Patent Application
20140336187
The present inventions relate to methods for inhibiting a parasite from the genus Leishmania, using an imido-substituted 1,4-napthoquinone compound.
Leishmaniasis is a parasitic infection caused by protozoan parasites of the genus Leishmania. The disease, named Leishman by the first person who described it in London in May 1903, is transmitted by the bite of a female sandfly (genus Phlebotomus) during a blood meal on its host. Clinical symptoms of the disease vary and include cutaneous, mucocutaneous, and visceral forms of the disease. Over 20 species and subspecies of Leishmania infect mammals, such as humans, causing a different spectrum of symptoms. These include Leishmania donovani complex with 2 species (Leishmania donovani, Leishmania infantum); the Leishmania mexicana complex with three main species (Leishmania mexicana, Leishmania amazonesis, and Leishmania venezuelensis); Leishmania tropica; Leishmania major; Leishmania aethiopica; and the subgenus Viannia with 4 main species Leishmania (V.) braziliensis, Leishmania (V.) guyanensis, Leishmania (V) panamensis, and Leishmania (V.) peruviana). Millions of people are at risk of disease and even death from the parasitic infections. It has been estimated approximately 12 million people worldwide are infected, 1.5 million with cutaneous leishmaniasis, 0.5 million with visceral leishmaniasis and that 350 million people are at risk of being infected. A conservative estimate of global yearly incidence is 1-1.5 million for cutaneous leishmaniasis and 0.5 million for visceral leishmaniasis.
Leishmaniasis is more common in tropical regions with warm climates making these regions most important areas for the historical development and concentration of zoonoses and related public health problems. In the western hemisphere (New World), it occurs in some parts of Mexico, Central America, and South America while in the Eastern Hemisphere (Old World) it is mostly found in regions of Asia, the Middle East, Africa, and Southern Europe (CDC, 2011). About 90% of visceral leishmaniasis (VL) cases occur in India, Bangladesh, Nepal, Sudan, Ethiopia, and Brazil while 90% of cutaneous leishmaniasis (CL) occurs in Afganisthan, Algeria, Iran, Saudi Arabia, Syria, Brazil, Colombia, Peru, and Bolivia. Cases of leishmaniasis found in the United States were mostly due to travel and immigration patterns. Cases in civilians are due to travelers acquiring the disease from tourist's destinations in Latin America. In the military, it is due to personnel becoming infected with leishmaniasis in Iraq and Afghanistan and returning home with infections (CDC, 2011).
In order to limit the number of cases of leishmaniasis in most endemic regions, the World Health Organization (WHO) in 2004 developed a plan of action in Afghanistan and its aim was to control debilitating leishmaniasis. WHO together with the Massoud Foundation and HealthNet International, in Kabul, Afghanistan, with the help of donations from the Belgian government, intended to reduce the incidence of leishmaniasis in less than two years. This initiative was again renewed in 2010 under the control of neglected tropical diseases and the major aim was to scale-up integrated interventions. This initiative, “working to overcome the global impact of neglected tropical diseases” covers 17 neglected tropical diseases mostly in poor settings where housing is below substandard, contamination of environments with filth is common, and insects and animals that spread disease are rampant (WHO, 2011). As a result of these initiatives, treatment with preventive chemotherapy reached 670 million people in 2008, however the data related to leishmaniasis have not been updated (WHO, 2010).
Even though some efforts to reduce vector and mammalian reservoir populations have been successful, no vaccines have been developed for leishmaniasis as yet (CDC, 2010). In some cases, there is reduced responsiveness. Patients who used to respond effectively to drugs suddenly fail to respond or relapse.
Treatment of leishmaniasis is by the use of pentavalent antimonials. Sodium stibogluconate is mostly used in several endemic regions for the treatment of all three types of leishmaniasis. However, it has a problem of drug resistance. Amphotericin B (Am.B), aminosidine (paromomycin, gabbromicina), pentamidine are used for all forms of leishmaniasis while miltefosine is an available treatment option for visceral leishmaniasis. The orphan drug Aminosidine, is mostly available in the United States while miltefosine is an approved first line drug in India.
Available drugs for the treatment of Leishmania are very expensive, present resistance, show less responsiveness with continuous use for treatment, and are highly cytotoxic to infected individuals throughout endemic regions. There are currently four to six available drugs for the treatment of leishmaniasis, but they are all toxic, expensive and most often, are not effective. Oftentimes the drugs are simply ineffective.
Even during treatment, infected individuals are required to take some time off from work to complete treatment due to toxic effects of current drugs and this is a big obstacle to economic growth.
Am. B in combination with miltefosine has resulted in greater than 90% cure rates of visceral leishmaniasis in north India. However, the major problem is that of toxicity, high cost, resistance, primary unresponsiveness, and lower sensitivity still exist.
Therefore, identification of alternative candidate compounds with anti-Leishmanial activities is of utmost urgency, and in particular there is a critical need to develop new therapeutic agents that have low cytotoxicity but high effectiveness against Leishmania parasites.
In its broadest aspect, a method for treating a mammalian patient at risk or suffering from a disease caused by a kinetoplastid parasite comprises administering to such subject an effective kinetoplasticidal amount of an imido-substituted 1,4-naphthoquinone to inhibit the kinetoplastid.
In an important aspect, a method of inhibiting Leishmania comprises administering to a patient for prophylaxis or to a patient in need of treatment an anti-Leishmanial effective amount of an imido-substituted 1,4-naphthoquinone.
In another important aspect, a method of inhibiting Leishmania comprises administering to a patient for prophylaxis or to a patient in need of treatment an anti-Leishmanial effective amount of an imido-substituted 1,4-naphthoquinone represented by the general formula:
wherein X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy; and Q represents the imido-substituent bonded to the 1,4-napthoquinone moiety through the imido nitrogen.
In an aspect of the method, in the general formula X is bromo, chloro, fluoro or iodo.
In an aspect of the method, in the general formula, X is bromo or chloro.
In an aspect of the method, Q is represented by:
wherein in Q each R is, independently, a substituted or unsubstituted hydrocarbon, provided that one R can, optionally, be hydrogen, and provided that, optionally, R can include at least one hetero atom.
In an aspect of the method, Q is represented by:
wherein in Q each R is, independently, cyclic or acyclic, substituted or unsubstituted, or the R groups bond together to form a cyclic imido-substituent.
In an aspect of the method, when Q is represented by:
wherein each R is independent of the other, and
In an aspect of the method, Q is an aryl-imido substituent.
In an aspect of the method, the imido-substituted 1,4-naphthoquinone in the general formula is represented by:
wherein each R is, independently, an optionally halo-substituted straight or branched C1 to C10 alkyl, preferably a C1 to C6 alkyl. X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy.
In an aspect of the method, Q in the general formula is represented by:
wherein the symbol ( ) designates —(CH2)— and n is 1 to 3. Preferably n is 1 or 2.
In an aspect of the method, Q is represented by:
wherein the aryl ring may, optionally, be substituted, such as substituted with halogen.
In an aspect of the method, an imido-substituted 1,4-naphthoquinone is represented by:
wherein X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy, to mention examples; each Y, independently, represents hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl or alkyl, and each m, independent of the other, is 0, 1, 2, 3, 4 or 5.
In an aspect of the method, on one of the aryl rings each Y can be hydrogen.
In an aspect of the method, when each m is 0, an imido-substituted 1,4-naphthoquinone is represented by:
wherein each Y is hydrogen.
In an aspect of the method, an imido-substituted 1,4-naphthoquinone is represented by:
wherein X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy, and each Y, independently, is alkoxy, aryloxy or halogen, and each m is 1. Y is ortho, meta or para-substituted. In another aspect, Y is meta-substituted. In a further aspect of this method, one Y can be hydrogen.
In an aspect of the method, an imido-substituted 1,4-naphthoquinone is represented by:
wherein X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy and each Y, independently, is halogen. X and Y are independent of each other. In a further aspect, Y is ortho, meta or para-substituted. In another aspect, Y is meta-substituted. In a further aspect of this method, one Y can be hydrogen.
In an aspect of the method, an imido-substituted 1,4-naphthoquinone having in vitro toxicity against both promastigote and amastigote forms of Leishmania donovani parasites is administered to a patient in need of treatment.
In an aspect of the method, an imido-substituted 1,4-naphthoquinone having a selectivity index against both promastigote and amastigote forms of Leishmania donovani parasites is administered to a patient in need of treatment.
In an aspect of the method, an imido-substituted 1,4-naphthoquinone having an IC50 cytoxicity value less than that for Amphotericin B, is administered to a patient in need of treatment against Leishmania parasites.
In an aspect of the method, an anti-Leishmanial effective amount of a compound selected from the group consisting of IMDNQ2, IMDNQ3, IMDNQ4, IMDNQ5, IMD8 and IMNDQ15 is administered to a patient to inhibit Leishmania donovani.
In an aspect of the method, IMDNQ2, IMDNQ3, IMDNQ4, IMDNQ5, IMD8 and IMDNQ15 had IC50 values of 2.27 μM, 10.81 μM, 6.81 μM, 31.28 μM, 4.3 μM, and 0.05 μM in promastigotes and 5.83 μM, 4.10 μM, 1.19 μM, 4.67 μM, 2.07 μM, 18.85 μM in amastigotes, respectively.
