MEDICINAL USE OF SERRULATANE DITERPENES

TECHNICAL FIELD

This invention relates to terpenes and uses thereof. In particular, the invention relates to serrulatane diterpenes and to the use of such compounds in the treatment of diseases and disorders such as cancer.

Throughout this application, various publications are referred to by first author and year of publication. Full citations for these publications, in the order they appear in the application, are presented in a References section immediately before the claims.

BACKGROUND OF THE INVENTION

Plant secondary metabolites have been a major source of medicinal substances providing lead compounds for drug discovery and development.

Plants produce resins and exudates containing secondary metabolites to protect young flower and leaf buds against bacteria and fungi, and damage from sunlight. Accordingly, compounds from plant resins and exudates frequently possess antimicrobial and antioxidant/free radical scavenging activities. Worker honey bees (Apis mellifera) collect plant resins/exudates of young shoots and buds from certain trees and shrubs. The resulting mixture is called propolis, or so called bee glue. Propolis is a complex resinous substance and a rich source of bioactive substances. It is used by the bees to mix with wax to seal cracks and holes in their hives and as a disinfectant to protect against microbial infections. The medicinal properties of propolis have been exploited by man since Ancient civilizations.

Currently, propolis is extensively available as a natural health product, and is widely used in cosmetics. However, its modern use in medicine is limited, largely due to the wide variations in chemical compositions arising from honey bees collecting from different or a mixture of plant sources. The composition of propolis is dependent upon the surrounding flora to which the bees have access, and as such, differences in flora may result in differences in propolis compositions. For example, it is known that flavonoids are the major pharmacologically active compounds in European propolis, polyprenylated benzophenones are the main substances in Cuban and Venuzuelan propolis, and prenylated cinnamic acid derivatives are predominant in Brazilian propolis.

Recognition of the botanical origin of the propolis produced by honey bees enables beehives to be placed in favourable locations such that propolis from a single botanical source may be produced to enable manufacture of medicines of high quality and efficacy.

The medicinal uniqueness of propolis is determined by the selective collecting ability of honey bees, as they can recognise natural materials that are relatively non-polar and have antibiotic properties. As reported, the common source of propolis is leaf and flower bud exudates, which are of high antibiotic character in order to protect the delicate growing of plant tissue from attack by microorganisms. It has also been reported that honey bees collect exudates from wounded or diseased plant tissues. Such sources are potentially rich in antibiotic substances produced by plants in response to wounding or attack from insects, microorganisms and viruses.

Medicinal properties of a propolis are associated with the biological activities of the individual plant resins/exudates. It is not clear from previous studies whether the bees simply collect a plant material that is known as propolis, or if there is metabolic modification or addition from the bees. However, there does not appear to be evidence of significant amounts of material added from honey bees, or strong evidence for metabolic transformation. Thus, a better understanding is required regarding the composition of propolis in specific geographical locations to be able to utilize it to its full benefit in the development of new agents in the treatment of diseases and disorders such as cancer.

SUMMARY OF THE INVENTION

In work leading up to the present invention a survey of propolis samples isolated from Kangaroo Island (South Australia) was conducted. Surprisingly, the Inventors found that unlike other propolis which commonly contains flavonoids as active constituents, Kangaroo Island propolis from the Myoporum genus of plants, in particular Myoporum insulare, contains serrulatane diterpenes. Thus, the present invention relates to compounds isolated from the resins/exudates of leaves and leaf buds of Myoporum insulare, propolis sourced from the same plant and uses thereof.

Myoporum insulare species is a shrub occurring on ridges and coastal cliffs in Australia. Common names include boobialla, native juniper and, in Western Australia, blueberry tree. Myoporum is a genus of approximately 30 species, of which sixteen are found in Australia. Myoporum insulare species has a variable growth habitat and may be a dense or an open shrub or a small tree up to 6 metres. The leaves are lance-shaped to elliptical, 30-100 mm long by 10-20 mm wide with glossy green colour. The flowers occur in groups of up to 8 in the leaf axils in late spring and summer and are usually white or occasionally pale pink.

The Inventors have isolated five major compounds, namely compounds 1 to 5, from propolis obtained from Myoporum insulare. Compounds 1 to 5 are shown in Table 1. The conventional numbering for serrulatane compounds is shown with respect to Compound 1. In general, serrulatane diterpenes have the (1R,4S)-configuration (Ghisalberti, 1994).

Compound 1 gives a derivative, Compound 6, on oxidation. Further in regard to derivatization, the compounds can also be acylated, alkylated, alkenylated or benzoylated through the free hydroxyl groups. For example, compounds 1 and 2 can be acetylated to give derivatives 7 and 8 while benzoylation can give derivatives 9 and 10.

TABLE 1

Representative serrulatane diterpenoid

compounds.

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TABLE 2

Serrulatane diterpenoid derivatives.

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Representative compounds were evaluated for pharmacological activity and were found to be useful in modulating a number of diseases or disorders. For example, the compounds were found to inhibit cancer cell proliferation and showed moderate to strong inhibition of various targets associated with cancer pathology. This indicates that the serrulatane diterpene compounds of the present disclosure may be useful as therapeutic agents e.g. for the treatment of cancer. The compounds were also found to effectively inhibit inflammatory activity and modulate immune response, showing moderate to strong inhibition of COX1 and COX2, the release of proinflammatory cytokines, lipoxygenase and the nuclear trasnscription factor NF-κB. Therefore, the serrulatane diterpenes described herein provide a potentially attractive lead for pharmaceutical research and development and as biological tools for further understanding the pathophysiology of diseases and disorders such as cancer, and diseases and disorders associated with inflammation, such as skin inflammatory conditions.

Accordingly, in a first aspect, there is provided a method of treating a cancer, the method comprising administering a therapeutically effective amount of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition including said compounds, to a subject in need thereof, wherein:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl, OC(O)C_4-5alkadienyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl, OC(O)C_4-5alkadienyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or OC(O)C_4-5alkadienyl;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or OC(O)C_4-5alkadienyl; and

A - - - B is CH═C or CH₂—CH.

According to a second aspect, there is provided use of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl, OC(O)C_4-5alkadienyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5-alkenyl, OC(O)C_4-5alkadienyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or OC(O)C_4-5alkadienyl;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or OC(O)C_4-5alkadienyl; and

A - - - B is CH═C or CH₂—CH,

in the preparation of a medicament for treating a cancer.

According to a third aspect, there is provided use of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

in treating a cancer.

According to a fourth aspect, there is provided a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

for use in treating a cancer.

According to a fifth aspect, there is provided a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

According to a sixth aspect, there is provided a pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to the fifth aspect and a pharmaceutically acceptable excipient.

According to a seventh aspect, there is provided a pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to the fifth aspect, or a mixture thereof, and a pharmaceutically acceptable excipient.

According to an eighth aspect, there is provided a compound as defined in the fifth aspect isolated from propolis, wherein the propolis originates from plants of the Myoporum genus.

According to a ninth aspect, there is provided a compound as defined in the fifth aspect isolated from the resin, gum or exudate of Myoporum genus.

According to a tenth aspect, there is provided a method of treating a disease or disorder, the method comprising administering a therapeutically effective amount of a compound according to the fifth aspect or a composition according to the sixth or seventh aspect.

According to an eleventh aspect, there is provided use of a compound according to the fifth aspect in the preparation of a medicament for treating a disease or disorder.

According to a twelfth aspect, there is provided use of a compound according to the fifth aspect or a composition according to the sixth aspect or seventh aspect in treating a disease or disorder.

According to a thirteenth aspect of the present invention, there is provided a compound according to the fifth aspect or a composition according to the sixth aspect or seventh aspect for use in treating a disease or disorder.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows representative ¹H NMR (400 MHz, CDCl₃) spectrum of the 4:1 mixture of mono-methylated products (compounds 11 and 12).

FIG. 2 shows representative ¹³C NMR (100 MHz, CDCl₃) spectrum of the 4:1 mixture of mono-methylated products (compounds 11 and 12).

FIG. 3 to FIG. 6 show example binding curves for Compound 1 against epigenetic targets.

FIG. 3 shows ASH1L (h) bromodomain binding curve.

FIG. 4 shows CECR2(h) bromodomain binding curve.

FIG. 5 shows SP140 (h) bromodomain binding curve.

FIG. 6 shows UHRF1(108-286) (h) binding curve.

FIG. 7 shows binding against 5-lipoxygenase for Compound 1 (blue circles, IC₅₀=2.31 μM) and a reference compound NGA (red squares, IC₅₀=0.64 μM).

FIG. 8 shows binding against lipid peroxidase for Compound 1 (blue circles, IC₅₀=1.47 μM) and a reference compound N-propyl gallate (red squares, IC₅₀=165 μM).

FIG. 9 shows binding against monoamine oxidase (MAO-A) for Compound 1 (blue circles, IC₅₀=6.74 μM) and a reference compound clorgyline (red squares, IC₅₀=1.43 nM).

FIG. 10 shows transcription response and binding against NF-κB for Compound 1 (blue circles, IC₅₀=1.36 μM) and a reference compound cyclosporin A (red squares, IC₅₀=0.029 μM).

FIG. 11 shows proliferation response when cell line MDA-MB-415 is treated with Compound 1.

FIG. 12 shows proliferation response when cell line RKO-AS45-1 is treated with Compound 1.

FIG. 13 shows proliferation response when cell line SW480 is treated with Compound 1.

FIG. 14 shows proliferation response when cell line 639-V is treated with Compound 1.

FIG. 15 shows proliferation response when cell line Hs 729 is treated with Compound 1.

FIG. 16 shows proliferation response when cell line Hs 852.T is treated with Compound 1.

FIG. 17 shows proliferation response when cell line HCT-8 is treated with Compound 1.

FIG. 18 shows proliferation response when cell line IM-9 is treated with Compound 1.

FIG. 19 shows the response when the cell line HREC is treated with Compound 1 in an OncoPanel Normal Cell Proliferation Assay.

FIGS. 20A to 20D show

Specific embodiments of the disclosure are described below. It will be appreciated that these embodiments are illustrative and not restrictive.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a method of treating a cancer, the method comprising administering a therapeutically effective amount of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition including said compounds, to a subject in need thereof,

wherein:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl, OC(O)C_4-5alkadienyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl, OC(O)C_4-5alkadienyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or OC(O)C_4-6alkadienyl;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC_4-5alkadienyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or OC(O)C_4-5alkadienyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O; and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl, OC(O)Ph or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl, OC(O)Ph or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O):

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

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Preferably, the compound is a compound of Formula (Ib):

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Preferably, the compound is a compound of Formula (Ic):

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Preferably, the compound is a compound of Formula (Id):

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Preferably, the compound is a compound of Formula (Ie):

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Preferably, the compound is a compound of Formula (If):

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Preferably, the compound is a compound of Formula (Ig):

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Preferably, the compound is a compound of Formula (Ih):

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Preferably, the compound is a compound of Formula (Ii):

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Preferably, A - - - B is CH═C.

The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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Preferably, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

The cancer of the central nervous system is preferably a glioma. Preferably, the cancer of the central nervous system is a medulloblastoma. The cancer of the central nervous system is preferably a neuroblastoma.

Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

While it is possible that, for use in therapy a therapeutically effective amount of the compounds as defined herein, or a pharmaceutically acceptable salt or solvate thereof, may be administered as the raw chemical; in the first aspect of the present invention the active ingredient is administered as a pharmaceutical composition. Thus, the present invention also contemplates a pharmaceutical composition comprising a compound of formula (I) to (Ii), or a pharmaceutically acceptable salt or a solvate thereof, and a pharmaceutically acceptable excipient. The excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

When applicable, the compounds of the present invention, including the compounds of formula (I) to (Ii) may be in the form of and/or may be administered as a pharmaceutically acceptable salt.

As used herein the term “pharmaceutically acceptable salt” refers to salts which are toxicologically safe for systemic administration. The pharmaceutically acceptable salts may be selected from alkali or alkaline earth metal salts, including, sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine and the like.

As used herein the term “pharmaceutically acceptable excipient” refers to a solid or liquid filler, carrier, diluent or encapsulating substance that may be safely used in administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers or excipients may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

As used herein, the term “solvate” refers to a complex of variable stoichiometry formed by a solute (in this disclosure, a compound of formula (I) to (Ii) or a salt or physiologically functional derivative thereof) and a solvent. Such solvents for the purpose of the disclosure may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid. In particular the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include, without limitation, water, ethanol, acetic acid, glycerol, liquid polyethylene glycols and mixtures thereof. A particular solvent is water.

Administration of compounds of the formula (I) to (Ii) may be in the form of a “prodrug”. A prodrug is an inactive form of a compound which is transformed in vivo to the active form. Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question.

The compounds of the present disclosure may be suitable for the treatment of diseases in a human or animal subject. In one example, the subject is a mammal including a human, horse, dog, cat, sheep, cow, or primate. In another example, the subject is a human. In a further example, the subject is not a human. The terms “subject” and “patient” are used interchangeably herein.

As used herein, the term “effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.

Furthermore, the term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. The term also includes within its scope amounts effective to prevent a disease, disorder, or side effect.

As used herein the term “treatment” refers to defending against or inhibiting a symptom, treating a symptom, delaying the appearance of a symptom, reducing the severity of the development of a symptom, and/or reducing the number or type of symptoms suffered by an individual, as compared to not administering a pharmaceutical composition comprising a compound of the invention. The term also includes within its scope prevention of a disease, disorder, or side effect. The term “treatment” encompasses use in a palliative setting

The antitumor effect of the compounds of the present disclosure may be applied as a sole therapy or as a combination therapy i.e. where two or more serrulatane diterpenoids may be administered in combination. Therapy may also involve administration of a mixture of two or more serrulatane diterpenoids. Therapy may involve, in addition, administration of one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. In the field of medical oncology it is normal practice to use a combination of different forms of treatment to treat each patient with cancer such as a combination of surgery, radiotherapy and/or chemotherapy. In particular, it is known that irradiation or treatment with antiangiogenic and/or vascular permeability reducing agents can enhance the amount of hypoxic tissue within a tumour. Therefore the effectiveness of the compounds of the present invention may be improved by conjoint treatment with radiotherapy and/or with an antiangiogenic agent.

The individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present disclosure is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other anti-neoplastic agents includes in principle any combination with any pharmaceutical composition useful for treating cancer.

When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation and may be formulated for administration. When formulated separately they may be provided in any convenient formulation, conveniently in such a manner as are known for such compounds in the art.

Pharmaceutical compositions of the present disclosure may be formulated for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Therefore, the pharmaceutical compositions of the invention may be formulated, for example, as tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions. Such pharmaceutical formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Such pharmaceutical formulations may be prepared as enterically coated granules, tablets or capsules suitable for oral administration and delayed release formulations.

When a compound is used in combination with a second therapeutic agent active against the same disease, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

Also disclosed herein is use of a compound of Formula (I),

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wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

in the preparation of a medicament for treating a cancer.

Preferably:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

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Preferably, the compound is a compound of Formula (Ib):

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Preferably, the compound is a compound of Formula (Ic):

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Preferably, the compound is a compound of Formula (Id):

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Preferably, the compound is a compound of Formula (Ie):

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Preferably, the compound is a compound of Formula (If):

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Preferably, the compound is a compound of Formula (Ig):

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Preferably, the compound is a compound of Formula (Ih):

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Preferably, the compound is a compound of Formula (Ii):

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Preferably, A - - - B is CH═C.

The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

Also disclosed herein is use of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt, or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

in treating a cancer.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O):

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O; and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph; or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

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Preferably, the compound is a compound of Formula (Ib):

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Preferably, the compound is a compound of Formula (Ic):

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Preferably, the compound is a compound of Formula (Id):

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Preferably, the compound is a compound of Formula (Ie):

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Preferably, the compound is a compound of Formula (If):

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Preferably, the compound is a compound of Formula (Ig):

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Preferably, the compound is a compound of Formula (Ih):

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Preferably, the compound is a compound of Formula (Ii):

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Preferably, A - - - B is CH═C.

The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

Also disclosed herein is a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

for use in treating a cancer.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5-alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph; or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

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Preferably, the compound is a compound of Formula (Ib):

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Preferably, the compound is a compound of Formula (Ic):

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Preferably, the compound is a compound of Formula (Id):

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Preferably, the compound is a compound of Formula (Ie):

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Preferably, the compound is a compound of Formula (If):

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Preferably, the compound is a compound of Formula (Ig):

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Preferably, the compound is a compound of Formula (Ih):

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Preferably, the compound is a compound of Formula (Ii):

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Preferably, A - - - B is CH═C.

The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

Also disclosed herein is a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

OC(O)C_2-5alkenyl; and
A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CH₂;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is CHOH;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably:

X is C(O);

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

Preferably, the compound is a compound of Formula (Ia):

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Preferably, the compound is a compound of Formula (Ib):

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Preferably, the compound is a compound of Formula (Ic):

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Preferably, the compound is a compound of Formula (Id):

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Preferably, the compound is a compound of Formula (Ie):

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Preferably, the compound is a compound of Formula (If):

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Preferably, the compound is a compound of Formula (Ig):

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Preferably, the compound is a compound of Formula (Ih):

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Preferably, the compound is a compound of Formula (Ii):

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Preferably, A - - - B is CH═CH.

The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably selected from the group consisting of:

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Preferably, the compound is selected from the group consisting of:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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The compound is preferably:

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Preferably, the compound is:

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Also disclosed herein is a pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, as described herein, and a pharmaceutically acceptable excipient.

The compounds or pharmaceutical compositions disclosed above are preferably for use as a medicament.

The compounds or pharmaceutical compositions disclosed above are preferably for use in therapy.

Also disclosed is a compound described above isolated from propolis, wherein the propolis originates from plants of the Myoporum genus. Preferably, the propolis originates from Myoporum insulare.

Also disclosed is a compound described above isolated from the resin, gum or exudate of Myoporum genus. Preferably, the compound is isolated from the resin, gum or exudate of Myoporum insulare.

Also disclosed is a method of treating a disease or disorder, the method comprising administering a therapeutically effective amount of a compound or a composition described herein.

Also disclosed is use of a compound described herein in the preparation of a medicament for treating a disease or disorder.

Also disclosed herein is use of a compound or a composition described herein in treating a disease or disorder.

Also disclosed herein is a compound or a composition described herein for use in treating a disease or disorder.

Preferably, the disease or disorder is selected from the group consisting of: acute coronary syndrome, an aging-related disease or disorder; an allergic disease or a related condition; Alzheimer's disease, atherosclerosis, an autoimmune disease; a bacterial infection, cancer; dementia, depression or a related condition; diabetes; dyslipidemia, hyperlipidemia, hypertension, itchytosis, an immune disease; a metabolic disease or disorder; a neurological disease or disorder; obesity; Parkinson's disease; pain; rheumatoid arthritis, a disease or disorder associated with inflammation, such as skin inflammatory conditions, and a skin disease or disorder.

The disease or disorder is preferably cancer.

Preferably, the lung cancer is a non-small cell lung cancer. The lung cancer is preferably a small cell lung cancer.

Preferably, the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

The sarcoma is preferably a liposarcoma, rhabdomyosarcoma, or fibrosarcoma. Preferably, the sarcoma is a soft tissue sarcoma. The soft tissue sarcoma is preferably a soft tissue osteosarcoma.

Preferably, the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

The skin disease or disorder is preferably selected from the group consisting of: eczema, psoriasis, acne, a wound, a scar, inflammation including psoriasis and atopic dermatitis, a burn, sunburn, skin damage and skin irritation.

EXAMPLE EMBODIMENTS

1. A method of treating a cancer, the method comprising administering a therapeutically effective amount of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition including said compounds, to a subject in need thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

2. Use of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

in the preparation of a medicament for treating a cancer.

3. Use of a compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

in treating a cancer.

4. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl; and

A - - - B is CH═C or CH₂—CH.

5. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

6. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

7. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CH₂;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

8. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is CHOH;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

9. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein:

X is C(O);

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

10. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ia):

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11. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ib):

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12. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ic):

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13. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Id):

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14. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ie):

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15. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (If):

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16. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ig):

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17. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ih):

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18. The method according to example embodiment 1 or the use according to example embodiment 2 or example embodiment 3, wherein the compound is a compound of Formula (Ii):

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19. The method or use according to any one of example embodiments 1 to 16, wherein A - - - B is CH═C.

20. The method according to any one of example embodiments 1 to 19 or the use according to any one of example embodiments 2 to 19, wherein the compound is selected from the group consisting of:

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21. The method or use according to example embodiment 20, wherein the compound is selected from the group consisting of:

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22. The method or use according to example embodiment 21, wherein the compound is selected from the group consisting of:

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23. The method or use according to example embodiment 22, wherein the compound is selected from the group consisting of:

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24. The method or use according to example embodiment 23, wherein the compound is:

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25. The method or use according to example embodiment 23, wherein the compound is:

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26. The method or use according to example embodiment 23, wherein the compound is:

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27. The method or use according to example embodiment 23, wherein the compound is:

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28. The method or use according to example embodiment 23, wherein the compound is:

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29. The method or use according to example embodiment 23, wherein the compound is:

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30. The method according to any one of example embodiments 1 to 29 or the use according to any one of example embodiments 2 to 29, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

31. The method or use according to example embodiment 30, wherein the cancer of the central nervous system is a glioma.

32. The method or use according to example embodiment 30, wherein the cancer of the central nervous system is a medulloblastoma.

33. The method or use according to example embodiment 30, wherein the cancer of the central nervous system is a neuroblastoma.

34. The method or use according to example embodiment 30, wherein the lung cancer is a non-small cell lung cancer.

35. The method or use according to example embodiment 30, wherein the lung cancer is a small cell lung cancer.

36. The method or use according to example embodiment 30, wherein the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

37. The method or use according to example embodiment 30, wherein the sarcoma is a liposarcoma, rhabdomyosarcoma, or fibrosarcoma.

38. The method or use according to example embodiment 30, wherein the sarcoma is a soft tissue sarcoma.

39. The method or use according to example embodiment 38, wherein the soft tissue sarcoma is a soft tissue osteosarcoma.

40. The method according to example embodiment 30, wherein the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

41. A compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

42. A compound of Formula (I),

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a geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof,

wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH,

for use in treating a cancer.

43. The compound according to example embodiment 41 or example embodiment 42, wherein:

X is CH₂, CHOH or C(O);

R₁is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph, OC(O)C_2-5alkenyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

where X is CH₂, CHOH or C(O), R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl, OCH₂Ph, OC_2-5alkenyl, OC(O)C_1-5alkyl, OC(O)Ph or OC(O)C_2-5alkenyl; and

A - - - B is CH═C or CH₂—CH.

44. The compound according to example embodiment 41 or example embodiment 42, wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

45. The compound according to example embodiment 41 or example embodiment 42, wherein:

X is CH₂, CHOH or C(O);

A - - - B is CH═C or CH₂—CH.

46. The compound according to example embodiment 41 or example embodiment 42, wherein:

X is CH₂;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

47. The compound according to example embodiment 41 or example embodiment 42, wherein:

X is CHOH;

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

48. The compound according to example embodiment 41 or example embodiment 42, wherein:

X is C(O);

R₁is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₂is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

R₃is independently H, OH, OC_1-5alkyl, OC(O)C_1-5alkyl or ═O;

and no more than one of R₁, R₂and R₃can be H;

R₄is H, OH, OC_1-5alkyl, OCH₂Ph, OC(O)C_1-5alkyl or OC(O)Ph;

or X and R₃form a six-membered ether ring or lactone ring, or a six membered ring containing a hemiacetal carbon atom or an acetal carbon atom wherein R₄is respectively OH or OC_1-5alkyl or OC(O)C_1-5alkyl; and

A - - - B is CH═C or CH₂—CH.

49. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ia):

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50. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ib):

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51. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ic):

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52. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Id):

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53. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ie):

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54. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (If):

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55. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ig):

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56. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ih):

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57. The compound according to example embodiment 41 or example embodiment 42, wherein the compound is a compound of Formula (Ii):

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58. The compound according to any one of example embodiments 41 to 57, wherein A - - - B is CH═C.

59. The compound according to any one of example embodiments 41 to 58, wherein the compound is selected from the group consisting of:

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60. The compound according to example embodiment 59, wherein the compound is selected from the group consisting of:

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61. The compound according to example embodiment 60, wherein the compound is selected from the group consisting of:

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62. The compound according to example embodiment 61, wherein the compound is selected from the group consisting of:

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63. The compound according to example embodiment 62, wherein the compound is:

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64. The compound according to example embodiment 62, wherein the compound is:

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65. The compound according to example embodiment 62, wherein the compound is:

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66. The compound according to example embodiment 62, wherein the compound is:

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67. The compound according to example embodiment 62, wherein the compound is:

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68. The compound according to example embodiment 62, wherein the compound is:

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69. The compound according to any one of example embodiments 42 to 68, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, a sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

70. The compound according to example embodiment 69, wherein the cancer of the central nervous system is a glioma.

71. The compound according to example embodiment 69, wherein the cancer of the central nervous system is a medulloblastoma.

72. The compound or use according to example embodiment 69, wherein the cancer of the central nervous system is a neuroblastoma.

73. The compound according to example embodiment 69, wherein the lung cancer is a non-small cell lung cancer.

74. The compound according to example embodiment 69, wherein the lung cancer is a small cell lung cancer.

75. The compound according to example embodiment 69, wherein the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

76. The compound according to example embodiment 69, wherein the sarcoma is a liposarcoma, rhabdomyosarcoma, or fibrosarcoma.

77. The compound according to example embodiment 69, wherein the sarcoma is a soft tissue sarcoma.

78. The compound according to example embodiment 77, wherein the soft tissue sarcoma is a soft tissue osteosarcoma.

79. The compound according to example embodiment 69, wherein the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

80. A pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to example embodiment 41 or any one of example embodiments 43 to 68, and a pharmaceutically acceptable excipient.

81. A pharmaceutical composition comprising a compound, geometric isomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, according to example embodiment 41 or any one of example embodiments 43 to 68, or a mixture thereof, and a pharmaceutically acceptable excipient.

82. The compound according to example embodiment 41 or any one of example embodiments 43 to 68, or the pharmaceutical composition according to example embodiment 80 or example embodiment 81, for use as a medicament.

83. The compound according to example embodiment 41 or any one of example embodiments 43 to 68, or the pharmaceutical composition according to example embodiment 80 or example embodiment 81, for use in therapy.

84. A compound as defined in any one of example embodiments 42 to 68 isolated from propolis, wherein the propolis originates from plants of the Myoporum genus.

85. The compound according to example embodiment 84, wherein the propolis originates from Myoporum insulare.

86. A compound as defined in example embodiment 41 or any one of example embodiments 43 to 68 isolated from the resin, gum or exudate of Myoporum genus.

87. The compound according to example embodiment 86 isolated from the resin, gum or exudate of Myoporum insulare.

88. A method of treating a disease or disorder, the method comprising administering a therapeutically effective amount of a compound example embodiment 41 or any one of example embodiments 43 to 68 or a composition according to example embodiment 80 or example embodiment 81.

89. Use of a compound according to example embodiment 41 or any one of example embodiments 43 to 68 in the preparation of a medicament for treating a disease or disorder.

90. Use of a compound according to example embodiment 41 or any one of example embodiments 43 to 68 or a composition according to example embodiment 80 or example embodiment 81 in treating a disease or disorder.

91. A compound according to example embodiment 41 or any one of example embodiments 43 to 68 or a composition according to example embodiment 80 or example embodiment 81 for use in treating a disease or disorder.

92. The method according to example embodiment 88 or use according to example embodiment 89 or example embodiment 90 or compound according to example embodiment 91, wherein the disease or disorder is selected from the group consisting of: acute coronary syndrome, an aging-related disease or disorder; an allergic disease or a related condition; Alzheimer's disease, atherosclerosis, an autoimmune disease; a bacterial infection, cancer; dementia, depression or a related condition; diabetes; dyslipidemia, hyperlipidemia, hypertension, itchytosis, an immune disease; a metabolic disease or disorder; a neurological disease or disorder; obesity; Parkinson's disease; pain; rheumatoid arthritis, a disease or disorder associated with inflammation, such as skin inflammatory conditions, and a skin disease or disorder.

93. The method or use or compound according to example embodiment 92, wherein the disease or disorder is cancer.

94. The method or use or compound according to example embodiment 93, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, a cancer of the central nervous system, cancer of the adrenal glands, cancer of the placenta, cancer of the testis, cervical cancer, colon cancer, kidney cancer, head and neck cancer, myeloma, leukemia, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, thyroid cancer, uterine cancer, a carcinoma, a lymphoma, sarcoma, eye cancer, esophageal cancer, bile duct cancer or vulva cancer.

95. The method or use or compound according to example embodiment 94, wherein the cancer of the central nervous system is a glioma.

96. The method or use or compound according to example embodiment 94, wherein the cancer of the central nervous system is a medulloblastoma.

97. The method or use or compound according to example embodiment 94, wherein the cancer of the central nervous system is a neuroblastoma.

98. The method or use or compound according to example embodiment 94, wherein the lung cancer is a non-small cell lung cancer.

99. The method or use or compound according to example embodiment 94, wherein the lung cancer is a small cell lung cancer.

100. The method or use or compound according to example embodiment 94, wherein the carcinoma is adenosquamous cell carcinoma or squamous cell carcinoma.

101. The method or use or compound according to example embodiment 94, wherein the sarcoma is a liposarcoma, rhabdomyosarcoma, or fibrosarcoma.

102. The method or use or compound according to example embodiment 94, wherein the sarcoma is a soft tissue sarcoma.

103. The method or use or compound according to example embodiment 102, wherein the soft tissue sarcoma is a soft tissue osteosarcoma.

104. The method or use or compound according to example embodiment 94, wherein the lymphoma is Burkitt's lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

105. The method or use or compound according to example embodiment 92, wherein the skin disease or disorder is selected from the group consisting of: eczema, psoriasis, acne, a wound, a scar, inflammation including psoriasis and atopic dermatitis, a burn, sunburn, skin damage and skin irritation.

EXAMPLES

In order to better understand the nature of the invention a number of examples will now be described as follows:

Experimental
Materials

Honey bees observed collecting from the boobialla trees and shrubs (Myoporum insulare) were captured in plastic tubes, capped and frozen. Sections of the bee hind legs holding propolis were cut and pooled. Plant samples were obtained and dried at 40° C. in a ventilated oven (Thermoline, NSW Australia) a few days to provide voucher specimens for identification by botanist, A/Prof Murray Henwood, John Ray Herbarium, University of Sydney, Sydney, Australia, and registered as Myoporum insulare voucher number Duke-131202-01. Resin extracted from plant samples was stored at −20° C. until analysis or further processing.

Propolis was obtained from apiary sites with from 1 to 10 colonies of European honey bee (Apis mellifera ligustica) in standard 10-frame box hives, each fixed with a propolis mat under the hive cover lid. The propolis samples were collected and provided by a few beekeepers in eight apiary sites located in different parts of Kangaroo Island, South Australia. These sites were located in the vicinity of Hanson Bay, Vivonne Bay, Flour Cask Bay, Island Beach, Kingscote, Brownlow, Rainy Creek and Eleanor River. These locations are known to be a habitat for the plant Myoporum insulare on Kangaroo Island. This provided 23 propolis samples which, based on TLC analysis and ¹H NMR techniques, were estimated to be at least 90% sourced from M. insulare. The samples were stored at −20° C. until further analysis and processing.

Thin layer chromatography sheets precoated with silica gel 60 F₂₅₄and silica gel 60H for normal-phase short-column vacuum chromatography (NPSCVC) were purchased from Merck. TLC plates were visualized with a UVGL-58 mineral-light lamp, Multiband UV-2544/366.

All the chemicals used in the isolation and synthesis, including deuterated NMR solvents such as d-chloroform, d₄-methanol and d₆-dimethyl sulfoxide were purchased from Novachem Pty Ltd (Collingwood, Vic, Australia). Solvents including hexane, dichloromethane, ethyl acetate, isopropanol, ethanol, methanol, and acetic acid were of analytical grade and purchased from Chem Supply, Gillman, SA, Australia.

General Methods

Rotavapor model R-114 rotary evaporator with a water bath temperature ranging between 40-60° C. was used to evaporate the solvent fraction. Vacuum pump V-700 or Vacuubrand MD 4C NT diaphragm pump (Vacuubrand GMBH, Wertheim, Germany) with vacuum controller V-800 or V-850 is used. Final drying is carried out by a Napco 5831 vacuum oven (NAPCO, Salt Lake City, USA) using a DirectTorr vacuum pump (Sargent-Welch, Buffalo, USA).

Isocratic analytical HPLC was performed on a Shimadzu UFLC, LC-20AD pump, SIL-20A HT autosampler, with a Hewlett-Packard Column, NUCLEOSIL 100 C₁₈, 5 μm, 4 mm×125 mm, injection volume 20 μl, eluted at 1 ml/min and detected at 230 mm with a UV-Vis detector (Shimadzu SPD-20A). The column was eluted with methanol-water-acetic acid (65:34.8:0.2).

Gradient analytical HPLC was performed on a Shimadzu Nexera X2 LC-30AD system, SIL-30 autosampler, with a Hewlett-Packard Column, NUCLEOSIL 100 C₁₈, 5 μm, 4 mm×125 mm, injection volume 10 μl, eluted at 1 ml/min and detected at 320 mm with a SPD-M30A UV-Vis diode array detector. The column was eluted with a gradient system made up of methanol (phase A), and methanol:water:acetic acid (40:59.8:0.2) (phase B). A solvent gradient was applied as follows: 0-2 min: 32-50% A, 2-9 min: 50-90% A, 9-12 min: 90-100% A, 12-14 min, 100% A, and finally 14-15 min: 100-32% A, maintained over 5 min.

¹H and ¹³C Nuclear magnetic resonance (NMR) analyses were carried out on Varian 400 MHz System with a SMS autosampler (Palo Alto, Calif., USA). Chemical shifts (6) of peaks in NMR spectra are reported in parts per million (ppm) and are referenced to tetramethylsilane (Si(CH₃)₄, TMS). ¹H NMR data is reported as chemical shift (6), relative integral, multiplicity (abbreviations: s=singlet, d=doublet, t=triplet, td=triplet of doublets, q=quartet, dd=doublet of doublets, ddd=doublet of doublet of doublets, bs=broad singlet, bt=broad triplet), coupling constant (J in Hz) and assignment. ¹³C NMR data is reported as chemical shift (δ) and assignment.

For low resolution ESI-MS, the samples were analyzed by Finnigan Polaris Ion Trap MS/MS system (Finnigan Corporation, San Jose, USA) using an Xcalibur 1.2 data system, in Electrospray Ionization (ESI) negative and positive modes using infusion technique.

High resolution ESI-MS were measured on a Bruker Daltonics Apex Ultra Fourier Transform Ion Cyclotron Resonance 7 Tesla Mass Spectrometer, mass spectrometry unit, School of Chemistry, The University of Sydney.

Determination of the ¹H-NMR Chemical Profiles of the Plant and Propolis Specimens

Young leaf shoots (25 g), bee hind leg (0.01 g) and beehive propolis (1.0 g) were extracted with dichloromethane at room temperature for 15 min. The extracts were filtered, dried under reduced pressure and analyzed by ¹H-NMR and HPLC. Samples were found to contain serrulatane diterpenes. Propolis and plant samples were subsequently selected for isolation of the components.

