WO2010018555A2 - Quinoline compounds containing a dibemethin group - Google Patents

Quinoline compounds containing a dibemethin group Download PDF

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WO2010018555A2
WO2010018555A2 PCT/IB2009/053578 IB2009053578W WO2010018555A2 WO 2010018555 A2 WO2010018555 A2 WO 2010018555A2 IB 2009053578 W IB2009053578 W IB 2009053578W WO 2010018555 A2 WO2010018555 A2 WO 2010018555A2
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Prior art keywords
methyl
mmol
compound
phenyl
chloro
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PCT/IB2009/053578
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French (fr)
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WO2010018555A4 (en
WO2010018555A3 (en
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Roger Hunter
Timothy John Egan
Vincent Kudakwashe Zishiri
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University Of Cape Town
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Priority to AP2011005602A priority Critical patent/AP2848A/en
Publication of WO2010018555A2 publication Critical patent/WO2010018555A2/en
Publication of WO2010018555A3 publication Critical patent/WO2010018555A3/en
Publication of WO2010018555A4 publication Critical patent/WO2010018555A4/en
Priority to ZA2011/01772A priority patent/ZA201101772B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/42—Nitrogen atoms attached in position 4
    • C07D215/46—Nitrogen atoms attached in position 4 with hydrocarbon radicals, substituted by nitrogen atoms, attached to said nitrogen atoms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • These compounds include calcium channel blockers such as verapamil, antidepressants such as imipramine (1), antihistamines such as azatadine and antipsychotics such as chlorpromazine among others. 5
  • antidepressants such as imipramine (1)
  • antihistamines such as azatadine
  • antipsychotics such as chlorpromazine among others. 5
  • chlorpheniramine 6 ' 7 these compounds have not been tested clinically because most require unacceptably high levels to exert significant chloroquine resistance reversing activity. This leaves development of new drugs as the only viable alternative at present. This includes re-engineered 4-amino-7-chloroquinolines such as ferroquine and isoquine which are currently in development and which do not show cross resistance with chloroquine. 8 ' 9
  • a 4-amino- 7-chloroquinoline compound comprising a dibenzyimethylamine side chain attached to the amino group of the quinoline group, the compound comprising formula (I):
  • X- I > X 2 , X3 and X 4 are independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl;
  • Y is CH or N; m, n, p, q, r and s are independently from 0 to 5; and
  • R1 , R2, R3 and R4 are independently selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein
  • R3 and R4 together with the carbon atoms to which they are joined optionally form a six membered ring.
  • the six membered ring formed by R3 and R4 may have one or more substituents independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl.
  • the compound may be any pharmaceutically acceptable salt of these compounds. Examples of the compound are:
  • the compound and any salts thereof may be used for preventing and/or treating malaria, in particular malaria caused by strains of Plasmodium falciparum. More particularly, the compound may be for use in preventing and/or treating malarial infection from chloroquine sensitive or chloroquine resistant Plasmodium strains.
  • a pharmaceutical composition including a therapeutically effective amount of a compound substantially as described . above and a pharmaceutically acceptable carrier.
  • the composition may be for treating malaria.
  • the composition may include a second antimalarial compound, such as chloroquine.
  • a compound substantially as described above in a method of making a medicament for use in a method of preventing and/or treating malaria.
  • the medicament may comprise the compound and a suitable carrier.
  • a method of preventing and/or treating malaria comprising administering an effective amount of a compound substantially as described above to an animal in need thereof.
  • the animal may be a human.
  • the compound may be administered together with a second antimalarial compound, such as chloroquine.
  • a second antimalarial compound such as chloroquine.
  • Figure 1 The crystal structure of 4.
  • Figure 2 Correlations between pK a values and resonance constants (R) of the group attached to the terminal phenyl ring (see Table 2).
  • Figure 3 Correlations of biological activity (IC 50 ) with vacuolar accumulation ratio (VAR), IC 50 for ⁇ -hematin inhibition activity (BHIA 50 ) and a molecular structure descriptor pos (ortho-, meta- or para- indicated by 2, 3 or 4 respectively and referring to compounds 4, 7, 10 and 13; 5, 8, 11 and 14; and 6, 9, 12 and 15, respectively),
  • VAR vacuolar accumulation ratio
  • BHIA 50 ⁇ -hematin inhibition activity
  • pos molecular structure descriptor pos
  • X- b * 2 ⁇ X3 and X 4 are independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl;
  • Y is CH or N; m, n, p, q, r and s are independently from 0 to 5; and
  • R1, R2, R3 and R4 are independently selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein
  • R3 and R4 together with the carbon atoms to which they are joined optionally form a six membered ring.
  • the six membered ring formed by R3 and R4 can have one or more substituents independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl.
  • the compound may be any pharmaceutically acceptable salt of these compounds
  • R 1 , R 2 , R3 and R 4 are:
  • R can be any one of the compounds shown in Table 1.
  • Z H, Cl 1 Br, I, F, CN, CONH 2 , NO 2 , NH 2 , CH 3 , CF 3 , CH 2 CH 3 , SO 2 NH 2 , SCF 3 , N(CH 3 ) 2 , OCH 3 , OH, OCF 3 and R 2 is the structure:
  • the compounds and any salts thereof can be used for preventing and/or treating malaria, in particular malaria caused by strains of Plasmodium falciparum, and more particularly, chloroquine sensitive or chloroquine resistant Plasmodium strains.
  • a pharmaceutical composition for this purpose can be prepared by adding a suitable carrier to the compound.
  • Therapeutically effective doses (or growth inhibitory amounts) of a compound or pharmaceutical composition of the invention can be determined by one of skill in the art, with a goal of achieving local (e.g., tissue) concentrations that are at least as high as the IC50 of the applicable compound disclosed in the examples herein.
  • An example of a dosage range is from about 0.1 to about 200 mg/kg body weight orally in single or divided doses.
  • a dosage range is from about 1.0 to about 100 mg/kg body weight orally in single or divided doses, including from about 1.0 to about 50 mg/kg body weight, from about 1.0 to about 25 mg/kg body weight, from about 1.0 to about 10 mg/kg body weight (assuming an average body weight of approximately 70 kg; values adjusted accordingly for persons weighing more or less than average).
  • the compositions are, for example, provided in the form of a tablet containing from about 50 to about 1000 mg of the active ingredient, particularly about 75 mg, about 100 mg, about 200 mg, about 400 mg, about 500 mg, about 600 mg, about 750 mg, or about 1000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject being treated.
  • a tablet containing from about 500 mg to about 1000 mg active ingredient is administered once (e.g., a loading dose) followed by administration of 1/2 dosage tablets (e.g., from about 250 to about 500 mg) each 6 to 24 hours for at least 3 days.
  • 1/2 dosage tablets e.g., from about 250 to about 500 mg
  • the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the subject undergoing therapy.
  • o-aminomethyldibemethin (4a) was synthesized from /V-benzylmethylamine (16) and benzyl bromide (17) in four steps as shown in Scheme 1. Coupling of benzylmethylamine and benzyl bromide produced dibemethin (18) which was then reacted with f-butyllithium followed by anhydrous dimethylformamide to produce the benzaldehyde (19). This benzaldehyde was converted to an oxime (20) by reaction with hydroxylammonium chloride and finally reduced to the desired product (4a) with LiAIH 4 .
  • the other two parent aminomethyl dibemethin compounds (5a and 6a) were prepared in a three step reaction from m- and p-dibromoxylene (22 and 23) and N, N- benzylmethylamine (16), while the remaining aminomethyl dibemethins (7a - 15a) were synthesized in four steps from either a benzyl chloride (27, 28) or p- (dimethylamino)benzaldehyde (29) as shown in Scheme 2.
  • Compounds (24 - 26) were prepared by reacting o-, m- or p-dibromoxylene (21 - 23) with sodium azide under an inert atmosphere overnight.
  • the benzyl chloride derivatives (27, 28) were reacted with nnethylamine to produce a series of benzyl methylamines (30 - 32) and the benzaldehyde derivative (29) was reductively aminated to produce the corresponding benzyl methylamine (32).
  • Reaction of the suitable benzyl methylamine with (24, 25 or 26) under reflux produced the azides (33 - 43) which were reduced by the Staudinger reaction to produce the desired dibemethin derivatives (5a - 15a).
  • reaction mixture was worked up by the addition of a saturated solution of aqueous Na 2 CO 3 and the product extracted into ethyl acetate (3 x 50 ml), the organic extracts dried over anhydrous MgSO 4 and the solvent removed under reduced pressure to give a crude product (17.05 g), which was chromatographed on silica-gel (170 g) using 10% ethyl acetate in hexane as eluent.
  • Target molecules (4 - 15) were synthesized in a single step by reaction of an appropriate dibemethin (4a - 15a) with excess commercially available 4,7- dichloroquinoline in anhydrous ⁇ /-methyl-2-pyrrolidone under N 2 in the presence of potassium carbonate and triethylamine. The reaction was conducted in sealed cycloaddition tubes at 90 - 130 0 C over periods ranging from 16 - 48 h. The products were then extracted from alkaline aqueous solution into ethyl acetate and purified by column chromatography to afford modest yields. All products were characterized by infrared, 1 H and 13 C NMR and mass spectrometry. Solids were further characterized by melting point determination and elemental combustion analysis, while oils were subjected to high resolution mass spectrometry.
  • the two pK a values of compounds 4 - 15 were determined by pH titration and are reported in Table 2.
  • the IC 50 values for ⁇ -hematin inhibition were determined using a pyridine based 96-well plate method. 13 The method relies on the fact that 5 % aqueous pyridine dissolves hematin, but not ⁇ -hematin at pH 7.5. The extent of inhibition was then characterized by measuring the intensity of the monomeric pyridine-hematin complex at 405 nm. Values are also reported in Table 4.
  • Table 4 The resonance constants (R), measured acid dissociation constant (pK s ) values, ⁇ -hematin inhibitory activities (BHIAgg), in vitro antimalarial activities (ICm) versus the D10 and K1 strains of P. falciparum and resistance index (Rl) of compounds 4 - 15.
  • IC 50 values for ⁇ -hematin inhibition covered a somewhat larger range, with the most potent inhibitor (6) having an IC 50 of 0.32, while the least active (12) exhibited a value of 1.44.
  • the samples were prepared to a 2mg/ml stock solution in 100% DMSO or 100% methanol and sonicated to enhance solubility. Samples were tested as a suspension if not completely dissolved. Stock solutions were stored at -2O 0 C. Further dilutions were prepared on the day of the experiment. Chloroquine (CQ) was used as the reference drug in all experiments. A full dose-response was performed for all compounds to determine the concentration inhibiting 50% of parasite growth (IC 50 - value). Test samples were tested at a starting concentration of 100 ng/ml or 10 ⁇ g/ml, which was then serially diluted 2-fold in complete medium to give 10 concentrations. The same dilution technique was used for all samples.
  • CQ Chloroquine
  • the MTT-assay is used as a colorimetric assay for cellular growth and survival, and compares well with other available assays. 19
  • the tetrazolium salt MTT was used to measure all growth and chemosensitivity. Compound 4 was tested in triplicate on three separate occasions.
  • the compound was dissolved in 10% DMSO.
  • the initial concentrations of stock solutions were 2mg/ml.
  • the compound was tested as a suspension and stored at -2O 0 C until use.
  • the highest concentration of solvent to which the cells were exposed to had no measurable effect on the cell viability (data not shown).
  • Emetine was used as the reference drug in all experiments.
  • the initial concentration of emetine was 100 ⁇ g/ml, which was serially diluted in complete medium with 10-fold dilutions to give 6 concentrations, the lowest being 0.001 ⁇ g/ml.
  • the same dilution technique was applied to the test sample with an initial concentration of 100 ⁇ g/ml to give 5 concentrations, with the lowest concentration being 0.01 ⁇ g/ml.
  • IC 50 50% inhibitory concentration
  • mice were challenged using the 4-day suppressive test as described by Peters et al, 20 with modifications. Identical procedures were used for both the 5 mg/kg and 20 mg/kg experiments. There were 5 mice in each test group.
  • mice Each mouse was infected with 1x10 7 parasitized erythrocytes by intraperitoneal injection in 200 ⁇ l PBS. Parasite stocks were made from a donor mouse which was sacrificed once parasite levels were deemed sufficient to infect the test mice; infected blood cells were obtained via cardiac puncture and placed in tubes containing PBS and EDTA to prevent clotting. Each compound was diluted to 5 mg/kg as described above; a fresh stock of CQ (5 mg/kg) was also produced. Mice were treated with 100 ⁇ l of 4, 6 or chloroquine via sub-cutaneous injection.
  • Days +1 to +3 Mice treated with 100 ⁇ l freshly-prepared drug stocks; drugs were administered at approximately the same time as on Day 0 to within half an hour.
  • the prototype (4) was subjected to cytotoxicity testing in Chinese hamster ovarian (CHO) cells.
  • the IC 50 in this system was found to be 32 ⁇ M, which is 232 times higher than the IC 50 in the D10 strain of P. falciparum and 170 times greater than that in the K1 strain.
  • compound 4 exhibits highly selective activity against the malaria parasite.
  • Compounds 4 and 6 were also tested against P. yoelii nigeriensis in a mouse model by intra-peritoneal injection at 5 and 20 mg/kg and compared to chloroquine diphosphate at the same doses. As indicated in Table 5, both compounds were less effective than chloroquine, although substantial activity was observed at 20 mg/kg.
  • Table 5 In vivo antimalarial activities of suspensions of compounds 4 and 6 administered to adult BaIbC white mice infected with P. voelii niaeriensis by intra-peritoneal injection.
  • the parent compounds 4, 5 and 6 were sent to the Research School of Biology, Australian National University in Canberra Australia and tested for inhibition of CQ transport by PfCRT. All three were found to inhibit CQ transport by PfCRT (the protein primarily responsible for chloroquine resistance), with 5 exhibiting an IC 50 of 65 ⁇ M. This concentration can be expected to be easily attained within the parasite digestive vacuole as a result of pH trapping.
  • mice were challenged using the suppressive test as described by Peters et al, 20 with modifications.
  • the in vivo activity of 4 was evaluated with the 4 days Peters' test, due to the physical properties and the available amounts of material, 5 was used for a three times treatment (day 0, day 1 and day 2) and 6 was used for a one times treatment (day 0).
  • parasitaemia was determined on day 4. Identical procedures were used for both the 30 mg/kg and 100 mg/kg experiments.
  • hepahnized blood was taken from a donor mouse with approximately 30% parasitaemia and diluted in physiological saline to 10 8 parasitized erythrocytes per ml.
  • 0.2ml was injected intravenously (i.v.) into experimental groups of 3 mice, and a control group of 3 mice. 4 hours post-infection the experimental groups were treated with a single dose 24, 48 and 72 hours postinfection, the experimental groups were treated with a single daily dose.
  • the survival of the animals was monitored up to 30 days. Mice surviving for 30 days were checked for parasitaemia. A compound is considered curative if the animal survives to day 30 post-infection with no detectable parasites. The results are expressed as reduction of parasitaemia on day 4 in % as compared to the untreated control group, and mean survival compared to the untreated control group.
  • Table 6 tabulates the results of the in vivo antimalarial activity of the parent compounds (4, 5 and 6) against P. berghei infected mice when the compounds were administered by oral dosing at 30 mg/kg and 100 mg/kg.
  • [Q] v is the concentration of the compound in the digestive vacuole of the parasite
  • [Q] e is its concentration in the extracellular medium
  • pH v is the digestive vacuole pH (taken as 5, the midpoint between two recent estimates of 4.8 and 5.2)
  • pH e is the pH of the external medium (7.4).
