A CRYSTALLINE FORM OF A SALT OF A MORPHOLINO SULFONYL INDOLE DERIVATIVE AND A PROCESS FOR ITS PREPARATION
FIELD OF THE INVENTION
The present invention relates to a crystalline form of a pharmaceutically acceptable
salt of a morpholino sulfonyl indole derivative (as described herein) that is capable of
inhibiting, modulating and/or regulating Insulin-Like-Growth Factor I Receptor (IGF-1R)
and Insulin Receptor (IR); and a process for its preparation.
BACKGROUND OF THE INVENTION
Protein kinases (PKs) are enzymes that catalyze the phosphorylation of hydroxy
groups on tyrosine, serine and threonine residues of proteins. The consequences of this
seemingly simple activity are staggering; cell growth, differentiation and proliferation; i.e.,
virtually all aspects of cell life, in one way or another depend on PK activity. Furthermore,
abnormal PK activity has been related to a host of disorders, ranging from relatively non lifethreatening
diseases such as psoriasis to extremely virulent diseases such as glioblastoma
(brain cancer). PKs can be broken into two classes, the protein tyrosine kinases (PTKs) and
the serine-threonine kinases (STKs).
Certain growth factor receptors exhibiting PK activity are known as receptor tyrosine
kinases (RTKs). They comprise a large family of transmembrane receptors with diverse
biological activity. At present, at least nineteen (19) distinct subfamilies of RTKs have been
identified. One RTK subfamily contains the insulin receptor (IR), insulin-like growth factor
I receptor (IGF-1R) and insulin receptor related receptor (IRR). IR and IGF-1R interact with
insulin to activate a hetero-tetramer composed of two entirely extracellular glycosylated a
subunits and two subunits which cross the cell membrane and which contain the tyrosine
kinase domain. The Insulin-like Growth Factor-1 Receptor (IGF-1R), and its ligands, IGF-1
and IGF-2, are abnormally expressed in numerous tumors, including, but not limited to,
breast, prostate, thyroid, lung, hepatoma, colon, brain, neuroendocrine, and others.
Numerous IGF-1R small molecule inhibitors have been found to inhibit cancer
growth in vitro, in vivo and in clinical trials. For example, BMS-754807 effectively inhibits
the growth of a broad range of human tumor types in vitro, including mesenchymal (Ewing's,
rhabdomyosarcoma, neuroblastoma, and liposarcoma), epothelial (breast, lung, pancreatic,
colon, gastric), and hematopoietic (multiple myeloma and leukemia) tumor cell lines.
Carboni et al., Mol Cancer Ther 2009; 8(12).
The association between abnormal PK activity and disease is not restricted to cancer.
For example, RTKs have been associated with diseases such as psoriasis, diabetes mellitus,
endometriosis, angiogenesis, atheromatous plaque development, Alzheimer's disease,
epidermal hyperproliferation, neurodegenerative diseases, age-related macular degeneration
and hemangiomas. Defects in Insulin-R and IGF-1R are indicated in type-II diabetes
mellitus. A more complete correlation between specific RTKs and their therapeutic
indications is set forth in Plowman et al., DN&P, 1994, 7:334-339.
The amorphous or non-crystalline form of a pharmaceutically acceptable salt, in
particular, methane sulfonate salt of the morpholino sulfonyl indole derivative, (S)-ethyl 4-
(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-lH-indol-7-ylamino)
piperidine-l-carboxylate, that is capable of inhibiting, modulating and/or regulating Insulin-
Like-Growth Factor I Receptor and Insulin Receptor has been disclosed in the applicant's co
pending PCT patent application. The amorphous or non-crystalline form had relatively
inadequate shelf-life due to stability problems under stress conditions, which caused
difficulty in reproducing its the pharmacological activity. Therefore, there was a need for
developing a process for the preparation of a stable crystalline form of said derivative for
overcoming the problems associated with the instability of the amorphous or non-crystalline
form of the specified compound, which problems have been addressed by the applicant in the
current patent application by providing a stable crystalline form of the compound, designated
herein as the Compound I .
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a crystalline form of a pharmaceutically
acceptable salt of a morpholino sulfonyl indole derivative, particularly, (S)-ethyl 4-(2-
carbamoyl-5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl) -lH-indol-7-ylamino)
piperidine-l-carboxylate methane sulfonate (herein after referred to as Compound I).
In another aspect, the present invention relates to a process for the preparation of the
crystalline form of Compound I .
