Patent Application
20220213030
The present invention relates to novel aminoguanidine hydrazone-derivatives which are effective as retromer stabilizers and useful as neuroprotecting drugs. The invention also relates to pharmaceutical compositions comprising thereof and their use in therapy and diagnostic.
The endocytic pathway (Huotari and Helenius 2011) includes a predominant recycling circuit for plasma membrane receptors and ligands, a degradative system for digestion of (macro) molecules and exogenous large particles, and a late endosome (LEs)-mediated feeder pathway for transport of non-sorted fluid and membrane components from the former to the latter circuit. Dysfunctional endocytic membrane trafficking in neurons is common in neurodegenerative diseases (NDDs), leading to the accumulation of dysfunctional proteins and organelles, to neuronal vulnerability and cell death (Schreij, Fon et al. 2016).
The retromer complex (Seaman 2005) is a multi-modular protein assembly involved in the retrieval of cargos from endosomes and in their delivery to the trans-Golgi network (TGN) (Seaman 2004); and in the recycling of cargos from endosomes back to the cell surface (Temkin, Lauffer et al. 2011). Its trimeric cargo recognition core (CRC) (Hierro, Rojas et al. 2007) binds to the trans-membrane proteins to be transported. CRC is made by vacuolar protein sorting-associated proteins 26 (VPS26) and VPS29, which assemble onto the VPS35 backbone.
Retromer dysfunctions in NDDs may cause abnormal processing of cargos into neurotoxic fragments (Bhalla, Vetanovetz et al. 2012), reduced protease-driven degradation of protein oligomers and aggregates in the endosomal-lysosomal system (Futerman and van Meer 2004), impaired autophagosome formation and functionality (Zavodszky, Seaman et al. 2014), increased seeding and cell-to-cell spread of intracellular neurotoxic aggregates (Walker, Diamond et al. 2013) and reduction of microglia phagocytic receptors (Lucin, O'Brien et al. 2013).
Increased retromer levels enhance cargo trafficking and transport in cells (Small, Kent et al. 2005). A stronger interaction among CRC proteins stabilizes the retromer (Norwood, Shaw et al. 2011) and should increase its functionality. An in silico screen recently targeted retromer stabilizers (Mecozzi, Berman et al. 2014) binding at the interface between VPS35 and VPS29. The thiophene-connected bis-isothiourea R55 (1) was identified as a pharmacological chaperone (Convertino, Das et al. 2016) being able to bind at the VPS35-VPS29 interface. R55 stabilizes CMC in vitro (≈10° C. increase in denaturation temperature of CMC), increases VPS35 (≈180%) and VPS26 levels (≈150%) in neuronal cultures, and is not toxic up to 50 μM (Mecozzi, Berman et al. 2014). R55 reduces the levels of Aβ40 (up to 44%) and Aβ42 (up to 39%) in wild type and APP-mutated hippocampal neurons (Mecozzi, Berman et al. 2014).
There is an existing need for novel retromer stabilizers which are effective and which can be valuably used in the treatment and prevention of several neurological diseases, such as for instance amyotrophic lateral sclerosis (ALS), Charcot-Marie Tooth disease, Alzheimer's disease (AD), Parkinson's disease (PD) and the like.
Objects of the Invention
It an object of the invention to provide novel compounds which are effective as retromer stabilizers.
It is a further object of the invention to provide the use of the novel compounds of the invention in diagnostic and in therapy, particularly in the treatment of neurological diseases such as amyotrophic lateral sclerosis (ALS), Charcot-Marie Tooth disease, Alzheimer's disease (AD), Parkinson's disease (PD) and the like.
It is a further object of the invention to provide pharmaceutical compositions comprising the novel compounds of the invention.
It is another object of the invention to provide a process for the preparation of the novel compounds of the invention.
According to one of its aspects, the invention relates to a novel compound of Formula (I)
and salts thereof,
wherein:
R1 is selected from hydrogen and halo;
R2 is selected from hydrogen, halo, hydroxy, alkoxy and alkyl;
R3 is selected from hydrogen, halo, hydroxy, alkoxy and alkyl, aralkyl, aryl, —COOR8, —NHCOR8, —NHSO2R8, —COR9, —NO2 and aminoguanidyl-hydrazone;
R4 is selected from hydrogen and alkyl;
R5 is selected from hydrogen and alkyl;
R6 is selected from hydrogen, alkyl, aryl and arylalkyl;
R7 is selected from hydrogen and alkyl; or
R5 and R6 together with the two nitrogen atoms to which they are bound, form a cycle selected from imidazolino, tetrahydropyrimidino and benzimidazolino; or
R6 and R7, each independently, together with the nitrogen atom to which they are bound, form a pyrrolidino or a piperidino group;
R8 is selected from hydrogen, substituted alkyl and aryl;
R9 is selected from groups of Formula (IIIa), (IIIb), (IIIc), (IIId) and (IIIe)
wherein the asterisk (*) indicates the linking bond,
and its salts and solvates.
The term “halo” herein indicates any of any of the halogen atoms, preferably chlorine and bromine, advantageously bromine.
The term “alkyl” herein indicates a linear or branched, saturated C1-C6 alkyl, preferably C1-C4 alkyl, such as methyl, ethyl, n-propyl, i-propyl and n-butyl, i-butyl, sec-butyland tert-butyl. The alkyl group may be optionally substituted, preferably by a halogen atom, a hydroxy, a C1-C6 alkoxy, or an amino group.
The term “alcoxy” herein indicates a linear or branched, saturated C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy, ethoxy, propoxy and butoxy groups.
The term “aryl” herein indicates an optionally substituted aryl group, preferably an optionally substituted monocyclic aryl, optionally substituted by a halogen atom, a linear or branched C1-C6 alkyl, a hydroxy, a C1-C6 alkoxy, or an amino group; said optionally substituted aryl group is preferably selected from phenyl and tolyl, such as p-tolyl.
The term “arylalkyl” herein indicates an optionally substituted aryl, preferably an optionally substituted monocyclic aryl, group which is bound to Formula (I) by an alkyl residue, preferably a methylene; a preferred arylalkyl group is benzyl.
According to a preferred embodiment, in the compound of Formula (I)
R1 is selected from hydrogen and bromine;
R2 is selected from hydrogen, bromine, hydroxy, methoxy, methyl, n-butyl and n-butyloxy;
R3 is selected from hydrogen, bromine, methoxy, —NO2, phenyl, —NHCOCH3, —NHCO-p-tolyl, —NHSO2CH3, —NHSO2-p-tolyl, COOCH3, —CO—R9 and aminoguanidyl-hydrazone;
R4 is selected from hydrogen and methyl;
R5 is hydrogen;
R6 is selected from hydrogen, methyl, n-butyl and benzyl;
R7 is hydrogen, or
R5 and R6, together with the two nitrogen atoms to which they are bound, form a cycle selected from imidazolino, tetrahydropyrimidino and benzimidazolino; or
R6 and R7, together with the nitrogen atom to which they are bound, form a pyrrolidino or a piperidino group;
R9 is selected from groups of Formula (IIIa), (IIIb), (IIIc), (IIId) and (IIIe)
wherein the asterisk (*) indicates the linking bond.
Preferred compounds according to the invention are compounds of Formula (I) wherein:
Additional, preferred compounds according to the invention are compounds of formula (I) wherein:
Most preferred compounds are compounds 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2i, 2l, 2v and 2z. Any type of salt of the compounds of Formula (I) can be prepared, for instance in synthetic steps or for purification reasons and all salts are included in the scope of protection of the present invention.
According to a preferred embodiment, the salt of the compound of Formula (I) is preferably a pharmaceutically acceptable salt.
The compounds of Formula (I) can form salts with a variety of organic and inorganic acids. Such salts include those formed with hydrochloric acid, hydrobromic acid, hydroiodic acid, methanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid and others (e.g., nitrates, phosphates, borates, tartrates, citrates, succinate, benzoates, ascorbates, salicylates and the like). Such salts may be formed as known to the skilled in the art.
Preferred salts are hydrochloric acid, hydrobromic acid, hydroiodic acid and acetic acid salts.
The compound of Formula (I) may be prepared by any suitable synthetic method.
In the present disclosure, even if not expressly indicated, the compound Formula (I) may always be in the form of one of its salts and/or solvates.
Any possible stereoisomer as well as possible crystalline forms of the compound of Formula (I) and its salts or solvates are included in the scope of the invention.
According to one of its aspects, the present invention relates to a process for the preparation of a compound of Formula (I) or one of its salts or solvates, which comprises reacting a compound of Formula (IV)
wherein R1, R2 and R3 are as above defined, with a compound of Formula (V)
or a salt thereof, wherein R4, R5, R6, and R3 are as above defined, in a suitable solvent, and isolating the compound of Formula (I) thus obtained, optionally purifying and/o optionally converting it in a salt or solvate thereof.
The starting compounds of Formula (IV) and (V) are known compounds or can be prepared by methods known in the art.
Examples of their preparation is given in the following Schemes and Experimental Section.
The compound of Formula (V) is used in a molar amount which is at least double of the amount of the compound of Formula (IV).
According to a preferred embodiment, the compound of Formula (V) is used in a salified form, for instance in the form of hydrobromide, hydrochloride or hydroiodide salt.
The above reaction may be carried out at a temperature ranging from 40° C. to the reflux temperature of the reaction mixture, preferably from 60° C. to the reflux temperature of the reaction mixture, advantageously at about 80° C.
The solvent is preferably a lower alcohol, preferably ethanol. Other solvents may however be used in the process of the invention.
Preferably, a catalytic amount of an acid may be used in the above reaction, preferably of hydrochloric or acetic acid.
The reaction is completed in a few hours, for instance 1-12 hours. The skilled in the art is anyway perfectly able to check the development of the reaction with conventional methods, for instance chromatographic techniques, such as thin layer chromatography (TLC).
The compound of Formula (I) may be isolated with common techniques, for instance by filtration or extraction with appropriate solvents from the reaction mixture.
If necessary or desired, the compound of Formula (I) may be purified by conventional techniques, such as chromatography, crystallization etc.
Compound of Formula (I) wherein R3 is an aminoguanidyl-hydrazone group, may be also prepared by reacting a compound of Formula (VI)
in lieu of the compound of Formula (IV), with the compound of Formula (V) as above defined, in the same reaction conditions above disclosed.
As a comparative example, the compound of Formula (VII) was prepared,
by reacting compound of Formula (VIII)
with the compound of Formula (V) as above defined, in the same reaction conditions above disclosed. Compound (VIII) is disclosed in WO95/19767 as an inflammation modulator.
In the following synthetic schemes, the dashed lines indicate the delocalization of the charge between the two salified nitrogens.
Reagents and conditions of synthesis are given in the Experimental Section of the description.
Compound 2aa (R═CH2Ph) can be prepared as shown in Scheme 3, starting from the corresponding 5-benzyl-phenyl 1,3-dicarboxylic acid 9aa.
Compounds 2bb (R═CH2CH2NH2) and 2cc (R=p-OMePh) can be prepared as shown in Scheme 7, using the corresponding acylating agents in step c, and obtaining respectively 10bb and 10cc.
Compounds 2dd (COOCH2CH2NH2 ester) and 2ee (R=2-morpholinylethyl ester) can be prepared as shown in Scheme 9, by hydrolysing compound 7p and esterifying it with N-protected ethanolamine or with N-hydroxyethyl morpholine.
Compounds 2ff (diethylamide), 2gg (morpholinoamide), 2hh (4′-methylpiperazinylamide), 2ii (4′-(3″-nitrophenyl)piperazinylamide), 2jj (4′-(4″-methoxyphenyl)piperazinylamide) and 2kk (4′-(4″-chlorophenyl)piperazinylamide)) can be prepared as shown in Scheme 10, by substituting N-phenylpiperazine with the appropriate amidation reagent.
Compound 2ll (RX═—(CH2)5—) can be prepared as shown in Scheme 12, by using piperidine as an amidating reagent in step a to yield 14ll.
Compound 2 mm (RX═—(CH2)5—) can be prepared as shown in Scheme 14, by using a tetrahydropyrimidine-containing aminoguanidine as an aminoguanidylating reagent in step a to yield 2 mm.
The use of compounds of Formula (I) or pharmaceutically acceptable salts thereof, in therapy and diagnostics is a further subject-matter of the invention.
Compounds of Formula (I), especially the preferred compounds as above defined, showed an interesting retromer stabilizer activity and are useful in the treatment and/or prevention of several neurological diseases, such as for instance amyotrophic lateral sclerosis (ALS), Charcot-Marie Tooth disease, Alzheimer's disease (AD), Parkinson's disease (PD) and other neurological diseases, wherein retromer stabilizing activity is beneficial.
For their use in therapy, compounds of Formula (I) or one of their pharmaceutically acceptable salts, are preferably administered in the form of pharmaceutical compositions.
According to one of its aspects, the invention relates to a pharmaceutical composition comprising at least one compound of Formula (I), or one of its pharmaceutically acceptable salts or solvates, preferably in combination with at least one pharmaceutically acceptable carrier and/or excipient.
The compositions of the invention include all the compositions where the compound of the present invention is comprised in an amount which is effective in achieving its intended aim. Although the individual needs vary, determining optimal ranges of effective amount per each component is known to persons skilled in the art. The administered dose will therefore depend on age, health and weight of the recipient, type of treatment, frequency of the treatment and nature of the desired effect.
Typically, the compound can be administered daily to mammals and in particular to humans, orally at a dose between 1 to 100 mg/kg, or an equivalent amount of one of its pharmaceutically acceptable salts, of the body weight of the treated mammal. Preferably, about 1 to 100 mg/kg are administered orally. For the intramuscular administration, the dose is generally half of the oral dose. For example, an acceptable intramuscular dose would be about 0.5 to 50 mg/kg, and more preferably, about 0.1 to 10 mg/kg.
Preferably the compositions, which can be administered by oral and parenteral route, are those known to the person skilled in the art, e.g., they will be in the form of: tablets, dragees, prolonged release pills and capsules, mouthwashes, gels, liquid suspension, hair dyes, hair gels, shampoos and other preparations which can be administered rectally, such as suppositories, as well as acceptable solutions for the parenteral, topical or oral administration, containing about 0.01 to 99%, preferably 0.25 to 75% of active ingredient(s).
The pharmaceutical compositions can be administered by any means to achieve the desired aim. For example, the administration can be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or oral.
The pharmaceutical preparations of the present invention are produced as is known to one skilled in the art, e.g., by conventional mixing, granulation, sugar-coating, dissolution, or freeze-drying processes. Therefore, the pharmaceutical preparations for oral use can be obtained by combining the active ingredient with solid excipients, optionally by grinding the resulting mixture and treating the granule mixture, after adding suitable excipients, if desired and necessary, in order to obtain tables or dragee cores.