  • Values are given in Table 7.
  • An accumulation normalized IC 50 for antimalarial activity can be calculated by multiplying the observed IC 50 by the VAR value for each compound.
  • 4-aminoquinolines are believed to act by inhibiting hemozoin formation in the digestive vacuole, these numbers may be more relevant than the observed IC 50 . They are therefore reported in Table 7.
  • VAR-ICs n Calculated vacuolar accumulation ratio (VAR) and vacuolar accumulation normalized IC Rn against the K1 strain of P. falciparum in vitro (VAR-ICs n ).

Abstract

4-Amino-7-chloroquinolines are described containing dibenzylmethylamine (dibemethin) side chains attached via a methylene bridge to the amino group of the quinoline showing strong antimalarial and resistance reversing activity. The compounds are of the general formula (I), wherein X1, X2, X3 and X4 are independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl; Y is CH or N; m, n, p, q, r and s are independently from 0 to 5; R1, R2, R3 and R4 are independently selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein R3 and R4 together with the carbon atoms to which they are joined optionally form a six membered ring; or pharmaceutically acceptable salts thereof. Pharmaceutical compositions containing at least one of these compounds are also described for treating or preventing malaria.

Description

QUINOLINE COMPOUNDS CONTAINING A DIBEMETHIN GROUP
BACKGROUND OF THE INVENTION
The development and spread of chloroquine resistance has been a major setback to malaria control worldwide.1 Although parasites have been reported to recover chloroquine sensitivity after an extended period of using other drugs,2 it has been shown that this arises from expansion of a chloroquine sensitive sub-population of parasites rather than back mutation.3 Thus drug rotation is not a viable option as chloroquine pressure is likely to simply result in rapid re-expansion of chloroquine resistant strains.4 Chemosensitizing agents, also called chloroquine resistance reversing agents are known. These compounds include calcium channel blockers such as verapamil, antidepressants such as imipramine (1), antihistamines such as azatadine and antipsychotics such as chlorpromazine among others.5 However, with the exception chlorpheniramine,6'7 these compounds have not been tested clinically because most require unacceptably high levels to exert significant chloroquine resistance reversing activity. This leaves development of new drugs as the only viable alternative at present. This includes re-engineered 4-amino-7-chloroquinolines such as ferroquine and isoquine which are currently in development and which do not show cross resistance with chloroquine.8'9
A recent development has been the proposal of the concept of a so-called "reversed chloroquine".10 This is a 4-amino-7-chloroquinoline which is linked to a resistance reversing agent such as imipramine via a linker (2). These compounds make use of the 4-amino-7-chloroquinoline antimalarial pharmacophore which is believed to act by inhibiting hemozoin formation in the digestive vacuole of the malaria parasite. WO 2006/088541 describes a wide range of such compounds in which the resistance reverser possesses the pharmacophore features identified by Bhattacharjee et al. for a resistance reverser. 12 This involves two suitably arranged aromatic rings with a three or four carbon chain linked to the bridging group between the rings and terminating in an amino group (3). The key requirements for resistance reversal in these imipramine-like resistance reversers are two hydrophobic, aromatic rings fused to a 7-membered ring containing a nitrogen atom which bears a three carbon chain terminated by a H-bond acceptor group, preferably N.
Figure imgf000003_0001
The applicant has now found that, surprisingly, resistance reversing activity does not require this structural motif.
SUMMARY OF THE INVENTION
According to a first embodiment of the invention, there is provided a 4-amino- 7-chloroquinoline compound comprising a dibenzyimethylamine side chain attached to the amino group of the quinoline group, the compound comprising formula (I):
Figure imgf000004_0001
(I) wherein
X-I > X2, X3 and X4 are independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl;
Y is CH or N; m, n, p, q, r and s are independently from 0 to 5; and
R1 , R2, R3 and R4 are independently selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein
R3 and R4 together with the carbon atoms to which they are joined optionally form a six membered ring.
The six membered ring formed by R3 and R4 may have one or more substituents independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl.
The compound may be any pharmaceutically acceptable salt of these compounds. Examples of the compound are:
/V-[{2-(W-Benzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine;
Λ/-[{3-(Λ/-Benzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine;
A/-[{4-(Λ/-Benzyl-A/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine;
Λ/-[{2-(Λ/-p-Chlorobenzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine;
A/-[{3-(Λ/-p-Chlorobenzyl-A/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine;
Λ/-[{4-(/V-p-Chlorobenzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chIoro-4- quinolinamine;
7-Chloro-Λ/-[{2-(Λ/-p-methoxybenzyl-Λ/-methylaminomethyl)phenyl}methyl]-4- quinolinamine;
7-Chloro-Λ/-[{3-(Λ/-p-methoxybenzyl-Λ/-methylaminomethyl)phenyl}methyl]-4- quinolinamine;
7-Chloro-A/-[{4-(Λ/-p-Methoxybenzyl-Λ/-methylaminomethyl)phenyl}methyl]-4- quinolinamine;
7-Chloro-Λ/-[{2-(/V-p-dimethylaminobenzyl-Λ/- methylaminomethyl)phenyl}methyl]- 4-quinolinamine;
7-Chloro-Λ/-[{3-(/V-p-Dimethylaminobenzyl-Λ/- methylaminomethyl)phenyl}methyl]- 4-quinolinamine; and
7-Chloro-Λ/-[{4-(Λ/-p-dimethylaminobenzyl-Λ/- methylaminomethyl)phenyl}methyl]-4-quinolinamine.
The compound and any salts thereof may be used for preventing and/or treating malaria, in particular malaria caused by strains of Plasmodium falciparum. More particularly, the compound may be for use in preventing and/or treating malarial infection from chloroquine sensitive or chloroquine resistant Plasmodium strains.
According to a second embodiment of the invention, there is provided a pharmaceutical composition including a therapeutically effective amount of a compound substantially as described . above and a pharmaceutically acceptable carrier. The composition may be for treating malaria.
The composition may include a second antimalarial compound, such as chloroquine.
According to a third embodiment of the invention, there is provided the use of a compound substantially as described above in a method of making a medicament for use in a method of preventing and/or treating malaria.
The medicament may comprise the compound and a suitable carrier.
According to a fourth embodiment of the invention, there is provide a method of preventing and/or treating malaria, the method comprising administering an effective amount of a compound substantially as described above to an animal in need thereof.
The animal may be a human.
The compound may be administered together with a second antimalarial compound, such as chloroquine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : The crystal structure of 4.
Figure 2: Correlations between pKa values and resonance constants (R) of the group attached to the terminal phenyl ring (see Table 2). (a) Statistically significant correlation for the quinoline N, pKai (r2 = 0.69, P = 0.0008). (b) Correlation for the dibemethin tertiary amino group, pKa2. In this case the correlation is not as good, but is still statistically significant if all of the data points are included (r2 = 0.35, P = 0.042). Omission of a single data point (open circle) for compound 7 improves the fit considerably (r2 = 0.65, P = 0.0028). Figure 3: Correlations of biological activity (IC50) with vacuolar accumulation ratio (VAR), IC50 for β-hematin inhibition activity (BHIA50) and a molecular structure descriptor pos (ortho-, meta- or para- indicated by 2, 3 or 4 respectively and referring to compounds 4, 7, 10 and 13; 5, 8, 11 and 14; and 6, 9, 12 and 15, respectively), (a) Linear correlation between log IC50 and log BHIA50 is not statistically significant (r2 = 0.30, P = 0.083) unless the point for compound 12 is omitted (open circle) which considerably improves the correlation (r2 = 0.50, P = 0.021). (b) Linear correlation between the vacuolar accumulation normalized log IC50 (log VAR-IC50) and log BHIA50 is also not statistically significant (r2 = 0.33, P = 0.064) unless the point for compound 12 is omitted (open circle) which again considerably improves the correlation (r2 = 0.51 , P = 0.012). (c) Statistically significant linear correlation between log IC5O and the structural descriptor (r2 = 0.52, P = 0.012). (d) Multiple linear correlation between the log of the vacuolar accumulation normalized IC50 (log VAR-IC50) and both log BHIA50 and the structural descriptor conform to the equation log VAR-IC50 = 0.84 x log BHIA50 - 0.34 x pos - 1.65. F = 10.3 > Fcrit = 8.65 at the 99 % confidence level, (e) Multiple linear correlation of log IC50 with VAR, log BHIA50 and pos. Here the data conform to the equation log IC50 = 0.95 x log BHIA50 - 0.35 x pos - 3.87 x log VAR + 10.24. F = 9.70 > 8.45 at the 95 % confidence level.
DETAILED DESCRIPTION OF THE INVENTION
4-amino-7-chloroquinolines containing dibenzylmethylamine (dibemethin) side chains attached via a methylene bridge to the amino group of the quinoline showing strong antimalarial and chloroquine resistance reversing activity are described herein. The compounds are of the general formula (I):
Figure imgf000008_0001
(I) wherein
X-b *2< X3 and X4 are independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl;
Y is CH or N; m, n, p, q, r and s are independently from 0 to 5; and
R1, R2, R3 and R4 are independently selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein
R3 and R4 together with the carbon atoms to which they are joined optionally form a six membered ring.
The six membered ring formed by R3 and R4 can have one or more substituents independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl.
The compound may be any pharmaceutically acceptable salt of these compounds
Some examples of the groups R1, R2, R3 and R4 are:
Figure imgf000009_0001
Figure imgf000009_0002
Dibemethin compounds that could be used to form suitable compounds of the invention include formulae (II) to (XIII):
Figure imgf000010_0001
Figure imgf000010_0002
Figure imgf000010_0003
Formulae (H)-(XIII)
where
X = Cl, Br, I, F, CN, CONH2, NO2, NH2, H, CH3, CF3, CH2CH3, SO2NH2, SCF3, N(CH3)2, OCH3, OH, OCF3, R1 = H, CH3, CH2CH3, CHCH2Or CCH. R can be any one of the compounds shown in Table 1.
Table 1 : Examples of the group R in the formulae (H)-(XIII) above
Figure imgf000010_0004
Figure imgf000011_0003
in Table 1, Z = H, Cl1 Br, I, F, CN, CONH2, NO2, NH2, CH3, CF3, CH2CH3, SO2NH2, SCF3, N(CH3)2, OCH3, OH, OCF3 and R2 is the structure:
Figure imgf000011_0001
(XIV) where examples of R3 are:
H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and examples of R4 are shown in Table 2.
Table 2: Specific examples of R4 (in formulae (XIV))
Figure imgf000011_0002
In Table 2, specific examples of the group E are H, Cl, Br, I, F, CN, CONH2, NO2, NH2, CH3, CF3, CH2CH3, SO2NH2, SCF3, N(CH3)2, OCH3, OH and OCF3.
More particular examples of compounds of the invention are:
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000013_0002
Figure imgf000014_0001
Figure imgf000014_0002
Figure imgf000014_0003
Figure imgf000014_0004
Figure imgf000015_0001
The compounds and any salts thereof can be used for preventing and/or treating malaria, in particular malaria caused by strains of Plasmodium falciparum, and more particularly, chloroquine sensitive or chloroquine resistant Plasmodium strains. A pharmaceutical composition for this purpose can be prepared by adding a suitable carrier to the compound.
Therapeutically effective doses (or growth inhibitory amounts) of a compound or pharmaceutical composition of the invention can be determined by one of skill in the art, with a goal of achieving local (e.g., tissue) concentrations that are at least as high as the IC50 of the applicable compound disclosed in the examples herein. An example of a dosage range is from about 0.1 to about 200 mg/kg body weight orally in single or divided doses. In particular examples, a dosage range is from about 1.0 to about 100 mg/kg body weight orally in single or divided doses, including from about 1.0 to about 50 mg/kg body weight, from about 1.0 to about 25 mg/kg body weight, from about 1.0 to about 10 mg/kg body weight (assuming an average body weight of approximately 70 kg; values adjusted accordingly for persons weighing more or less than average). For oral administration, the compositions are, for example, provided in the form of a tablet containing from about 50 to about 1000 mg of the active ingredient, particularly about 75 mg, about 100 mg, about 200 mg, about 400 mg, about 500 mg, about 600 mg, about 750 mg, or about 1000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject being treated. In one exemplary oral dosage regimen, a tablet containing from about 500 mg to about 1000 mg active ingredient is administered once (e.g., a loading dose) followed by administration of 1/2 dosage tablets (e.g., from about 250 to about 500 mg) each 6 to 24 hours for at least 3 days. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the subject undergoing therapy.
A series of twelve 4-amino-7-chloroquinolines containing dibenzylmethylamine side chains attached to the amino group of the quinoline were synthesized by reacting aminomethyldibemethins (4a - 15a) with 4,7- dichloroquinoline.
Figure imgf000016_0001
X X X
4a H 5a H 6a H
7a Cl 8a Cl 9a Cl
10a OCH3 11a OCH 12a OCH3
13a N(CH3)2 14a N(Ch '3'2 15a N(CH3 3)/2 Linking these molecules to a 7-chloroquinoline nucleus resulted in compounds (4 - 15) that have been found to be both active antimalarials in chloroquine sensitive parasites and capable of reversing resistance. Thus, it is expected that such molecules will combat resistance in the malaria parasite both to chloroquine and themselves.
Figure imgf000017_0001
X X X
4 H 5 H 6 H
7 Cl 8 Cl 9 Cl
10 OCH3 11 OCH3 12 OCH3
13 N(CH '33V/2 14 N(CH '33V/2 15 N(CH33V/2
These compound are:
Λ/-[{2-(Λ/-Benzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (4);
Λ/-[{3-(A/-Benzyl-A/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (5);
Λ/-[{4-(Λ/-Benzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (6);
Λ/-[{2-(Λ/-p-Chlorobenzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (7);
Λ/-[{3-(Λ/-p-Chlorobenzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (8);
Λ/-[{4-(A/-p-Chlorobenzyl-Λ/-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (9);
7-Chloro-Λ/-[{2-(Λ/-p-methoxybenzyl-Λ/-methylaminomethyl)phenyI}methyl]- 4- quinolinamine (10); 7-Chloro-Λ/-[{3-(W-p-methoxybenzyl-A/-methylaminomethyl)phenyl}πnethyl]- A- quinolinamine (11);
7-Chloro-Λ/-[{4-(Λ/-p-Methoxybenzyl-Λ/-methylaminomethyl)phenyl}methyl]- A- quinolinamine (12);
7-Chloro-N-[{2-(/V-p-dimethylaminobenzyl-Λ/- methylaminomethyl)phenyl}methyl]- 4-quinolinamine (13);
7-Chloro-N-[{3-(Λ/-p-Dimethylaminobenzyl-W- methylaminomethyl)phenyl}methyl]- 4-quinolinamine (14); and
7-Chloro-N-[{4-(Λ/-p-dimethylaminobenzyl-Λ/- methylaminomethyl)phenyl}methyl]-4-quinolinamine (15).
Examples
The invention will now be described in more detail by way of the following non- limiting examples.
Materials
Solvents, acids, and common salts were obtained from Sarchem, Krugersdorp, South Africa. All other starting materials, including 4,7- dichloroquinoline, were obtained from Sigma-Aldrich, Vorna Valley, South Africa. The synthesis of the dibemethin side chains is described below. Pre-coated silica gel plates as well as silica and alumina for column chromatography were obtained from Merck, South Africa. 1H and 13C NMR spectra were recorded on a Varian Mercury spectrometer at 300 MHz, and a Varian Unity spectrometer at 400 MHz. All spectra were recorded in d1- or d3-chloroform or d4-methanol. Infrared spectra were recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrophotometer in the range 3600-800 cm"1. All mass spectra were obtained using electron-impact techniques.