In yet another aspect, the present invention relates to a pharmaceutical composition
comprising a therapeutically effective amount of the crystalline form of Compound I and one
or more pharmaceutically acceptable excipients or carriers.
In a further aspect, the present invention relates to a pharmaceutical composition
comprising a therapeutically effective amount of the crystalline form of Compound I, and a
pharmaceutically acceptable carrier and optionally other therapeutic agents.
In another aspect, the present invention relates to a crystalline form of the Compound
I for use in the treatment of an Insulin-Like-Growth Factor I Receptor (IGF-1R) or Insulin
Receptor (IR) mediated disease or disorder by administering to a subject in need thereof, a
therapeutically effective amount of the crystalline form of Compound I .
In yet another aspect, the present invention relates to a crystalline form of the
Compound I for use in the treatment of cancer, by administering to a subject in need thereof,
a therapeutically effective amount of the crystalline form of Compound I .
In another aspect, the present invention relates to a method for the treatment of an
Insulin-Like-Growth Factor I Receptor (IGF-1R) or Insulin Receptor (IR) mediated disease
or disorder by administering to a subject in need thereof, a therapeutically effective amount
of the crystalline form of Compound I .
In yet another aspect, the present invention relates to a method of treatment of cancer
by administering to a subject in need thereof, a therapeutically effective amount of the
crystalline form of Compound I .
In a further aspect, the present invention relates to use of the crystalline form of
Compound I for the treatment of an Insulin-Like-Growth Factor I Receptor (IGF-1R) or
Insulin Receptor (IR) mediated disease or disorder.
In a still further aspect, the present invention relates to use of the crystalline form of
Compound I for the treatment of cancer.
In another aspect, the present invention relates to use of the crystalline form of
Compound I for the manufacture of a medicament for use in the treatment of an Insulin-Like-
Growth Factor I Receptor (IGF-1R) or Insulin Receptor (IR) mediated disease or disorder.
In yet another aspect, the present invention relates to use of the crystalline form of
Compound I for the manufacture of a medicament for use in the treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows characteristic X-Ray powder diffraction spectrum (diffractogram) of the
crystalline form of Compound I obtained when solvent used for crystallization is isopropyl
acetate.
Figure 2 shows characteristic differential scanning calorimetric (DSC) thermogram for the
crystalline form of Compound I obtained when solvent used for crystallization is isopropyl
acetate.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a crystalline form of a pharmaceutically
acceptable salt of a morpholino sulfonyl indole derivative, particularly, (S)-ethyl 4-(2-
carbamoyl-5-chloro-3-(2-(phenoxymethyl)morpholinosulfonyl)-lH-indol-7-
ylamino)piperidine-l-carboxylate methane sulfonate (Compound I) and a process for its
preparation.
The crystalline form of compound I is useful in the inhibition of Insulin-Like-Growth
Factor I Receptor (IGF-1R) and Insuli
Compound I (as methane sulfonate)
Unless otherwise indicated, the term "compound I" as used herein and the appended
claims refers to, (S)-ethyl 4-(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl)
morpholinosulfonyl)-1H-indol-7-ylamino)piperidine- 1-carboxylate methane sulfonate.
However, the free base namely (S)-ethyl 4-(2-carbamoyl-5-chloro-3-(2-
(phenoxymethyl)morpholinosulfonyl)- 1H-indol-7-ylamino)piperidine- 1-carboxylic acid is
referred to herein as the free base of compound I or compound I free base.
The term "amorphous form of compound I" encompasses within its scope, the
amorphous form of compound I or its mixture with one or more crystalline form(s) of
compound I .
Many pharmaceutically active compounds have been found to exist in more than one
polymorphic form, such as one or more crystalline forms, an amorphous form, and/or
sometimes one or more solvated forms. Frequently it is found that the different forms have
different physical or chemical properties, such as solubility, hygroscopicity, etc., or have
properties that render some form easier to formulate into a pharmaceutical product. In
addition, certain forms can have a greater stability than the other forms, as shown by a
decreased tendency to spontaneously convert into a different polymorphic form or to entrap
impurity causing instability. Unfortunately, predicting the potential number, or even the
existence, of polymorphs for a given molecule is not possible. However, regulatory agencies
desire that the various polymorphic forms of a compound be identified before a
pharmaceutical product is approved for marketing, because it is essential that a product will
remain stable and have predictable properties during its entire shelf life.
