R41 and R42 are independently H, C1-4 fluoroalkyl or optionally substituted C1-4 alkyl, or R41 and R42 together with the carbon atom to which they are attached form a 5-8 membered heterocycle, any carbon atom of which is optionally substituted; or
In the case of formula (IIIb):
Q is an oxime of formula=NHOR43, wherein R43 is
(i) selected from H, C1-4 fluoroalkyl or optionally substituted C1-4 alkyl, or
(ii) -A-R1 wherein
A is a direct bond, —C(O)O*—, C(R3)(R4)O*—, —C(O)NH* wherein the atom marked * is directly connected to R1;
R3 and R4 are selected independently from H, fluoro, C1-4 alkyl, or C1-4 fluoroalkyl, or R3 and R4 together with the atom to which they are attached form a cyclopropyl group,
R1 is selected from groups [1], [2], [3], [4], [5], [6], [7], [8], [9] or [10] wherein the atom marked ** is directly connected to A:
R5 is hydrogen;
R6 is selected from —CH2CH(OH)CH2OH, or —CH2CH2R9;
R7 and R7b are independently selected from H, C1-4 alkyl, or C1-4 fluoroalkyl;
R8 and R8b are selected from:
(i) H, C1-4 alkyl, or C1-4 fluoroalkyl, or
(ii) the side chain of a natural or unnatural alpha-amino acid;
or R7 and R8 together with the atom to which they are attached form a C3-7 carbocyclic ring;
R9 is selected from —N(R11)(R12), or —N+(R11)(R12)(R13)X−, N(R11)C(O)R14, —SO3H or —OP(O)(OH)2;
wherein R11, R12, and R13 are independently selected from H, C1-4 alkyl, or C1-4 fluoroalkyl, or
R11 and R12 together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring optionally substituted with one or more groups selected from H, fluoro, C1-4 alkyl, C1-4 fluoroalkyl, C1-4 alkoxy, or —C(O)R3;
or in the case where R1 is group [7], R9 is —NR11R12, wherein R11 is hydrogen C1-4 alkyl, or C1-4 fluoroalkyl, and R12 is C1-4 alkyl, or C1-4 fluoroalkyl, and wherein R12 joins together with R8b such that R12 and R8b together with the nitrogen to which R12 is attached form a 5 or 6 membered cyclic amine group;
R14 is H, C1-4 alkyl, or C1-4 fluoroalkyl;
X− is a pharmaceutically acceptable anion;
R15 is 3-pyridyl or 1,4-dihydro-1-methyl-pyridin-3-yl;
In an embodiment R43 is C1-4 alkyl optionally substituted with a phosphate group (—P(O)OR61OR62). In an example of such an embodiment OR43 is —OCH2P(O)OR61OR62, wherein R61 and R62 are independently H or C1-4 alkyl.
In another embodiment R43 is an amino acid derivative having the structure —C(O)CH(R100)NH2 wherein the group R100 is the side chain of a natural or unnatural amino acid. In an embodiment OR43 is —OC(O)CH(CH(CH3)2)NH2.
In an embodiment of the third aspect of the invention, the groups Q and/or OR41 and/or OR42 is/are any solubilising group, including but not limited to the group B—R21 as defined above.
Preferably the invention is as set out in the claims.
As used herein, the term “includes” means including the following integers, but not limited thereto.
As used herein, the term “(Ca-Cb)alkyl” wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms. Thus when a is 1 and b is 6, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.
As used herein, the term “(Ca-Cb)fluoroalkyl” has the same meaning as “(Ca-Cb)alkyl” except that one or more of the hydrogen atoms directly connected to the carbon atoms forming the alkyl group is replaced by the corresponding number of fluorine atoms.
The term “C1-6-alkoxy” refers to a straight or branched C1-6-alkyl group which is attached to the remainder of the molecule through an oxygen atom. For parts of the range C1-6-alkoxy, all subgroups thereof are contemplated such as C1-5-alkoxy, C1-4-alkoxy, C1-3-alkoxy, C1-2-alkoxy, C2-6-alkoxy, C2-5-alkoxy, C2-4-alkoxy, C2-3-alkoxy, etc. Examples of said C1-6-alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy.
As used herein the unqualified term “carbocyclic” refers to a mono-, bi- or tricyclic radical having up to 16 ring atoms, all of which are carbon, and includes aryl and cycloalkyl.
As used herein the unqualified term “cycloalkyl” refers to a monocyclic saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
As used herein the unqualified term “aryl” refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical, and includes radicals having two monocyclic carbocyclic aromatic rings which are directly linked by a covalent bond. Illustrative of such radicals are phenyl, biphenyl and napthyl.
As used herein the unqualified term “heteroaryl” refers to a mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and O, and includes radicals having two such monocyclic rings, or one such monocyclic ring and one monocyclic aryl ring, which are directly linked by a covalent bond. Illustrative of such radicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, triazinyl, indolyl and indazolyl.
As used herein the unqualified term “heterocyclyl” or “heterocyclic” includes “heteroaryl” as defined above, and in addition means a mono-, bi- or tri-cyclic non-aromatic radical containing one or more heteroatoms selected from S, N and O, and to groups consisting of a monocyclic non-aromatic radical containing one or more such heteroatoms which is covalently linked to another such radical or to a monocyclic carbocyclic radical. Illustrative of such radicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.
When the term cyclic amino group is used the cyclic amino groups can have 3-8 ring atoms, 3-7 ring atoms, 5-7 ring atoms, 5-6 ring atoms. When the terms 3-8 or 3-7 cyclic amino group is used all ranges within those ranges are disclosed, for example 3-8 includes 3-7. Both 3-8 and 3-7 include 4-7 and 5-7 and 5-6. Examples of 5 and 6 membered cyclic amino groups include morpholine, piperidine, piperazine, pyrrolidine.
Unless otherwise specified in the context in which it occurs, the term “substituted” as applied to any moiety herein means substituted with up to four compatible substituents, each of which independently may be, for example, (C1-C6)alkyl, (C1-C6)alkoxy, hydroxy, hydroxy(C1-C6)alkyl, mercapto, mercapto(C1-C6)alkyl, (C1-C6)alkylthio, halo (including fluoro, bromo and chloro), fully or partially fluorinated (C1-C3)alkyl, (C1-C3)alkoxy or (C1-C3)alkylthio such as trifluoromethyl, trifluoromethoxy, and trifluoromethylthio, nitro, nitrile (—CN), oxo, phenyl, phenoxy, monocyclic heteroaryl or heteroaryloxy with 5 or 6 ring atoms, tetrazolyl, —COORA, —CORA,
—OCORA, —SO2RA, —CONRARB, —SO2NRARB, —NRARB, OCONRARB, —NRBCORA,
—NRBCOORA, —NRBSO2ORA or —NRACONRARB wherein RA and RB are independently hydrogen or a (C1-C6)alkyl group or, in the case where RA and RB are linked to the same N atom, RA and RB taken together with that nitrogen may form a cyclic amino ring, such as a morpholine, piperidinyl or piperazinyl ring. Where the substituent is phenyl, phenoxy or monocyclic heteroaryl or heteroaryloxy with 5 or 6 ring atoms, the phenyl or heteroaryl ring thereof may itself be substituted by any of the above substituents except phenyl, phenoxy, heteroaryl or heteroaryloxy. An “optional substituent” may be one of the foregoing substituent groups.
As used herein the term “salt” includes base addition, acid addition and quaternary salts. Compounds of the invention which are acidic can form salts, including pharmaceutically acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine and the like. Those compounds of formula (I), (II), (IIIa) or (IIIb) which are basic can form salts, including pharmaceutically acceptable salts with inorganic acids, e.g. hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p-toluenesulphonic, benzoic, benzenesunfonic, glutamic, lactic, and mandelic acids and the like.