Synthesis and characterization of dibemethin side chains
o-aminomethyldibemethin (4a) was synthesized from /V-benzylmethylamine (16) and benzyl bromide (17) in four steps as shown in Scheme 1. Coupling of benzylmethylamine and benzyl bromide produced dibemethin (18) which was then reacted with f-butyllithium followed by anhydrous dimethylformamide to produce the benzaldehyde (19). This benzaldehyde was converted to an oxime (20) by reaction with hydroxylammonium chloride and finally reduced to the desired product (4a) with LiAIH4. The other two parent aminomethyl dibemethin compounds (5a and 6a) were prepared in a three step reaction from m- and p-dibromoxylene (22 and 23) and N, N- benzylmethylamine (16), while the remaining aminomethyl dibemethins (7a - 15a) were synthesized in four steps from either a benzyl chloride (27, 28) or p- (dimethylamino)benzaldehyde (29) as shown in Scheme 2. Compounds (24 - 26) were prepared by reacting o-, m- or p-dibromoxylene (21 - 23) with sodium azide under an inert atmosphere overnight. The benzyl chloride derivatives (27, 28) were reacted with nnethylamine to produce a series of benzyl methylamines (30 - 32) and the benzaldehyde derivative (29) was reductively aminated to produce the corresponding benzyl methylamine (32). Reaction of the suitable benzyl methylamine with (24, 25 or 26) under reflux produced the azides (33 - 43) which were reduced by the Staudinger reaction to produce the desired dibemethin derivatives (5a - 15a).
Figure imgf000019_0001
Figure imgf000019_0003
Figure imgf000019_0002
4a 20
Scheme 1 : Synthesis of 11. (i) 2h under N2, dichloromethane + triethylamine, 0 0C then satd. Na2CO3; (ii) t-butyllithium under N2 in anhydrous Et2O, - 78 0C, then warmed to 0 0C then anhydrous DMF and reacted at rt. for 3 h; (iii) hydroxylammonium chloride in absolute ethanol, then NaOH and refluxed under N2 overnight; (iv) LiAIH4 in anhydrous Et2O refluxed overnight under N2.)
Figure imgf000020_0001
XVlII - XIX
Figure imgf000020_0003
Figure imgf000020_0002
33, 34
5a - 15a
Scheme 2: Synthesis of 5a - 15a. (i) o- (21), (ii) m- (22) and (iii) p-a,a- dibromoxylene (23) reacted with NaN3 in anhydrous DMF under N2 at rt. Overnight gave 24, 25 and 26 respectively; (iv) 25 and (v) 26 in anhydrous acetonitrile and K2CO3 mixed with N-benzylmethylamine (16) at 0 0C and then refluxed overnight yielded 5a and 6a respectively; (vi) to 4-chlorobenzylchloride (27) and (vii) A- methoxybenzylchloride (28) 40 % aq. CH3NH2 in THF added under N2 at 0 0C and reacted at rt. overnight to yield 30 and 31 respectively; (viii) A-N, N- dimethylaminobenzaldehyde (29) 40% aq. CH3NH2 in acetonitrile added under N2 at 0 0C and reacted for 2 h, then NaCNBH3 added and reacted at r.t. for 4 h to yield 32; (ix) 24 (x) 25 and (xi) 26 in anhydrous acetonitrile and K2CO3 mixed with 30 under N2 at 0 °C then refluxed for 6 h yielded 35, 36 and 37 respectively; (xii) 24, (xiii) 25 and (xiv) 26 in anhydrous acetonitrile and K2CO3 mixed with 31 under N2 at 0 0C then refluxed for 6 h yielded 38, 39 and 40 respectively; (xv) 24, (xvi) 25 and (xvii) 26 in anhydrous acetonitrile and K2CO3 mixed with 32 under N2 at 0 0C then refluxed for 6 h yielded 41 , 42 and 43 respectively; (xviii - xxviii) 33 - 43 in THF under N2 mixed with PPh3 and stirred for 30 min at rt. then H2O added and refluxed for 6 h yielded 5a - 15a respectively. Yields: 30 - 34 (from starting materials) 81 , 87, 73, 57 and 59 % respectively; 35 - 43 58, 61 , 67, 64, 61 , 57, 57, 61 and 63 % respectively; 5a - 15a 88, 89, 93, 93, 93, 83, 88, 89, 83, 81 and 89 % respectively.) Λf,Λ/-DibenzyImethylamine (dibemethin) (18)
To a stirred solution of Λ/-benzylmethylamine (10.6 ml, 82.5 mmol) in DCM (50 ml) and triethylamine (17.2 ml, 123.8 mmol) at 0 0C under an atmosphere of nitrogen, benzyl bromide (9.8 ml, 82.5 mmol) was added slowly and the reaction allowed to progress for 2 hours after which time a white precipitate had formed. The reaction mixture was worked up by the addition of a saturated solution of aqueous Na2CO3 and the product extracted into ethyl acetate (3 x 50 ml), the organic extracts dried over anhydrous MgSO4 and the solvent removed under reduced pressure to give a crude product (17.05 g), which was chromatographed on silica-gel (170 g) using 10% ethyl acetate in hexane as eluent. The product was dried in vacuo to give 18 as a pale yellow oil (11.62 g, 67%): 1H NMR (300 MHz, CDCI3) δ 7.57 - 7.38 (1OH, m, ArH), 3.70 (4H, s, CH2), 2.37 (3H, s, CH3); 13C NMR (CDCI3, 75 MHz) δ 139.2 (Arqua, ), 128.8 (Arortho ), 128.1 (Armeta ), 126.8 (Arpara ), 61.8 (CH2), 42.1 (CH3).
o - (Λ/-Benzyl-Λ/-methylamino)methyl-benzaldehyde (19) f-Butyllithium (10.0 ml, 1.7 M, 17.0 mmol) was slowly added drop-wise to a stirred solution of 18 (3.00 g, 14.2 mmol) in anhydrous diethyl ether (100 ml) at -78 0C under an atmosphere of nitrogen. The mixture was warmed up to 0 0C and allowed to stir until the colour of the mixture changed from orange to pale-yellow and a white precipitate of the lithium-salt had formed. Anhydrous DMF (1.1 ml, 14.3 mmol) was then slowly added drop-wise at 0 0C and the reaction slowly warmed to room temperature while stirring for 3 hours after which time the reaction mixture was clear of precipitate. Diethyl ether was evaporated off on a rotary evaporator, the resulting solid worked up with deionised water and the product extracted into ethyl acetate (3 x 100 ml). The organic extracts were dried over anhydrous MgSO4 and the solvent removed under reduced pressure to give a crude product (3.2 g), which was chromatographed on silica-gel using mixtures of ethyl acetate: hexane (0:100) to (5: 95) as eluent. The product was dried in vacuo to give a yellow oil, 19, (2.61 g, 77%): IR (CHCI3) vmax (cm"1) 3066, 3029, 3010, 2949, 2880, 2843, 2793, 1689 (C=O), 1600 (Arc=c); 1H NMR (CDCI3, 300 MHz) δ 10.42 (1 H, s), 7.92 (1 H, dd, J = 1.5, 7.5 Hz), 7.50 - 7.23 (8H, m), 3.84 (2H, s), 3.53 (2H, s), 2.13 (3H, s);13C NMR (CDCI3, 75 MHz) δ 192.0, 141.7 (IV)1 138.5 (IV), 134.9 (IV), 133.0, 130.4, 128.9, 128.8 (2C), 128.1 (2C), 127.6, 127.0, 61.8, 58.9, 41.6; HRMS (ES): Found 239.13034 (M+): C16H17NO (M+) requires 239.13101. o-(Λ/-Benzy!-Λ/-methyIamino)methyl-benzaldehyde oxime (20)
To a stirred solution of 19 (2.50 g, 10.4 mmol) in absolute ethanol (50 ml), was added hydroxylammonium chloride (1.97 g, 28.3 mmol) followed by a solution of NaOH (2.26 g, 56.5 mmol) in deionised water (3 ml). The reaction was allowed to reflux overnight under an atmosphere of nitrogen. The mixture was then worked up with deionised water and extracted with ethylacetate (3 x 50 ml). The organic extracts were dried over anhydrous MgSO4 and the solvent evaporated under reduced pressure. The crude product was chromatographed on silica-gel using a mixture of ethyl acetate: hexane (10:90) as eluent. The product was dried in vacuo to give 20 as a pale-yellow oil (2.17 g, 85%): IR (CHCI3) vmax (cm"1) 3305, 3065, 3028, 3011 , 2950 (ArCH), 2881, 2841 , 2791 , 1601 (Arc=c); 1H NMR (CDCI3, 300 MHz) δ 8.80 (1 H, br s), 8.69 (1H, s), 7.80 (1H, dd, J = 1.7, 6.5 Hz)1 7.35 - 7.25 (8H, m), 3.64 (2H, s), 3.54 (2H, s), 2.15 (3H, s);13C NMR (CDCI3, 75 MHz) δ 149.2, 138.8 (IV), 137.7 (IV), 131.4 (IV), 130.7, 129.4, 129.1 (2C), 128.3 (2C), 127.6, 127.0, 126.4, 62.1, 60.0, 41.8; HRMS (ES): Found 254.14271 , (M+): C16H18N2O (M+) requires 254.14191.
o-[(Λ/-Benzyl-Λ/-methyl)aminomethyl]-benzylamine (4a)
To a stirred solution of 20 (1.00 g, 3.90 mmol) in anhydrous diethyl ether (10 ml), was added LiAIH4 (75 mg, 1.96 mmol) slowly and the reaction allowed to reflux overnight under an atmosphere of nitrogen. After the reaction mixture was allowed to cool to room temperature, a saturated solution of aqueous Na2SO4 (10 ml) with a few drops of triethylamine was added to the reaction mixture. This mixture was stirred for 30 minutes, the resultant precipitate filtered through Celite and the residue washed with a solution of 5% triethylamine in THF (100 ml). The solvent was removed under reduced pressure with a small volume of toluene being added to facilitate the azeotropic removal of any water. The crude product was purified by column chromatography using 100% ethyl acetate followed by EtOAc: MeOH: NEt3 (94: 5: 1) as eluent to give 4a as a dark-orange oil, (0.83 g, 89%): IR (DMSO) vmax(cm"1) 3523 - 3422, 3287, 2791 , 2143, 2050, 1973, 1711 , 1663 (Arc=c), 1579, 1491 ; 1H NMR (CDCI3, 300 MHz) δ 7.32 - 7.23 (9H, m), 6.88 (2H, br s), 3.94 (2H, s), 3.58 (2H, s), 3.57 (2H, s), 2.07 (3H, s);13C NMR (CDCI3, 75 MHz) δ 137.5 (IV), 137.1 (IV), 136.8 (IV), 131.3, 130.7, 129.5(2C); 128.5 (2C); 128.4, 128.0, 127.4, 62.3, 60.8, 43.0, 40.9 ; HRMS (ES): Found 240.16025 (M+): C16H20N2 (M+) requires 240.16265. a, or-2, 3 and 4 azido-bromo xylenes (24, 25, 26)
Sodium azide (1.0 equiv.) was added to a stirred solution of the appropriate a, α'-dibromoxylene (o, m or p) in anhydrous DMF under N2, and the reaction allowed to stir at room temperature overnight. The reaction mixture was diluted with ethyl acetate (100 ml) and the organic layer washed with saturated brine (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude products were used without further purification.
N-[{3-(Azidomethyl)phenyϊ}methyl]-A/-benzyImethylamine (33)
To a stirred solution of crude 25 (0.22 g, 0.99 mmol) and K2CO3 (0.27 g, 2.00 mmol.) in anhydrous acetonitrile (25 ml) at 0 0C, was added Λ/-benzylmethylamine (0.19 ml, 1.49 mmol) and the reaction heated and allowed to progress under reflux overnight. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (10:90) to (20:80) as eluent to give 33 as a yellow oil (0.15 g, 57%);Η NMR (CDCI3, 300 MHz) δ 7.40 - 7.14 (9H, m), 4.35 (1 H, s), 3.57 (2H, s), 3.57 (2H, s), 2.22 (3H, s);13C NMR (CDCI3, 75 MHz) δ 140.0 (IV), 138.9 (IV)1 135.3(IV) , 128.9, 128.9 (2C), 128.7, 128.7, 128.2 (2C), 127.0, 126.8, 61.8, 61.5, 54.8, 42.1 ; HRMS (ES): Found 266.14947(M+): C16H18N4 (M+) requires 266.15315.
m-[(/V-BenzyI-/V-methyl)aminomethyl]-benzyIamine (5a)
To a stirred solution of 33 (0.44g, 1.65 mmol) in THF (4.50 ml) under N2, was added PPh3 (0.44, 1.66 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water was added (1.50 ml, 83.33 mmol) and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (94: 5: 1) as eluent to give 5a as a dark-yellow oil (0.35 g, 88%): IR (DMSO) vmax 3567 - 3374, 3273, 2840, 2784, 2149, 2056, 1968, 1711 , 1661 (Ar0=C), 1579; 1H NMR (CD3OD, 300 MHz) δ 7.60 (1 H, s), 7.44 - 7.24 (8H, m), 4.10 (2H, s), 3.67 (2H, s), 3.67 (2H, s), 2.23 (3H, s);13C NMR (CD3OD, 75 MHz) δ 139.6, 138.3, 136.1 , 131.0, 131.0, 130.7, 130.1 , 129.5, 129.1 , 128.7, 62.6, 62.1 , 44.5, 41.9; HRMS (ES): Found 239.13034 (M+). C16H17NO (M+) requires 239.13101.
W-[{4-(Azidomethyl)phenyl}methyl]-Λ/-benzylmethylamine (34)
To a stirred solution of 26 (0.50 g, 2.25 mmol) and K2CO3 (0.61 , 4.50 mmol) in anhydrous acetonitrile (50 ml) at 0 0C, was added Λ/-benzylmethylamine (0.43 ml, 3.40 mmol) and the reaction heated and allowed to progress under reflux overnight. Acetonitrile was then removed under reduced pressure and the reaction mixture partitioned between ethyl acetate (100 ml) and saturated Na2CO3 (3 x 50 ml). The organic extracts dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (10:90) to (20:80) as eluent to give 34 as a yellow oil (0.35 g, 59%); 1H NMR (CDCI3, 400 MHz) δ 7.43 - 7.26 (9H, m), 4.35 (1H, s), 3.56 (2H, s), 3.56 (2H, s), 2.22 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 139.7 (IV), 139.2 (IV), 134.1 (IV), 129.4 (2C), 129.0 (2C), 128.3 (2C), 128.2 (2C), 127.0, 61.9, 61.5, 54.7, 42.1 ; HRMS (ESI): Found 267.1605(M+ +1). C16H18N4 (M+ +1) requires 267.1610.
p-[(Λ/-Benzyl-N-methyl)aminomethyl]-benzylamine (6a)
To a stirred solution of 34 (0.70 g, 2.63 mmol) in THF (7.20 ml) under N2, was added PPh3 (0.44 g, 1.66 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water (2.39 ml, 132.60 mmol) was added and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (94: 5: 1) as eluent to give 6a as a dark-yellow oil (0.56 g, 89%): IR (DMSO) vmax 3531 - 3408, 3273, 2131 , 2057, 1973, 1659 (Arc=c), 1612, 1512; 1H NMR (CD3OD, 400 MHz) δ 7.21 - 7.10 (9H, m), 3.73 (2H, s), 3.38 (2H, s), 3.37 (2H, s, 2.03 (3H, s); 13C NMR (CD3OD, 75 MHz) δ 140.3, 139.8, 139.2, 130.7 (2C), 130.3 (2C), 129.3 (2C), 128.8 (2C), 128.3 , 62.7, 62.4, 45.9, 42.4; HRMS (ES): Found 240.16265 (M+). C16H20N2 (M+) requires 240.16164.