Preparation of Myoporum insulare Resin Extract

Fresh young leaves and young shoot tips were harvested from several Myoporum insulare trees. The leaves were stored in plastic bags on ice until processing within 5 days. To extract the external resin/exudate the leaves and shoot tips (4 Kg) were mixed with dichloromethane (4 litres) and after 1 hour standing the dichloromethane extract was filtered and the dichloromethane was removed from the filtrate by evaporation under reduced pressure using a rotary evaporator to give a brown partially solid dried resin (12.5 g, yield 0.31%).

Isolation and Identification of Serrulatane Diterpenes from Myoporum insulare Propolis from Kangaroo Island.

General Method

Propolis (100 g) was extracted with dichloromethane at room temperature with stirring for 1 hr. The extract was subjected to purification using normal-phase short column vacuum chromatography (NPSCVC). A step-wise gradient of mobile phase (2×100 mL) consisting of dichloromethane (DCM) and ethyl acetate (EtOAc) at 0, 1, 2, 4, 8, 10, 15, 20, 50 and 100% was employed to elute the components which were analysed by TLC and NMR. Further purification of the compounds, if required, was subsequently carried out on the same NPSCVC with different mobile phases consisting of either hexane and EtOAc or hexane and isopropanol. Structures and identity of these purified compounds were characterized by ¹H- and ¹³C-NMR and mass spectrometry including high resolution mass spectrometry. Detailed structural analyses of the isolated compound were also carried out when needed by 2D-NMR using Gradient Heteronuclear Multiple Bond Coherence (GHMBC). The major serrulatane diterpenoid was determined to be 7,8,18-trihydroxyserrulat-14-ene (1).

Isolated Serrulatane Diterpenes from the Plant, Myoporum insulare
7,8,18-trihydroxyserrulat-14-ene (1)

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Yellowish liquid, yield 50 mg, [α]_D²⁰−27.3° (C=1.0, CHCl₃). UV (CH₃OH) λ_maxnm (log ε) 279 (3.37) and 320 (2.49). IR (ν): 3381, 2924, 2864, 1625, 1581, 1448 cm⁻¹. HRESIMS: 341.2089 m/z [M+Na]⁺ (calcd 341.2087) C₂₀H₃₀O₃. ¹H NMR (400 MHz, CDCl₃) δ: 6.56, (1H, s, H-5), 5.04 (1H, bt, J=7.0 Hz, H-14), 3.63 (2H, d, J=6.2 Hz, H-18), 3.07 (1H, pentet of d, J=6.8, 1.6 Hz, H-1), δ 2.76 (1H, td, J=5.6, 2.6 Hz, H-4), 2.20 (3H, s, H-19), 1.99 (1H, m, H-13A), 1.90 (1H, m, H-13B), 1.90 (1H, m, H-3A), 1.86 (1H, m, H-2A), 1.83 (1H, m, H-11), 1.68 (3H, s, H-16), 1.66 (1H, m, H-2B), 1.58 (3H, s, H-17), 1.51 (1H, m, H-3B), 1.25 (1H, m, H-12A), 1.36 (2H, m, H-12B), 1.21 (3H, d, J=7.0 Hz, H-20). ¹³C NMR (100 MHz, CDCl₃) δ: 15.54 (C-19), 17.68 (C-17), 20.51 (C-2), 21.11 (C-20), 25.70 (C-16), 26.16 (C-13), 26.47 (C-3), 26.98 (C-1), 29.85 (C-12), 37.91 (C-4), 45.45 (C-11), 64.38 (C-18), 121.31 (C-6), 122.00 (C-5), 124.43 (C-14), 127.18 (C-9), 130.70 (C-10), 131.75 (C-15), 139.74 (C-7), 141.61 (C-8).

5,18-epoxyserrulat-14-en-8,18-diol (2)

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Red liquid, yield 84 mg, [α]_D²⁰−21.5° (C=1.0, CHCl₃). UV (CH₃OH) λ_maxnm (log ε) 293 (3.36) and 334 (2.91). IR (ν): 3327, 2970, 2926, 2860, 1647, 1558, 1448, 1043 cm⁻¹. HRESIMS: 315.1970 m/z [M−H]⁻ (calcd 315.1966). C₂₀H₂₈O₃. ¹H NMR (400 MHz, CDCl₃) δ 6.48 (1H, s, H-7), 5.59 (1H, bs, H-18), 5.14 (1H, bt, J=7.0 Hz, H-14), 3.11 (1H, sextet, J=7.6, H-1), 2.54 (1H, td, J=11.4, 3.6 Hz, H-4), 2.25 (1H, m, H-2A), 2.20 (1H, m, H-13A), 2.13 (3H, s, H-19), 2.09 (1H, m, H-13B), 2.08 (1H, m, H-3A), 1.69 (1H, m, H-12A), 1.70 (3H, s, H-16), 1.63 (3H, s, H-17), 1.52 (1H, m, H-11), 1.41 (1H, m, H-2B), 1.28 (3H, d, J=6.9 Hz, H-20), 1.27 (1H, m, H-12B), 1.05 (1H, m, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ 15.88 (C-19), 17.75 (C-17), 22.89 (C-20), 25.36 (C-13), 25.70 (C-16), 26.62 (C-3), 27.79 (C-1), 28.90 (C-12), 31.75 (C-2), 31.99 (C-4), 40.48 (C-11), 91.88 (C-18), 115.94 (C-7), 122.94 (C-6), 124.09 (C-14), 124.29 (C-10), 125.21 (C-9), 131.99 (C-15), 141.71 (C-5), 146.85 (C-8).

5,18-epoxy-8-hydroxyserrulat-14-ene (3)

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Shiny yellow liquid, yield 87 mg, [α]_D²⁰+8.9° (C=1.0, CHCl₃). UV (CH₃OH) λ_maxnm (log ε) 296 (3.55) and 334 (2.66). IR (ν): 3502, 2922, 2856, 1622, 1597, 1421, 1230 cm⁻¹. HRESIMS: 301.21635 m/z [M+H]⁺ (calcd 301.21621) C₂₀H₂₈O₂. ¹H NMR (400 MHz, CDCl₃) δ: 6.46 (1H, s, H-7), 5.13 (1H, bt, J=6.9 Hz, H-14), 4.35 (1H, dd, J=10.5, 3.7 Hz, H-18A), 3.70 (1H, t, J=10.8 Hz, H-18B), 3.13 (1H, sextet, J=7.4 Hz, H-1), 2.31 (1H, m, H-4), 2.24 (1H, m, H-2A), 2.16 (1H, m, H-13A), 2.15 (1H, m, H-3A), 2.12 (3H, s, H-19), 2.02 (1H, m, H-13B), 1.74 (1H, m, H-12A), 1.70 (3H, s, H-16), 1.63 (3H, s, H-17), 1.55, (1H, m, H-11), 1.40 (1H, m, H-2B), 1.28 (3H, d, J=7.0 Hz, H-20), 1.18 (1H, m, H-12B), 1.06 (1H, td, J=12.2, 4.4 Hz, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ: 15.82 (C-19), 17.73 (C-17), 23.12 (C-20), 25.44 (C-13), 25.70 (C-16), 26.97 (C-3), 27.61 (C-1), 29.97 (C-12), 31.47 (C-2), 37.17 (C-11), 39.00 (C-4), 69.52 (C-18), 115.79 (C-7), 122.64 (C-6), 124.12 (C-14), 124.26 (C-9), 125.70 (C-10), 131.99 (C-15), 145.27 (C-5), 146.16 (C-8).

7,8-dihydroxyserrulat-14-ene (4)

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Yellowish liquid, yield 56 mg, [α]_D²⁰−21.1° (C=1.0, CHCl₃). UV (CH₃OH) λ_maxnm (log ε) 278 (3.36) and 329 (2.95). IR (ν): 3396, 2954, 2924, 2868, 1635, 1577, 1456 cm⁻¹. HRESIMS: 325.21413 m/z [M+Na]⁺ (calcd 325.21380) C₂₀H₃₀O₂. ¹H NMR (400 MHz, CDCl₃) δ 6.53 (1H, s, H-5), 5.00 (1H, bt, J=7.0 Hz, H-14), 3.06 (1H, pentet of d, J=6.7, 2.8 Hz, H-1), 2.53 (1H, td, J=5.5, 3.4 Hz, H-4), 2.22 (3H, s, H-19), 2.00 (1H, m, H-13A), 1.95 (1H, m, H-2A), 1.84 (1H, m, H-11), 1.82 (1H, m, H-3A), 1.80 (1H, m, H-13B), 1.70 (1H, m, H-3B), 1.67 (3H, s, H-16), 1.59 (3H, s, H-17), 1.48 (1H, m, H-2B), 1.29 (1H, m, H-12A), 1.12 (3H, d, J=6.8 Hz, H-20), 1.05 (1H, m, H-12B), 0.95 (3H, d, J=6.8, H-18). ¹³C NMR (100 MHz, CDCl₃) δ: 15.53 (C-19), 17.62 (C-17), 18.66 (C-18), 19.63 (C-3), 21.10 (C-20), 25.71 (C-16), 26.25 (C-13), 27.1 (C-1), 27.43 (C-2), 33.35 (C-12), δ 37.8 (C-11), δ 41.88 (C-4), δ 120.50 (C-6), δ 122.46 (C-5), δ 124.96 (C-14), δ 127.09 (C-9), δ 131.10 (C-15), δ 131.90 (C-10), δ 139.19 (C-7), δ 140.93 (C-8).

Serrulat-14-en-5,8-dione (5)

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Light orange liquid, yield 30 mg. ¹H NMR (400 MHz, CDCl₃) δ 6.68 (1H, s, H-5), 5.02 (1H, bt, J=7.1 Hz, H-14), 2.85 (1H, m, H-1), 2.20 (1H, m, H-4), 2.12 (1H, m, H-3A), 2.03 (1H, m, H-13A), 1.95 (3H, s, H-19), 1.88 (1H, m, H-11), 1.85 (1H, m, H-13B), 1.69 (1H, m, H-2A), 1.67 (3H, s, H-16), 1.58 (3H, s, H-17), 1.34 (1H, m, H-2B), 1.30 (1H, m, H-12A), 1.20 (1H, m, H-3B), 1.12 (1H, m, H-12B), 1.07 (6H, d, J=6.9 Hz, H-18 and H-20). ¹³C NMR (100 MHz, CDCl₃) δ 15.21 (C-19), 17.69 (C-17), 18.67 (C-20), 18.75 (C-18), 20.55 (C-3), 25.71 (C-16), 26.10 (C-13), 26.56 (C-1), 26.59 (C-2), 33.41 (C-12), 34.9 (C-11), 43.83 (C-4), 124.13 (C-14), 132.00 (C-15), 135.36 (C-6), 140.04 (C-7), 140.70 (C-9), 150.13 (C-10), 179.85 (C-5), 181.55 (C-8).

5,18-epoxyserrulat-14-en-7,8-dione (6)

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Purple liquid, yield 50 mg, HRESIMS: 315.19573 m/z [M+H]+ (calcd 315.19547) C₂₀H₂₆O₃. ¹H NMR (400 MHz, CDCl₃) δ 5.07 (1H, bt, J=6.8 Hz, H-14), 4.49 (1H, dd, J=11.0, 3.8 Hz, H-18A), 3.86 (1H, t, J=11.1 Hz, H-18B), 2.82 (1H, m, H-1), 2.14 (1H, m, H-4), 2.15 (1H, m, H-2A), 2.13 (1H, m, H-3A), 2.09 (1H, m, H-13A), 1.97 (1H, m, H-13B), 1.81 (3H, s, H-19), 1.76 (1H, m, H-12A), 1.74 (1H, m, H-11), 1.71 (3H, s, H-16), 1.63 (3H, s, H-17), 1.23 (1H, m, H-2B), 1.19 (1H, m, H-12B), 1.13 (3H, d, H-20) 1.11 (1H, m, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ 7.57 (C-19), 17.78 (C-17), 21.67 (C-20), 24.66 (C-13), 25.32 (C-3), 25.71 (C-16), 28.74 (C-1), 29.15 (C-12), 30.77 (C-2), 37.07 (C-11), 40.09 (C-4), 71.85 (C-18), 114.44 (C-6), 123.06 (C-14), 132.87 (C-15), 139.43 (C-9), 149.67 (C-10), 162.76 (C-5), 179.38 (C-7), 181.91 (C-8).

Compound 6 was observed to be formed from oxidation of Compound 1.

Acetylation of Serrulatane Diterpenes
7,8,18-triacetyloxyserrulat-14-ene (7)

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The serrulatane (1) (400 mg) was dissolved in acetic anhydride (1 mL) and in dry pyridine (1 mL) and stirred at room temperature overnight (Davis et al., 1999). The reaction mixture was quenched with distilled water (30 mL), and was extracted with dichloromethane (3×40 mL). The dichloromethane solution was extracted with aqueous 0.1 M hydrochloric acid (30 mL) to remove the residual trace of pyridine. The dichloromethane solutions were dried with Na₂SO₄, filtered and concentrated on a rotary evaporator to give a yellow oily residue. Finally, using normal-phase short-column vacuum chromatography (NPSCVC) with dichloromethane and ethyl acetate (100:0 to 90:10, v/v) the compound (7) (434 mg, 77%) was purified and isolated as a pale yellow oil, yield 27 mg. HRESIMS: 467.2404 m/z [M+Na]⁺ (calcd 467.2206). C₂₆H₃₆O₆. ¹H NMR (400 MHz, CDCl₃) δ 6.92 (1H, s, H-5), 4.97 (1H, bt, J=7.1 Hz, H-14), 4.11 (2H, d, J=6.3 Hz, H-18), 2.92 (1H, m, H-1), 2.88 (1H, m, H-4), 2.31 (3H, s, H-26), 2.29 (3H, s, H-24), 2.14 (3H, s, H-19), 2.12 (1H, m, H-11), 2.06 (3H, s, H-22), 1.99 (1H, m, H-13A), 1.87 (1H, m, H-13B), 1.87 (1H, m, H-3A), 1.87 (1H, m, H-2A), 1.68 (1H, m, H-3B), 1.66 (3H, s, H-16), 1.54 (3H, s, H-17), 1.50 (1H, m, H-2B), 1.33 (1H, m, H-12A), 1.23 (1H, m, H-12B), 1.15 (3H, d, J=7.0 Hz, H-20). ¹³C NMR (100 MHz, CDCl₃) δ 16.08 (C-19), 17.60 (C-17), 19.16 (C-3), 20.36 (C-24), 20.51 (C-26), 20.97 (C-22), 21.45 (C-20), 25.68 (C-16), 26.11 (C-13), 26.96 (C-2), 27.58 (C-1), 28.32 (C-12), 36.92 (C-4), 41.92 (C-11), 65.88 (C-18), 123.94 (C-14), 128.09 (C-5), 128.31 (C-6), 132.04 (C-15), 139.26 (C-7), 140.56 (C-8), 134.06 (C-9), 137.11 (C-10), 168.1 (C-23), 168.34 (C-25), 171.18 (C-21).

5,18-epoxyserrulat-14-ene-8,18-diacetate (8)

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To acetylate compound (2), 80 mg of serrulatane (2) was dissolved in acetic anhydride (1 mL) and in dry pyridine (1 mL) and stirred at room temperature overnight, and with the same workup procedure and using short-column vacuum chromatography with hexane and ethyl acetate (100:0 to 80:20, v/v) the compound (8) (57.8 mg, 58%) was purified and isolated. Red coloured liquid, yield 18 mg, HRESIMS: m/z 423.2143 [M+Na]⁺ calcd 423.2142. C₂₄H₃₂O₅. ¹H NMR (400 MHz, CDCl₃) δ: 6.69 (1H, s, H-7), 6.53 (1H, d, J=1.7 Hz, H-18), 5.08 (1H, bt, J=6.5 Hz, H-14), 2.96 (1H, sextet, J=7.4 Hz, H-1), 2.54 (1H, td, J=11.6, 3.8 Hz, H-4), 2.3 (3H, s, H-24), 2.19 (1H, m, H-13A), 2.19 (1H, m, H-2A), 2.14 (3H, s, H-19), 2.12 (1H, m, H-3A), 2.09 (3H, s, H-22), 2.02, (1H, m, H-13B), 1.70 (1H, m, H-12A), 1.69 (3H, s, H-16), 1.62 (3H, s, H-17), 1.65 (1H, m, H-11), 1.42 (1H, m, H-2B), 1.30 (1H, m, H-12B), 1.23 (3H, d, J=6.8 Hz, H-12), 1.10 (1H, m, H-3B). ¹³C NMR (100 MHz, CDCl₃) δ: 15.88 (C-19), 17.68 (C-17), 21.09 (C-22), 21.23 (C-24), 22.78 (C-20), 25.15 (C-13), 25.70 (C-16), 26.22 (C-3), 28.26 (C-1), 28.29 (C-12), 31.41 (C-2), 32.3 (C-4), 38.93 (C-11), 90.18 (C-18), 41.92 (C-11), 122.53 (C-7), 123.48 (C-14), 123.77 (C-10), 123.96 (C-6), 130.9 (C-9), 132.57 (C-15), 142.44 (C-8), 145.63 (C-5), 170.05 (C-23), 170.19 (C-21).

Methylation of Serrulatane Diterpenes
8-methoxy-7,18-dihydroxyserrulat-14-ene (11) and 7-methoxy-8,18-dihydroxyserrulat-14-ene (12)

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Compound 1 (140 mg) was treated with diazomethane (excess equiv) in diethyl ether at 0° C. to room temperature overnight to give an undetermined 4 to 1 (¹H-NMR) mixture of mono-methylated products 11 and 12 (35 mg, 88% purity by HPLC).

Compounds 11 and 12

Yellow oil Mass Spectrum (positive mode) m/z=333.2 (12%, (M+H)⁺), 315.2 (100%, (M+H-H₂O)⁺), 205.2 (18%, (M+H-C₈H₁₆O)⁺). HPLC analysis: Wavelength, 210 nm, bandwidth 4; Column, SorbTech C₁₈AQ, 2.1×50 mm, 3 μm; Retention time 7.474 min; Mobile phase acetonitrile/formic acid/water; Gradient method, 5-95% CAN+0.1% Formic acid in Water+0.1% Formic acid in 14 min, hold at 95% CAN+Formic acid for 4 min. UV_max, 275 nm and 210 nm, shoulder at 225 nm. ¹H-NMR analysis indicated a 4:1 undetermined mixture of 11 and 12 (interchangeable major and minor isomers). ¹H NMR (400 MHz, CDCl₃) δ: 6.74 (1H, s, H-5)[minor], 6.56, (1H, s, H-5)[major], 5.78 (1H, OH)[major], 5.57 (1H, OH)[minor], 5.01 (1H, bt, J=6.3 Hz, H-14), 3.78 (3H, s, CH₃O)[minor], 3.77 (3H, s, CH₃O)[major], 3.63 (2H, m, H-18), 3.14 (1H, m, H-1)[major], 3.07 (1H, m, H-1)[minor], δ 2.81 (1H, m, H-4), 2.25 (3H, s, H-19)[major], 2.21 (3H, s, H-19)[minor], 2.0-1.8 (5H, m, H-13, H-3A, H-2A, H-11), 1.7-1.6 (1H, m, H-2B), 1.66 (3H, bs, H-16), 1.55 (3H, bs, H-17), 1.52-1.46 (1H, m, H-3B), 1.35-1.23 (2H, m, H-12), 1.21 (3H, d, J=6.8 Hz, H-20)[major] 1.20 (3H, d, J=6.8 Hz, H-20)[minor]. ¹³C NMR (100 MHz, CDCl₃) δ: 15.50 (C-19)[minor], 15.77 (C-19)[major], 17.62 (C-17), 20.22 (C-2)[major], 20.44 (C-2)[minor], 21.08 (C-20)[major], 22.22 (C-20)[minor], 25.66 (C-16), 26.27 (C-13), 26.81 (C-3), 27.14 (C-1)[major], 27.87 (C-1)[minor], 29.14 (C-12)[minor], 29.26 (C-12)[major], 37.53 (C-4)[minor], 37.55 (C-4)[major], 45.43 (C-11)[minor], 45.53 (C-11)[major], 60.64 (OCH₃)[major], 60.89 (OCH₃)[minor], 64.17 (C-18)[major], 64.30 (C-18)[minor], 121.89 (C-5), 124.55 (C-14), 126.39, 126.70, 127.69, 130.42, 131.63, 131.66, 133.17, 135.11, 143.15, 144.85, 145.26, 146.27.

FIG. 1 and FIG. 2 show the ¹H NMR (400 MHz, CDCl₃) and the ¹³C NMR (100 MHz, CDCl₃) spectra of the 4:1 mixture of compounds 11 and 12.

Biological Evaluation of Serrulatane Terpene Compounds

The serrulatane diterpenes 1 to 3 were evaluated for in vitro biological activities using assays for epigenetics (Tables 3 to 12). Table 3 shows the experimental conditions used in epigenetic binding assays. Table 4 shows the results of inhibition of compounds 1 to 3 on binding of epigenetic interacting protein modules. Tables 5 to 8 show representative inhibition data for Compound 1 on binding example epigenetic targets (Table 5: ASH1L (h) bromodomain; Table 6: CECR2(h) bromodomain; Table 7: SP140 (h) bromodomain; Table 8: UHRF1(108-286) (h)). Corresponding binding curves for the data displayed in Tables 5 to 8 are shown in FIGS. 3 to 6. Table 9 shows IC₅₀values from inhibition/concentration-response curves of compounds 1 and 2 for binding of epigenetic interacting protein modules against reference compounds.

Table 10 shows the inhibition of compounds 1 to 3 at 10 μM on activity of epigenetic enzymes against references.

Table 11 shows IC₅₀values from inhibition/concentration-response curves of compounds 1, 2 and 4 for inhibition of epigenetic enzymes against reference compounds. Table 12 shows the pharmacological activity of compounds 1 to 3. Diterpenes 1 to 3 were also evaluated for in vitro pharmacological activity (Tables 13 to 16). Table 13 shows pharmacology assay methods. Table 14 shows pharmacological activity of compounds 1 to 3 at 10 μM. Table 15 shows activity of Compound 1 against 5-lipoxygenase, lipid peroxidase and monoamine oxidase (MAO-A) IC₅₀(μM) and reference compounds. Table 16 shows the effect of compounds 1 to 4 at 10 μM on 2-deoxy-D-glucose uptake into muscle cells.

Table 17 shows dose response and IC₅₀values for the pharmacological activity of representative serrulatane diterpenes.

Diterpenes 1 to 4 and Myoporum insulare resin were evaluated in a cell viability assay (Table 18).

Diterpenes 1, 2, 4 and Myoporum insulare resin were assayed for cell cancer cell proliferation in an OncoPanel assay (Table 19 to 29). Table 21 shows Oncopanel cell proliferation results for Compounds 11 and 12.

Epigenetic Assays

Epigenetic binding assays were carried out by Eurofins CEREP (France). The conditions used in the assays including: enzymes, source of enzymes, substrate, substrate concentration, ligand, ligand concentration, tracer, incubation, detection method, and reference compound and concentration are detailed in Table 3. Appropriate literature references are also provided. The AlphaScreen detection method refers to an Amplified Luminescent Proximity Homogeneous Assay used to study biomolecular interactions (see Ullman et al. 1994). LANCE and LanthaScreen assays are based on the principle of time-resolved fluorescence energy transfer (TR-FRET) and are described, for example, in Ma et al. 2008.

The IC₅₀values (concentration causing a half-maximal inhibition of control specific activity) were determined by non-linear regression analysis of the inhibition/concentration-response curves generated with mean replicate values using Hill equation curve fitting. Inhibition/concentration-response curve concentrations were 30, 3, 0.3, 0.03 and 0.003 μM.

TABLE 3

Epigenetic binding assay details

Assay
Main Feature

Bromodomain

ATAD2B bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

Ligand concentration
75 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
Ischemin sodium salt (IC50: 76.6 μM)

ASH1L bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H4 di acetyl Lys 5/8

Ligand concentration
75 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 13.8 μM)

BAZ2A bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
25 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
G5K2801 (IC50: 1.8 μM)

BRPF1-1 bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

Ligand concentration
10 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
Bromosporine (IC50: 0.026 μM)

CECR2 bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

Ligand concentration
10 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
Bromosporine (IC50: 0.062 μM)

EP300 bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H3 acetyl Lys56

2012]
Ligand concentration
25 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
SGC-CBP30 (IC50: 0.050 μM)

KAT2A (GCN5L2) bromodo
Source
human recombinant (E. coli)

main [Filippakopoulos et al.
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
30 nM

Incubation
15 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 45 μM)

PCAF bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
100 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 12 μM)

PB1(2) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H3 acetyl Lys 14

Ligand concentration
60 nM

Incubation
15 min/RT

Detection method
AlphaScreen

Reference:
PFI-3 (IC50: 68 μM)

PB1(3) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H3 acetyl Lys14

Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 47 μM)

PB1(4) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H3 acetyl Lys14

Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
Ischemin sodium salt (IC50: 6.8 μM)

PHIP(2) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H4 diacetyl Lys5/8

Ligand concentration
100 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
SGC-CBP30 (IC50: 0.130 μM)

SP140 bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
biotin-H3 acetyl Lys 9

Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
Ischemin (IC50: 86 μM)

SMARCA2 bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
Biotin H3 mono acetyl Lys 14

Ligand concentration
5 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
PFI-3 (IC50: 0.47 μM)

TAF1(1) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
100 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
G5K2801 (IC50: 4.4 μM)

SMARCA4 bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos et al. 2012]
Ligand
Biotin -H3 mono acetyl Lys14

Ligand concentration
15 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
PFI-3 (IC50: 3 μM)

TAF1(2) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
100 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
G5K2801 (IC50: 10 μM)

BAZ2B bromodomain
Source
human recombinant (E. coli)

[Philpott et al. 2011]
Ligand
biotin-H3 acetyl Lys14

Ligand concentration
25 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
G5K2801 (IC50: 1.8 μM)

ATAD2A bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
100 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 70 μM)

BRD2(1) bromodomain
Source
human recombinant (E. coli)

[Philpott et al. 2011]
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-11 (IC50: 0.510 μM)

BRD2(2) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 0.017 μM)

BRD3(1) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
25 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 0.040 μM)

BRD3(2) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 di acetyl Lys 5/8

2012]
Ligand concentration
100 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
PFI-1 (IC50: 0.79 μM)

BRD4(1) bromodomain
Source
human recombinant (E. coli)

[Philpott et al. 2011]
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 0.018 μM)

BRD4(2) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
PFI-1 (IC50: 1.3 μM)

BRDT(1) bromodomain
Source
human recombinant (E. coli)

[Filippakopoulos and Knapp
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

2012]
Ligand concentration
25 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
JQ-1 (IC50: 0.110 μM)

CREBBP bromodomain
Source
human recombinant (E. coli)

[Philpott et al. 2011]
Ligand
biotin-H3 acetyl Lys56

Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
SGC-CBP30 (IC50: 0.120 μM)

FALZ bromodomain
Source
human recombinant (E. coli)

[Philpott et al. 2011]
Ligand
biotin-H4 tetra acetyl Lys 5/8/12/16

Ligand concentration
50 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
I-CBP112 (IC50: 24.5 μM)

Chromodomain

CBX1 [Kaustov et al. 2011]
Source
human recombinant (E. coli)

Ligand
biotin- H3 trimethylated Lys9

Ligand concentration
5 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

CBX2 [Kaustov et al. 2011]
Source
human recombinant (E. coli)

Ligand
biotin- H3 trimethylated Lys27

Ligand concentration
20 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

CBX4 [Kaustov et al. 2011]
Source
human recombinant (E. coli)

Ligand
biotin- H3 trimethylated Lys27

Ligand concentration
8 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

CBX6 [Kaustov et al. 2011]
Source
human recombinant (E. coli)

Ligand
biotin- H3 trimethylated Lys27

Ligand concentration
5 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

CBX5 [Kaustov et al. 2011]
Source
human recombinant (E. coli)

Ligand
biotin- H3 trimethylated Lys9

Ligand concentration
6 nM

Incubation
15 min/RT

Detection method
AlphaScreen

Reference:
NA

CBX7 [Kaustov et al. 2011]
Source
human recombinant (E. coli)

Ligand
biotin- H3 trimethylated Lys27

Ligand concentration
10 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

CBX8 [Kaustov et al. 2011]
Source
human recombinant (E. coli)

Ligand
biotin- H3 trimethylated Lys27

Ligand concentration
55 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

MBT domain

L3MBTL1 [Kim et al. 2006]
Source
human recombinant (E. coli)

Ligand
biotin- H3 methylated Lys4

Ligand concentration
50 nM

Incubation
15 min/RT

Detection method
AlphaScreen

Reference:
NA

L3MBTL3 [Kim et al. 2006]
Source
human recombinant (E. coli)

Ligand
biotin-H4 dimethylated lysine 20

Ligand concentration
60 nM

Incubation
15 min/RT

Detection method
AlphaScreen

Reference:
NA

PHD Domain

SP140 [Org et al. 2008]
Source
human recombinant (E. coli)

Ligand
Biotin H3K4me

Ligand concentration
15 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

TRIM 33 [Venturini et al.
Source
human recombinant (E. coli)

1999]
Ligand
biotin- H3 trimethylated Lys9

Ligand concentration
8 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

UHRF1(108-286) [Xie et al.
Source
human recombinant (E. coli)

2012]
Ligand
biotin-H3 trimethylated Lys9

Ligand concentration
15 nM

Incubation
15 min/RT

Detection method
AlphaScreen

Reference:
NA

PHF20(1) [Adams-Cioba et al.
Source
human recombinant (E. coli)

2012]
Ligand
biotin- H4 dimethylated Lys20

Ligand concentration
100 nM

Incubation
30 min/RT

Detection method
AlphaScreen

Reference:
NA

Cell-based detection methyl modifications

H3K27 ac [Hayashi-Takanaka
Source
HeLa Cells

et al. 2011]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

H3K27 me2-1 [Kubicek, S.
Source
MCF7 cells

et al. 2007]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

H3K27 me3 [Kubicek, S.
Source
SU-DHL-6 cells

et al. 2007]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

H3K36 me2 [Kubicek, S.
Source
HeLa cells

et al. 2007]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

H3K4 me2 [Ken-ichi Noma
Source
HeLa cells

and Grewal 2002]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

H3K79 me2 [Kubicek, S.
Source
MCF7 cells

et al. 2007]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

H3K9 ac [Hayashi-Takanaka
Source
HeLa cells

et al. 2011]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

H3K9 me2 [Kubicek, S.
Source
HeLa cells

et al. 2007]
Incubation
24 h/37° C.

Detection method
AlphaScreen

Reference:
NA

Demethylases (KDMs)

FBXL10 (h) [Rotili and Mai
Source
human recombinant (Sf9 cells)

2011]
Substrate
biotin-H3K36me2 (24 nM)

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.61 μM)

FBXL11 (h) [Chowdhury
Source
human recombinant (E. coli)

2011]
Ligand
biotin- H3K36me2

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 1.5 μM)

JARID1A (h) [Nottke et al.
Source
human recombinant (Sf9 cells)

2009]
Ligand
biotin-H3K4me3

Ligand concentration
100 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.18 μM)

JARID1B (h) [Kristensen
Source
human recombinant (Sf9 cells)

2012]
Ligand
biotin-H3K4me3

Ligand concentration
60 nM

Incubation
30 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.15 μM)

JARID1C (h) [Nottke et al.
Source
human recombinant (Sf9 cells)

2009]
Substrate
biotin-H3K4me3

Substrate concentration
15 nM

Incubation
30 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.23 μM)

JMJD1A (h) [Heightman
Source
human recombinant (E. coli)

2011]
Substrate
biotin-H3K4me1

Substrate concentration
25 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.76 μM)

JMJD2A (h) [King et al.
Source
human recombinant (E. coli)

2010]
Ligand
biotin-H3K9me3

Ligand concentration
100 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.083 μM)

JMJD2B (h) [King et al.
Source
human recombinant (E. coli)

2010]
Ligand
biotin- H3K9me3

Ligand concentration
100 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.14 μM)

JMJD2C (h) [King et al.
Source
human recombinant (E. coli)

2010]
Ligand
biotin- H3K9me3

Ligand concentration
100 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.091 μM)

JMJD2D (h) [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.091 μM)

JMJD2E (h) [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin-H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
2,4 PDCA (IC50: 0.061 μM)

JMJD3 (h) [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE 1198

Reference:
2,4 PDCA (IC50: 52 μM)

LSD1 (h) [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
Tranylcypromine (IC50: 22 μM)

PHF8(h) [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE 1198

Reference:
Daminozide (IC50: 0.28 μM)

UTX (h) [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin-H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
IOX1 (0.15 μM)

DNA methyltransferases (DNMTs)

DNMT1 (h) [Pradhan et al.
Source
human recombinant (E. coli)

1999]
Substrate
Poly (dI-dC)-Poly(dIdC) [6 mU/mL]

Tracer
[3H] SAM (50 nM)

Incubation
30 min/37° C.

Detection method
Scintillation counting

Reference:
SAH (IC50: 0.23 μM)

DNMT3b [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
SAH (IC50: 0.094 μM)

DNMT3B/DNMT3L (h)
Source
human recombinant (Sf9 cells)

[Suetake et al. 2004]
Substrate
Poly (dI-dC)-Poly(dIdC) [0.15 mU/mL]

Tracer
[3H] SAM (200 nM)

Incubation
20 min/37° C.

Detection method
Scintillation counting

Reference:
SAH (IC50: 0.045 μM)

hDNMT3a [Aoki et al. 2001]
Source
human recombinant (Sf9 cells)

Substrate
Poly (dI-dC)-Poly(dIdC) [0.15 mU/mL]

Tracer
[3H] SAM (200 nM)

Incubation
20 min/37° C.