The formation of specific salt forms can provide compounds of the invention with improved physicochemical properties. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
The term ‘solvate’ is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water.
Compounds with which the invention is concerned which may exist in one or more stereoisomeric form, because of the presence of asymmetric atoms or rotational restrictions, can exist as a number of stereoisomers with R or S stereochemistry at each chiral centre or as atropisomers with R or S stereochemistry at each chiral axis. The invention includes all such enantiomers and diastereoisomers and mixtures thereof. In particular the carbon atom to which the R8 or R8b substituent is attached may be in either the R or the S stereochemical configuration.
The compounds of the invention include compounds of formula (I), (II), (IIIa) or (IIIb) as hereinbefore defined, including all polymorphs and crystal habits thereof, and isomers thereof (including optical, geometric and tautomeric isomers) as hereinafter defined and isotopically-labeled compounds of formula (I), (II), (IIIa) or (IIIb).
For use in accordance with the invention, the following structural characteristics are currently contemplated, in any compatible combination, in the compounds of formula (I):
The groups Z1, Z2, and Z3 are each independently selected from H, F, or Cl. In an embodiment Z1 is Cl, Z2 is F, and Z3 is H. In another embodiment Z1 is Cl, Z2 and Z3 are H. In another embodiment Z1 is H, Z2 is F, and Z3 is H. In another embodiment Z1 is F, Z2 is H, and Z3 is F. The above definitions of Z1, Z2, and Z3 is H are applicable to compounds of formula (I), (II), (IIIa), and (IIIb). As an illustration, the preferred definition of Z1, Z2, and Z3 applied to the compounds of formula (I) is as follows:
In a preferred embodiment Z1 is Cl, Z2 is F or H, and Z3 is H.
Examples of side chains of natural alpha amino acids include those of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, α-aminoadipic acid, α-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, α-methylserine, ornithine, pipecolic acid, and thyroxine.
Natural alpha-amino acids which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups in their characteristic side chains include arginine, lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, and cysteine. When R8 or R8b in the compounds of the invention is one of those side chains, the functional substituent may optionally be protected.
The term “protected” when used in relation to a functional substituent in a side chain of a natural alpha-amino acid means a derivative of such a substituent which is substantially non-functional. For example, carboxyl groups may be esterified (for example as a C1-C6 alkyl ester), amino groups may be converted to amides (for example as a NHCOC1-C6 alkyl amide) or carbamates (for example as an NHC(═O)OC1-C6 alkyl or NHC(═O)OCH2Ph carbamate), hydroxyl groups may be converted to ethers (for example an OC1-C6 alkyl or a O(C1-C6 alkyl)phenyl ether) or esters (for example a OC(═O)C1-C6 alkyl ester) and thiol groups may be converted to thioethers (for example a tert-butyl or benzyl thioether) or thioesters (for example a SC(═O)C1-C6 alkyl thioester).
Examples of side chains of non-natural alpha amino acids include:
an optional substituent, C1-C6 alkyl, phenyl, 2,-3-, or 4-hydroxyphenyl, 2,-3-, or 4-methoxyphenyl, 2,-3-, or 4-pyridylmethyl, benzyl, phenylethyl, 2-, 3-, or 4-hydroxybenzyl, 2,-3-, or 4-benzyloxybenzyl, 2,-3-, or 4-C1-C6 alkoxybenzyl, and benzyloxy(C1-C6alkyl)-groups, wherein any of the foregoing non-natural amino acid side chains is optionally substituted in the alkyl, phenyl or pyridyl group; or
groups -[Alk]nR50 where Alk is a (C1-C6)alkyl or (C2-C6)alkenyl group optionally interrupted by one or more —O—, or —S— atoms or —N(R51)— groups [where R51 is a hydrogen atom or a (C1-C6)alkyl group], n is 0 or 1, and R50 is an optionally substituted cycloalkyl or cycloalkenyl group; or
a heterocyclic(C1-C6)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (C1-C6)alkoxy, cyano, (C1-C6)alkanoyl, trifluoromethyl (C1-C6)alkyl, hydroxy, formyl, amino, (C1-C6)alkylamino, di-(C1-C6)alkylamino, mercapto, (C1-C6)alkylthio, hydroxy(C1-C6)alkyl, mercapto(C1-C6)alkyl or (C1-C6)alkylphenylmethyl; and
A is a direct bond, —C(O)O*—, C(R3)(R4)O*— such as —CH2O—, CH(CH3)O—, or C(CH3)2O—, —C(O)NH* wherein the atom marked * is directly connected to R1,
R3 and R4 are selected independently from H, fluoro, C1-4 alkyl such as methyl, ethyl or isopropyl, or C1-4 fluoroalkyl such as trifluoromethyl, or R3 and R4 together with the atom to which they are attached form a cyclopropyl group. In an embodiment R3 and R4 are both hydrogen.
R1 is selected from any one of the groups [1], [2], [3], [4], [5], [6], [7], [8], [9] or [10] wherein the atom marked ** is directly connected to A:
R5 is hydrogen
R6 is selected from —CH2CH(OH)CH2OH, or —CH2CH2R9. In an embodiment R6 is —CH2CH(OH)CH2OH, —CH2CH2NR11R12, or —CH2CH2NR11R12R13X−. In an embodiment R11 and R12 together with the nitrogen atom to which they are attached form a 5, 6, or 7 membered cyclic amino group such as pyrrolidine, piperidine, homopiperazine, piperazine, homopiperazine, morpholine, or homomorpholine.
R7 and R7b are independently selected from hydrogen, C1-4 alkyl such as methyl, ethyl, isopropyl, or C1-4 fluoroalkyl such as trifluoromethyl. In an embodiment R7 and R7b are both hydrogen.
R8 and R8b are selected from:
(iii) H, C1-4 alkyl, or C1-4 fluoroalkyl, or
(iv) the side chain of a natural or unnatural alpha-amino acid;
or R7 and R8 together with the atom to which they are attached form a C3-7 carbocyclic ring;
R9 is selected from —N(R11)(R12) such as —N(CH3)2, or —N+(R11)(R12)(R13)X−, N(R11)C(O)R14, —SO3H or —OP(O)(OH)2;
wherein R11, R12, and R13 are independently selected from H, C1-4 alkyl, or C1-4 fluoroalkyl. In an embodiment R11, R12, and R13 are methyl or ethyl.
In an embodiment the carbon atom(s) bearing group R8 and/or R8b has (have) the stereochemical configuration of a natural amino acid, which is the L-configuration.
In an embodiment R11 and R12 together with the nitrogen atom to which they are attached form a 4 to 7 membered heterocyclic ring optionally substituted with one or more groups selected from H, fluoro, C1-4 alkyl such as methyl or isopropyl, C1-4 fluoroalkyl, C1-4 alkoxy such as methoxy, or —C(O)R3 such as —O(O)OH3.
In an embodiment R1 is group [7]. In a preferred embodiment, R1 is group [7], and R9 is —NR11R12, wherein R11 is hydrogen, C1-4 alkyl, or C1-4 fluoroalkyl, and R12 is C1-4 alkyl, or C1-4 fluoroalkyl, and the group R12 joins together with R8b or the carbon atom to which R8b is attached such that R12 and R8b, together with the nitrogen atom to which R12 is attached, form a 5 or 6 membered cyclic amine group. In an embodiment that ring formed by R8b and R12 is a 5-membered ring such that the amino acid proline is formed. The ring of the proline amino acid is optionally substituted with one or more groups selected from H, fluoro, C1-4 alkyl such as methyl or isopropyl, C1-4 fluoroalkyl, C1-4 alkoxy such as methoxy, or —C(O)R3 such as —O(O)OH3.