(4-Chlorobenzyl)methyIamine (30)
To a stirred solution of 4-chlorobenzylchloride (3.00 g, 18.63 mmol) in THF (300 ml) under N2 at 0 0C, was added methylamine solution in water (40% v/v) (9.03 mi, 93.48 mmol) and the reaction allowed to progress at room temperature overnight. The solvent was removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (92: 5: 3) as eluent to give 30 as a yellow oil (2.49 g, 81%); 1H NMR (CDCi3, 300 MHz) δ 7.25 - 7.18 (4H, m), 3.66 (2H, s), 2.38 (3H, s), 1.65 ( 1H, s); 13C NMR (CDCI3, 100 MHz) δ 138.6 (IV), 132.6 (IV), 129.5 (2C), 128.4 (2C), 55.2, 35.8; HRMS (ESI): Found 156.0580 (M+). C8H10NCI (M+) requires 156.0556.
Λ/-[{2-(Azidomethyl)phenyl}methyl]-Λ/-(4-chlorobenzyl)methylamine (35)
To a stirred solution of 30 (0.60 g, 3.85 mmol) and K2CO3 (0.71 g, 5.14 mmol.) in anhydrous acetonitrile (150 ml) under N2 at 0 0C, was added crude 24 (0.58 g, 2.57 mmol) and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (5:95) to (20:80) as eluent to give 35 as a yellow oil (0.45 g, 58%); 1H NMR (CDCI3, 400 MHz) δ 7.39 - 7.26 (9H, m), 4.55 (2H, s), 3.58 (2H, s), 3.50 (2H, s), 2.13 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 137.4 (IV), 137.1 (IV), 134.7 (IV), 132.7 (IV), 130.7 , 130.2 (2C), 129.6, 128.3 (2C), 128.1 , 127.7, 61.5, 59.9, 52.0, 41.9; HRMS (ESI): Found 301.1220 (M+). C16H18N4 CI (M+) requires 301.1211.
o-[(/V-4-Chlorobenzyl-W-methyl)aminomethyI]-benzylamine (7a)
To a stirred solution of 35 (0.4Og, 1.33 mmol) in THF (3.60 ml) under N2, was added PPh3 (0.42 g, 1.60 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water was added (1.20 ml, 66.67 mmol) and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (94: 5: 1) as eluent to give 7a as a yellow oil (0.34 g, 93%); 1H NMR (CDCI3, 300 MHz) δ 7.35 - 7.16 (9H, m), 3.87 (2H, s), 3.55 (2H, s), 3.52 (2H, s), 2.07 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 141.3 (IV), 136.7 (IV), 136.4 (IV), 133.0 (IV), 131.1 , 130.6 (2C), 129.4, 128.5 (2C), 128.2 , 127.1 , 61.7, 60.7, 44.1 , 41.4; HRMS (ESI): Found 275.1315 (M+). C16H20N2CI (M+) requires 275.1324.
W-[{3-(AzidomethyI)phenyl}methyl]-W-(4-chlorobenzyl)methylamine (36)
To a stirred solution of 30 (0.80 g, 5.13 mmol) and K2CO3 (0.95 g, 6.85 mmol) in anhydrous acetonitrile (200 ml) at 0 0C, was added crude 25 (0.77 g, 3.43 mmol), and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (5:95) to (20:80) as eluent to give 36 as a yellow oil (0.63 g, 61%); 1H NMR (CDCI3, 300 MHz) δ 7.36 - 7.31 (7H, m), 7.22 (1H, m), 4.35 (1 H, s), 3.54 (2H, s), 3.50 (2H, s), 2.19 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 139.4 (IV), 137.7 (IV), 135.3 (IV), 132.6 (IV), 130.1 (2C), 128.8 (2C), 128.7, 128.6, 128.3, 126.9, 61.5, 61.0, 54.7, 42.1 ; HRMS (ESI): Found 301.1220 (M+). C16H18N4 CI (M+) requires 301.1221.
m-[(A/-4-Chlorobenzyl-Λ/-methyl)aminomethyl]-benzylamine (8a)
To a stirred solution of 36 (0.60 g, 2.00 mmol) in THF (5.40 ml) under N2, was added PPh3 (0.63 g, 2.40 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water was added (1.80 ml, 100.00 mmol) and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (94: 5: 1) as eluent to give 8a as a light-orange oil (0.56 g, 93%): IR (DMSO) vmax (cm"1) 3547- 3399, 3271 , 2835, 2783, 2144, 2048, 1707, 1663 (Arc=c), 1605, 1491 ; 1H NMR (CDCI3, 300 MHz) δ 7.31 - 7.18 (8H, m), 3.85 (2H, s), 3.49 (2H, s), 3.46 (2H, s), 3.02 (2H, br s), 2.15 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 142.0 (IV), 139.3 (IV), 137.7 (IV), 132.4 (IV), 130.0 (2C), 128.3 (IV), 125.8, 128.2 (2C), 127.6, 127.4, 61.6, 61.0, 45.8, 42.0; HRMS (ESI): Found 275.1315 (M+). C16H20N2CI (M+) requires 275.1316.
W-[{4-(Azidomethyl)phenyl}methyI]-W-(4-chlorobenzyl)methylamine (37)
To a stirred solution of 30 (1.00 g, 6.41 mmol) and K2CO3 (1.19 g, 8.60 mmol.) in anhydrous acetonitrile (300.00 ml) at 0 0C, was added crude 26 (0.96 g, 4.25 mmol) and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (5:95) to (20:80) as eluent to afford 37 as a light-yellow oil (0.85 g, 67%); 1H NMR (CDCI3, 400 MHz) δ 7.44 - 7.31 (8H, m), 4.35 (2H, s), 3.56 (2H, s), 3.52 (2H, s), 2.22 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 139.3 (IV), 137.7 (IV), 134.0 (IV), 132.4 (IV), 130.0 (2C), 129.1 (2C), 128.2 (2C), 128.0 (2C), 61.3, 61.0, 54.4, 42.0; HRMS (ESI): Found 301.1220 (M+). C16H18N4 Cl (M+) requires 301.1223.
p-[(ΛC-4-ChIorobenzyI-W-methyl)aminomethyl]-benzyIamine (9a)
To a stirred solution of 37 (0.8Og, 2.67 mmol) in THF (7.20 ml) under N2, was added PPh3 (0.92 g, 3.20 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water (2.40 ml, 133.33 mmol) was added and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (94: 5: 1) as eluent to give 9a as a dark-orange oil (0.68 g, 93%): IR (DMSO) vmax (cm~1) 3551- 3388, 3287, 2790, 2138, 2063, 1957, 1708, 1662 (Arc=c), 1513, 1490; 1H NMR (CDCI3, 300 MHz) δ 7.32 - 7.24 (8H, m), 3.83 (2H, s), 3.48 (2H, s), 3.44 (2H, s), 3.16 (2H, br s), 2.14 (2H, s); 13C NMR (CDCI3, 75 MHz) δ 137.7 (IV), 132.3 (IV), 131.7 (IV), 129.9 (2C), 128.9 (2C), 128.1 (2C), 127.7 (IV), 127.0 (2C), 61.3, 60.7, 45.5, 41.9; HRMS (ESI): Found 275.1315 (M+). C16H20N2CI (M+) requires 275.1326.
(4-Methoxybenzyl)methylamine (31)
To a stirred solution of 4-methoxybenzylchloride (3.00 g, 19.18 mmol) in THF (350 ml) under N2 at 0 0C, was added methylamine solution in water (40% v/v) (9.30 ml, 95.86 mmol) and the reaction allowed to progress at room temperature overnight. The solvent was removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (92: 5: 3) as eluent to give 31 as a yellow oil (2.72 g, 87%); 1H NMR (CDCI3, 300 MHz) δ 7.27 (2H, d, J = 6.6 Hz1), 6.87 (2H, d, J = 6.6 Hz), 3.75 (3H, s), 3.64 (2H, s), 2.33 ( 3H, s); 13C NMR (CDCI3, 100 MHz) δ 159.0 (IV), 132.5 (IV), 129.6 (2C), 113.8 (2C), 54.9 (2C), 35.1; HRMS (ESi): Found 152.1070 (M+). C9H14NO (M+) requires 152.1070.
Λ/-[{2-(Azidomethyl)phenyl}methyl]-Λ/-(4-lViethoxybenzyl)methylanriine (38)
To a stirred solution of 31 (1.00 g, 6.61 mmol) and K2CO3 (1.22 g, 8.87 mmol.) in anhydrous acetonitrile (300 ml) at 0 0C, was added crude 24 (0.99 g, 4.38 mmol, synthesized as outlined in GP1) and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (10:90) to (25:75) as eluent to give 38 as a yellow oil (0.83 g, 64%); 1H NMR (CDCI3, 300 MHz) δ 7.42 - 7.20 (6H, m), 6.90 (2H, dd, J = 2.3, 6.8 Hz), 4.56 (2H, s), 3.83 (3H, s), 3.57 (2H, s), 3.51 (2H, s), 2.15 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 159.1 (IV), 137.8 (IV), 135.1 (IV), 131.3 (IV), 131.1, 130.5 (2C), 129.7, 128.3, 128.3, 114.0 (2C), 62.1 , 60.0, 55.5, 52.2, 42.3; HRMS (ESI): Found 297.1715 (M+). C17H2-(N4O (M+) requires 297.1727.
o-[(W-4-Methoxybenzyl-W-methyl)am[nomethyl]-benzylamine (1 Oa)
To a stirred solution of 38 (0.80 g, 2.70 mmol) in THF (7.29 ml) under N2, was added PPh3 (0.85 g, 3.24 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water (2.43 ml, 135.00 mmol) was added and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (93: 5: 2) as eluent to give 10a as a light-brown oil (0.61 g, 83%): IR (DMSO) vmax (cm"1) 3592- 3324, 2593, 2142, 2061 , 1981 , 1664 (Arc=c), 1510; 1H NMR (CDCI3, 300 MHz) δ 7.32 - 7.15 (6H, m), 6.83 (2H, dd, J = 2.0, 6.5 Hz), 3.85 (2H, s), 3.77 (3H, s), 3.53 (2H, s), 3.49 (2H, s), 3.40 (2H, br s), 2.09 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 158.7 (IV), 142.1 (IV), 136.7 (IV), 131.0, 130.5 (IV), 130.4 (2C), 129.1 , 128.0, 127.0, 113.6 (2C), 62.0, 60.5, 55.1 , 44.3, 41.4. /V-[{3-(Azidomethyl)phenyl}methyl]-W-(4-methoxybenzyI)methylamine (39)
To a stirred solution of 31 (1.00 g, 6.61 mmol) and K2CO3 (1.22 g, 8.87 mmol.) in anhydrous acetonitrile (300 ml) at 0 0C, was added crude 25 (0.99 g, 4.38 mmol) and the reaction heated and allowed to progress under reflux for 5 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (10:90) to (25:75) as eluent to give 39 as a yellow oil (0.79 g, 61%); 1H NMR (CDCI3, 300 MHz) δ 7.35 - 7.18 (4H, m), 7.28 (2H, dd, J = 2.1 , 6.4 Hz), 6.87 (2H, dd, J = 2.1 , 6.4 Hz), 4.34 (2H, s), 3.81 (3H, s), 3.53 (2H, s), 3.49 (2H, s), 2.18 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 158.7 (IV), 140.1 (IV), 135.3 (IV), 131.0 (IV), 130.1 (2C), 128.9, 128.7, 128.7, 126.8, 113.6 (2C), 61.3, 61.2, 55.2, 54.8, 42.0; HRMS (ESI): Found 297.1715 (M+). C17H21N4 O (M+) requires 297.1716.
m-[(N-4-Methoxybenzy!-N-methyl)aminomethyl]-benzylamine (11 a)
To a stirred solution of 39 (0.75 g, 2.53 mmol) in THF (6.83 ml) under N2, was added PPh3 (0.80 g, 3.04 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water was added (2.28 ml, 126.56 mmol) and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (93: 5: 2) as eluent to give 11a as a dark-yellow oil (0.60 g, 88%): IR (DMSO) vmax (cm"1) 3531- 3410, 3286, 2137, 2060, 1986, 1661 (Arc=c), 1611 , 1511 (NH); 1H NMR (CDCI3, 400 MHz) δ 7.29 - 6.80 (8H, m), 4.53 (2H, br s), 3.73 (2H, s), 3.77 (3H, s), 3.46 (2H, s), 3.43 (2H, s), 2.13 (3H, s); HRMS (ESI): Found 271.1810 (M+). C17H23N2 O (M+) requires 271.1813.
W-[{4-(AzidomethyI)phenyl}methyI]-Λ/-(4-methoxyben2yI)methylamine (40)
To a stirred solution of 31 (1.00 g, 6.61 mmol) and K2CO3 (1.22 g, 8.87 mmol.) in anhydrous acetonitrile (300 ml) at 0 0C, was added crude 26 (0.99 g, 4.38 mmol) and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (10:90) to (25:75) as eluent to afford 40 as a yellow oil ( 0.74 g, 57%); 1H NMR (CDCI3, 400 MHz) δ 7.45 - 6.91 (8H, m), 4.37 (2H, s), 3.77 (3H, s), 3.52 (2H, s), 3.48 (2H, s), 2.15 (3H1 s); 13C NMR (CDCI3, 75.5 MHz) δ 159.2 (IV), 140.2 (IV), 134.6 (IV), 131.6 (IV), 130.2 (2C), 128.5 (2C), 129.4 (2C), 113.9 (2C), 61.4, 61.4, 54.9, 54.4, 41.8; HRMS (ESI): Found 297.1715 (M+). C17H2iN4O (M+) requires 297.1706.
p-[(ΛM-MethoxybenzyI-W-methyI)aminomethyl]-benzylamine (12a)
To a stirred solution of 40 (0.70 g, 2.36 mmol) in THF (6.39 ml) under N2, was added PPh3 (0.74 g, 2.84 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water (2.13 ml, 118.13 mmol) was added and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed by rotary evaporation with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (92: 5: 3) as eluent to give 12a as a light-brown oil (0.57 g, 89%): IR (DMSO) vmax (cm~1) 3374- 3272, 2833, 2785, 2145, 2056, 1978, 1903, 1662 (Arc=c), 1610, 1583; 1H NMR (CDCI3, 400 MHz) δ 7.32 - 7.27 (4H, m), 7.23 (2H, dd, J = 2.0, 6.8 Hz), 6.85 (2H, dd, J = 2.0, 6.8 Hz), 4.53 (2H, br s), 3.83 (2H, s), 3.77 (3H, s), 3.46 (2H, s), 3.43 (2H, s), 2.13 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 158.5 (IV), 138.6 (IV)1 138.4 (IV), 131.0 (IV), 129.9 (2C), 129.1 (2C), 127.4 (2C), 113.5 (2C), 61.1 , 61.0, 55.1 , 44.9, 41.8.