The preparation of the amorphous form of Compound I has been described in a co
pending patent application of the applicant. The amorphous form of Compound I ((S)-ethyl
4-(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl)mo holinosulfonyl)-lH-indol-7-
ylamino)piperidine-l-carboxylate methane sulfonate) was found to be unstable under stress
conditions, due to its tendency to entrap methane sulfonic acid used for the salt preparation.
The said compound I in its amorphous form was found to have a relatively inadequate shelf
life due to a slow rate of degradation caused by an entrapped acid, which caused difficulty in
reproducing its pharmacological activity. Therefore, there was a need for developing a
process for preparation of a stable form of the Compound I with a view to obtain
reproducibility of the compound's pharmacological activity. The synthesis provided in the
current invention affords a crystalline form of Compound I, which is stable with reproducible
pharmacological activity even under stress conditions or after elapse of long duration of time.
The current synthesis facilitates a large-scale or commercial synthesis by incorporating a
sequence of techniques known in the art, as well as the methods set forth below, from readily
available starting materials.
Polymorphs of compounds are generally prepared by crystallization of the compounds
under different conditions. The different conditions are, for example, using different
commonly used solvents or their mixtures for crystallization; crystallization at different
temperatures; various modes of cooling, ranging from very fast to very slow cooling during
crystallizations. Polymorphs can also be obtained by heating or melting the compound
followed by gradual or fast cooling. The presence of polymorphs can be determined by IR
(Infra-red) spectroscopy, solid probe NMR (Nuclear Magnetic Resonance) spectroscopy,
differential scanning calorimetry, powder X-ray diffraction or such other techniques.
In an embodiment, the present invention relates to a process for the preparation of
the crystalline form of Compound I, comprising:
Step 1) purifying the free base of Compound I by treating a solution of said free base in a
solvent selected from isopropyl acetate, THF, 2-methyl tetrahydrofuran, toluene, heptane,
methylethylketone, ethyl acetate, isopropyl acetate or combinations thereof; with activated
charcoal and Si-thiol (silicycle), filtering the resulting mixture through a celite bed,
repeatedly washing the celite bed with the same solvent and evaporation of the filtrate; and
Step 2) reacting the purified free base of compound I with methanesulfonic acid in said
solvent at a temperature range of 70-80 °C for about 4-5 h to initiate crystallization of the
Compound I as methane sulfonate salt, followed by cooling the resulting reaction mixture to
room temperature and further to a temperature range of 0-5 °C to afford a crystalline mass,
which is optionally washed with the same chilled solvent to obtain the required crystalline
form of Compound I .
In an embodiment, the solvent used in step 1) and step 2) above may be selected from
THF, 2-methyl tetrahydrofuran, a mixture of 2-methyl tetrahydrofuran and toluene, a mixture
of 2-methyl tetrahydrofuran and heptane, methylethylketone, ethyl acetate or isopropyl
acetate.
In a futher embodiment, the solvent used in step 1) and step 2) above is isopropyl
acetate.
It has been found that the crystalline form of the compound I obtained with each of
the above-mentioned solvents is the same.
In another embodiment, the present invention relates to a process for the preparation
of the crystalline form of Compound I, comprising crystallizing the amorphous form of the
compound I( (S)-ethyl 4-(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-
lH-indol-7-ylamino)piperidine-l-carboxylate methane sulfonate) with a solvent selected
from isopropyl acetate, THF, 2-methyl tetrahydrofuran, toluene, heptane, methylethylketone,
ethyl acetate, isopropyl acetate or combinations thereof. The amorphous form of the
compound I is obtained by reacting the free base of Compound I with methanesulphonic acid
in THF as the solvent at room temperature for about 30 min. to 2 h, according to the process
for preparation of the amorphous form of compound I, as disclosed by the applicant in a co
pending PCT patent application.
In an embodiment, the solvent used for crystallization of the amorphous form of the
compound I may be selected from THF, 2-methyl tetrahydrofuran, a mixture of 2-methyl
tetrahydrofuran and toluene, a mixture of 2-methyl tetrahydrofuran and heptane,
methylethylketone, ethyl acetate or isopropyl acetate.
In another embodiment, the solvent used for crystallization of the amorphous form of
the compound I is isopropyl acetate.