In an aspect, IC50 values of IMDNQ2, IMDNQ3, IMDNQ4, IMDNQ5, IMD8 and IMNDQ15 in mice fibroblasts cells were much higher (78.75 μM, 24.83 μM, 168.1 μM, 4.7 μM, 14197.35 μM, and 2.94 μM, respectively) compared to that of Amphotericin B (known drug for treating leishmaniasis) which was 1.18 μM.
In an aspect, IMD7, IMD8, IMDNQ4, IMDNQ2, and IMDNQ3 exhibited very low cytotoxicity, with IC50 values being 1448.56 μM, 14197.35 μM, 168.1 μM, 78.75 μM, and 24.83 μM against mouse fibroblasts cells.
In an aspect, the method comprises treating a patient in need of treatment for leishmaniasis with a therapeutically effective amount of an active ingredient that is a compound represented by the general formula.
In an aspect, the method comprises treating a patient in need of treatment for leishmaniasis with a therapeutically effective amount of an active ingredient that is a compound selected from the group consisting of IMDNQ1, IMDNQ2, IMDNQ3, IMDNQ4, IMDNQ5, IMDNQ6, IMD7, IMD8, IMDNQ9, IMNDQ10, IMNDQ11. IMNDQ12, IMNDQ13, IMNDQ14 and IMNDQ15.
In an aspect, the method comprises prophylaxis against Leishmania genus by administering an effective anti-Leishmanial amount of a compound represented by the general formula to a patient.
In an aspect, the method comprises prophylaxis against Leishmania genus by administering an effective anti-Leishmanial amount of an active ingredient that is a compound selected from the group consisting of IMDNQ1, IMDNQ2, IMDNQ3, IMDNQ4, IMDNQ5, IMDNQ6, IMD7, IMD8, IMDNQ9, IMNDQ10, IMNDQ11. IMNDQ12, IMNDQ13, IMNDQ14 and IMNDQ15 or a derivative thereof.
In an aspect, the method comprises inhibiting tublin polymerization in a Leishmania parasite by administering an effective anti-tublin polymerization amount of an imido-substituted 1,4-naphthoquinione compound. In a further aspect the compound is represented by the general formula. In a further aspect the parasite is Leishmania donovani.
The methods described herein advantageously utilize imido-substituted 1,4-naphthoquinones as a novel class of anti-Leishmanial agents to inhibit proliferation of Leishmania parasites. The methods can provide prophylaxis or treatment for a vertebrate against a parasite in the Leishmania genus. The methods can provide treatment against the various stages of Leishmania parasite infections. Thus, administering an imido-substituted 1,4-naphthoquinone can provide prophylaxis or treatment to a patient against the proliferation of Leishmania parasites.
In particular, the method can provide treatment for a human against Leishmania disease.
Administering an imido-substituted 1,4-naphthoquinone to a patient in a stage of infection with Leishmania genus can treat against cutaneous, mucocutaneous, and visceral forms of the disease.
Administering an imido-substituted 1,4-naphthoquinone to a patient in a stage of infection with Leishmania donovani can treat against visceral leishmaniasis.
Administering an imido-substituted 1,4-naphthquinone to a patient in a stage of infection with Leishmania donovani can treat against promastigote and/or amastigote forms of Leishmania donovani parasites.
Administering an imido-substituted 1,4-naphthoquinone can be used to treat Leishmania infections where the parasites are susceptible in promastigote and/or amastigote forms of Leishmania donovani within the life cycle.
Administering for prophylaxis may help break the life cycle of leishmaniasis disease and reduce the patient's chances of becoming infected or infecting another through a vector.
Administering an imido-substituted 1,4-naphthoquinone to a patient can, in principle, lead to inhibiting proliferation of Leishmania, by directly affecting the parasite's life cycle. Once a vector ingests blood from the patient whose blood plasma contains a imido-substituted 1,4-naphthoquinone, further development of Leishmania parasite in the vector may be inhibited.
The imido-substituted 1,4-naphthoquinones include, for example, 2-imido 3-halo-1,4-naphthoquinones.
An aspect of the method is inhibiting proliferation of Leishmania in a patient in need of treatment by administering a cyclic-imido-substituted 1,4-naphthoquinone, to the patient.
Administering includes sublingual administration, oral administration, and, in principle, intravenous administration. A pharmaceutical composition can contain the active pharmaceutical ingredient and may additionally comprise a pharmaceutically acceptable vehicle or adjuvant. A pharmaceutical composition can be in the form of a solid pharmaceutical dosage form (tablet, caplet, capsule, or deliverable from an osmotic pump as examples) or syrup. Remington, The Science and Practice of Pharmacy, provides general information regarding pharmaceutical dosage forms.
An anti-Leishmanial effective amount of the imido-substituted 1,4 naphthoquinone refers to an amount effective in inhibiting proliferation of a parasite in the Leishmania genus and includes an leishmaniocidal amount against a parasite from the Leishmania genus.
A therapeutically effective amount means an amount of the imido 1,4-naphthoquinone that can provide a therapeutic benefit to a patient against leishmaniasis.
Patient includes human. A patient in need of treatment includes a human patient in need of treatment against Leishmania parasites. Thus, methods of treating a mammal other than human (veterinary treatments) against Leishmania parasites are also within the scope of our inventions, and in particular canines.
A method for inhibiting proliferation of Leishmania with imido-substituted 1,4-naphthoquinones, such as imido-substituted 3-halo 1,4-napthoquinones, can exhibit greater anti-Leishmanial efficacy against Leishmania than the presently clinically used standard drug, Amphotericin B. For example, compared to Amphotericin B (IC50=5.26 μM in promastigotes and 22.26 μM in amastigotes), some imido-naphthoquinone analogs (as examples) are significantly more potent against Leishmania, such as IC50 values ranging from IMDNQ4 having 1.19 μM in amastigotes and IMDNQ15 having 0.5 pM in promastigotes. Thus, in one of its aspects the method for inhibiting proliferation of Leishmania comprises administering an imido-substituted 1,4-naphthoquinone, such as an imido-substituted 3-halo 1,4-napthoquinone, having an acceptable IC50 value, preferably an IC50 value equal to or lower than Amphotericin B.
A method for inhibiting proliferation of Leishmania, and in particular Leishmania donovani with an imido-substituted 1,4-naphthoquinone, especially an imido-substituted 3-halo 1,4-napthoquinone, can exhibit greater selectivity against Leishmania than the presently clinically used Amphotericin B. Thus, in another of its aspects the method for inhibiting proliferation of Leishmania comprises administering an imido-substituted 1,4-naphthoquinone, such as an imido-substituted 3-halo 1,4-napthoquinone, having an acceptable selectivity index, preferably a selectivity index better than Amphotericin B.
A method for inhibiting proliferation of Leishmania with an imido-substituted 1,4-naphthoquinone, such as an imido-substituted 3-halo 1,4-napthoquinone, exhibiting better cyotoxicity characteristics than Amphotericin B. In vitro testing has demonstrated representative imido-naphthoquinone analogs were relatively non-cytotoxic to Balb/C 3T3 mouse fibroblast cell line with IC50 values of well over the value for Amphotericin B. For example, cytotoxicity study on Balb/C 3T3 mouse fibroblast cell line showed that IMDNQ4, IMD7, and IMD8 are far less cytotoxic than Amphotericin B (
In the synthesis of the imido-substituted 1,4-naphthoquiniones, compounds represented by the formula:
wherein X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy, are suitable starting materials that provide the 1,4-napthoquinone skeleton. For example, a 2-amino-3-halo-1,4-naphthoquinone is a suitable starting material for preparing imido-substituted 1,4, naphthoquiones having a 3-halo 1,4-naphthoquinone skeleton. The 2-amino-3-chloro-1,4-naphthoquinone is commercially available. It can also be facilely obtained from 2,3-dichloro-1,4-naphthoquinone and ammonia in a mixture of concentrated ammonium hydroxide and ethanol. A 2-amino-3-bromo-1,4-naphthoquinone starting material can be prepared by refluxing commercially available 2,3-dibromo-1,4-naphthoquinone with ammonia/ammonium hydroxide mixture in ethanol. A 2-amino-3-iodo-1,4-naphthoquinone starting material can be prepared as described in Perez et al., Synthesis of Iodinated Naphthoquinones Using Morpholine-Iodine Complex, Synthetic Communications, 34(18):3389-3397 (2004) (compound (14)), the complete disclosure of which is incorporated herein by reference. For imido-substituted 1,4, naphthoquiones having a 2-alkoxy 1,4-naphthoquinone skeleton, a 2-amino-3-alkoxy-1,4-naphthoquinone or 2-amino 3-aryloxy-1,4-naphthoquinone are representative classes of starting material. It will be appreciated that X can also be halo-alkyl, such as trifluoro methyl, or halo-alkoxy, such as trifluoromethoxy or a halo-alkyl, such as trifluoro methyl as an example.
The above-mentioned starting materials are suitable for reacting with a selected acid halide(s) to obtain the imido-substituted 1,4-naphthoquinone compound. Exemplary acid halides are shown in
In the following description, the imido substitutent may be shown as being ‘symmetrical’ for illustrative purposes and it should be understood that the imido substitutent can be mixed. For example, in a “mixed” imido compound useful in the present methods, the “R” groups in the imido substitutent can be the same or different, and each Y can be the same or different, in which case an “unsymmetrical” or mixed imido substituent is provided.