R14 is H, C1-4 alkyl such as methyl, or C1-4 fluoroalkyl;
X− is a pharmaceutically acceptable anion;
R15 is 3-pyridyl or 1,4-dihydro-1-methyl-pyridin-3-yl;
R27 is selected from H, C1-4 alkyl such as methyl, ethyl, propyl or isopropyl, or C1-4 fluoroalkyl such as trifluoromethyl. In an embodiment R27 is hydrogen or methyl.
R28 is selected from H, C1-4 alkyl such as methyl, ethyl, propyl or isopropyl, or C1-4 fluoroalkyl such as trifluoromethyl. In an embodiment R28 is hydrogen or methyl.
In an embodiment, R1 is selected from [71] and [101]:
In an embodiment R1 is selected from any one of groups [4A], [4B], [4C], [4D], [5A], or [5B]:
Specific compounds of the invention include those of the Examples herein.
In an embodiment, A is a direct bond and R1 has the formula (7A):
wherein R21 is hydrogen, or C1-6 alkyl such as methyl; and
R8 and R8b are each independently a side chain of a natural amino acid; preferably the side chains are selected from the side chains of alanine, valine, and leucine; preferably the carbon atoms bearing the R8 and R8b groups are in the natural amino acid stereochemical configuration, which is the L-configuration, as in formula (7AA):
In an embodiment A is a direct bond and R1 has the formula (7A) or (7AA) wherein R8 is methyl or isopropyl and R8b is isopropyl or —CH2CH(CH3)2, and R21 is hydrogen or methyl.
It will be understood that the compounds of formula (I), (II), (IIIa) or (IIIb) may be further modified by adding one or more prodrug groups Q, -AR1 or R2. For example the compounds of formula (I) or (II) may be modified by exchanging the oxygen atom Q for a prodrug Q group as defined in (IIIa) or (IIIb). Alternatively, the compounds of formula (I) could be modified by replacing the hydrogen atom R2 by the prodrug group R2 as defined in formula (II), and vice versa.
The present invention makes available a pharmaceutical composition comprising a compound of formula (I), (II), (IIIa) or (IIIb) together with one or more pharmaceutically acceptable carriers and/or excipients.
The present invention makes available a compound of formula (I), (II), (IIIa) or (IIIb) for use in medicine.
In an embodiment the inventions encompasses the use of a compound of formula (I), (II), (IIIa) or (IIIb) treatment of a disease or medical condition which benefits from inhibition of gap junction activity. Inhibition of gap junction activity may be achieved by blocking the gap junction as a whole or by blocking one or more hemichannels.
In an embodiment the inventions encompasses a method of treatment of a disease or medical condition which benefits from inhibition of gap junction activity, comprising administering to a subject suffering from such disease or condition and effective amount of a compound of formula (I), (II), (IIIa) or (IIIb).
In an embodiment the disease or condition which benefits from inhibition of gap junction activity is selected from among migraine, aura with or without migraine, epilepsy, non-epileptic seizures, cerebrovascular accidents including stroke, intracranial haemorrhage (including traumatic brain injury, epidural hematoma, subdural hematoma and subarachnoid haemorrhage), and intra-cerebral haemorrhage, spinal cord vascular accidents arising from trauma, epidural hematoma, subdural hematoma or subarachnoid haemorrhage, pain including pain arising from hyperalgesia caused by damage to sensory neurons (i.e. neuropathic pain including but not limited to diabetic neuropathy, polyneuropathy, cancer pain, fibromyalgia, myofascial pain, post herpetic neuralgia, spinal stenosis, HIV pain, post-operative pain, post-trauma pain) or inflammation (including pain associated with osteoarthritis, rheumatoid arthritis, sciatica/radiculopathy, pancreatitis, tendonitis), neurodegenerative disease (including but not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Disease and Amyotrophic Lateral Sclerosis) and cardiovascular disease including myocardial infarction, coronary revascularization or angina.
It will be understood that the pharmacology of the brain is a complex and constantly evolving area of research. Without wishing to be bound by theory, it is currently hypothesised that the claimed compounds exert their therapeutic effect by inhibiting gap junction activity. However, it is anticipated that the claimed compounds may exert their therapeutic effect by additional and/or alternative mechanisms of action. For the avoidance of doubt, the claimed compounds are expected to be useful for treatment of any one of the diseases selected from among migraine, aura with or without migraine, epilepsy, non-epileptic seizures, cerebrovascular accidents including stroke, intracranial haemorrhage (including traumatic brain injury, epidural hematoma, subdural hematoma and subarachnoid haemorrhage), and intra-cerebral haemorrhage, spinal cord vascular accidents arising from trauma, epidural hematoma, subdural hematoma or subarachnoid haemorrhage, pain including pain arising from hyperalgesia caused by damage to sensory neurons (i.e. neuropathic pain including but not limited to diabetic neuropathy, polyneuropathy, cancer pain, fibromyalgia, myofascial pain, post herpetic neuralgia, spinal stenosis, HIV pain, post-operative pain, post-trauma pain) or inflammation (including pain associated with osteoarthritis, rheumatoid arthritis, sciatica/radiculopathy, pancreatitis, tendonitis), neurodegenerative disease (including but not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Disease and Amyotrophic Lateral Sclerosis) and cardiovascular disease including myocardial infarction, coronary revascularization or angina
It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Optimum dose levels and frequency of dosing will be determined by clinical trial, as is required in the pharmaceutical art. However, for administration to human patients, the total daily dose of the compounds of the invention may typically be in the range 1 mg to 1000 mg depending, of course, on the mode of administration. For example, oral administration may require a total daily dose of from 10 mg to 1000 mg, while an intravenous dose may only require from 1 mg to 500 mg. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein. These dosages are based on an average human subject having a weight of about 60 kg to 100 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly, and especially obese patients.
The compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties. Suitable routes for administration include oral, intravenous, buccal, intranasal, inhalation, rectal, and intradermal. The orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
The pro-drug may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. The person skilled in the art is aware of many excipients useful for IV formulation.
The compounds of formula (I) above may be prepared by, or in analogy with, conventional methods. The preparation of intermediates and compounds according to the Examples of the present invention may in particular be illuminated by the following Schemes. Definitions of variables in the structures in Schemes herein are commensurate with those of corresponding positions in the formulas delineated herein.
wherein A, Q, Z1, Z2, Z3, R1 and R2 are as defined in formula (I);
Compounds of general formula (I) can easily be prepared from the alcohols of general formula (IV) by either using the alcohol directly or pre-forming the alkoxide using a suitable base/reagent (e.g. NaH) and coupling to a suitably activated A-R1 or R1 group (or protected A-R1 or R1 group). Activated A-R1 or R1 group functionalities typically used for the formation of phosphates, esters, carbonates and carbamates include, but not limited to, phosphoryl chlorides, acid chlorides, activated carboxylic acids, chloroformates, activated carbonates and isocyanates. Alternatively, the A-R1 or R1 group can be introduced in a step-wise manner using standard methodologies. Suitable protecting group strategies can be employed where necessary. The formation of (la) from (IV) using 2-dimethylaminoethyl carbonochloridate as an activated R group is representative of this approach.
The synthesis of Tonabersat, and other structurally related compounds, is disclosed in WO 95/34545. The present invention encompasses compounds prepared by applying the pro-drug groups -AR1, R2 and Q taught herein to the specific Examples disclosed in WO 95/34545. The methods proposed for the synthesis of compounds of general formula (I) are known to those skilled in the art, for example in Rautio et al., Nature Reviews Drug Discovery, 7, 255-270, 2008.