In an embodiment, the crystalline form of the Compound I of the present invention is
characterized by X-Ray diffraction peaks at an angle of refraction 2-theta of 9.22, 11.92,
13.58, 15.74, 18.37, 18.65, 18.95, 19.37, 19.59, 20.33, 20.92, 22.48, 22.79, 23.97, 24.19,
24.59, 28.48 ± 0.2°.
In another embodiment, the crystalline form of the Compound I of the present
invention is characterized by the melting temperature onset of the crystalline form of
Compound I was determined by differential scanning calorimetry (DSC) which is found to be
224.08 ± 0.5 °C at 20 deg/min under nitrogen, with a peak melting temperature of 226.83+
0.5 °C.
According to the present invention, process for the preparation of the free base of the
compound I from which the crystalline form of Compound I (as methane sulfonate salt) is
prepared, employs reaction steps as shown in the following scheme 1.
Scheme 1:
Compound I (free base)
Accordingly, a process for the preparation of the free base of compound I comprises
the following steps:
Step la: Diazotising compound 1 (which is commercially available or may be prepared by
methods, well-known in the art):
by reacting it with NaNC>2 and HC1 at a temperature range of -10 to 5 °C, followed by a
dropwise addition of the diazotized mixture to an alkaline solution of the reagent, ethyl 2-
methyl-3-oxobutanoate in a base selected from NaOEt, KOH or NaOH in a solvent selected
from methanol or ethanol at a to -15 °C to obtain compound 2.
In an embodiment, the step l a is carried out using NaOEt as the base in ethanol as the
solvent.
Step lb: Cyclising compound 2 obtained in step l a by reaction with a Lewis acid such as
nCl2, AICI 3, BF3, P2O5 or polyphosphoric acid at a temperature range of 80 - 120 °C for 5-
12 h to obtain compound 3.
3
In an embodiment, cyclization of the compound 2 is carried out using polyphosphoric acid in
o-phosphoric acid as the Lewis acid at a temperature range of 80 - 85 °C for 2-3 h.
Step lc: Sulphonating compound 3 obtained in step l b by reaction with sulphuric acid and
acetic anhydride at a temperature range of 0-30 °C for 10-20 h to obtain compound 4.
4
Step Id: Reacting compound 4 as obtained in step l c with oxalyl chloride or thionyl chloride
in the presence of an organic base selected from triethylamine or pyridine in a solvent
selected from DMF, methylene dichloride or a mixture thereof at a temperature range of 25 -
50 °C for 2-4 h to obtain the corre oride 4A:
4A
which is optionally isolated; and is then reacted with compound E:
E
in the presence of an organic base selected from pyridine or triethylamine in a solvent
selected from dichloromethane or chloroform at room temperature (25-30 °C) for 1-4 h to
obtain compound 5.
5
In an embodiment, in the step Id, compound 4A is isolated prior to reaction with the
reagent E.
In another embodiment, the crude compound 5 obtained in step Id is purified with an
alcohol selected from methanol, ethanol, n-propanol, isopropanol or n-butanol to obtain
substantially pure compound 5.
In yet another embodiment, the crude compound 5 obtained in step Id is purified with
methanol.
Step le: Reducing compound 5 obtained in step Id by reacting it with a reducing agent
selected from Fe and NH4C1, Zn and HC1 or SnCk, for 2-8 h in a solvent selected from
methanol, ethanol, THF, water or a mixture thereof, to obtain compound 6.
6
In an embodiment, in step l e reduction of compound 5 is carried out using Fe and
NH4CI as the reducing agent in a mixture of THF, water and ethanol as solvent at a
temperature range of 70-80 °C for 2-4 h.
In an embodiment, the residual iron and iron oxides obtained during reduction using
Fe and NH4C 1 as reducing agent were removed by using EDTA and chloroform.
In another embodiment, the residual iron and iron oxides obtained during reduction
using Fe and NH4CI as reducing agent were removed by filtration.
In an embodiment, the crude compound 6 obtained is purified with an alcohol
selected from methanol, ethanol, n-propanol, isopropanol or n-butanol to obtain substantially
pure compound 6.
In another embodiment, the crude compound 6 obtained is purified with isopropanol.
Step If: Reacting compound 6 obtained in step l e with isopropyl alcohol and ammonia at a
temperature range of 80 - 120 °C at a pressure of 0.5 - 10 kg/cm2 for 10-18 h in an autoclave
or in a microwave for 10-15 min to obtain compound 7 :
In an embodiment, the crude compound 7 is purified with an alcohol selected from
methanol, ethanol, n-propanol, isopropanol or n-butanol to obtain substantially pure
compound 7.