In the following description, various syntheses and compounds are shown in which X is chloro. It will be appreciated that X is not restricted to chloro. X can be a halogen other than chloro.
In an aspect of the method, Q is represented by:
wherein in Q each R is, independently, a substituted or unsubstituted hydrocarbon, provided that one R can, optionally, be hydrogen, and provided that, optionally, R can include at least one hetero atom.
A sub-class of imido-substituted 1,4-naphthoquinones includes those represented by the formula:
In general, each R is independently a cyclic or acyclic group. Each R includes acyclic, such as straight chain alkyl —(CH2)nCH3) or branched alkyl, or cyclic, such as cyclo alkyl, or aryl. The expression open-chain imide derivative connotes the case where R is straight or branched alkyl. In another aspect, R can include unsaturation, e.g, an alkenyl. R can be cyclo alkyl, which includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. By preference, cyclo alkyl is a C5 to C7 cyclo alkyl. The R groups may be bonded to each other to form an alkylene bridge, such as a divalent alkylene bridge, although it will be appreciated that the R groups can, together, comprise a polycyclic moiety.
Depending on the R group, the imido compounds can adapt an anti-conformation. For instance, when R is acyclic, the acyclic imido groups can adapt anti-orientation or are capable of some form of staggered orientation, whereas when R is cyclic, the imido groups tend to adapt the syn orientation.
Compounds represented by the foregoing formula can be synthesized by adapting the following representative reaction scheme:
wherein RCOCl is selected to provide the desired R group, and purification of the reaction product yields the intended compound(s) within the general formula for the imido-substituted 1,4-naphthoquinone. Although X is chloro in the example, it will be appreciated that by selecting the appropriate starting material, X can be, for instance, another substituent.
When R is a straight chain alkyl —(CH2)nCH3, n is generally 0 to 10, preferably 2 to 5, including methyl, ethyl, propyl, butyl, pentyl, and hexyl, such as
as examples. Longer R groups are possible. Although X is chloro in the example, it will be appreciated that by selecting the appropriate starting material, X can be, for instance, another substituent.
When R is branched, the branching can be along the chain or can be terminal branching. For terminal branching, an R group can be represented by —(CH2)nCH(CH3)2 as an example, where n is from 0 to 6, preferably from 1 to 4, such as
as an example. Although X is chloro in the example, it will be appreciated that by selecting the appropriate starting material, X can be, for instance, another substituent.
Within the foregoing sub-classes of the above imido-substituted 1,4-naphthoquinones are those in which the open chain imido derivatives have halogen substitution. In one aspect, in a halo-substituted alkylene derivative according to the general formula, the R groups have terminal halo-substitution. Suitable compounds can be synthesized as shown in the following representative exemplary reaction scheme:
An exemplary chloroacyl chloride reagent is shown for illustrative purposes. Other suitable reagents, such as another acyl dihalide can be selected so that the alkyl group has different halo-substitution, such as a terminally bromo-substituted alkyl group (such as by using bromoacetyl bromide). Mono-halogenation is illustrated but it will be appreciated that other multi-halogenated derivatives are included within the scope of the present methods. Other suitable acyl halides include 2-bromopropionyl chloride, 2-chloropropionyl chloride, 2,3-dibromopropionyl chloride, 2,3-dichloropropionyl chloride, bromoacetyl chloride, 3-bromopropionyl chloride, 4-chloropropionyl chloride, 4-bromopropionyl chloride, 4-bromobutryl chloride, 4-chlorobutryl chloride, 2,4-dibromobutryl chloride, 5-chlorovaleroyl chloride, 5-bromovaleroyl chloride, dichloroacetyl chloride, trichloroacetyl chloride, 6-chloroheanoyl chloride, and the like by examples. Although X is chloro in the example, it will be appreciated that by selecting the appropriate starting material, X can be, for instance, another substituent.
The R groups can also be bonded together to form an alkylene bridge —(CH2)n- in which case n is an integer of 1 to 3, preferably n is 2 or 3, so that Q represents a cyclic imido-substitutent (a nitrogen-containing ring having dione substitution) such as
to mention examples. The 3-cyclic-imido-substituted 2-halo 1,4-napthoquinone compounds can be synthesized by adapting the following representative reaction scheme:
wherein ( ) designates —(CH2)— and n is an integer of 1 to 3. 2-chloro-3-(N-succinimidyl)-1,4-naphthoquinone is obtained when n is 1. As shown, the succinimidyl derivative (IMDNQ1) has a surprisingly beneficial combination of properties. 2-chloro-3-(N-glutaimidyl)-1,4-napthoquinone is obtained when n is 2. Although X is shown as chloro in the exemplary formulas and in the representative synthesis, it will be appreciated that by selecting the appropriate starting material, X can be, for instance, another substitutent.
A further sub-class of 2-imido-substituted 1,4-naphthoquinones includes derivatives in which the 2-imido-substitution comprises a heterocyclic ring having dione substitution in which the additional hetero atom is preferably oxygen. For instance, the ring can be a five or six member ring with oxygen as an additional hetero atom. An exemplary derivative is a morpholine dione analog, such as IMDNQ14. Morpholine dione analogs can be synthesized as shown in the following exemplary reaction scheme:
X is not restricted to chloro. X can be another halogen, to mention examples. When X is halogen, the microwave treatment can vary in duration and intensity, as seen from Berhe, S., et al., Microwave-assisted synthesis of imido-substituted 2-chloro-1,4-naphthoquinone derivatives and their cytotoxic activities on three human prostate cancer cell lines, Lett. Drug Des. Discov., 5, 485-488 (2008), but typically on lab scale synthesis the duration is on the order of minutes.
A sub-class of imido-substituted 1,4-naphthoquinones includes phthalimidyl derivatives. The compound IMDNQ12 is an example.
A sub-class of imido-substituted 1,4-naphthoquinones includes the cyclic imido-substituted derivatives, which include diarylimido-substituted derivatives. Diarylimido-substituted derivatives, which may be optionally substituted, include those represented by the formula:
wherein X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy, Y, independently, is hydrogen, halogen, alkoxy, alkyl, halo-alkyl, or halo-alkoxy and m is 0, 1, 2, 3, 4, or 5. When an m=0, Y is hydrogen. X and Y arc independent of each other.
Exemplary diarylimido derivatives having Y halogen substitution include those when m is 1 represented by the formula:
Examples include those compounds denoted herein as IMDNQ1 through IMDNQ6.
Mono-halogen-substituted diarylimido derivatives can be synthesized as shown in the following exemplary reaction scheme:
In the exemplary reaction scheme, X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy, and each Y, independently, is H, halogen, alkyl or alkoxy. Co-produced is a mixed imido compound having R hydrogen and the other R is an aryl-imido substituent. X includes bromo, chloro, fluoro, and iodo. Bromo, chloro and fluoro may be preferred when X is halogen. Y may be at the meta, ortho and/or para position of the aryl ring. Meta-halogen substitution on each aryl ring in the imido moiety may be preferred when Y is halogen. Y includes bromo, chloro, fluoro, and iodo. More particularly, Y can be bromo, chloro or fluoro. When Y is a halogen, Y may preferably be chloro or fluoro, with chloro being preferred. IMDNQ4 is a member of this sub-class of imido-substituted 1,4-naphthoquinones. Y can be alkyl, including branched alkyl, or alkoxy as shown in the Examples. When Y is hydrogen, benzoyl chloride can be used.