Detection method
Scintillation counting

Reference:
SAH (IC50: 0.052 μM)

Histone acetyltransferases (HATs)

CREBBP(h) [Hong et al.
Source
human recombinant (E. coli)

2007]
Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
Garcinol (IC50: 1.5 μM)

GCN5L2(h) [Hong et al.
Source
human recombinant (Sf9 cells)

2007]
Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
LANCE

Reference:
Anacardic acid (IC50: 4.7 μM)

HAT1(h) [Zhang et al. 2012]
Source
human recombinant (E. coli)

Ligand
biotin-H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Garcinol (IC50: 1.5 μM)

MYST3(h) [Zhang et al. 2012]
Source
human recombinant (E. coli)

Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Garcinol (IC50: 1.8 μM)

MYST4 (h) [Zhang et al. 2012]
Source
human recombinant (E. coli)

Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
Scintillation counting 1293

Reference:
Curcumin (IC50: 11 μM)

pCAF(h) [Zhang et al. 2012]
Source
human recombinant (E. coli)

Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
Scintillation counting 1293

Reference:
Garcinol (IC50: 2.9 μM)

TIP60 (h) [Zhang et al. 2012]
Source
human recombinant (Sf 21 cells)

Ligand
biotin- H3K27me3

Ligand concentration
50 nM

Incubation
10 min/RT

Detection method
Scintillation counting 1293

Reference:
Garcinol (IC50: 1.5 μM)

Histone deacetylase (HDACs) [Strahl and Allis 2000]

HDAC1 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate

Substrate concentration
20 μM

Incubation
10 min/RT

Detection method
fluoro-lysine fluorimetry

Reference:
trichostatin A (IC50: 0.0052 μM)

HDAC2 (h)
Source
human recombinant fluorogenic HDAC substrate

Ligand
NA

Substrate concentration
20 μM

Incubation
15 min/RT

Detection method
fluoro-lysine fluorimetry 896

Reference:
trichostatin A (IC50: 0.024 μM)

HDAC3 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate

Substrate concentration
50 μM

Incubation
10 min/RT

Detection method
fluoro-lysine fluorimetry

Reference:
trichostatin A (IC50: 0.0068 μM)

HDAC4 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate

Substrate concentration
20 μM

Incubation
30 min/RT

Detection method
fluoro-lysine fluorimetry

Reference:
trichostatin A (IC50: 4.0 μM)

HDAC5 (h)
Source
human recombinant

Ligand
fluorogenic HDAC substrate

Substrate concentration
20 μM

Incubation
30 min/RT

Detection method
fluoro-lysine fluorimetry

Reference:
trichostatin A (IC50: 1.0 μM)

HDAC6 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate

Substrate concentration
25 μM

Incubation
30 min/RT

Detection method
fluoro-lysine fluorimetry

Reference:
trichostatin A (IC50: 0.0074 μM)

HDAC7 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate class 2a

Substrate concentration
50 μM

Incubation
45 min/RT

Detection method
fluoro-lysine fluorimetry

Reference:
trichostatin A (IC50: 1.8 μM)

HDAC8 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate class 2a

Substrate concentration
50 μM

Incubation
45 min/RT

Detection method
fluoro-lysine fluorimetry 896

Reference:
trichostatin A (IC50: 0.41 μM)

HDAC9 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate class 2a

Substrate concentration
50 μM

Incubation
30 min/RT

Detection method
fluoro-lysine fluorimetry 896

Reference:
trichostatin A (IC50: 9.1 μM)

HDAC10 (h)
Source
human recombinant

Substrate
fluorogenic HDAC substrate class 2a

Substrate concentration
50 μM

Incubation
45 min/RT

Detection method
fluoro-lysine fluorimetry 896

Reference:
trichostatin A (IC50: 0.009 μM)

HDAC11 (h)
Source
human recombinant (E. coli)

Substrate
fluorogenic HDAC substrate class 2a

Substrate concentration
50 μM

Incubation
30 min/37° C.

Detection method
fluoro-lysine fluorimetry 896

Reference:
Scriptaid (IC50: 8.9 μM)

Sirtuins

sirtuin 1 (h) (inhibitor effect)
Source
human recombinant (E. coli)

[Strahl and Allis 2000]
Substrate
fluorogenic HDAC substrate class 2a

Substrate concentration
50 μM

Incubation
30 min/37° C.

Detection method
fluoro-lysine fluorimetry

Reference:
Suramin (IC50: 7.9 μM)

sirtuin 2 (h) (inhibitor effect)
Source
human recombinant (E. coli)

[Michan and Sinclair 2007]
Substrate
fluoro-lysine sirtuin 2 deacetylase substrate

Substrate concentration
150 μM

Incubation
60 min/RT

Detection method
fluoro-lysine fluorimetry

Reference:
Suramin (IC50: 21 μM)

sirtuin 3 (h) (inhibitor effect)
Source
human recombinant (E. coli)

[Strahl and Allis 2000]
Substrate
fluorogenic HDAC substrate class 2a

Substrate concentration
50 μM

Incubation
30 min/37° C.

Detection method
fluoro-lysine fluorimetry

Reference:
Niacinamide (IC50: 21 μM)

Sirtuin 6 (h) [Michishita et al.
Source
human recombinant (E. coli)

2008]
Substrate
Fluorogenic HDAC

Substrate concentration
50 μM

Incubation
180 min/RT

Detection method
fluoro-lysine fluorimetry

Reference
EX-527 (IC50: 550 μM)

Sirtuin 7 (h) [Kim and Kim
Source
human recombinant (Sf9 cells)

2013]
Substrate
fluorogenic HDAC

Substrate concentration
25 μM

Incubation
60 min/37° C.

Detection method
fluoro-lysine fluorimetry

Reference:
JFD00244 (IC50: 1300 μM)

Methyltransferases (MTs)

ASH1L [An et al. 2011]
Source
human recombinant (E. coli)

Substrate
polynucleosome (1.5 μg/mL)

Tracer
[3H] SAM (150 nM)

Incubation
15 min/RT

Detection method
Scintillation counting

Reference:
SAH (IC50: 9.1 μM)

DOT1L (h) [An et al. 2011]
Source
human recombinant (E. coli)

Substrate
polynucleosome (2.5 μg/mL)

Tracer
[3H] SAM (150 nM)

Incubation
15 min/RT

Detection method
Scintillation counting

Reference:
SAH (IC50: 0.14 μM)

EHMT1 (h) [Yost et al. 2011]
Source
human recombinant (E. coli)

Substrate (concentration)
histone H3 full length (10 nM)

Co-substrate (concentration)
[3H] SAM (25 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (IC50: 0.18 μM)

EZH1/EED/SUZ12 (h) [Shen
Source
human recombinant

et al. 2008]
Substrate (concentration)
histone H3 full length (70 nM)

Co-substrate (concentration)
[3H] SAM (120 nM)

Incubation
90 min/RT

Detection method
Scintillation counting

Reference:
SAH (IC50: 8.4 μM)

EZH2/EED/SUZ12 (h)
Source
human recombinant (Sf9 cells)

(PRC2 complex) [Philpott et
Substrate (concentration)
histone H3 full length (50 nM)

al. 2011]
Co-substrate (concentration)
[3H] SAM (35 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (IC50: 22 μM)

G9a (h) [Yost et al. 2011]
Source
human recombinant (E. coli)

Substrate
histone H3 full length (5 nM)

Co-substrate
[3H] SAM (25 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (2.1 μM)

hSMYD2 [An et al. 2011]
Source
human recombinant (Sf9 cells)

Substrate
histone H4 full length (25 nM)

Co-substrate
[3H] SAM 150 nM

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.16 μM)

MLL complex (h) [Jiang et al.
Source
human recombinant (E. coli)

2013]
Substrate
Histone H3 full length (35 nM)

Tracer
[3H] SAM (200 nM)

Incubation
30 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.97 μM)

MLL2(h) complex [Jiang et al.
Source
human recombinant (E. coli)

2013]
Substrate
Histone H3 full length (35 nM)

Tracer
[3H] SAM (200 nM)

Incubation
30 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.97 μM)

MLL3(h) complex [Ali et al.
Source
human recombinant (E. coli)

2014]
Substrate
Core histone (20 nM)

Tracer
[3H] SAM (200 nM)

Incubation
20 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.97 μM)

MLL4(h) complex [Ali et al.
Source
human recombinant (E. coli)

2014]
Substrate
Core histone (30 nM)

Tracer
[3H] SAM (200 nM)

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.97 μM)

NSD1 (h) [Allali-Hassani et
Source
human recombinant (E. coli)

al. 2014]
Substrate
Histone H3 full length (35 nM)

Tracer
[3H] SAM (200 nM)

Incubation
30 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.97 μM)

NSD3/WHSC1L1 (h) [Selvi
Source
human recombinant (E. coli)

et al. 2010]
Substrate
Histone H3 full length (35 nM)

Tracer
[3H] SAM (200 nM)

Incubation
30 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.97 μM)

PRDM9 (h) [Munoz-Fuentes
Source
human recombinant (E. coli)

et al. 2011]
Substrate
Histone H3 full length (250 nM)

Co-Substrate
[3H] SAM (60 nM)

Incubation
30 min/RT

Detection method
Scintillation counting

Reference:
SAH (370 μM)

PRMT1 (h) [Cheng et al.
Source
human recombinant (E. coli)

2004]
Substrate
histone H4 full length (25 nM)

Co-substrate
[3H] SAM (60 nM)

Incubation
90 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.094 μM)

PRMT3 (h) [Li et al. 2012]
Source
human recombinant (E. coli)

Substrate
histone H4 full length (25 nM)

Co-substrate
[3H] SAM (60 nM)

Incubation
90 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.86 μM)

PRMT4 (h) [Selvi et al. 2010]
Source
human recombinant (Sf9 cells)

Substrate
histone H4 full length (25 nM)

Co-substrate
[3H] SAM (60 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.094 μM)

PRMT5 complex (h) [Yost et al.
Source
human recombinant (Sf9 cells)

2011]
Substrate
histone H4 full length (250 nM)

Co-substrate
[3H] SAM (600 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.65 μM)

PRMT6 (h) [Iberg et
Source
human recombinant (Sf9 cells)

al. 2007]
Substrate
histone H4 full length (250 nM)

Co-substrate
[3H] SAM (600 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.65 μM)

PRMT7 (h) [Zurita-Lopez
Source
human recombinant (Sf9 cells)

et al. 2012]
Substrate
histone H2B (21-41) biotin labeled (4 nM)

Co-substrate
[3H] SAM (250 nM)

Incubation
90 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.65 μM)

PRMT8 (h) [Lee et al. 2005]
Source
human recombinant (Sf9 cells)

Substrate
histone H4 full length (250 nM)

Co-substrate
[3H] SAM (600 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (0.65 μM)

SETD2(h) [Du et al. 2008]
Source
human recombinant (E. coli)

Substrate
Nucleosome (0.5 μg/mL)

Co-substrate
[3H] SAM (250 nM)

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
SAH (1.2 μM)

SETD7 (h) [Li et al. 2012]
Source
human recombinant (E. coli)

Substrate
histone H3 full length (120 nM)

Co-substrate
[3H] SAM (850 nM)

Incubation
30 min/RT

Detection method
Scintillation counting

Reference:
SAH (24 μM)

SETD8 (h) [Yost et al. 2011]
Source
human recombinant (E. coli)

Substrate
histone H3 full length (120 nM)

Co-substrate
[3H] SAM (850 nM)

Incubation
30 min/RT

Detection method
Scintillation counting

Reference:
SAH (24 μM)

SETDB1 (h) [Schultz et al.
Source
human recombinant (Sf9 cells)

2002]
Substrate
histone H3 full length (15 nM)

Co-substrate
[3H] SAM (25 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (1.8 μM)

SUV39H2 (h) [Yost et al.
Source
human recombinant (Sf9 cells)

2011]
Substrate
histone H3 full length (500 nM)

Co-substrate
[3H] SAM (350 nM)

Incubation
120 min/RT

Detection method
Scintillation counting

Reference:
SAH (24 μM)

SUV4-20H2 (h) [Li et al.
Source
human recombinant (E. coli)

2012]
Substrate
Nucleosome (1.5 μg/ml)

Co-substrate
[3H] SAM (75 nM)

Incubation
15 min/RT

Detection method
Scintillation counting

Reference:
SAH (24 μM)

WHSC1(h) (NSD2(h))
Source
human recombinant (E. coli)

[Kang et al. 2009]
Substrate
Core Histone (1500 nM)

Tracer
[3H] SAM (250 nM)

Incubation
15 min/RT

Detection method
Scintillation counting

Reference:
Chaetocin (IC50: 0.48 μM)

Kinases

Aurora B (h) (substrate
Source
Baculovirus

histone H3 full length)
Substrate
Histone H3 full length (150 nM)

[Sabbattini et al. 2007]
Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (0.038 μM)

DAPK3/ZIP (substrate
Source
Baculovirus

histone H3 full length)
Substrate
Histone H3 full length (50 nM)

[Preuss et al. 2003]
Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.0073 μM)

Haspin (h) (substrate histone
Source
human recombinant (E. coli)

H3 full length) [Han et al.
Substrate
baculovirus Histone H3 full length (250 nM)

2011]
Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.0073 μM)

IKK alpha(h) (substrate histone
Source
human recombinant (Sf21 cells)

H3 full length) [Baek 2011]
Substrate
Histone H3 full length (50 nM)

Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.03 μM)

MSK1(h) (substrate histone
Source
human recombinant insect cells

H3 full length) [Baek 2011]
Substrate
Histone H3 full length (100 nM)

Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.015 μM)

MSK2(h) (substrate histone
Source
human recombinant (insect cells)

H3 full length) [Baek 2011]
Substrate
Histone H3 full length (20 nM)

Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.0058 μM)

PIM1(h) (histone H3 full
Source
human recombinant (Sf21 cells)

length substrate) [Baek 2011]
Substrate
Histone H3 full length (50 nM)

Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.03 μM)

PKBalpha/AKT1(h)
Source
human recombinant (Sf21 cells)

(substrate histone H3 full length)
Substrate
Histone H3 full length (50 nM)

[Barnett et al. 2005]
Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.03 μM)

PKBbeta/AKT2(h)
Source
human recombinant (baculovirus)

(substrate histone H3 full
Substrate
Histone H3 full length (100 nM)

length) [Barnett et al. 2005]
Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.03 μM)

PKBgamma/AKT3(h)
Source
human recombinant (baculovirus)

(substrate histone H3 full
Substrate
Histone H3 full length (150 nM)

length) [Baek 2011]
Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.03 μM)

Rsk2(h) (substrate histone H3
Source
human recombinant (baculovirus)

full length) [Baek 2011]
Substrate
Histone H3 full length (250 nM)

Tracer
[33P] ATP

Incubation
10 min/RT

Detection method
Scintillation counting

Reference:
Staurosporine (IC50: 0.007 μM)

Small molecule methyltransferases

Catechol O-methyltransferase
Source
human recombinant (E. coli)

(h)
Substrate
Pyrocatechol (250 nM)

Tracer
SAM (10 μM)

Incubation
15 min/37° C.

Detection method
MS

Reference:
SAH-d4 (IC50: 14 μM)

Glycine N-
Source
human recombinant (E. coli)

methyltransferase (h)
Substrate
Glycine (100 μM)

Tracer
SAM (20 μM)

Incubation
30 min/22° C.

Detection method
MS

Reference:
SAH-d4 (IC50: 17 μM)

Guanidinoacetate N-
Source
human recombinant (E. coli)

methyltransferase (h)
Substrate
Guanidineacetic acid (4 μM)

Tracer
SAM (7 μM)

Incubation
30 min/22° C.

Detection method
MS

Reference:
SAH-d4 (IC50: 2.3 μM)

Histamine N-
Source
human recombinant (E. coli)

methyltransferase (h)
Substrate
Histamine (4 μM)

Tracer
SAM (4 μM)

Incubation
30 min/22° C.

Detection method
MS

Reference:
SAH-d4 (IC50: 13 μM)

Nicotinamide N-
Source
human recombinant (E. coli)

methyltransferase (h)
Substrate
Nicotinamide (8 μM)

Tracer
SAM (6 μM)

Incubation
15 min/22° C.

Detection method
MS

Reference:
SAH-d4 (IC50: 13 μM)

Phenylethanolamine N-
Source
human recombinant (E. coli)

methyltransferase (h)
Substrate
DL-Normetanephrine SAM (35 μM)

Tracer
SAM (6 μM)

Incubation
45 min/22° C.

Detection method
MS

Reference:
SAH-d4 (IC50: 13 μM)

Thiopurine S-
Source
human recombinant (E. coli)

methyltransferase (h)
Substrate
6-mercaptopurine (1.5 μM)

Tracer
SAM (1.5 μM)

Incubation
30 min/22° C.

Detection method
MS

Reference:
SAH-d4 (IC50: 2.3 μM)

Ubiquitin modifying enzymes

BAP1 (h) [Misaghi et al.
Source
human recombinant (E. coli)

2009]
Substrate
LanthaScreen DUB substrate

Substrate concentration
20 nM

Incubation
180 min/RT

Detection method
LanthaScreen

Reference:
Ubiquitin aldehyde (IC50: 0.23 μM)

USP 10 (h) [Horton et al.
Source
human recombinant (Sf9 cells)

2007]
Substrate
LanthaScreen DUB substrate

Substrate concentration
10 nM

Incubation
60 min/22° C.

Detection method
LanthaScreen

Reference:
Ubiquitin aldehyde (IC50: 0.23 μM)

USP 14 (h) [Hu et al. 2005]
Source
human recombinant (Sf9 cells)

Substrate
LanthaScreen DUB substrate

Substrate concentration
10 nM

Incubation
240 min/RT

Detection method
LanthaScreen

Reference:
Ubiquitin Aldehyde (0.004 μM)

USP 16 (h) [Horton et al.
Source
Human recombinant HEK 293 cells

2007]
Substrate
LanthaScreen DUB substrate

Substrate concentration
20 nM

Incubation
180 min/22° C.

Detection method
LanthaScreen

Reference:
Iodoacetamide (3.5 μM)

USP 21(h) [Ye et al. 2011]
Source
human recombinant (E. coli)

Substrate
LanthaScreen DUB substrate

Substrate concentration
10 nM

Incubation
15 min/RT

Detection method
LanthaScreen

Reference:
Ubiquitin aldehyde (IC50: 0.17 μM)

USP 7 (h) [Tian et al. 2011]
Source
human recombinant (Sf21 cells)

Substrate
LanthaScreen DUB substrate

Substrate concentration
10 nM

Incubation
60 min/RT

Detection method
LanthaScreen

Reference:
Ubiquitin Aldehyde (0.11 μM)

Results for the binding assays are shown below in Table 4.

TABLE 4

Inhibition of Compounds 1 to 3 at 10 μM on binding of epigenetic

interacting protein modules against reference compounds.

% Inhibition^A
Reference

Comp
Comp
Comp

IC₅₀

Binding Assay
1
2
3
Compound
μM

Bromodomain

ATAD2B (h)
52
25
28
Ischemin sodium
76.6

salt

ASH1L (h)
95
59
42
JQ-1
13.8

BAZ2A (h)
56
17
16
GSK2801
1.8

BRPF1-1 (h)
−25
−25
−4
Bromosporine
0.026

CECR2 (h)
100
25
15
Bromosporine
0.062

EP300 (h)
89
37
40
SGC-CBP30
0.050

KAT2A
76
19
18
JQ-1
45

(GCN5L2) (h)

PCAF (h)
37
16
3
JQ-1
12

PB1(2) (h)
31
22
13
PFI-3
68

PB1(3) (h)
38
13
6
JQ-1
47

PB1(4) (h)
41
−3
16
Ischemin sodium
6.8

salt

PHIP(2) (h)
61
30
23
SGC-CBP30
0.130

SP140 (h)
84
43
1
Ischemin
86

SMARCA2 (h)
83
21
18
PFI-3
0.47

TAF1(1) (h)
48
17
17
GSK2801
4.4

SMARCA4 (h)
53
16
13
PFI-3
3

TAF1(2)(h)
62
28
22
GSK2801
10

BAZ2B (h)
58
23
21
GSK2801
1.8

ATAD2A (h)
100
16
9
JQ-1
70

BRD2(1) (h)
60
89
19
JQ-1
0.510

BRD2(2) (h)
58
22
18
JQ-1
0.017

BRD3(1) (h)
47
17
13
JQ-1
0.040

BRD3(2) (h)
35
25
1
PFI-1
0.79

BRD4(1) (h)
49
11
8
JQ-1
0.018

BRD4(2) (h)
76
16
13
PFI-1
1.3

BRDT(1) (h)
43
23
29
JQ-1
0.110

CREBBP (h)
65
21
17
SGC-CBP30
0.120

FALZ (h)
42
18
5
I-CBP112
24.5

Chromodomain

CBX1 (h)
15
0
−1
NA

CBX2 (h)
17
6
3
NA

CBX4 (h)
25
29
40
NA

CBX6 (h)
−1
27
58
NA

CBX5 (h)
25
24
25
NA

CBX7 (h)
25
33
43
NA

CBX8 (h)
27
31
23
NA

MBT domain

L3MBTL1
41
61
59
NA

L3MBTL3 (h)
22
29
33
NA

PHD domain

SP140 (h)
74
0
2
NA

TRIM 33 (h)
59
3
−2
NA

UHRF1(108-
73
34
29
NA

286) (h)

Tudor domain

PHF20(1)
7
2
2
NA

^A% Inhibition of Control Specific Binding

NA Not-applicable

Representative inhibition data for Compound 1 on binding example epigenetic targets is shown in Tables 5 to 8.

TABLE 5

ASH1L (h) bromodomain

Conc.
1^st
2^nd
Mean

3.0E−09M
−2.6
−8.2
−5.4

3.0E−08M
−12.3
1.8
−5.2

3.0E−07M
−0.2
−2.9
−1.6

3.0E−06M
17.0
28.0
22.5

3.0E−05M
100.6
99.6
100.1

TABLE 6

CECR2(h) bromodomain binding curve

Conc.
1^st
2^nd
Mean

3.0E−09M
5.9
2.4
4.2

3.0E−08M
−0.9
−1.7
−1.3

3.0E−07M
−1.5
0.9
−0.3

3.0E−06M
7.3
8.6
7.9

3.0E−05M
99.4
99.6
99.5

TABLE 7

SP140 (h) bromodomain binding curve.

Conc.
1^st
2^nd
Mean

3.0E−09M
−6.0
−8.6
−7.3

3.0E−08M
−9.7
−14.6
−12.1

3.0E−07M
−14.1
−11.0
−12.6

3.0E−06M
8.4
11.3
9.9

3.0E−05M
102.3
102.3
102.3

TABLE 8

UHRF1(108-286) (h) binding curve.

Conc.
1^st
2^nd
Mean

3.0E−09M
−11.9
−13.1
−12.5

3.0E−08M
−13.3
−12.6
−12.9

3.0E−07M
−12.1
−13.2
−12.7

3.0E−06M
−1.8
−0.1
−0.9

3.0E−05M
81.5
83.3
82.4

TABLE 9

IC₅₀values from inhibition/concentration-response curves

of compounds 1 and 2 for binding of epigenetic interacting

protein modules against reference compounds.

Compound

1
2

Reference

IC₅₀
IC₅₀
Reference
IC₅₀

(μm)
(μm)
Comp.
(μm)

Bromodomain

ASH1L (h)
4.2

JQ-1
8.7

ATAD2A (h)
3.1

Ischemin
5.2

BRD2(1) (h)

22
1-BET 151
0.028

BRD4(2) (h)
5.6

PFI-1
1.1

CECR2(h)
5

Bromosporine
0.14

CREBBP (h)
3.4

SGC-CBP30
0.046

EP300 (h)
4.5

SGC-CBP30
0.068

KAT2A (GCN5L2)
3.9

GSK2801
10

(h)

SP140 (h)
3.8

Ischemin
11

PHD domain

SP140 (h)
4.6

NA
NA

UHRF1(108-286) (h)
11

NA
NA

TABLE 10

Inhibition of compounds 1 to 3 at 10 μM on

activity of epigenetic enzymes against references.

% Inhibition^A
Reference

Comp
Comp
Comp

IC₅₀

Assay
1
2
3
Compound
μm

Cell-based detection methyl modifications

H3K27 ac
−16
−12
−11
(Not-applicable) NA

H3K27 me2-1
−2
3
2
NA

H3K27 me3
−8
−7
0
NA

H3K36 me2
10
7
−8
NA

H3K4 me2
−5
−1
−9
NA

H3K79 me2
−2
−16
−5
NA

H3K9 ac
17
3
−1
NA

H3K9 me2
1
11
20
NA

Demethylases (KDMs)

FBXL10 (h)
37
7
22
2,4 PDCA
0.61

FBXL11 (h)
34
2
11
2,4 PDCA
1.5

JARID1A (h)
64
−9
3
2,4 PDCA
0.18

JARID1B (h)
70
6
−2
2,4 PDCA
0.15

JARID1C (h)
49
−13
25
2,4 PDCA
0.23

JMJD1A (h)
80
37
−1
2,4 PDCA
0.76

JMJD2A (h)
29
−10
12
2,4 PDCA
0.083

JMJD2B (h)
65
16
8
2,4 PDCA
0.14

JMJD2C (h)
35
2
8
2,4 PDCA
0.091

JMJD2D (h)
36
−17
18
2,4 PDCA
0.091

JMJD2E (h)
65
10
6
2,4 PDCA
0.061

JMJD3 (h)
91
9
11
2,4 PDCA
52

LSD1 (h)
10
10
−3
Tranylcypromine
22

PHF8(h)
36
−11
13
Daminozide
0.28

UTX (h)
68
5
27
IOX1
0.15

DNA methyltransferases (DNMTs)

DNMT1 (h)
10
4
−5
SAH
0.23

DNMT3b
−3
−17
−4
SAH
0.094

DNMT3B/
23
27
−20
SAH
0.045

DNMT3L (h)

hDNMT3a
3
3
−31
SAH
0.052

Histone acetyltransferases (HATs)

CREBBP(h)
74
78
43
Garcinol
1.5

GCN5L2(h)
11
5
1
Anacardic Acid
4.7

HAT1(h)
−3
3
−3
Garcinol
1.5

MYST3(h)
18
11
3
Garcinol
1.8

MYST4(h)
25
39
71
Curcumin
11

pCAF(h)
−25
−26
−20
Garcinol
2.9

TIP60(h)
20
10
−3
Garcinol
1.5

Histone deacetylase (HDACs)

HDAC1 (h)
−2
−1
−1
trichostatin A
0.0052

HDAC2 (h)
1
0
−2
trichostatin A
0.024

HDAC3 (h)
1
1
3
trichostatin A
0.0068

HDAC4 (h)
−73
−20
−11
trichostatin A
4.0

HDAC5 (h)
−3
−2
−1
trichostatin A
1.0

HDAC6 (h)
12
2
−18
trichostatin A
0.0074

HDAC7 (h)
−111
−6
16
trichostatin A
1.8

HDAC8 (h)
−8
−19
0
trichostatin A
0.41

HDAC9 (h)
−50
−1
−9
trichostatin A
9.1

HDAC10 (h)
1
3
6
trichostatin A
0.0090

HDAC11 (h)
27
−47
−130
scriptaid
8.9

Sirtuins

sirtuin 1 (h)
2
−3
−5
suramin
7.9

(inhibitor effect)

sirtuin 2 (h)
−4
6
−19
suramin
21

(inhibitor effect)

sirtuin 3 (h)
−14
−8
−3
niacinamide
21

(inhibitor effect)

Sirtuin 6 (h)
2
6
−4
EX-527
550

Sirtuin 7 (h)
7
−2
−8
JFD00244
1300

Methyltransferases (MTs)

ASH1L
3
20
−54
SAH
9.1

DOT1L (h)
−5
18
13
SAH
0.14

EHMT1 (h)
13
9
63
SAH
0.18

EZH1/EED/SUZ12 (h)
−1
3
−12
SAH
8.4

EZH2/EED/SUZ12 (h)
19
26
4
SAH
22

(PRC2 complex)

G9a (h)
86
64
49
SAH
2.1

hSMYD2
12
5
−12
SAH
0.16

MLL complex (h)
2
22
11
SAH
0.97

MLL2(h) complex
6
3
−15
SAH
24

MLL3(h) complex
−22
−21
−33
SAH
9.0

MLL4(h) complex
−16
−18
−4
SAH
0.55

NSD1 (h)
69
29
−10
chaetocin
0.13

NSD3/WHSC1L1 (h)
−17
11
−35
Suramin
1.5

PRDM9 (h)
9
5
4
SAH
370

PRMT1 (h)
16
26
−1
SAH
0.094

PRMT3 (h)
22
50
14
SAH
0.86

PRMT4 (h)
90
84
77
SAH
0.094

PRMT5 complex (h)
8
−8
9
SAH
0.65

PRMT6 (h)
49
57
34
SAH
0.053

PRMT7 (h)
−2
0
−36
SAH
0.86

PRMT8 (h)
5
24
−26
SAH
0.14

SETD2(h)
−9
−4
−11
SAH
1.2

SETD7 (h)
12
16
−2
SAH
24

SETD8 (h)
4
25
−10
mercurochrome
2.4

SETDB1 (h)
53
43
52
SAH
1.8

SUV39H2 (h)
−8
−16
14
SAH
24

SUV4-20H2 (h)
11
20
−45
SAH
4.7

WHSC1(h) (NSD2(h))
3
14
7
Chaetocin
0.48

Kinases

Aurora B (h) (substrate
6
4
−1
Staurosporine
0.038

histone H3 full length)

DAPK3/ZIP (substrate
4
9
8
Staurosporine
0.0073

histone H3 full length)

Haspin (h) (substrate
2
11
7
Staurosporine
0.032

histone H3 full length)

IKK alpha(h) (substrate
26
28
14
staurosporine
0.03

histone H3 full length)

MSK1(h) (substrate
6
2
10
Staurosporine
0.015

histone H3 full length)

MSK2(h) (substrate
9
12
−1
Staurosporine
0.0058

histone H3 full length)

PIM1(h) (histone H3
22
11
5
Staurosporine
0.025

full length substrate)

PKBalpha/AKT1(h)
−1
−19
−15
Staurosporine
0.0073

(substrate histone H3

full length)

PKBbeta/AKT2(h)
13
11
12
Staurosporine
0.021

(substrate histone H3

full length)

PKBgamma/AKT3(h)
5
−7
3
Staurosporine
0.03

(substrate histone H3

full length)

Rsk2(h) (substrate
5
7
−4
Staurosporine
0.007

histone H3 full length)

Small molecule methyltransferases

Catechol O-
6
1
0
SAH-d4
14

methyltransferase (h)

Glycine N-
55
5
−11
SAH-d4
17

methyltransferase (h)

Guanidinoacetate N-
8
−1
−1
SAH-d4
2.3

methyltransferase (h)

Histamine N-
18
−2
18
SAH-d4
13

methyltransferase (h)

Nicotinamide N-
13
−2
−2
SAH-d4
13

methyltransferase (h)

Phenylethanolamine N-
23
9
5
SAH-d4
3.9

methyltransferase (h)

Thiopurine S-
40
28
19
SAH-d4
2.3

methyltransferase (h)

Ubiquitin modifying enzymes

BAP1 (h)
1
−9
24
Ubiquitin
0.23

Aldehyde

USP 10 (h)
7
2
−7
N-Methylmaleimide
1000

USP 14 (h)
1
0
−24
Ubiquitin
0.004

Aldehyde

USP 16 (h)
11
14
12
Iodoacetamide
3.5

USP 21 (h)
−18
3
3
Ubiquitin
0.17

aldehyde

USP 7 (h)
−18
−14
9
Ubiquitin
0.11

Aldehyde

^A% Inhibition of Control Values

TABLE 11

IC₅₀values from inhibition/concentration-response

curves of compounds 1, 2 and 4 for inhibition of

epigenetic enzymes against reference compounds.

Compound

1
2
4

Reference

Enzyme and Cell-
IC₅₀
IC₅₀
IC₅₀
Reference
IC₅₀

based assays
(μM)
(μM)
(μM)
Compound
(μM)

Demethylases

JARID1A (h)
9.6

2,4 PDCA
0.099

JARID1B (h)
3.6

2,4 PDCA
0.12

JMJD1A (h)
4.5

2,4 PDCA
3.9

JMJD2B (h)
12

2,4 PDCA
0.095

JMJD2E (h)
4.7

2,4 PDCA
0.11

JMJD3 (h)
14

2,4 PDCA
73

UTX (h)
6.7

IOX1
0.056

Histone acetyltransferases

CREBBP(h)
3.9
6.1

Garcinol
5.4

MYST4 (h)

4.2
Curcumin
8.9

Histone methyltransferases

G9a (h)
11
5.4

SAH
4.6

NSD1 (h)
4.9

chaetocin
0.24

PRMT4 (h)
4.7
5.4
4.7
SAH
0.056

Results: Epigenetic Targets, Diseases and Cancer

Epigenetic modifications can have a role in the development of a variety of diseases. Epigenetic regulation involves hierarchical covalent modification of DNA and proteins that package such as histones. DNA is regulated by methylation and demethylation on the cytosine residues. DNA methylation is mediated by members of the DNA-methyl transferase family (DNMT1, DNMT3A and DNMT3B) whereas demethylation is mediated by the family ten-eleven translocation (TET1-3). Besides methylation and demethylation, histone is also regulated by acetylation and deacetylation. The key proteins that mediate epigenetic signaling though acetylation and methylation of histones comprise histone acetyltransferases (HATs), histone deacetylases (HDMs), protein methyltransferases (PMTs) including lysine methyltransferases (KMTs) protein arginine methyltransferases (PRMTs), and bromodomain-containing proteins and proteins that bind to methylated histones (Arrowsmith et al., 2012; Plass et al., 2013; Tough et al., 2014; Biggar and Li, 2015).

Bromodomain Containing Proteins

Compound 1 showed significant inhibitory activity towards many of the bromodomain containing proteins (Table 10), in particular ASH1L, CECR2, EP300, KAT2A, PHIP(2), SP140, SMARCA2, TAF1(2), ATAD2A, BRD2(1), BRD4(2) and CREBBP. Compound 2 only showed significant activity towards BRD2(1). Recent studies have implicated bromodomain (BRD) containing proteins in a wide range of human diseases, including cancer (Taverna et al., 2007; Prinjha et al., 2012; Biggar and Li, 2015). The most investigated member of BRD containing proteins (BCPs) as drug targets is the BET family proteins. Currently, there are several BET inhibitors in various stages of clinical trials including RVX-208, I-BET 762, OTX 015, CPI-0610 and TEN-010 (see Table 14 in Tough et al., 2014).

In addition, JQ1 and I-BET have been shown to interact with NF-κB and induce apoptosis in drug resistant leukemia (Ciceri et al., 2014). NF-κB plays a central role inflammation and unresolved inflammation is involved in many disease states including cancer.

Several bromodomain containing proteins, including ASH1L, ATAD2A/B, BAZ2A/B, CECR2, EP300, KAT2A, PHIP(2), PB1, SMARCA2/4, BRD2/4 and CREBBP, have been shown to be up-regulated in many types of cancers (Tough et al., 2014; Fu et al., 2015). Hence, BRDs are therefore therapeutic targets for cancer. BRD-containing domains have been linked to the development of a number of extremely aggressive tumours containing BRD4-NUT and BRD3-NUT. CREBBP mutations have been identified in relapsed acute lymphoblastic leukaemia and are very common in diffuse large B-cell lymphoma and follicular lymphoma, Hodgkin's lymphoma. CREBBP and the related HAT are highly expressed in advanced prostate cancer and expression levels have been linked with patient survival. ATAD2 is over expressed in more than 70% of breast tumours and higher protein levels correlate with poor overall survival and disease recurrence (Ciro et al., 2009). ATAD2B is highly expressed in glioblastoma and oligodendroglioma as well as breast carcinoma (Krakstad et al., 2015). BRD4 is linked to development of cervical cancer (Weidner-Glunde et al.; 2010).

Compound 1 showed good inhibitory activity against domain PHD (plant homology domain) containing domain proteins SP140, TRIM 33, UHRF1 (108-286)).

Methyltransferases

Compound 1 was a strong inhibitor of the lysine methyltransferase enzyme G9a whereas compounds 2 and 3 were weaker (Table 10). G9a is a multipotent regulator of gene expression which has been shown to be over expressed in many different types of cancers (See reviews for details: Shankar et al., 2013; Casciello et al., 2015). Compounds 1 to 3 strongly inhibited the activity of PRMT4 and moderately inhibited the activity of PRMT6. Dysregulated PRMT expression and activity have been observed in a variety of cancers and PRMT1-5 and 7 have been shown to be overexpressed or otherwise contribute to tumorigenesis whereas PRMT8, and 9 have not been implicated in oncogenesis (Fuhrmann et al., 2015). PRMT4 (CARMI) is necessary for NFκB target gene expression. Further study showed a link between PRMT4 and p300 acetyltransferase activity in NFκB recruitment and gene activation.