Suitable excipients are, in particular, diluents and fillers such as sugars, celluloses and calcium phosphate, as well as binders such as, e.g., starch, gelatin, cellulose and polyvinyl pyrrolidone. If desired, disaggregating agents can be added as the above mentioned starches, cross-linked polyvinyl pyrrolidone, agar and alginic acids and the salts thereof. Other useful excipients are free-flowing agents and lubricants, e.g., silica, talc, stearic acid and salts thereof and polyethylene glycol. The cores of the pills and the tablets can have suitable coating that, if desired, is resistant to digestive juices. In order to produce the coatings resistant to digestive juices, it is possible to use suitable polymers as it is well known to the person skilled of the art.
Other pharmaceutical compositions that can be used orally include hard capsules made of gelatin, as well as sealed, soft capsules made of gelatin and a plasticizer such as, e.g., glycerol or sorbitol. Hard capsules can contain the active ingredient in the form of granules which can be mixed with diluents, binders and/or lubricants and, optionally, stabilizers. In the soft capsules, the active ingredient is preferably dissolved or suspended in a suitable liquid, such as fatty oils or liquid paraffin. Moreover, stabilizers can be added.
Possible pharmaceutical compositions that can be used rectally include, e.g., suppositories, consisting of a combination of one or more active ingredients together with suitable excipients. Such suitable excipients are, e.g., natural or synthetics triglycerides, or paraffin hydrocarbons.
Formulations suitable for the parenteral administration include aqueous solutions of the active ingredient in soluble form, preferably in water. Moreover, suspensions of the active ingredients can be administered, such as suspensions of suitable injectable oils. Suitable lipophilic solvents or carriers include fatty oils or esters of synthetic fatty acids. Aqueous injections can contain substances increasing the viscosity of the suspension, such as sorbitol and dextran. Optionally, the suspension can also contain stabilizers.
In addition to be administered in pharmaceutical compositions, the compounds of Formula (I) as described above may also be administered as pure compounds.
According to another of its aspects, the invention relates to the use of a compound of Formula (I) or one of its pharmaceutically acceptable salts or solvates, in the treatment and/or prevention of several neurological diseases, such as for instance amyotrophic lateral sclerosis (ALS), Charcot-Marie Tooth disease, Alzheimer' s disease (AD), Parkinson's disease (PD) and other neurological diseases, wherein retromer stabilizing activity is beneficial.
According to another of its aspects, the invention relates to the use of a pharmaceutical composition of the invention in the treatment and/or prevention of several neurological diseases, such as for instance amyotrophic lateral sclerosis (ALS), Charcot-Marie Tooth disease, Alzheimer's disease (AD), Parkinson's disease (PD) and other neurological diseases, wherein retromer stabilizing activity is beneficial.
According to another of its aspects, the invention relates to a compound of Formula (I) or of a pharmaceutically acceptable salt or solvate thereof, for its use as a retromer stabilizer and as a neuroprotectant.
According to another of its aspects, the invention relates to a method for the treatment and/or prevention of several neurological diseases, such as for instance amyotrophic lateral sclerosis (ALS), Charcot-Marie Tooth disease, Alzheimer' s disease (AD), Parkinson's disease (PD) and other neurological diseases, wherein retromer stabilizing activity is beneficial, said method comprising administering, to a subject in need thereof, an effective amount of a compound of Formula (I) or one of its pharmaceutically acceptable salts or solvates, or of a pharmaceutical composition according to the invention.
In therapy, especially in the treatment and/or prevention of neurological diseases such as those above disclosed, a compound of Formula (I) may be either administered alone, or in combination with another compound of Formula (I) and/or with another therapeutic agent useful in said treatment and/or prevention.
According to another of its aspects, the invention relates to the use of a compound of Formula (I) or one of its salt or solvates in diagnostics.
According to another of its aspects, the invention relates to a diagnostic tool comprising a compound of Formula (I) or one of its pharmaceutically acceptable salts. The invention is further disclosed in the following Experimental Section for illustrative and not limiting purposes.
Experimental Section
Biochemical Characterization of Amino Guanidine Hydrazones
Several experimental evidence shows that retromer has a neuroprotective role, especially in age-related neurodegenerative disorders. For example, the disruption of this ancient multimeric complex has been observed in a number of diseases including Parkinson's disease (PD) and Alzheimer's disease (AD) (Small and Petsko 2015). In PD a mutation of VPS35 (D620N) is dominant for a late onset form of the disease (Vilarino-Guell, Wider et al. 2011). Furthermore, VPS35 levels are significantly reduced in brain regions selectively vulnerable to AD (Small, Kent et al. 2005).
Thus, novel pharmacological chaperones were synthesized according to the invention, which are capable to significantly increase the levels of VPS35, and consequently of the retromer complex, would be promising compounds to be tested for their neuroprotective effects. To functionally screen the majority of synthetized amino guanidine hydrazones of the invention and determine their effects on the retromer complex, we incubated Neuro2a cells with each synthetized molecule at the concentration of 10 μM. We included controls in each experiment using the standard Compound R55 (Mecozzi, Berman et al. 2014) that efficiently and dose-dependently increased VPS35 levels after 48 hours of incubation (
The test was executed in triplicate for each tested compound of the invention, cells lysates were established after 48 hours' incubation, and levels of VPS35 were normalized on the housekeeping gene β-Actin by Western blot. We observed a preliminary SAR among the compounds.
Most of the tested compound of the invention were able to increase the levels of VPS35 levels, although with a certain variability. In particular, Compound 2a was able to consistently increase VPS35 levels with a limited variability among replicates (Table 1).
Table 1 shows mean fold changes±s.d. from independent cultures (n=3) receiving each compound at 10 μM for 48 hours. Fold changes are calculated over normalized VPS35 levels measured in vehicle-treated cells.
As a comparison, compound (VIII) was also tested in the same conditions above. The result of the test are reported in Table 2
Retromer Levels in Amyotrophic Lateral Sclerosis
While the role of retromer is assessed in several neurodegenerative diseases (Small, Kent et al. 2005; Vilarino-Guell, Wider et al. 2011) nothing is known about any putative role in ALS. Misfolded aggregated proteins are thought to participate to pathogenic mechanisms leading motor neurons (MNs) cell death (Cleveland 1999). For example, TAR DNA binding protein-43 (TDP-43) are identified as a core component of ubiquitinated inclusions in a large number sporadic ALS patients (Arai, Hasegawa et al. 2006). Retromer has been implicated in AD which is featured by amyloid beta plaques and aggregates of Aβ protein in the brain of patients, as well as mutations in VPS35 are identified in autosomal dominant PD patients (MacLeod, Rhinn et al. 2013).
We determined VPS35 levels in MNs of mice carrying the hSOD1G93A (mouse strain B6.Cg-Tg (SOD1G93A) 1Gur/J, here after G93A) and their wild type (WT) litters (Gurney, Pu et al. 1994). We scored VPS35 immunoreactivity in lumbar spinal cords (SCs) of preclinical G93A (day 30, 60 and 90) mice. Sections were double labelled with marker NeuN to identify MNs (
Retromer Stabilization Increases Cell Survival of G93A Transfected Cells
Pathological alteration of the Golgi apparatus, such as its fragmentation, has been described in many neurodegenerative disorders including ALS. Neuro2a cells transfected with plasmids encoding the G93A mutant form of the SOD1 show alteration of the Golgi transport (Soo, Halloran et al. 2015). Along the same lines, Golgi fragmentation precedes neuromuscular denervation and axon retraction in ALS mice (van Dis, Kuijpers et al. 2014). We transfected Neuro2a cells with plasmids encoding the G93A mutant form of the human SOD1 gene (hereafter G93A) in presence or in absence of compound 2a (10 μM). Cells were harvested after 48 hours, labelled for Golgin-97 and scored for the distribution of trans-Golgi. Untreated cells and cells receiving the control plasmid encoding the GFP showed a compact distribution of Golgin-97, while percentages of cells showing partial or complete fragmentation of the Golgi complex were greatly increased in cells transfected with the G93A plasmid. However, administration of the compound 2a significantly diminished these percentages, reconstituting the Golgin-97 distribution that we observed in control cells (
We next used further plasmids to perform a VPS35 short interference in Neuro2a cells, with the final goal of reducing VPS35 levels in cells that were also receiving G93A plasmids. We firstly established the efficacy of plasmids to inhibit VPS35 expression. We transfected 3 different commercial plasmids designed to interfere Vps35 mRNA in Neuro2a cells. While all of them were able to reduce VPS35 levels, the Sh56 plasmid resulted in 60% reduction of VPS35 mRNA and protein levels (
We next transfected Neuro2a cells with G93A along with Sh56 or scramble plasmids. The simple reduction of VPS35 levels in Neuro2a cells did not affect their survival. As expected, cell survival was reduced in Neuro2a cells receiving G93A plasmids (
We next assayed cytotoxicity by measuring lactate dehydrogenase (LDH) release with a LDH-Glo™ cytotoxicity assay. Cells were treated with plasmids encoding G93A and the Sh56-RNAi sequence with or without compound 2a, while supernatants were used to measure LDH contents. Overexpression of G93A increased the release of LDH by Neuro2a cells, when compared with cells transfected with LacZ-encoding plasmids or with LacZ+Sh56 plasmids (
Compound 2a increases the stability of the retromer complex in vitro. We further validated this concept by measuring VPS35 degradation rates by a cycloheximide (CHX) chase assay. Neuro2a cells received vehicle or lead 2a (10 μM) for 24 hours, then CHX was added to the medium, and VPS35 levels were measured by western blot. We observed that vehicle-treated cells exhibited a drop of VPS35 levels (˜40%) after 8 hours of CHX treatment. Conversely, pre-treating cells with lead 2a protected VPS35 from degradation (
We next asked whether the compound 2a can cross the blood brain barrier (BBB) of the brain. We run a pharmacokinetic assay on C57BL6J mice that received daily intraperitoneal injections of compound 2a (10 mg/Kg/day). Mice were sacrificed after 1, 7, 15 and 30 days saline perfused to avoid the CNS contamination of compounds contained in blood circulation and brains used for the determination of compound 2a levels by mass spectrometry. We detected a significant amounts of compound 2a in the brain confirmed the ability of compound 2a to access the CNS (
In all these experiments we did not observe cell toxicity in Neuro2a as well as any adverse events in mice injected with the compound 2a. Nonetheless, we assessed the cytotoxicity of compound 2a, measuring cell survival of Neuro2a cells exposed to increasing concentrations of 2a. The calculated LD50 was ˜260 μM (
We tested the specificity of compound 2a in an assay based on H2O2-induced chromosomal breaks that lead to DNA fragmentation and cell death. In principle such DNA lesions are the cause of cell death according to a process that is not influenced by the retromer. Thus, our leads should not be able to modulate rates cell survival in Neuro2a cells receiving H2O2. We incubated Neuro2a cells with 200 μM of H2O2 along with compounds 2a, 2c, 2d and 2g (10 μM). Comparing cell survival between cells receiving H2O2 with or without our compounds did not result in any significant difference (
Compound 2a Attenuates Clinical Impairment and MN Cells Death in G93A Mice
Having established that compound 2a crosses the BBB we started to treat mice carrying the G93A mutant form of the SOD1. Starting from day 30, G93A mice and their WT litters were daily injected with compound 2a (10 mg/kg) or vehicle. Mice receiving 2a displayed a small drop of the body weight when compared with mice receiving the vehicle treatment, although such reduction did not reach statistical significance (
Mice were sacrificed at day 100, and lumbar SCs assayed for studying MNs cell morphology and numbers. We initially scored NeuN+ neurons in the ventral horn of the SC to monitor bona fide motor neurons. Mice receiving compound 2a displayed significantly higher numbers of motor neurons than vehicle-treated G93A mice (
We next performed histopathological analysis of sciatic nerves from WT control and G93A mice receiving compound 2a or vehicle. Consistent with the advanced stage of the disease, G93A mice at day 100 displayed axonal degeneration in sciatic nerves when compared with their WT litters (
We next assayed retromer proteins labelling sections of mice sacrificed at day 100 for VPS35 and NeuN. The analysis of confocal stacks of lumbar ventral horns revealed that the fluorescent intensity levels of VPS35 (measured as MFI) were significantly increased in G93A mice receiving compound 2a, if compared with the intensity measured in vehicle-treated G93A mice (
We next investigated Cathepsin D (CTSD) expression and distribution in G93A mice, since it is known that the trafficking of this lysosomal enzyme involves the retromer complex (Seaman 2004). In humans, maturation of CTSD starts with a pre-pro-CTSD that is converted to pro-CTSD (52 kDa) in the endoplasmic reticulum. Recycling of pro-CTSD, and its downstream migration to acidic compartments involves CI-MPR and the retromer pathway. In late endosomes, pro-CTSD is further processed to an intermediate form (48 kDa), and finally converted to mature CTSD (34 kDa) in lysosomes (Laurent-Matha 2006). The inactivation of VPS35 or the knockdown of VPS26 affect the maturation of CTSD. We scored CTSD levels in equal amounts of lumbar SC extracts obtained from vehicle-treated WT, G93A mice receiving the vehicle, and G93A treated with lead 2a. We observed that the mature CTSD/pro-CTSD ratio was slightly, but not significantly, reduced in SC extracts of vehicle-treated G93A mice when compared with WT litters. Possibly, we did not see a reduction of mature CTSD in total protein lysates because at day 100 glial cells of G93A mice express high amounts of mature CTSD (
To finally prove that compound 2a can increase the stabilization of the retromer complex we probed SCs extracts for VPS35 and VPS26. These experiments further confirmed the ability of 2a to increase VPS35 and VPS26b levels in G93A mice (
Ubiquitinated inclusions accumulate in MNs of sALS and fALS patients and in lumbar SCs of ALS mice (Kabashi 2001). High levels of ubiquitinated proteins may represent an overwhelmed response of MNs to protein misfolding and aggregation, as well as a reduction of lysosome functionality. We characterized poly-ubiquitinated proteins in lumbar SCs protein extracts by 10% denaturating PAGE. We included in our experiments protein extracts from Neuro2a cells treated with the proteasome inhibitor Bortezomib (BTZ), able to increase poly-ubiquitination, as an experimental control (
SOD1 mutations increase the propensity of this protein to aggregate in cells (Johnston 2000). We checked whether compound 2a could reduce the accumulation of G93A aggregates in vivo. Starting at post-natal day 83, we injected G93A mice and WT littermates daily with compound 2a (10 mg/kg). We scored motor performances in both vehicle- and compound 2a-treated G93A mice until day 103. Mice receiving compound 2a displayed a general attenuation of motor deficits that confirmed our previous observations (
These results indicate that ubiquitinated proteins (
Neuro2a-7B11 cells were incubated with increasing concentrations of compound 2a (0.1-100 μM) as well as with the control molecules Bafilomycin Al and Torin-1. After 48 hours the GFP/phluorin signal, which is ph-sensitive, was measured by flow cytometry and fold changes calculated over untreated cells. As expected, we observed augmented fluorescence blocking the fusion of autophagosomes with lysosomes by Bafilomycin A1 treatment. On the other hand, fluorescence signals were significantly dampened by the mTORC1/2 inhibitor Torin 1 (
We next determined whether a reduction of VPS35 levels in Neuro2a cells could affect lysosomal homeostasis, mimicking what we and others observed in the ALS mouse model (Cheng 2018). We transfected Neuro2a cells with plasmids encoding the Sh56 RNAi to downregulate VPS35. After 24 hours of starvation, we analyzed the lysosomes using the Lyso-tracker. We observed an enlarged and abnormal distribution of lysosomes in cells with VPS35 interference by Sh56 (
We observed that the molecular structure featuring compound 2a is reminiscent of the patented compound CNI-1493 (N,N′-bis [3,5 -bis [1(aminoiminomethyl) hydrazono]ethyl]phenyl]decanediamide tetrahydrochloride (CAS Reg. No. 164301-51-3)), (
Loss of VPS35 Expression in Post Mortem ASL Samples and in iPSCs-Derived MNs from Familiar ALS Fibroblasts
We next investigated VPS35 in post mortem cerebral alpha-MNs located in the ventral horn of the human SC (
We next generated iPSC lines from fALS patients carrying three SOD1 gene variants (ALS 8: p.Asn65Ser; ALS 13: p.Leu144Phe; ALS 27: p.Asp97Asn), and from three age and sex-matched healthy volunteers. Efficient reprogramming was assessed labelling cultures for OCT3/4, SOX2, NANOG, TRA1-60 and SSEA4 (not shown). According to a published protocol we induced iPSCs lines to acquire the phenotype of caudal neuroepithelial progenitors expressing OLIG2+PAX6− (Du 2015). We differentiated these progenitors to acquire the OLIG2−ISLET1+ cell phenotype. We scored the percentages of ISLET1+ out the total number of nuclei, and we observed similar rates of cell differentiation among controls and ALS samples (controls: 81.6%±9; ALS: 81.8%±12). Using immunofluorescence and WBs, we observed at day 33 a substantial reduction of VPS35 levels in ISLET1+ MNs deriving from ALS patients (
Conclusion
The retromer complex is highly conserved in eukaryotes and it has been associated to several neurodegenerative disorders, including PD and AD, although, never investigated in ALS. Using G93A transgenic mice we observed that CRC protein levels are substantially reduced in ALS motor neurons. Interestingly, such reduction occurs before the appearance of any signs of the disease and, above all before motor neurons degeneration. Parallel experiments performed on post mortem SCs from ALS patients further indicate the reduction of VPS35 and VPS26 levels in ventral horn bona fide motor neurons. These results suggest that retromer failure can anticipate the degeneration of these cells and therefore strategies fostering retromer stabilization can be neuroprotective in ALS. Compound 2a is able to significantly increase VPS35 levels and, as consequence, the retromer functionality. Such compound rescues Golgi fragmentation in vitro and in vivo and, above all, reduced rates of cell death in Neuro2a cell line receiving G93A plasmids. In a preclinical experiment in which we injected G93A mice with compound 2a, we observed that by fostering retromer functionality in vivo we were able to reduce MNs degeneration, demyelination and peripheral fibers loss. Consequently, G93A mice receiving compound 2a display increased locomotion performances. Furthermore, the increased locomotion performances were mirrored by increased levels of VPS35 and VPS26 that we scored in MNs as well as by increased levels of two retromer cargos, namely CI-MPR and Sortilin. We also observed a substantial increase of mature CTSD in spinal cords of G93A treated with compound 2a. The general increase of CTSD and possibly of other lysosomial enzymes decreased the accumulation of polyubiquitianted proteins, a pathological hallmark of ALS. Interestingly, we also observed a reduction of aggregated SOD1 in these mice. These findings open a new avenue for the treatment of ALS.