In general, for the compounds in which an arylimido group is (are) substituted with one or more Y substituent, a synthesis, such as a synthesis above or in the Examples, can be adapted and
may be used where m is 1, 2, 3, 4 or 5. Acyl halides include 3,5-bis(trifluoromethyl)benzoyl chloride, 2-bromobenzoyl chloride, 2-chlorobenzoyl chloride, 2-fluorobenzoyl chloride, 2-iodobenzoyl chloride, 2-methoxybenzoyl chloride, 2-ethoxybenzoyl chloride, 2-(trifluoromethoxy)benzoyl chloride, 2,4-difluorobenzoyl chloride, 2,6-difluorobenzyol chloride, 2,4-dichlorobenzoyl chloride, 2,6-dichlorobenzoyl chloride, O-acetylsalicyloyl chloride, 2-methoxybenzoyl chloride, 2,6-dimethoxybenzoyl chloride, 2-(trifluoromethyl)benzoyl chloride, 3-bromobenzoyl chloride, 3-chlorobenzoyl chloride, 3-fluorobenzoyl chloride, 3-iodobenzoyl chloride, 3,4-bromobenzoyl chloride, 3,4-di-chlorobenzoyl chloride, 3-methoxybenzoyl chloride, 3,4-dimethoxybenzoyl chloride, 3,4-dimethylbenzoyl chloride, 3,4-difluorobenzoyl chloride, 3,4,5-trimethoxybenzoyl chloride, 3-(trifluoro)benzoyl chloride, 3-(chloromethyl)benzoyl chloride, 4-bromobenzoyl chloride, 4-chlorobenzoyl chloride, 4-fluorobenzoyl chloride, 4-iodobenzoyl chloride, 4-methoxybenzoyl chloride, 4-ethoxybenzoyl chloride, 4-butoxybenzoyl chloride, 4-(hexyloxy)benzoyl chloride, 4-(heptyloxy)benzoyl chloride, 4-(trifluoromethyl)benzoyl chloride, 4-(tert-butyl)benzoyl chloride, 4-(trifluoromethoxy)benzoyl chloride, 4-ethoxybenzoyl chloride, 4-propylbenzoyl chloride, 4-butylbenzoyl chloride, 5-pentylbenzoyl chloride, 4-hexylbenzoyl chloride, 4-heptylbenzoyl chloride, 3,5-dichlorobenzoyl chloride, 2,3-dichlorobenzoyl chloride, 2,3-difluorobenzoyl chloride, 2,5-dichlorobenzoyl chloride, 2,5-difluorobenzoyl chloride, 3,5-dimethoxybenzoyl chloride, 2,4,6-trimethylbenzoyl chloride, 2,4,6,-trichlorobenzoyl chloride, 2,4,6,-trifluorobenzoyl chloride, 2,4,5-trifluorobenzoyl chloride, 2,3,4-trifluorobenzoyl chloride, 2,4-dimethoxybenzoyl chloride, 2,5-dimethoxybenzoyl chloride, 2-fluoro-3-(trifluoromethyl)benzoyl chloride, 2-fluoro-4-(trifluoromethyl)benzoyl chloride, 2-fluoro-5-(trifluoromethyl)benzoyl chloride, 3-fluoro-5-(trifluoromethyl)benzoyl chloride, 4-fluoro-2-(trifluoromethyl)benzoyl chloride, 4-fluoro-3-(trifluoromethyl)benzoyl chloride, 5-fluoro-2-(trifluoromethyl)benzoyl chloride, 2-fluoro-6-(trifluoromethyl)benzoyl chloride, 2,4-bis(trifluoromethyl)benzoyl chloride, 2,6-bis(trifluoromethyl)benzoyl chloride, 3-(tri fluoromethoxy)benzoyl chloride, 2,3,4,5-fluorobenzoyl chloride, 2,4-dichloro-5-fluorobenzoyl chloride, 3-(dichloromethyl)benzoyl chloride, 2,3,5-trifluorobenzoyl chloride, 3,4,5-trifluorobenzoyl chloride, 2-chloro-6-fluorobenzoyl chloride, 3-chloro-4-fluorobenzoyl chloride, 4-chloro-2,5-difluorobenzoyl chloride, 5-fluoro-2-methylbenzoyl chloride, 3-fluoro-4-methylbenzoyl chloride, 2,6-difluoro-3-methylbenzoyl chloride, 3-chloro-2-6-(trifluoromethyl)benzoyl chloride, 5-chloro-2-(trifluoromethyl)benzoyl chloride, and 2-chloro-6-fluoro-3-methylbenzoyl chloride, 6-chloro-2fluoro-3-methylbenzoyl chloride, 2-chloro-5-fluorobenzoyl chloride, 4-fluoro-3-methyl chloride, 5-chloro-2-fluorobenzoyl chloride, 2-chloro-3,6-fluorobenzoyl chloride, 3-chloro-2,4-fluorobenzoyl chloride, 3-chloro-2-fluoro-5(trifluoromethyl)benzoyl chloride, 4-methoxy-3-(trifluromethyl)benzoyl chloride, 4-methyl3-(trifluoromethyl)benzoyl chloride, 2-chloro-5-(trifluoromethyl)benzoyl chloride, 2,3-difluoro-4-methylbenzoyl chloride, 3,5-dichloro-4-methoxybenzoyl chloride, 2,4,5-trifluoro-3-methoxybenzoyl chloride, 2,3,4,6-tetrafluorobenzoyl chloride, 5-bromo-2,3,4-trimethylbenzoyl chloride, 4-bromo-2,6-difluorobenzoyl chloride, 2-fluoro-5-iodobenzoyl chloride, 2-fluoro-6-iodobenzoyl chloride, 4-bromo-2-fluorobenzoyl chloride, and 2-bromo-6-chlorobenzoyl chloride by way of example.
Other imido-substituted 1,4-naphthoquinones with different Q moieties can be obtained with other acid halides, including those disclosed in
It will be appreciated that when an R group is aryl or aryloxy or cyclo alkyl (which includes polycyclic alkyl), there may be an intervening linking group (sometimes called a spacer group) between the aryl or aryloxy or cyclo alkyl group to the imido-functional group. An exemplary such linking group would be an alkylene group, as an example.
In each of the various aspects of the present inventions, a Y substituent can be substituted alkyl, such as halogen-substituted alkyl, including trifluoro methyl, or substituted alkoxy, such as halogen-substituted alkoxy, including trifluoromethoxy, to mention examples.
The imido-substituted 1,4-naphthoquinone compound can be symmetrical or mixed, such as shown in the Examples. An exemplary reaction scheme for preparing a sub-class of imido-substituted 1,4-naphthoquinones having mixed Y group(s) can be represented as follows:
It will be appreciated that an unsymmetrical imido substituent, e.g., “mixed” as to an aryl ring(s), the Y substituent(s), and/or in the position(s) of a Y substituent(s) may be achieved by selecting a desired member from the class of acid chlorides from the class of benzoyl chlorides for the first step, and a different member for the second step. It will also be appreciated that a “mixed” imido-substituted 1,4-naphthoquinone is obtained in the first step wherein, for instance, the imido-nitrogen is bonded to hydrogen (one of the R groups) and the other R is substituted aryl. Other “mixed” compounds are obtained by adapting an appropriate synthesis and using an appropriate acid halide, which includes the exemplary acid chlorides in
Another sub-class of imido-substituted 1,4 includes naphthoquinones unsymmetrical alkyl aryl imido-substituted naphthoquinones which can be synthesized by adapting the following representative reaction scheme. An aminonaphthoquinone analog is first converted to the alkyl amido derivative which is subsequently reacted with an aryl acid chloride in the presence of an alkalin hydride, such as sodium hydride, in anhydrous THF to furnish the unsymmetrical alkyl aryl imidonaphthoquinone derivative.
An R group can be alkyl, such as described elsewhere herein, which includes C1-C6 alkyl. The other R group can be an aromatic group, such as an aryl group, such as described elsewhere herein. X can be a substitutent as described elsewhere herein, which includes, for example, hydrogen, halogen, alkyl, alkoxy (such as lower alkoxy, methoxy or the like).
A 1,4-naphthoquinone starting material as shown in various reaction schemes herein is chloro substituted (X=chloro) only for illustrative purposes. It will be appreciated that the 1,4-naphthoquinone starting material can have a 3-substitution so that X in the general formula and in the various formulas can be other than chloro.
A difference in anti-Leishmanial activity against Leishmania donovani promastigotes vs. amastigotes is observed. The differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates.
The ratio between the toxic dose and the therapeutic dose of a drug is a selectively index. It is used as a measure of the relative safety of the drug for a particular treatment. The selectivity index (SI) herein is the ratio of IC50 for fibroblast cells/IC50 for parasites and was calculated to compare the toxicity for mammalian cells and the activity against Leishmania donovani. The presently prescribed Amphotericin B has a selectivity index of 0.05 in amastigotes and 0.22 in promastigotes. In one aspect of the method, the selectivity index of all compounds used except IMDNQ5 (in promastigotes) is greater than the selectivity index for Amphotericin B in promastigotes and amastigotes. Further, in an aspect of the invention, some representative compounds are relatively non-cytotoxic to Balb/C 3T3 mouse fibroblast cell line with IC50 values of well below the value compared to Amphotericin B.
The in vitro testing shows the present method should have in vivo efficacy in inhibiting proliferation of Leishmania donovani, and thus indicating a disease caused by the Leishmania genus may be treated by administering a compound according to the general formula.
Inhibitory concentration is typically evaluated at the 50% inhibitory concentration (IC50). Inhibition of proliferation may be attained at a lower concentration in practice, but an IC50 concentration may be desirable.
Representative imido-substituted 1,4-napthoquinone compounds, and their synthesis, are described in Bakare, O., et al, Synthesis and MEK1 inhibitory activities of imido-substituted 3-chloro-1,4-naphthoquinones. Bioorg. Med. Chem., 11, 3165-3170 (2003); Berhe, S., et al., Microwave-assisted synthesis of imido-substituted 3-chloro-1,4-naphthoquinone derivatives and their cytotoxic activities on three human prostate cancer cell lines, Lett. Drug Des. Discov., 5, 485-488 (2008); Akinboye et al., Acta Cryst. E65, o24 (2009), and Akinboye et al., Acta Cryst. E65, o277 (2009), the complete disclosures of which are incorporated herein by reference.
A patient in need of treatment may be diagnosed by testing and by physical examination. Testing includes serological tests, immunoassays and PCR methods to diagnose for the presence of Leishmania parasites infection in an individual. The testing is sometimes performed in tandem. Serological testing of blood samples from an individual can yield negative and positive sero results. A so-called sero-positive result is indicative of infection. So-called sero-negative results may or may not indicate the absence of infection. The primary limitation of this technique revolves around interpretation of a positive titer which may only indicate exposure to the parasite as opposed to active infection. However, due to the disease progression more than a single test with a single sero-negative result is preferred. PCR methods can be used in determining a patient in need of treatment. The most reliable diagnostic test relies on demonstration of Leishmania parasites either cytologically or histopathologically, in stained preparations of bone marrow, lymph node, spleen, skin or other tissues and organs (skeletal muscle, peripheral nerves, renal interstitium, and synovial membranes. Leishmania parasites most commonly reside in macrophages, but have been observed in other cell lines including neutrophils, eosinophilis, endothelial cells and fibroblasts. While microscopic visualization of parasites provide a definitive diagnosis, this technique may be only 60% effective for bone marrow samples and 30% effective for lymph node specimens, making it less sensitive than other testing strategies.