Demethylases

Compound 1 showed significant inhibitory activity towards many of the lysine demethylase enzymes (Table 10), in particular JARID1A, JARID1B, JMJD1A, JMJD2B, JMJD2E, JMJD3 and UTX. Aberrant expression and mutations of lysine demethylases have been linked to various cancers (Hojfeldt et al., 2013; Tough et al., 2014). Mutation of lysine demethylases including FBXL10, JMJD2A, JMJDB, JMJD2C, JARID1B and PHF2 have been shown to be overexpressed in breast, colorectal, lung, prostrate, bladder and other tumours; the functional significance of JMJD2C overexpression is further suggested by the presence of the JMJD2C gene within an amplified region of a chromosome in multiple cancers (Xiang et al., 2007; Couvelard et al., 2008; Roesch et al., 2010; He et al., 2011a; Berry and Janknecht, 2013; Kogure et al., 2013; Tzatsos et al., 2013).

Histone Acetyltransferases

The serrulatanes diterpenes showed significant inhibitory activity on histone acetyltransferases CREBBP and MYST4 (Table 10). Misregulation of histone acetyltransferase activity has been linked to many different pathogenic states including cancers, neurodegenerative disorders, and metabolic, respiratory, inflammatory and cardiovascular diseases (Adcock and Lee, 2006; Avvakumov and Cote, 2007; Grabiec et al., 2008; Ghizzo et al., 2011, Iyer et al., 2011; Pirooznia and Elefant, 2013).

TABLE 12

Enzyme and Cell-based stimulation assays for

compounds 1 to 3 at 10 μM concentration

Reference

% Stimulation Relative to Control

EC₅₀

Assay
Comp 1
Comp 2
Comp 3
Compound
(μM)

H3K9 ac
−21
−26
−8
(Not-applicable) NA

(increase)

noneH3K27 ac
−28
−17
−25
NA

(increase)

sirtuin 1 (h)
−5
−3
−4
Resveratrol
27

(activator

effect)

Pharmacology Assays

The in vitro pharmacological studies were conducted by Eurofins PanLab (Taiwan) using the parameters shown in Table 13 with respect to appropriate literature references.

TABLE 13

Pharmacology assay methods.

Angiotensin system

Angiotensin AT2 [Lee et al. 2001]

Source
Human recombinant CHO-K1 cells

Vehicle
1.00% DMSO

Ligand
0.050 nM [¹²⁵I] CGP-42112A

Non-Specific Ligand
10.0 μM (Sar¹, Ile⁸)-Angiotensin II

Incubation
3 hours @ 37° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4, 5 mM

MgCl₂, 1 mM EDTA, 0.1% BSA

Quantitation Method
Radio ligand Binding

Angiotensin, AT₁/ACE [Rubin et al. 1978]

Source
Dunkin Hartley Guinea pig 600 ±

80.0 g ileum

Vehicle
0.10% DMSO

Incubation
5 minutes @ 32° C.

(Time/Temperature)

Incubation Buffer
Krebs, pH 7.4

Quantitation Method
Isotonic (cm changes)

Peptidase, Angiotensin Converting Enzyme [Bunning et al. 1983]

Source
Rabbit lung

Substrate
500 μM (N-3[2-furyl] acryloyl)-Phe-Gly-Gly

(FAPGG)

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
30 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM HEPES, pH 7.5, 300 mM NaCl

Quantitation Method
Spectrophotometric quantitation of FAPGG

Chemokines

Chemokine CCR1 [Hesselgesser et al. 1998]

Source
Human recombinant Chem-2 cells

Vehicle
1.00% DMSO

Ligand
0.10 nM [¹²⁵I] MIP-1α

Non-Specific Ligand
0.10 μM MCP-3

Incubation
3 hours @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM HEPES, pH 7.4, 5 mM MgCl2,

1 mM CaCl2, 0.2% BSA

Quantitation Method
Radio ligand Binding

Chemokine CCR2B [Gong et al. 1997]

Source
Human recombinant CHO-K1 cells

Vehicle
1.00% DMSO

Ligand
0.10 nM [¹²⁵I] MCP-1

Non-Specific Ligand
0.030 μM MCP-1

Incubation
60 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4, 5 mM

MgCl2, 1 mM EDTA, 0.1% BSA

Quantitation Method
Radio ligand Binding

Chemokine CX3CR1 [Combadiere et al. 1998]

Source
Human recombinant Chem-1 cells

Vehicle
1.00% DMSO

Ligand
20.0 pM [¹²⁵I] Fractalkine

Non-Specific Ligand
10 nM Fractalkine

Incubation
90 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM HEPES, pH 7.5, 1 mM CaCl2,

5 mM MgCl2, 0.5% BSA, 0.1% NaN3

Quantitation Method
Radio ligand Binding

Chemokine CXCR1/2 (IL-8, Non-Selective) [Grob et al. 1990]

Source
Human neutrophils

Vehicle
1.00% DMSO

Ligand
15.0 pM [¹²⁵I] IL-8

Non-Specific Ligand
10 nM IL-8

Incubation
2 hours @ 4° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4 1 mM EDTA, 5 mM

MgCl2, 5 mg/ml BSA

Quantitation Method
Radio ligand Binding

Chemokine CXCR2 (IL-8RB) [Ahuja and Murphy 1996]

Source
Human recombinant CHO-K1 cells

Vehicle
1.00% DMSO

Ligand
15.0 pM [¹²⁵I] IL-8

Non-Specific Ligand
10 nM IL-8

Incubation
60 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
25 mM HEPES, pH 7.4, 2 mM CaCl2,

1 mM MgCl2, 0.2% BSA

Quantitation Method
Radio ligand Binding

Chemokine CXCR4 [Valenzuela-Fernandez 2002]

Source
Human recombinant Chem-1 cells

Vehicle
1.00% DMSO

Ligand
0.030 nM [¹²⁵I] SDF-1α

Non-Specific Ligand
0.030 μM SDF-1α

Incubation
90 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM HEPES, pH 7.4, 5 mM MgCl2,

1 mM CaCl2, 0.2% BSA

Quantitation Method
Radio ligand Binding

Histone Deacetylases (HDACs)

Deacetylase, Histone 1 [Strahl and Allis 2000]

Source
Human recombinant Insect Sf9 cells

Substrate
25.0 μM Fluor-de-Lys deacetylase

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
60 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
25 mM Tris-HCl, pH 8.0, 2.7 mM KCl,

1 mM MgCl2, 137 mM NaCl

Quantitation Method
Spectrofluorimetric quantitation of Fluor- de-Lys

deacetylsubstrate

Deacetylase, Histone 2 [Strahl and Allis 2000]

Source
Human recombinant Insect Sf9 cells

Substrate
25.0 μM Fluor-de-Lys deacetylase

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
60 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
25 mM Tris-HCl, pH 8.0, 2.7 mM KCl,

1 mM MgCl2, 137 mM NaCl

Quantitation Method
Spectrofluorimetric quantitation of Fluor- de-Lys

deacetylsubstrate

Growth Factor

Epidermal Growth Factor (EGF) [Dittadi et al. 1990]

Source
Human A431 cells

Vehicle
1.00% DMSO

Ligand
0.080 nM [¹²⁵I] EGF (human)

Non-Specific Ligand
0.10 μM EGF (human)

Incubation
60 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM HEPES, pH 7.7, 0.1% BSA, 1.2 mM

CaCl2, 5 mM KCl, 1.2 mM MgSO4, 138 mM

NaCl

Quantitation Method
Radio ligand Binding

Other classes

Catechol-O-Methyl Transferase (COMT) [Muller-Enoch et al. 1976]

Source
Porcine liver

Substrate
1.0 μM Esculetin

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
30 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
36 mM Tris-HCl, pH 8.0, 1.35 mM DTT,

0.54 mM MgCl2, 30.6 μM SAM

Quantitation Method
Spectrofluorimetric quantitation of scopoletin

Phospholipase sPLA2-V [Tietge et al. 2005]

Source
Human E. coli

Substrate
250 μM diheptanoyl thio-PC

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
30 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
16.25 mM Tris-HCl, 6.5 mM CaCl2, 65 mM

KCl, 0.2 mM Triton X-100

Quantitation Method
Spectrophotometric quantitation of heptanoyl

thio-PC

Protein Tyrosine Phosphatase, PTPN1 (PTP1B) [Montalibet et al. 2005]

Source
Human recombinant E. coli

Substrate
10.0 μM DiFMUP

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
60 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
50 mM HEPES, pH 7.2, 0.1% BSA, 1 mM DTT

Quantitation Method
Spectrofluorimetric quantitation of DiFMU

Steroid 5α-Reductase [Sun and Tu 1998]

Source
Wistar Rat liver

Substrate
0.90 μM Testosterone

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
30 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
40 mM Potassium Phosphate, pH 6.5 containing 1

mM DTT, 50 μM NADPH

Quantitation Method
EIA quantitation of Testosterone

Thioredoxin Reductase [Becker et al. 2000]

Source

E. coli

Substrate
1.0 mM 5,5′-Dithiobis (2-nitrobenzoic acid)

(DTNB)

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
60 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
100 mM Potassium Phosphate, pH 7.4,

2 mM EDTA, 0.2 mg/ml BSA

Quantitation Method
Spectrophotometric quantitation of

5-Mercapto-2-nitrobenzoic acid

Xanthine Oxidase [Hatano et al. 1990]

Source
Bovine buttermilk

Substrate
165 μM Xanthine

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
30 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
33.3 mM Potassium Phosphate, pH 7.5

Quantitation Method
Spectrophotometric quantitation of Uric acid

Histamine H1 [De Backer et al., 1993]

Source
Human recombinant Chem-1 cells

Substrate
1.00% DMSO

Vehicle
1.20 nM [³H] Pyrilamine

Pre incubation
1.0 μM Pyrilamine

(Time/Temperature)

Incubation
3 hours @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4, 5 mM MgCl2

Quantitation Method
Radio ligand Binding

Histamine H2 [Ruat et al., 1990]

Source
Human recombinant CHO-K1 cells

Vehicle
1.00% DMSO

Ligand
0.10 nM [¹²⁵I] Aminopotentidine

Non-Specific Ligand
3.0 μM Tiotidine

Incubation
2 hours @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM Phosphate, pH 7.4

Quantitation Method
Radio ligand Binding

Histamine H3 [Krueger et al., 2005]

Source
Human recombinant Chem-1 cells

Substrate
1.00% DMSO

Vehicle
0.40 nM [³H] N-α-Methylhistamine (NAMH)

Pre incubation
1.0 μM R(−)-α-Methylhistamine (RAMH)

(Time/Temperature)

Incubation
2 hours @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4, 5 mM

MgCl2, 0.1% BSA

Quantitation Method
Radio ligand Binding

Histamine H4 [Liu et al., 2001]

Source
Human recombinant Chem-1 cells

Substrate
1.00% DMSO

Vehicle
8.20 nM [³H] Histamine

Pre incubation
1.0 μM Histamine

(Time/Temperature)

Incubation
90 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4, 1.25 mM EDTA

Quantitation Method
Radio ligand Binding

HMG-CoA Reductase [Kubo and Strott, 1987]

Source
Human recombinant E. coli

Substrate
2.50 μM [¹⁴C]HMG-CoA

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
20 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
100 mM KH2PO4, pH 7.5, 8 mM G-6-P,

1 mM NADP, 4 mM EDTA, 2 mM DTT,

0.6 U/ml G-6-P-DH

Quantitation Method
Quantitation of [¹⁴C] Mevalonate

5-Lipoxygenase [Pufahl et al., 2007]

Source
Human recombinant Insect Sf9 cells

Substrate
25.0 μM Arachidonic acid

Vehicle
1.00% DMSO

Pre incubation
5 minutes @ 25° C.

(Time/Temperature)

Incubation
20 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4, 5 mM CaCl2, 2 mM

EDTA, 1 μM ATP

Quantitation Method
Spectrofluorimetric quantitation of rhodamine 123

Lipid Peroxidase [Mansuy et al., 1986]

Source
Dunkin Hartley Guinea pig liver microsomes

Substrate
Polyunsaturated fatty acid

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
20 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
0.25M Potassium Phosphate, pH 7.4,

0.1 mM EDTA

Quantitation Method
Spectrophotometric quantitation of

Malondialdehyde

Lipoxygenase 12-LO [Romano et al., 1993]

Source
Human platelets

Substrate
30.0 μM Arachidonic acid

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100

Quantitation Method
Spectrophotometric quantitation of 12-HETE

Monoamine Oxidase MAO-A [Urban et al., 1991]

Source
Human recombinant Insect Hi5 cells

Substrate
50.0 μM Kynuramine

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
60 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
100 mM Potassium Phosphate, pH 7.4

Quantitation Method
Spectrofluorimetric quantitation of

4-hydroxyquinoline

Monoamine Oxidase MAO-B [Urban et al., 1991]

Source
Human recombinant Insect Hi5 cells

Substrate
50.0 μM Kynuramine

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
60 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
100 mM Potassium Phosphate, pH 7.4

Quantitation Method
Spectrofluorimetric quantitation of

4-hydroxyquinoline

Myeloperoxidase [Svensson et al., 1987]

Source
Human PMN leukocytes

Substrate
20.0 mM Guaiacol

Vehicle
1.00% DMSO

Pre incubation
15 minutes @ 25° C.

(Time/Temperature)

Incubation
5 minutes @ 25° C.

(Time/Temperature)

Incubation Buffer
0.1M Sodium Phosphate, pH 7.4

Quantitation Method
Spectrophotometric quantitation of Tetraguaiacol

[3H]-2-Deoxy-D-glucose uptake[Yamamoto et al., 2006]

Source
Rat L6 skeletal muscle cells

Substrate
0.10% DMSO

Vehicle
24 hours @ 37° C.

Pre incubation
15 minutes @ 37° C.

(Time/Temperature)

Incubation
KRPH, pH 7.4

(Time/Temperature)

Incubation Buffer
Radiometric quantitation of insulin-induced 2-DG

uptake

Quantitation Method
Rat L6 skelet al muscle cells

Adhesion and Transcription response

Adhesion, ICAM-1-Mediated [Cobb et al. 1992]

Source
Human

Vehicle
0.40% DMSO

Ligand
0.030 nM [¹²⁵I] SDF-1α

Non-Specific Ligand
0.030 μM SDF-1α

Incubation
30 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
25 mM HEPES, pH 7.4, RPMI-1640,

1% FBS

Quantitation Method
Spectrofluorimetric quantitation of adhesion

Adhesion, VCAM-1-Mediated [Stoltenborg et al. 1994]

Source
Human

Vehicle
0.40% DMSO

Incubation
60 minutes @ 37° C.

(Time/Temperature)

Incubation Buffer
25 mM HEPES, pH 7.4, RPMI-1640,

1% FBS

Quantitation Method
Spectrofluorimetric quantitation of adhesion

Transcription Response, NF-κB [Lenardo and Baltimore 1989]

Source
Human

Vehicle
0.50% DMSO

Incubation
4 hours @ 37° C.

(Time/Temperature)

Incubation Buffer
RPMI-1640, pH 7.4

Quantitation Method
Spectrofluorimetric quantitation of β-

galactosidase

The results of pharmacological assays are shown in Table 14.

TABLE 14

Pharmacological activity of compounds 1 to 3 at 10 μM.

% Control Response
Assay

Ascii Assay Name
1
2
3
Year

Angiotensin system

Angiotensin AT2
29
18
9
2015

Angiotensin, AT 1/ACE - Agonist
0
0
0
2015

Angiotensin, AT 1/ACE - Antagonist
34
10
17
2015

Peptidase, Angiotensin Converting
0
−3
−3
2015

Enzyme

Chemokines

Chemokine CCR1
33
−3
−5
2015

Chemokine CCR2B
1
−23
−10
2015

Chemokine CX3CR1
−1
5
7
2015

Chemokine CXCR1/2 (IL-8, Non-
30
−28
−1
2015

Selective)

Chemokine CXCR2 (IL-8RB)
12
−7
1
2015

Chemokine CXCR4
14
−27
−13
2015

Histone Deacetylases (HDACs)

Deacetylase, Histone 1
17
23
NA
2014

Deacetylase, Histone 1
−27
−18
−26
2015

Deacetylase, Histone 2
56
44
NA
2014

Deacetylase, Histone 2
34
21
12
2015

Deacetylase, Histone 3
−5
25
NA
2014

Deacetylase, Histone 4
4
−5
NA
2014

Deacetylase, Histone 5
−9
−11
NA
2014

Deacetylase, Histone 6
0
−17
NA
2014

Deacetylase, Histone 7
1
−16
NA
2014

Deacetylase, Histone 8
29
13
NA
2014

Deacetylase, Histone 9
−14
−13
NA
2014

Deacetylase, Histone 10
11
9
NA
2014

Deacetylase, Histone 11
9
5
NA
2014

Sirtuins

Deacetylase, Sirtuin SIRT1
6
−3
NA
2014

Deacetylase, Sirtuin SIRT2
0
26
NA
2014

Deacetylase, Sirtuin SIRT3
−6
−9
NA
2014

Deacetylase, Sirtuin SIRT5
−16
24
NA
2014

Deacetylase, Sirtuin SIRT6
35
18
NA
2014

Growth Factor

Epidermal Growth Factor (EGF)
−8
−9
4
2015

Nitric Oxide Synthases

Nitric Oxide Synthase, Endothelial
6
5
NA
2014

(eNOS)

Nitric Oxide Synthase, Inducible (iNOS)
16
7
NA
2014

Nitric Oxide Synthase, Neuronal (nNOS)
14
5
NA
2014

Other classes

Catechol-O-Methyl Transferase (COMT)
0
−1
3
2015

Histamine H1
85
66
NA
2014

Histamine H1
97
60
50
2015

Histamine H2
67
24
NA
2014

Histamine H2
73
−5
66
2015

Histamine H3
−10
−5
NA
2014

Histamine H3
12
24
1
2015

Histamine H4
50
16
NA
2014

Histamine H4
12
−1
10
2015

HMG-CoA Reductase
29
27
24
2015

5-Lipoxygenase
79
53
67
2015

Lipid Peroxidase
98
53
74
2015

Lipoxygenase 12-LO
60
40
29
2015

Monoamine Oxidase MAO-A
91
34
27
2015

Monoamine Oxidase MAO-B
75
39
18
2015

Myeloperoxidase
52
5
NA
2014

Myeloperoxidase
27
1
−7
2015

Phospholipase sPLA2-V
4
3
0
2015

Protein Tyrosine Phosphatase,
20
3
−3
2015

PTPN1 (PTP1B)

Steroid 5alpha-Reductase
2
−6
3
2015

Thioredoxin Reductase
71
6
NA
2014

Thioredoxin Reductase
35
12
9
2015

Xanthine Oxidase
14
20
27
2015

Pharmacological Assays: Further Lipoxygenase, Lipid Peroxidase and Monoamine Oxidase Assays

Further in vitro pharmacological studies were conducted by Eurofins Panlabs (Taiwan) Ltd. using the parameters shown in Table 3 with respect to appropriate literature references.

For the Compound 1 5-lipoxygenase, lipid peroxidase and monoamine oxidase MAO-A assays, the inhibition/concentration-response curve concentrations were 10, 3, 1, 0.3, 0.01, 0.03 and 0.01 μM (Table 15).

TABLE 15

Activity of Compound 1 against 5-lipoxygenase,

lipid peroxidase and monoamine oxidase (MAO-A)

IC₅₀(μM) and reference compounds

Compound

1
Reference

Assay
IC₅₀μM
Compound
IC₅₀μM

5-Lipoxygenase
2.31
NDGA
0.64

Lipid Peroxidase
1.47
N-Propyl Gallate
206

Monoamine oxidase
6.74
Clorgyline
0.00143

A (MAO-A)

Compounds 1 to 3 are potent to moderately potent inhibitors of lipid oxidation (Tables 14 and 15). Therefore, they can be used in blocking inflammation induced by oxidative damage in cancer.

5-Lipoxygenase promotes lipid oxidation and produces leukotrienes. Compounds 1 to 3 are potent to moderately potent inhibitor of 5-lipoxygenase (Tables 14 and 15). Therefore, they can be used in blocking inflammation induced by oxidative damage and inflammatory pathophysiology in cancer.

12-Lipoxygenase promotes lipid oxidation and produces leukotrienes. Compounds 1 to 3 are moderately potent inhibitor of 12-lipoxygenase (Table 14). Therefore, they can be used in blocking inflammation induced by oxidative damage and inflammatory pathophysiology in cancer.

Compound 1 is a potent inhibitor of monoamine oxidase A and B (Tables 14 and 15). Therefore, it can be used in blocking oxidative damage induced by oxidation of monoamines and as an antidepressant in cancer.

Compounds 1 to 3 inhibit binding of the radioligand to histamine H1 and H2 receptors (Table 14). Therefore, they can be used in blocking inflammation induced by histamine resulting in anti-inflammatory action useful in cancer treatment.

Compound 1 inhibited the activity of thioredoxin reductase (Table 14). Inhibition of thioredoxin reductase activity can be used to selectively increasing oxidative stress in cancer cell while reducing oxidative stress in normal cells and

Compound 1 inhibited the activity of myeloperoxidase (Table 14). Inhibition of myeloperoxidase activity can be used to selectively reduce oxidative stress in immune cells during cancer therapy.

Curves showing binding against 5-lipoxygenase; lipid peroxidase; and monoamine oxidase (MAO-A) for Compound 1 are presented in FIGS. 7 to 9.

Pharmacology Assays: 2-Deoxy-D-Glucose Uptake into Muscle Cells

TABLE 16

Effect of compounds 1 to 4 at 10 μM on

2-deoxy-D-glucose uptake into muscle cells.

% Control Response
Assay

Ascii Assay Name
1
2
3
4
Year

[3H]-2-Deoxy-D-glucose
−61
15
−52
−45
2016

uptake - agonist*

[3H]-2-Deoxy-D-glucose
152
67
195
151
2016

uptake - antagonist*

*agonist - Increase in 2-DG uptake relative to insulin response

*antagonist - Inhibition of insulin-induced 2-DG uptake

Pharmacology Assays: Adhesion and Transcription Response

TABLE 17

Dose response and IC₅₀values for the pharmacological

activity of representative serrulatane diterpenes.

Compound 1
Compound 2
Compound 3

Conc
Resp
IC₅₀
Resp
IC₅₀
Resp
IC₅₀

Ascii Assay Name
μM
Av %
μM
Av %
μM
Av %
μM

Adhesion, ICAM-1-
10
6
>10
5
>10
7
>10

Mediated

Adhesion, ICAM-1-
1
3
>10
4
>10
6
>10

Mediated

Adhesion, ICAM-1-
0.1
−2
>10
3
>10
−4
>10

Mediated

Adhesion, ICAM-1-
0.01
1
>10
0
>10
−5
>10

Mediated

Adhesion, ICAM-1-
0.001
−4
>10
7
>10
−7
>10

Mediated

Adhesion, VCAM-1-
10
−7
>10
−18
>10
−4
>10

Mediated - Antagonist

Adhesion, VCAM-1-
1
−9
>10
−18
>10
7
>10

Mediated - Antagonist

Adhesion, VCAM-1-
0.1
3
>10
−8
>10
13
>10

Mediated - Antagonist

Adhesion, VCAM-1-
0.01
0
>10
−17
>10
18
>10

Mediated - Antagonist

Adhesion, VCAM-1-
0.001
13
>10
−7
>10
13
>10

Mediated - Antagonist

Transcription Response,
10
103
1.4
34
>10
−19
>10

NF-kappaB - Antagonist

Transcription Response,
1
17
1.4
10
>10
1
>10

NF-kappaB - Antagonist

Transcription Response,
0.1
0
1.4
−3
>10
−7
>10

NF-kappaB - Antagonist

Transcription Response,
0.01
−5
1.4
−7
>10
−11
>10

NF-kappaB - Antagonist

Transcription Response,
0.001
−25
1.4
−7
>10
−16
>10

NF-kappaB - Antagonist

Nuclear factor kappa B (NF-κB) was first discovered as a factor in the nucleus of B lymphocytes that binds to the enhancer of kappa light chain of immunoglobulin and is also a lymphoid specific (Sen and Baltimore, 1986). The NF-κB family includes ReWAp65, NF-κB1 p50/p105, NF-κB2 p52/p100, C-Rel and RelB. These proteins contain an N-terminal Rel homology domain (RHID) that is responsible for binding to DNA and other proteins and harbour a nuclear leading sequence (NLS). NF-κB proteins function as a dimeric transcription factor that regulates the expression of genes influencing a broad range of biological processes including innate and adaptive immunity, inflammation, stress responses, B-cell development and lymphoid organogenesis (Pantano et al., 2006; Brigelius-Fohê and Fohê 2011; Ghosh et al., 2012; Akdis et al., 2017).

NF-κB has been shown to have diverse and complex roles in cancer and immune modulation (Perkins, 2012; Sethi et al., 2012; Parri and Chiarugi, 2013; D'Ignazio et al., 2015). Based on significant inhibitory activity against NF-κB (IC₅₀0.69 μM) and cytokines (IC₅₀6.6, 8.8, 4.3, 8.9 μM, for IL1β, IL-6, IL10, TNFα respectively, Compound 1 can potentially influence the expression of the aforementioned biological processes as well as modulation of immune responses. In support for these results, Eurofins Oncopanel Genomic analysis showed Compound 1 clustered near to ABT-199, a BH3 domain inhibitor of BCL-2. ABT-199 blocked anti-apoptotic of BCL-2 leading to programmed cell death. In addition to antitumor effect of BCL-2 therapeutics, the BH3 mimetics have been proposed in the context of immune modulation (Ludwig et al., 2016). Taken together, Compound 1 could target cancer cell death as well as immune modulation. FIG. 10 shows transcription response and binding against NF-κB for Compound 1.

In Vitro Cell Viability Assay of Serrulatane Terpenes

Four serrulatane diterpenes, namely compounds 1 to 4, and Myoporum insulare resin were evaluated for in vitro inhibition of cell growth in 43 cancer cell lines and one normal cell line as shown in Table 19. The assay is a 72 hour (three day) assay.

Methodology of the In Vitro Cell Viability Assay

Cell viability assays were carried out by Eurofins PanLab (Taiwan). The assays are based on the established principle that cell viability (survival) can be evaluated by measuring the intracellular levels of adenosine triphosphate (ATP) by bioluminescence in metabolically active cells (Xia, M et al. 2008). Assays are carried out by seeding cells in two plates (T₀and T₇₂). At time “zero” cell plate (T₀) is harvested, treated, and incubated for 72 h (three days) when the T₇₂plate will be harvested. The intracellular levels of ATP are measured and represent the amount of viable cells.

Cell viability assay results for compounds 1 to 4 and Myoporum insulare resin/exudate extract towards 43 cancer cell lines and 1 normal cell line are shown in Table 18.

TABLE 18

Inhibition of cell growth at 10.5 μg/mL of Myoporum insulare

resin and 10 μM of compounds 1 to 4.

% Cell growth

Myoporum

Cell lines

insulare resin
1
2
3
4

Normal

HUVEC,
32
−90
97
91
37

Endothelium

Brain

U-87 MG
36
7
101
111
81

SK-N-MC
4
−87
73
82
−92

Leukemia

HL-60 (TB)
13
−62
−35
138
−83

K-562
−20
−55
69
93
60

MV-411
−96
−96
37
76
−94

MOLT-4
−97
−94
38
77
−93

Non-Small Cell

Lung Cancer

A549/ATCC
60
−92
102
106
93

LL/2
41
−62
105
111
98

NCI-H460
63
−37
88
100
93

PC-6
30
−62
92
96
4

Colon Cancer

CT26.WT
36
2
120
103
33

SW-620
−94
−89
97
107
74

DLD-1
29
−65
95
101
68

HCT-15
15
−62
94
116
69

HCT-116
45
−74
81
99
64

HT-29
69
−66
107
101
91

Melanoma

B16-F0
84
7
123
116
56

SK-MEL-5
−69
−96
95
118
88

Ovarian

Cancer

OVCAR-3
−33
−93
67
86
60

SK-OV-3
6
−35
82
98
74

Prostrate

Cancer

LNCaP
−5
−86
73
84
56

PC-3

−87
70
99
64

Breast Cancer

BT474
56
−67
118
108
64

MCF7
−51
−71
114
112
77

MCF-7 AdrR
−43
−90
92
101
77

MDA-MB-231
−38
−92
97
100
84

MDA-MB-468
−69
−92
37
85
52

T-47D
102
20
104
119
47

4T1
75
52
60
99
26

Kidney Cancer

A-498
43
−43
91
92
89

ACHN
34
−41
101
105
105

Liver Cancer

HC-4
80
−91
104
105
91

Hep3B
20
−84
87
84
93

HepG2
27
−23
95
103
90

Lymphoma

Ramos
−94
−92
33
129
−82

H33HJ-JA1
−73
−90
57
90
−87

U937
28
−86
102
105
20

Pancreas

MIA PaCa-2
20
3
92
106
92

PANC-1
64
36
87
116
82

Skin

A-431
64
−89
100
102
107

A375
9
−5
74
100
65

Stomach

KATO III
12
−91
84
105
83

Uterus

MES-SA
−57
−88
75
94
68

Cell Viability Assay Results

At 10 μM Compound 1 showed potent reduction in cell viability (−80 to −100%) towards 20 cancer cell lines. Moderate reduction in cell viability (−40 to −80%) was observed towards 12 cancer cell lines and weak effect towards cell viability for 4 cell lines. Growth inhibition was observed for the remaining cell lines.

At 10 μM Compound 2 showed weak reduction in cell viability (−35%) towards the HL-60 leukemia cells and no significant effect on the remaining cell lines. At 10 μM Compound 3 showed no significant effect on all 44 cell lines. At 10 μM Compound 4 showed potent reduction in cell viability (−82 to −94%) towards six lymphoma, leukemia and brain cancer cell lines: Ramos and H33HJ-JA1 (lymphoma); HL-60 (TB), MOLT-4 and MV-411 (leukemia), and SK-N-MC (brain). Compound 4 was selective with no significant reduction in the growth of HUVEC endothelium cells (normal cell line).

At 10.5 μg/mL Myoporum insulare resin showed potent reduction in cell viability (−80 to −100%) towards four cancer cell lines: SW-620 (colon); Ramos (lymphoma); and MV-411 and MOLT4 (leukemia). Moderate reduction in cell viability (−40 to −80%) was observed towards six cancer cell lines: MCF-7, MCF7 AdrR, MDA-MB-468 (breast); H33HJ-JA1 (lymphoma); SK-MEL-5 (melanoma); and MES-SA (uterus). Weak cell viability reduction (−5 to −38%) towards five cell lines: K-562 (leukemia); OVCAR-3 (ovarian); LNCaP (prostrate); MDA-MB-231 (breast); and A375 (skin). No significant effect was observed towards HUVEC endothelium cells (normal cell line).

OncoPanel Cell Proliferation Assay

The OncoPanel cell proliferation assay measures the proliferation response of cancer cell lines to drug treatments through high-content fluorescence imaging or bioluminescence.

Whereas the cell viability assay described above is a three-day assay, the OncoPanel assay is a 10-day assay.

Experimental Procedure

Cells were grown in RPMI 1640, 10% FBS, 2 mM L-alanyl-L-glutamine, 1 mM Na pyruvate or a special medium. Cells were seeded into 384-well plates and incubated in a humidified atmosphere of 5% CO₂at 37° C. Compounds were added the day following cell seeding. At the same time, a time zero untreated cell plate was generated. After a 10-day incubation period, cells were fixed and stained to allow fluorescence imaging of nuclei. At 7 days post-seeding, the growth media were replaced and the plates were re-dosed with the test compound.

Compounds were serially diluted in half-log steps from the highest test concentration specified in the above table, and assayed over 10 concentrations with a maximum assay concentration of 0.1% DMSO. Automated fluorescence microscopy was carried out using a Molecular Devices ImageXpress Micro XL high-content imager, and images were collected with a 4× objective. 16-bit TIFF images were acquired and analyzed with MetaXpress 5.1.0.41 software.

Data Analysis

Cell proliferation was measured by the fluorescence intensity of an incorporated nuclear dye. The output is referred to as the relative cell count, where the measured nuclear intensity is transformed to percent of control (POC) using the following formula:

$POC = \frac{I_{x}}{I_{0}} \times 100$

Where I_xis the nuclear intensity at concentration x, and I₀is the average nuclear intensity of the untreated vehicle wells.

Cellular response parameters were calculated using nonlinear regression to a sigmoidal single-site dose response model:

$y = A + \frac{B - A}{1 + {(C / x)}^{D}}$

Where y is a response measured at concentration x, A and B are the lower and upper limits of the response, C is the concentration at the response midpoint (EC₅₀), and D is the Hill Slope (Fallahi-Sichani, M., S. et al. 2013).

Time zero non-treated plates were used to determine the number of doublings during the assay period, using the formula:

$Doublings = \log_{2} (\frac{N}{N_{T 0}})$

Where N is the cell number in untreated wells at the assay end point and N_T0is the cell number at the time of compound addition.

Cell count IC₅₀is the test compound concentration at 50% of maximal possible response. EC₅₀is the test compound concentration at the curve inflection point or half the effective response (parameter C of the fitted curve solution). GI₅₀is the concentration needed to reduce the observed growth by half (midway between the curve maximum and the time zero value). Activity area is an estimate of the integrated area above the curve (Barretina, J. G. et al. 2012). Activity area values range from 0-10, where a value of zero indicates no inhibition of proliferation at all concentrations, and a value of 10 indicates complete inhibition of proliferation at all concentrations. In rare instances, values<0 or >10 may be observed. In these instances, values<0 should be considered as equivalent to 0, whereas values>10 should be considered equivalent to 10.

Curve-fitting, calculations, and report generation were performed using a custom data reduction engine and MathIQ based software (AIM).

The OncoPanel cell proliferation results (OncoPanel, Eurofins PanLab, USA, across 280 human cancer cell lines) for compounds 1, 2 and 4 are shown below in Table 19. Tables 21 to 28 show representative proliferation response data (Table 21: MDA-MB-415; Table 22: RKO-AS45-1; Table 23: SW480; Table 24: 639-V; Table 25: Hs 729; Table 26: Hs 852.T; Table 27: HCT-8; Table 28: IM-9) and the cell proliferation results for Myoporum insulare resin are shown below in Table 29.

TABLE 19

OncoPanel cell proliferation results for compounds 1, 2 and 4.