In an embodiment, the crude compound 7 is purified with isopropanol.
Step lg: Reacting compound 7 obtaine If with compound F :
in the presence of trifluoroacetic acid in a base such as sodium triacetoxy borohydride in a
solvent selected from dichloromethane or ethyl acetate at room temperature for 0.5 - 2 h to
obtain Compound I as a free base.
Compound I (free base)
Process for the preparation of the compound E used in step Id above employs
reaction steps depicted in the following scheme 2 :
Scheme 2 :
Accordingly, a process for the preparation of Compound E used in step Id above, comprises
the following steps:
Step 2a:
Reacting commercially available phenol with (R)-2-(chloromethyl)oxirane in the presence of
a base selected from aqueous NaOH or aqueous KOH and a phase transfer catalyst such as
tetrabutylammonium hydrogen sulphate at a temperature range of 80-120 °C for 1-4 h to
obtain Compound A;
Step 2b:
Reacting Compound B ;
H 0 H
IB
n
B
with chlorosulfonic acid in a solvent selected from chloroform, carbon tetrachloride, or
dichloromethane at 0-10 °C during addition of the acid over a period of 15-30 min, followed
by at room temperature for 10-16 h to obtain Compound C;
HN ° S0 3H
Bn
Step 2c:
Reacting the compound A obtained in step 2a with the compound C obtained in step 2b in
the presence of an aqueous base such as NaOH or aqueous KOH in a solvent selected from
toluene, dioxane or THF in the presence of a phase transfer catalyst such as
tetrabutylammoniun hydrogen sulfate at a temperature range of 30-50 °C for 10-16 h to
obtain Compound D;
Step 2d:
Carrying out debenzylation of the compound D by refluxing the said compound D with
ammonium formate and 10 % Pd/C in an atmosphere of carbon dioxide in a solvent selected
from ethanol or methanol at 50-70 °C for 1-3 h to obtain the compound E.
UTILITY
In one aspect, this present invention relates to a method of modulating the catalytic
activity of PKs (protein kinases) in a subject in need thereof comprising contacting the PK
with the crystalline form of compound I . As used herein, the term "modulation" or
"modulating" refers to the alteration of the catalytic activity of receptor tyrosine kinases
(RTKs), cellular tyrosine kinases (CTKs) and serine-threonine kinases (STKs). In particular,
modulating refers to the activation of the catalytic activity of RTKs, CTKs and STKs,
preferably the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs,
depending on the concentration of the compound or salt to which the RTKs, CTKs or STKs
is exposed or, more preferably, the inhibition of the catalytic activity of RTKs, CTKs and
STKs.
The term "catalytic activity" as used herein refers to the rate of phosphorylation of
tyrosine under the influence, direct or indirect, of RTKs and/or CTKs or the phosphorylation
of serine and threonine under the influence, direct or indirect, of STKs.
The term "contacting" as used herein refers to bringing the crystalline form of
compound 1 and a target PK together in such a manner that the compound can affect the
catalytic activity of the PK, either directly; i.e., by interacting with the kinase itself, or
indirectly; i.e., by interacting with another molecule on which the catalytic activity of the
kinase is dependent. Such "contacting" can be accomplished "in vitro," i.e., in a test tube, a
petri dish or the like. In a test tube, contacting may involve only a compound and a PK of
interest or it may involve whole cells. Cells may also be maintained or grown in cell culture
dishes and contacted with the compound in that environment. In this context, the ability of
the compound to affect a PK related disorder; i.e., the IC50 of the compound, defined below,
can be determined before use of the compound in vivo with more complex living organisms
is attempted. For cells outside the organism, multiple methods exist, and are well known to
those skilled in the art, to get the PKs in contact with the compound including, but not limited
to, direct cell microinjection and numerous transmembrane carrier techniques.
The above-referenced PK is selected from the group comprising an RTK, a CTK or
an STK in another aspect of this invention. Preferably, the PK is an RTK.
Furthermore, it is an aspect of this invention that the receptor tyrosine kinase (RTK)
whose catalytic activity is modulated by the crystalline form of compound I is selected from
the group comprising EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRa, PDGFRp,
TrkA, TrkB, TrkC, HGF, CSFIR, C-Kit, C-fms, Flk-IR, Flk4, KDR/Flk- 1, Flt-1, FGFR-1R,
FGFR-1R, FGFR-3R and FGFR-4R. Preferably, the receptor protein kinase is selected from
IR, IGF-1R, or IRR.