Diagnosis of a patient in the acute phase of leishmaniasis disease who is in need of treatment may include physical examination. The acute stage may extend for a few weeks or months following initial infection. Many symptoms may not be unique to leishmaniasis disease.
The methods described herein advantageously utilize an active ingredient such as 2-imido-substituted 3-halo-1,4-naphthoquinones, as a novel class of selective anti-Leishmanial agents effective against Leishmania parasites.
The expression imido-substituted 1,4-naphthoquinone includes a compound according to the general formula.
The complete disclosure of each reference cited herein is incorporated by reference.
Those skilled in the art will recognize that modifications and variations may be made without departing from the true spirit and scope of the invention. The invention, therefore, is not to be limited to the embodiments described and illustrated in the following non-limiting examples but is to be determined from the appended claims.
The following non-limiting Examples illustrate the invention without limiting its scope.
In the Examples, reactions were carried out using laboratory grade materials and solvents. Melting points were determined in open capillary tubes on a Mel-Temp melting point apparatus and are uncorrected. The IR spectra were recorded on a Perkin Elmer PE 100 spectrometer with an Attenuated Total Reflectance (ATR) window. The and 13C-NMR spectra were obtained on a Bruker Advance 400 MHz spectrometer in deuterated chloroform (CDCl3). Chemical shifts are in δ units (ppm) with TMS (0.00 ppm) or CHCl3 (7.26 ppm), as internal standard for 1H-NMR, and CDCl3 (77.00 ppm) for 13C-NMR. Electrospray ionization mass spectrometry was recorded on a Thermo LTQ Orbitrap XL mass spectrometer and compounds dissolved in acetonitrile with 0.1% formic acid. The known intermediates were prepared according to procedures that are reported in the literature.
Exemplary imido-substituted naphthoquinone compounds IMDNQ 1 through IMDNQ15 were prepared in the Examples.
A general procedure for the synthesis of aryl-imido-substituted 1,4-naphthoquinones represented by the formula
wherein X is hydrogen, halogen, alkoxy (cyclic or alicyclic), trifluoro methyl, aryloxy, or benzyloxy and Y is halogen is exemplified with respect to IMDNQ1-IMDNQ6, respectively in Examples 1 through 6. 2-Amino-3-chloro-1,4-naphthoquinone (1.47 mmol) or the 3-bromo-analog was dissolved in THF (15 mL). NaH (3.08 mmol) was added and the mixture stirred at room temperature for 15 min. Appropriate acid chloride (3.08 mmol) was added drop wise, and the resulting mixture stirred at room temperature for 24 hours. The THF was then evaporated under vacuum and ice-cooled water added to the residual mixture. The resulting aqueous mixture was extracted with CH2Cl2 (2×30 mL) and the combined organic phase washed with water (3×15 mL), saturated NaCl solution (15 mL) and dried over anhydrous MgSO4. The crude product was purified by triturating in hot ethanol followed by recrystallization in ethyl acetate and/or column chromatography on silica gel.
Yellow solid. (27%). Mp 212-213° C. IR (cm−1) 1737.38, 1714.75, 1696.91, 1673.62, 1589.20, 1571.10. 1H NMR (CDCl3) 7.33-7.36 (m, 4H), 7.68-7.72 (m, 4H), 7.79-7.85 (m, 2H), 8 09-8.11 (m, 1H), 8.20-8.22 (m, 1H). 13C NMR (CDCl3) 127.72, 127.80, 129.18, 130.34, 130.50, 131.32, 132.52, 134.92, 134.95, 139.67, 142.46, 143.87, 170.40, 177.05, 178.59. ESI MS m/z 505.975 ([M+Na]+ calcd 505.973).
Yellow solid. (49%). Mp 217-218° C. IR (cm−1) 1720.19, 1681.22, 1618.00, 1588.02. 1H NMR (CDCl3) 7.10-7.16 (m, 2H), 7.21-7.31 (m, 4H), 7.79-7.89 (m, 4H), 8.17-8.20 (m, 1H), 8.20-8.26 (m, 1H). 13C NMR (CDCl3) 126.76, 126.93, 127.60, 127.80, 129.69, 130.54, 130.85, 131.37, 132.36, 132.52, 132.56, 132.75, 134.14, 134.75, 134.91, 142.65, 144.43, 177.13, 178.22. ESI MS m/z 505.9741 ([M+Na]+ calcd 505.9730).
Yellow solid. (56%). Mp 284-286° C. IR (cm−1) 3074.94, 1719.61, 1689.97, 1672.75, 1591.10. 1H NMR (CDCl3) 7.00-7.06 (m, 1H), 7.75-7.85 (m, 1H), 8.10-8.12 (m, 6H), 8.20-8.22 (m, 4H). 13C NMR (CDCl3) 115.97, 116.19, 116.41, 126.65, 126.96, 127.67, 127.77, 130.53, 130.57, 130.58, 131.35, 131.62, 131.71, 132.75, 134.88, 142.42, 144.13, 164.14, 166.68, 170.33, 177.13, 178.63. ESI MS m/z 474.034 ([M+Na]+ calcd 474.032).
Yellow solid. (31%). Mp 258-260° C. IR (cm−1) 3075.36, 1713.64, 1698.11, 1672.50, 1591.48, 1571.31. 1H NMR (CDCl3) 7.27-7.31 (t, J=7.85 Hz, 2H), 7.42-7.45 (ddd, J=1.03, 2.09, 8.07 Hz, 2H), 7.60-7.63 (td, J=1.07, 7.68 Hz, 2H), 7.70-7.71 (t, J=1.82, 2H), 7.80-7.85 (m, 2H), 8.11-8.14 (m, 1H), 8.21-8.23 (m, 1H). 13C NMR (CDCl3) 127.02, 127.93, 128.03, 129.34, 130.21, 130.75, 131.55, 133.29, 135.14, 135.21, 136.06, 143.00, 143.84, 170.20, 177.25, 178.66. ESI MS m/z 505.9741 ([M+Na]+ calcd 505.9730).
Yellow crystal (34%). Mp 232-234° C. IR (cm−1) 1728.78, 1688.76, 1671.10, 1588.22, 1468.70. 1H NMR (CDCl3) 7.13 (2H, J=7.9 Hz), 7.21 (2H, J=1.6, 7.9 Hz), 7.28 (dd, 2H, J=1.3, 7.5 Hz), 7.78-7.86 (m, 2H), 7.94 (d, 2H, 5.2 Hz), 8.15-8.21 (m, 1H), 8.22-8.28 (m, 2H). 13C NMR (CDCl3) 126.65, 127.66, 128.08, 129.63, 130.49, 130.81, 131.23, 132.25, 134.68, 134.80, 140.78, 145.81, 167.57, 177.26, 177.81. ESI MS m/z 549.9240 ([M+Na]+ calcd 549.9224).
Yellow solid (66%). Mp: 170-172° C. IR (cm−1) 1719.23, 1670.56, 1596.69, 1505.57. 1H NMR (CDCl3) 7.05 (t, 4H, J=12.0 Hz), 7.77-7.88 (m, 6H), 8.08-8.15 (m, 1H), 8.21-8.27 (m, 1H). 13C NMR (CDCl3) 115.57, 115.79, 127.4, 127.71, 130.18, 130.30, 130.33, 130.85, 131.35, 131.44, 134.46, 134.50, 138.52, 146.90, 165.03, 169.91, 176.96, 177.97. ESI MS m/z 517.9785 ([M+Na]+ calcd 517.9815).
2-Amino-3-chloro-1,4-naphthoquinone (IMD7) was prepared by refluxing commercially available 2,3-dichloro-1,4-naphthoquinone with ammonia/ammonium hydroxide mixture in ethanol.
2-Amino-3-bromo-1,4-naphthoquinone (IMD8) was prepared by refluxing commercially available 2,3-dibromo-1,4-naphthoquinone with ammonia/ammonium hydroxide mixture in ethanol.
2-amino-3-chloro-1,4-naphthoquinone was dissolved in THF (15 mL). NaH was added and the mixture was stirred at room temperature for 15 mins. The 4-methoxybenzoyl chloride was added, drop wise, and the mixture was stirred for 24 hours. (Mole ratio of Substrate:NaH:Acid Chloride (1:2.3:2.3)) THF was evaporated under vacuum and the mixture was washed with ice-water (10 g ice and 10 mL water). The ice-water mixture was extracted with CH2Cl2 (30 mL, 20 mL consecutively) and the combined organic phase washed with water (3×20 mL), saturated NaCl solution (3×20 mL), then dried over anhydrous MgSO4. The crude was purified via triturating in hot ethanol, recrystallization in ethyl acetate and/or via column chromatography.