Compound 1
Compound 2
Compound 4

Cell
Cell
Cell
Cell
Cell
Cell
Cell
Cell
Cell

Count
Count
Count
Count
Count
Count
Count
Count
Count

EC₅₀
IC₅₀
GI₅₀
EC₅₀
IC₅₀
GI₅₀
EC₅₀
IC₅₀
GI₅₀

Cell Line
Type
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)

5637
Bladder
3.38
3.38
3.32
>30
>30
>30
15.20
15.20
15.10

639-V
Bladder
2.01
2.01
2.01
6.54
8.00
7.98
12.00
12.00
12.00

647-V
Bladder
3.12
3.12
3.11
11.30
11.30
11.20
5.99
6.00
5.99

BFTC-905
Bladder
1.79
1.79
1.78
8.03
8.03
8.01
9.70
9.70
9.68

HT-1197
Bladder
2.31
2.34
2.28
10.70
10.70
10.30
15.60
15.60
15.20

HT1376
Bladder
1.45
1.46
1.43
4.71
4.71
4.47
14.10
14.10
13.80

J82
Bladder
1.06
1.06
1.05
12.90
12.90
12.70
6.22
6.22
6.09

SCaBER
Bladder
3.12
3.12
3.10
11.50
11.50
11.40
16.90
16.90
16.80

T24
Bladder
1.44
1.44
1.44
20.40
20.40
20.30
12.20
12.20
12.20

TCCSUP
Bladder
1.75
1.75
1.72
8.20
8.20
7.78
8.91
8.91
8.67

UM-UC-3
Bladder
1.85
1.85
1.84
5.30
5.75
5.74
3.83
3.83
3.82

AU565
Breast
1.62
1.62
1.61
6.40
6.40
6.29
10.10
10.10
10.00

BT20
Breast
1.59
1.61
1.53
12.20
12.70
12.30
14.10
14.30
13.90

BT474
Breast
5.41
5.46
4.95
>30
>30
>30
25.50
25.70
25.00

BT-549
Breast
3.14
3.14
3.12
25.80
25.90
25.80
15.30
15.30
15.10

CAMA-1
Breast
3.51
3.51
3.40
>30
>30
>30
>30
>30
>30

EFM-19
Breast
1.61
1.61
1.54
11.20
11.20
10.60
11.90
11.90
11.30

Hs 578T
Breast
4.74
4.76
4.68
28.70
28.70
28.20
15.00
15.00
14.90

KPL-1
Breast
0.81
0.81
0.81
4.70
4.73
4.70
4.18
4.18
4.13

MCF7
Breast
1.37
1.37
1.35
10.40
10.40
9.96
12.10
12.10
11.90

MDA MB 231
Breast
1.47
1.48
1.47
4.39
4.39
4.36
11.40
11.40
11.30

MDA MB 453
Breast
0.78
0.78
0.77
3.68
3.69
3.65
3.76
3.76
3.68

MDA MB 468
Breast
2.75
2.76
2.74
8.07
8.08
8.00
7.41
7.43
7.34

MDA-MB-415
Breast
1.02
1.02
1.00
11.50
11.50
10.80
28.50
28.50
27.40

MDA-MB-436
Breast
2.68
2.69
2.67
9.72
9.77
9.73
10.30
10.30
10.20

SK-BR-3
Breast
1.79
1.80
1.76
12.20
12.20
11.90
10.30
10.30
9.97

T47D
Breast
4.63
4.63
4.28
10.20
10.20
9.40
3.28
3.28
2.92

A172
CNS - Glioma
1.12
1.15
1.14
9.02
9.02
8.93
8.08
8.08
8.03

CCF-STTG1
CNS - Glioma
5.79
6.60
5.24
>30
>30
>30
19.70
19.70
16.00

DBTRG-05MG
CNS - Glioma
2.65
2.66
2.64
12.20
13.00
12.80
17.60
17.60
17.40

DK-MG
CNS - Glioma
6.16
6.41
6.12
1.64
>30
>30
17.20
17.20
16.40

H4
CNS - Glioma
2.77
2.77
2.77
3.06
>30
>30
13.00
13.00
13.00

Hs 683
CNS - Glioma
4.90
4.91
4.76
29.80
29.80
28.30
24.50
24.60
24.30

M059J
CNS - Glioma
3.09
3.09
3.02
9.98
15.80
14.20
13.00
13.00
12.90

PFSK-1
CNS - Glioma
4.32
4.33
4.31
14.70
14.70
14.60
3.82
3.82
3.80

SNB-19
CNS - Glioma
1.66
1.66
1.65
17.30
17.30
16.80
13.00
13.00
12.90

SW1088
CNS - Glioma
2.44
2.44
2.43
10.70
10.70
10.50
8.77
8.77
8.69

SW1783
CNS - Glioma
3.19
3.19
3.09
10.20
11.00
10.50
12.30
12.30
12.10

T98G
CNS - Glioma
2.05
2.05
2.05
19.10
19.10
19.00
24.50
24.50
24.50

U-118 MG
CNS - Glioma
4.56
4.58
4.49
29.60
29.60
29.40
13.90
13.90
13.60

U-138MG
CNS - Glioma
6.29
6.33
6.15
>30
>30
>30
25.00
25.00
24.90

U-87 MG
CNS - Glioma
2.12
2.12
2.04
>30
>30
>30
13.80
13.80
13.60

D341 Med
CNS -
9.09
9.46
9.37
19.20
19.20
18.70
8.57
8.57
8.40

Medulloblastoma

Daoy
CNS -
1.06
1.06
1.06
8.64
8.65
8.64
3.19
3.19
3.19

Medulloblastoma

BE(2)C
CNS -
4.23
4.23
4.18
6.91
7.52
7.48
13.10
13.10
13.00

Neuroblastoma

CHP-212
CNS -
1.43
1.43
1.41
11.40
11.40
11.10
9.77
9.78
9.74

Neuroblastoma

MC-IXC
CNS -
4.01
4.01
4.00
16.90
16.90
16.90
6.88
6.88
6.88

Neuroblastoma

SK-N-AS
CNS -
3.58
3.58
3.54
6.23
7.92
7.70
5.86
5.86
5.75

Neuroblastoma

SK-N-DZ
CNS -
3.16
3.16
2.94
>30
>30
>30
11.90
11.90
11.50

Neuroblastoma

SK-N-FI
CNS -
7.31
7.31
6.32
>30
>30
>30
21.50
21.50
20.60

Neuroblastoma

Colo 201
Colon
0.92
0.92
0.92
8.51
8.51
8.45
4.07
4.07
4.04

Colo 205
Colon
1.31
1.31
1.31
10.70
10.70
10.70
11.90
11.90
11.90

Colo 320 HSR
Colon
3.09
3.13
3.13
11.60
11.60
11.60
5.15
5.16
5.15

Colo 320DM
Colon
3.20
3.20
3.18
7.72
7.72
7.67
7.91
7.91
7.87

DLD-1
Colon
2.96
2.96
2.96
10.70
11.20
11.20
15.70
15.70
15.70

HCT-116
Colon
1.75
1.75
1.75
6.68
6.68
6.67
10.30
10.30
10.30

HCT-15
Colon
1.84
1.84
1.83
8.64
9.49
9.48
13.40
13.40
13.40

HCT-8
Colon
1.23
1.23
1.23
6.27
6.27
6.24
12.90
12.90
12.90

HT-29
Colon
4.01
4.01
4.00
19.50
19.50
19.40
17.00
17.00
17.00

LS1034
Colon
1.87
1.87
1.83
12.70
12.70
12.00
23.60
23.60
23.50

LS123
Colon
1.13
1.16
1.03
6.57
18.40
13.40
12.00
12.00
10.70

LS411N
Colon
8.72
8.72
8.51
23.90
23.90
23.40
23.10
23.10
22.60

MT-3
Colon
3.67
3.67
3.66
16.90
16.90
16.80
23.90
23.90
23.80

NCI-H508
Colon
14.40
14.40
14.30
>30
>30
>30
>30
>30
>30

NCI-H747
Colon
3.09
3.11
3.10
12.30
>30
>30
28.10
28.20
28.10

RKO
Colon
1.69
1.69
1.69
7.22
7.92
7.92
12.00
12.00
12.00

RKO-AS45-1
Colon
1.50
1.50
1.50
9.64
9.64
9.62
12.80
12.80
12.80

RKOE6
Colon
1.72
1.72
1.72
6.95
6.95
6.93
6.79
6.79
6.76

SW1417
Colon
3.15
3.15
3.07
22.80
22.80
21.80
>30
>30
29.30

SW1463
Colon
3.73
3.75
3.71
>30
>30
>30
29.40
29.60
29.50

SW403
Colon
8.09
8.11
7.89
>30
>30
>30
>30
>30
>30

SW48
Colon
1.33
1.33
1.32
8.73
8.73
8.65
13.00
13.00
13.00

SW480
Colon
1.21
1.21
1.20
10.10
10.10
9.93
12.30
12.30
12.30

SW620
Colon
1.48
1.48
1.48
13.20
13.20
13.20
11.30
11.30
11.30

SW837
Colon
2.35
2.37
2.33
>30
>30
>30
18.90
18.90
18.70

SW948
Colon
2.21
2.21
2.20
14.70
14.70
14.50
29.70
>30
>30

WiDr
Colon
2.32
2.32
2.31
14.50
14.50
14.40
12.80
12.80
12.80

NCI-H295R
Endocrine -
14.70
14.70
13.50
>30
>30
>30
25.60
25.60
21.60

Adrenal gland

BHT-101
Endocrine -
2.16
2.17
2.17
14.00
14.00
13.90
6.80
6.80
6.77

Thyroid

CAL-62
Endocrine -
1.69
1.69
1.69
5.60
5.79
5.78
5.36
5.36
5.36

Thyroid

CGTH-W-1
Endocrine -
1.20
1.20
1.20
5.83
5.83
5.81
3.45
3.48
3.47

Thyroid

SW579
Endocrine -
1.71
1.71
1.69
12.30
12.30
12.20
11.00
11.00
11.00

Thyroid

Y79
Eye
5.60
5.60
5.33
6.57
7.65
7.41
5.65
5.65
5.34

C-33A
Female GU -
7.18
7.18
7.08
12.00
12.00
11.90
9.57
9.57
9.42

Cervix

C-4 II
Female GU -
1.09
1.10
1.09
8.94
9.03
9.00
12.60
12.60
12.50

Cervix

HeLa
Female GU -
2.99
2.99
2.98
16.60
16.60
16.50
21.10
21.10
21.10

Cervix

HT-3
Female GU -
3.88
3.89
3.84
17.60
19.30
18.60
14.50
14.50
14.30

Cervix

SiHa
Female GU -
5.22
5.23
5.17
28.30
28.30
25.80
11.30
13.50
13.20

Cervix

Ca Ski
Female GU -
5.28
5.29
5.24
>30
>30
>30
17.60
17.60
17.60

Ovary

CaOV3
Female GU -
1.91
1.92
1.85
10.20
10.20
9.61
10.20
10.30
10.20

Ovary

ME-180
Female GU -
2.25
2.26
2.21
>30
>30
>30
25.30
25.30
25.20

Ovary

MS751
Female GU -
2.23
2.23
2.21
7.55
8.78
8.72
12.10
12.10
12.00

Ovary

OVCAR3
Female GU -
1.53
1.57
1.52
14.40
14.40
13.70
15.60
15.60
14.90

Ovary

PA-1
Female GU -
4.21
4.21
4.21
11.60
16.30
16.20
7.89
7.90
7.89

Ovary

SKOV3
Female GU -
1.58
1.58
1.56
15.40
15.40
15.20
8.43
8.43
8.33

Ovary

AN3 CA
Female GU -
1.52
1.52
1.49
11.30
11.30
11.10
9.62
9.62
9.43

Uterus

HEC-1-A
Female GU -
2.57
2.57
2.56
12.10
12.10
12.00
12.30
12.30
12.20

Uterus

KLE
Female GU -
3.70
3.82
3.56
26.30
26.30
20.50
18.20
18.20
16.60

Uterus

SW954
Female GU -
7.63
7.63
7.60
>30
>30
>30
21.70
21.70
21.60

Vulva

BV-173
Leukemia
3.80
3.80
3.79
9.33
9.33
9.33
3.56
3.56
3.55

CCRFCEM
Leukemia
4.73
4.73
4.71
11.50
11.50
11.40
4.92
4.93
4.91

CEM-C1
Leukemia
1.96
1.96
1.95
3.26
3.26
3.26
1.47
1.47
1.47

CML-T1
Leukemia
1.93
1.93
1.93
3.69
3.69
3.68
3.93
3.93
3.93

EM-2
Leukemia
2.39
2.39
2.37
10.80
10.80
10.80
3.99
4.00
4.00

HEL-92-1-7
Leukemia
13.90
13.90
13.90
13.40
13.40
13.40
4.05
4.05
4.05

J-RT3-T3-5
Leukemia
0.01
0.01
0.010
2.08
2.08
2.06
2.71
2.71
2.71

Jurkat
Leukemia
2.25
2.25
2.24
2.66
2.66
2.65
3.59
3.59
3.58

K562
Leukemia
4.60
4.60
4.58
10.20
10.20
10.10
12.20
12.20
12.20

KG-1
Leukemia
0.12
0.12
0.11
11.30
12.00
11.90
13.30
13.30
13.10

KU812
Leukemia
2.81
2.81
2.57
6.52
6.52
5.66
9.50
9.50
9.13

MEG01
Leukemia
7.35
7.35
7.22
13.80
13.80
13.70
8.39
8.39
8.37

MHH-PREB-1
Leukemia
6.17
6.17
6.17
7.85
7.85
7.85
5.71
5.71
5.71

MOLT-16
Leukemia
1.84
1.84
1.84
13.30
13.30
13.30
4.02
4.02
4.01

MOLT-3
Leukemia
2.13
2.13
2.11
1.27
1.27
1.26
3.15
3.15
3.13

MV-4-11
Leukemia
3.58
3.58
3.57
8.56
8.56
8.52
3.81
3.81
3.80

MX1
Leukemia
1.29
1.29
1.29
10.80
10.80
10.80
2.46
2.46
2.46

NALM-6
Leukemia
1.81
1.81
1.81
1.67
1.68
1.68
2.61
2.61
2.61

RS4; 11
Leukemia
1.77
1.77
1.71
6.43
6.43
6.30
4.15
4.23
4.16

TF-1
Leukemia
2.11
2.11
1.98
2.75
2.75
2.44
6.57
6.57
6.26

Thp1
Leukemia
1.38
1.38
1.34
5.32
5.69
5.61
12.00
12.00
11.90

BC-1
Lymphoma
1.52
1.52
1.51
7.95
7.95
7.93
4.16
4.16
4.16

BCP-1
Lymphoma
3.18
3.18
3.14
8.48
8.48
8.40
8.82
8.82
8.75

CA46
Lymphoma
2.27
2.27
2.26
3.83
3.83
3.82
4.06
4.06
4.06

CRO-AP2
Lymphoma
5.72
5.72
5.66
6.38
6.38
6.36
9.94
9.94
9.88

Daudi
Lymphoma
1.84
1.84
1.84
3.93
3.93
3.92
2.28
2.28
2.28

DB
Lymphoma
2.10
2.10
2.09
3.23
3.25
3.24
3.85
3.85
3.84

DOHH-2
Lymphoma
1.41
1.41
1.41
3.24
3.24
3.24
2.24
2.24
2.24

DoTc2 4510
Lymphoma
1.03
1.03
1.01
6.07
6.07
5.84
9.23
9.23
9.09

EB2
Lymphoma
6.47
6.49
6.45
23.30
23.30
23.00
8.35
8.38
8.32

EB-3
Lymphoma
3.56
3.56
3.56
9.72
9.72
9.71
3.93
3.93
3.93

GA-10
Lymphoma
1.93
1.94
1.94
3.72
3.73
3.73
1.23
1.23
1.23

Hs 445
Lymphoma
1.30
1.32
1.31
4.34
4.34
4.23
5.80
5.80
5.74

Hs 611.T
Lymphoma
2.92
2.94
2.91
10.70
10.70
10.60
5.11
5.11
5.05

HT
Lymphoma
1.52
1.53
1.51
6.51
6.51
6.43
4.49
4.49
4.46

JeKo-1
Lymphoma
5.23
5.23
5.21
4.48
4.48
4.47
3.91
3.91
3.91

Jiyoye
Lymphoma
5.88
5.88
5.87
17.80
17.80
17.70
3.55
3.55
3.55

L-428
Lymphoma
1.08
1.09
1.08
8.87
8.87
8.83
2.07
2.07
2.06

MC116
Lymphoma
3.79
3.79
3.78
13.80
13.80
13.80
12.50
12.50
12.40

NAMALWA
Lymphoma
3.39
3.39
3.39
5.71
5.71
5.70
3.91
3.91
3.90

Raji
Lymphoma
4.60
4.60
4.59
8.04
8.04
8.04
3.92
3.92
3.92

Ramos (RA 1)
Lymphoma
2.96
2.96
2.96
4.39
4.39
4.39
4.17
4.17
4.17

RPMI 6666
Lymphoma
3.53
3.53
3.44
10.40
10.70
10.70
23.10
23.10
23.00

SR
Lymphoma
3.80
3.80
3.80
10.70
10.70
10.70
4.39
4.39
4.39

ST486
Lymphoma
1.09
1.09
1.08
3.87
3.87
3.86
2.66
2.66
2.65

SU-DHL-10
Lymphoma
3.94
3.94
3.93
9.92
9.92
9.90
7.68
7.68
7.67

SU-DHL-4
Lymphoma
2.01
2.01
2.01
4.20
4.20
4.20
5.74
5.74
5.74

SU-DHL-5
Lymphoma
1.20
1.20
1.19
4.28
4.28
4.27
3.80
3.80
3.80

SU-DHL-8
Lymphoma
1.77
1.77
1.77
7.40
7.40
7.40
3.90
3.90
3.90

SUP-T1
Lymphoma
3.96
3.96
3.95
12.10
12.10
12.00
6.51
6.51
6.49

TUR
Lymphoma
5.09
5.09
5.08
2.63
>30
>30
8.98
8.98
8.98

ARH-77
Myeloma
1.95
1.97
1.95
9.50
9.60
9.57
13.50
13.50
13.40

IM-9
Myeloma
0.42
0.42
0.42
3.92
3.92
3.92
6.71
6.71
6.71

RPMI 8226
Myeloma
0.68
0.68
0.67
2.02
2.02
1.95
3.03
3.04
3.03

SKO-007
Myeloma
0.99
0.99
0.93
1.59
1.59
1.42
4.66
4.71
4.55

U266B1
Myeloma
3.22
3.22
3.08
9.15
9.28
9.01
10.10
10.10
9.81

A-253
Head and Neck
4.13
4.14
4.13
28.50
28.70
28.70
18.20
18.20
18.10

A388
Head and Neck
1.66
1.67
1.66
13.50
13.50
13.40
7.66
7.66
7.59

A431
Head and Neck
2.80
2.81
2.80
>30
>30
>30
24.80
24.80
24.80

Cal 27
Head and Neck
2.16
2.16
2.15
7.64
7.64
7.62
12.30
12.30
12.20

Detroit 562
Head and Neck
1.96
1.97
1.96
14.50
14.50
14.40
25.00
25.00
25.00

FaDu
Head and Neck
0.87
0.87
0.87
8.66
8.66
8.66
8.28
8.28
8.28

OE19
Head and Neck
3.23
3.23
3.21
>30
>30
>30
20.70
20.70
20.50

OE21
Head and Neck
3.68
3.68
3.67
>30
>30
>30
12.70
12.70
12.70

SCC-25
Head and Neck
3.94
3.96
3.95
>30
>30
>30
25.00
25.00
25.00

SCC-4
Head and Neck
4.66
4.72
4.63
>30
>30
>30
15.80
15.80
15.50

SCC-9
Head and Neck
3.44
3.45
3.44
26.70
27.30
27.30
16.50
16.50
16.40

769-P
Kidney
0.85
0.85
0.85
6.95
6.95
6.94
3.72
3.72
3.71

786-O
Kidney
2.05
2.05
2.05
27.90
27.90
27.90
14.00
14.00
13.90

A498
Kidney
1.29
1.29
1.29
13.30
13.30
13.20
12.30
12.30
12.20

A-704
Kidney
1.57
1.60
1.54
>30
>30
>30
5.70
5.70
5.41

ACHN
Kidney
1.74
1.74
1.74
8.79
8.79
8.76
9.50
9.50
9.47

Caki-1
Kidney
2.86
2.86
2.75
0.17
>30
>30
14.40
14.40
14.10

Caki-2
Kidney
4.38
4.38
4.35
28.80
29.10
28.90
13.10
13.60
13.50

G-401
Kidney
4.00
4.00
3.99
26.00
26.20
26.20
8.08
8.08
8.05

SK-NEP-1
Kidney
0.01
0.01
0.006
0.06
0.06
0.06
9.62
9.63
9.62

HepG2
Liver
0.82
0.82
0.81
6.74
6.95
6.86
25.60
25.70
25.60

HLE
Liver
4.62
4.63
4.59
10.00
16.40
16.00
7.24
7.24
7.15

HLF
Liver
4.47
4.48
4.45
13.70
15.70
15.60
8.72
8.74
8.70

HuCCT1
Liver
2.48
2.48
2.48
22.60
22.60
22.60
24.80
24.80
24.80

HUH-6 Clone 5
Liver
1.52
1.52
1.51
15.50
15.50
15.30
9.63
9.63
9.46

OCUG-1
Liver
4.30
4.30
4.29
>30
>30
29.90
15.60
15.60
15.50

SNU-423
Liver
1.48
1.49
1.46
5.29
5.31
5.26
9.30
9.31
9.28

A427
Lung - NSCLC
4.52
4.52
4.50
11.20
11.20
11.20
10.00
10.00
9.98

A549
Lung - NSCLC
1.66
1.67
1.66
22.00
22.00
21.90
10.90
10.90
10.80

Calu1
Lung - NSCLC
3.10
3.10
3.07
13.90
13.90
13.80
8.88
8.88
8.77

Calu6
Lung - NSCLC
9.24
9.24
8.59
14.50
16.60
15.70
25.00
25.00
24.50

ChaGoK1
Lung - NSCLC
1.53
1.54
1.53
8.32
8.32
8.26
8.92
8.92
8.82

COR-L105
Lung - NSCLC
1.44
1.45
1.42
4.76
4.76
4.61
8.66
8.66
8.47

COR-L23
Lung - NSCLC
7.18
7.18
7.14
23.80
23.80
23.60
13.30
13.30
13.20

Hs 229.T
Lung - NSCLC
2.88
2.94
2.89
29.30
29.80
28.60
25.90
26.30
25.20

NCI-H292
Lung - NSCLC
3.20
3.20
3.18
16.70
16.70
16.60
9.21
9.21
9.19

NCIH441
Lung - NSCLC
12.10
12.10
11.70
>30
>30
>30
4.76
>30
>30

NCI-H460
Lung - NSCLC
2.34
2.34
2.33
5.57
5.92
5.91
12.20
13.00
13.00

NCI-H520
Lung - NSCLC
2.43
2.43
2.41
9.03
9.20
9.17
20.80
20.80
20.60

NCI-H596
Lung - NSCLC
8.78
8.78
8.41
12.00
15.10
14.40
28.20
>30
>30

NCI-H661
Lung - NSCLC
2.28
2.29
2.28
9.51
9.51
9.51
5.94
5.94
5.86

SKMES1
Lung - NSCLC
1.06
1.06
1.06
7.80
7.80
7.67
11.60
11.60
11.50

DMS114
Lung - SCLC
2.77
2.77
2.57
11.50
13.80
13.10
11.50
11.60
11.40

DMS53
Lung - SCLC
0.81
0.81
0.77
5.91
5.95
5.75
8.95
8.95
8.60

NCIH446
Lung - SCLC
10.30
10.30
10.00
9.75
9.85
9.77
7.81
7.81
7.62

NCI-H69
Lung - SCLC
6.30
6.44
5.84
16.20
16.20
14.90
15.90
15.90
15.10

SHP-77
Lung - SCLC
0.89
0.90
0.89
8.01
8.01
7.90
6.83
6.83
6.75

SW900
Lung - SCLC
3.29
3.33
3.24
26.60
26.80
26.70
23.30
23.30
23.10

AsPC-1
Pancreas
2.87
2.87
2.84
14.10
14.10
13.80
14.70
14.70
14.50

BxPC-3
Pancreas
3.29
3.30
3.29
13.00
>30
>30
13.40
13.40
13.40

Capan-1
Pancreas
>30
>30
>30
>30
>30
>30
>30
>30
>30

Capan-2
Pancreas
1.33
1.33
1.32
8.64
8.64
8.48
24.80
24.80
24.70

CFPAC-1
Pancreas
3.65
3.66
3.65
12.30
13.50
13.40
25.20
25.20
25.10

HPAF-II
Pancreas
1.38
1.38
1.37
9.46
9.62
9.60
12.40
12.50
12.40

Hs 766T
Pancreas
1.61
1.61
1.54
28.40
28.40
26.20
9.34
9.34
9.08

HuP-T4
Pancreas
4.52
4.54
4.53
>30
>30
>30
13.70
13.70
13.60

Mia PaCa-2
Pancreas
2.97
2.97
2.97
16.30
16.30
16.20
11.00
11.00
11.00

PANC-1
Pancreas
2.87
2.87
2.83
7.06
7.06
7.01
11.70
11.70
11.60

PSN-1
Pancreas
2.32
2.32
2.32
12.00
12.00
12.00
5.04
5.04
5.04

SU.86.86
Pancreas
7.61
7.63
7.58
13.10
>30
>30
>30
>30
>30

YAPC
Pancreas
1.91
1.91
1.90
13.00
13.00
12.90
18.00
18.20
18.20

BeWo
Placenta
9.06
9.06
8.92
7.68
>30
>30
15.60
15.60
15.50

JAR
Placenta
12.20
12.20
12.20
>30
>30
>30
6.53
6.53
6.49

JEG-3
Placenta
4.33
4.33
4.32
>30
>30
>30
6.50
6.51
6.48

22Rv1
Prostate
4.08
4.08
4.06
11.40
11.40
11.30
9.08
9.08
9.00

BM-1604
Prostate
2.58
2.58
2.56
19.10
19.10
18.90
>30
>30
>30

BPH1
Prostate
5.24
5.24
5.20
>30
>30
>30
17.30
17.30
17.30

DU145
Prostate
2.80
2.80
2.78
21.00
21.00
20.70
18.10
18.10
18.10

LNCaP
Prostate
3.90
3.90
3.47
0.86
0.86
0.28
11.50
12.90
12.40

PC-3
Prostate
3.88
3.88
3.86
12.00
12.00
11.80
7.71
7.71
7.64

A101D
Skin (Melanoma)
8.42
8.42
8.25
>30
>30
>30
>30
>30
>30

A375
Skin (Melanoma)
1.05
1.05
1.05
6.45
6.45
6.44
5.74
5.74
5.74

A7
Skin (Melanoma)
0.94
0.94
0.94
5.54
5.63
5.60
10.50
10.50
10.50

C32
Skin (Melanoma)
2.11
2.11
1.81
19.20
19.20
15.50
7.84
7.84
6.37

CHL-1
Skin (Melanoma)
1.37
1.37
1.36
14.30
14.30
14.20
11.90
11.90
11.80

COLO 829
Skin (Melanoma)
0.68
0.68
0.62
5.35
5.35
4.93
3.71
3.71
3.29

G-361
Skin (Melanoma)
1.79
1.79
1.77
11.70
11.80
11.80
13.80
13.90
13.80

HMCB
Skin (Melanoma)
1.37
1.37
1.36
6.68
6.68
6.57
9.49
9.49
9.38

Hs 294T
Skin (Melanoma)
2.64
2.64
2.59
7.01
7.01
6.72
8.33
8.33
8.23

Hs 688(A).T
Skin (Melanoma)
1.33
1.44
1.27
11.60
13.20
12.20
7.43
7.68
6.88

Hs 695T
Skin (Melanoma)
1.18
1.27
1.16
9.07
9.48
8.99
5.00
5.18
4.02

Hs 852.T
Skin (Melanoma)
2.49
2.56
2.42
15.40
15.40
14.70
12.00
12.00
11.60

Hs 934.T
Skin (Melanoma)
2.53
3.27
2.34
>30
>30
24.10
11.10
11.60
10.20

Hs 936.T(C1)
Skin (Melanoma)
2.00
2.00
1.97
3.62
3.76
3.74
9.10
10.40
10.20

MALME3M
Skin (Melanoma)
1.79
1.79
1.66
19.80
19.80
17.30
20.60
20.60
17.80

MeWo
Skin (Melanoma)
1.32
1.32
1.31
9.45
9.97
9.91
9.66
9.66
9.51

RPMI-7951
Skin (Melanoma)
0.70
0.70
0.70
5.77
6.15
6.13
9.98
9.98
9.96

SH-4
Skin (Melanoma)
1.95
1.95
1.94
9.60
11.90
11.80
11.60
11.60
11.50

SK-MEL-1
Skin (Melanoma)
4.29
4.41
4.11
15.70
15.70
14.70
>30
>30
>30

SK-MEL-28
Skin (Melanoma)
1.54
1.54
1.53
9.46
9.89
9.85
11.20
11.20
11.10

SK-MEL-3
Skin (Melanoma)
4.37
4.38
4.28
22.00
22.00
20.80
>30
>30
>30

WM-266-4
Skin (Melanoma)
1.15
1.15
1.14
6.39
6.39
6.25
10.90
10.90
10.80

G-292, clone
Soft Tissue -
4.55
4.57
4.41
8.80
8.80
8.57
6.23
6.25
6.07

A141B1
Osteosarcoma

HOS
Soft Tissue -
0.96
0.96
0.96
4.82
4.82
4.81
3.08
3.08
3.08

Osteosarcoma

Hs 888.Sk
Soft Tissue -
2.20
2.38
2.05
28.30
28.50
27.90
12.30
12.70
12.10

Osteosarcoma

KHOS-240S
Soft Tissue -
0.95
0.95
0.95
4.59
4.59
4.58
4.96
4.96
4.96

Osteosarcoma

MG-63
Soft Tissue -
9.24
9.24
9.16
>30
>30
>30
11.20
11.20
11.10

Osteosarcoma

SaOS2
Soft Tissue -
1.98
2.10
2.04
15.70
15.70
15.10
10.60
10.70
10.70

Osteosarcoma

SJSA1
Soft Tissue -
1.41
1.41
1.41
7.11
7.11
7.02
7.45
7.45
7.45

Osteosarcoma

SW1353
Soft Tissue -
1.00
1.00
0.99
9.49
9.49
9.46
4.15
4.15
4.14

Osteosarcoma

U2OS
Soft Tissue -
1.66
1.66
1.65
12.70
12.70
12.70
4.21
4.21
4.20

Osteosarcoma

A204
Soft Tissue -
0.52
0.54
0.52
8.46
8.54
8.50
0.72
0.72
0.68

Sarcoma

A-673
Soft Tissue -
1.54
1.55
1.54
10.80
11.10
11.10
2.88
2.88
2.87

Sarcoma

Hs 729
Soft Tissue -
2.77
2.78
2.67
23.30
23.30
21.80
11.90
11.90
11.60

Sarcoma

Hs 821.T
Soft Tissue -
4.22
4.22
1.80
>30
>30
13.90
12.60
12.90
9.99

Sarcoma

HT-1080
Soft Tissue -
0.96
0.96
0.96
3.31
3.31
3.30
4.42
4.42
4.42

Sarcoma

MES-SA
Soft Tissue -
1.23
1.23
1.22
9.26
9.26
9.22
6.49
6.49
6.44

Sarcoma

RD
Soft Tissue -
0.81
0.81
0.81
2.98
2.98
2.96
5.19
5.19
5.17

Sarcoma

SJRH30
Soft Tissue -
1.42
1.42
1.41
5.09
5.10
5.07
3.68
3.68
3.68

Sarcoma

SK-LMS-1
Soft Tissue -
1.57
1.57
1.56
6.79
11.30
11.20
9.04
9.04
8.98

Sarcoma

SK-UT-1
Soft Tissue -
2.23
2.23
2.22
11.70
11.70
11.60
5.49
5.49
5.46

Sarcoma

SW684
Soft Tissue -
9.37
9.37
8.28
>30
>30
>30
>30
>30
>30

Sarcoma

SW872
Soft Tissue -
0.96
0.96
0.96
4.46
4.46
4.45
4.39
4.39
4.38

Sarcoma

SW982
Soft Tissue -
1.07
1.07
1.07
7.63
7.63
7.53
7.29
7.30
7.26

Sarcoma

TE 125.T
Soft Tissue -
6.47
9.21
1.48
>30
>30
28.70
23.60
23.60
17.00

Sarcoma

TE 381.T
Soft Tissue -
1.62
1.62
1.59
8.14
8.14
7.99
6.28
6.28
6.08

Sarcoma

VA-ES-BJ
Soft Tissue -
3.67
3.67
3.55
>30
>30
>30
15.20
15.20
15.00

Sarcoma

AGS
Stomach
0.70
0.70
0.70
4.69
4.69
4.69
5.25
5.25
5.24

HS 746T
Stomach
5.58
5.74
4.64
>30
>30
>30
20.00
20.00
17.10

KATO III
Stomach
3.28
3.28
3.27
8.98
16.80
16.60
10.90
11.10
11.10

SK-PN-DW
Stomach
4.66
4.66
4.63
10.80
10.90
10.90
4.91
4.91
4.88

SNU-1
Stomach
1.25
1.25
1.25
8.51
9.42
9.41
6.99
6.99
6.98

SNU-16
Stomach
1.31
1.31
1.31
9.96
9.96
9.91
11.00
11.00
11.00

SNU-5
Stomach
3.92
3.92
3.87
>30
>30
>30
14.40
14.40
14.20

NTERA-2 cl.D1
Testis
2.25
2.25
2.20
12.50
12.50
12.30
7.41
7.41
7.39

TABLE 20

Oncopanel cell proliferation results for Compounds 11 and 12.

Compound 11 and 12

Cell
Cell
Cell

Count
Count
Count

EC₅₀
IC₅₀
GI₅₀

Cell Line
Type
(μM)
(μM)
(μM)

647-V
Bladder
>30
>30
>30

AU565
Breast
16.4
17.1
16.9

CAMA-1
Breast
>30
>30
>30

MCF7
Breast
27.5
27.5
27

MDA MB 231
Breast
>30
>30
>30

MDA MB 453
Breast
10.6
10.6
10.4

MDA-MB-415
Breast
17.8
17.8
15.7

Hs 683
CNS - Glioma
27
27
25.6

M059J
CNS - Glioma
>30
>30
>30

HuTu 80
Colon
8.61
8.7
8.65

LS-174T
Colon
>30
>30
>30

HeLa
Female GU - Cervix
>30
>30
>30

OVCAR3
Female GU - Ovary
>30
>30
>30

AN3 CA
Female GU - Uterus
10.8
13.3
13.1

MEG01
Leukemia
17.5
17.5
17.3

MOLT-16
Leukemia
21.8
21.8
21.7

MOLT-3
Leukemia
11.8
14.1
14

MV-4-11
Leukemia
>30
>30
>30

MX1
Leukemia
14.9
14.9
14.9

A427
Lung - NSCLC
16
16
15.8

Calu1
Lung - NSCLC
>30
>30
>30

Calu6
Lung - NSCLC
>30
>30
>30

Hs 229.T
Lung - NSCLC
>30
>30
>30

NCI-H292
Lung - NSCLC
12.1
>30
>30

NCI-H520
Lung - NSCLC
>30
>30
>30

NCI-H596
Lung - NSCLC
>30
>30
>30

DOHH-2
Lymphoma
12.2
13.4
13.4

EB2
Lymphoma
>30
>30
>30

Jiyoye
Lymphoma
>30
>30
>30

L-428
Lymphoma
21.3
21.3
21.1

NAMALWA
Lymphoma
>30
>30
>30

Raji
Lymphoma
>30
>30
>30

SUP-T1
Lymphoma
>30
>30
>30

PSN-1
Pancreas
>30
>30
>30

Hs 934.T
Skin (Melanoma)
21.9
>30
16.3

HOS
Soft Tissue - Osteosarcoma
>30
>30
>30

KHOS-240S
Soft Tissue - Osteosarcoma
>30
>30
>30

SW1353
Soft Tissue - Osteosarcoma
>30
>30
>30

Hs 729
Soft Tissue - Sarcoma
26.4
>30
>30

HT-1080
Soft Tissue - Sarcoma
12.7
12.7
12.7

TABLE 21

Representative data for proliferation response when

cell line MDA-MB-415 is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
90.2
1.3

3.02E−03
100.0
6.4

9.53E−03
98.0
8.7

3.01E−02
93.4
2.7

9.52E−02
98.1
10.3

3.01E−01
100.2
8.6

9.51E−01
62.3
5.3

3.00E+00
1.3
0.2

9.49E+00
0.3
0.0

3.00E+01
0.2
0.0

The data displayed in Table 21 is graphed in FIG. 11.