(A-N, Λ/-Dimethylaminobenzyl)-methylamine (32)
To a stirred solution of 4-Λ/, Λ/-dimethylaminobenzaldehyde (3.00 g, 18.3 mmol) (29) in acetonitrile (400 ml) under N2 at 0 0C was added methylamine solution in water (40% v/v) (10.0 ml, 103 mmol) and the reaction allowed to progress for 2 hours. Then NaCNBH3 (3.46 g, 64.1 mmol) was added and the reaction allowed to progress at room temperature for 4 hours. The solvent was removed on the rotorevaporator with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (92: 5: 3) as eluent to give 32 as a yellow oil (2.20 g, 73%); 1H NMR (CDCI3, 300 MHz) δ 7.17 (2H, d, J = 8.7 Hz), 6.70 (2H, d, J = 8.7 Hz), 3.59 (2H, s), 2.88 (6H, s), 2.34 (3H, s); 13C NMR (CDCI3, 100 MHz) δ 150.0 (2CIV)), 129.1 (2C), 112.7 (2C), 55.6, 40.3, 35.6 (2C); HRMS (ESI): Found 165.1392 (M+): C10H17N2 (M+) requires 165.1390.
W-[{2-(Azidomethyl)phenyl}rnethyI]-W-(4-DirnethyIaminobenzyI)methyIarnine (41)
To a stirred solution of 32 (1.00 g, 6.06 mmol) and K2CO3 (1.25 g, 9.09 mmol) in anhydrous acetonitrile (150 ml) at 0 0C, was added crude 24 (1.14 g, 5.05 mmol), synthesized as outlined in GP1 and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (05:95) to (30:70) as eluent to give 41 as a yellow oil (0.89 g, 57%): 1H NMR (CDCI3, 400 MHz) δ 7.48 - 7.42 (4H, m), 7.40 (2H, d, J = 8.7 Hz), 6.90 (2H, d, J = 8.7 Hz), 4.69 (2H, s), 3.69 (2H, s), 3.64 (2H, s), 3.09 (6H, s), 2.31 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 149.7 (IV), 137.4 (IV), 134.7 (IV),130.5, 129.8 (2C), 129.7, 127.6, 127.2, 126.4 (IV), 112.2 (2C), 61.8, 59.4 , 51.6, 41.7, 40.3 (2C); HRMS (ESI): Found 310.2032 (M+). C18H24N5 (M+) requires 310.2047.
o-[(Λ/-4-DimethyIaminobenzyl-Λ/-methyl)aminomethyl]-benzylamine (13a)
To a stirred solution of 41 (0.80 g, 2.58 mmol) in THF (7.20 ml) under N2, was added PPh3 (0.81 g, 3.10 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water (2.40 ml, 133 mmol) was added and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed on the rotorevaporator with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (93: 5: 2) as eluent to give 13a as a light-brown oil (0.61 g, 83%): IR (DMSO) vmax (cm""1) 3550 - 3401 , 3293, 2132, 2068, 1980, 1663, 1613, 1522; 1H NMR (CDCI3, 300 MHz) δ 7.31 - 7.17 (6H, m), 6.71 (2H, d, J = 9.0 Hz), 4.05 (2H, br s), 3.85 (2H, s), 3.53 (2H, s), 3.48 (2H, s), 2.92 (6H, s), 2.07 (3H, s);13C NMR (CDCI3, 75.5 MHz) δ 149.9 (IV), 141.3 (IV), 137.0 (IV), 131.1 , 130.3 (2C), 129.5, 128.0, 127.0, 125.8 (IV), 112.5 (2C), 62.1 , 60.5, 44.2, 41.2, 40.6 (2C); HRMS (ESI): Found 284.2127 (M+): C18H26N3 (M+) requires 284.2123. W-[{3-(A2idomethyl)phenyI}methyI]-W-(4-DimethylaminobenzyI)methylamine (42)
To a stirred solution of 32 (1.00 g, 6.06 mmol) and K2CO3 (1.25 g, 9.09 mmol) in anhydrous acetonitrile (150 ml) at 0 0C, was added crude 25 (1.14 g, 5.05 mmol), synthesized as outlined in GP1 and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (05:95) to (30:70) as eluent to give 42 as a yellow oil (0.95 g, 61%): 1H NMR (CDCI3, 300 MHz) δ 7.35 - 7.26 (4H, m), 7.23 (2H, d, J = 8.9 Hz), 6. 73 (2H, d, J = 8.9 Hz), 4.34 (2H, s), 3.52 (2H, s), 3.47 (2H, s), 2.94 (6H, s), 2.19 (3H, s); 13C NMR (CDCi3, 75 MHz) δ 1.49.9 (IV), 140.4 (IV), 135.2 (IV), 129.9 (2C), 128.9, 128.7, 128.6, 126.8 (IV), 126.7, 113.6 (2C), 61.4, 61.2, 54.8, 42.0, 40.7 (2C); HRMS (ESI): Found 310.2032 (M+). C18H24N5 (M+) requires 310.2042.
/77-[(Λ/-4-DimethylaminobenzyI-N-methyI)aminomethyl]-benzylamine (14a)
To a stirred solution of 42 (0.80 g, 2.58 mmol) in THF (7.20 ml) under N2, was added PPh3 (0.81 g, 3.10 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water (2.40 ml, 133 mmol) was added and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed on the rotorevaporator with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (93: 5: 2) as eluent to give 14a as a light-brown oil (0.59 g, 81%): IR (DMSO) vmax (cm"1) 3531- 3410, 3286, 2137, 2060, 1986, 1661 , 1611 , 1511 ; 1H NMR (CDCI3, 300 MHz) δ 7.29 - 7.18 (6H, m), 6.86 (2H, d, J = 8.7 Hz), 3.86 (2H, s), 3.79 (6H, s), 3.49 (2H, s), 3.47(2H, s), 2.50 (2H, br s), 2.16 (3H, s);13C NMR (CDCI3, 75 MHz) δ 158.6 (IV), 142.4 (IV), 139.6 (IV), 131.1 (IV), 130.0 (2C), 128.3, 127.6, 127.5, 125.7, 113.5 (2C), 61.5, 61.2, 55.1 (2C), 46.1, 42.0.
N-[{4-(Azidomethyl)phenyl}methyl]-W-(4-Dimethylaminobenzyl)methylamine (43)
To a stirred solution of 32 (1.00 g, 6.06 mmol) and K2CO3 (1.25 g, 9.09 mmol) in anhydrous acetonitrile (150 ml) at 0 0C, was added crude 26 (1.14 g, 5.05 mmol), synthesized as outlined in GP1 and the reaction heated and allowed to progress under reflux for 6 hours. Acetonitrile was then removed under reduced pressure and the reaction mixture worked up with ethyl acetate (100 ml) and saturated aqueous Na2CO3 (3 x 50 ml), dried (MgSO4) and concentrated in vacuo. The crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (05:95) to (30:70) as eluent to give 43 as a yellow oil (0.99 g, 63%): 1H NMR (CDCI3, 400 MHz) δ 7.40 - 7.25 (4H, m), 7.23 (2H, d, J = 8.6 Hz)1 6.72 (2H, d, J = 8.6 Hz), 4.32 (2H, s), 3.51 (2H, s), 3.46 (2H, s), 2.92 (6H, s), 2.15 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 149.8 (IV), 139.9 (IV), 133.8 (IV), 129.8, 129.3 (2C), 128.1 , 126.9 (IV), 112.5 (2C), 61.4, 61.1, 54.6, 42.1 , 40.7 (2C); HRMS (ESI): Found 310.2032 (M+): C18H24N5 (M+) requires 310.2024.
p-[(Λ/-4-Dimethylanninobenzyl-/V-methyi)aminomethyl]-benzyIamine (15a)
To a stirred solution of 43 (0.80 g, 2.58 mmol) in THF (7.20 ml) under N2, was added PPh3 (0.81 g, 3.10 mmol) and the reaction mixture allowed to stir for 30 minutes at room temperature. Water (2.40 ml, 133 mmol) was added and the reaction heated and allowed to reflux for 6 hours. The reaction was then cooled to room temperature and solvent removed on the rotorevaporator with water removed by azeotroping with toluene. The crude product was purified directly by column chromatography using EtOAc (100%) followed by EtOAc: MeOH: NEt3 (93: 5: 2) as eluent to give 15a as a light-brown oil (0.65 g, 89%): IR (DMSO) Vm3x (Cm"1) 3545 - 3385, 3285, 2582, 2141, 2066, 1985, 1904, 1661, 1521; 1H NMR (CDCI3, 400 MHz) δ 7.31 - 21 (4H1 m), 7.19 (2H, d, J = 8.8 Hz), 6.69 (2H, d, J = 8.8 Hz), 3.81 (2H, s), 3.46 (2H, s), 3.43 (2H, s), 2.91 (6H, s), 2.49 (2H, br s), 2.14 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 149.8 (IV),141.2 (IV), 138.0 (IV), 129.8 (2C), 129.2 (2C), 127.0 (2C); 126.8 (IV), 112.5 (2C), 61.2, 61.1 , 46.0, 41.9, 40.7 (2C).
Synthesis of target molecules
Target molecules (4 - 15) were synthesized in a single step by reaction of an appropriate dibemethin (4a - 15a) with excess commercially available 4,7- dichloroquinoline in anhydrous Λ/-methyl-2-pyrrolidone under N2 in the presence of potassium carbonate and triethylamine. The reaction was conducted in sealed cycloaddition tubes at 90 - 130 0C over periods ranging from 16 - 48 h. The products were then extracted from alkaline aqueous solution into ethyl acetate and purified by column chromatography to afford modest yields. All products were characterized by infrared, 1H and 13C NMR and mass spectrometry. Solids were further characterized by melting point determination and elemental combustion analysis, while oils were subjected to high resolution mass spectrometry.
W-[{2-(W-BenzyI-W-methylaminomethyI)phenyl}methyl]-7-chIoro-4- quinolinamine (4)
To a stirred solution of 4a (0.42 g, 1.73 mmol) in anhydrous /V-methyl-2- pyrrolidone (2 ml) under N2, were added triethylamine (1.21 ml, 8.67 mmol), K2CO3 (0.48 g, 3.47 mmol) and 4, 7-dichloroquinoline (1.72, 8.67 mmol) and the mixture was heated at 90 0C for 48 Hours. After the mixture was allowed to cool to room temperature it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The combined organic extracts were further washed 5 times with saturated brine to ensure removal of any traces of the pyrrolidone, before being dried over anhydrous Na2SO4 and filtered. Following solvent removal, the resulting crude product was dried under reduced pressure and purified by silica-gel chromatography using mixtures of ethyl acetate: hexane (30:70) to (80:20) as eluent to give 4 as a white crystalline solid (0.26 g, 37%): m.p. (EtOAc: Hexane) 101-103 0C; IR (DMSO) vmax 3583, 3384 - 3256, 2595, 2140, 2050, 1975, 1659, 1580; 1H NMR (CDCI3, 300 MHz) δ 8.53 (1H, d, J = 5.1 Hz), 7.89 (1 H, d, J = 2.0 Hz), 7.49 - 7.19 (10H, m), 6.85 (1 H, dd, J = 2.0, 9.0 Hz), 6.52 (1 H, d, J = 5.1 Hz), 4.43 (2H, d, J = 5.1 Hz), 3.61 (2H, s), 3.58 (2H, s), 2.14 (3H, s), 1.27 (1 H, s);13C NMR (CDCI3, 75 MHz) δ 152.0, 150.2, 149.3, 137.4, 137.2, 137.1, 134.5, 132.0, 130.5, 129.9, 129.9, 128.4, 128.4, 127.9, 127.6, 124.7, 122.2, 117.8, 99.0, 61.9, 61.0, 46.6, 41.9; (Found: C, 74.56; H, 6.07; N, 10.08%. C25H24N3CI requires C, 74.71; H, 6.02; N, 10.45%).
Λ/-[{3-(W-Benzyl-N-methyIaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (5)
To a stirred solution of 5a (0.50 g, 2.08 mmol) in anhydrous Λ/-methyl-2- pyrrolidone (5 ml) under N2, were added triethylamine (1.45 ml, 10.4 mmol), K2CO3 (0.57 g, 4.16 mmol) and 4,7-dichloroquinoline (2.06 g, 10.4 mmol). The mixture was heated under pressure in a cyclo-addition tube at 130 0C overnight. After the mixture was allowed to cool to room temperature, it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in vacuo to afford a crude product, which was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (90:10) as eluent to give 5 as a white solid (0.19 g, 23%): M.p. (EtoAc:Hex) 103-104 0C; IR (DMSO) vmax 3582, 3395 - 3279, 2596, 2143, 2060, 1972, 1660, 1579; 1H NMR (CDCI3, 300 MHz) δ 8.51 (1 H, d, J = 5.1 Hz, 7.98 (1 H, d, J = 2.6 Hz, 7.69 (1 H, d, J = 9.0 Hz), 7.37 (1 H, dd, J = 2.6, 9.0 Hz), 7.34 - 7.20 (9H, m), 6.45 (1 H, d, J = 5.1 Hz), 5.43 (1 H, br t, J = 5.1 Hz), 4.51 (2H, d, J = 5.1 Hz) , 3.53 (2H, s), 3.51 (2H1 s), 2.19 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 152.1 , 149.5, 149.2, 140.4, 139.1 , 137.2, 134.9, 128.9, 128.8, 128.8, 128.5, 128.2, 127.9, 126.9, 126.1 , 125.4, 120.9, 117.2, 99.7, 61.9, 61.6, 47.6, 42.3; (Found: C, 74.53; H, 5.76; N, 9.87%. C25H24N3CI requires C, 74.71; H, 6.02; N, 10.45%).