Obtain a yellow solid. (47.9%). Mp 283-287° C. IR (cm−1) 3019.56, 1698.7, 1668.81, 1599.28, 1574.26, 1508.65. 1H NMR (CDCl3). 3.81 (s, 6H), 6.80-6.84 (td, J=2.86, 8.95 Hz, 4H), 7.73-7.82 (m, 6H), 8.09-8.11 (m, 1H), 8.19-8.21 (m, 1H). 13C NMR (CDCl3) 55.48, 113.98, 114.21, 126.76, 127.57, 127.64, 130.38, 130.79, 131.41, 131.48, 134.61, 134.66, 141.69, 144.95, 163.34, 171.03, 177.48, 178.77.
IMDNQ10 derivative was synthesized from 2-amino-3-chloro-1,4-naphthoquinone and the appropriate acid chloride in accordance with Bakare, O., et al, Synthesis and MEK1 inhibitory activities of imido-substituted 2-chloro-1,4-naphthoquinones. Bioorg. Med. Chem., 11, 3165-3170 (2003); and Berhe, S., et al., Microwave-assisted synthesis of imido-substituted 2-chloro-1,4-naphthoquinone derivatives and their cytotoxic activities on three human prostate cancer cell lines, Lett. Drug Des. Discov., 5, 485-488 (2008).
2-amino-3-chloro-1,4-naphthoquinone was dissolved in THF (15 mL). NaH was added and the mixture was stirred at room temperature for 15 mins. The 3,4,5-(trimethoxy)benzoyl chloride was added, drop wise, and the mixture was stirred for 24 hours. (Mole ratio of Substrate:NaH:Acid Chloride (1:2.3:2.3)) THF was evaporated under vacuum and the mixture was washed with ice-water (10 g ice and 10 mL water). The ice-water mixture was extracted with CH2Cl2 (30 mL, 20 mL consecutively) and the combined organic phase washed with water (3×20 mL), saturated NaCl solution (3×20 mL), then dried over anhydrous MgSO4. The crude was purified via triturating in hot ethanol, recrystallization in ethyl acetate and/or via column chromatography. 2-bis-(3,4,5-trimethoxybenzoyl)amino-3-chloro-1,4-naphthoquinone: (IMDNQ 11)
Obtain orange crystals (51.6%). Mp 171-172° C. IR (cm−1) 3015.37, 2939.81, 2838.40, 1704.21, 1675.26, 1586.02, 1122.75. 1H NMR (CDCl3) 3.81 (s, 12H), 3.84 (s, 6H), 7.02 (s, 4H), 7.81-7.84 (m, 2H), 8.12-8.14 (m, 1H), 8.22-8.24 (m, 1H). 13C NMR (CDCl3) 56.27, 56.35, 60.93, 127.62, 127.77, 129.31, 130.69, 131.39, 134.86, 142.06, 142.27, 144.45, 153.01, 171.08, 177.27, 178.84.
The phthalimidyl (IMDNQ12) derivative was synthesized from 2-amino-3-chloro-1,4-naphthoquinone and the appropriate acid chloride in accordance with Bakare, O., et al, Synthesis and MEK1 inhibitory activities of imido-substituted 2-chloro-1,4-naphthoquinones. Bioorg. Med. Chem., 11, 3165-3170 (2003); and Berhe, S., et al., Microwave-assisted synthesis of imido-substituted 2-chloro-1,4-naphthoquinone derivatives and their cytotoxic activities on three human prostate cancer cell lines, Lett. Drug Des. Discov., 5, 485-488 (2008).
The mono-butryl derivative (IMDNQ13) was synthesized from 2-amino-3-chloro-1,4-naphthoquinone and the appropriate acid chloride in accordance with Bakare, O., et al, Synthesis and MEK1 inhibitory activities of imido-substituted 2-chloro-1,4-naphthoquinones. Bioorg. Med. Chem., 11, 3165-3170 (2003); and Berhe, S., et al., Microwave-assisted synthesis of imido-substituted 2-chloro-1,4-naphthoquinone derivatives and their cytotoxic activities on three human prostate cancer cell lines, Lett. Drug Des. Discov., 5, 485-488 (2008).
The morpholine dione analog (IMDNQ14) was synthesized by microwave irradiation of a mixture of 2-amino-3-chloro-1,4-naphthoquinone and diglycolyl chloride as depicted in scheme 1 in Berhe, S., et al., Microwave-assisted synthesis of imido-substituted 2-chloro-1,4-naphthoquinone derivatives and their cytotoxic activities on three human prostate cancer cell lines, Lett. Drug Des. Discov., 5, 485-488 (2008).
The dibutryl (IMDNQ15) derivative was synthesized from 2-amino-3-chloro-1,4-naphthoquinone and the appropriate acid chloride in accordance with Bakare, O., et al, Synthesis and MEK1 inhibitory activities of imido-substituted 2-chloro-1,4-naphthoquinones. Bioorg. Med. Chem., 11, 3165-3170 (2003); and Berhe, S., et al., Microwave-assisted synthesis of imido-substituted 2-chloro-1,4-naphthoquinone derivatives and their cytotoxic activities on three human prostate cancer cell lines, Lett. Drug Des. Discov., 5, 485-488 (2008).
Activity and Potency of 15 Compounds Against Leishmania donovani Parasites
The activity and/or potency of naphthoquinione compounds (
The 15 compounds were screened using both promastigote and amastigote forms of Leishmania donovani parasites. A cut off of at least 50% growth inhibition in the screening was an initial screening criteria to make the evaluation more facile. Six active compounds met this initial screening criteria. All 15 compounds were screened using the Resazurin assay. Low levels of cellular toxicity and selectivity indices of a compound were factors in screening that led to the six compounds. IC50 values were different in promastigotes and amastigotes but had a similar pattern in most of the screened compounds. Amphotericin B had an IC50 value of 5.26 μM in promastigotes and 22.26 μM in amastigotes, while IMDNQ2, IMDNQ3, IMDNQ4, IMDNQ5, IMD8 and IMDNQ15 had IC50 values of 2.27 μM, 10.81 μM, 6.81 μM, 31.28 μM, 4.3 μM, and 0.05 μM in promastigotes and 5.83 μM, 4.10 μM, 1.19 μM, 4.67 μM, 2.07 μM, 18.85 μM in amastigotes, respectively. The six screened compounds had very low cytotoxicity in mice fibroblast cells compared with the standard drug, Amphotericin B. The IC50 values of these compounds in mice fibroblasts cells were much higher (78.75 μM, 24.83 μM, 168.1 μM, 4.7 μM, 14197.35 μM, and 2.94 μM) compared to that of Amphotericin B which was 1.18 μM. Selected candidate compounds have demonstrated high leishmanicidal activities on either promastigotes, amastigotes or on both forms of the parasites and have proven to have acceptable toxicity at limited dose.
The naphthoquinone compounds used in the current disclosure have limited cytotoxicity levels to human cancer cell lines and confirmed in mice fibroblast cells. Fibroblast cells have been documented as natural host cells in Latent leishmaniasis (Bogdan ct al. J. Expermental Medicine Vol 191 (12), pp 2121-2130, 2000).
the Present Naphthoquinone Compounds have Low Cytotoxicity on Mice Fibroblast Cells
Fibroblasts cells (4×104 cells/nil and adding 100 μl per well) were incubated with different concentrations of the naphthoquinone compounds (
In vitro anti-Leishmanial activities of the compounds (
Naphthoquinone compounds (
The exposure to the compounds in relation to time was done to be able to evaluate the effect of time on each of the compounds. Exposure of promastigotes to the compounds was done for 2 hrs, 4 hrs, 6 hrs, and 24 hrs. Exposure time that had a significant effect was at 24 hrs of exposure.
Table 1 below indicates the tabulated anti-Leishmanial activities in promastigotes and amastigotes of Leishmania donovani, cytotoxicities in mice fibroblast cells, and the selectivity indices of the listed compounds.
indicates data missing or illegible when filed
Leishmania donovani isolated from human, Khartum, Sudan (ATCC#30030) were purchased from the American Type Culture Collection (ATCC). This clone was isolated in 1959. Balb/C 3T3 mouse fibroblasts used were the NIH 3T3 mouse embryonic fibroblast cells isolated from NIH Swiss mouse embryos and initiated in 1962 by G. Todaro and H. Green (Kuwahara et al., 1971; Todaro and Green, 1963).
Culturing Leishmania donovani Promastigotes
Approximately 5×105 promastigotes/ml of L. donovani Khartum strain were inoculated into Yeager's Liver infusion tryptose (YLIT) medium or Brain Heart Infusion (BHI) broth (Difco), supplemented with glucose (2.5 mg/ml), 50 U/ml Pen-Strep and 0.005 mM Hemin at 25-26° C. and subcultured bi-weekly, Mikus et al., Parasitol Int., 48(3):265-9 (2000) (Mikus and Steverding, 2000).
Culturing Leishmania donovani Amastigotes
Amastigotes were grown in MAA/20 medium (Amastigote medium consisted of: M199 medium supplemented with 0.5% Tryptone broth, 0.01 mM bathocuproinedisulfonic acid, 0.108 mM L-Cysteine, 15 mM D-glucose, 0.685 mM L-glutamine, and 0.025 mM Hemin and 20% fetal calf serum (FCS) at a pH of 5.5). Amastigotes were maintained at 37° C. in the presence of 5% CO2 and passaged bi-weekly (Mikus and Steverding, 2000).