TABLE 22

Representative data for proliferation response when

cell line RKO-AS45-1 is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
95.4
2.0

3.02E−03
83.7
7.7

9.53E−03
88.3
3.7

3.01E−02
86.2
5.6

9.52E−02
80.5
4.7

3.01E−01
90.0
6.9

9.51E−01
72.1
9.7

3.00E+00
7.6
3.5

9.49E+00
0.1
0.0

3.00E+01
0.1
0.0

The data displayed in Table 22 is graphed in FIG. 12.

TABLE 23

Representative data for proliferation response

when cell line SW480 is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
111.8
7.0

3.02E−03
116.5
19.3

9.53E−03
103.7
9.4

3.01E−02
99.5
0.5

9.52E−02
94.1
10.2

3.01E−01
93.5
6.4

9.51E−01
69.9
17.0

3.00E+00
4.1
4.6

9.49E+00
0.2
0.0

3.00E+01
0.4
0.4

The data displayed in Table 23 is graphed in FIG. 13.

TABLE 24

Representative data for proliferation response

when cell line 639-V is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
92.0
14.3

3.02E−03
96.1
1.9

9.53E−03
101.2
5.0

3.01E−02
103.6
8.6

9.52E−02
91.6
9.6

3.01E−01
92.6
14.1

9.51E−01
91.9
12.2

3.00E+00
15.8
4.2

9.49E+00
0.3
0.1

3.00E+01
0.0
0.0

The data displayed in Table 24 is graphed in FIG. 14.

TABLE 25

Representative data for proliferation response when

cell line Hs 729 is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
100.6
7.6

3.02E−03
103.0
10.5

9.53E−03
108.7
4.1

3.01E−02
107.0
2.1

9.52E−02
99.9
0.7

3.01E−01
99.4
7.7

9.51E−01
98.9
1.2

3.00E+00
46.0
8.8

9.49E+00
3.2
0.2

3.00E+01
1.0
0.3

The data displayed in Table 25 is graphed in FIG. 15.

TABLE 26

Representative data for proliferation response when

cell line Hs 852.T is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
104.3
9.1

3.02E−03
104.5
5.1

9.53E−03
100.2
6.2

3.01E−02
94.5
5.6

9.52E−02
92.2
2.4

3.01E−01
95.3
2.9

9.51E−01
85.5
4.3

3.00E+00
41.6
5.1

9.49E+00
13.1
1.7

3.00E+01
2.7
0.5

The data displayed in Table 26 is graphed in FIG. 16.

TABLE 27

Representative data for proliferation response

when cell line HCT-8 is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
102.6
3.7

3.02E−03
104.1
9.7

9.53E−03
91.0
10.1

3.01E−02
91.8
3.5

9.52E−02
100.8
2.6

3.01E−01
94.9
4.8

9.51E−01
72.6
2.3

3.00E+00
1.9
0.5

9.49E+00
0.0
0.0

3.00E+01
0.0
0.0

The data displayed in Table 27 is graphed in FIG. 17.

TABLE 28

Representative data for proliferation response

when cell line IM-9 is treated with Compound 1

Relative cell count (%)

Concentration (microM)
Mean
StdDev

9.55E−04
94.9
34.7

3.02E−03
102.8
9.5

9.53E−03
103.1
2.4

3.01E−02
107.3
9.3

9.52E−02
97.7
3.4

3.01E−01
97.5
8.6

9.51E−01
0.0
0.0

3.00E+00
0.1
0.0

9.49E+00
0.1
0.1

3.00E+01
0.0
0.0

The data displayed in Table 28 is graphed in FIG. 18.

TABLE 29

Oncopanel cell proliferation results for

Myoporum insulare resin extract.

M. insulare resin extract

Cell
Cell
Cell

Count
Count
Count

EC₅₀
IC₅₀
GI₅₀

Cell Line
Type
(μg/mL)
(μg/mL)
(μg/mL)

5637
Bladder
3.71
3.75
3.75

^A

639-V
Bladder
3.18
3.18
3.17

^B

647-V
Bladder
2.69
2.69
2.69

^C

BFTC-905
Bladder
3.28
3.28
3.28

^B

HT-1197
Bladder
3.18
3.20
3.17

^C

HT1376
Bladder
1.31
1.31
1.27

^C

J82
Bladder
1.26
1.26
1.26

^D

SCaBER
Bladder
2.74
2.74
2.73

^C

T24
Bladder
1.70
1.70
1.70

^A

TCCSUP
Bladder
2.76
2.77
2.73

^B

UM-UC-3
Bladder
0.98
0.98
0.97

^C

AU565
Breast
1.62
1.62
1.61

^A

BT20
Breast
1.57
1.61
1.50

^B

BT474
Breast
5.60
5.60
4.94

^A

BT-549
Breast
3.25
3.25
3.16

^A

CAMA-1
Breast
3.57
3.57
3.50

^A

EFM-19
Breast
1.59
1.59
1.50

^A

Hs 578T
Breast
8.96
9.00
8.96

^A

KPL-1
Breast
0.61
0.61
0.61

^A

MCF7
Breast
1.72
1.73
1.71

^C

MDA MB 231
Breast
2.03
2.03
2.03

^A

MDA MB 453
Breast
0.53
0.53
0.52

^A

MDA MB 468
Breast
2.86
2.86
2.84

^A

MDA-MB-415
Breast
1.12
1.12
1.05

^A

MDA-MB-436
Breast
2.70
2.70
2.67

^A

SK-BR-3
Breast
2.15
2.16
2.10

^C

T47D
Breast
4.80
4.80
4.59

^B

A172
CNS - Glioma
1.46
1.46
1.45

^B

CCF-STTG1
CNS - Glioma
9.00
9.00
7.35

^C

DBTRG-
CNS - Glioma
3.23
3.24
3.22

^C

05MG

DK-MG
CNS - Glioma
5.99
6.23
6.06

^C

H4
CNS - Glioma
3.32
3.32
3.32

^C

Hs 683
CNS - Glioma
5.01
5.01
4.87

^B

M059J
CNS - Glioma
4.62
4.62
4.55

^B

PFSK-1
CNS - Glioma
3.89
3.89
3.85

^B

SNB-19
CNS - Glioma
1.39
1.41
1.41

^C

SW1088
CNS - Glioma
1.79
1.79
1.78

^C

SW1783
CNS - Glioma
3.06
3.06
2.96

^B

T98G
CNS - Glioma
3.06
3.06
3.06

^B

U-118 MG
CNS - Glioma
4.20
4.24
4.20

^B

U-138MG
CNS - Glioma
5.92
5.92
5.74

^C

U-87 MG
CNS - Glioma
2.46
2.50
2.45

^C

D341 Med
CNS -
3.78
3.96
3.92

^B

Medulloblastoma

Daoy
CNS -
1.10
1.11
1.10

^A

Medulloblastoma

BE(2)C
CNS -
3.07
3.07
3.05

^B

Neuroblastoma

CHP-212
CNS -
1.19
1.19
1.14

^B

Neuroblastoma

MC-IXC
CNS -
3.32
3.32
3.30

^B

Neuroblastoma

SK-N-AS
CNS -
3.18
3.20
3.19

^A

Neuroblastoma

SK-N-DZ
CNS -
3.35
3.42
3.39

^B

Neuroblastoma

SK-N-FI
CNS -
3.37
5.39
4.66

^C

Neuroblastoma

Colo 201
Colon
1.08
1.08
1.08

^D

Colo 205
Colon
1.46
1.46
1.46

^D

Colo 320 HSR
Colon
2.45
2.45
2.44

^B

Colo 320DM
Colon
3.19
3.19
3.17

^B

DLD-1
Colon
3.19
3.19
3.19

^A

HCT-116
Colon
1.44
1.44
1.44

^B

HCT-15
Colon
1.92
1.92
1.92

^C

HCT-8
Colon
2.56
2.56
2.55

^D

HT-29
Colon
4.31
4.31
4.31

^A

LS1034
Colon
1.96
1.96
1.93

^B

LS123
Colon
1.28
1.38
1.29

^B

LS411N
Colon
6.41
6.41
6.30

^B

MT-3
Colon
4.10
4.10
4.10

^A

NCI-H508
Colon
>10.5
>10.5
>10.5

^B

NCI-H747
Colon
3.64
3.71
3.68

^B

RKO
Colon
1.88
1.88
1.87

^C

RKO-AS45-1
Colon
2.78
2.78
2.78

^C

RKOE6
Colon
2.49
2.49
2.49

^B

SW1417
Colon
3.27
4.03
3.92

^D

SW1463
Colon
3.64
3.68
3.64

^B

SW403
Colon
9.10
9.42
9.31

^B

SW48
Colon
1.14
1.14
1.14

^C

SW480
Colon
1.04
1.04
1.03

^C

SW620
Colon
2.32
2.32
2.32

^B

SW837
Colon
2.92
3.20
3.14

^C

SW948
Colon
>10.5
>10.5
>10.5

^D

WiDr
Colon
1.12
1.12
1.11

^B

NCI-H295R
Endocrine -
>10.5
>10.5
>10.5

^A

Adrenal gland

BHT-101
Endocrine -
2.72
2.72
2.71

^C

Thyroid

CAL-62
Endocrine -
1.18
1.18
1.18

^A

Thyroid

CGTH-W-1
Endocrine -
1.03
1.03
1.03

^B

Thyroid

SW579
Endocrine -
3.01
3.01
3.00

^C

Thyroid

Y79
Eye
3.08
3.10
3.02

^D

C-33A
Female GU -
4.27
4.27
4.24

^C

Cervix

C-4 II
Female GU -
1.19
1.19
1.19

^B

Cervix

HeLa
Female GU -
3.75
3.75
3.71

^C

Cervix

HT-3
Female GU -
3.99
4.03
3.99

^B

Cervix

SiHa
Female GU -
5.60
5.60
5.53

^B

Cervix

Ca Ski
Female GU -
5.53
5.53
5.46

^A

Ovary

CaOV3
Female GU -
2.59
2.60
2.52

^B

Ovary

ME-180
Female GU -
3.22
3.22
3.20

^B

Ovary

MS751
Female GU -
2.65
2.65
2.63

^B

Ovary

OVCAR3
Female GU -
2.01
2.08
2.02

^D

Ovary

PA-1
Female GU -
4.73
4.73
4.73

^B

Ovary

SKOV3
Female GU -
1.73
1.73
1.72

^B

Ovary

AN3 CA
Female GU -
1.61
1.61
1.58

^B

Uterus

HEC-1-A
Female GU -
1.60
1.68
1.67

^A

Uterus

KLE
Female GU -
3.28
3.28
2.92

^C

Uterus

SW954
Female GU -
8.51
8.51
8.47

^C

Vulva

BV-173
Leukemia
2.17
2.17
2.17

^B

CCRFCEM
Leukemia
3.85
3.85
3.82

^A

CEM-C1
Leukemia
1.13
1.13
1.12

^A

CML-T1
Leukemia
1.43
1.43
1.43

^A

EM-2
Leukemia
2.58
2.58
2.56

^A

HEL-92-1-7
Leukemia
5.29
5.29
5.25

^A

J-RT3-T3-5
Leukemia
1.29
1.29
1.29

^D

Jurkat
Leukemia
0.91
0.91
0.91

^B

K562
Leukemia
3.75
3.75
3.75

^A

KG-1
Leukemia
4.73
5.39
5.32

^B

KU812
Leukemia
4.52
4.52
4.45

^C

MEG01
Leukemia
4.55
4.55
4.55

^A

MHH-PREB-1
Leukemia
3.82
3.82
3.82

^C

MOLT-16
Leukemia
2.49
2.49
2.48

^A

MOLT-3
Leukemia
1.42
1.42
1.42

^C

MV-4-11
Leukemia
2.76
2.76
2.75

^A

MX1
Leukemia
1.28
1.28
1.28

^C

NALM-6
Leukemia
1.03
1.03
1.03

^C

RS4; 11
Leukemia
1.86
1.86
1.82

^A

TF-1
Leukemia
4.55
4.55
4.45

^C

Thp1
Leukemia
1.78
1.78
1.74

^A

BC-1
Lymphoma
1.73
1.73
1.73

^A

BCP-1
Lymphoma
3.26
3.26
3.23

^A

CA46
Lymphoma
2.03
2.03
2.03

^A

CRO-AP2
Lymphoma
3.99
3.99
3.99

^A

Daudi
Lymphoma
1.33
1.33
1.33

^A

DB
Lymphoma
1.09
1.10
1.10

^B

DOHH-2
Lymphoma
1.01
1.01
1.01

^A

DoTc2 4510
Lymphoma
1.38
1.38
1.36

^A

EB2
Lymphoma
5.60
5.60
5.57

^A

EB-3
Lymphoma
1.68
1.68
1.68

^B

GA-10
Lymphoma
1.42
1.42
1.42

^A

Hs 445
Lymphoma
1.46
1.46
1.44

^C

Hs 611.T
Lymphoma
3.29
3.29
3.28

^D

HT
Lymphoma
1.61
1.61
1.60

^C

JeKo-1
Lymphoma
2.58
2.58
2.57

^A

Jiyoye
Lymphoma
4.24
4.24
4.24

^A

L-428
Lymphoma
1.24
1.24
1.24

^A

MC116
Lymphoma
3.15
3.15
3.15

^A

NAMALWA
Lymphoma
1.68
1.69
1.68

^D

Raji
Lymphoma
3.92
3.92
3.92

^A

Ramos (RA 1)
Lymphoma
2.33
2.33
2.33

^C

RPMI 6666
Lymphoma
3.28
3.32
3.27

^C

SR
Lymphoma
1.74
1.74
1.74

^A

ST486
Lymphoma
1.08
1.08
1.08

^A

SU-DHL-10
Lymphoma
4.17
4.17
4.17

^A

SU-DHL-4
Lymphoma
1.82
1.82
1.82

^C

SU-DHL-5
Lymphoma
1.02
1.02
1.02

^B

SU-DHL-8
Lymphoma
1.74
1.74
1.74

^A

SUP-T1
Lymphoma
3.75
3.75
3.75

^A

TUR
Lymphoma
4.48
4.48
4.48

^C

ARH-77
Myeloma
2.66
2.67
2.65

^A

IM-9
Myeloma
0.68
0.68
0.68

^B

RPMI 8226
Myeloma
0.99
1.00
0.99

^C

SKO-007
Myeloma
0.99
1.00
0.99

^B

U266B1
Myeloma
2.82
2.82
2.71

^B

A-253
Head and Neck
8.23
8.23
8.23

^B

A388
Head and Neck
1.92
1.94
1.93

^B

A431
Head and Neck
3.29
3.30
3.30

^A

Cal 27
Head and Neck
2.17
2.17
2.17

^B

Detroit 562
Head and Neck
2.99
3.00
2.99

^B

FaDu
Head and Neck
0.90
0.90
0.90

^B

OE19
Head and Neck
2.97
2.98
2.96

^C

OE21
Head and Neck
3.89
3.89
3.89

^C

SCC-25
Head and Neck
5.99
5.99
5.95

^C

SCC-4
Head and Neck
8.51
8.51
8.47

^B

SCC-9
Head and Neck
7.35
7.42
7.39

^B

769-P
Kidney
1.05
1.05
1.05

^B

786-O
Kidney
2.81
2.81
2.81

^B

A498
Kidney
1.80
1.80
1.79

^B

A-704
Kidney
2.99
3.01
2.98

^B

ACHN
Kidney
3.26
3.26
3.25

^A

Caki-1
Kidney
4.66
4.66
4.59

^C

Caki-2
Kidney
4.76
4.80
4.80

^A

G-401
Kidney
4.13
4.13
4.13

^B

SK-NEP-1
Kidney
3.99
3.99
3.99

^D

HepG2
Liver
1.54
1.54
1.54

^C

HLE
Liver
3.92
3.92
3.89

^B

HLF
Liver
3.99
3.99
3.96

^C

HuCCT1
Liver
3.64
3.64
3.64

^B

HUH-6 Clone
Liver
1.70
1.70
1.69

^B

5

OCUG-1
Liver
3.61
3.64
3.64

^B

SNU-423
Liver
1.47
1.47
1.45

^B

A427
Lung - NSCLC
4.13
4.13
4.10

^A

A549
Lung - NSCLC
2.98
2.98
2.98

^C

Calu1
Lung - NSCLC
2.72
2.72
2.69

^A

Calu6
Lung - NSCLC
9.56
9.77
9.59

^B

ChaGoK1
Lung - NSCLC
1.69
1.69
1.68

^A

COR-L105
Lung - NSCLC
2.85
2.86
2.84

^A

COR-L23
Lung - NSCLC
4.59
4.62
4.59

^D

Hs 229.T
Lung - NSCLC
3.09
3.31
3.15

^A

NCI-H292
Lung - NSCLC
3.50
3.50
3.49

^A

NCIH441
Lung - NSCLC
>10.5
>10.5
>10.5

^A

NCI-H460
Lung - NSCLC
2.99
2.99
2.99

^A

NCI-H520
Lung - NSCLC
3.02
3.02
3.01

^A

NCI-H596
Lung - NSCLC
8.89
8.93
8.86

^A

NCI-H661
Lung - NSCLC
1.70
1.70
1.69

^A

SKMES1
Lung - NSCLC
1.26
1.26
1.25

^A

DMS114
Lung - SCLC
4.62
4.69
4.55

^C

DMS53
Lung - SCLC
0.51
0.51
0.48

^A

NCIH446
Lung - SCLC
3.75
3.75
3.75

^B

NCI-H69
Lung - SCLC
5.01
5.01
4.73

^A

SHP-77
Lung - SCLC
1.03
1.03
1.03

^B

SW900
Lung - SCLC
3.64
3.68
3.61

^C

AsPC-1
Pancreas
2.51
2.51
2.45

^A

BxPC-3
Pancreas
4.48
4.48
4.48

^A

Capan-1
Pancreas
>10.5
>10.5
>10.5

^C

Capan-2
Pancreas
1.34
1.34
1.33

^A

CFPAC4
Pancreas
3.57
3.57
3.57

^A

HPAF-II
Pancreas
2.30
2.31
2.29

^C

Hs 766T
Pancreas
2.44
2.47
2.42

^A

HuP-T4
Pancreas
>10.5
>10.5
>10.5

^C

Mia PaCa-2
Pancreas
3.15
3.15
3.15

^A

PANC-1
Pancreas
2.20
2.20
2.17

^A

PSN-1
Pancreas
2.70
2.70
2.70

^B

SU.86.86
Pancreas
>10.5
>10.5
>10.5

^C

YAPC
Pancreas
3.37
3.37
3.34

^A

BeWo
Placenta
10.05
>10.5
>10.5

^B

JAR
Placenta
8.65
8.65
8.65

^C

JEG-3
Placenta
4.38
4.38
4.34

^B

22Rv1
Prostate
4.13
4.13
4.10

^A

BM-1604
Prostate
3.89
3.89
3.85

^C

BPH1
Prostate
4.10
8.82
7.98

^A

DU145
Prostate
3.61
3.61
3.57

^A

LNCaP
Prostate
3.17
3.17
2.62

^C

PC-3
Prostate
4.06
4.06
4.03

^A

A101D
Skin
6.23
6.23
6.09

^B

(Melanoma)

A375
Skin
1.41
1.41
1.41

^B

(Melanoma)

A7
Skin
0.92
0.92
0.91

^B

(Melanoma)

C32
Skin
1.71
1.71
1.36

^B

(Melanoma)

CHL-1
Skin
1.59
1.59
1.59

^D

(Melanoma)

COLO 829
Skin
1.01
1.03
0.98

^D

(Melanoma)

G-361
Skin
2.70
2.70
2.69

^B

(Melanoma)

HMCB
Skin
1.46
1.47
1.46

^C

(Melanoma)

Hs 294T
Skin
1.73
1.73
1.67

^B

(Melanoma)

Hs 688(A).T
Skin
2.11
2.22
1.99

^B

(Melanoma)

Hs 695T
Skin
0.22
0.22
0.11

^C

(Melanoma)

Hs 852.T
Skin
3.64
3.64
3.50

^B

(Melanoma)

Hs 934.T
Skin
3.01
4.52
3.02

^B

(Melanoma)

Hs 936.T(C1)
Skin
2.26
2.26
2.22

^B

(Melanoma)

MALME3M
Skin
2.69
2.69
2.53

^C

(Melanoma)

MeWo
Skin
1.93
1.93
1.91

^B

(Melanoma)

RPMI-7951
Skin
0.74
0.74
0.73

^C

(Melanoma)

SH-4
Skin
2.60
2.60
2.58

^B

(Melanoma)

SK-MEL-1
Skin
3.96
4.27
4.13

^B

(Melanoma)

SK-MEL-28
Skin
2.38
2.39
2.37

^A

(Melanoma)

SK-MEL-3
Skin
5.25
5.25
5.15

^C

(Melanoma)

WM-266-4
Skin
1.19
1.19
1.18

^B

(Melanoma)

G-292, clone
Soft Tissue -
2.26
2.26
2.16

^B

A141B1
Osteosarcoma

HOS
Soft Tissue -
1.06
1.06
1.06

^B

Osteosarcoma

Hs 888.Sk
Soft Tissue -
3.09
3.19
3.10

^B

Osteosarcoma

KHOS-240S
Soft Tissue -
2.18
2.18
2.18

^D

Osteosarcoma

MG-63
Soft Tissue -
9.66
9.70
9.66

^A

Osteosarcoma

SaOS2
Soft Tissue -
3.36
3.43
3.33

^B

Osteosarcoma

SJSA1
Soft Tissue -
1.51
1.51
1.50

^B

Osteosarcoma

SW1353
Soft Tissue -
1.76
1.76
1.76

^B

Osteosarcoma

U2OS
Soft Tissue -
2.35
2.35
2.34

^C

Osteosarcoma

A204
Soft Tissue -
0.65
0.65
0.64

^A

Sarcoma

A-673
Soft Tissue -
1.15
1.15
1.15

^B

Sarcoma

Hs 729
Soft Tissue -
3.07
3.11
3.01

^C

Sarcoma

Hs 821.T
Soft Tissue -
2.79
5.43
2.56

^B

Sarcoma

HT-1080
Soft Tissue -
0.59
0.59
0.59

^A

Sarcoma

MES-SA
Soft Tissue -
1.53
1.53
1.53

^B

Sarcoma

RD
Soft Tissue -
0.69
0.69
0.69

^C

Sarcoma

SJRH30
Soft Tissue -
1.46
1.46
1.46

^A

Sarcoma

SK-LMS-1
Soft Tissue -
2.23
2.23
2.21

^C

Sarcoma

SK-UT-1
Soft Tissue -
2.39
2.39
2.38

^B

Sarcoma

SW684
Soft Tissue -
9.14
9.14
7.95

^C

Sarcoma

SW872
Soft Tissue -
0.35
0.35
0.35

^A

Sarcoma

SW982
Soft Tissue -
1.20
1.20
1.19

^B

Sarcoma

TE 125.T
Soft Tissue -
2.05
6.34
1.31

^B

Sarcoma

TE 381.T
Soft Tissue -
1.56
1.56
1.55

^A

Sarcoma

VA-ES-BJ
Soft Tissue -
4.17
4.17
4.13

^B

Sarcoma

AGS
Stomach
0.85
0.85
0.85

^B

HS 746T
Stomach
3.99
6.51
4.94

^A

KATO III
Stomach
4.62
4.62
4.62

^C

SK-PN-DW
Stomach
2.16
2.16
2.15

^B

SNU-1
Stomach
1.51
1.51
1.51

^C

SNU-16
Stomach
1.44
1.44
1.44

^D

SNU-5
Stomach
4.80
4.80
4.73

^D

NTERA-2
Testis
1.98
1.99
1.96

^B

cl.D1

^A14 Jul. 2016;

^B22 Aug. 2016;

^C22 Sep. 2016;

^D5 Oct. 2016

For Compound 1, a broad spectrum of activity was observed over the 280 cell types with very potent to strong (GI₅₀=0.006 to 2.98 μM) activity towards 174 cell lines. Very potent activity was observed towards SK-NEP-1 kidney (GI₅₀=0.00618 μM) and J-RT3-T3-5 leukemia (GI₅₀=0.0101 μM) cell lines. Moderately potent activity was observed (GI₅₀=0.11 μM) towards KG-1 leukemia cell line. Moderately potent activity (GI₅₀=0.4 to 1.0 μM) was observed towards the following cell lines: 2 breast cell lines KPL-1, MDA-MB-453; 3 myeloma cell lines IM-9, RPMI8226, SKO-007; 3 melanoma cell lines A7, COLO829, RPMI-7951; 7 soft tissue cell lines HOS (osteosarcoma), KHOS-240S (osteosarcoma), SW1353 (osteosarcoma), A204 (sarcoma), HT-1080 (sarcoma), RD (sarcoma), SW 872 (sarcoma); 2 small cell lung carcinoma cell lines DMS53, SHP-77; and also Colo 201 (colon); FaDu (head and neck), 769-P (kidney), HepG2 (liver), and AGS (stomach). Strong activity (GI₅₀=1 to 2.98 μM) was shown towards 149 cell lines: 16 melanoma; 15 lymphoma; 9 female genitourinary; 14 soft tissue (sarcoma); 16 colon; 12 leukemia; 8 non-small cell lung carcinoma; 8 pancreas; 9 breast; 10 CNS; 8 bladder; 5 kidney; 4 head and neck, 4 endocrine, 1 myeloma, 3 liver, 2 small cell lung carcinoma, 2 prostrate, 2 stomach and 1 testis.

For Compound 2, a narrow spectrum of activity was observed over the 280 cell types with potent to strong (GI₅₀=0.06 to 2.96 μM) activity towards 10 cell lines. Potent activity was observed towards SK-NEP-1 kidney (GI₅₀=0.0606 μM) Strong activity (GI₅₀=1 to 2.96 μM) was observed towards the following cell lines: 5 leukemia cell lines J-RT3-T3-5, Jurkat, MOLT-3, NALM-6, TF-1; 2 myeloma cell lines RPMI8226, SKO-007; 1 soft tissue cell line RD (sarcoma). For Compound 2 activities towards Jurkat, MOLT-3, NALM-6, TF-1 (leukemia) and SKO-007 (myeloma) were similar in magnitude to Compound 1.

For Compound 4, a narrow spectrum of activity was observed over the 280 cell types with moderately potent to strong (GI₅₀=0.68 to 2.92 μM) activity towards 12 cell lines. One strong activity (GI₅₀=0.684 μM) was observed towards A204 (soft tissue sarcoma) similar in magnitude to that observed for Compound 1. Strong activity (GI₅₀=1.23 to 2.92 μM) for Compound 4 was observed towards the following cell lines: 5 lymphoma (Daudi, DOHH-2, GA-10, L-428, ST486); 2 leukemia (MX1, NALM-6); also for T47D (breast), CEM-C1 (leukemia), A-673 (soft tissue sarcoma). The activity towards these cell lines is comparable in magnitude to Compound 1, however, Compound 4 show strong activity towards the J-RT3-T3-5 leukemia cell line (GI₅₀=2.71 μM) approximately 270 times less activity than the very potent activity observed for Compound 1 (GI₅₀=0.0101 μM) towards this cell line.

The plant resin extract showed strong activity (GI₅₀=0.11 to 1.03 μg/mL) towards 24 cell lines: 4 soft tissue sarcoma (A204, HT-1080, RD, SW 872); 4 melanoma (A7, COLO829, Hs695T, RPMI-7951); 3 myeloma (IM-9, RPMI8226, SKO-007); 2 breast (KPL-1, MDA-MB-453); 2 lymphoma (DOHH-2, SU-DHL-5); 2 small cell carcinoma (DMS53, SHP-77); 2 leukemia (Jurkat, NALM-6) and also bladder (UM-UC-3); colon (SW480); thyroid (CGTH-W-1); head and neck (FaDu); and stomach (AGS). For the Hs695T melanoma cell line, calculated on a weight-weight basis, the plant resin extract (GI₅₀=0.11 μg/mL) was approximately 3 times more active than Compound 1 (GI₅₀=0.37 μg/mL) towards this particular cell line. From weight comparison for all other cell lines the plant extract was less active than Compound 1.

Overall, Compound 1 provides a broad spectrum of anticancer activity towards a wide range of cell lines. Compound 2 shows specificity for the LNCaP prostrate cell line, also specific activity towards SK-NEP-1 (kidney), Jurkat, MOLT-3, NALM-6, TF-1 (leukemia) and SKO-007, RPMI8226 (myeloma) similar in magnitude to Compound 1. Compound 4 showed moderately potent to strong activity (GI₅₀=0.68 to 2.92 μM) towards a narrow range of cell lines (12 out of 280) compared with Compound 1 (174 out of 280).

TABLE 30

Number of cancer cell types for levels of growth

inhibitory activity for Compounds 1, 2 and 4

Number of cell types

GI₅₀range
Comp
Comp
Comp

GI Activity
(μM)
1
2
4

very potent
0.001-0.029
2
0
0

Potent
0.03-0.099
0
1
0

moderately potent
0.1-0.99
23
1
1

Strong
1-2.99
149
8
11

moderately strong
3-9.99
99
110
125

weak
10-30.00
6
118
130

very weak
>30
1
42
13

TOTAL

280
280
280

TABLE 31

Number of cancer cell types for levels of growth

inhibitory activity for M. insulare extract

GI₅₀range
Number of cell types

GI Activity
(μg/mL)

M. insulare extract

very potent
0.00035-0.0104
0

Potent
0.0105-0.034
0

moderately potent
0.035-0.34
1

Strong
0.35-1.04
23

moderately strong
1.05-3.49
165

weak
3.5-10.49
83

very weak
>10.5
8

TOTAL

280

Inhibition of Toll-Like Receptor 4 (TLR4) Activation on Human Peripheral Blood Mononuclear Cells (PBMC's).
Study Objective

To evaluate compound capability to inhibit Toll-like receptor 4 (TLR4) activation on human peripheral blood mononuclear cells (PBMC's).

Experimental Protocol

Compound 1 was evaluated for ability to inhibit TLR4 in human PBMC's, as measured by release of specific cytokines. Evaluation was conducted by Eurofins Panlabs Inc., St Charles, Mo., USA.

TLR4 Inhibition Assay Protocol

- a) Cryopreserved human PBMC's were drip-thawed.
- b) Cells were diluted to the appropriate density (1×105 per well) and seeded into 96-well polypropylene plates with 150 μL per well of culture medium (RPMI 1640, 10% heat-inactivated FBS, 1% penicillin/streptomycin, 2 mM L-glutamine).
- c) Cells were incubated at 37° C., 5% CO₂for 1 hour prior to the addition of test compounds or controls.
- d) Test compounds were solubilized in DMSO to make stock solutions, and then diluted further with cell culture medium. The positive control, LPS (from S. minnesota R595), was resuspended in endotoxin-free water at 0.5 mg/mL to make working stock solutions. Dexamethasone was used as a reference control compound.
- e) Compounds and controls, including appropriate vehicle controls, were added to the PBMC's in volumes of 10 μL and incubated for 1 hour at 37° C., 5% CO₂. Compounds were tested in duplicate at final assay concentrations of 10, 3, 1, 0.3, 0.1, and 0.03 μM. Vehicle control wells simply received 10 μL of the appropriate vehicle.
- f) After a 1-hour incubation, 40 μL of diluted working stock LPS were added to the test compound and control wells to give final assay volumes of 200 μL. Plates were incubated for 24 hours at 37° C., 5% CO₂.
- g) Plates were centrifuged at 200×g for 10 minutes. Cell culture supernatants were collected and stored at −80° C. until needed for analysis.
- h) Cytokine levels in each sample were determined using Luminex methodology, per the manufacturer's protocol.

PBMC Donor: Human donor 1 (lot #100)

Results and Discussion

Stimulation of human PBMC's with LPS for 24 hours elicited the measurable release of appropriate cytokines within expected ranges. Conversely, dexamethasone inhibited the LPS-stimulated release of cytokines from human PBMC's, also within expected ranges. Compound 1 elicited measurable inhibition of the LPS-induced release of specific cytokines from human PBMC's, following a 24-hour stimulation.

As seen from the results shown in Table 32, Compound 1 elicited a moderate to strong inhibition of the secretion of all cytokines at the top test concentration, giving measured IC₅₀values of 4.3 μM, 6.6 μM, 8.8 μM, and 8.9 μM for IL-10, IL-1Iβ, IL-6, and TNFα, respectively. (IC₅₀values could not be measured for IL-8 and MIP-1α.)

TABLE 32

Compound 1 activity against certain LPS-induced cytokines

Compound 1

LPS-induced cytokine

Assay
IC₅₀(μM)

I-1β
6.6

IL-6
8.8

IL-8
NA

IL-10
4.3

MIP-1α
NA

TNF-α
8.9

Bioprint Assays

The Bioprint assay (conducted by Eurofins Cerep, France) provides a profile that is designed with the dual purpose of assessing safety at targets with known safety liabilities as well as providing a rich enough profile to search for compounds with similar profiles.

Methodology

Compound 1 was tested at 10 μM. For Compound 1 at 10 μM, the agonist radioligand assays for control specific inhibition of interest for biological activity or safety are as follows:—

GPCR Family:—adenosine A3 (h), 92%; adrenergic alpha 2C (h), 92%; cannabinoid CB2 (h), 71%; cholecystokinin CCK1 (CCKA) (h), 81%; dopamine D1 (h), 67%; dopamine D3 (h), 85%; histamine H1 (h), 64%; melatonin MT1 (ML1A) (h), 91%.

Transporters:—norepinephrine transporter (h), 76%.

Non-steroid Nuclear Receptors:—PPARgamma (h), 91%.

For Compound 1 at 10 μM, the enzyme assays for control specific inhibition of interest for biological activity or safety are as follows:—

AA Metabolism:—COX1 (h), 81%; COX2 (h), 74%.

Compound binding was calculated as a % inhibition of the binding of a radioactively labeled ligand specific for each target. Compound enzyme inhibition effect was calculated as a % inhibition of control enzyme activity. Results showing an inhibition or stimulation higher than 50% are considered to represent significant effects of the test compounds.