Synthetic Methods
General Procedures. 1H NMR spectra were recorded on a Bruker Avance 400 MHz instrument in CDCl3, CD3OD or D2O as solvent at 400 MHz. 13C NMR spectra were recorded in CDCl3, CD3OD or D2O as solvent at 101 MHz. Coupling constants are given in Hertz and are rounded to the nearest 0.1 Hz. LC-MS data were collected with a Waters Acquity™ Ultra performance LC equipped with an Acquity UPLC™ HSS T3 column (2.1 mm×50 mm, 1.8 μm) and a SQD detector. Purifications were carried out either by flash chromatography on silica gel (particle size 60 μm, 230-400 mesh), on Kieselgel, or by Biotage™ flash chromatography [Biotage columns Si-25-M (150×25 mm; silica gel (40-63 μm), flow rate 25 mL/min)], or by Biotage™ C18 reverse phase chromatography [Biotage column C18HS (150×25 mm; KP-C18-HS (35-70 μm), flow rate 25 mL/min)]. Some final compounds were purified by C18 reverse phase semi-preparative HPLC using a Waters X-Bridge column (19 mm×15.0 cm, 5 μm). Melting points were determined with a Stuart Scientific SMP3 melting point apparatus. Solvents were distilled and dried according to standard procedures, and reactions requiring anhydrous conditions were performed under nitrogen or argon atmosphere.
Substituted isophthalic acids, reduction to alcohols, general procedure A (8c, 8g-j, 8p). A suspension of substituted isophthalic acid (2.4-3.3 mmoles, 1 eq.) in anhydrous THF (≈1 mL/mmol) was vigorously stirred and cooled to 0° C. under nitrogen atmosphere. A solution of 1M BH3 in THF (9.6-13.2 mL, 4 eqs.) was added dropwise in 1 hr. The reaction mixture was allowed to warm slowly to rt, and was stirred for additional 48 hrs. MeOH (7-10 mL) was added dropwise to the reaction mixture and the solvent was evaporated under reduced pressure. Addition of MeOH and evaporation was repeated (7-10 mL×3 times). Then, the crude was dissolved in EtOAc (40 mL), washed with saturated NaHCO3 (2×20 mL) and brine (10 mL). The organic layer was dried with anhydrous Na2SO4, filtered and evaporated at reduced pressure. The resulting solid was purified by flash chromatography on silicagel (eluant mixture: n-hexane/EtOAc) to afford the corresponding pure substituted 1,3-bis-benzylic alcohol.
Substituted 1,3-bis-benzylic alcohols, oxidation to aldehydes, general procedure B (7c, 7g-j, 7k-p). Manganese dioxide (2.5-10 mmoles, 5 eqs.) was added under vigorous stirring to a solution of a substituted 1,3-bis-benzylic alcohol (0.5-2 mmoles, 1 eq.) in chloroform (≈5 mL/mmol), and the reaction mixture was heated at reflux. After overnight reflux/stirring, the reaction mixture was cooled to rt, and filtered through a Celite pad. The pad was thoroughly washed with CH2Cl2 (40 mL), the combined organic phase was concentrated in vacuo, and the residue was purified by flash chromatography on silicagel (eluant mixture: n-hexane/EtOAc) to afford the corresponding pure substituted isophthalaldehyde.
Substituted isophthalaldehydes, condensation with aminoguanidine, general procedure C (2a-z,). Aminoguanidine hydrochloride (1-2 mmoles, 1 eq.) and aqueous 1N HCl (3-5 drops, catalytic) were sequentially added to a warm, vigorously stirred solution of a substituted isophthalaldehyde (0.5-1 mmoles, 1 eq.) in absolute EtOH (≈10 mL/mmol). The reaction mixture was refluxed for 2-8 hours, with TLC monitoring (8:2 CH2Cl2:MeOH with a few AcOH drops). The reaction mixture was then cooled to rt, the solid product was filtered, washed with cold EtOH (10 mL), with Et2O (10 mL), and dried in vacuum to yield the corresponding pure N-unsubstituted 1,3-bis-amino guanidine phenyl hydrazine as a dihydrochloride salt.
Substituted isophthalaldehydes, condensation with N-substituted aminoguanidines, general procedure D (2a-z). A N-substituted aminoguanidine salt (0.6-1.8 mmoles, 1 eq.) was added to a warm, vigorously stirred solution of substituted isophthalaldehyde (0.3-0.9 mmoles, 1 eq.) in absolute EtOH (≈7.5 mL/mmol). The reaction mixture was refluxed for 8 hours, with TLC monitoring (8:2 CH2Cl2:MeOH with a few AcOH drops). The reaction mixture was then cooled to rt, the solid product was filtered, washed with cold EtOH (10 mL), with Et2O (10 mL), and dried in vacuum to yield the corresponding pure N-substituted 1,3-bis-amino guanidine phenyl hydrazine as a dihydrohalide salt (n some cases, a preparative HPLC separation was needed).
1,3-Bis-amino guanidine phenyl hydrazone, dihydrochloride salt (2a). The title compound (280 mg, 0.88 mmoles, 88% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from isophthalaldehyde (134 mg, 1 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (221 mg, 2 mmoles, 1 eq.) in absolute ethanol (10 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.26 (bs, 2H, NH), 8.32 (s, 1H, H2), 8.22 (s, 2H, H1′, H3′), 8.15-7.58 (bs, 6H, NH), 7.96 (d, J=7.6 Hz, 2H, H4), 7.53 (t, J=7.6 Hz, 1H, H5). 13C_NMR (100 MHz, D2O): δ(ppm) 154.7, 147.3, 133.2, 129.2, 126.6. MS (ESI+): m/z 247.30 [M+H+]. Calculated MS, C10H14N8: 246.28.
2-Bromo-1,3-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2b). The title compound (280 mg, 0.88 mmoles, 88% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 2-bromo isophthalaldehyde (197 mg, 0.93 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (205 mg, 1.86 mmoles, 1 eq.) in absolute ethanol (9 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.22 (bs, 2H, NH), 8.65 (s, 2H, H1′, H3′), 8.38 (d, J=7.7 Hz, 2H, H4), 8.30-7.60 (bs, 6H, NH), 7.51 (t, J=7.7 Hz, 1H, H5). 13C_NMR (100 MHz, D2O): δ(ppm) 155.6, 146.7, 132.9, 130.0, 126.9. MS (ESI+): m/z 325.29 and 327.29 [M+H+]. Calculated MS, C10H13BrN8: 324.18 and 326.18.
1,3-Bis-(hydroxymethyl)-4-bromobenzene (8c). The title compound (455 mg, 2.10 mmol, 86% yield) was prepared from 4-bromoisophthalic acid (590 mg, 2.41 mmoles, 1.0 eq.) and 1M BH3 in THF (10 mL, 10.00 mmoles, ≈4 eqs.) in THF (2.5 mL), following the experimental protocol of general procedure A. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 7.39 (m, 3H, H2, H5, H6), 5.39 (t, J=5.6 Hz, 2H, OH), 4.52 (d, J 5.6 Hz, 4H, H1′, H3′). MS (ESI+): m/z 218.12 [M+H+]. Calculated MS, C8H9BrO2: 217.04.
4-Bromo isophthalaldehyde (7c). The title compound (108 mg, 0.51 mmol, 87% yield) was prepared from 8c (128 mg, 0.59 mmoles, 1.0 eq.) and manganese dioxide (257 mg, 2.95 mmoles, 5 eqs.) in chloroform (3 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 10.40 (s, 1H, H1′), 10.06 (s, 1H, H3′), 8.42 (d, J=2.09 Hz, 1H, H2), 7.98 (dd, J=2.09 Hz, J=7.88 Hz, 1H, H6), 7.72 (d, J=7.88 Hz, 1H, H5). MS (ESI+): m/z 214.16 [M+H+]. Calculated MS, C8H5BrO2: 213.01.
4-Bromo-1,3-bis-amino guanidine phenyl hydrazone, dihydrochloride salt (2c). The title compound (178 mg, 0.44 mmoles, 88% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 4-bromo isophthalaldehyde (108 mg, 0.51 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (112 mg, 1.02 mmoles, 1 eq.) in absolute ethanol (5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.40 (bs, 2H, NH), 8.58 (d, J=2.0 Hz, 1H, H2), 8.54 (s, 1H, H1′), 8.20 (s, 1H, H3′), 8.20-7.60 (bs, 6H, NH), 7.98 (dd, J=2.0 Hz, J=8.4 Hz, H6), 7.78 (d, J=8.4 Hz, 1H, H5). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 157.0, 156.8, 146.3, 145.8, 134.8, 134.7, 133.8, 131.1, 128.5, 126.5. MS (ESI+): m/z 315.33 and 317.33 [M+H+]. Calculated MS, C10H13BrN8: 324.18 and 326.18.
1-Hydroxy-2,4-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2d). The title compound (224 mg, 0.67 mmoles, 77% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7d (132 mg, 0.87 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (193 mg, 1.75 mmoles, 1 eq.) in absolute ethanol (8 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.05 (bs, 2H, NH), 10.95 (bs, 1H, OH), 8.52 (s, 1H, H, H1′), 8.48 (d, J=1.8 Hz, 1H, H2), 8.12 (s, 1H, H3′), 8.20-7.40 (bs, 6H, NH), 7.95 (dd, J=8.6 Hz, J=1.8 Hz, 1H, H6), 7.05 (d, J=8.6 Hz, 1H, H5). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 158.8, 155.7, 155.4, 148.4, 147.4, 131.6, 129.4, 126.1, 119.3, 117.7. MS (ESI+): m/z 263.37 [M+H+]. Calculated MS, C10H14N8O: 262.28.
4-Methoxy isophthalaldehyde (7e). MeI (0.13 mL, 2 mmoles, 1 eq.) was added to a stirred mixture of 4-hydroxy isophthalaldehyde 7d (150 mg, 1 mmol, 1 eq.) and K2CO3 (420 mg, 3 mmoles, 1.5 eqs.) in dry DMF (5 mL) under a nitrogen atmosphere. The reaction mixture was stirred at r.t. for 24 hrs, then the solvent was removed at reduced pressure. The crude was dissolved in EtOAc (30 mL), washed with 5% aqueous NaOH (3×20 mL) and with brine (10 mL). The combined organic layer was dried on anhydrous Na2SO4, filtered and concentrated at reduced pressure. 4-Methoxy isophthalaldehyde 7e (105 mg, 0.60 mmoles, 60% yield) was obtained as a white solid pure enough to be used without further purification. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.50 (s, 1H, H1′), 10.01 (s, 1H, H3′), 8.35 (d, J=2.0 Hz, 1H, H2), 8.12 (dd, J=2.0 Hz, J=9.1 Hz, 1H, H6), 7.15 (d, J=9.1 Hz, 1H, H5), 4.00 (s, 3H, OMe). MS (ESI+): m/z 179.21 [M+H+]. Calculated MS, C9H8NO3: 178.17.