NIH 3T3 mouse fibroblasts were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% Fetal calf serum, 50 units/ml penicillin, 50 units/ml streptomycin B in a humidified incubator at 37° C. in the presence of 5% CO2 (Scala et al., 2010).
L. donovani
Parasites were collected by centrifugation for 10 minutes at 800×g, supernatant discarded, and parasites resuspended in 1× phosphate-buffered saline (PBS). The pelleted parasites were then resuspended in fresh medium and counted using a hemocytometer, Sen et al., Cell Death Diff., 11:934-936 (Sen et al., 2004a).
Medium from the culturing T-75 flask was discarded, washed with 1×PBS 2× to remove medium completely. 3 ml of 1× Trypsin-EDTA was added to flask making sure that all the cells were covered with the Trypsin and the flask was incubated at 37° C. for ˜5 minutes. When more than 90% of the cells are detached, the trypsinization reaction was neutralized by the addition of 5 ml fresh culturing medium (Scala et al., 2010).
Antileishmanial Activity on L. donovani Promastigotes
The Leishmanicidal properties of fifteen naphthoquinone compounds (
Antileishmanial Activity on L. donovani Amastigotes
Amastigotes were plated at a concentration of 2×105 cells/2000 μl 24 hrs before treatment with various compounds. Amastigotes were treated with dilutions of compounds in 96-well microtitre plates. Dilutions of compounds ranging from 0.03 μM to 44 μM were prepared and added to amastigotes. Amastigotes were exposed to compounds for 48 hrs and were incubated at 37° C. During the period of exposure to compounds, plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. After the incubation period, 20 μl Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilled water) (Mikes and Steverding, 2000) were added to each well and plates were incubated for another 2 hrs. The plates were read with a microplate reader using a wavelength of 570 nm. Decrease of fluorescence (inhibition) was expressed as percentage of the fluorescence of control cultures and was plotted against the concentrations of compounds. The IC50 values were calculated from the sigmoidal inhibition curves. Amphotericin B was used as a reference drug.
Assays were performed to assess the cytotoxicity of these compounds in 96-well microtiter plates, with each well containing 100 μl of RPMI medium supplemented with 10% bovine calf scrum with 50 U/ml Pen-Strep. The cells were plated at a concentration of 4×103 cells/well (4×104 cells/ml). Fibroblast cells were then incubated with compounds for 48 hrs and inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 μl of resazurin solution (12.5 mg resazurin dissolved in 100 nil distilled water) were added to each well and the plates were incubated for another 2 hrs. The plates were read with a microplate reader at 570 nm. Experiments were done at least 3 times in triplicates (Scala et al., 2010).
BALB/cAn NHsd (female, 10-11 weeks) mice were obtained from Charles Rivers Laboratories International Inc., Ballardvale, Wilmington, Mass., USA and weighed ˜20 g each at the time of infection. A standard mouse diet and regular clean tap water were supplied ad libitum. All animals were ‘specific pathogen free’. Experiments were conducted in accordance with Howard University's Institutional Animal Care and Use Committee (HU-IACUC) office approval.
Below are the compounds selected for use in this in vivo mice study.
Balb/c mice 10-11 weeks old were infected with L. donovani inoculated subcutaneously (SC) at the base of the tail (maximum 0.2 ml) with 1×106 freshly harvested promastigotes. These represented a VL model which has been described previously (Yardley and Croft, 1999). After infection, mice were marked for individual identification and randomly allocated into groups of two. Seven days after infection, a mouse was sacrificed, and parasitic burden assessed based on microscopic enumeration of amastigotes (Leishman donovan bodies) against host cell nuclei on liver and spleen impression smears (Stauber et al., 1958). Blood was collected through cardiac puncture for use in all serum based assays.
Dosing began on the seventh day post-infection for 4 continuous days. As a positive control, one group did not receive any treatment. Dosing was administered subcutaneously at the base of the tail.
Table 2 shows four of the naphthoquinone compounds that were used in an in vivo study. It includes the different groups of mice and the concentrations of compounds used for the treatment of infections. A Representation of infections and treatments of BALB/c Mice is in Table 2. All mice were infected with 106 log phase promastigotes in PBS. In each group with different concentrations, there were two mice (n=2). Four compounds (IMDNQ4, IMD8, IMDNQ10, and IMDNQ15) were used for the treatment of infected mice including the reference drug Amphotericin B. Two sets of controls were used. The positive controls that were infected but were not treated with any drug except PBS, and the negative controls that were not infected with the parasites, but injected with only PBS.
Mice were housed in cages kept in an animal room with controlled temperature and humidity. Mice were anesthetized each time doses were administered. Isofluoroethane was delivered in a precision vaporizer. Observation of mice for purposeful movements in cages after each dosing was done. Mice were euthanized during each blood and organ collection. Collected blood was spun after 1 hr. and the serum was stored in a −80° C. freezer for later testing.
Preparation of Antigens (L. donovani)
To make L. donovani crude antigens, L. donovani promastigotes and amastigotes were counted using a hemocytometer, spun at 800×g for 10 min, and washed three times with 1× phosphate buffered saline (PBS). After the final wash, parasites were resuspended in 1×PBS at a concentration of 1×108 promastigotes (amastigotes)/ml and lysed by 5 freeze and thaw cycles or by sonication at maximum speed for 5 cycles of 10 seconds with cooling, and frozen at −80° C. until needed for assay (Burns et al., 1993; Zijlstra et al., 1998). Protein concentration was determined by the BIORAD assay.
5 μg/ml of antigen in 100 mM carbonate-bicarbonate buffer pH 7.4, were added to the wells of a 96 well polyvinyl plate (100 μl/well) and incubated at room temperature for 1 hr or overnight at 4° C. Wells were emptied, blocking buffer was added (200 μl/well), and wells were incubated at room temperature for 1 hr. Wells were emptied and 50 or 100 μl/well of diluted samples added (5× mice serum), and incubated for 1.5-2 hrs. Plates were washed 4× and an anti-mouse-IgG was diluted 1:500, and added (100 μl/well); these plates were incubated for 30 min in the dark and secondary antibody tagged to HRP was added at a dilution of 1:500. TMB-HRP developer was added 100 μl/well for 30 min in the dark and plates were read at 630 nm on a spectrophotometer (Zijlstra et al., 1998).
The efficacy of compounds was assessed by microscopically determining the reduction in amastigotes burden Leishman donovan bodies (LDBs) within the liver and spleen. Impression smears were taken 14 days post infection (7 days after the start of treatment). Mice liver and spleen imprints were made by touching a freshly cut surface of the mice liver and spleen many times with a Poly-L-Lysine coated slide. The slides were air dried, fixed in “Diff Quick” fixative for 30 seconds, drained, stained with “Quick Diff” solution II for 30 seconds, drained, and then finally counterstaining with “Diff Quick” solution I for 30 seconds and then drained. Slides were rinsed in tap water to remove excess stain and rapidly dehydrated in absolute alcohol. Slides were examined by light microscopy, using ×1000 oil immersion. LDBs were determined by dividing the number of LDBs/number of cells nuclei x weight of organ in milligrams. This gave the number of “Leishman donovan units” (LDUs).
Serum was collected from infected, treated and non-treated mice 14 days post infection and 7 days from the start of treatment for the determination of alanine amino transferase (ALT) and aspartate amino transferase (AST) activities. Assays were done in 96 well microtiter plates and samples and standards were run in duplicates or triplicates. For ALT, 2 μl of serum from infected treated and untreated samples were incubated with 10 μl of ALT substrate solution (1.78 g DL-alanine and 30 mg α-keto glutarate in 20 ml of phosphate buffer containing 1.25 ml of 0.4 M NaOH. Volume made up to 100 ml with buffer, pH 7.4. 1 ml of chloroform as preservative. Stable for 2 months at 2-8° C.), mixed and incubated at 37° C. for 30 minutes. 10 μl of dinitrophenolhydrazine (2,4 DNPH) was added. Blanks (ddH2O) and negative (not infected) samples were then added. Samples were mixed and incubated at room temperature for 20 minutes. 100 μl of 0.4 M NaOH was added, mixed and incubated at room temperature for 5 min. Plates were read at 490 nm. For AST, 2 μl of test and positive (infected, treated and untreated) samples were incubated with 10 μl of AST substrate solution (2.66 g DL-aspartic acid and 30 mg α-keto glutarate in 20.5 ml of 1 M NaOH pH 7.4, volume made up to 100 ml with phosphate buffer. 1 ml of chloroform was added as preservative. This was stable for 2 months at 2-8° C.) and incubated at 37° C. for 1 hr. 10 μl of 2,4 DNPH added and mixed. 2 μl of negative controls (not infected) and blanks (ddH2O) added and mixed and then incubated at room temperature for 20 min. 100 μl of 0.4 M NaOH was added, mixed and incubated at room temperature for 5 min and read at 490 nm. The enzyme activity was determined from the standard curve drawn using sodium pyruvate as standard solution (2 mM/ml pyruvate). Enzyme activities were expressed as units/ml. Reference ranges by this method are: AST; 0.096-3.8 U/ml and ALT; 0.112-3.0 U/ml. (Mallick et al., 2003; Reitman and Frankel, 1957).