/V-[{4-(W-Benzyl-W-methylaminomethyl)phenyl}methyl]-7-chloro-4- quinolinamine (6)
To a stirred solution of 6a (0.35 g, 1.46 mmol) in anhydrous Λ/-methyl-2- pyrrolidone (3.5 ml) under N2, were added triethylamine (1.02 ml, 7.25 mmol), K2CO3 (0.57 g, 4.16 mmol) and 4,7-dichloroquinoline (2.06 g, 10.4 mmol). The mixture was heated under pressure in a cyclo-addition tube at 90 0C for 48 hours. After the mixture was allowed to cool to room temperature it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (90:10) as eluent to give 6 as a colourless solid (0.19 g, 32%): M.p. (EtoAc:Hex)120-122 0C; IR (DMSO) vmax 3603, 3394 - 3225, 2598, 2349, 2140, 2056, 1969, 1903, 1657; 1H NMR (CDCI3, 300 MHz) δ 8.53 (1H, d, J = 5.1 Hz), 7.98 (1 H, d, J = 1.9 Hz), 7.68 (1H, d, J = 8.3 Hz), 7.41 - 7.22 (9H, m), 7.28 (1 H, dd, J = 1.9, 8.3 Hz), 6.46 (1H, d, J = 5.1 Hz), 5.29 (1H, br s), 4.49 (2H, d, J = 5.1 Hz), 3.53 (2H, s), 3.51 (2H, s), 2.19 (3H, s);13C NMR (CDCI3, 75 MHz) δ 152.1 , 149.5, 149.2, 139.5, 139.3, 135.8, 134.9, 129.5, 129.0, 128.8, 128.2, 127.6, 127.0, 125.5, 120.9, 117.1 , 99.6, 61.9, 61.4, 47.4, 42.3; (Found: C1 74.35; H, 5.88; N, 10.00%. C25H24N3CI requires C, 74.71 ; H, 6.02; N, 10.45%). /^-^-(W-p-Chlorobenzyl-W-methylaminomethylJpheny^methyll-T-chloro^- quinolinamine (7)
To a stirred solution of 7a (0.20 g, 0.73 mmol) in anhydrous Λ/-methyl-2- pyrrolidone (2 ml) under N2, were added triethylamine (0.58 ml, 4.17 mmol), K2CO3 (0.23 g, 1.46 mmol) and 4,7-dichloroquinoline (0.82 g, 4.14 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature, it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in vacuo to afford a crude product, which was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (80:10) as eluent to give 7 as a yellow oil (98 mg, 31%): IR (DMSO)
Figure imgf000036_0001
(cm"1) 3595, 3378-3231, 2142, 2067, 1975, 1657; 1H NMR (CDCI3, 300 MHz) δ 8.53 (1 H1 d, J = 5.4 Hz), 7.91 (1 H, d, J = 2.4 Hz), 7.41 - 7.12 (8H, m), 7.26 (1 H, d, J = 9.0 Hz), 6.98 (1 H, dd, J = 2.4, 9.0 Hz), 6.52 (1 H, d, J = 5.4 Hz), 5.29 (1H, s), 4.44 (2H, d, J = 3.0 Hz), 3.60 (2H, s), 3.54 (2H, s), 2.16 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 151.9, 150.1 , 149.1 , 137.1 , 136.9, 135.9, 134.8, 133.5, 132.0, 131.1 , 130.5, 128.6, 128.5, 128.4, 128.0, 124.9, 121.8, 118.0, 99.1 , 61.4, 60.8, 46.5, 42.0; HRMS (ESI): Found 436.1347 (M+). C25H24N3CI2 (M+) requires 436.1344.HRMS
W-[{3-(W-p-ChIorobenzyl-W-methylaminomethyI)phenyI}methyl]-7-chloro-4- quinolinamine (8)
To a stirred solution of 8a (0.45 g, 1.64 mmol) in anhydrous Λ/-methyl-2- pyrrolidone (4 ml) under N2, were added triethylamine (1.16 ml, 8.32 mmol), K2CO3 (0.46 g, 3.33 mmol) and 4,7-dichloroquinoline (1.65 g, 8.32 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature, it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in vacuo to afford a crude product, which was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (90:10) as eluent to give 8 as a white solid (0.19 g, 27%): M.p. (DCM:Hex) 103-104 0C; IR (DMSO) vmax (cm"1) 3620, 3372-3193, 2594, 2345, 2150, 2054, 1971 , 1911 , 1676, 1626; 1H NMR (CDCI3, 300 MHz) δ 8.48 (1H, d, J = 5.3 Hz), 7.97 (1 H, d, J = 2.4 Hz), 7.16 (1 H, d, J = 8.7 Hz), 7.37 - 7.23 (9H, m), 6.42 (1H, d, J = 5.3 Hz), 5.62 (1H, br s), 4.51 (2H, d, J = 4.8 Hz), 3.50 (2H, s), 3.45 (2H, s), 2.15 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 151.5, 149.8, 149.8, 140.1 , 137.6, 137.2, 135.1 , 132.6, 130.0, 128.9, 128.4, 128.4, 128.3, 127.8, 126.2, 125.5, 121.2, 117.1 , 99.6, 61.6, 61.0, 47.5, 42.2; (Found: C, 68.76; H, 6.14; N, 6.33%. C25H24N3CI2 requires C, 68.81 ; H, 5.31; N, 9.63%).
/^-^-(N-p-Chlorobenzyl-W-methylaminonnethylJphenyllmethylJ-T-chloro^- quinolinamine (9)
To a stirred solution of 9a (0.57 g, 2.08 mmol) in anhydrous Λ/-methyl-2- pyrrolidone (6 ml)) under N2, were added triethylamine (1.45 ml, 10.4 mmol), K2CO3 (0.57 g, 4.16 mmol) and 4,7-dichloroquinoline (2.06 g, 10.40 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (90:10) as eluent to give 9 as a colourless crystalline solid (0.19 g, 21%): M.p. (dichloromethane: hexane) 101-104 0C; IR (DMSO) vmax (cm"1) 3607, 3362-3195, 2344, 2146, 2059, 1947, 1905, 1677, 1627; 1H NMR (CDCI3, 300 MHz) δ 8.47 (1 H, d, J = 5.4 Hz), 7.95 (1 H, d, J = 1.8 Hz), 7.37 - 7.25 (1 H, d, J = 9.0 Hz), 7.32 (9H, m), 6.40 (1 H, d, J = 5.4 Hz), 5.73 (1 H, br s), 4.48 (2H, d, J = 3.9 Hz), 3.50 (2H, s), 3.47 (2H, s), 2.17 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 151.6, 149.8, 149.8, 139.0, 137.8, 135.8, 135.0, 130.1, 129.4, 128.4, 128.3, 127.5, 127.5, 125.4, 121.2, 117.1 , 99.5, 61.4, 61.0, 47.3, 42.2; Found: C, 67.69; H, 6.32; N, 6.32%: C25H24N3CI2 requires C, 68.81 ; H, 5.31; N, 9.63%.
7-Chloro-Λ/-[{2-(W-p-methoxybenzyI-Λ/-methylaminomethyl)phenyl}methyI]- 4- quinolinamine (10)
To a stirred solution of 10a (0.35g, 1.30 mmol) in anhydrous Λ/-methyl-2- pyrrolidone (3.50 ml) under N2, were added triethylamine (0.91 ml, 6.50 mmol), K2CO3 (0.54 g, 3.90 mmol) and 4,7-dichloroquinoline (2.06 g, 10.4 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature, it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (100:0) as eluent to give 10 as a dark- yellow oil (0.12 g, 21%): IR (DMSO) vmax (cm~1) 3618, 3376-3177, 2633, 2595, 2548, 2149, 2059, 1972, 1903, 1681 ; 1H NMR (CDCI3, 400 MHz) δ 8.53 (1 H, d, J = 5.4 Hz), 7.91 (1 H, d, J = 2.3 Hz), 7.62 (1 H, s), 7.34 - 7.31 (4H, m), 7.27 (1 H1 d, J = 9.0 HzS)1 7.09 (2H, d, J = 8.9 Hz), 6.92 (1 H, dd, J = 2.3, 9.0 Hz), 6.75 (2H, d, J = 8.9 Hz), 6.53 (1H, d, J = 5.4 Hz), 4.42 (2H, s), 3.77 (3H, s), 3.61 (2H, s), 3.53 (2H, s), 2.16 (3H1 s; 13C NMR (CDCI3, 75 MHz) δ 159.1 , 151.7, 150.3, 149.0, 137.2, 137.1 , 134.6, 132.0, 131.0, 130.5, 129.3, 128.3, 128.1 , 127.9, 124.7, 122.3, 117.8, 113.8, 99.0, 61.5, 60.9, 55.2, 46.5, 41.7; HRMS (ESI): Found 432.1860 (M+): C26H27N3OCI (M+) requires 432.1860.
7-Chloro-/V-[{3-(W-p-methoxybenzyl-W-methyIaminomethyl)phenyl}methyI]- 4- quinolinamine (11)
To a stirred solution of 11a (0.40 g, 1.48 mmol) in anhydrous Λ/-methyl-2- pyrrolidone (4.00 ml) under N2, were added triethylamine (1.04 ml, 7.40 mmol), K2CO3 (0.61 g, 4.44 mmol) and 4,7-dichloroquinoline (1.47 g, 7.40 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature, it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (90:10) as eluent to give 11 as a light-brown oil (0.13 g, 20%): IR (DMSO) vmax (crrf1) 3600, 3363 - 3169, 2599, 2350, 2148, 2059, 1973, 1907, 1679, 1618; 1H NMR (CDCI3, 300 MHz) δ 8.51 (1H, d, J = 5.4 Hz), 7.98 (1 H, d, J = 1.8 Hz), 7.70 (1 H, d, J = 9.0 Hz), 7.40 - 7.24 (4H, m), 7.36 (1 H, dd, J = 2.1 , 9.0 Hz), 7.21 (2H, d, J = 8.7 Hz), 6.80 (2H, d, J = 8.7 Hz), 6.45 (1 H, d, J = 5.4 Hz), 5.44 (1 H, br s), 4.51 (2H, d, J = 5.4 Hz), 3.78 (3H, s), 3.50 (2H, s), 3.45 (2H, s), 2.17 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 158.6, 152.0, 149.6, 149.1, 140.4, 137.2, 134.9, 131.0, 130.0, 128.8, 128.7, 128.5, 128.0, 126.1 , 125.4, 121.1 , 117.2, 113.6, 99.6, 61.4, 61.2, 55.2, 47.5, 42.1 ; HRMS (ESI): Found 432.1843 (M+): C26H27N3OCI (M+) requires 432.1825. 7-Chloro-W-[{4-(W-p-Methoxybenzyl-W-methylaminomethyl)phenyl}methyl]- 4- quinolinamine (12)
To a stirred solution of 12a (0.50 g, 1.85 mmol) in anhydrous N-methyl-2- pyrrolidone (5.00 ml) under N2, were added triethylamine (1.30 ml, 9.25 mmol), K2CO3 (0.77 g, 5.55 mmol) and 4,7-dichloroquinoline (1.84 g, 9.25 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of ethyl acetate: hexane (50:50) to (100:0) as eluent to give 12 as a dark- yellow oil (0.19 g, 24%): IR (DMSO) vmax (cm"1) 3589, 3365 - 3255, 2148, 2059, 1977, 1910, 1661 ; 1H NMR (CDCI3, 400 MHz) δ 8.46 (1H, d, J = 5.7 Hz), 7.94 (1 H, d, J = 2.1 Hz), 7.69 (1 H, d, J = 9.0 Hz), 7.38 - 7.25 (7H, m), 6.86 (2H, d, J = 8.7 Hz), 6.40 (1 H, d, J = 5.7 Hz), 5.57 (1H, br s), 4.49 (2H, d, J = 4.8 Hz), 3.76 (3H, s), 3.47 (2H, s), 3.44 (2H, s), 2.15 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 158.7, 151.5, 149.9, 148.4, 139.4, 135.7, 135.2, 131.1, 130.1, 129.6, 128.3, 127.6, 125.6, 121.2, 117.1, 113.7, 99.5, 61.2, 61.2, 55.3, 47.4, 42.1; HRMS (ESI): Found 432.1843 (M+): C26H27N3OCI (M+) requires 432.1835.
7-Chloro-W-[{2-(W-p-dimethylaminobenzyI-W- methylaminomethyl)phenyl}methyl]- 4-quinolinamine (13)
To a stirred solution of 13a (0.40 g, 1.41 mmol) in anhydrous /V-methyl-2- pyrrolidone (4.00 ml) under N2, were added triethylamine (1.00 ml, 7.19 mmol), K2CO3 (0.58 g, 4.23 mmol) and 4,7-dichloroquinoline (1.39 g, 7.04 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of hexane: ethyl acetate: methanol (50:50:0) to (0:90:10) as eluent to give 13 as a dark-yellow oil (112 mg, 18%): IR (DMSO) vmax (cm"1) 3633, 3360 - 3104, 2706, 2641 , 2598, 2342, 2147, 2056, 1970, 1901 , 1H NMR (CDCI3, 300 MHz) δ 8.54 (1H, d, J = 5.4 Hz, 7.89 (1H, d, J = 2.1 Hz, 7.79 (1H, br s), 7.40 - 7.10 (4H, m), 7.04 (2H, d, J = 8.6 Hz), 6.89 (1 H, d, J = 2.4 Hz), 6.86 (1 H, d, J = 2.1 Hz), 6.57 (2H, d, J = 8.6 Hz), 6.54 (1 H, d, J = 5.4 Hz), 4.41 (2H, s), 3.61 (2H1 s), 3.50 (2H, s), 2.92 (6H, s), 2.14 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 152.0, 150.3, 150.0, 149.2, 137.4, 134.4, 132.0, 130.8, 130.5, 128.2, 128.0, 127.8, 124.8, 124.7, 122.6, 117.9, 112.3, 98.8, 61.6, 61.0, 46.5, 41.5, 40.4; HRMS (ESi): Found 445.2159 (M+): C27H30N4 CI(M+) requires 445.2172.
7-Chloro-W-[{3-(W-p-Dimethylaminobenzyl-W- methylaminomethyl)phenyl}methyl]- 4-quinolinamine (14)
To a stirred solution of 14a (0.40 g, 1.41 mmol) in anhydrous W-methyl-2- pyrrolidone (4.00 ml) under N2, were added triethylamine (1.00 ml, 7.19 mmol), K2CO3 (0.58 g, 4.23 mmol) and 4,7-dichloroquinoline (1.39 g, 7.04 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of hexane: ethyl acetate: methanol (50:50:0) to (0:90:10) as eluent to give 14 as a dark-yellow oil (0.14 g, 22%): IR (DMSO) vmax (cm"1) 3363 - 3169, 2599, 2350, 2148, 2059, 1973, 1907, 1679, 1618; 1H NMR (CDCI3, 400 MHz) δ 8.47 (1H, d, J = 5.2 Hz), 7.95 (1 H, d, J = 2.0 Hz), 7.70 (1 H, d, J = 8.8 Hz), 7.38 - 7.21 (4H, m), 7.31 (1H, dd, J = 2.0, 8.8 Hz), 7.13 (2H, d, J = 8.6 Hz), 6.61 (2H, d, J = 8.6 Hz), 6.41 (1H, d, J = 5.2 Hz), 5.58 (1H, br s), 4.47 (2H, d, J = 4.8 Hz), 3.47 (2H, s), 3.40 (2H, s), 2.88 (6H, S,), 2.15 (3H, s); 13C NMR (CDCI3, 75 MHz) δ 152.4, 152.0, 149.9, 149.6, 140.7, 137.2, 135.0, 130.1 , 129.8, 128.8, 128.8, 128.6, 128.1 , 126.1 , 125.5, 121.0, 117.2, 112.5, 99.7, 61.3, 61.3, 47.7, 42.2, 40.7; HRMS (ESI): Found 445.2159 (M+). C27H30N4CI (M+) requires 445.2138.
7-Chloro-W-[{4-(W-p-dimethylaminobenzyl-/V- methylaminomethyl)phenyl}methyl]-4-quinolinamine (15)
To a stirred solution of 15a (0.40 g, 1.41 mmol) in anhydrous A/-methyl-2- pyrrolidone (4.00 ml) under N2, were added triethylamine (1.00 ml, 7.19 mmol), K2CO3 (0.58 g, 4.23 mmol) and 4,7-dichloroquinoiine (1.39 g, 7.04 mmol). The mixture was heated under pressure in a cyclo-addition tube at 120 0C overnight. After the mixture was allowed to cool to room temperature it was poured into saturated brine (20 ml) and extracted with ethyl acetate (3 x 50 ml). The organic layer was further washed with saturated brine (5 x 50 ml) to ensure removal of any traces of the pyrrolidone. The organic layer was dried (Na2SO4), concentrated in vacuo and the resulting crude product was purified by column chromatography using mixtures of hexane: ethyl acetate: methanol (50:50:0) to (0:90:10) as eluent to give 15 as a dark-yellow oil (0.16 g, 26%): IR (DMSO) vmax (cm"1) 3616, 3378 - 3212, 2596, 2150, 2062, 1977, 1904, 1674, 1635; 1H NMR (CDCI3, 400 MHz) δ 8.43 (1 H, d, J = 5.4 Hz), 7.92 (1 H, d, J = 2.0 Hz), 7.70 (1 H, d, J = 9.2 Hz), 7.34 - 7.28 (4H, m), 7.26 (1H, dd, J = 2.4, 6.4 Hz), 7.19 (2H, d, J = 8.4 Hz), 6.68 (2H, d, J = 8.4 Hz), 6.37 (1 H, d, J = 5.4 Hz), 5.82 (1 H, br s), 4.43 (2H1 d, J = 4.8 Hz), 3.46 (2H, s), 3.42 (2H, s), 2.89 (6H, s), 2.15 (3H, s);13C NMR (CDCI3, 75 MHz) δ 151.7, 149.7, 149.7, 148.8, 139.2, 135.6, 134.8, 129.7, 129.3, 128.3, 127.3, 126.7, 125.2, 121.3, 117.1 , 112.4, 99.5, 61.2, 61.0, 47.1, 42.0, 40.6; HRMS (ESI): Found 445.2159 (M+). C27H30N4CI (M+) requires 445.2171.