1-Methoxy-2,4-bis-amino guanidine phenyl hydrazone, dihydrochloride salt (2e). The title compound (126 mg, 0.36 mmoles, 79% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 4-methoxy isophthalaldehyde (75 mg, 0.46 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (101 mg, 0.92 mmoles, 1 eq.) in absolute ethanol (4.5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.12 (bs, 2H, NH), 8.51 (s, 1H, H1′), 8.49 (s, 1H, H2), 8.19 (s, 1H, H3′), 8.20-7.40 (m, 6H, NH), 8.00 (d, J=6.6 Hz, 1H, H6), 7.12 (d, J=6.6 Hz, 1H, H5), 3.92 (s, 3H, OMe). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 160.1, 156.9, 147.2, 142.7, 132.2, 127.4, 127.0, 123.0, 114.2, 56.2. MS (ESI+): m/z 277.44 [M+H+]. Calculated MS, C11H16N8O: 276.31.
4-(n-Butoxy) isophthalaldehyde (7f). n-BuBr (0.22 mL, 2 mmoles, 1 eq.) was added to a stirred mixture of 7d (150 mg, 1 mmol, 1 eq.) and K2CO3 (420 mg, 3 mmoles, 1.5 eqs.) in dry DMF (5 mL) under a nitrogen atmosphere. The reaction mixture was heated to 85° C. and stirred for 24 hrs, then cooled at r.t. After solvent removal at reduced pressure, the crude was dissolved in EtOAc (30 mL), washed with 5% aqueous NaOH (3×20 mL) and with brine (10 mL). The combined organic layer was dried on anhydrous Na2SO4, filtered and concentrated at reduced pressure. 4-(n-Butoxy) isophthalaldehyde 7f (155 mg, 0.75 mmoles, 75% yield) was obtained as a white solid pure enough to be used without further purification. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.50 (s, 1H, H′), 10.00 (s, 1H, H3′), 8.35 (d, J=2.0Hz, 1H, H2), 8.12 (dd, J=2.0 Hz, J=9.1 Hz, 1H, H6), 7.15 (d, J=9.1 Hz, 1H, H5), 4.23 (t, J=2.4 Hz, 2H, H4′), 1.91 (m, 2H, H4″), 1.56 (m, 2H, H4′″), 1.04 (t, J=7.8 Hz, 3H, H4″″). MS (ESI+): m/z 221.35 [M+H+]. Calculated MS, C12H14NO3: 220.23.
1-(n-Butoxy)-2,4-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2f). The title compound (166 mg, 0.42 mmoles, 80% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7f (110 mg, 0.53 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (118 mg, 1.06 mmoles, 1 eq.) in absolute ethanol (5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.12 (bs, 2H, NH), 8.51 (s, 2H, H1′, H1), 8.16 (s, 1H, H3′), 8.20-7.40 (bs, 6H, NH), 8.00 (d, J=8.7 Hz, 1H, H6), 7.12 (d, J=8.7 Hz, 1H, H5), 4.15 (t, J=2.4 Hz, 2H, H4′), 1.80 (m, 2H, H4″), 1.52 (m, 2H, H4′″), 0.99 (t, J=7.8 Hz, 3H, H4″″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 160.1, 156.9, 147.2, 142.7, 132.2, 127.4, 127.0, 123.0, 114.2, 69.1, 31.3, 19.3, 14.3. MS (ESI+): m/z 319.66 [M+H+]. Calculated MS, C14H22N8O: 318.38.
1,3-bis-(hydroxymethyl)-5-methyl benzene (8g). The title compound (410 mg, 2.70 mmol, 81% yield) was prepared from 5-methyl isophthalic acid (600 mg, 3.33 mmoles, 1.0 eq.) and 1M BH3 in THF (13.3 mL, 13.3 mmoles, 4 eqs.) in THF (3 mL), following the experimental protocol of general procedure A. 1H_NMR (400 MHz, CDCl3): δ(ppm) 6.96 (s, 1H, H2), 6.80 (s, 2H, H4), 4.62 (s, 4H, H1′, H3′), 3.81 (s, 3H, Me), 2.11 (s, 2H, OH). MS (ESI+): m/z 153.21 [M+H+]. Calculated MS, C9H12O2: 152.19.
5-Methyl isophthalaldehyde (7g). The title compound (151 mg, 1.02 mmol, 77% yield) was prepared from 8g (200 mg, 1.32 mmoles, 1.0 eq.) and manganese dioxide (575 mg, 6.60 mmoles, 5 eqs.) in chloroform (6.5 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.15 (s, 2H, CHO), 8.25 (s, 1H, H2), 8.00 (s, 2H, H4), 2.53 (s, 3H, Me). MS (ESI+): m/z 149.29 [M+H+]. Calculated MS, C9H8O2: 148.16.
5-Methyl-1,3-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2g). The title compound (179 mg, 0.54 mmoles, 77% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7g (104 mg, 0.70 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (155 mg, 1.40 mmoles, 1 eq.) in absolute ethanol (7 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.25 (bs, 2H, NH), 8.21 (s, 2H, H1′, H3′), 8.10 (s, 1H, H2), 8.00-7.50 (bs, 6H, NH), 7.82 (s, 2H, H4), 2.41 (s, 3H, Me). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 156.9, 147.4, 139.9, 135.2, 130.9, 125.5, 21.4. MS (ESI+): m/z 261.45 [M+H+]. Calculated MS, C11H16N8: 260.28.
1,3-bis-(hydroxymethyl)-5-bromo benzene (8h). The title compound (475 mg, 2.19 mmol, 88% yield) was prepared from 5-bromo isophthalic acid (610 mg, 2.49 mmoles, 1.0 eq.) and 1M BH3 in THF (10 mL, 10 mmoles, ≈4 eqs.) in THF (2.5 mL), following the experimental protocol of general procedure A. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 7.43 (s, 2H, H4), 7.28 (s,1H, H2), 4.67 (s, 4H, H1′, H3′), 1.79 (s, 2H, OH). MS (ESI+): m/z 217.20 and 219.20 [M+H+]. Calculated MS, C8H9BrO2: 216.15 and 218.15.
5-Bromo isophthalaldehyde (7h). The title compound (97 mg, 0.45 mmol, 76% yield) was prepared from 8h (128 mg, 0.59 mmoles, 1.0 eq.) and manganese dioxide (257 mg, 2.95 mmoles, 5 eqs.) in chloroform (3 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.06 (s, 2H, CHO), 8.30 (t, J=1.4 Hz, 1H, H2), 8.26 (d, J=1.4 Hz, 2H, H4). MS (ESI+): m/z 213.24 and 215.24 [M+H+]. Calculated MS, C8H5BrO2: 212.12 and 214.12.
5-Bromo-1,3-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2h). The title compound (133 mg, 0.33 mmoles, 91% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7h (75 mg, 0.35 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (78 mg, 0.70 mmoles, 1 eq.) in absolute ethanol (3.5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.31 (bs, 2H, NH), 8.27 (d, J=1.2 Hz, 2H, H4), 8.22 (t, J=1.2 Hz, 1H, H2), 8.17 (s, 2H, H1′, H3′), 8.20-7.60 (bs, 6H, NH). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 156.9, 145.8, 137.5, 132.0, 127.8, 124.1. MS (ESI+): m/z 327.45 and 329.45 [M+H+]. Calculated MS, C10H13BrN8: 324.25 and 326.25.
3,5-bis-(hydroxymethyl)-1-methoxybenzene (8i). The title compound (377 mg, 2.24 mmol, 80% yield) was prepared from 5-methoxy isophthalic acid (550 mg, 2.80 mmoles, 1.0 eq.) and 1M BH3 in THF (11.2 mL, 11.2 mmoles, 4 eqs.) in THF (2.8 mL), following the experimental protocol of general procedure A. 1H_NMR (400 MHz, CDCl3): δ(ppm) 6.96 (s, 1H, H2), 6.80 (s, 2H, H4), 4.62 (s, 4H, H1′, H3′), 3.81 (s, 3H, Me), 2.11 (s, 2H, OH). MS (ESI+): m/z 169.21 [M+H+]. Calculated MS, C9H12O3: 168.19.
5-methoxy isophthalaldehyde (7i). The title compound (297 mg, 1.81 mmol, 92% yield) was prepared from 8i (330 mg, 1.97 mmoles, 1.0 eq.) and manganese dioxide (825 mg, 9.95 mmoles, 5 eqs.) in chloroform (10 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.30 (s, 2H, H1′, H3′), 7.96 (s, 1H, H2), 7.61 (s, 2H, H4), 3.99 (s, 3H, Me). MS (ESI+): m/z 165.29 [M+H+]. Calculated MS, C9H8O3: 164.16.
1-Methoxy-3,5-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (21). The title compound (161 mg, 0.46 mmoles, 81% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7i (93 mg, 0.57 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (125 mg, 1.14 mmoles, 1 eq.) in absolute ethanol (6 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.25 (bs, 2H, NH), 8.22-7.61 (bs, 6H, NH), 8.17 (s, 2H, H1′, H3′), 7.83 (t, J=1.08 Hz, 1H, H2), 7.59 (d, J=1.08 Hz, 2H, H4), 3.82 (s, 3H, OMe). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 160.3, 155.9, 146.3, 135.7, 120.6, 114.7, 56.2. MS (ESI+): m/z 277.47 [M+H+]. Calculated MS, C11H16N8O: 276.30.
3,5-bis-(hydroxymethyl)-1-nitro benzene (8j). The title compound (504 mg, 2.75 mmol, 83% yield) was prepared from 5-nitro isophthalic acid (700 mg, 3.32 mmoles, 1.0 eq.) and 1M BH3 in THF (13.2 mL, 13.2 mmoles, 4 eqs.) in THF (3 mL), following the experimental protocol of general procedure A. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 8.16 (s, 2H, H4), 7.72 (s, 1H, H2), 4.83 (s, 4H, H1′, H3′). MS (ESI+): m/z 184.24 [M+H+]. Calculated MS, C8H9NO4: 183.17.
5-nitro isophthalaldehyde (7j). The title compound (94 mg, 0.53 mmol, 78% yield) was prepared from 8j (123 mg, 0.67 mmoles, 1.0 eq.) and manganese dioxide (291 mg, 3.395 mmoles, 5 eqs.) in chloroform (4 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.21 (s, 2H, H1′, H3′), 8.96 (s, 2H, H4), 8.72 (s, 1H, H2). MS (ESI+): m/z 180.29 [M+H+]. Calculated MS, C8H5NO4: 179.14.
1-Nitro-3,5-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2j). The title compound (154 mg, 0.42 mmoles, 80% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7j (94 mg, 0.53 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (117 mg, 1.06 mmoles, 1 eq.) in absolute ethanol (5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.44 (bs, 2H, NH), 8.80 (d, J=1.6 Hz, 2H, H4), 8.75 (t, J=1.6 Hz, 1H, H2), 8.34 (s, 2H, H1′, H3′), 8.25-7.75 (bs, 6H, NH). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 156.0, 149.4, 144.5, 136.3, 132.7, 123.2. MS (ESI+): m/z 292.47 [M+H+]. Calculated MS, C10H13N9O2: 291.29.
1,3-Bis-(hydroxymethyl)-5-phenylbenzene (8k). A 2M aqueous Na2CO3 solution (1.86 mL, 3.72 mmoles, 3 eqs.) was added under vigorous stirring at r.t. under nitrogen atmosphere to a mixture of 8h (270 mg, 1.24 mmoles, 1 eq.), phenylboronic acid (177 mg, 1.45 mmoles, 1.2 eqs.) and Pd(PPh3)4 (143 mg, 0.124 mmoles, 0.1 eqs.) in 1,4-dioxane (8 mL). The mixture was heated at reflux and stirred for 6 hrs. After cooling to r.t., the reaction mixture was then diluted with EtOAc (40 mL), washed with sat. aq. NaHCO3 (20 mL) and brine (15 mL). The organic layer was concentrated at reduced pressure, and the crude product was purified by flash chromatography (eluant: 75:25 n-hexane/EtOAc) to yield pure 1,3-bis-(hydroxymethyl)-5-phenylbenzene 8k (220 mg, 1.02 mmoles, 86% yield) as a white solid. 1H_NMR (400 MHz, CDCl3): δ(ppm) 7.62 (d, J=7.2 Hz, 2H, H2″), 7.54 (s, 2H, H4), 7.46 (t, J=7.2 Hz, 2H, H3″), 7.38 (m, 2H, H2, H4″), 4.79 (s, 4H, H1′, H3′), 1.86 (bs, 2H, OH). MS (ESI+): m/z 215.43 [M+H+]. Calculated MS, C14H14O2: 214.26.
5-Phenyl isophthalaldehyde (7k). The title compound (135 mg, 0.64 mmol, 81% yield) was prepared from 8k (170 mg, 0.79 mmoles, 1.0 eq.) and manganese dioxide (343 mg, 3.95 mmoles, 5 eqs.) in chloroform (4 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.21 (s, 2H, H1′, H3′), 8.39 (dd, J=1.5 Hz, 2H, H4), 8.37 (t, J=1.5 Hz, 1H, H2), 7.71 (dd, J=8.6 Hz, J=1.5 Hz, 2H, H2″), 7.54 (dt, J=8.6 Hz, J=7.3 Hz, 2H, H3″), 7.48 (dt, J=7.3 Hz, J=1.5 Hz, 1H, H4″). MS (ESI+): m/z 211.31 [M+H+]. Calculated MS, C14H10O2: 210.23.
5-Phenyl-1,3-bis-amino guanidine phenyl hydrazone, dihydrochloride salt (2k). The title compound (190 mg, 0.48 mmoles, 79% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7k (127 mg, 0.61 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (134 mg, 1.22 mmoles, 1 eq.) in absolute ethanol (6 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.35 (bs, 2H, NH), 8.30-8.25 (m, 5H, H2, H4, H1′, H3′), 8.20-7.60 (bs, 6H, NH), 7.83 (d, J=8.4 Hz, 2H, H2″), 7.53 (t, J=8.4 Hz, 2H, H3″), 7.44 (t, J=8.4 Hz, 1H, H4″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 156.0, 146.4, 1411.7, 139.3, 135.1, 129.4, 128.5, 127.5, 126.4. MS (ESI+): m/z 323.53 [M+H+]. Calculated MS, C16H18N8: 322.38.