Results

Compound 1 results for inhibition of control specific binding to receptors are shown in Table 33 whilst results for inhibition of control specific binding inhibition of enzymes in enzyme and cell-based assays are shown in Table 34.

TABLE 33

Bioprint results for Compound 1 tested at 10 μM for inhibition of control specific binding to receptors.

Compound

1

% Inhibition

of Control
Reference

Family
Binding Assay
Specific Binding
Compound
IC₅₀(μM)

GPCR

ADENOSINE
A1 (h)
10
CPA
0.0021

(agonist radioligand)

ADENOSINE
A2A (h)
18
NECA
0.036

(agonist radioligand)

ADENOSINE
A2B (h)
8
NECA
0.59

(antagonist radioligand)

ADENOSINE
A3 (h)
92
IB-MECA
4.5E−04

(agonist radioligand)

ADRENERGIC
alpha 1A (h)
17
WB 4101
2.7E−04

(antagonist radioligand)

ADRENERGIC
alpha 1B (h)
41
prazosin
1.7E−04

(antagonist radioligand)

ADRENERGIC
alpha 2A (h)
35
yohimbine
0.0065

(antagonist radioligand)

ADRENERGIC
alpha 2B (h)
7
yohimbine
0.0065

(antagonist radioligand)

ADRENERGIC
alpha 2C (h)
92
yohimbine
0.004

(antagonist radioligand)

ADRENERGIC
beta 1 (h)
19
atenolol
0.34

(agonist radioligand)

ADRENERGIC
beta 2 (h)
2
ICI 118551
7.8E−04

(agonist radioligand)

ADRENERGIC
Adrenergic beta3
33
Alprenolol
0.25

ANGIOTENSIN II
AT1 (h)
60
saralasin
0.0019

(antagonist radioligand)

ANGIOTENSIN II
AT2 (h)
54
angiotensin-II
1.4E−04

(agonist radioligand)

APELIN
APJ (apelin) (h)
24
apelin-13, TFA
3.4E−04

(agonist radioligand)

BOMBESIN
BB3 (h)
26
Bn(6-14)
0.0048

(agonist radioligand)

BRADYKININ
B2 (h)
−37
NPC 567
0.038

(agonist radioligand)

CANNABINOID
CB1 (h)
46
CP 55940
0.001

(agonist radioligand)

CANNABINOID
CB2 (h)
71
WIN 55212-2
0.0022

(agonist radioligand)

CYTOKINES
TNF-alpha (h)
−23
TNF-alpha
1.6E−04

(agonist radioligand)

CHOLECYSTOKININ
CCK1 (CCKA) (h)
81
CCK-8s
1.5E−04

(agonist radioligand)

CHOLECYSTOKININ
CCK2 (CCKB) (h)
6
CCK-8s
1.7E−04

(agonist radioligand)

CORTICOTROPIN
CRF1 (h)
34
sauvagine
2.5E−04

RELEASING
(agonist radioligand)

FACTOR

DOPAMINE
D1 (h)
67
SCH 23390
3.9E−04

(antagonist radioligand)

DOPAMINE
D2S (h)
59
7-OH-DPAT
0.0033

(agonist radioligand)

DOPAMINE
D3 (h)
85
(+)butaclamol
0.0024

(antagonist radioligand)

ENDOTHELIN
ETA (h)
6
endothelin-1
4.9E−05

(agonist radioligand)

ENDOTHELIN
ETB (h)
40
endothelin-3
1.7E−05

(agonist radioligand)

GABA
GABAB(1b) (h)
−17
CGP 54626
0.0022

(antagonist radioligand)

GLUCAGON
glucagon (h)
−20
glucagon
0.001

(agonist radioligand)

CHEMOKINES
CCR2 (h)
48
MCP-1
8.7E−05

(agonist radioligand)

HISTAMINE
H1 (h)
64
pyrilamine
0.0023

(antagonist radioligand)

HISTAMINE
H2 (h)
37
cimetidine
0.66

(antagonist radioligand)

HISTAMINE
H3 (h)
10
(R)alpha-Me-histamine
0.0016

(agonist radioligand)

HISTAMINE
H4 (h)
23
imetit
0.0047

(agonist radioligand)

LEUKOTRIENES
BLT1 (LTB4) (h)
27
LTB4
3.7E−04

(agonist radioligand)

LEUKOTRIENES
CysLT1 (LTD4) (h)
10
LTD4
6.3E−04

(agonist radioligand)

MELANIN-
MCH1 (h)
−5
human MCH
7.8E−05

CONCENTRATING-
(agonist radioligand)

HORMONE

MELANOCORTIN
MC1
11
NDP-alpha-MSH
1.2E−04

(agonist radioligand)

MELANOCORTIN
MC3 (h)
60
NDP-alpha-MSH
2.7E−04

(agonist radioligand)

MELANOCORTIN
MC4 (h)
37
NDP-alpha-MSH
4.0E−04

(agonist radioligand)

MELATONIN
MT1 (ML1A) (h)
91
melatonin
2.8E−04

(agonist radioligand)

MOTOLIN
motilin (h)
−12
[Nleu13]-motilin
8.9E−04

(agonist radioligand)

MUSCARINIC
M1 (h)
8
pirenzepine
0.031

(antagonist radioligand)

MUSCARINIC
M2 (h)
1
methoctramine
0.032

(antagonist radioligand)

MUSCARINIC
M3 (h)
−3
4-DAMP
0.001

(antagonist radioligand)

MUSCARINIC
M4 (h)
19
4-DAMP
9.2E−04

(antagonist radioligand)

NEUROKININ
NK1 (h)
62
[Sar9, Met(O2)11]-SP
3.7E−04

(agonist radioligand)

NEUROKININ
NK2 (h)
52
[Nleu10]-NKA (4-10)
0.0029

(agonist radioligand)

NEUROPEPTIDE Y
Y1 (h)
29
NPY
1.6E−04

(agonist radioligand)

OPIOD & OPIOID-
delta (DOP) (h)
62
DPDPE
0.002

LIKE
(agonist radioligand)

OPIOD & OPIOID-
kappa (KOP)
39
U 50488
0.0012

LIKE
(agonist radioligand)

OPIOD & OPIOID-
mu (MOP) (h)
43
DAMGO
5.3E−04

LIKE
(agonist radioligand)

OPIOD & OPIOID-
NOP (ORL1) (h)
−4
nociceptin
8.4E−04

LIKE
(agonist radioligand)

PLATELET
PAF (h)
39
C16-PAF
0.0034

ACTIVATED
(agonist radioligand)

FACTOR

PROSTANOID
EP2 (h)
53
PGE2
0.0027

(agonist radioligand)

PROSTANOID
FP (h)
54
PGF2alpha
0.003

(agonist radioligand)

PROSTANOID
IP (PGI2) (h)
16
iloprost
0.017

(agonist radioligand)

SEROTONIN
5-HT1A (h)
−18
8-OH-DPAT
8.3E−04

(agonist radioligand)

SEROTONIN
5-HT1B
42
serotonin
0.013

(antagonist radioligand)

SEROTONIN
5-HT1D
18
serotonin
0.0026

(agonist radioligand)

SEROTONIN
5-HT2A (h)
9
(±)DOI
4.7E−04

(agonist radioligand)

SEROTONIN
5-HT2B (h)
70
(±)DOI
0.0067

(agonist radioligand)

SEROTONIN
5-HT2C (h)
32
(±)DOI
2.4E−04

(agonist radioligand)

SEROTONIN
5-HT4e (h)
2
serotonin
0.19

(antagonist radioligand)

SEROTONIN
5-HT6 (h)
42
serotonin
0.18

(agonist radioligand)

SEROTONIN
5-HT7 (h)
14
serotonin
4.4E−04

(agonist radioligand)

SOMASTATIN
sst1 (h)
18
somatostatin-28
5.5E−04

(agonist radioligand)

SOMASTATIN
sst4 (h)
40
somatostatin-14
0.0042

(agonist radioligand)

UROTENSIN-II
UT (h)
46
urotensin-II
9.5E−04

(agonist radioligand)

VASOACTIVE
VPAC1 (VIP1) (h)
−9
VIP
2.7E−04

INTESTINAL
(agonist radioligand)

PEPTIDE

VASOPRESSIN
V1a (h)
12
[d(CH2)51, Tyr(Me)2]-
0.0015

(agonist radioligand)

AVP

VASOPRESSIN
V2 (h)
11
AVP
0.0013

(agonist radioligand)

TRANSPORTERS

CHOLINE
choline transporter
−41
hemicholinium-3
0.0046

(CHT1) (h)

(antagonist radioligand)

DOPAMINE
dopamine transporter (h)
63
BTCP
0.012

(antagonist radioligand)

GABA
GABA transporter
−10
nipecotic acid
2.3

(antagonist radioligand)

NOREPINEPHRINE
norepinephrine
76
protriptyline
0.0039

transporter (h)

(antagonist radioligand)

SEROTONIN
5-HT transporter (h)
56
imipramine
0.0044

(antagonist radioligand)

ION-CHANNELS

GABA
GABAA1 (h) (alpha
−36
muscimol
0.053

1, beta 2, gamma 2)

(agonist radioligand)

GABA CHANNELS
BZD (central)
−31
diazepam
0.0094

(agonist radioligand)

GABA CHANNELS
Cl- channel (GABA-
42
picrotoxinin
0.33

gated)

(antagonist radioligand)

GLUTAMATE
PCP
−11
MK 801
0.0096

CHANNELS
(antagonist radioligand)

GLUTAMATE
AMPA
4
L-glutamate
0.37

CHANNELS
(agonist radioligand)

GLUTAMATE
kainate
15
kainic acid
0.023

CHANNELS
(agonist radioligand)

GLUTAMATE
NMDA
9
CGS 19755
0.35

CHANNELS
(antagonist radioligand)

GLYCINE
glycine (strychnine-
4
glycine
0.28

CHANNELS
insensitive)

(antagonist radioligand)

NICOTINIC
N neuronal alpha 4beta 2 (h)
−3
nicotine
0.0046

CHANNELS
(agonist radioligand)

NICOTINIC
N muscle-type (h)
1
alpha -bungarotoxin
0.0019

CHANNELS
(antagonist radioligand)

SEROTONIN
5-HT3 (h)
−2
MDL 72222
0.0086

CHANNELS
(antagonist radioligand)

Ca²⁺ CHANNELS
Ca2+ channel (L,
55
nitrendipine
1.3E−04

dihydropyridine site)

(antagonist radioligand)

Ca²⁺ CHANNELS
Ca2+ channel (L,
−30
diltiazem
0.057

diltiazem site)

(benzothiazepines)

(antagonist radioligand)

Ca²⁺ CHANNELS
Ca2+ channel (L,
−4
D 600
0.027

verapamil site)

(phenylalkylamine)

(antagonist radioligand)

Ca²⁺ CHANNELS
Ca2+ channel (N)
5
omega -conotoxin
1.7E−06

(antagonist radioligand)

GVIA

K⁺ CHANNELS
SKCa channel
14
apamin
9.7E−06

(antagonist radioligand)

Na⁺ CHANNELS
Na+ channel (site 2)
48
veratridine
5.9

(antagonist radioligand)

NUCLEAR RECEPTORS

NON-STEROID
PPARgamma (h)
91
rosiglitazone
0.015

NUCLEAR
(agonist radioligand)

RECEPTORS

STEROID
AR (h)
−9
mibolerone
0.0021

NUCLEAR
(agonist radioligand)

RECEPTORS

STEROID
Estrogen ER alpha (h)
40
Diethylstilbestrol
4.5E−04

NUCLEAR
(agonist radioligand)

RECEPTORS

STEROID
GR (h)
47
dexamethasone
0.0035

NUCLEAR
(agonist radioligand)

RECEPTORS

OTHER RECEPTORS

SIGMA
sigma (non-selective) (h)
11
haloperidol
0.045

(agonist radioligand)

THYROID
Thyroid Hormone
9
Triiodothyronine
3.9E−05

HORMONE

TABLE 34

Bioprint results for Compound 1 tested at 10 μM for inhibition of control

specific binding inhibition of enzymes in enzyme and cell-based assays.

Compound

1

% Inhibition

Enzyme and
of Control
Reference

Family
Cell-based Assays
Values
Compound
IC₅₀(μM)

KINASES

RTK
FLT-1 kinase (h)
36
staurosporine
0.0098

(VEGFR1)

RTK
IRK (h) (InsR)
−15
staurosporine
0.052

CTK
Abl kinase (h)
14
staurosporine
0.11

CTK
Fyn kinase (h)
6
PP1
0.11

CTK
Lyn A kinase (h)
9
staurosporine
0.012

CTK
ZAP70 kinase (h)
33
staurosporine
0.021

CMGC
CDK2 (h) (cycA)
12
staurosporine
0.011

CMGC
ERK2 (h) (P42mapk)
6
staurosporine
1

CMGC
p38alpha kinase (h)
4
SB202190
0.033

CAMK
CaMK2alpha (h)
2
AIP
0.092

OTHER NON-KINASE ENZYMES

AA METABOLISM
COX1(h)
81
Diclofenac
0.0078

AA METABOLISM
COX2(h)
74
NS398
0.19

ATPASE
ATPase (Na+/K+)
22
ouabain
0.25

MONOAMINE &
acetylcholinesterase (h)
32
galanthamine
0.57

NEUROTRANSMITTER

MONOAMINE &
COMT (catechol-O-methyl
−6
Ro 41-0960
0.061

NEUROTRANSMITTER
transferase)

MONOAMINE &
MAO-A
15
clorgyline
0.0012

NEUROTRANSMITTER
(antagonist radioligand)

NO SYNTHASES
inducible NOS
−1
1400 W
0.029

PHOSPHODIESTERASES
PDE2A1 (h)
43
EHNA
1.2

PHOSPHODIESTERASES
PDE3B (h)
−3
milrinone
1.2

PHOSPHODIESTERASES
PDE4D2 (h)
55
Ro 20-1724
0.15

PHOSPHODIESTERASES
PDE5 (h) (non-selective)
11
dipyridamole
0.84

PHOSPHODIESTERASES
PDE6 (non-selective)
4
zaprinast
0.16

SERINE PROTEASES
caspase-3 (h)
−3
Ac-DEVD-CHO
0.0029

ASPARTIC PROTEASES
BACE-1 (h) (beta -
0
OM 99-2
0.081

secretase)

ASPARTIC PROTEASES
HIV-1 protease
9
pepstatin A
2.4

METALLOPROTEASES
ACE (h)
14
captopril
5.8E−04

METALLOPROTEASES
ACE-2 (h)
−3
Ac-GG-26-NH2
0.2

METALLOPROTEASES
MMP-1 (h)
−2
GM6001
0.0054

METALLOPROTEASES
MMP-2 (h)
0
GM6001
7.9E−04

METALLOPROTEASES
MMP-9 (h)
1
GM6001
3.6E−04

MISCELLANEOUS
MT3 (ML2)
31
melatonin
0.16

ENZYMES
(agonist radioligand)

MISCELLANEOUS
xanthine oxidase/
−140
allopurinol
7.8

ENZYMES
superoxide O2-

scavenging

In Vitro Toxicity: Normal Cell Panel Toxicity Assay

The OncoPanel (Eurofins Panlabs Inc., St Charles, Mo., USA) normal cell proliferation assay measures the proliferation response of normal cells to drug treatments through high-content fluorescence imaging.

Methodology

Normal cells (see description in Table 35) were grown in special medium for each cell type. Cells were seeded into 384-well plates and incubated in a humidified atmosphere of 5% CO₂at 37° C. Compounds (information in Table 36) were added the day following cell seeding. At the same time, a time zero untreated cell plate was generated. After a 3-day incubation period, cells were fixed and stained to allow fluorescence imaging of nuclei.

TABLE 35

Description of cells used in normal cell panel toxicity assay

Normal cell name
Cell type
Source
Catalog number

CCD 841 CoN
Colon epithelial
ATCC
CRL-1790

HMEC
Mammary epithelial
ATCC
PCS-600-010

HREC
Renal mixed epithelial
ATCC
PCS-400-012

Wi38
Lung fibroblast
ATCC
CCL-75

TABLE 36

Information for compounds used in normal cell panel toxicity assay

Amount
Stock
Stock
Max final

received
conc.
volume
test conc.

Compound ID
F.W.
(mg)
(mM)
(microL)
(microM)

Carbonyl
204.62
5.70
10.00
2786
10.00

cyanide 3-chloro-

phenylhydrazone

Compound 1
318.00
4.80
30.00
500
30.00

Cycloheximide
281.35
5.40
10.00
1919
10.00

Paclitaxel
853.91
N/A
0.30
1000
0.30

Staurosporine
466.50
1.00
1.00
2144
1.00

Compounds were serially diluted in half-log steps from the highest test concentration specified in the above table, and assayed over 10 concentrations with a maximum assay concentration of 0.1% DMSO.

The experimental procedure and calculations are similar to the Oncopanel 10-day incubation cell proliferation assay described previously.

Results

Compound 1 activity (EC₅₀IC₅₀and GI₅₀(μM)) is shown against the reference compound paclitaxel in Table 37. Data on other reference compounds for comparison purposes is shown in Table 38.

TABLE 37

Compound 1 activity (EC₅₀IC₅₀and GI₅₀(μM)) against paclitaxel

Compound 1
Paclitaxel

Cell
Cell
Cell
Cell
Cell
Cell

Count
Count
Count
Count
Count
Count

normal human
EC₅₀
IC₅₀
GI₅₀
EC₅₀
IC₅₀
GI₅₀

Cell Line
primary cells
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)

CCD 841
colon epithelial
4.86
14.3
5.27
0.0119
N/A
0.0221

CoN

HMEC
mammary
30
30
6.39
>30
>30
>30

epithelial

HREC
renal mixed
6.45
7.98
4.3
0.00172
0.0484
0.0016

epithelial

Wi38
lung fibroblast
5.45
10.4
5.92
0.0039
0.00941
0.0036

TABLE 38

Further reference compound activity

Carbonyl cyanide 3-

Cycloheximide
chlorophenylhydrazone
Staurosporine

Cell
Cell
Cell
Cell
Cell
Cell
Cell
Cell
Cell

Count
Count
Count
Count
Count
Count
Count
Count
Count

EC₅₀
IC₅₀
GI₅₀
EC₅₀
IC₅₀
GI₅₀
EC₅₀
IC₅₀
GI₅₀

Cell Line
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)
(μM)

CCD 841
0.505
2.77
0.588
10
10
6.21
0.00024
0.00057
0.00019

CoN

HMEC
3.65
10
0.306
>10
>10
0.175
0.00229
>1
0.00091

HREC
0.117
0.593
0.0369
0.553
1.52
0.308
0.00055
0.0027
0.0005

Wi38
0.285
0.871
0.369
8.84
8.84
6.15
0.00049
0.00074
0.00033

Table 39 shows representative data for the response when the cell line HREC is treated with Compound 1 in an OncoPanel Normal Cell Proliferation Assay.

TABLE 39

Compound 1 against HREC

Concentration
Relative cell count (%)

(microM)
Mean
StdDev

9.55E−04
98.5
8.8

3.02E−03
94.0
7.4

9.53E−03
93.3
6.6

3.01E−02
96.0
8.3

9.52E−02
95.8
4.5

3.01E−01
96.7
8.7

9.51E−01
89.3
4.7

3.00E+00
85.1
14.4

9.49E+00
40.4
3.3

3.00E+01
20.9
5.7

The data displayed in Table 39 is graphed in FIG. 19.

Discussion

Compound 1 and reference compounds, paclitaxel, cycloheximide, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and staurosporine, were tested on 4 normal cell types, CCD 841 CoN, HMEC, HREC and Wi38. In summary, Compound 1 showed relatively low toxicity towards all 4 cell lines. Paclitaxel showed low toxicity towards the HMEC cell line and high toxicity towards the other three cell lines. Based on average GI₅₀for the 4 normal cell lines, the order of toxicity is staurosporine>paclitaxel>cycloheximide>CCCP>Compound 1.

In Vitro Toxicity: ADME-Toxicity Study

ADME-Toxicity study of compounds 1, 2 and 4 was conducted by Eurofins Panlabs Inc, St Charles, Mo., USA using a CYP panel.

Methodology

Cytochrome P450 Inhibition was monitored through HPLC-UV/VIS and HPLC-MS/MS detection. Peak areas corresponding to the metabolite of each substrate were recorded. The percent of control activity was then calculated by comparing the peak area obtained in the presence of the test compound to that obtained in the absence of the test compound. Subsequently, the percent inhibition was calculated by subtracting the percent control activity from 100 for each compound. IC₅₀values (concentration causing a half-maximal inhibition of control values) were determined by non-linear regression analysis of the concentration-response curve using Hill equation curve fitting [Dierks, E. A. et al. (2001)].

Results

TABLE 40

Inhibition of CYPs by compounds 1, 2 and 4 against reference compounds

% Inhibition of Control Values

Human liver microsomes
Compound 1
Compound 2
Compound 4
Reference

In Vitro Metabolism
1 μM
10 μM
1 μM
10 μM
1 μM
10 μM
Compound
IC₅₀μM

CYP1A (phenacetin sub.)
28
92
38
63
14
85
Furafylline
16

CYP2B6 (bupropion sub.)
13
55
15
38
16
49
Clopidogrel
0.39

CYP2C8 (paclitaxel sub.)
14
76
12
52
24
61
Montelukast
0.57

CYP2C9 (diclofenac sub.)
5
77
20
72
31
91
Sulfaphenazole
0.28

CYP2C19 (omeprazole sub.)
37
90
34
73
25
78
Oxybutynin
6.7

CYP2D6 (dextromethorphan sub.)
13
45
18
44
21
45
Quinidine
0.12

CYP3A (midazolam sub.)
16
65
15
37
15
50
Ketoconazole
0.18

CYP3A (testosterone sub.)
7
77
17
57
20
48
Ketoconazole
0.18

Summary of Results
Enzyme—Substrates
CYP1A (Phenacetin Substrate)

Compounds 1, 2 and 4 are strong inhibitors at 10 μM (63-92%). With IC₅₀values estimated to be <10, Compounds 1, 2 and 4 are stronger inhibitors the reference standard furafylline (IC₅₀16 μM). Compounds 1 and 2 showed moderate inhibition at 1 μM (28% and 38% respectively).

CYP2B6 (Bupropion Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (13-16% at 1 μM) compared with the reference compound clopidogrel (IC₅₀0.39 μM).

CYP2C8 (Paclitaxel Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (12-24% at 1 μM) compared with the reference compound montelukast (IC₅₀0.57 μM).

CYP2C9 (Diclofenac Substrate)

Compounds 1 was a very weak at 1 μM (5%) and Compounds 2 and 4 relatively weak inhibitors (20 and 31%) compared with the reference compound sulfaphenazole (IC₅₀0.28 μM).

CYP2C19 (Omeprazole Substrate)

Compounds 1, 2 and 4 are moderately strong inhibitors (25-37% at 1 μM) with activity comparable with oxybutynin (IC₅₀6.7 μM).

CYP2D6 (Dextromethorphan Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (12-21% at 1 μM) compared with the reference compound quinidine (IC₅₀0.12 μM).

CYP3A (Midazolam Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (15-16% at 1 μM) compared with the reference compound ketoconazole (IC₅₀0.18 μM).

CYP3A (Testosterone Substrate)

Compounds 1, 2 and 4 are relatively weak inhibitors (7-20% at 1 μM) compared with the reference compound ketoconazole (IC₅₀0.18 μM).

In Vivo Toxicity: Compound 1 Maximum Tolerated Dose for Mice

Two studies were conducted by Eurofins Panlabs Taiwan, Ltd. The purpose of Study 1 was to assess the possible adverse effects of Compound 1 alone or in combination with carboplatin in Maximum Tolerated Dose (MTD) assay. The purpose of Study 2 is a follow-up study performed at escalating doses.

The vehicle information and dosing volume used in the studies are shown in Table 41. Table 42 and Table 43 show results for Study 1 in terms of mortality and body weight observations. In regard to Study 2, Table 44 and Table 45 show results for mortality and body weight observations and behavioral, symptomatic i.e. neurological and autonomic signs for Compound 1, alone and in combination with carboplatin, are shown in Table 46 and Table 47. Corresponding signs for the vehicles are shown in Table 48 and Table 49.

TABLE 41

Vehicle information and dosing volume for MTD assay studies

Concen-
Dosing

tration
volume
Dosage

Study
Test article
Vehicle
(mg/mL)
(mL/kg)
(mg/kg)

1
Compound 1
10% Tween 20/
10
10
100

90% PBS

2
Compound 1
20% Tween 20/
20
10
200

80% PBS

Test Substance and Dosing Pattern

Compound 1 was dissolved in 10% Tween 20/90% PBS or 20% Tween 20/80% PBS for IP injections. Compound 1 was administrated alone or in combination with carboplatin at a dosing volume of 10 or 20 mL/kg for each substance.

Study 1

For mortality, test substance (Compound 1; 100 mg/kg) alone or in combination with carboplatin (8.75, 17.5, or 35 mg/kg; given on the same day) was administered to mice by IP injection to evaluate the possible adverse effects in MTD assay. The mice were a group of 3 female NOD/SCID mice at 6-7 weeks of age. The mortality was observed at 30 min and again at 3, 24, 48 and 72 hours after compound administrations.

For bodyweight, test substance (Compound 1; 100 mg/kg) alone or in combination with carboplatin (8.75 or 17.5 mg/kg; given on the same day) was administered to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The body weight of each animal was measured and recorded daily for 3 days.

Compound 1 alone did not elicit significant adverse effects and were considered tolerated. All test animals survived over the 72-hour observation period (Table 42). However, Compound 1 in combination with carboplatin was associated with slight to moderate limb tone, and resulted in a 33% mortality after 72 hours of dosing, reflecting the dose level was not well-tolerated (Table 42).

Compound 1, in combination with the lower doses of carboplatin (8.75 and 17.5 mg/kg), was further investigated in the MTD study. Compound 1, injected with two doses of carboplatin on the same day, was associated with slight to moderate abdominal tone during the first 30 minutes of administrations (Table 44 and Table 45). However, no mortality and body weight losses were observed in test animals over the experimental period, signifying the dose levels were tolerated (Table 44 and Table 45).

TABLE 42

MTD in mice (Study 1, Round 1 & 2)

Response (death/test)

Dose
30
3
Day
Day
Day

Compound
Route
(mg/kg)
min
hrs
1
2
3

Vehicle^a
IP
10
mL/kg
0/3
0/3
0/3
0/3
0/3

Vehicle^a+
IP
10 mL/kg +
0/3
0/3
0/3
0/3
0/3

Vehicle^b

10 mL/kg

Compound 1
IP
100
mg/kg
0/3
0/3
0/3
0/3
0/3

Carboplatin
IP
35
mg/kg
0/3
0/3
0/3
0/3
0/3

Compound
IP
100 mg/kg +
0/3
0/3
0/3
0/3
1/3

1 +

35 mg/kg

carboplatin

Vehicle^a+
IP
10 mL/kg +
0/3
0/3
0/3
0/3
0/3

Vehicle^b

10 mL/kg

Compound
IP
100 mg/kg +
0/3
0/3
0/3
0/3
0/3

1 +

17.5 mg/kg

carboplatin

Compound
IP
100 mg/kg +
0/3
0/3
0/3
0/3
0/3

1 +

8.75 mg/kg

carboplatin

Legend:

^a10% Tween 20/90% PBS

^b0.9% NaCl

TABLE 43

MTD in mice (Study 1; Round 2)

Body Weight (g)

Dose

Day
Day
Day
Day

Compound
Route
(mg/kg)
No.
0
1
2
3

Vehicle^a+
IP
10 mL/kg +
1
20
20
19
19

Vehicle^b

10 mL/kg
2
20
20
20
19

3
22
21
21
21

Compound
IP
100 mg/kg +
1
22
21
21
20

1 +

17.5 mg/kg
2
22
22
22
21

carboplatin

3
21
21
21
21

Compound
IP
100 mg/kg +
1
22
20
20
20

1 +

8.75 mg/kg
2
21
21
22
21

carboplatin

3
21
21
21
20

Legend:

^a10% Tween 20/90% PBS

^b0.9% NaCl

Study 2

A follow-up MTD study was performed to evaluate for the adverse effects of the test substance (Compound 1) at escalating doses alone or in combination with carboplatin (17.5 mg/kg) via IP in a group of 3 female NOD/SCID mice at 6-7 weeks of age.

For mortality, treatment with Compound 1 at escalating doses (200 mg/kg) alone and in combination with carboplatin (17.5 mg/kg; given on the same day) were administered by IP injection to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The mortality was monitored at 30 min and again at 3, 24, 48 and 72 hours after compound administrations.

For body weight, test substance (Compound 1) at escalating doses (200 mg/kg) alone or in combination with carboplatin (17.5 mg/kg; given on the same day) were administered by IP injection to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The body weight of each animal was measured and recorded daily for 3 days.

On treatment with Compound 1 (200 mg/kg) alone and in combination with carboplatin (17.5 mg/kg) exhibited a 33-100% mortality, but no marked weight loss during the study period (Table 30 and Table 31).

TABLE 44

MTD in mice (Study 2)

Response (death/test)

Dose
30
3
Day
Day
Day

Compound
Route
(mg/kg)
min
hrs
1
2
3

Vehicle^a
IP
10
mL/kg
0/3
0/3
0/3
0/3
0/3

Vehicle^a+
IP
10 mL/kg +
0/3
0/3
0/3
0/3
0/3

Vehicle^b

10 mL/kg

PT# 1206425
IP
200
mg/kg
0/3
0/3
1/3
3/3
3/3

(UOS-70)

(Compound 1)

PT# 1206425
IP
200 mg/kg +
0/3
0/3
0/3
1/3
1/3

(UOS-70)

17.5 mg/kg

(Compound

1) +

carboplatin

Vehicle^a
IP
20
mL/kg
0/3
0/3
1/3
2/3
2/3

(20%

Tween20/

80% PBS)

Legend:

^a20% Tween 20/80% PBS

^b0.9% NaCl

TABLE 45

MTD in mice (Study 2; Round 1 & 2)

Body Weight (g)

Dose

Day
Day
Day
Day

Compound
Route
(mg/kg)
No.
0
1
2
3

Vehicle^a
IP
10
mL/kg
1
23
22
23
23

(20%

2
24
23
23
23

Tween20/

3
23
22
22
22

80% PBS)

Vehicle^a+
IP
10 mL/kg +
1
22
21
20
19

Vehicle^b

10 mL/kg
2
22
22
21
19

3
22
20
20
20

Compound 1
IP
200
mg/kg
1
22
21
NA
NA

2
21
NA
NA
NA

3
21
21
NA
NA

Compound
IP
200 mg/kg +
1
21
20
20
21

1 +

17.5 mg/kg
2
22
22
NA
NA

carboplatin

3
21
20
19
20

Vehicle^a
IP
20
mL/kg
1
23
NA
NA
NA

2
22
22
NA
NA

3
23
22
22
21

Legend:

^a20% Tween 20/80% PBS

^b0.9% NaCl

For behavioral, symptomatic i.e. neurological and autonomic signs, test substance (Compound 1; 200 mg/kg) alone or in combination with carboplatin (17.5 mg/kg; given on the same day) were administered by IP injection to a group of 3 female NOD/SCID mice at 6-7 weeks of age. The animals were then observed for presences of acute toxic symptoms and autonomic effects for 30 min after the first dose.

Pronounced behavioral effects such as decreased startle, touch response, pinna and placing were observed on Compound 1 treatment, alone and in combination with carboplatin (see Table 46). Decreased neurologic signs, such as spontaneous activity, righting, ataxia and low limb post were observed on Compound 1 treatment (Table 46). Hypothermia (decreased body temperature) was observed as an autonomic sign (Table 47).

TABLE 46

Behavioral and neurologic signs in mice

for Compound 1 (Study 2; Round 1)

Treatment

Compound 1
Compound 1 + carboplatin

Route

IP
IP

Dosage

200 mg/kg
200 mg/kg + 17.5 mg/kg

No. 1
No. 2
No. 3
No. 1
No. 2
No. 3

BEHAVIORAL

B.W. (g)
22
21
21
21
22
21

Irritability
−
−
−
−
−
−

Hyperactivity
−
−
−
−
−
−

Inc. Startle
−
−
−
−
−
−

Inc. Touch
−
−
−
−
−
−

Dec. Startle Response
+
+
+
+
+
+

Dec. Touch Response
+
+
+
+
+
+

Inc. Exploration
−
−
−
−
−
−

Dec. Exploration
+
+
+
+
+
+

Pinna
+
+
+
+
+
+

Placing
±
±
±
±
+
±

NEUROLOGIC

Tremor
−
−
−
+
−
+

Dec. Spont. Activity
+
+
+
+
+
+

Straub Tail
−
−
−
−
−
−

Reactivity
−
+
+
+
±
+

Righting
+
+
±
±
+
+

Ataxia
+
+
±
±
+
+

Convulsion C.T.C-T
−
−
−
−
−
−

Low Limb Post
+
+
+
+
+
+

Abdominal Tone
+
+
+
+
+
+

Limb Tone
+
+
+
+
+
+

Grip Strength
−
±
+
−
+
±

TABLE 47

Autonomic signs in mice for Compound 1 (Study 2; Round 1)

Treatment

Compound 1
Compound 1 + carboplatin

Route

IP
IP

Dosage

200 mg/kg
200 mg/kg + 17.5 mg/kg

AUTONOMIC
No. 1
No. 2
No. 3
No. 1
No. 2
No. 3

Skin Color
−
−
−
−
−
−

Respiration
D±
D+
D+
D+
D+
D+

Salivation F.V.
−
−
−
−
−
−

Lacrimation
−
−
−
−
−
−

Diarrhea
−
−
−
−
−
−

Body Temperature
↓+
↓+
↓+
↓+
↓+
↓+

Piloerection
−
−
−
−
−
−

Inc. Palpebral Size
−
−
−
−
−
−

Dec. Palpebral Size
−
−
−
−
±
−

Others
−
−
−
−
−
−

Death
−
−
−
−
−
−

TABLE 48

Behavioral and neurologic signs in mice

for vehicles (Study 2; Round 1)

Treatment

Vehicle^a
Vehicle^a+ Vehicle^b

Route

IP
IP

Dosage

10 mL/kg
10 mL/kg + 10 mL/kg

No. 1
No. 2
No. 3
No. 1
No. 2
No. 3

BEHAVIORAL

B.W. (g)
23
24
23
22
22
22

Irritability
−
−
−
−
−
−

Hyperactivity
−
−
−
−
−
−

Inc. Startle
−
−
−
−
−
−

Inc. Touch
−
−
−
−
−
−

Dec. Startle Response
−
−
−
−
−
−

Dec. Touch Response
−
−
−
−
−
−

Inc. Exploration
−
−
−
−
−
−

Dec. Exploration
−
−
−
−
−
−

Pinna
−
−
−
−
−
−

Placing
−
−
−
−
−
−

NEUROLOGIC

Tremor
−
−
−
−
−
−

Dec. Spont. Activity
−
−
−
−
−
−

Straub Tail
−
−
−
−
−
−

Reactivity
−
−
−
−
−
−

Righting
−
−
−
−
−
−

Ataxia
−
−
−
−
−
−

Convulsion C.T.C-T
−
−
−
−
−
−

Low Limb Post
−
−
−
−
−
−

Abdominal Tone
−
−
±
±
±
±

Limb Tone
−
−
−
−
−
−

Grip Strength
−
−
−
−
−
−

TABLE 49

Autonomic signs in mice for vehicles (Study 2; Round 1)

Treatment

Vehicle^a
Vehicle^a+ Vehicle^b

Route

IP
IP

Dosage

10 mL/kg
10 mL/kg + 10 mL/kg

AUTONOMIC
No. 1
No. 2
No. 3
No. 1
No. 2
No. 3

Skin Color
−
−
−
−
−
−

Respiration
−
−
−
−
−
−

Salivation F.V.
−
−
−
−
−
−

Lacrimation
−
−
−
−
−
−

Diarrhea
−
−
−
−
−
−

Body Temperature
−
−
−
−
−
−

Piloerection
−
−
−
−
−
−

Inc. Palpebral Size
−
−
−
−
−
−

Dec. Palpebral Size
−
−
−
−
−
−

Others
−
−
−
−
−
−

Death
−
−
−
−
−
−

Legend for Tables 46 to 49

- ^a20% Tween 20/80% PBS
- ^b0.9% NaCl
- -: no effects
- ±: Slight to moderate effects
- +: Severe effects
- Inc.: Increased
- Dec.: Decreased
- Spont.: Spontaneous
- C.: Chronic
- T.: Tonic
- C-T: Chronic-Tonic
- ↓: Low
- D: Deep

In Vivo Anti-Cancer Activity: Compound 1 and Gemcitabine

An in vivo study indicates that Compound 1 is effective in combination with gemcitabine. The study design is detailed below and in Table 50. There is evidence for synergy from the study results which are shown in Table 51 and Table 52. Monotherapy and combinations were well tolerated.