All experiments were done in duplicate or triplicates and three different reproducible experiments were considered. The means and standard errors (S.E) were determined. Data were analyzed by one way ANOVA and t-test for multiple comparisons using Microsoft 2010 excel and GraphPad PRISM software version 5.0. P<0.05 was considered significant.
In Vitro Antileishmanial Activities of Naphthoquinone Compounds Against L. donovani Promastigotes and Amastigotes
The inhibitory concentrations (IC50) of 15 naphthoquinone compounds (
The level of antileishmanial activities (promastigotes killing or viabilities) may be indicated by variation in IC50 values plotted in y-axis versus treatments (x-axis) with different concentrations of each tested compound ranging from 0.03 to 44 μM using Resazurin Assay. IC50 values were calculated and expressed as the means±S.E (standard error) of three different experiments. The IC50 value represents the concentration of tested naphthoquinone compound at which 50% promastigotes are killed after treatment.
In this test, the remaining 10 tested naphthoquinone compounds that showed lower anti-leishmanial activities against L. donovani promastigotes than Amphotericin B with IC50 value of 5.26 μM are as follows: (1) IMDNQ3 with IC50 value of 10.81±5.4 μM, (2) IMDNQ4 with IC50 value of 6.82±2.3 μM, (3) IMDNQ5 with IC50 value of 31.28±3.9 μM, (4) IMDNQ6 with IC50 value of 9.66±30.0 μM, (5) IMDNQ9 with IC50 value of 5.64±0.9 μM, (6) IMDNQ10 with IC50 value of 7.0±1.0 μM, (7) IMDNQ11 with IC50 value of 22.9±0.9 μM, (8) IMDNQ12 had an IC50 value of 9.80±3.7 μM, (9) IMDNQ13 with IC50 value of 5.37±0.001 μM, (10) IMDNQ11 with IC50 value of 11.85±0.001 μM.
In vitro antileishmanial activities of the fifteen naphthoquinone compounds (
The level of antileishmanial activities (amastigotes killing or viabilities) may be indicated by variation in IC50 values plotted in y-axis versus treatments (x-axis) with different concentrations of each tested compound ranging from 0.03 to 44 μM using Resazurin Assay. IC50 values were calculated and expressed as the means±S.E (standard error) of three different experiments. The IC50 value represents the concentration of tested naphthoquinone compound at which 50% amastigotes are killed after treatment.
Selectivity Indices of Naphthoquinone Compounds Against L donovani Promastigotes and Amastigotes.
The fifteen naphthoquinone compounds, which include imido-substituted 1,4-naphthoquinone compounds as seen from
Data in Table 3 were used to generate
Data from Table 4 were used to generate
L. donovani Promastigotes and Amastigotes.
Dose and Time-Dependent Inhibitory Effect of Naphthoquinone Compounds on L. donovani Promastigotes.
In order to determine the effect of the 15 naphthoquinone compounds (
Results of the 2-hour duration of observation are shown in
Results of the 4-hour duration of observation are shown in
Results of the 6-hour duration of observation are shown in
Results of the 24-hour duration of observation are shown in
The data in Table 5 summarize the observations shown in
The purpose of the test was to show the presence of amastigotes in the liver and spleen of infected BALB/c mice. BALB/c mice models of visceral leishmaniasis were generated by infecting mice with 106 log phase promastigotes in 200 μl 1×PBS. Infectivity was confirmed by making liver and spleen imprints of infected mice seven days post infection before the start of treatment. Slides were stained using the “Diff Quick” stain. Positive infectivity was confirmed by the presence of amastigotes in stained slides compared to that of the uninfected slides.
The purpose of the test was to confirm positive infectivity through levels of serum IgG in infected BALB/c mice. A high serum IgG level is predictive of an infection with visceral leishmaniasis. The results are expressed as percent increase or percent decrease in IgG levels. Four compounds with three different concentrations each were used for the tests. Amphotericin B had only one concentration.
Evaluation of Splenic and Liver Parasite Burden after Treatment with Selected Compounds
The purpose of the test was to evaluate splenic and liver parasite burden after treatment with selected compounds. This was done by making imprints and calculating the number of Leishman Donovan Units (LDUs) in cells. LDUs are calculated by counting the number of parasites per cell nuclei (number of amastigotes in cells÷number of cells) multiplied by the weight of the organ in milligrams. The lower the LDU value compared to that of untreated controls, the more effective the compound is at that concentration for the treatment of VL. Effectiveness of compounds in the treatment of VL is expressed as LDU numbers and as percent increases or percent decrease in LDUs. Percent increase in LDU means that the number of parasites increased compared to the untreated controls and percent decrease means that the number of parasites decreased compared to the untreated controls.
s A and B show suppressions in the liver parasite burden. All four compounds were effective in the treatment of VL. For IMDNQ4: 5 mg/kg had LDU value of 5823.8±3378, 31.79% reduction in parasite burden; 20 mg/kg had LDU value of 4266.0±6033, a 50.04% reduction in parasite burden; 50 mg/kg had LDU value of 1003.8±1420, an 88.24% reduction in parasite burden. For IMD8: 5 mg/kg had LDU value of 4752.5±293, a 56.66% reduction in parasite burden; 20 mg/kg had LDU value of 3099.9±2197, a 44.34% reduction in parasite burden; 50 mg/kg had LDU value of 1869.0±420 an 81.89% reduction in parasite burden. For IMDNQ 10: 5 mg/kg had LDU value of 2313.0±1521 a 72.91% reduction in parasite burden; 20 mg/kg had LDU value of 1794.0±2537, a 78.99% reduction in parasite burden; 50 mg/kg had an LDU value of 2478.0±229, a 70.98% reduction in parasite burden. For IMDNQ15: 5 mg/kg had LDU value of 2267.5±191, a 73.44% reduction in parasite burden; 20 mg/kg had LDU value of 2598.5±89, a 69.57% reduction in parasite burden; 50 mg/kg had LDU value of 5681.0±1785, a 45.92% reduction in parasite burden. Amphotericin B had LDU value of 5823.8 compared to 8538.5±2423 for the untreated control, a 33.47% reduction in parasite burden.
Table 6 is a summary of
In order to assess in vivo cytotoxicity of four naphthoquinone compounds (IMDNQ4, IMD8, IMDNQ 10, and IMDNQ 15), serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were analyzed and compared to those in untreated controls. The assay range for AST is 0.096-3.8 U/ml (mean assay range is 1.95 U/ml) and for ALT assay, 0.112-3.0 U/ml (mean assay range is 1.56 U/ml). The results are expressed as percent increase or decrease in AST or ALT levels.
Table 6 is a summary of
The data in Table 7 are as follows: for each of the three concentrations (5 mg/kg, 20 mg/kg, and 50 mg/kg) levels of serum AST and serum ALT in all test specimens; and percent change in the levels of serum AST and serum ALT in IMDNQ4, IMD8, IMDNQ10, IMDNQ15, and Amphotericin B, compared to the levels of serum AST and serum ALT in untreated controls. As shown in Table 8, a downward arrow indicates a decrease and an upward arrow indicates an increase in the levels of serum AST and serum ALT in the five test specimens compared the levels in untreated controls.
Compared to the untreated controls, for a 14.4% change in the level of serum AST in Amphotericin B, relative changes in the levels of AST in the four compounds were as follows: (1) IMDNQ4: 5 mg/kg had an 18.8% decrease, 20 mg/kg had an 18.2% decrease, and 50 mg/kg had a 28.6% decrease. (2) IMD8: 5 mg/kg had a 40.9% decrease, 20 mg/kg had a 30.9% decrease, and 50 mg/kg had a 40.9% decrease. (3) IMDNQ10: 5 mg/kg had a 12.3% decrease, 20 mg/kg had a 17.1% decrease, and 50 mg/kg had a 44.98% decrease. (4) IMDNQ15: 5 mg/kg had a 15.0% decrease, 20 mg/kg had a 5.5% increase, and 50 mg/kg had a 21.0% decrease.
Compared to the untreated controls, for a 47.4% change in the level of serum ALT in Amphotericin B, relative changes in the levels of ALT in the four compounds were as follows: (1) IMDNQ4: 5 mg/kg had a 40.0% increase, 20 mg/kg had a 0% increase, and 50 mg/kg had a 2.6% decrease. (2) IMD8: 5 mg/kg had an 11.2% decrease, 20 mg/kg had a 69.2% decrease, and 50 mg/kg had an 893% decrease. (3) IMDNQ10: 5 g/kg had a 41.4% increase, 20 mg/kg had a 24.4% decrease, and 50 mg/kg had an 89.7% decrease. (4) IMDNQ15: 5 mg/kg had a 50.0% increase, 20 mg/kg had a 46.6% increase, and 50 mg/kg had a 66.7% increase.
Therefore, the therapeutic effects of these compounds are not only comparable with that of amphotericin B, but are even more effective in the reduction of parasite burden compared to Amphotericin B.
The complete disclosure of each reference hereinbelow listed is incorporated by reference.
This PCT application claims priority from U.S. Provisional Applications 61/561,437, filed Nov. 18, 2011 and 61/676,735, filed Jul. 27, 2012, the complete disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/065525 | 11/16/2012 | WO | 00 | 5/16/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61561437 | Nov 2011 | US | |
| 61676735 | Jul 2012 | US |