3,5-Bis-(t-butyldimethylsilyloxymethyl)-1-nitro benzene (10j). 1H-imidazole (500 mg, 7.35 mmoles, 1.5 eqs.) and 8j (450 mg, 2.45 mmoles, 1 eq.) were dissolved in CH2Cl2 (12 mL) at r.t under stirring. Then the solution was cooled at 0° C., and t-butyldimethylsilyl chloride (930 mg, 6.12 mmoles, 1.25 eq.) was added in a single portion. The reaction mixture was slowly warmed to rt and stirred for additional 3 hrs. The reaction mixture was then diluted with CH2Cl2 (15 mL), washed with 5% aqueous citric acid (15 mL), 5% aqueous NaOH (15 mL) and brine (15 mL). The combined organic layer was dried on anhydrous Na2SO4, filtered and evaporated at reduced pressure. Crude 3,5-bis-(t-butyldimethylsilyloxymethyl)-1-nitro benzene 10j (900 mg, 2.19 mmoles) was obtained as a yellow oil, used without further purification in the next reaction step. 1H_NMR (400 MHz, CDCl3): δ(ppm) 8.00 (s, 2H, H4), 7.62 (s, 1H, H2), 4.81 (s, 4H, H1′, H3′), 0.98 (s, 18H, t-Bu), 0.1 (s, 12H, Me). MS (ESI+): m/z 412.84 [M+H+]. Calculated MS, C20H37Si2NO4: 411.69.
3,5-bis-(t-butyldimethylsilyloxymethyl)-1-amino benzene (11). 10% Pd/C (80 mg, catalytic) was added to a solution of 10j (797 mg, 1.93 mmol, 1 eq.) in absolute EtOH (10 mL), and the reaction mixture was vigorously stirred at r.t. under hydrogen atmosphere for 24 hrs. The reaction mixture was then filtered through a path of celite using MeOH (50 mL) for repeated washings. After concentration at reduced pressure, the crude was purified by flash chromatography on silica gel (eluant mixture: neat CH2Cl2 to 95:5 CH2Cl2/EtOAc), to afford pure 3,5-bis-(t-butyldimethylsilyloxymethyl)-1-amino benzene 11 (591 mg, 1.559 mmoles, 78% yield for two steps) as a light yellow oil. 1H_NMR (400 MHz, CDCl3): δ(ppm) 6.68 (s, 1H, H2), 6.59 (s, 2H, H4), 4.65 (s, 4H, H1′, H3′), 4.00 (bs, 2H, NH), 0.98 (s, 18H, t-Bu), 0.1 (s, 12H, Me). MS (ESI+): m/z 382.82 [M+H+]. Calculated MS, C20H39Si2NO2: 381.70.
3,5-bis-(t-butyldimethylsilyloxymethyl)-1-acetamido benzene (101). TEA (0.55 mL, 4 mmoles, ≈5 eqs.) and 11 (300 mg, 0.79 mmoles, 1 eq.) were dissolved under stirring in CH2Cl2 (8 mL). Then, acetic anhydride (0.22 mL, 2.36 mmoles, 3 eqs.) and dimethylaminopyridine (spatula tip, catalytic) were added, and the solution was stirred at r.t. for 2 additional hrs. After dilution with CH2Cl2 (15 mL), the solution was washed with 5% aqueous citric acid (10 mL), saturated aqueous NaHCO3 (10 mL) and brine (10 mL). The combined organic layer was dried with anhydrous Na2SO4, filtered and evaporated at reduced pressure. 3,5-Bis-(t-butyldimethylsilyloxymethyl)-1-acetamido benzene 101 was obtained as a crude yellow oil (350 mg), and was used as such in the next reaction step. 1H_NMR (400 MHz, CDCl3): δ(ppm) 7.33 (s, 2H, H4), 7.28 (bs, 1H, NH), 7.08 (s, 1H, H2), 4.74 (s, 4H, H1′, H3′), 2.22 (s, 3H, CO-Me), 0.98 (s, 18H, t-Bu), 0.1 (s, 12H, Si-Me). MS (ESI+): m/z 446.88 [M+Na+]. Calculated MS, C22H41Si12NO3: 423.74.
3,5-bis-(hydroxymethyl)-1-acetamido benzene (8l). Compound 101 (350 mg, theor. 0.79 mmoles, 1 eq.) was dissolved in dry MeOH (8 mL) under nitrogen atmosphere. The solution was cooled at 0° C., and acetyl chloride (30 μL, 0.39 mmoles, ≈0.5 eqs.) was added under stirring. The reaction mixture was stirred at 0° for additional 30 min. The solvent was then removed under reduced pressure, and the residue was triturated in acetone (10 mL), and filtered. The solid was washed with cold acetone (3×8 mL) and dried, to yield pure 3,5-bis-(hydroxymethyl)-1-acetamido benzene 8l (143 mg, 0.67 mmol, 84% yield over two steps) as a white solid. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 9.92 (s, 1H, NH), 7.45 (s, 2H, H4), 6.90 (s, 1H, H2), 5.18 (bs, 2H, OH), 4.41 (s, 4H, H1′, H3′), 2.00 (s, 3H, CO-Me). MS (ESI+): m/z 196.31 [M+H+]. Calculated MS, C10H13NO3: 195.22.
5-Acetamido isophthalaldehyde (7l). The title compound (104 mg, 0.54 mmoles, 88% yield) was prepared from 8l (120 mg, 0.62 mmoles, 1.0 eq.) and manganese dioxide (268 mg, 3.10 mmoles, 5 eqs.) in chloroform (10 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, CD3CN): δ(ppm) 10.01 (s, 2H, H1′, H3′), 8.65 (bs, 1H, NH), 8.72 (s, 2H, H4), 8.02 (s, 1H, H2), 2.09 (s, 3H, CO-Me). MS (ESI+): m/z 192.23 [M+H+]. Calculated MS, C10H9NO3: 191.19.
1-Acetamido-3,5-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (21). The title compound (163 mg, 0.43 mmoles, 85% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7l (97 mg, 0.51 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (113 mg, 1.02 mmoles, 1 eq.) in absolute ethanol (5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.18 (bs, 2H, NH), 10.22 (s, 1H, NH), 8.19 (s, 2H, H1′, H3′), 8.17 (s, 1H, H2), 8.00-7.75 (m, 6H, NH), 7.94 (s, 2H, H4), 2.08 (s, 3H, CO-Me). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 170.2, 156.8, 147.6, 141.2, 135.6, 122.1, 121.4, 24.4. MS (ESI+): m/z 326.40 [M+Na+]. Calculated MS, C12H17N9O: 303.33.
3,5-bis-(t-butyldimethylsilyloxymethyl)-1-p-tolylamido benzene (10m). TEA (0.23 mL, 1.63 mmoles, 2 eq.) and 11 (310 mg, 0.82 mmoles, 1 eq.) were dissolved under stirring at 0° C. in CH2Cl2 (8 mL). Then, 4-methylbenzoyl chloride (0.14 mL, 1.06 mmoles, 1.3 eqs.) was added, and the solution was stirred at 0° C. for 1 additional hr. After quenching with MeOH (1 mL) and dilution with CH2Cl2 (15 mL), the solution was washed with 5% aqueous citric acid (10 mL), saturated aqueous NaHCO3 (10 mL) and brine (10 mL). The combined organic layer was dried with anhydrous Na2SO4, filtered and evaporated at reduced pressure. The crude was purified by flash chromatography on silica gel (eluant mixture: neat CH2Cl2 to 95:5 CH2Cl2 /EtOAc), to afford pure 3,5-bis-(t-butyldimethylsilyloxymethyl)-1-amino(p-methylbenzoyl) benzene 10m (265 mg, 0.53 mmol, 67% yield) as a pale yellow oil. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 9.29 (bs, 1H, NH), 7.75 (d, J=8.2Hz, 2H, H7) 7.59 (s, 2H, H2), 7.18 (d, J=8.2 Hz, 2H, H8), 7.01 (s, 1H, H1), 4.64 (s, 4H, H3), 2.27 (s, 3H, H6), 0.82 (s, 18H, H5), 0.00 (s, 12H, H4). MS (ESI+): m/z 523.06 [M+Na+]. Calculated MS, C28H45Si2NO3: 499.84.
3,5-bis-(hydroxymethyl)-1-p-tolylamido benzene (8m). Compound 10m (260 mg, 0.52 mmol, 1eq) was dissolved in dry MeOH (10 mL) under nitrogen atmosphere. The solution was cooled at 0° C., and acetyl chloride (20 μL, 0.26 mmoles, ≈0.5 eqs.) was added under stirring. The reaction mixture was stirred at 0° for additional 30 min. The solvent was then removed under reduced pressure to yield the crude 3,5-bis-(hydroxymethyl)-1-p-tolylamido benzene 8m (140 mg), that was used in the next step without further purification. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 9.40 (bs, 1H, NH), 7.95 (d, J=8.2 Hz, 2H, H2″) 7.75 (s, 2H, H4), 7.33 (d, J=8.2 Hz, 2H, H3″), 7.12 (s, 1H, H2), 4.64 (s, 4H, H1′, H3′), 2.42 (s, 3H, H4″). MS (ESI+): m/z 272.48 [M+H+]. Calculated MS, C16H17NO3: 271.32.
5-p-tolylamido isophthalaldehyde (7m). The title compound (113 mg, 0.42 mmoles, 83% yield) was prepared from 8m (140 mg, 0.52 mmoles, 1.0 eq.) and manganese dioxide (340 mg, 5.20 mmoles, 10 eqs.) in chloroform (10 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, CDCl3): δ(ppm) 10.18 (s, 2H, H1′, H3′), 9.96 (bs, 1H, NH), 8.73 (d, J=4.1 Hz, 2H, H4), 8.22 (t, J=4.1 Hz, 1H, H2), 8.00 (d, J=8.2 Hz, 2H, H2″), 7.38 (d, J=8.2 Hz, 2H, H3″), 2.44 (s, 3H, H4″). MS (ESI+): m/z 268.38 [M+H+]. Calculated MS, C16H13NO3: 267.29.
1-p-Tolylamido-3,5-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2m). The title compound (69 mg, 0.15 mmoles, 83% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7m (47 mg, 0.18 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (41 mg, 0.37 mmoles, 1 eq.) in absolute ethanol (4 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.15 (bs, 2H, NH), 10.43 (bs, 1H, NH), 8.23 (s, 2H, H1′, H3′), 8.20 (s, 1H, H2), 8.17 (s, 2H, H4), 7.95 (d, J=8.2 Hz, 2H, H2″), 7.83 (bs, 6H, NH), 7.37 (d, J=8.2 Hz, 2H, H3″), 2.41 (s, 3H, H4″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 165.9, 155.9, 146.6, 142.4, 140.3, 134.8, 131.9, 129.5, 128.2, 122.9, 121.6, 21.5. MS (ESI+): m/z 380.50 [M+H+]. Calculated MS, C18H21N9O: 379.43.
3,5-bis-(t-butyldimethylsilyloxymethyl)-1-methylsulfonamido benzene (10n). Pyridine (0.72 mL, 0.87 mmoles, 2 eqs.) and 11 (166 mg, 0.43 mmoles, 1 eq.) were dissolved under stirring at 0° C. in CH2Cl2 (4 mL). Then, 4-methylsulfonyl chloride (50 μL, 0.65 mmoles, 1.5 eqs.) was added, the solution was stirred at 0° C. for 1 hr, and at r.t. for 15 hrs. After dilution with CH2Cl2 (15 mL), the solution was washed with water (10 mL) and brine (10 mL). The combined organic layer was dried with anhydrous Na2SO4, filtered and evaporated at reduced pressure. The crude was quickly eluted on silica gel (eluant mixture: 98:2 CH2Cl2/EtOAc), to afford crude 3,5-bis-(t-butyldimethylsilyloxymethyl)-1-methylsulfonamido benzene 10n (200 mg) as a pale yellow oil, that was used in the next step without further purification. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 8.44 (bs, 1H, NH), 7.12 (s, 2H, H4), 7.01 (s, 1H, H2), 4.64 (s, 4H, H1′, H3′), 2.84 (s, 3H, SO2-Me), 0.83 (s, 18H, t-Bu), 0.00 (s, 12H, Si-Me). MS (ESI+): m/z 460.94 [M+H+]. Calculated MS, C21H41Si2NO4S: 459.80.
3,5-bis-(hydroxymethyl)-1-methylsulfonamido benzene (8n). Compound 10n (200 mg, theoretical 0.43 mmol, 1 eq.) was dissolved in dry MeOH (8 mL) under nitrogen atmosphere. The solution was cooled at 0° C., and acetyl chloride (20 μL, 0.26 mmoles, ≈0.7 eqs.) was added under stirring. The reaction mixture was stirred at 0° for additional 30 min. The solvent was then removed under reduced pressure to yield the crude 3,5-bis-(hydroxymethyl)-1-methylsulfonamido benzene 8n (100 mg) as a pale brown solid, that was used in the next step without further purification. MS (ESI+): m/z 232.41 [M+H+]. Calculated MS, C9H13NO4S: 231.27.
5-methylsulfonamido isophthalaldehyde (7n). The title compound (65 mg, 0.29 mmoles, 68% yield over three steps) was prepared from 8n (100 mg, theoretical 0.43 mmoles, 1.0 eq.) and manganese dioxide (300 mg, 4.33 mmoles, ≈10 eqs.) in chloroform (6 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 10.02 (s, 2H, H3′), 9.02 (bs, 1H, NH), 8.09 (s, 1H, H2), 8.01 (s, 2H, H4), 3.01 (s, 3H, SO2-Me). MS (ESI+): m/z 228.30 [M+H+]. Calculated MS, C9H9NO4S: 227.24.
1-Methylsulfonamido-3,5-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2n). The title compound (100 mg, 0.24 mmoles, 83% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7n (65 mg, 0.29 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (63 mg, 0.58 mmoles, 1 eq.) in absolute ethanol (3.5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.25 (bs, 2H, NH), 10.01 (s, 1H, NH), 8.24 (s, 1H, H2), 8.21 (s, 2H, H1′, H3′), 7.86 (bs, 6H, NH), 7.60 (s, 2H, H4), 3.10 (s, 3H, SO2-Me). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 155.9, 146.3, 149.6, 135.4, 122.1, 121.4, 39.98. MS (ESI+): m/z 340.50 [M+H+]. Calculated MS, C11H17N9O2S: 339.37.
3,5-bis-(t-butyldimethylsilyloxymethyl)-1-p-tolylsulfonamido benzene (10o). Pyridine (0.72 mL, 0.87 mmoles, 2 eqs.) and 11 (166 mg, 0.43 mmoles, 1 eq.) were dissolved under stirring at 0° C. in CH2Cl2 (5 mL). Then, p-tosylsulfonyl chloride (125 mg, 0.65 mmoles, 1.5 eqs.) was added, the solution was stirred at 0° C. for 1 hr, and at r.t. for 6 hrs. After dilution with CH2Cl2 (15 mL), the solution was washed with saturated aqueous NaHCO3 (10 mL) and brine (10 mL). The combined organic layer was dried with anhydrous Na2SO4, filtered and evaporated at reduced pressure. The crude was purified by flash chromatography on silica gel (eluant mixture: neat CH2Cl2 to 95:5 CH2Cl2/EtOAc), to afford pure 3,5-bis-(t-butyldimethylsilyloxymethyl)-1-p-tolylsulfonamido benzene 10o (195 mg, 0.37 mmoles, 86% yield) as a white solid. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 8.87 (bs, 1H, NH), 7.61 (d, J=8.2 Hz, 2H, H2″), 7.22 (d, J=8.2Hz, 2H, H3″), 7.06 (s, 2H, H4), 6.93 (s, 1H, H2), 4.60 (s, 4H, H1′, H3′), 2.28 (s, 3H, Ar-Me), 0.85 (s, 18H, t-Bu), 0.00 (s, 12H, Si-Me). MS (ESI+): m/z 536.94 [M+H+]. Calculated MS, C27H45Si2NO4S: 535.89.