Optionally, a compound of formula (I) can also be transformed into another compound of formula (I) in one or more synthetic steps.
The following abbreviations have been used:
Reactions were conducted at room temperature unless otherwise specified. Preparative chromatography was performed using a Flash Master Personal system equipped with Isolute Flash II silica columns or using a CombiFlash Companion system equipped with GraceResolv silica column, unless otherwise stated. The purest fractions were collected, concentrated and dried under vacuum. Compounds were typically dried in a vacuum oven at 40° C. prior to purity analysis. Compound analysis was performed by HPLC/LCMS using an Agilent 1100 HPLC system/Waters ZQ mass spectrometer connected to an Agilent 1100 HPLC system with a Phenomenex Synergi, RP-Hydro column (150×4.6 mm, 4 μm, 1.5 mL per min, 30° C., gradient 5-100% MeCN (+0.085% TFA) in water (+0.1% TFA) over 7 min, 200-300 nm). The compounds prepared were named using IUPAC nomenclature. Accurate masses were measured using a Waters QTOF electrospray ion source and corrected using Leucine Enkephalin lockmass. Spectra were acquired in positive and negative electrospray mode. The acquired mass range was m/z 100-1000. Samples were dissolved in DMSO to give 1 mg/mL solutions which were then further diluted with Acetonitrile (50%)/Water (50%) to 1 μg/mL solutions prior to analysis. The values reported correspond either to the protonated or deprotonated molecular ions [MH]+ or [MH]−.
tert-Butyl 2-aminoacetate (7.50 g, 44.7 mmol) was dissolved in DCM (150 mL) and sat aq NaHCO3 (150 mL), and cooled to 0° C. Triphosgene (4.40 g, 14.8 mmol) was added portion-wise and the reaction mixture was stirred at 0-5° C. for 45 min. The aqueous fraction was extracted with DCM (2×) and the combined organic fractions were dried (MgSO4) and concentrated in vacuo. The residue was purified by distillation (boiling point 35-37° C./2 mm Hg) to give the title compound (3.41 g, 48.2%) as a colourless liquid.
1-[(3S,4S)-4-Amino-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-6-yl]ethan-1-one sulfuric acid hydrate (1.20 g, 3.42 mmol) was dissolved in THF (70 mL) and water (6 mL), and 2M aq NaOH (3.40 mL, 6.84 mmol) and Boc2O (760 mg, 3.48 mmol) were added. The reaction mixture was stirred for 23 h and partitioned between water (180 mL) and EtOAc (120 mL). The aqueous fraction was extracted with EtOAc (120 mL) and the combined organic fractions were dried (MgSO4) and concentrated in vacuo to give the crude title compound (1.12 g, 97.8%). LCMS (ES+): 236.1 [MH-Boc]+.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (400 mg, 1.02 mmol) was dissolved in DCM (8 mL) and Boc-Gly-OSu (full name: 2,5-dioxopyrrolidin-1-yl 2-{[(tert-butoxy)carbonyl]amino}acetate) (556 mg, 2.04 mmol), DIPEA (3914, 2.25 mmol) and DMAP (12 mg, 0.10 mmol) were added. The reaction mixture was stirred overnight and concentrated in vacuo. The residue was partitioned between EtOAc (15 mL) and 10% aq citric acid solution (10 mL). The organic fraction was washed with water (10 mL) and concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with heptane/EtOAc mixtures. The (3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl 2-{[(tert-butoxy)carbonyl]amino}acetate intermediate was dissolved in 4M HCl in dioxane (4 mL) and stirred for 90 min. The solvents were removed in vacuo and the residue partitioned between EtOAc (10 mL) and sat aq Na2CO3 solution (5 mL). The aqueous fraction was extracted with EtOAc (10 mL) and the combined organic fractions were concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with heptane/ethyl acetate mixtures. To each pure fraction was added 1.25M HCl in EtOH (200 μL). The pure fractions were combined and dried in vacuo to give the title compound (93 mg, 18.8%) as a white foam. LCMS (ES+): 449.0 [MH]+. HPLC: Rt 4.95 min, 96.9% purity.
Boc-Val-OH (full name: (2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutanoic acid) (521 mg, 2.40 mmol), EDC.HCl (537 mg, 2.80 mmol), HOBt (429 mg, 2.80 mmol) and DMAP (733 mg, 6.00 mmol) were dissolved in DCM (15 mL) and the reaction mixture was stirred for 15 min. N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (784 mg, 2.00 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was washed with 10% aq citric acid, water, 10% aq NaHCO3 and brine, dried (MgSO4) and concentrated in vacuo to give crude intermediate (3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl (2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutanoate (1.20 g). This material (1.20 g) was dissolved in MeOH (2 mL) and 4M HCl in dioxane (20 mL) and the reaction mixture was stirred for 1 h and concentrated in vacuo. The residue was triturated from MTBE, washed with hexane and purified by column chromatography on normal phase silica eluting with DCM/MeOH/NH4OH (100:2.5:0.5). The residue was dissolved in Et2O and 2M HCl in Et2O was added. The resulting precipitate was collected by filtration and washed with Et2O and hexane to give the title compound (172 mg, 16.3%) as a white solid. LCMS (ES+): 491.1 [MH]+. HPLC: Rt 5.38 min, 98.1% purity.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (5.00 g, 12.8 mmol), Boc-Ala-OH (3.38 g, 17.9 mmol) and DMAP (160 mg, 1.31 mmol) were dissolved in DCM (150 mL) and a solution of DCC (3.95 g, 19.1 mmol) in DCM (20 mL) was added drop-wise at 0° C. The reaction mixture was stirred for 3 h, filtered through Celite and concentrated in vacuo. The residue was purified by column chromatography and triturated from hexane. The resulting Boc intermediate (7.15 g) was dissolved in MeOH (10 mL), a solution of 4M HCl in dioxane (100 mL) was added and the reaction mixture was stirred for 3.5 h. The reaction mixture was concentrated in vacuo and the residue was triturated from hexane/Et2O (1:1) to give the crude title compound (6.30 g). LCMS (ES+): 463.1 [MH]+.
Intermediates 6-8 were prepared similarly to Intermediate 5, using the appropriate Boc-protected amino acid; see Table 1 below.
4-Nitrophenylchloroformate (2.38 g, 11.8 mmol) was dissolved in Et2O (50 mL), cooled to 0° C. and a solution of 1-(2-hydroxyethyl)piperidine (1.45 g, 11.3 mmol) in Et2O (40 mL) was added drop-wise. The resulting solution was stirred for 1 h and the precipitate was collected by filtration, washed with Et2O and dried in vacuo to give the crude title compound as an off-white solid. LCMS (ES+): 295.1 [MH]+.
Triphosgene (198 mg, 0.67 mmol) was dissolved in DCM (10 mL) and a solution of 2-dimethylaminoethanol (201 uL, 2.00 mmol) and DMAP (244 mg, 2.00 mmol) in DCM (10 mL) was added. The reaction mixture was stirred for 4 h. A solution of N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (784 mg, 2.00 mmol) and DMAP (488 mg, 4.00 mmol) in DCM (10 mL) was added and the reaction mixture was stirred overnight. The solution was absorbed onto silica and purified by column chromatography on normal phase silica eluting with EtOAc to give the title compound (308 mg, 30.4%) as a white solid. HPLC: Rt 5.27 min, 97.5% purity. HRMS (ESI+) calcd for C25H28CIFN2O6 507.170 found 507.171.