Alternative synthetic routes
Schemes 3 and 4 outline examples of other potential synthetic routes to obtain the compounds of the invention.
Figure imgf000041_0001
Figure imgf000041_0002
and NMe2
Scheme 3: Alternative route to the synthesis of 4-H, 4-CI, 4-MeO and 4-NMe2- analogues of the dibemequines. Reagents and conditions: (i) excess o, m or p - methyl-benzylamine, reflux (ii) Λ/-bromosuccinimide, benzoylperoxide, CCI4; (iii) K2CO3, CH3CN, reflux
Figure imgf000042_0001
Figure imgf000042_0002
Scheme 4: The proposed general reaction scheme for the synthesis of -7- substituted quinolines analogues of dibemequine, where X is H, hydroxyl, alkoxy (e.g. OMe), optionally substituted amino (e.g. NMe2), halo (e.g. I), haloalkyl (eg. CH2Br), nitro, or cyano; Reagents and conditions: (i) heat to reflux; (ii) Ph2O, reflux; (iii) reflux in NaOH (aq); (iv) Ph2O, reflux (v) POCI3, heat; (vi). excess o, m or p - methyl-benzylamine; reflux (vii) Λ/-bromosuccinimide, benzoylperoxide, CCI4; (viii) K2CO3, CH3CN, reflux.
Compounds (4) was selected as a prototype for this series of compounds. It was crystallized and the structure determined by single crystal X-ray diffraction analysis. The structure is shown in Figure 1 and crystallographic data are presented in Table 3. Table 3: Crystal structure data for compound 4
Empirical formula C25 H24 Cl N3
Formula weight 401.92
Crystal system, space group Monoclinic, P21/c a (A) 13.1065(2) b (A) 15.7570(3) c (A) 21.7792(5) α (°) 90
P C) 90.5650(10)
Y (°) 90
V (A)3 4497.60(15
Z 8 λ (Mo-Ka) (A) 0.71073
F (000) 1696
Crystal size (mm) 0.16 x 0.11 x 0.09
Range scanned θ (°) 3.02 - 25.37
Range of indices h: ±15 k: ±18
I: ±26
No. reflections collected 60271
No. unique reflections 8207
S 1.039
Figure imgf000043_0001
wR2 0.1118
Δp excursions /e.A3 -0.2, 0.242
Determination of pKg values and β-hematiπ inhibition activity
The two pKa values of compounds 4 - 15 were determined by pH titration and are reported in Table 2. The IC50 values for β-hematin inhibition were determined using a pyridine based 96-well plate method.13 The method relies on the fact that 5 % aqueous pyridine dissolves hematin, but not β-hematin at pH 7.5. The extent of inhibition was then characterized by measuring the intensity of the monomeric pyridine-hematin complex at 405 nm. Values are also reported in Table 4. Table 4: The resonance constants (R), measured acid dissociation constant (pKs) values, β-hematin inhibitory activities (BHIAgg), in vitro antimalarial activities (ICm) versus the D10 and K1 strains of P. falciparum and resistance index (Rl) of compounds 4 - 15.
Code R pKa1 pKa2 BHIA50 IC50 DIO/ IC50 K1/ Rlα nM nM
4 VZ1 0 7.57 9.85 1.12 138.3 187.9 1.4
5 VZ2 0 7.63 9.77 0.69 53.3 65.0 1.2
6 VZ3 0 7.56 9.90 0.32 26.5 24.5 0.9
7 2VZ1 -0.15 7.44 9.60 1.35 NDb 1127.5 -
8 2VZ2 -0.15 7.55 9.89 0.66 131.4 84.9 0.6
9 2VZ3 -0.15 7.44 9.74 0.34 NDb NDC -
10 3VZ1 -0.51 7.47 9.85 0.46 175.2 134.3 0.8
11 3VZ2 -0.51 7.38 9.67 0.52 90.5 116.8 1.3
12 3VZ3 -0.51 7.44 9.70 1.44 129.9 87.8 0.7
13 4VZ1 -0.92 7.40 9.71 0.48 177.7 153.3 0.9
14 4VZ2 -0.92 7.36 9.67 0.45 NDb 280.9 -
15 4VZ3 -0.92 7.33 9.66 0.41 47.8 37.0 0.8
CCf - - 8.4 10.8 1.91 29.4 285 9.7
' Equivalents relative to hematin, D not determined, IC50 > 100 ng/ml, c not determined, IC50 > 10 μg/ml, d Rl = IC50 (K1) / IC50 (D10), e chloroquine.
As expected, all of the compounds exhibit two pKa values. The lower one, corresponding to the quinoline heteraromatic N atom, was found to lie in a narrow range from 7.33 (15) to 7.63 (5). The higher, corresponding to the basic tertiary amino group in the dibemethin side chain, ranged from 9.6 (7) to 9.9 (6).
The IC50 values for β-hematin inhibition covered a somewhat larger range, with the most potent inhibitor (6) having an IC50 of 0.32, while the least active (12) exhibited a value of 1.44. Biological testing
In vitro antimalarial testing via the parasite lactate dehydrogenase assay
Continuous in vitro cultures of asexual erythrocyte stages of P.falciparum were maintained using a modified method of Trager and Jensen.16 Quantitative assessment of antiplasmodial activity in vitro was determined via the parasite lactate dehydrogenase assay using a modified method described by Makler.17
The samples were prepared to a 2mg/ml stock solution in 100% DMSO or 100% methanol and sonicated to enhance solubility. Samples were tested as a suspension if not completely dissolved. Stock solutions were stored at -2O0C. Further dilutions were prepared on the day of the experiment. Chloroquine (CQ) was used as the reference drug in all experiments. A full dose-response was performed for all compounds to determine the concentration inhibiting 50% of parasite growth (IC50 - value). Test samples were tested at a starting concentration of 100 ng/ml or 10 μg/ml, which was then serially diluted 2-fold in complete medium to give 10 concentrations. The same dilution technique was used for all samples. CQ was tested at a starting concentration of 1.15 μg/ml. The highest concentration of solvent to which the parasites were exposed to had no measurable effect on the parasite viability (data not shown). The IC50-values were obtained using a non-linear dose- response curve fitting analysis via Graph Pad Prism v.4.0 software.18
In vitro testing using chloroquine sensitive D10 and chloroquine resistant K1 strains
Compounds 4 - 15 were all tested against the chloroquine sensitive D10 and chloroquine resistant K1 strains of parasite in vitro (Table 4). In the D10 strain, activities were not determined above 100 ng/ml, while in the K1 strain measurements were not performed above 10 μg/ml. With these cutoff values, three compounds (7, 9 and 14) were inactive against the D10 strain, while one (9) was inactive in the K1 strain. Compounds 7 and 14 had the weakest measured IC50 values in the K1 strain. The IC50 values ranged from 27 nM (6) to 178 nM (13) in the D10 strain and from 24 nM (6) to 1178 nM (7). The resistance index (ratio of IC50 in the K1 strain to that in the D10 strain) ranged between 0.7 (12) and 1.4 (4). This variation is probably not statistically significant and reflects the essentially complete retention of activity in resistant parasites by these compounds.
Cytotoxicity testing
The MTT-assay is used as a colorimetric assay for cellular growth and survival, and compares well with other available assays.19 The tetrazolium salt MTT was used to measure all growth and chemosensitivity. Compound 4 was tested in triplicate on three separate occasions.
The compound was dissolved in 10% DMSO. The initial concentrations of stock solutions were 2mg/ml. The compound was tested as a suspension and stored at -2O0C until use. The highest concentration of solvent to which the cells were exposed to had no measurable effect on the cell viability (data not shown). Emetine was used as the reference drug in all experiments. The initial concentration of emetine was 100μg/ml, which was serially diluted in complete medium with 10-fold dilutions to give 6 concentrations, the lowest being 0.001 μg/ml. The same dilution technique was applied to the test sample with an initial concentration of 100μg/ml to give 5 concentrations, with the lowest concentration being 0.01 μg/ml.
The 50% inhibitory concentration (IC50) values for these samples were obtained from dose-response curves, using a non-linear dose-response curve fitting analysis via GraphPad Prism v.4 software.18
Evaluation of compounds 4 and 6 against Plasmodium yoelii nigeriensis (NS) in vivo in adult BaIbC white mice
Compounds 4 and 6 were stored at 4°C until evaluated. The compounds were diluted in DMSO to make stock solutions; thereafter, both compounds were diluted to test doses. These were made up to 5mg/kg for the initial evaluation and 20 mg/kg for the second evaluation. These doses were chosen since the compound proved as effective as chloroquine (CQ) in vitro and thus the same test doses as CQ were used in vivo. Typically, with Plasmodium yoelii NS, a 5 mg/kg dose of CQ in 100μl phosphate-buffered saline (PBS) produces a moderate parasite clearance in the bloodstream; a 20 mg/kg dose produces an almost complete clearance of circulating parasites.
An initial dilution of stock into PBS caused both compounds to precipitate. A small amount of TweenδO and ethanol was thus used to form a fine suspension of the compound which we could dilute satisfactorily in PBS to administer to the mice, since DMSO is highly toxic to the animals. Initially, 50 μl of TweenδO and 100 μl of ethanol were used to re-suspend each compound for the 5 mg/kg experiment at a total volume of 1ml; this was further diluted to 25 μl TweenδO and 50 μl ethanol when making the 20 mg/kg stocks in a total volume of 1 ml. These stock solutions were such that 100 μl of each stock would yield 5 mg of compound per kg of mouse. Mouse weight is approximately 20 g/animal.
Mice were challenged using the 4-day suppressive test as described by Peters et al,20 with modifications. Identical procedures were used for both the 5 mg/kg and 20 mg/kg experiments. There were 5 mice in each test group.
Day 0: Each mouse was infected with 1x107 parasitized erythrocytes by intraperitoneal injection in 200μl PBS. Parasite stocks were made from a donor mouse which was sacrificed once parasite levels were deemed sufficient to infect the test mice; infected blood cells were obtained via cardiac puncture and placed in tubes containing PBS and EDTA to prevent clotting. Each compound was diluted to 5 mg/kg as described above; a fresh stock of CQ (5 mg/kg) was also produced. Mice were treated with 100μl of 4, 6 or chloroquine via sub-cutaneous injection.
Days +1 to +3: Mice treated with 100μl freshly-prepared drug stocks; drugs were administered at approximately the same time as on Day 0 to within half an hour.
Day +4: No treatments. Mice evaluated and parasitaemia (percentage infected erythrocytes) determined. If necessary, mice were killed owing to significantly high parasite levels.
The prototype (4) was subjected to cytotoxicity testing in Chinese hamster ovarian (CHO) cells. The IC50 in this system was found to be 32 μM, which is 232 times higher than the IC50 in the D10 strain of P. falciparum and 170 times greater than that in the K1 strain. Thus, compound 4 exhibits highly selective activity against the malaria parasite. Compounds 4 and 6 were also tested against P. yoelii nigeriensis in a mouse model by intra-peritoneal injection at 5 and 20 mg/kg and compared to chloroquine diphosphate at the same doses. As indicated in Table 5, both compounds were less effective than chloroquine, although substantial activity was observed at 20 mg/kg. As both compounds were administered as suspensions of their free bases rather than solutions, as opposed to the chloroquine salt which was fully dissolved, the somewhat lower activity of these two compounds in vivo may just reflect lower bioavailability owing to the method of administration. Neither compound appeared to exert any obvious acute toxicity on the mice.
Table 5: In vivo antimalarial activities of suspensions of compounds 4 and 6 administered to adult BaIbC white mice infected with P. voelii niaeriensis by intra-peritoneal injection.
Mean parasitaemia % Range Mean parasitaemia % Range
5 mg/kg 5 mg/kg 20 mg/kg 20 mg/kg
CCf 6.1% 4-8 <1 % ND
4 23.45% 8.75-34. 72 2.76%* 1.30-4.05*
6 20.07% 17.71-32 .85 3.13% 1.99-3.86
*Total range from 4 mice only, following one death overnight after infection on Day 0.
The parent compounds 4, 5 and 6 were sent to the Research School of Biology, Australian National University in Canberra Australia and tested for inhibition of CQ transport by PfCRT. All three were found to inhibit CQ transport by PfCRT (the protein primarily responsible for chloroquine resistance), with 5 exhibiting an IC50 of 65 μM. This concentration can be expected to be easily attained within the parasite digestive vacuole as a result of pH trapping.
In vivo antimalarial activity of parent compounds administered to mice infected with P. berghei via oral dosing
Mice were challenged using the suppressive test as described by Peters et al,20 with modifications. The in vivo activity of 4 was evaluated with the 4 days Peters' test, due to the physical properties and the available amounts of material, 5 was used for a three times treatment (day 0, day 1 and day 2) and 6 was used for a one times treatment (day 0). In all cases parasitaemia was determined on day 4. Identical procedures were used for both the 30 mg/kg and 100 mg/kg experiments.
In summary, hepahnized blood was taken from a donor mouse with approximately 30% parasitaemia and diluted in physiological saline to 108 parasitized erythrocytes per ml. Of this suspension, 0.2ml was injected intravenously (i.v.) into experimental groups of 3 mice, and a control group of 3 mice. 4 hours post-infection the experimental groups were treated with a single dose 24, 48 and 72 hours postinfection, the experimental groups were treated with a single daily dose. 1 μl tail blood was taken 24 hours after the last drug treatment and the parasitaemia was determined with a FACScan. The difference between the mean value of the control group and those of the experimental groups was calculated and expressed as a percent relative to the control group (= activity). The survival of the animals was monitored up to 30 days. Mice surviving for 30 days were checked for parasitaemia. A compound is considered curative if the animal survives to day 30 post-infection with no detectable parasites. The results are expressed as reduction of parasitaemia on day 4 in % as compared to the untreated control group, and mean survival compared to the untreated control group.
Table 6 below tabulates the results of the in vivo antimalarial activity of the parent compounds (4, 5 and 6) against P. berghei infected mice when the compounds were administered by oral dosing at 30 mg/kg and 100 mg/kg.
Table 6: In vivo antimalarial activities of free-bases of compounds 4, 5 and 6 administered to mice infected with P. berphei by oral dosing
Mean Clearance of Mean Mean % Clearance Mean parasitaemia parasitaemia survival parasitaemia of survival
(100 mg/kg) (100 mg/kg) in days (30 mg/kg) parasitaemia in days
(100 (30 mg/kg) (30 mg/kg) mg/kg)
4 <0.1 % 99.9 % 30 0.3 % 99.6 % 20.7
5 <0.1 % 99.9 % 30 0.1 % 99.9 % 16.7
6 0.12 % 99.8 % 10 2.16 % 97.2 % 6.7
"Mean parasitaemia at day 4 for control group was 78.4% All the parent compounds demonstrated very good in vivo activity after oral dosing and reduced parasitaemia up to 99.9 % at 30 mg/kg. Compounds 4 and 5 were able to cure the mouse model at 100 mg/kg doses (Table 6).