3,5-bis-(hydroxymethyl)-1-p-tolylsulfonamido benzene (8o). Compound 10o (195 mg, 0.36 mmoles, 1 eq.) was dissolved in dry MeOH (8 mL) under nitrogen atmosphere. The solution was cooled at 0° C., and acetyl chloride (10 μL, 0.13 mmoles, ≈0.4 eqs.) was added under stirring. The reaction mixture was stirred at 0° for additional 2 hrs. The solvent was then removed under reduced pressure, and the residue was chromatographed on silica gel (eluant mixture: 7:3 n-hexane/EtOAc) to yield pure 3,5-bis-(hydroxymethyl)-1-p-tolylsulfonamido benzene 8o (90 mg, 0.29 mmoles, 81% yield) as a white solid. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 8.72 (bs, 1H, NH), 7.57 (d, J=8.2 Hz, 2H, H2″), 7.18 (d, J=8.2Hz, 2H, H3″), 7.00 (s, 2H, H4), 6.89 (s, 1H, H2), 4.39 (s, 4H, H1′, H3′), 4.03 (s, 2H, OH), 2.22 (s, 3H, Me). MS (ESI+): m/z 330.41 [M+Na+]. Calculated MS, C15H17NO4S: 307.36.
5-p-tolylsulfonamido isophthalaldehyde (7o). The title compound (79 mg, 0.26 mmoles, 90% yield over three steps) was prepared from 8o (90 mg, 0.29 mmoles, 1.0 eq.) and manganese dioxide (200 mg, 2.9 mmoles, 10 eqs.) in chloroform (6 mL), following the experimental protocol of general procedure B. 1H_NMR (400 MHz, acetone-d6): δ(ppm) 9.95 (s, 2H, H1′, H3′), 8.02 (s, 1H, H2), 7.90 (s, 2H, H4), 7.63 (d, J=8.2 Hz, 2H, H2″), 7.22 (d, J=8.2Hz, 2H, H3″), 2.22 (s, 3H, Me). MS (ESI+): m/z 304.39 [M+H+]. Calculated MS, C15H13NO4S: 303.33.
1-p-Tolylsulfonamido-3,5-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (2o). The title compound (80 mg, 0.16 mmoles, 76% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7o (64 mg, 0.21 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (46 mg, 0.42 mmoles, 1 eq.) in absolute ethanol (3 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.15 (bs, 2H, NH), 10.56 (s, 1H, NH), 8.17 (s, 1H, H2), 8.12 (s, 2H, H1′, H3′), 7.82 (bs, 6H, NH), 7.71 (d, J=8.2 Hz, 2H, H2″), 7.46 (s, 2H, H4), 7.36 (d, J=8.2 Hz, 2H, H3″), 2.34 (s, 3H, Me). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 155.4, 145.8, 143.5, 138.8, 136.6, 134.9, 129.8, 126.8, 121.2, 120.7, 21.0. MS (ESI+): m/z 416.56 [M+H+]. Calculated MS, C17H21N9O2S: 415.47.
1,3,5-Benzene tricarboxylate monomethyl ester (9p). 1.8M Aqueous NaOH (9.1 mL, 16.4 mmoles, ≈2 eqs.) was added dropwise to a vigorously stirred suspension of 1,3,5-benzene tricarboxylate trimethyl ester (2.0 g, 8.0 mmoles, 1 eq.) in MeOH (64 mL) at r.t. Stirring at r.t. was continued for 5 hrs, obtaining a clear solution. After solvent evaporation at reduced pressure, water (25 mL) was added and the pH was brought to 2.0 by dropwise addition of aqueous 6N HCl. The resulting suspension was extracted with EtOAc (2×25 mL). After washing with water (2×10 mL) and brine (2×10 mL), the combined organic phase was concentrated at reduced pressure. The crude 1,3,5-benzene tricarboxylate monomethyl ester 9p (1.5 g) was used in the next reaction step without further purification. MS (ESI+): m/z 225.22 [M+H+]. Calculated MS, C10H8O6: 224.17.
Methyl 3,5-bis-(hydroxymethyl)-benzene carboxylate (8p). The title compound (775 mg, 3.94 mmol, 54% yield over two steps) was prepared from 9p (1.5 g, theoretical 6.7 mmoles, 1.0 eq.) and 1M BH3 in THF (33 mL, 33 mmoles, ≈5 eqs.) in THF (3 mL), following the experimental protocol of general procedure A. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 7.80 (s, 2H, H4), 7.49 (s, 1H, H2), 4.58 (s, 4H, H1′, H3′), 3.45 (s, 2H, OH). MS (ESI+): m/z 197.27 [M+H+]. Calculated MS, C10H12O4: 196.20.
5-Carbomethoxy isophthalaldehyde ('7p). The title compound (645 mg, 3.35 mmoles, 85% yield) was prepared from 8p (775 mg, 3.94 mmoles, 1.0 eq.) and manganese dioxide (1.9 g, 19.5 mmoles, ≈5 eqs.) in chloroform (20 mL), following the experimental protocol of general procedure B. MS (ESI+): m/z 193.26 [M+H+]. Calculated MS, C10H8O4: 192.17.
1-Carbomethoxy-3,5-bis-amino guanidine phenyl hydrazone, dihydrochloride salt (2p). The title compound (109 mg, 0.29 mmoles, 71% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7p (78 mg, 0.41 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (90 mg, 0.81 mmoles, 1 eq.) in absolute ethanol (4 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.30 (bs, 2H, NH), 8.64 (s, 1H, H2), 8.44 (s, 2H, H1′, H3′), 8.30 (s, 2H, H4), 7.91 (bs, 6H, NH), 3.92 (s, 3H, OMe). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 166.1, 156.0, 145.7, 135.1, 131.5, 130.7, 130.0, 53.0. MS (ESI+): m/z 305.33 [M+H+]. Calculated MS, C12H16N8O2: 304.26.
5-Carboxy isophthalaldehyde (12). 2M aqueous NaOH (0.47 mL, 0.94 mmoles, 1.05 eqs) was added to a suspension of 7p (174 mg, 0.90 mmoles, 1 eq.) in MeOH (10 mL) under vigorous stirring at r.t. The resulting suspension was stirred at r.t. for 24 After 24 h, the solvent was removed at reduced pressure, and crude 5-carboxy isophthaldehyde (160 mg) was used in the next step without further purification. MS (ESI+): m/z 169.21 [M+H+]. Calculated MS, C9H6O4: 168.14.
5-(N4-phenylpiperazinyl)carboxamido isophthalaldehyde (7q). HOBt (162 mg, 1.2 mmoles, 1.25 eqs.) and EDC (230 mg, 1.2 mmoles, 1.25 eqs.) were sequentially added to a suspension of crude 12 (160 mg, theoretical 0.90 mmoles) in dry CH2Cl2 (10 mL) under vigorous stirring at r.t. under nitrogen atmosphere. The resulting mixture was stirred at r.t. for 15 minutes, then 4-phenyl-1-piperazine (195 mg, 1.2 mmoles, 1.25 equivand TEA (0.28 mL, 1.8 mmoles, 2 eqs.) were sequentially added. The resulting solution was stirred at r.t. for 24 hrs, and was then concentrated under reduced pressure. The residue was taken up in EtOAc (20 mL), washed with 5% citric acid solution (5 mL), saturated aqueous NaHCO3 (5 mL) and brine (5 mL). After drying over Na2SO4, the combined organic phase was filtered and concentrated under reduce pressure. Pure 5-(N4-phenylpiperazinyl)carboxamido isophthalaldehyde 7q (90 mg, 0.28 mmoles, 30% yield over 2 steps) was obtained after chromatographic purification (eluant mixture: 4:6 n-hexane/AcOEt+1% TEA). 1H NMR (400 MHz, acetone-d6): δ: 10.15 (s, 2H, H1′, H3′), 8.46 (s, 1H, H2), 8.23 (s, 2H, H4), 7.32 (m, 2H, H3′″), 6.94 (m, 3H, H2′″, H4′″), 3.99 (bs, 3H, H2″), 3.61 (bs, 1H, H2″), 3.30 (bs, 3H, H3″), 3.18 (bs, 1H, H3″). MS (ESI+): m/z 323.46 [M+H+]. Calculated MS, C19H18N2O3: 322.36.
5-(N4-phenylpiperazinyl)carboxamido-3,5-bis-amino guanidine phenyl hydrazone, dihydrochloride salt (2q). The title compound (110 mg, 0.20 mmoles, 72% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 7q (90 mg, 0.28 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (62 mg, 0.56 mmoles, 1 eq.) in absolute ethanol (5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.32 (bs, 2H, NH), 8.35 (s, 1H, H2), 8.25 (s, 2H, H1′, H3′), 8.09 (s, 2H, H4), 7.85 (bs, 6H, NH), 7.32 (m, 2H, H3′″), 7.21 (m, 2H, H2′″), 6.98 (m, 1H, H4′″), 3.92 (bs, 1H, H2″), 3.57 (bs, 1H, H2″), 3.28 (bs, 1H, H3″), 3.17 (bs, 1H, H3″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 168.5, 156.0, 145.7, 137.3, 134.9, 129.7, 128.2, 127.4, 121.2. MS (ESI+): m/z 435.63 [M+H+]. Calculated MS, C21H26N10O: 434.58.
1-3,5-tris-amino guanidine phenyl hydrazone, trihydrochloride salt (2r). The title compound (105 mg, 0.24 mmoles, 78% yield, ≥95% purity, white solid) was prepared as a trihydrochloride salt from 1,3,5-benzene tricarboxaldehyde (50 mg, 0.31 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (105 mg, 0.94 mmoles, 1 eq.) in absolute ethanol (5 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, D2O): δ(ppm) 7.57 (s, 3H, H1′), 7.30 (s, 3H, H2). 13C_NMR (100 MHz, D2O): δ(ppm) 154.5, 146.3, 133.4, 127.6. MS (ESI+): m/z 331.53 [M+H+]. Calculated MS, C12H18N12: 330.36.
N1-n-Butylamino guanidine hydroiodide (14t). S-methyl isothiosemicarbazide dihydroiodide 13 (510 mg, 2.19 mmoles, 1 eq.) was dissolved in MeOH (6 mL), and n-butylamine (325 μL, 3.28 mmoles, 1.5 eqs.) were added under stirring at r.t. The reaction mixture was concentrated at reduced pressure after 72 hours. Crude N1-n-butylamino guanidine hydroiodide 14t was obtained as a red oil (565 mg) that was used in the next step without purification. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 8.46 (bs, 1H, NH), 7.25 (bs, 3H, NH), 4.69 (bs, 2H, NH), 3.13 (s, 2H, H1), 1.48 (m, 2H, J=7 Hz, H2) 1.28 (m, 4H, J=7 Hz, H3), 0.92 (m, 3H, J=7 Hz, H4). MS (ESI+): m/z 131.26 [M+H+]. Calculated MS, C5H14N4: 130.19.
1,3-bis-(N3-n-butylamino) guanidine phenyl hydrazone, dihydroiodide salt (2t). The title compound (265 mg, 0.44 mmoles, 53% yield, ≥95% purity, pale yellow solid) was prepared as a dihydroiodide salt from isophthalaldehyde (110 mg, 0.82 mmoles, 1.0 eq.) and 14t (490 mg, theoretical 1.90 mmoles, 1.15 eqs.) in absolute ethanol (5 mL), following the experimental protocol of general procedure D, and a preparative HPLC separation (eluant mixture: neat water to neat CH3CN). 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 11.44 (bs, 2H, NH), 8.25 (m, 3H, H1′, H3′, H2), 8.21-7.90 (bs, 4H, NH), 8.03 (d, J=8.1 Hz, 2H, H4), 7.57 (t, J=8 Hz, 1H, H5), 3.37 (m, 4H, H6), 1.54 (m, 4H, H7), 1.35 (m, 4H, H8), 0.94 (t, J=7.12 Hz, 6H, H9). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 154.4, 146.5, 134.5, 129.6, 127.5, 41.3, 31.1, 19.7, 14.1.MS (ESI+): m/z 359.69 [M+H+]. Calculated MS, C18H30N8: 358.51.
N1-Benzylamino guanidine hydroiodide (14u). S-methyl isothiosemicarbazide hydroiodide 13 (995 mg, 4.28 mmoles, 1 eq.) was dissolved in MeOH (8 mL), and benzylamine (470 μL, 4.28 mmoles, 1 eq.) were added under stirring at r.t. The reaction mixture was concentrated at reduced pressure after 72 hours. N1-Benzylamino guanidine hydroiodide 14u was obtained after recrystallization from EtOH/diethyl ether as an orange solid (425 mg, 1.46 mmoles, 34% yield). 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 8.69 (bs, 1H, NH), 7.46-7.20 (m, 8H, NH, H2, H3, H4), 4.76 (s, 2H, NH2), 4.42 (s, 2H, H1′). MS (ESI+): m/z 165.27 [M+H+]. Calculated MS, C8H12N4: 164.21.
1,3-bis-(N3-benzylamino) guanidine phenyl hydrazone, dihydroiodide salt (2u). The title compound (177 mg, 0.26 mmoles, 70% yield, ≥95% purity, pale yellow solid) was prepared as a dihydroiodide salt from isophthalaldehyde (50 mg, 0.37 mmoles, 1.0 eq.) and 14u (250 mg, 0.86 mmoles, 1.3 eqs.) in absolute ethanol (5 mL), following the experimental protocol of general procedure D, and a preparative HPLC separation (eluant mixture: neat water to neat CH3CN). 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 11.60 (bs, 2H, NH), 8.57 (m, 2H, H4), 8.27 (m, 3H, H2, H1′, H3′), 8.05 (bs, 4H, NH), 7.55 (t, J=7.8 Hz, 1H, H5), 7.35 (m, 10H, H2″, H3″, H4″), 4.60 (d, J=5.4 Hz, 4H, H1″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 154.7, 146.9, 137.5, 134.5, 129.6, 129.0, 1128.0, 127.7, 127, 5, 44.5. MS (ESI+): m/z 449.69 [M+Na+]. Calculated MS, C24H26N8: 426.53.