EXAMPLE 1 (150 mg, 0.30 mmol) was dissolved in Et2O/DCM (16 mL, 3:1) and iodomethane (300 uL, 4.82 mmol) was added. The reaction mixture was allowed to stand over the weekend and the resulting precipitate was collected by filtration and washed with Et2O to give the title compound (109 mg, 56.9%) as an off-white solid, in two batches. LCMS (ES+): 521.1 [M]+. HPLC: Rt 5.37 min, 99.0% purity.
2-Diethylaminoethanol (1.30 g, 11.1 mmol) was dissolved in Et2O (50 mL) and added drop-wise at 0° C. to a solution of 4-nitrophenylchloroformate (2.24 g, 11.1 mmol) in Et2O (40 mL). The reaction mixture was stirred over the weekend and the resulting precipitate was collected by filtration and washed with Et2O to give a white solid (2.90 g, 82.2%). The 2-(diethylamino)ethyl 4-nitrophenyl carbonate hydrochloride intermediate (1.43 g, 4.50 mmol) and N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (1.18 g, 3.00 mmol) were dissolved in DCM (50 mL), DMAP (1.10 g, 9.00 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was washed with 2% aq NaOH (2×50 mL), sat aq NaHCO3 (3×50 mL), dried (MgSO4), absorbed onto silica and purified by column chromatography on normal phase silica eluting with pentane/EtOAc (1:1) then EtOAc to give the title compound (360 mg, 22.4%) as a white solid. HPLC: Rt 5.60 min, 97.6% purity. HRMS (ESI+) calcd for C27H32CIFN2O6 535.201 found 535.200.
2-Hydroxyethylmorpholine (727 mg, 5.54 mmol) was dissolved in Et2O (20 mL) and added drop-wise at 0° C. to a solution of 4-nitrophenylchloroformate (1.17 g, 5.81 mmol) in Et2O (25 mL). The reaction mixture was stirred overnight and the resulting precipitate was collected by filtration and washed with Et2O to give a white solid (1.84 g, 91.8%). The 2-(morpholin-4-yl)ethyl 4-nitrophenyl carbonate hydrochloride intermediate (998 mg, 3.00 mmol) and N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (1.18 g, 3.00 mmol) were dissolved in DCM (50 mL), DMAP (806 mg, 6.60 mmol) was added and the reaction mixture was stirred over the weekend. The reaction mixture was absorbed onto silica and purified by column chromatography on normal phase silica eluting with hexane/EtOAc (1:1) then EtOAc. The residue was dissolved in EtOAc, washed with 2% aq NaOH (2×50 mL), sat aq NaHCO3 (3×50 mL), dried (MgSO4) and concentrated in vacuo. The residue was triturated from hexane (50 mL) and washed with pentane to give the title compound (558 mg, 33.9%) as a white solid. HPLC: Rt 5.42 min, 98.8% purity. HRMS (ESI+) calcd for C27H30CIFN2O7 549.180 found 549.179.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (2.64 g, 6.74 mmol) and pyridine (1.20 mL, 14.8 mmol) were dissolved in DCM (50 mL) and triphosgene (669 mg, 2.23 mmol) was added. The reaction mixture was stirred for 1 h and a solution of tert-butyl (2S)-2-{[(tert-butoxy)carbonyl]amino}-3-hydroxypropanoate (1.76 g, 6.74 mmol) in DCM (30 mL) was added. The reaction mixture was stirred for 19 h, diluted with water (70 mL) and extracted into DCM (70 mL), dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with hexane/EtOAc (2:1) to give intermediate tert-butyl (2S)-3-[({[(3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl]oxy}carbonyl)oxy]-2-{[(tert-butoxy)carbonyl]amino}propanoate (4.58 g, 68.8%). This material (1.50 g, 2.21 mmol) was dissolved in DCM, 4M HCl in dioxane (15 mL) was added and the reaction mixture was stirred for 2 d. The reaction mixture was concentrated in vacuo and the residue was triturated from hexane (40 mL). The residue was suspended in Et2O (10 mL) and stirred overnight. The resulting precipitate was collected by filtration to give the title compound (1.06 g, 85.8%) as a cream solid. LCMS (ES+): 523.0 [MH]+. HPLC: Rt 4.92 min, 94.3% purity.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (3.92 g, 10.0 mmol) and pyridine (2.5 mL) were dissolved in DCM (125 mL) and triphosgene (980 mg, 3.33 mmol) was added. The reaction mixture was stirred for 2 h. Sodium isethionate (1.48 g, 10.0 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was diluted with DCM (50 mL) and EtOAc (100 mL) and washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was dissolved in EtOAc (50 mL), filtered and passed through a plug of silica. The residue was triturated from diisopropyl ether then hexane to give the title compound (133 mg, 2.3%) as a cream solid. LCMS (ES+): 544.0 [MH]+.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (392 mg, 1.00 mmol) and Et3N (349 uL, 2.50 mmol) were dissolved in PhMe (7 mL), Intermediate 1 (393 mg, 2.50 mmol) was added and the reaction mixture was heated under reflux for 4 h. Further Intermediate 1 (100 mg, 0.64 mmol) was added and the reaction mixture was heated under reflux overnight. The reaction mixture was concentrated in vacuo. The reaction was similarly repeated on 2.5 times scale and the combined residues were purified by column chromatography on normal phase silica eluting with hexane/EtOAc (2:1) and triturated from hexane to give a white solid (1.50 g, 78%). The tert-butyl 2-[({[(3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl]oxy}carbonyl)amino]acetate intermediate (1.50 g, 2.73 mmol) was dissolved in DCM (6 mL), cooled to 0° C. and TFA (6 mL) was added. The reaction mixture was stirred overnight and concentrated in vacuo. The residue was dissolved in EtOAc and extracted into 1M aq NaOH. The aqueous fraction was acidified with 1M aq HCl and extracted into EtOAc, washed with brine, dried (MgSO4) and concentrated in in vacuo. The residue was purified by column chromatography on normal phase silica eluting with DCM/MeOH/Et3N (100:5:1), dissolved in water and acidified with 1M aq HCl. The resulting precipitate was collected by filtration and washed with water to give the title compound (680 mg, 50%) as a white solid. HPLC: Rt 5.54 min, 100% purity. HRMS (ESI+) calcd for C23H22CIFN2O7 493.118 found 493.119.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (784 mg, 2.00 mmol) and pyridine (440 uL, 5.44 mmol) were dissolved in DCM (15 mL) and the reaction mixture was cooled to 0° C. Triphosgene (196 mg, 0.66 mmol) was added and the reaction mixture was stirred for 1 h. A solution of L-valine tert-butyl ester hydrochloride (419 mg, 2.00 mmol) in DCM (10 mL) was added at 0° C. and the reaction mixture was stirred overnight. The reaction mixture was washed with 2M aq HCl, dried (MgSO4), absorbed onto silica and purified by column chromatography on normal phase silica eluting with hexane/EtOAc (3:1) to give a white solid (561 mg, 47.5%). The tert-butyl (2S)-2-[({[(3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl]oxy}carbonyl)amino]-3-methylbutanoate intermediate (561 mg, 0.95 mmol) was dissolved in 4M HCl in dioxane (10 mL), stirred over the weekend and concentrated in vacuo. The residue was dissolved in DCM and washed with 1M aq NaOH (100 mL). The organic fraction was acidified with 2M aq HCl and extracted into EtOAc (50 mL×4), dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with hexane/EtOAc (1:1) and trituration from pentane/diisopropyl ether (10:1). The residue was dissolved in water and acidified with 1M aq HCl and the resulting precipitate was collected by filtration and washed with water to give the title compound (158 mg, 31.1%) as a pale green solid. HPLC: Rt 6.04 min, 100% purity. HRMS (ESI+) calcd for C26H28CIFN2O7 535.165 found 535.167.