TABLE 50

Tumor, Xenograft, Pancreas, MIA PaCa-2

in female nu/nu mice study design

Test
Conc.
Dosage
Mice^a

Group
Article
Route
mg/mL
mL/kg
mg/kg
(female)

1^#
Vehicle^b
IP
NA
10
NA
8

qwk x 3^c

2
Gemcitabine
IP
8
10
80
8

q4d x 4^d

4
Compound 1
IP
10
10
100
8

qwk x 3^c

6
Compound
IP +
10 + 8
10
100,
8

1 +
IP

qwk x 3^c+

Gemcitabine

80,

q4d x 4^d

^#Negative control group

^aFemale nu/nu mice aged 7-8 weeks are implanted with 1 × 10⁷MIA PaCa-2 cells (0.2 mL/mouse), and dosing is initiated when group mean tumor volumes reach ~80-150 mm³.

^bTest substance vehicle (10% Tween20/90% Saline)

^cDoses are administered once weekly for 3 weeks.

^dDoses are administered once every four days (four total administrations).

Body weight and tumor volumes are recorded twice weekly beginning on Day 1 and continuing until study completion.

Tumor growth inhibition (% TGI) is determined twice weekly during the dosing period by the formula: % TGI = (1 − [(T − T0)/(C − C0)]) × 100 where T = mean tumor volume of treated group, T0 = mean tumor volume of treated group at study start, C = mean tumor volume of control group and C0 = mean tumor volume of control group at study start.

Study is designed to continue for 28 days, or until the mean tumor volume within negative control group reaches 2000 mm³, whichever comes first.

After TGI is determined, study may be converted to determine Tumor Growth Delay (TGD).

Animals are to be sacrificed if/when:

1. Body weight loss >25%

2. Severe tumor ulceration

Estimated Timeline

Animal Arrival: Within one month receiving test compounds

Tumor cells implantation: Within two weeks after animals are obtained

Dosing: Approximately 1-2 weeks after tumor cell implantation

Sample Requirements

Compound 1 150 mg; Gemcitabine 256 mg.

Protocol
Tumor, Xenograft, Pancreas, MIA PaCa-2

Procedure: Groups of (8) female nu/nu mice (7-8 weeks old), bred in an animal isolator (IVC racks) under specific pathogens free (SPF) condition at 22 plus or minus 2° C. are used. Viable human pancreatic carcinoma MIA PaCa-2 (ATCC CRL-1420) cells (1.0×107 in 0.2 mL) are injected subcutaneously into the right flank of experimental mice. When tumor volumes reach ˜80-150 mm³(about 8-10 days post implant), the animals are randomly assigned into groups of eight, and test compounds and/or vehicle dose administrations are initiated (denoted as Day 1). Test compounds are administered as detailed in the “Study Design” section (Table 50). Tumor volumes and body weights are measured and recorded twice weekly over the course of the study period. Animals are monitored as a group. The endpoint of the experiment is a mean tumor volume in the negative control group of 2000 mm³or 28 days, whichever comes first.

Tumor volume (mm³) is estimated according to the formula for a prolate ellipsoid: length (mm)×[width (mm)]2×0.5. Tumor growth inhibition (% TGI) will be determined twice weekly during the dosing period by the formula: % TGI=(1−[(T−T0)/(C−C0)])×100 where T=mean tumor volume of treated group, T0=mean tumor volume of treated group at study start, C=mean tumor volume of control group and C0=mean tumor volume of control group at study start [Mohammed et al. 1998].

All aspects of this work including housing, experimentation, and animal disposal are performed in general accordance with the “Guide for the Care and Use of Laboratory Animals: Eighth Edition” (2011) in an AAALAC-accredited laboratory animal facility.

TABLE 51

Tumor volume change in a pancreas MIA PaCa-2

tumor xenograft model in response to treatment with

Compound 1 alone or in combination with Gemcitabine.

Assay Tumor Xenograft, Pancreas, MIA PaCa-2

WO#1060729 (AB67928) Female nu/nu mice

Dose

Tumor Volume (mm3)

(mg/kg)

Day
Day
Day
Day
Day

Gr.
Treatment
(Route)
No.
1
4
8
11
15

1
Vehicle
NA
1
64
125
186
326
574

(mg/kg)
2
74
65
87
128
151

QWK x 3
3
103
97
175
238
476

IP
4
122
111
142
179
422

5
76
63
135
160
258

6
90
79
81
96
171

7
90
84
73
140
326

8
103
125
180
315
629

Mean
90
94
132
198
376

SEM
7
9
17
31
63

2
Gemcitabine
80
1
65
65
114
165
213

mg/kg
2
74
82
104
97
160

Q4D x 4
3
104
128
151
215
419

IP
4
115
123
162
228
335

5
76
73
75
103
130

6
88
100
70
85
128

7
91
58
70
94
115

8
101
119
113
127
156

Mean
89
94
107
139
207

SEM
6
10
13
20
39

%
99
100
81
70
55

T/C

%
1
0
19
30
45

TGI

4
Compound 1
100
1
67
56
126
368
600

mg/kg
2
72
59
95
110
244

QWK x 3
3
104
105
125
192
276

IP
4
115
103
107
121
197

5
81
129
187
266
414

6
87
70
111
188
309

7
93
92
177
251
409

8
100
107
138
188
312

Mean
90
90
133
211
345

SEM
6
9
12
30
45

%
100
96
101
107
92

T/C

%
0
4
−1
−7
8

TGI

6
Compound
100
1
69
46
48
57
80

1 +
mg/kg
2
70
111
94
104
116

Gemcitabine
QWK x 3
3
108
86
101
97
103

IP +
4
111
132
124
157
265

80
5
84
68
71
35
95

mg/kg
6
86
67
77
105
123

Q4D x 4
7
96
55
28
25
23

IP
8
98
94
98
127
210

Mean
90
82
80
88
127

SEM
6
10
11
16
27

%
100
87
61
44
34

T/C

%
0
13
39
56
66

TGI

TABLE 52

Body weight change in a pancreas MIA PaCa-2 tumor xenograft model in

response to treatment with Compound 1 alone or in combination with Gemcitabine.

Assay # 581500 Tumor Xenograft, Pancreas, MIA PaCa-2

WO#1060729 (AB67928) Female nu/nu mice

Dose

(mg/kg)

Body Weight (g)

Gr.
Treatment
(Route)
No.
Day 1
Day 4
Day 8
Day 11
Day 15

1
Vehicle
NA (mg/kg)
1
28
27
27
27
26

QWK × 3
2
27
26
27
27
27

IP
3
25
24
25
26
27

4
24
24
24
24
24

5
26
25
25
25
25

6
25
25
25
26
26

7
25
23
23
24
24

8
25
25
24
25
26

Mean
25.6
24.9
25.0
25.5
25.6

SEM
0.5
0.4
0.5
0.4
0.4

2
Gemcitabine
80 mg/kg
1
24
23
25
25
26

Q4D × 4
2
30
28
30
31
31

IP
3
25
22
25
26
27

4
26
25
27
27
27

5
24
24
25
25
25

6
25
24
25
25
25

7
25
24
24
25
25

8
26
25
25
25
25

Mean
25.6
24.4
25.8
26.1
26.4

SEM
0.7
0.6
0.7
0.7
0.7

4
Compound 1
100 mg/kg
1
29
27
29
29
29

QWK × 3
2
26
24
26
25
25

IP
3
25
24
26
25
26

4
25
23
25
25
24

5
25
25
25
27
26

6
23
23
23
25
23

7
25
25
25
27
26

8
26
26
26
27
26

Mean
25.5
24.6
25.6
26.3
25.6

SEM
0.6
0.5
0.6
0.5
0.6

6
Compound 1 +
100 mg/kg
1
25
24
24
24
24

Gemcitabine
QWK × 3
2
24
23
23
24
24

IP +
3
27
27
26
26
26

80 mg/kg
4
27
25
26
26
26

Q4D × 4
5
23
22
23
23
23

IP
6
26
25
25
25
24

7
27
26
26
26
26

8
25
24
24
23
23

Mean
25.5
24.5
24.6
24.6
24.5

SEM
0.5
0.6
0.5
0.5
0.5

Evaluation of Compound 1 to Treat Skin Diseases
Aim

To evaluate compounds 1 with skin disease relevant in vitro assays. Evaluation was conducted by LEO Pharma Open Innovation, USA (openinnovation.leo-pharma.com).

Specific Objectives

- 1. Determination of inhibition of CCL2 release from IL-4, IL-13, IL-22 and IFN-γ-stimulated primary human keratinocytes [assay number 1280] and determination of the effect on cell viability [assay number 1243].
- 2. Determination of inhibition of IL-8 release from IL-17A and TNFα-induced primary human keratinocytes [assay number 1250] and determination of the effect on cell viability [assay number 1244].
- 3. Determination of inhibition of IL-17A secretion from human PBMC stimulated with antiCD3/antiCD28-coated beads [assay number 1321] and determination of the effect on cell viability [assay number 1322].
- 4. Determination of inhibition of IL-4 and IL-2 secretion from human CD4-positive T-cells stimulated with antiCD2/antiCD3/antiCD28-coated beads [assay number 1297, 1298] and determination of the effect on cell viability [assay number 1299].

Disease Relevance Summary

- 1. Compounds which inhibit keratinocyte CCL2 secretion may be expected to have efficacy in atopic dermatitis.
- 2. Compounds which inhibit keratinocyte IL-8 secretion may be expected to have efficacy in psoriasis.
- 3. Compounds which inhibit IL-17A secretion may be expected to have efficacy in psoriasis.
- 4. Compounds which inhibit IL-4 secretion may be expected to have efficacy in atopic dermatitis. Compounds which also inhibit IL-2 secretion have a broader immunosuppressive effect since IL-2 is an autocrine growth factor for T-cells.

Results

- 1. Effect of Compound 1 on monocyte chemoattractant protein-1 (MCP-1/CCL2) release in human keratinocytes induced by IL-4, IL-13, IL-22 and IFN-γ. FIG. 20A shows dose-responsive effect of Compound 1 on chemokine CCL2 release in human keratinocytes induced by IL-4, IL-13, IL-22 and IFN-γ. Inhibition of CCL2 release: Rel EC₅₀=92.2 nM, Max Effect=101%. Inhibition of cell viability: Rel EC₅₀=708.0 nM, Max Effect=103%. Compound 1 showed medium potency in reduction of CCL2 release without significant reduction of cell viability as shown in FIG. 1.
- 2. Effect of Compound 1 on IL-8 release in human keratinocytes induced by IL-17 and TNF-α.
  - FIG. 20B shows dose-responsive effect of Compound 1 on IL-8 release in human keratinocytes induced by IL-17 and TNF-α.
  - Inhibition of IL-8 release: Rel EC₅₀=157 nM, Max Effect=110%.
  - Inhibition of cell viability: Rel EC₅₀=246 nM, Max Effect=101%.
  - There is similar reduction of IL-8 release and cell viability.
- 3. Effect of Compound 1 on IL-17A release in Human PBMC inflammation induced by CD3, CD28 and IL-23.
  - FIG. 20C shows dose-responsive effect of Compound 1 on IL-17A release in Human PBMC inflammation induced by CD3, CD28 and IL-23.
  - Inhibition of IL-17A release: Rel EC₅₀=320 nM, Max Effect=100%.
  - Inhibition of cell viability: Rel EC₅₀=542 nM, Max Effect=101%.
  - There is similar reduction of IL-17 release and cell viability.
- 4. Effect of Compound 1 on IL-2 and IL-4 releases in T-cell inflammation induced by CD2, CD3 and CD28.
  - FIG. 20D. Dose-responsive effect of Compound 1 on IL-2 and IL-4 releases in T-cell inflammation induced by CD2, CD3 and CD28.
  - Inhibition of IL-2 release: Rel EC₅₀=327 nM, Max Effect=98%.
  - Inhibition of IL-4 release: Rel EC₅₀=523 nM, Max Effect=100%.
  - Inhibition of cell proliferation: Rel EC₅₀=587 nM, Max Effect=123%.
  - There is similar effect on IL-2, IL-4 and cell proliferation.

Discussion

Compound 1 showed pronounced effect in CCL2-release without significant effect on cell viability (FIG. 20A) compared to IL-8, IL-17A, IL-2 and IL-4 where the effect on release and cell viability are similar (FIGS. 20B to 20D). These results indicate that Compound 1 is a potential candidate for effective treatment of skin inflammatory diseases such as atopic dermatitis.

Methods
1. Keratinocyte CCL2 Release

Inhibition of CCL2 release from IL-4, IL-13, IL-22 and IFN-γ-stimulated primary human keratinocytes LEO Pharma Open Innovation assay number 1280

Disease Relevance

The inflammatory skin disease atopic dermatitis (AD) is characterized by the T-cell cytokines including IL-4, IL-13, IL-22 and IFN-γ. In the present assay, keratinocytes are stimulated with a mixture of these cytokines, and the release of CCL2 (C-C chemokine ligand 2, also called monocyte chemoattractant protein 1 (MCP-1)) is measured in the culture supernatant by proximity homogenous time-resolved fluorescence (HTRF). The purpose of the assay is to measure if test compounds inhibit the levels of CCL2 released by the keratinocytes. Compound which inhibit keratinocyte CCL2 secretion may be expected to have efficacy in atopic dermatitis.

A known steroid, Betamethasone, inhibits CCL2 release in this assay with an EC₅₀of approximately 15 nM, and an Emax (plateau of the fitted curve) of approximately 60%. A high Emax shows that the compound inhibits a large proportion of the secreted CCL2. A low EC₅₀value indicates that the compound is potent and to perform the inhibition at low concentrations.

Key assay parameters
Description

Human primary
Cells are frozen in passage 2.

keratinocytes (HEKa)
Cells are used in passage 3-6

IL-4, IL-13, IL-22
Atopic dermatitis-like stimulation:

and IFN-γ
Recombinant human interleukin (IL) 4, 13 and

22 at 10 ng/mL and Interferon-γ at 1 ng/mL

Effect
Inhibition of AD stimuli induced CCL2

release from HEKa cells

Positive control
AD-like stimulation + Terfenadine

(~100% effect)
(CAS: 50679-08-8) at 10 microM

Negative control
AD-like stimulation and 0.1% DMSO

(~0% effect)

Reference compound
Betamethasone (CAS: 378-44-9)

Incubation time
48 h

Capacity, run-time
The assay takes 3 days to perform.

and requirements

Method Description
Cell Culture:

HEKa are human epidermal keratinocytes isolated from adult skin. The cells have been cryopreserved at the end of the primary culture stage in a medium containing 10% DMSO. Sterile cell culture work applies.

Initiating Cultures from Cryopreserved HEKa Cells:

- Thaw a vial of the frozen HEKa stock in a 37° C. water/bead bath.
- Transfer the cell suspension into 30 ml T75 cell culture flask.
- Take 15 ml out and dispense over into 2×T75 (15 ml each).
- Set up for assay 5-6 days later or pass it on.

Maintenance of Stock Cultures

- Change the culture medium to freshly supplemented medium, 24 to 36 hours after establishing a secondary culture from cryopreserved cells. For subsequent subcultures, change the medium 48-72 hours after establishing the subculture.
- Only passage up to 3-5 in the assay.

Subculture of HEKa

- View and confirm 80% confluence.
- Remove all of the culture medium from the flask.
- Add 3 ml of TrypLE Express solution to the flask. Rock the flask to ensure that the entire surface is covered.
- Immediately remove all 3 ml of TrypLE Express solution from the flask.
- Add 1½ ml (T75) (3 ml T175) of fresh TrypLE Express solution to the flask.
- View the culture under a microscope. Incubate the flask at room temperature until the cells have become completely round, approximately 8-10 minutes.
- Tap the flask very gently to dislodge cells from the surface of the flask.
- Add 3-4 ml (T75) (7 ml T175) of Trypsin Neutralizer solution to the flask and transfer the detached cells to a sterile conical tube.
- Centrifuge the cells at 170×g for 7-10 minutes. Observe the cell pellet.
- Remove the supernatant from the tube, being careful not to dislodge the cell pellet.
- Resuspend the cell pellet in 15 ml medium with HKGS supplemented (cat no S-001-K) but without hydrocortisone (when use for assay). Pipette the cells up and down with a 10 ml pipette to ensure a homogeneous cell suspension.
- Determine the concentration of cells in the suspension.
- Dilute the cells in supplemented medium and seed new culture in a T175 flask. Incubate the cultures in a 37° C., 5% CO₂/95% air, humidified cell culture incubator.

Assay Day 0:

- Trypsinize the cells as described above.
- Resuspend pellet in 10 ml assay medium (EpiLife Medium with HKGS Kit but without hydrocortisone)
- Filter the cells though a 37 μm strainer.
- Count cells using a cell counter.
- Seed HEKa cells in 384-well view plates 3500 c/w/40 μL.
- Place the plates with lids on in a humidity chamber with lid (24.5×24.5 cm box added H₂O in the bottom).
- Incubate plates over night at 37° C., 5% CO₂/95% air

Assay Day 1:

- Prepare compound-containing source plate, i.e. titrations in DMSO to be transferred to the assay plate.
- Add 80 nL compounds and controls from the source plate to the assay plate (with cells).
- Add 40 μL stimulation (recombinant human interleukin (IL) 4, 13, 22 at 10 ng/mL and Interferon-gamma at 1 ng/mL) in medium with supplement, but without hydrocortisone.
- Incubate plates at 37° C., 5% CO₂/95% air for 2 days.

Assay Day 3:

- Remove plates from incubation and equilibrate to room temperature for 30 minutes.
- Transfer 8 μL supernatant to white proxi 384-well plates for CCL2 detection.
- Add 8 μL media to the detection plate.
- Add 4 μL CCL2 HTRF detection reagent (to the white proxi 384w).
- Seal and incubate for 4 hours and read in a plate reader.

Data Analysis and Calculations

CCL2 concentration in the supernatants is measured using homogeneous time-resolved fluorescence resonance (TR-FRET). The assay is quantified by measuring fluorescence at 665 nm (proportional to CCL2 concentration) and 620 nm (control). A ratio 665/620*1000 is calculated.

% Effect:

The capacity of the test compound to inhibit CCL2 release is normalized to the signal in the control wells with keratinocytes incubated with 0.1% DMSO (0%) and 10 μM Terfenadine (100%), which fully inhibits the CCL2 release.

2. Keratinocyte IL-8 Release

Inhibition of IL-8 release from IL-17A and TNFα-induced primary human keratinocytes LEO Pharma Open Innovation assay number 1250.

Disease Relevance

The inflammatory skin disease psoriasis is characterized by cytokines IL-17A and TNFα. In the present assay, primary human keratinocytes are stimulated with a mixture of these cytokines, and the release of IL-8 (also called CXCL8) is measured in the culture supernatant by proximity homogenous time-resolved fluorescence (HTRF). The purpose of the assay is to measure if the test compound is able to inhibit the levels of IL-8 released by the keratinocytes, indicating that it is able to inhibit part of the inflammation occurring in cells.

Compounds which inhibit keratinocyte IL-8 secretion may be expected to have efficacy in psoriasis.

A known steroid, Betamethasone, inhibits IL-8 release in this assay with an EC₅₀of approximately 10 nM, and an Emax (plateau of the fitted curve) of approximately 50%. A high Emax indicates that the compound inhibits a large proportion of the IL-8 secretion. A low EC₅₀value indicates that the compound is potent and performs the inhibition at low concentrations.

Key assay parameters
Description

Human epidermal
Cells are frozen in passage 2.

keratinocytes,
Cells are used in passage 3-5

adult (HEKa)

IL-17A and TNFα
Psoriasis-like stimulation:

Recombinant human interleukin (IL) 17A and

tumor necrosis factor alpha (TNFα) both

at 10 ng/mL

Effect
Inhibition of IL-17A/TNFα-induced IL-8

release from HEKa

Positive control
No stimulation and 0.1% DMSO

(~100% effect)

Negative control
IL-17A/TNFα stimulation and 0.1% DMSO

(~0% effect)

Reference compound
Betamethasone (CAS: 378-44-9)

Incubation time
72 h

Capacity, run-time
The assay takes 3 days to perform.

and requirements

Method Description
Cell Culture:

HEKa are human epidermal keratinocytes isolated from adult skin (See Cell Culture Method described for Keratinocyte CCL2 release above).

Assay Day 0:

- Detach the cells as described above.
- Resuspend pellet in 10 ml EpiLife Medium with HKGS Kit but without hydrocortisone
- Filter the cells though a 37 μm strainer.
- Count cells using a cell counter.
- Seed Human Epidermal Keratinocytes, adult (HEKa) cells in 384-well view plates 3500 c/w/40 μL. Use cell media with supplement but without hydrocortisone as assay medium.
- Place the plates with lids on in a humidity chamber with lid (24.5×24.5 cm box added H₂O in the bottom).
- Incubate plates over night at 37° C., 5% CO₂/95% air.

Assay Day 1:

- Prepare compound-containing source plate, i.e. titrations in DMSO to be transferred to the assay plate.
- Empty the assay plate, and re-add 25 μL assay medium.
- Add 75 nL compounds and controls from the source plate to the assay plate (with cells).
- Add 25 μL assay medium and incubate for 2 hours.
- Add 25 μL stimulation mix in assay medium (recombinant human interleukin 17A and Tumor necrosis factor-alpha) to give a final concentration of 10 ng/mL of both cytokines.
- Incubate plates at 37° C., 5% CO₂/95% air for 3 days.

Assay Day 4:

- Remove plates from incubation and equilibrate to room temperature for 30 minutes.
- Transfer 2 μL supernatant to white proxi 384-well plates for IL-8 detection.
- Add 2 μL assay medium
- Prepare IL-8 HTRF mix (according to manufactory protocol) and add 2 μL pr well.
- Seal and incubate for 3 hours and read fluorescence in a plate reader.

Data Analysis and Calculations

IL-8 concentration in the supernatant is measured using homogeneous time-resolved fluorescence resonance (TR-FRET). The assay is quantified by measuring fluorescence at 665 nm (proportional to IL8 concentration) and 620 nm (control). A ratio 665/620*1000 is calculated as a representation of the amount of IL-8 in the supernatant.

% Effect:

The capacity of the test compound to inhibit IL-8 release is normalized to the signal in the negative control wells with keratinocytes incubated with 0.1% DMSO (0%) and 10 μM Terfenadine (100%), which fully inhibits the release of IL-8.

3. PBMC IL-17 Assay

Inhibition of IL-17A secretion from human PBMC stimulated with antiCD3/antiCD28-coated beads. LEO Pharma Open Innovation assay number 1321

Disease Relevance

The inflammatory skin disease psoriasis is characterized by cytokines IL-17A and TNFα. In the present assay, human peripheral blood mononuclear cells (PBMC) are stimulated with beads coated with antibodies against CD3 and CD28 to activate the T-cell receptor, and stimulated with IL-23 to promote T-helper17 activity. The cells are incubated for three days, then the secreted level of IL-17A is measured in the culture supernatant using AlphaLISA and the amount of living cells is measured by addition of resazurin (PrestoBlue®) to identify compounds that inhibit cell proliferation or viability. The purpose of the assay is to measure if test compounds inhibit the secretion of IL-17A by the PBMC. Compounds which inhibit IL-17A secretion may be expected to have efficacy in psoriasis.

The reference compound in this assay is the calcineurin inhibitor Tacrolimus and inhibits IL-17A release with an EC₅₀of approximately 0.34 nM, and with an Emax (plateau of the fitted curve) of approximately 90%. A high Emax shows that the compound inhibits a large proportion of the secreted IL-17. A low EC₅₀value indicates that the compound is potent and performs the inhibition at low concentrations.

Key Assay Parameters

Parameter
Description

Cells
Human PBMC isolated from buffy coats and

kept at −150° C. until day of assay

Stimulation
Beads coated with antibodies to CD3 and CD28

(T-cell activation/expansion kit Miltenyi

Biotec cat no 130-091-441) One bead/cell.

Effect
Inhibition of IL-17A release

0% effect of
Bead stimulation, 0.1% DMSO

test compound

100% effect of
Bead stimulation, 0.1% DMSO,

test compound
Terfenadine 10 uM

Reference compound
Tacrolimus (Cas no 104987-11-3-P)

Incubation time
3 days

Capacity, run-time
The assay takes 3 days to perform.

and requirements

Method Description
PBMC Isolation:

- PBMC are isolated from fresh human buffy coats by centrifugation on Lymphoprep® following instructions from Sebmate®
- Cells are spun down and resuspended in freezing medium (RPMI1640+20% FBS+5% DMSO)
- Cells are frozen in aliquots of 8×10⁷cells/vial matching one 384 plate. Cells are placed in Coolcell box and placed in −80° C. freezer for one day. Vials are moved to the −150° C. freezer in a prechilled box.

Assay Day 1:

- Prepare assay medium: RPMI1640+10% heat inactivated FBS and 1% pen/strep, supplemented with human IL-23, 20 ng/mL (R&D systems, cat no 1290-IL-010)
- Test compounds: Prepare compound-containing source plate, i.e. titrations in DMSO to be transferred to the assay plate.
- Add 70 nL compounds and controls from the source plate to the assay plate.
- Thaw the cells by adding 1 vial to 20 ml warm assay medium
- Centrifuge the cells down at 170G in 10 minutes, remove the supernatant and resuspend in new medium.
- Filter the cells though a 37 μm strainer.
- Count the cells in the cell counter.
- Beads (T Cell Activation/Expansion Kit—human, Miltenyi Biotech, cat. no. 130-091-441): Coat beads with antibodies (antiCD3 and antiCD28) according to the kit procedure.
- Add the beads to the cells, one bead per cell, 2.1e+06 cells/ml. Use a pipette to mix the cells and beads carefully.
- Seed the cell/bead mixture into the assay plate, 70 μL/well (1.3e+05 cells/well)
- Place the plates in a humidity chamber with lid (24.5×24.5 cm box added H₂O in the bottom).
- Incubate plates at 37° C., 5% CO₂/95% air for 3 days.

Assay Day 4:

- IL-17A concentration in the supernatants is measured using an alphaLISA kit (Perkin Elmer, cat no. AL219F).
- Pretest to find optima dilution of samples: Test 5 μL supernatant from control wells and run IL-17A alphaLISA assay with only 2 hours of incubation.
- When data are available, remove assay plates from incubation and equilibrate to room temperature for 30 minutes.
- Transfer 5 μL or corrected amounts of supernatant to a 384-proxy plate
- Add 5 μL alphaLISA acceptor beads.
- Incubate 1 hour at room temperature
- Add 5 μL donor beads
- Incubate overnight at room temperature protected from light.
- Add 7 μL PrestoBlue reagent (resazurin)/well to the assay plate and incubated for 2-4 hours at 37° C./5% CO₂. The fluorescence in the wells is measured (Ex615/Em 535).

Assay day 5:

- Read alphaLISA plate in a plate reader

Data Analysis and Calculations

IL-17A concentration in the supernatants is measured using an alphaLISA kit (Perkin Elmer, cat no. AL219F).

% Effect IL-17:

The capacity of the test compound to inhibit IL-17A release is normalized to the signal in the negative control wells treated with a toxic compound, Terfenadine.

The level of living cells in the wells is measured by the addition of PrestoBlue® and the conversion of Resazurin to Resorufin (fluorescent) in living cells is measured.

% Effect Viability/Proliferation:

The effect of the test compounds on viability/proliferation is normalized to the PrestoBlue signal in the negative control wells treated with a toxic compound, Terfenadine.

4 and 5. T-Cell IL-2 & IL-4 Assay

Inhibition of IL-4 and IL-2 secretion from human CD4-positive T-cells stimulated with antiCD2/antiCD3/antiCD28-coated beads. LEO Pharma Open Innovation assay number 1297, 1298, 1299

Disease Relevance

The inflammatory skin disease atopic dermatitis is characterized by the T-cell cytokines IL-4, IL-13 and IL-22. In the present assay, human T-cells are stimulated with beads coated with antibodies against CD2, CD3 and CD28 to activate the T-cell receptor. Then the level of secreted of IL-4 is measured in the culture supernatant using electrochemiluminescence (MSD kits, Meso Scale Discovery), IL-2 is measured by proximity homogenous time-resolved fluorescence (HTRF) and the amount of living cells is measured by addition of resazurin (PrestoBlue®) to allow identifying compounds that inhibit T-cell proliferation or viability. The purpose of the assay is to measure if a test compound is able to inhibit the secretion of IL-4 by the T-cells, indicating that it is able to inhibit part of the inflammation occurring in the skin. Compounds which inhibit IL-4 secretion may be expected to have efficacy in atopic dermatitis. Compounds which also inhibit IL-2 secretion have a broader immunosuppressive effect since IL-2 is an autocrine growth factor for T-cells. A known steroid, Betamethasone, inhibits IL-4 release in this assay with an EC₅₀of approximately 15 nM, and with an Emax (plateau of the fitted curve) of approximately 80%. A high Emax shows that the compound is able to inhibit a large proportion of the secreted IL-4. A low EC₅₀value indicates that the compound is potent and is able to perform the inhibition at low concentrations.

Key Assay Parameters

Human CD4-postive T-cells isolated from buffy coats

and kept at −150 degree C. until day of assay

Stimulation
Beads coated with antibodies to CD2, CD3

and CD28

(T-cell activation/expansion kit Miltenyi

Biotec cat no 130-091- 441) One bead/cell.

Effect
Inhibition of IL-4 release from T-cells

0% effect of
Bead stimulation, 0.1% DMSO

test compound

100% effect of
Unstimulated cells, 0.1% DMSO

test compound

Reference compound
Betamethasone (CAS: 378-44-9)

Incubation time
48 h

Capacity, run-time
The assay takes 3 days to perform.

and requirements

Method Description
CD4+ T-Cell Isolation:

- Peripheral blood mononuclear cells (PBMC) are isolated from fresh human buffy coats by centrifugation on Lymphoprep® following instructions from Sebmate™ (Stemcell).
- The cells are resuspended in 30 ml of PBS+2% Serum and counted.
- CD4 positive T-cells are isolated from freshly isolated PBMC using EasySep™ humane CD4+ T-cell Isolation Kit (Catalog #17952) and following the manufacturer's protocol.
- Cells are spun down and resuspended in freezing medium (X-vivo 15+10% FBS+5% DMSO)
- Cells are frozen in aliquots of 3×10⁷cells/vial matching one 384 plate. Cells are place in Coolcell box and place in −80° C. freezer for one day then transferred to the −150° C. freezer in a prechilled box.

Assay Day 1:

- Prepare assay medium: 500 ml X-Vivo 15+5 ml Glutamax (2 mM final)+5 ml pen/strep (100 U/ml final).
- Add 5 μL medium to the wells of a 384 well plate (Greiner cat. No 788986).
- CD4+ Cells: Thaw frozen cells from −150° C. freezer in 37° C. bath, and pipette the cells into 10 ml medium. Spin 10 min @ 170 g.
- Resuspend pellet in 3.5 ml per 2.5×10⁷cells and add 10 μL per well to the plate (40000 cells/well)
- Test compounds: Prepare compound-containing source plate, i.e. titrations in DMSO to be transferred to the assay plate.
- Add 25 nL compounds and controls from the source plate to the assay plate (with cells).
- Beads (T Cell Activation/Expansion Kit—human, Miltenyi Biotech, cat. no. 130-091-441): Coat beads with antibodies according to the kit procedure.
- Add 10 μL beads to each well except for the negative controls. One bead per cell. Add 10 μL medium to the negative controls.
- Incubate plates at 37° C., 5% CO₂/95% air for 2 days.

Assay Day 3:

- Remove plates from incubation and equilibrate to room temperature for 30 minutes.
- IL-4 concentration in 10 μL supernatant is measured using an MSD IL-4 384-plate kit (Custom made) according to manufacturer protocol.
- IL-2 concentrations in the supernatant is measured by an IL-2 HTRF kit, CisBio, cat. No 62HIL02PEH.
- Add 4 μL medium to white proxi 384-well plates
- Add 1 μL supernatant.
- Add 4 μL mHTRF mix (each diluted 1:300 in ⅔ reconstitution buffer and 1/3 assay medium).
- Spin the plates for 1 minute at 170 g
- Seal and incubate overnight and read fluorescence in a plate reader
- The amount of viable cells is measured by PrestoBlue® reagent
- Add 15 μL diluted PrestoBlue® reagent (resazurin) corresponding to 2.5 μL Prestoblue/well to the assay plate.
- Incubate overnight at 37° C./5% CO₂. The fluorescence in the wells is measured (Ex615/Em 535).

Data Analysis and Calculations

IL-4 concentration in the supernatants is measured using electrochemiluminescence (MSD kits, Meso Scale Discovery). IL-2 concentration in the supernatants is measured using homogeneous time-resolved fluorescence resonance (TR-FRET). The assay is quantified by measuring fluorescence at 665 nm (proportional to IL-2 concentration) and 620 nm (control). A ratio 665/620*1000 is calculated.

% Effect for IL-2 and IL-4:

The capacity of the test compound to inhibit IL-4 and IL-2 release is normalized to the signal in the negative control wells with un-stimulated T-cells.

The level of living cells in the wells is measured by the addition of PrestoBlue® and the conversion of Resazurin to Resorufin (fluorescent) in living cells is measured.

% Effect for Viability/Proliferation:

The effect of the test compounds on viability/proliferation is normalized to the PrestoBlue signal in the negative control wells treated with a toxic compound, Terfenadine.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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	Number	Date	Country
Parent	16330676	Mar 2019	US
Child	17123539		US

MEDICINAL USE OF SERRULATANE DITERPENES

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