N1-Pyrrolidinyl guanidine hydroiodide (14v). S-methyl isothiosemicarbazide hydroiodide 13 (1.21 g, 5.21 mmoles, 1 eq.) was dissolved in MeOH (10 mL), and pyrrolidine (650 μL, 7.81 mmoles, 1.5 eqs.) were added under stirring at r.t. The reaction mixture was concentrated at reduced pressure after 72 hours. Crude N1-pyrrolidinyl guanidine hydroiodide 14v was obtained from trituration with EtOAc (20 mL), solvent concentration under reduced pressure, trituration with diethyl ether (20 mL), filtration and washing with diethyl ether (3×5 mL). Crude 14v (1.22 g, pink solid) was used as such in the next reaction step. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 8.74 (bs, 1H, NH), 7.22 (bs, 3H, NH), 4.78 (bs, 2H, NH2), 3.30 (t, 4H, J=6.4, H1), 1.90 (m, 4H, J=6.4, H2). MS (ESI+): m/z 129.22 [M+H+]. Calculated MS, C5H12N4: 128.18.
1,3-bis-(N3-pyrrolidinyl) guanidine phenyl hydrazone, dihydroiodide salt (2v). The title compound (128 mg, 0.21 mmoles, 70% yield, ≥95% purity, pale yellow solid) was prepared as a dihydroiodide salt from isophthalaldehyde (40 mg, 0.30 mmoles, 1.0 eq.) and 14v (168 mg, 0.66 mmoles, 1.1 eqs.) in absolute ethanol (3 mL), following the experimental protocol of general procedure D. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 11.31 (bs, 2H, NH), 8.43 (s, 2H, H1′, H3′), 8.28 (s, 1H, H2), 8.06 (d, J=7.84 Hz, 2H, H4), 7.97 (bs, 4H, NH), 7.57 (t, J=7.84 Hz, 1H, H5), 3.50 (t, J=6.4 Hz, 8H, H1″), 2.00 (t, J=6.4 Hz, 8H, H2″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 152.3, 147.1, 134.5, 129.8, 129.7, 127.4, 48.1, 25.1.MS (ESI+): m/z 355.58 [M+H+]. Calculated MS, C18H26N8: 354.46.
1-p-Tolylamido-3,5-bis-(N3-n-butylamino) guanidine phenyl hydrazone, diformate salt (2w). The title compound (170 mg, 0.29 mmoles, 69% yield, ≥95% purity, pale pink solid) was prepared as a diformate salt from 7m (110 mg, 0.42 mmoles, 1.0 eq.) and 14t (254 mg, 0.98 mmoles, 1.2 eqs.) in absolute ethanol (7 mL), following the experimental protocol of general procedure D, and a preparative HPLC separation (eluant mixture: neat water to neat MeOH+0.3% formic acid). 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 10.32 (bs, 1H, NH), 8.64 (bs, 2H, NH), 8.31 (s, 2H, NH), 8.13 (s, 2H, H1′, H3′), 8.08 (s, 1H, H2), 8.05 (s, 2H, H4), 7.93 (d, J=8.2 Hz, 2H, H2″), 7.83 (bs, 4H, NH), 7.36 (d, J=8.2 Hz, 2H, H3″), 3.24 (bs, 4H, H1′″), 2.40 (s, 3H, H4″), 1.53 (m, 4H, H2′″), 1.36 (m, 4H, H3′″), 0.92 (t, J=8.3 Hz, 6H, H4′″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 166.2, 165.9, 144.9, 142.3, 140.1, 132.1, 129.4, 128.2, 122.1, 121.8, 40.4, 31.3, 21.5, 19.9, 14.1. MS (ESI+): m/z 492.70 [M+H+]. Calculated MS, C26H37N9O: 491.64.
1,3-Bis-(N2N3-imidazolidinyl) guanidine phenyl hydrazone, dihydrobromide salt (2x). The title compound (196 mg, 0.43 mmoles, 82% yield, ≥95% purity, white solid) was prepared as a dihydrobromide salt from isophthalaldehyde (70 mg, 0.52 mmoles, 1.0 eq.) and imidazolidinamino guanidine hydrobromide (198 mg, 1.10 mmoles, 1.05 eqs.) in absolute ethanol (3.5 mL), following the experimental protocol of general procedure D. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 12.42 (bs, 2H, NH), 8.75 (bs, 4H, NH), 8.27 (s, 2H, H1′, H3′), 8.22 (s, 1H, H2), 7.99 (d, J=7.79 Hz, 2H, H4), 7.59 (t, J=7.79 Hz, 1H, H5), 3.77 (s, 8H, H1″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) δ157.9, 148.1, 132.9, 130.0, 129.6, 128.0, 43.0. MS (ESI+): m/z 295.47 [M+H+]. Calculated MS, C14H18N8: 294.35.
1,3-Bis-(N1-methyl-N2N3-imidazolidinyl) guanidine phenyl hydrazone, dihydrobromide salt (2y). The title compound (205 mg, 0.42 mmoles, 80% yield, ≥95% purity, white solid) was prepared as a dihydrobromide salt from isophthalaldehyde (70 mg, 0.52 mmoles, 1.0 eq.) and N3-methyl imidazolidinamino guanidine hydrobromide (213 mg, 1.10 mmoles, 1.05 eqs.) in absolute ethanol (3.5 mL), following the experimental protocol of general procedure D. 1H_NMR (400 MHz, DMSO-d6/D2O mixture): δ(ppm) 8.38 (s, 1H, H2), 8.19 (s, 2H, H1′, H3′), 8.05 (d, J=7.75 Hz, 2H, H4), 7.60 (t, J=7.75 Hz, 1H, H5), 3.81 (s, 6H, N-Me), 3.75 (s, 8H, H1″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 159.5, 144.4, 134.5, 130.0, 129.7, 128.3, 43.6, 22.2. MS (ESI+): m/z 327.49 [M+H+]. Calculated MS, C16H22N8: 326.41.
1,3-Bis-(N2N3-benzimidazolidinyl) guanidine phenyl hydrazone, diacetate salt (2z). N,N′-Benzimidazolyl amidinoguanidine (110 mg, 0.74 mmoles, 1.05 eqs.) and AcOH (50 μL) were added under stirring at r.t. to a solution of isophthalaldehyde (46 mg, 0.34 mmoles, 1 eq.) in absolute EtOH (5 mL). The reaction mixture was heated and refluxed for 2 hrs. After cooling to r.t and filtration, the solid precipitate was collected, washed with water (2×5 mL), and dried to afford pure 1,3-bis-N2N3-benzimidazolyl guanidine phenyl hydrazone, diacetate salt 2z (87 mg, 0.22 mmoles, 66% yield) as a yellow solid. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 11.68 (bs, 4H, NH), 8.36 (s, 1H, H2), 8.15 (s, 2H, H1′, H3′), 7.75 (d, J=7.7 Hz, 2H, H4), 7.59 (t, 1H, J=7.7 Hz, H5), 7.33 (m, 4H, H1″), 7.05 (m, 4H, H2″). 13C_NMR (100 MHz, DMSO-d6): δ(ppm) 149.8, 146.4, 134.8, 131.6, 129.7, 128.9, 127.4, 123.6, 112.6. MS (ESI+): m/z 395.55 [M+H+]. Calculated MS, C22H18N8: 394.44.
Reference Example
1′,3′-Dimethyl-1,3-bis-aminoguanidine phenyl hydrazone, dihydrochloride salt (Compound (VIII)). The title compound (274 mg, 0.73 mmoles, 70% yield, ≥95% purity, white solid) was prepared as a dihydrochloride salt from 1,3-diacetylbenzene (170 mg, 1.05 mmoles, 1.0 eq.) and aminoguanidine hydrochloride (232 mg, 2.10 mmoles, 1 eq.) in absolute ethanol (10 mL), following the experimental protocol of general procedure C. 1H_NMR (400 MHz, DMSO-d6): δ(ppm) 11.40 (bs, 2H, NH), 8.35 (s, 1H, H2), 8.06 (d, J=9.2 Hz, 2H, H4), 8.15-7.60 (bs, 6H, NH), 7.50 (t, J=9.2 Hz, 1H, H5), 2.41 (s, 6H, Me). 13C_NMR (100 MHz, D2O): δ(ppm) 155.3, 152.7, 136.0, 128.6, 127.6, 124.0, 13.3. MS (ESI+): m/z 275.45 [M+H+]. Calculated MS, C12H18N8: 274.33.
Biology
Cell Cultures
Mouse albino neuroblastoma Neuro2a cells were maintained in standard medium (DMEM+2 mM Glutamine+10% Fetal Bovine Serum (FBS)). Split sub-confluent cultures (70-80%) 1:3 to 1:8 i.e. seeding at 4×10 000 cells cm2 and kept in 5% CO2; 37° C. Transfections were performed using Lipofectamine® LTX Transfection Reagent (Thermofisher) according manufacturer' instruction. The following plasmids were used: pcDNA3.1(+)SOD1G93A, pCAGG GFP, p CMV-LacZ, CMV-VPS35, Sigma Mission shRNA: TRCN000011556 (Sh56), TRCN000011559 (Sh59), TRCN000011558 (Sh58), scramble.
Cell survival experiments were run on 96-well plates using Cell Counting Kit-8 (CCK8) survival kit (96992-Sigma) according the manufacturer's recommendations. Compound 2a cytotoxicity was tested on Neuro2a cells receiving increasing amount of compound 2a (1, 10, 50, 100, 200, 500 and 1000 μM) for 48 hours.
LDH cytotoxicity was run on 96 well plates using LDH-Glo™ Cytotoxicity Assay (Promega). Briefly, Neuro2a cells were transfected with plasmids encoding pCMV-LacZ; pcDNA3.1(+)SOD1G93A with or without VPS35 short interfering plasmids (Sh56) or compound 2a (10 μM). After 48 hours, 5 μl of supernatants were 100 fold diluted in LDH storage buffer (Tris-HCl 200 mM, 10% Glycerol, 1% BSA, Sigma-Aldrich) and used to determine LDH, according to manufacturer's recommendations. Maximum LDH Release Control was obtained lysing untreated Neuro2a cells with 2 μL of 10% Triton X-100 (Sigma-Aldrich). In each experiment we calculated % Cytotoxicity=(Exp.LDH Release-Medium Background)/(Max LDH Release Control-Medium Background), and we run parallel positive controls treating cells with 200 μM H2O2.
Cycloheximide (CHX) assay was done on Neuro2a cells seeded in 6-well plates and incubated with compound 2a (10 μM) or vehicle for 24 hours. We next added CHX (Sigma) at 10 μg/mL and cells were collected at 0, 2, 4 and 8 hours for the time-course assay of VPS35 levels.
Autophagy assay were done using a stable Neuro2a cell clone expressing the pHluorin-mKate2-tagged human LC3 plasmids, Cell clones were generated according our published methods (Mazzocchi 2016). Upon transfection, cells were selected by 400 μg/mL G418 Disulfate Salt Solution (Sigma-Aldrich, St. Louis, Mo.) and further cloned by limiting dilution. Candidate cell clones were evaluated by flow cytometry (Cytoflex-S) and the clone 7B11 was selected. Quality controls of the assay were done calculating Z-factor (>0.5) and SSMD (>4.7 for Bafilomycin A1 and <4.7 for Torin 1) values in Neuro2a 7B11 cells treated BafilomycinA1 (200 nM) and Torin-1 (250 nM), (7 independent experiments). Neuro2a 7B11 cells were treated with Compound 2a (0.1-100 μM) for 48 hours then MFI levels of the pHfluorin were measured in living cells by flow cytometry.
Screening the Library of Amino Guanidine Hydrazones 2a-2mm in Neuro2a Cells
Neuro2a cells were seeded on 12 wells multiwell at the concentration of 100 000 cells/well. Cells were incubated with each compound, including the standard R55, at 10 μM for 48 hours. Cells were then washed in PBS 1× and lysed in a buffer containing Tris-HCl 10 mM pH8, EDTA 1 mM pH8, NaCl 100 mM, NP40 1%, with protease inhibitor cocktail (Sigma) 1×. Using a cell scraper total lysates were collected from each well, quantified with BCA (BCA protein assay, Thermofisher). Five μg were resolved on precast 10% acrylamide gel (Biorad). Proteins were transferred to a PVDF membrane (Millipore) and western blot were performed using the following antibodies: goat αVPS35 (1:1000), mouse α-βActin (1:10000). Detection of proteins was obtained by a chemiluminescence detection kit (PerkinElmer).
Immunoblotting
Lumbar spinal cords were dissected from saline perfused mice and rapidly homogenized in the following lysis buffer: Tris-HCl 10 mM pH 8, EDTA pH 8 1 mM, NaCl 100 mM, NP40 1% and protease inhibitor cocktail (Sigma). Protein extracts were obtained using a tight-fitting glass Potter tissue grinder (1 ml; Wheaton) and then sonicated at a frequency of 20 kHz (10 times-1s). Protein lysates from cells cultures were obtained from plates receiving PBS1× washing and subsequently the lysis buffer followed by sonication. Protein concentrations were measured by BCA protein assay kit (Thermofisher) according to the manufacturer's recommendations. Ten μg of protein extract was loaded on 10% Mini-PROTEAN TGX Stain-Free precast gels for PAGE (Bio-Rad). Gel electrophoresis was performed at 100 V for 2 hours. Gels were transferred on Immobilon®-P PVDF membrane according to the manufacturer's instructions (Millipore). Blots were blocked in 5% BSA in Tris buffered saline plus 0.1% Tween-20 (TBS-T) for 1 hour before receiving primary antibodies: goat α-VPS35 1:800 (ab10099, Abcam); rabbit α-VPS26b 1:1000 (15915-1-AP, ProteinTech); rabbit α-VPS29 1:1000 (ab236796 Abcam); mouse α-βActin 1:25000 (Sigma); mouse α-Golgin97 1:1000 (Invitrogen); mouse α-Ubiquitin 1:500 (Merk) overnight at 4° C. on shaker. Following several washes with TBS-T 1×, blots were incubated in appropriate HRP-labeled secondary antibodies (Bio-Rad) for 1 hour at room temperature. Visualization of protein bands was obtained using ClarityMax-ECL (Bio-Rad) according to the manufacturer's instructions and images were acquired on ChemiDoc (Bio-Rad). Quantifications were done using the Image Lab 6.0.1 software (Bio-Rad).
Blue-Native Gel Electrophoresis
G93A mice and their WT litters were injected daily with vehicle or compound 2a (10 mg/kg, from day 83 to day 103). Lumbar spinal cords dissected from saline perfused mice were lysed in Tris-HCl 10 mM pH 8, EDTA 1 mM pH 8, NaCl 100 mM, NP40 1% and protease inhibitor cocktail (Sigma). Extracts were obtained using a tight-fitting glass Potter tissue grinder (1 ml; Wheaton). We incubated 250 μl of each extract with 100 mM Iodoacetamide (Sigma). Samples were mixed with 4× Sample NativePAGE (Thermofisher) sample buffer and the G-250 additive (Thermofisher) and proteins were resolved on native 4-15% Bis-Tris gel (Bio-Rad) with Dark Blue Cathode buffer (Thermofisher) at 150 V for ⅓ of the gel length. We replaced the running buffer with Light Blue Cathode Buffer to complete the run. At the end of the electrophoresis, gels were washed with 5 mM tris(2-carboxyethyl)phosphine (Sigma) for 15 min and then proteins were transferred to nitrocellulose and fixed on the membrane by 8% acetic acid for 15 min. Rabbit α-SOD1 (1:2000, Genetex) was incubated overnight as above described. Visualization of proteins was performed using ClarityMax-ECL (Bio-Rad) according to the manufacturer's instructions and images were acquired on ChemiDoc (Bio-Rad).