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (784 mg, 2.00 mmol) and pyridine (440 uL, 5.44 mmol) were dissolved in DCM (15 mL), triphosgene (196 mg, 0.66 mmol) was added and the reaction mixture was stirred for 1 h. A solution of (2S)-2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid hydrochloride (678 mg, 2.00 mmol) in DCM (10 mL) was added and the reaction mixture was stirred overnight. Water (20 mL) was added and the aqueous fraction was extracted with DCM (20 mL). The combined organic fractions were dried (MgSO4), concentrated in vacuo and purified by column chromatography on normal phase silica eluting with hexane/EtOAc (1:1) to give intermediate tert-butyl (2S)-2-[({[(3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl]oxy}carbonyl)amino]-6-{[(tert-butoxy)carbonyl]amino}hexanoate (1.21 g, 84.0%). This material (990 mg, 1.37 mmol) was dissolved in TFA (4 mL) and stirred for 6 h. The reaction mixture was partitioned between EtOAc (25 mL) and water (40 mL) and the aqueous fraction was extracted with EtOAc (25 mL). The combined organic fractions were dried (MgSO4) and concentrated in vacuo to give the title compound (912 mg, 96.0%) as an off white solid. LCMS (ES+): 564.0 [MH]+. HPLC: Rt 4.85 min, 93.2% purity.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (784 mg, 2.00 mmol), DMAP (20 mg, 0.16 mmol) and 2-{[(2S)-1-[(tert-butoxy)carbonyl]pyrrolidin-2-yl]formamido}acetic acid (544 mg, 2.00 mmol) were dissolved in DCM (10 mL), a solution of DCC (619 mg, 3.05 mmol) in DCM (10 mL) was added and the reaction mixture was stirred for 3 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo and purified by column chromatography on normal phase silica eluting with hexane/EtOAc (1:1) to give intermediate tert-butyl (2S)-2-[(2-{[(3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl]oxy}-2-oxoethyl)carbamoyl]pyrrolidine-1-carboxylate (886 mg, 68.6%). This material (886 mg, 1.36 mmol) was dissolved in DCM (4 mL), 4M HCl in dioxane (10 mL) was added and the reaction mixture was stirred for 3 h and concentrated in vacuo (930 mg crude residue). 390 mg of the residue was triturated from hexane (10 mL) to give the title compound (265 mg, 79.6%) as a white solid. HPLC: Rt 5.14 min, 96.5% purity. HRMS (ESI+) calcd for C27H29CIFN3O6 546.181 found 546.181.
Boc-Leu-OH (full name: (2S)-2-{[(tert-butoxy)carbonyl]amino}-4-methylpentanoic acid) (463 mg, 2.00 mmol) and HATU (913 mg, 2.40 mmol) were dissolved in DCM (20 mL) and DMF (2 mL) and the reaction mixture was stirred for 30 min. Intermediate 3 (971 mg, 2.00 mmol) and NMM (607 mg, 6.00 mmol) were added and the reaction mixture was stirred for 5 h and concentrated in vacuo. The residue was dissolved in EtOAc and washed with 10% aq citric acid. The organic fraction was washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with hexane/EtOAc (65:35) to give intermediate (3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl 2-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-4-methylpentanamido]acetate (1.07 g, 80.8%). This material (1.00 g, 1.51 mmol) was dissolved in MeOH (15 mL), 4M HCl in dioxane (15 mL) was added and the reaction mixture was stirred for 3.5 h and concentrated in vacuo. The residue was triturated from hexane and washed with Et2O and hexane to give the title compound (758 mg, 83.8%) as an off white solid. HPLC: Rt 5.38 min, 97.5% purity. HRMS (ESI+) calcd for C28H33CIFN3O6 562.212 found 562.213.
Boc-Gly-OH (full name: 2-{[(tert-butoxy)carbonyl]amino}acetic acid) (389 mg, 2.22 mmol), EDC.HCl (511 mg, 2.67 mmol) and HOBt (409 mg, 2.67 mmol) were dissolved in DCM (20 mL), Intermediate 3 (1.08 g, 2.22 mmol) and DIPEA (1.42 mL, 8.19 mmol) were added and the reaction mixture was stirred over the weekend. The reaction mixture was diluted with DCM, washed with 2M aq HCl and sat aq NaHCO3, dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with hexane/EtOAc (2:1 then 1:1) to give intermediate (3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl 2-(2-{[(tert-butoxy)carbonyl]amino}acetamido)acetate (887 mg, 65.8%). This material (887 mg, 1.46 mmol) was dissolved 4M HCl in dioxane (20 mL) and the reaction mixture was stirred for 1 h and concentrated in vacuo. The residue was partitioned between EtOAc and 1M aq NaOH and the organic fraction was dried (MgSO4) and concentrated in vacuo. The residue was dissolved in Et2O and EtOAc, and 2M HCl in Et2O (4 mL) was added. The resulting precipitate was collected by filtration and washed with Et2O to give the title compound (529 mg, 66.6%) as a beige solid. HPLC: Rt 4.91 min, 96.9% purity. HRMS (ESI+) calcd for C24H25CIFN3O6 506.149 found 506.149.
Boc-Ile-OH (full name: (2S,3S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylpentanoic acid) (477 mg, 2.06 mmol), EDC.HCl (474 mg, 2.47 mmol) and HOBt (379 mg, 2.47 mmol) were dissolved in DCM (20 mL) and the reaction mixture was cooled to 0° C. Intermediate 3 (1.00 g, 2.06 mmol) and DIPEA (1.32 mL, 7.60 mmol) were added and the reaction mixture was stirred overnight. The reaction mixture was diluted with DCM (30 mL), washed with 2M aq HCl (50 mL) and sat aq NaHCO3 (50 mL), dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with hexane/EtOAc (2:1) to give intermediate (3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl 2-[(2S,3S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylpentanamido]acetate (1.36 g, 56.9%). This material (777 mg, 1.17 mmol) was dissolved in 4M HCl in dioxane (10 mL) and the reaction mixture was stirred overnight and concentrated in vacuo. The residue was partitioned between EtOAc and 1M aq NaOH and the organic fraction was dried (MgSO4) and concentrated in vacuo. The residue was dissolved in Et2O and 2M HCl in Et2O was added. The resulting precipitate was collected by filtration and washed with Et2O to give the title compound (537 mg, 76.5%) as a beige solid. HPLC: Rt 5.61 min, 96.1% purity. HRMS (ESI+) calcd for C28H33CIFN3O6 562.212 found 562.213.
Boc-L-Valine hydroxysuccinimide ester (408 mg, 1.30 mmol), Intermediate 3 (350 mg, 0.72 mmol) and DIPEA (553 uL, 3.17 mmol) were dissolved in DCM (25 mL) and the reaction mixture was stirred for 20 h, diluted with DCM (10 mL) and washed with sat aq NH4Cl (2×25 mL). The organic fraction was dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography, dissolved in 1M HCl (20 mL) and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography to give the title compound as an off white solid (262 mg, 62.1%). HPLC: Rt 5.20 min, 98.0% purity. HRMS (ESI+) calcd for C27H31CIFN3O6 548.196 found 548.197.
Boc-L-Valine hydroxysuccinimide ester (220 mg, 0.70 mmol), Intermediate 4 (307 mg, 0.58 mmol) and DIPEA (446 uL, 2.56 mmol) were dissolved in DCM (25 mL) and the reaction mixture was stirred overnight, diluted with DCM (10 mL) and washed with sat aq NH4Cl (2×25 mL). The organic fraction was dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography, dissolved in 1M HCl (20 mL) and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography to give the title compound as a white solid (129 mg, 35.4%). HPLC: Rt 5.85 min, 99.4% purity. HRMS (ESI+) calcd for C30H37CIFN3O6 590.243 found 590.242.