Discussion
Dependence ofpKa on the identity of the terminal group on the side chain
Although the pKa values for compounds 4 - 15 vary over only a very small range of about 0.3 log units, a statistically significant correlation with the physical characteristics of the functional group attached to the terminal phenyl ring of the side chain is observed (Figure 2). Unexpectedly, both pKa-ι and pKa2 increase as the functional group becomes less resonance releasing (i.e. as the parameter R becomes more positive). One would expect that an electron releasing group would strengthen the N-H+ bonds, making deprotonation more difficult and raising rather than decreasing the pKa. Furthermore, the quinoline N atom is separated from the functional group on the terminal phenyl ring by fifteen bonds and even the dibemethin N atom is separated from this group by six bonds. This means that the influence of the group on pKa must be a through space interaction. The crystal structure of 4 illustrates that these molecules can adopt a folded structure in which the quinoline comes into relatively close contact with the terminal phenyl ring of the side chain. Without a more detailed investigation of such intramolecular interactions, the trend in pKa values is unlikely to be rationalized.
Using equation 1 the predicted accumulation ratio of 4 - 15 in the digestive vacuole of the parasite (VAR) can be calculated. 14
Figure imgf000050_0001
Here [Q]v is the concentration of the compound in the digestive vacuole of the parasite, [Q]e is its concentration in the extracellular medium, pHv is the digestive vacuole pH (taken as 5, the midpoint between two recent estimates of 4.8 and 5.2) and pHe is the pH of the external medium (7.4). Values are given in Table 7. An accumulation normalized IC50 for antimalarial activity can be calculated by multiplying the observed IC50 by the VAR value for each compound. As 4-aminoquinolines are believed to act by inhibiting hemozoin formation in the digestive vacuole, these numbers may be more relevant than the observed IC50. They are therefore reported in Table 7.
Table 7: Calculated vacuolar accumulation ratio (VAR) and vacuolar accumulation normalized ICRn against the K1 strain of P. falciparum in vitro (VAR-ICsn).
VARa VAR-IC50 / mM
4 15029 2.82
5 15843 1.03
6 14892 0.37
7 13173 14.85
8 14753 1.25
10 13620 1.83
11 12318 1.44
12 13181 1.16
13 12608 1.93
14 12031 3.38
15 11601 0.43 according to eq. 1 , assuming a vacuolar pH of 5.0
Correlations of biological activity and β-hematin inhibition
In the case of compounds 4 - 9, the β-hematin inhibition activity increases markedly in going from the ortho- to meta- to para- derivatives. However, this pattern is reversed in the series 10 - 12 and no significant differences are seen in compounds 13 - 15. Thus, the abilities of these compounds to inhibit hemozoin formation appears to depend on both the site of attachment of the aminoquinoline to the dibemethin side chain and the identity of the group on the terminal phenyl ring of the dibemethin.
As IC50 values were not determined for three of the compounds against the D10 strain of parasite, while values were obtained for eleven of the twelve compounds against the K1 strain, the K1 IC50 data were used for structure activity relationship analysis. Plots of log IC50 for in vitro antimalarial activity versus log IC50 for inhibition of β-hematin formation (log BHIA50) indicate that there is a general decrease in biological activity as β-hematin inhibition activity decreases (Fig. 3a). However, the trend is not statistically significant unless the data for compound 12 is omitted. The correlation is only marginally improved if the antimalarial activity is normalized for accumulation in the digestive vacuole (Fig. 3b). The correlation is again only statistically significant if compound 12 is omitted. While this may suggest that either the BHIA50 or the IC50 value for 12 is an outlier, it is more likely an indication that other factors play a role in biological activity in addition to vacuolar accumulation and hemozoin inhibition. By contrast to a previous study of short-chain analogues of chloroquine in which vacuolar accumulation and β-hematin inhibition alone correlated with activity,15 in the present study the structure of the side chain is not kept constant. Therefore, correlation analyses were also performed in which a structural descriptor (the position number 2, 3 and 4 for ortho-, mete- and para- positions respectively) was included. The log IC50 is significantly correlated with position in the order ortho- > meta- > para (Fig. 3c). This correlation improves slightly if the accumulation normalized IC50 is used (r2 = 0.55 and P = 0.0087 versus r2 = 0.52 and P = 0.012 without normalization). When the structural descriptor was used in the correlation together with the log BHIA50 the correlation with accumulation normalized log IC50 is greatly improved (Fig. 3d). When vacuolar accumulation is treated as a third variable in multiple correlation analysis with the observed log IC50 values, the correlation improves even further (Fig. 3e).
The factors affecting the biological activities of this series of compounds are in agreement with a previous study on short chain chloroquine analogs,15 with the additional observation of an independent influence of side-chain structure. The origin of this effect is unknown. It could indicate enhanced uptake of the para- substituted compounds, or more likely the a decrease in the concentration of the quinoline available to inhibit hemozoin formation arising from interaction with constituents of the cell or culture medium which binds the ortho- compounds most strongly. In any event, both hemozoin inhibition and vacuolar accumulation are confirmed to be significant for biological activity.
Finally, it is noteworthy that there is no indication of any significant cross resistance with chloroquine in this series of compounds in the K1 strain of parasite. When the available IC50 values are plotted on the same correlation graph as those of the K1 strain (Fig. 3e), the points clearly fall on the same correlation line. The maintenance of activity against chloroquine resistant parasites could be a result of their ability to inhibit transport by PfCRT (i.e. resistance reversal), or merely owing to the altered structure of the side chain, which makes it no longer susceptible to the resistance machinery of the digestive vacuole.
Conclusions
The applicant has shown that the series of novel 4-amino-quinolines with dibemethin side chains attached via a methylene bridge at the ortho-, meta- or para- position on the dibemethin exhibit activity against chloroquine sensitive and resistant parasites and inhibit chloroquine transport by PfCRT. Three compounds, 5, 6 and 15 show promising levels of in vitro antimalarial activity, with compound 5 in particular being more active than chloroquine against chloroquine sensitive parasites. Both the prototype compound 4 and compound 6 have been shown to have significant in vivo antimalarial activity in the mouse model P. yoelii nigeriensis, although neither was as active as chloroquine diphosphate when administered i.p. Preparation of salts of these compounds and improved formulation are expected to improve in vivo activity considerably, as both 5 and 6 were administered as suspensions, whereas chloroquine diphosphate was a solution. A preliminary cytotoxicity investigation of compound 4 suggests that toxicity is acceptably low relative to antimalarial activity. Structure activity studies show that, as with other chloroquine analogs, the activity of this class of compound results from their ability to accumulate in the parasite digestive vacuole and inhibit hemozoin formation.
References
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(2) Nkhoma, S.; Molyneux, M.; Ward, S. A. In vitro antimalarial susceptibility profile and pfcrt/pfmdr-1 genotypes of Plasmodium falciparum field isolates from Malawi. Am. J. Trop. Med. Hyg. 2007, 76, 1107-1112.
(3) Mita, T.; Kaneko, A.; Lum, J. K.; Zungu, I. L.; Tsukahara, T.; Eto, H.; Kobayakawa, T.; Bjόrkman, A.; Tanabe, K. Expansion of wild-type allele rather than back mutation in pfcrt explains the recent recovery of chloroquine sensitivity of Plasmodium falciparum in Malawi. MoI. Biochem. Parasitol. 2004, 135, 159-163. (4) Walliker, D.; Hunt, P.; Babiker, H. A. Fitness of drug-resistant malaria parasites. Acta Trop. 2005, 94, 251-259.
(5) van Schalkwyk, D.; Egan, T. J. Quinoline-resistance reversing agents for the malaria parasite Plasmodium falciparum. Drug Resist. Update 2006, 9, 211-226.
(6) Abok, K. Re: Malaria treatment in peri-urban area of Kisumu town. East Afr. Med. J. 1997, 74, 757.
(7) Sowunmi, A.; Oduola, A. M. J. Comparative efficacy of chloroquine/chlorpheniramine combination and mefloquine for the treatment of chloroquine-resistant Plasmodium falciparum malaria in Nigerian children. Trans R. Soc. Trop. Med. Hyg. 1997, 91, 689-693.
(8) O' Neill, P. M.; Mukhtar, A.; Stocks, P. A.; Randle, L. E.; Hindley, S.; Ward, S. A.; Storr, R. C; Bickley, J. F.; O'Neil, I. A.; Maggs, J. L.; Hughes, R. H.; Winstanley, P. A.; Bray, P. G.; Park, B. K. Isoquine and related amodiaquine analogues: a new generation of improved 4- aminoquinoline antimalarials. J. Med. Chem. 2003, 46, 4933-4945.
(9) Biot, C. Ferroquine: A new weapon in the fight against malaria. Curr. Med. Chem. 2004, 3, 135- 147.
(10) Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe, M. K.; Peyton, D. H. A chloroquine-like molecule designed to reverse resistance in Plasmodium falciparum. J. Med. Chem. 2006, 49, 5623-5625.
(11) WO 2006/088541 A2; Portland State Univ., 2006.
(12) Bhattacharjee, A. K.; Kyle, D. E.; Vennerstrom, J. L.; Milhous, W. K. A 3D QSAR pharmacophore model and quantum chemical structure-activity analysis of chloroquine(CQ)- resistance reversal. J. Chem. Inf. Comput Sci. 2002, 42, 1212-1220.
(13) Ncokazi, K. K.; Egan, T. J. A colorimetric high-throughput b-hematin inhibition screening assay for use in the search for antimalarial compounds. Anal. Biochem. 2005, 338, 306-319.
(14) Warhurst, D. C; Craig, J. C; Adagu, I. S.; Meyer, D. J.; Lee, S. Y. The relationship of physico- chemical properties and structure to the differential antiplasmodial activity of the cinchona alkaloids. Malaria J. 2003, 2:26.
(15) Kaschula, C. H.; Egan, T. J.; Hunter, R.; Basilico, N.; Parapini, S.; Taramelli, D.; Pasini, E.; Monti, D. Structure-activity relationships in 4-aminoquinoline antiplasmodials. The role of the group at the 7-position. J. Med. Chem. 2002, 45, 3531-3539.
(16) Trager, W.; Jensen, J. B. Human malaria parasites in continuous culture. Science 1976, 193, 673-675.
(17) Makler, M. T.; Ries, J. M.; Williams, J. A.; Bancroft, J. E.; Piper, R. C; Gibbins, B. L.; Hinrichs, D. J. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg. 1993, 48, 739-741.
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Claims

CLAIMS:
1. A compound having formula (I):
Figure imgf000055_0001
(I) wherein
Xiι X2. X3 and X4 are independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl;
Y is CH or N; m, n, p, q, r and s are independently from 0 to 5;
R1 , R2, R3 and R4 are independently selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein
R3 and R4 together with the carbon atoms to which they are joined optionally form a six membered ring; or a pharmaceutically acceptable salt thereof.
A compound according to claim 1 , wherein the six membered ring formed by R3 and R4 has one or more substituents independently selected from the group consisting of H, alkoxy, amido, optionally substituted amino, cyano, halo, haloalkyl, hydroxyl, nitro, sulphonamide and trifluoromethyl.
A compound according to claim 1 , which is Λ/-[{2-(Λ/-Benzyl-/V- methylaminomethyl)phenyi}methyl]-7-chloro-4-quinolinamine.
4. A compound according to claim 1, which is Λ/-[{3-(Λ/-Benzyl-/V- methylaminomethyl)phenyl}methyl]-7-chloro-4-quinolinamine.
5. A compound according to claim 1, which is /V-[{4-(Λ/-Benzyl-Λ/- methylaminomethyl)phenyl}methyl]-7-chloro-4-quinolinamine.
6. A compound according to claim 1 , which is Λ/-[{2-(Λ/-p-Chlorobenzyl-/V- methylaminomethyl)phenyl}methyl]-7-chloro-4-quinolinamine.
7. A compound according to claim 1 , which is Λ/-[{3-(Λ/-p-Chlorobenzyl-/V- methylaminomethyl)phenyl}methyl]-7-chloro-4-quinolinamine.
8. A compound according to claim 1 , which is Λ/-[{4-(Λ/-p-Chlorobenzyl-/V- methylaminomethyl)phenyl}methyl]-7-chloro-4-quinoiinamine.
9. A compound according to claim 1 , which is 7-Chloro-Λ/-[{2-(Λ/-p- methoxybenzyl-Λ/-methylaminomethyl)phenyl}methyl]-4-quinolinamine.
10. A compound according to claim 1 , which is 7-Chloro-Λ/-[{3-(Λ/-p- methoxybenzyl-Λ/-methylaminomethyl)phenyl}methyl]-4-quinolinamine.
11. A compound according to claim 1 , which is 7-Chloro-Λ/-[{4-(Λ/-p- Methoxybenzyl-/V-methylaminomethyl)phenyl}methyl]-4-quinolinamine.
12. A compound according to claim 1 , which is 7-Chloro-Λ/-[{2-(Λ/-p- dimethylaminobenzyl-Λ/-methylaminomethyl)phenyl}methyl]- 4-quinolinamine.
13. A compound according to claim 1, which is 7-Chloro-/V-[{3-(Λ/-p- Dimethylaminobenzyl-Λ/-methylaminomethyl)phenyl}methyl]- 4-quinolinamine.
14. A compound according to claim 1, which is 7-Chloro-Λ/-[{4-(Λ/-p- dimethylaminobenzyl-A/-methylaminomethyl)phenyl}methyl]-4-quinolinamine.
15. A compound according to any one of claims 1 to 14, for use in a method of preventing and/or treating malaria.
16. A compound according to claim 15, wherein the malaria is caused by strains of Plasmodium falciparum.
17. A compound according to claim 16, wherein the P. falciparum strains are chloroquine sensitive or chloroquine resistant.
18. A pharmaceutical composition including a compound as claimed in any one of claims 1 to 17 and a pharmaceutically acceptable carrier.
19. A pharmaceutical composition according to claim 18, which includes a second antimalarial compound.
20. A pharmaceutical composition according to claim 19 wherein the second antimalarial compound is chloroquine.
21. Use of a compound as claimed in any one of claims 1 to 17 in a method of making a medicament for use in a method of preventing and/or treating malaria.
22. A method of preventing and/or treating malaria, the method comprising administering an effective amount of a compound as claimed in any one of claims 1 to 17 to an animal in need thereof.
23. A method according to claim 22, wherein the animal is a human.
24. A method according to either of claims 22 or 23, wherein the compound is administered with a second antimalarial compound.
25. A method according to claim 24, wherein the second antimalarial compound is chloroquine.
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US10865214B2 (en) 2015-10-05 2020-12-15 The Trustees of Columbia University in they City of New York Activators of autophagic flux and phospholipase D and clearance of protein aggregates including tau and treatment of proteinopathies
US11008341B2 (en) 2015-10-05 2021-05-18 The Trustees Of Columbia University In The City Of New York Activators of autophagic flux and phospholipase D and clearance of protein aggregates including tau and treatment of proteinopathies
US11230558B2 (en) 2015-10-05 2022-01-25 The Trustees Of Columbia University In The City Of New York Activators of autophagic flux and phospholipase D and clearance of protein aggregates including tau and treatment of proteinopathies
US11261199B2 (en) 2015-10-05 2022-03-01 The Trustees Of Columbia University In The City Of New York Activators of autophagic flux and phospholipase d and clearance of protein aggregates including tau and treatment of proteinopathies

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