Immunohistochemistry, Immunofluorescence and Morphometric Analyses Paraffin embedded SCs were obtained from ALS patients and non-neurological controls from the Target ALS Human Postmortem Tissue Core. Sections were stored at the INSPE tissue Bank. Sections were incubated with a polyclonal VPS35 antibody (1:400 Abcam ab97545) and with the polyclonal VPS26 antibody (ab23892 Abcam) after rehydration and antigen retrieval (slides were warmed-up at 97° C. in TRIS EDTA pH=9 by water bath). Peroxidase-diaminobenzidine (DAB) reaction was used for detecting primary antibody, biotinylated anti-rabbit IgG (H+L) (1:500 Vector laboratories) and avidin-biotin complex (ABC 1:100 Vector laboratories). An operator that was blind to the disease status and demographic data subsequently evaluated these sections.
Sections from mice (frozen cryo sections) or glass covers containing cells were washed three times in PBS 1× (Lonza, 5 min each), and incubated in the following blocking mix: PBS 1×/FBS 10% (Invitrogen), BSA 1 mg/mL (Sigma), TritonX100 0.1% (Sigma), for 1 h at room temperature. Antibodies were diluted in blocking mix and incubated at +4° C. overnight according manufacturer's instructions. The following day, sections were rinsed in PBS and fluorescent secondary antibodies—i.e. never deriving from the species from which the primary antibodies are derived—(Alexafluor conjugated) diluted in blocking mix, were used according to the manufacturer's instructions. Slides were incubated in Hoechst 33342 (Sigma) for nuclei counterstaining. When necessary, we performed antigens retrieval by boiling samples in 10 mM sodium citrate (pH 6) for 5 min. The following antibodies and working concentrations were used: rabbit α-VPS35 1:1000 (ab97545 Abcam); rabbit α-VPS26 1:1000 (ab23892 Abcam); rabbit α-Sortilin 1:1000 (ab16640 Abcam); mouse α-CI-MPR 1:100 (Novus bio. NB-300-514), mouse α-NeuN 1:800 (Millipore MAB377); mouse α-Ubiquitin 1:500 (Millipore MAB1510); mouse α-Golgin97 1:700 (Thermofisher A21270); rabbit α-ISLET 1:1000 (ab20670 Abcam); goat α-ChAT 1:200 (Millipore ab144p); chicken α-GFP 1:1000 (Invitrogen); chicken α-Neurofilament-medium 1:500 (Biolegend PCK-593P); rat α-MBP 1:100 (kindly provided by Dr. A. Bolino); mouse α-GM130 1:100 (BD biosciences, 610823); rat α-CTSD 1:100 (R&D system MAB1029). Tyramide Signal Amplification (TSA, PerkinElmer) was used, when appropriate, to improve fluorescent intensity in single and double immunofluorescence
Imaging
We used Olympus, BX51 with the following objectives: X20, X40 and X63 equipped with the following cameras: Leica CCD Microscope DFC3000 G and DMC2900 to obtain 16-bit light and fluorescence images (1296×966 pixels). Fluorescence images were merged using Photoshop (Adobe) CS4 or NHI-Image J software (US National Institutes of Health). We obtained confocal images using Leica SP8 with X40 and X64 objectives and equipped with super-sensitive HyD detectors. We recorded each fluorescence signal as square 8-bit images (1024×1024 pixels). Images were post processed to generate maximal projections of Z-stacks (acquired with a 0.4-0.8 μm step) and cross-sectional profiles of the Z-stack (acquired with a 0.3 μm step) and pseudo-colored using Las-X software.
ELISA Assay
Raw 264.7 cells (105/well) were treated with compound 2a, CNI-1493 or vehicle for 1 hour then we applied LPS (100 ng/ml) and supernatants were collected after 4 hours. We used the Enzyme-linked immunosorbent assays (ELISA) procedure with OptEIA™ set for the detection of mouse TNFα (BD-Biosciences) according to manufacturer's instructions. Concentrations of TNFα were calculated according to a standard curve and expressed as nanograms per microliter. When concentrations of the cytokine were below the detection threshold, they were assumed to be 0 ng/μl.
Quantification of MFI Levels in Tissue MNs
Fluorescence intensity levels of VPS35, VPS26, Sortilin, CTSD and Ubiquitin were calculated using NHI-Image J software (US National Institutes of Health) according to the following protocol: stacks of confocal images were used to generate the maximal projections (Las-X) and then post-processed by NHI-Image J to crop single NeuN+ MNs from the ventral horn of the spinal cord. We next performed background (calculated on adjacent slices only receiving secondary antibodies) subtraction for each channel, before applying the NHI-Image J mean filter (with radius 2.0 pixels) to calculate mean NeuN fluorescence levels in individual MN. Using this approach, we established a region of interest (ROI) that gated single MN. On this selected ROI, we calculated mean fluoresce levels (MFI) of each protein.
Morphological Assessment of Golgi Apparatus in Neuro2a
Golgi morphology was assessed in control or in Neuro2a cells receiving G93A plasmids with or without compound 2a (10 μM) according to published methods (Stafa 2012). In each experiment, we included GFP-transfected cells as additional control. We used Golgin97 immunofluorescence to label trans-Golgi network membranes. We classified Golgi morphology as follow: normal (polarized network), intermediate (partially fragmented) or fragmented (severely fragmented with Golgi stacks dispersed in the cytoplasm throughout), (Stafa 2012). In each experiment, cells were randomly sampled across three coverslips. Golgi subclasses were expressed as a percent of the total number of Golgi scored for each experimental condition.
Micro Electrode Array (MEA) Recordings
Primary neuronal cultures were established from the cerebral cortex of E16.5 C57BL6J embryos. Upon dissection, cells were plated on MEA biochips (60 electrode MEA biochips with 200 μm electrode spacing and 30 μm electrodes diameter with an integrated reference electrode, Multichannel Systems, GmbH) at the density of 3×105 cells/MEA and kept in Neurobasal medium supplemented with B27 for 14 days. At day 14 neuronal cultures were exposed to increasing concentration of lead 2a (1, 3, 10, 30 and 100 μM) in Krebs-Ringer-Hepes buffer (KRH, 130 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 25 mM Hepes, 0.6% glucose, 1% glutamine, Sigma). Firing activity was recorded using a pre-amplifier stage (MEA-1060-Inv-BC-Standard, gain: 55, bandwidth: 300 Hz-8 kHz, MCS GmbH), an amplification and filtering stage (FA64S, gain 20, bandwidth: 10 Hz-6 kHz, MCS GmbH) and a data acquisition device (sampling frequency: 25 kHz). Then, an off-line signal processing was performed and raw data were analyzed by MC-Rack Software (MCS GmbH).
Real Time PCR
Lumbar spinal tracts dissected from saline perfused mice or Neuro2a cells were lysed to extract total RNAs using the RNeasy Mini Kit (Qiagen) according to the manufacturer's recommendations, including DNase (Promega) digestion. We synthetized the cDNA using ThermoScript RT-PCR System (Invitrogen) and Random Hexamer (Invitrogen), according to the manufacturer's instructions. The LightCycler 480 System (Roche) and LightCycler 480 SYBR Green I Master Mix (Roche) were used for real-time PCR experiments. Normalization was obtained using the housekeeping gene Histone H3
(H3 F: 5′-GGTGAAGAAACCTCATCGTTACAGGCCTGGTAC-3′ H3 R: 5′-CTGCAAAGCACCAATAGCTGCACTCTGGAAGC-3′). The following specific primers were used for gene expression analysis:
Pharmacology—In Vivo
Detection of Compound 2a in Brain and Blood Samples Isolated from Mice
Healthy mice were daily injected with vehicle or compound 2a (10 mg/kg) and sacrificed after 1, 7, 15 and 30 days. Upon saline perfusion, brains were dissociated from skulls, carefully weighted and rapidly stored in dry ice. Then, brains (˜400 mg) were checked for the presence of Compound 2a. The extraction will be performed with MeOH according to the protocol reported in Yuan et al., 2012. After the extraction, each biological sample was re-suspended in 60 μl of LC/MS grade water and 5 μl analyzed by LC-MS/MS using the UPLC 1290 (Agilent Technologies) coupled to the TripleTOF 5600+ mass spectrometer (SCIEX) in positive mode. A Waters ACQUITY UPLC BEH HILIC column (2.1×10 mm, 1.7 μm) was used for the chromatographic separation through a gradient of solvent A (ACN+0.1% FA) and solvent B (H2O+0.1% FA). Calibration curves were constructed by plotting the peak area versus the corresponding concentrations using MultiQuant 2.1 software (SCIEX).
Generation of MNs from Human Inducible Pluripotent Stem Cells(hiPSCs)
Skin biopsies from ALS patients and healthy volunteers were performed under local anesthesia, after informed consent at San Raffaele Hospital. The protocol was approved by the Local Etic Committee. Primary fibroblasts were grown in Dulbecco's modified Eagle's medium with Glutamax I (GIBCO). Human iPS cell lines were generated using non integrating Sendai virus, maintained in feeder-free conditions in mTeSR1 (Stem Cell Technologies) on embryonic stem cell (HESC) Matrigel (Corning). MNs were generated following the protocol published by Du and colleagues with minimal modification (Du et al., 2015). Briefly, when hiPSCs reached confluence, they have been detached using EDTA and plated 1:4 on Matrigel ES coated dishes in mTeSR-1 supplemented with ROCK inhibitor (StemMACS, Miltenyi Biotec). On the following day the medium was replaced with a chemically defined neural medium: DMEM/F12, Neurobasal medium at 1:1 ratio, 1% B27, 0.5% N2 (all from Gibco, ThermoFisher Scientific), 1% P/S (Gibco), 1% Glutamax (Gibco) and 0.1 mM Ascorbic Acid (Sigma). CHIR99021 (3 μM, Tocris), DMH1 (2 μM, Tocris) and SB431542 (2 μM, Miltenyi Biotec) were added to the medium. The culture medium was changed every other day, until day 6. On day 7, cells have been dissociated with Dispase (1 U/mL), split 1:4 and kept on Matrigel growth factor reduced (MaGR) for 6 days in the basal neural medium described above, supplemented with 1 μM CHIR99021, 2μM DMH1, 2 μM SB431542, RA (Retinoic Acid, 0.1 μM, Sigma) and SAG (Smoothened Agonist, 0.5 μM, Calbiochem). On day 13, cells have been dissociated with Dispase (1 U/mL), split 1:4, and kept for 6 days in neural medium supplemented with 3 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM RA, 0.5 μM SAG and 0.5 mM Valproic Acid (VPA, Tocris). To induce MN differentiation, cells were dissociated with Dispase (1 U/mL) and cultured in suspension for 6 days in neural medium with 0.5 μM RA and 0.1 μM SAG. On day 25, cells were dissociated into single-cells with Accumax (Sigma) and plated on MaGR coated plates, in neural medium with 0.5 μM RA, 0.1 μM SAG, 0.1 μM Compound E (Calbiochem) until day 35. Half of the medium has usually been changed every other day.
Animals
Mice were maintained under pathogen-free conditions at San Raffaele Hospital mouse facility (Milan, Italy). All efforts were made to minimize animal suffering and to reduce the number of mice used in accordance with the European Communities Council Directive of 24 Nov. 1986 (86/609/EEC). All animal experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of the Italian Ministry of Health. Procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee of the San Raffaele Scientific Institute (protocol number 693/2015PR). Transgenic mutant SOD1 mice carrying the SOD1G93A allele (strain B6.Cg-Tg (SOD1G93A) 1Gur/J) were purchased from Jackson laboratories, while C57BL6J used for breeding and experimental purposes were purchased from Charles River (Italy). Compound 2a (10 mg/kg) was daily intraperitoneally injected by an operator blind to the treatment and to the genotype. At the sacrifice, mice were given an overdose of anesthetic drugs and transcardially perfused with saline followed by 80/100 ml 4% paraformaldehyde in PBS, pH 7.2 (Sigma). Spinal cords were coronally sliced at ˜6-mm thickness and post fixed in 4% paraformaldehyde (Sigma) in PBS, pH 7.2 for 12 h at +4° C. Tissues were cryoprotected in PBS/30% Sucrose (Sigma), embedded in OCT inclusion media and stored at −80° C. before processing. Lumbar spinal cords were 12 μM sectioned, labeled and digital images were acquired every 350 μm in a region encompassing 2.5 mm of the spinal cord. Animal cohort numbers were determined by power analysis based on preliminary results.
Comparative studies on sciatic nerves were done on tissues removed upon saline perfusion and rapidly fixed with 2% glutaraldehyde in 0.12 mol/L phosphate buffer, post fixed with 1% osmium tetroxide, and embedded in Epon (Fluka). Semi-thin sections (1 μm thick) were stained with toluidine blue and examined by light microscopy (Olympus BX51). Digitized not overlapping semi-thin section images from corresponding levels of the sciatic nerve were obtained with a digital camera (Leica DFC300F) using a 40X objective. Quantifications were done on at least 5 acquired images from each mouse. The numbers of degenerating nerve fibers were quantified using ImageJ software (National Institutes of Health, Bethesda, Md.).
Motor Function
Motor activity of G93A mice was assessed by rotarod. Mice were habituated for 1 min on a static rotor and 1 min at constant speed (4 rpm) for two times and then tested for motor function over three trials performed over three consecutive days (one per day). Each trial consisted of three test sessions with 15 min interval between sessions. For each session five mice were placed on an accelerating rotor (4-40 rpm) and the latency to fall was recorded, with a maximum limit for individual animal set at 900 s.
Statistics
We express data as the mean value (±standard error of the mean (s.e.m.) or ±standard deviation (s.d.)) of independent experiments. We assessed normality of data applying either Kolmogorov-Smirnov test (with Dallas-Wilkinson-Lille for P value) or D'Agostino & Pearson omnibus normality test. Comparisons were made using the following tests: unpaired t-test, one-way or two-way analysis of variance (ANOVA) tests, followed by Tukey's multiple Comparison test or by Bonferroni multiple comparison test. Statistical analyses were performed using PRISM5.01 (GraphPad Software, La Jolla, Calif., USA). Values lower than 0.05 were considered statistically significant.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 19166534.8 | Apr 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/059207 | 4/1/2020 | WO | 00 |