Boc-Gly-OH (full name: 2-{[(tert-butoxy)carbonyl]amino}acetic acid) (164 mg, 0.95 mmol), EDC.HCl (218 mg, 1.14 mmol) and HOBt (174 mg, 1.14 mmol) were dissolved in DCM (10 mL) and the reaction mixture was cooled to 0° C. Intermediate 4 (500 mg, 0.95 mmol) and DIPEA (0.6 mL, 3.45 mmol) were added and the reaction mixture was stirred overnight. The reaction mixture was diluted with DCM, washed with 2M aq HCl and sat aq NaHCO3, dried (MgSO4) and concentrated in vacuo to give crude intermediate (3S,4S)-6-acetyl-4-[(3-chloro-4-fluorobenzene)amido]-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl (2S)-2-(2-{[(tert-butoxy)carbonyl]amino}acetamido)-3-methylbutanoate (614 mg, 82.3%). This material (506 mg, 0.78 mmol) was dissolved in 4M HCl in dioxane (10 mL) and the reaction mixture was stirred for 1.5 h and concentrated in vacuo. The residue was triturated from MTBE and washed with MTBE. The residue was suspended in Et2O and the reaction mixture was stirred for 1 h. The precipitate was collected by filtration and washed with Et2O. The residue was partitioned between EtOAc and 1M aq NaOH and the organic fraction was dried (MgSO4) and concentrated in vacuo. The residue was dissolved in Et2O and 2M HCl in Et2O was added. The resulting precipitate was collected by filtration and washed with Et2O to give the title compound (191 mg, 42.0%) as a pale yellow solid. H PLC: Rt 5.33 min, 92.5% purity. HRMS (ESI+) calcd for C27H31CIFN3O6 548.190 found 548.197.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (1.33 g, 3.39 mmol), 4-(morpholin-4-yl)butanoic acid (1.00 g, 4.77 mmol) and DMAP (41.0 mg, 0.34 mmol) were dissolved in DCM (40 mL) and the reaction mixture was cooled to 0° C. and a 1M solution of DCC in DCM (5.1 mL) was added drop-wise. The reaction mixture was stirred for 4 h and filtered and the filtrate was concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with DCM/MeOH/NH4OH (100/2.5/0.5). The residue was dissolved in Et2O and 2M HCl in Et2O was added. The resulting precipitate was collected by filtration and washed with Et2O. The residue was dissolved in water, filtered and the filtrate washed with ether, basified with 10% aq NaHCO3 and extracted into EtOAc. The organic fraction was washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was dissolved in Et2O and 2M HCl in Et2O was added. The resulting precipitate was collected by filtration and washed with Et2O to give the title compound (1.00 g, 50.5%) as a white solid. HPLC: Rt 5.29 min, 95.1% purity. HRMS (ESI+) calcd for C28H32CIFN2O6 547.201 found 547.202.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (784 mg, 2.00 mmol) was dissolved in THF (25 mL) and the reaction mixture was cooled to 0° C. NaH (100 mg, 60% dispersion in mineral oil, 2.50 mmol) was added and the reaction mixture was stirred at 0° C. for 20 min. 4-Piperidinopiperidine-1-carbonyl chloride (461 mg, 2.00 mmol) was added portion-wise and the reaction mixture was stirred overnight and concentrated in vacuo. The residue was partitioned between 15% aq NH4Cl and EtOAc and the organic fraction was washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography on normal phase silica eluting with DCM/MeOH/NH4OH (100/2.5/0.5) and dissolved in Et2O and 2M HCl in Et2O was added. The resulting precipitate was collected by filtration and washed with Et2O to give the title compound (900 mg, 72.3%) as a white solid. HPLC: Rt 5.41 min, 99.8% purity. HRMS (ESI+) calcd for C31H37CIFN3O5 586.248 found 586.250.
Intermediate 2 (566 mg, 1.69 mmo), DMAP (20.0 mg, 0.16 mmol) and 3-chloro-4-fluorobenzoic acid (295 mg, 1.69 mmol) were dissolved in DCM (10 mL) and a solution of DCC (522 mg, 2.53 mmol) in DCM (10 mL) was added. The reaction mixture was stirred overnight and purified by column chromatography on normal phase silica eluting with hexane/EtOAc (3:1) to give the title compound (784 mg, 94.4%). The (3S,4S)-6-acetyl-4-{[(tert-butoxy)carbonyl]amino}-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran-3-yl 3-chloro-4-fluorobenzoate intermediate (784 mg, 1.59 mmol) was dissolved in DCM (6 mL) and 4M HCl in dioxane (4 mL) was added. The reaction mixture was stirred for 20 h and concentrated in vacuo. The residue was suspended in hexane (40 mL), stirred for 1 h and the resulting precipitate was collected by filtration to give the title compound (174 mg, 25.5%) as an off white solid. LCMS (ES+): 392.0 [MH]+. HPLC: Rt 5.44 min, 95.8% purity.
Boc-Val-OH (435 mg, 2.00 mmol) and HATU (913 mg, 2.40 mmol) were dissolved in DCM (20 mL) and DMF (2 mL) and the reaction mixture was stirred for 30 min. Intermediate 5 (1.00 g, 2.00 mmol) and NMM (0.61 g, 6.00 mmol) were added and the reaction mixture was stirred for 5 h and concentrated in vacuo. The residue was dissolved in EtOAc and washed with 10% aq citric acid, brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography, dissolved in MeOH (1 mL) and 4M HCl in dioxane (7.6 mL) was added. The reaction mixture was stirred for 2.5 h and concentrated in vacuo. The residue was triturated from Et2O to give the title compound (700 mg, 77%) as a white solid. HPLC: Rt 5.32 min, 95.4% purity. HRMS (ESI+) calcd for C28H33CIFN3O6562.212 found 562.213.
Examples 21-29 were prepared similarly to Example 20 using Intermediates 5-8 and the appropriate Boc-protected amino acid; see Table 2 below.
N-[(3S,4S)-6-Acetyl-3-hydroxy-2,2-di methyl-3,4-dihydro-2H-1-benzopyran-4-yl]-3-chloro-4-fluorobenzamide (1.18 g, 3.00 mmol) and Intermediate 9 (1.49 g, 4.50 mmol) were dissolved in DCM (50 mL), DMAP (1.10 g, 9.00 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was washed with 2% aq NaOH (2×100 mL), sat aq NaHCO3 (2×100 mL), dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography and triturated from hexane/diisopropyl ether to give the title compound (259 mg, 16%) as a white solid. HPLC: Rt 5.65 min, 99.1% purity. HRMS (ESI+) calcd for C28H32CIFN2O6 547.201 found 547.202.
wherein Q, Z1, Z2, Z3 and R2 are as defined in the section entitled “detailed description of the invention” and P2 is a suitable protecting group.
Compounds of general formula (IIa) can easily be prepared from the alcohols of general formula (IVa) by protecting the hydroxyl functionality with a suitable protecting group P2 to give compounds of general formula (VI) and then coupling the prodrug functionality onto the amide nitrogen atom in one or more steps using synthetic strategies analogous to those used for the synthesis of compounds of general formula (I). The final step is to remove the protecting group P2 to give compounds of general formula (IIa).
wherein A, Z1, Z2, Z3, R1 and R2 are as defined in the section entitled “detailed description of the invention”
Compounds of general formula (IIIa) can easily be prepared from the ketones of general formula (Id) by either using an alcohol or diol in the presence of an acid and removal of the water generated to prepare acyclic or cyclic ketals respectively. Such methods proposed for the synthesis of compounds of general formula (IIIa) are known to those skilled in the art, for example in T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis (2nd edition) J. Wiley & Sons, 1991 and P. J. Kocienski, Protecting Groups, Georg Thieme Verlag, 1994.
wherein A, Q, Z1, Z2, Z3, R1, R2, and R43 are as defined in the section entitled “detailed description of the invention”.
Compounds of general formula (IIIb) can easily be prepared from the ketones of general formula (I) where Q=O by using the appropriate hydroxylamine and removal of the water generated to prepare the ketoxime. Such methods proposed for the synthesis of compounds of general formula (IIIb) are known to those skilled in the art, for example in T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis (2nd edition) J. Wiley & Sons, 1991
Without wishing to be bound by theory, the general mode of action of the claimed pro-drugs is as follows. For IV administration the high solubility conferred by the solubilising pro-moiety to the parent Tonabersat-like drug is expected to allow a rapid bolus injection whereupon the pro-drug will be quickly cleaved by plasma esterases/phosphatases to reveal the parent drug. For PO administration the mode of action is either where the solubilising pro-drug is predominantly cleaved in the gut by esterases/phosphatases prior to absorption of the parent drug into the systemic circulation, or where the solubilising pro-drug is absorbed intact and then quickly cleaved by plasma esterases/phosphatases to reveal the parent drug.
In an embodiment prodrugs of the present invention are suitable for oral administration. The skilled person understands that the pH of the gastrointestinal tract changes along its length. For example, the stomach has a pH of around pH 1.5 and the GI tract after the stomach has a pH of around 5 to 7.5. For more detail see, for example, Measurement of gastrointestinal pH profiles in normal ambulant human subjects, Gut. 1988 Aug.; 29(8): 1035-1041. Improved solubility is expected to result in improved absorption, and therefore improved oral bioavailability. Thus improved solubility at any pH value between around pH 1.5 to 8 is expected to improve oral bioavailability. Compounds of the invention were assessed for solubility in aqueous solutions having a pH of from 2 to 10. In an embodiment prodrugs of the invention have a solubility of >0.5 mg/mL in an aqueous solution having a pH of from 2 to 8. In an embodiment prodrugs have a solubility of >5.0 mg/mL, or >10.0 mg/mL, >100.0 mg/mL, or >200.0 mg/mL. In an embodiment the prodrugs have the aforementioned aqueous solubility at a pH within the range of from 4 to 8, or from 6 to 8.
In an embodiment prodrugs of the invention are administered intravenously. High prodrug solubility is advantageous in order to reduce the volume of solution administered to the patient, and to reduce the risk of damage to the circulatory system. Solubility of >10 mg/mL is preferred. Yet more preferred is solubility of >30 mg/mL or >100.0 mg/mL. Yet more preferred is solubility of >200.0 mg/mL. The solubility is measured in an aqueous solution having a pH of from 2 to 10, which pH range is advantageous for intravenous prodrug delivery. See, for example, A guide on intravenous drug compatibilities based on their pH, Nasser S C et al. /Pharmacie Globale (IJCP) 2010, 5 (01)). In an embodiment the prodrugs of the claimed invention have solubility of >10 mg/mL in an aqueous solution having a pH of from 2 to 10.
The solubility of certain Examples is shown in Table 3.
Example Prodrugs of the claimed invention were dosed either intravenously or orally to fasted male Sprague Dawley rats. The rats underwent surgery for jugular vein cannulation 48 h prior to dosing. Following dosing, 0.25 mL blood samples were taken via the cannulae at 0, 5, 10, 20, 30, 45, 60, 120, 240 & 360 min in EDTA coated tubes. Tubes were spun at 13,000 rpm for 4 min and 100 ul of supernatant taken immediately and stored at −80° C. prior to analysis. Plasma samples were analysed by LC-MS/MS following extraction by protein precipitation, and levels of parent prodrug and tonabersat were measured by MRM (Multiple Reaction Monitoring) analysis against an extracted calibration curve of plasma samples spiked with the Example prodrug and tonabersat.
The exposure of tonabersat in plasma following dosing of the prodrugs of the invention was compared directly to the exposure observed following dosing of an equimolar amount of tonabersat under analogous assay conditions (5.00 mg/kg oral dosing or 0.78 mg/kg intravenous dosing). In an embodiment prodrugs of the present invention have >10% exposure of tonabersat obtained following either oral or intravenous dosing of the prodrug to a human or animal subject, compared to the exposure obtained from dosing an equimolar amount of tonabersat itself. In an embodiment the exposure of tonabersat following dosing of the prodrugs is >20%, or >30%, or >40%, or >50%, or preferably >70% compared to the exposure obtained from dosing an equimolar amount of tonabersat itself.
Scheme 6 shows the in vivo hydrolysis of the prodrug compounds of the invention of formula (Va) to the corresponding drug of formula (Vb).
Prodrug compounds of formula (Va) where Z1 is Chloro, Z2 is Fluoro, and Z3 is hydrogen are hydrolysed in vivo to tonabersat. It is expected that all prodrugs compounds of formula (Va) having Z1, Z2, and Z3 groups as set out in claim 1 will similarly hydrolyse to the corresponding drugs of formula (Vb).
Example 1 was dosed according to this protocol at 6.43 mg/kg PO. Plasma levels of tonabersat were determined to be 53 ng/mL at 5 min and 576 ng/mL at 6 hrs showing conversion of the prodrug to tonabersat over this timecourse following oral dosing. This corresponds to an exposure of tonabersat following dosing of the prodrugs of 53% compared to the exposure obtained from dosing an equimolar amount of tonabersat itself.
Example 2 was dosed according to this protocol at 1.04 mg/kg IV. The plasma level of tonabersat was determined to be 2212 ng/mL at 5 min showing conversion of the prodrug to tonabersat following intravenous dosing. This corresponds to an exposure of tonabersat following dosing of the prodrugs of 45% compared to the exposure obtained from dosing an equimolar amount of tonabersat itself.
Table 4 shows the exposure of tonabersat obtained following either oral or intravenous dosing of prodrug Examples 1-30, compared to the exposure obtained from dosing an equimolar amount of tonabersat itself.
Compounds of the invention were tested for inhibition of the human ether a go-go related gene (hERG) K+ channel using IonWorks patch clamp electrophysiology. 8 Point concentration-response curves were generated on two occasions using 3-fold serial dilutions from the maximum assay concentration (33 uM). Electrophysiological recordings were made from a Chinese Hamster Lung cell line stably expressing the full length hERG channel. Single cell ion currents were measured in the perforated patch clamp configuration (100 ug/mL amphoterocin) at room temperature using an IonWorks Quattro instrument. The internal solution contained 140 mM KCl, 1 mM MgCl2, 1 mM EGTA and 20 mM HEPES and was buffered to pH 7.3. The external solution contained 138 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 8 mM Na2HPO4 and 1.5 mM KH2PO4, and was buffered to pH 7.3. Cells were clamped at a holding potential of 70 mV for 30 s and then stepped to +40 mV for 1 s. This was followed by a hyperpolarising step of 1 s to 30 mV to evoke the hERG tail current. This sequence was repeated 5 times at a frequency of 0.25 Hz. Currents were measured from the tail step at the 5th pulse, and referenced to the holding current. Compounds were incubated for 6-7 min prior to a second measurement of the hERG signal using an identical pulse train. A minimum of 17 cells were required for each pIC50 curve fit. A control compound (quinidine) was used.
Example 1 was tested in line with the preceding experimental procedure and shown to have a hERG 1050 of >20 uM.
In an embodiment the compounds of the invention have a hERG 1050 of >11 uM. Table 5 shows the hERG IC50 values of certain Examples.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1304814.5 | Mar 2013 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2013/053423 | 12/23/2013 | WO | 00 |