In addition, it is an aspect of this invention that the cellular tyrosine kinase whose
catalytic activity is modulated by the crystalline form of compound I is selected from the
group consisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn,
Lck, Blk, Hck, Fgr and Yrk.
Another aspect of this invention is that the serine-threonine protein kinase whose
catalytic activity is modulated by the crystalline form of compound I is selected from the
group consisting of CDK2 and Raf.
In another aspect, this invention relates to a method for treating or preventing a PKrelated
disorder in a subject in need of such treatment comprising administering to the subject
a therapeutically effective amount of the crystalline form of compound I .
The term "subject" as used herein refers to an animal, preferably a mammal, and most
preferably a human.
The term "mammal" used herein refers to warm-blooded vertebrate animals of the
class Mammalia, including humans, characterized by a covering of hair on the skin and, in
the female, milk-producing mammary glands for nourishing the young. The term mammal
includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig as well as
human.
As used herein, "PK-related disorder," "PK driven disorder," and "abnormal PK
activity" all refer to a condition characterized by inappropriate (i.e., diminished or, more
commonly, exessive) PK catalytic activity, where the particular PK can be an RTK, a CTK or
an STK. Inappropriate catalytic activity can arise as the result of either: (1) PK expression in
cells which normally do not express PKs; (2) increased PK expression leading to unwanted
cell proliferation, differentiation and/or growth; or, (3) decreased PK expression leading to
unwanted reductions in cell proliferation, differentiation and/or growth. Excessive-activity of
a PK refers to either amplification of the gene encoding a particular PK or its ligand, or
production of a level of PK activity which can correlate with a cell proliferation,
differentiation and/or growth disorder (that is, as the level of the PK increases, the severity of
one or more symptoms of a cellular disorder increase as the level of the PK activity
decreases).
"Treat," "treating" or "treatment" with regard to a PK-related disorder refers to
alleviating or abrogating the cause and/or the effects of a PK-related disorder.
As used herein, the terms "prevent", "preventing" and "prevention" refer to a method
for barring a mammal from acquiring a PK-related disorder in the first place.
The term "administration" and variants thereof (e.g., "administering" a compound) in
reference to the crystalline form of compound I means introducing the compound into the
system of the animal in need of treatment. When the crystalline form of compound I is
provided in combination with one or more other therapeutically active agents (e.g., a
cytotoxic agent, etc.), "administration" and its variants are each understood to include
concurrent and sequential introduction of the compound or prodrug thereof and other agents.
The term "therapeutically effective amount" as used herein means that amount of
active compound or pharmaceutical agent (i.e. the crystalline form of Compound I) that
elicits the biological or medicinal response in a tissue, system, animal or human that is being
sought by a researcher, veterinarian, medical doctor or other clinician.
The term "treating cancer" or "treatment of cancer" refers to administration to a
mammal afflicted with a cancerous condition and refers to an effect that alleviates the
cancerous condition by killing the cancerous cells, but also to an effect that results in the
inhibition of growth and/or metastasis of the cancer.
The protein kinase-related disorder may be selected from the group comprising an
RTK, a CTK or an STK-related disorder in a further aspect of this invention. Preferably, the
protein kinase-related disorder is an RTK-related disorder.
In yet another aspect of this invention, the above referenced PK-related disorder may
be selected from the group consisting of an EGFR-related disorder, a PDGFR-related
disorder, an IGFR-related disorder and a flk-related disorder.
The above referenced PK-related disorder may be a cancer selected from, but not
limited to astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder
cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer,
adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma,
erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma,
glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non-small cell lung
cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell
carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular
cancer or osteosarcoma in a further aspect of this invention.
More preferably, the PK-related disorder is a cancer selected from breast cancer,
colon carcinoma, colorectal cancer, Ewing's sarcoma or rhabdosarcoma.
The present invention therefore relates to a crystalline form of Compound I for use in
the treatment of diseases or disorders mediated by Insulin-Like-Growth Factor I Receptors
(IGF -IR) or Insulin Receptors (IR) comprising administering to a subject in need thereof, a
therapeutically effective amount of the crystalline form of Compound I .
In an embodiment, the present invention relates to the crystalline form of Compound I
for use in the treatment of diseases or disorders mediated by Insulin-Like-Growth Factor I
Receptors or Insulin Receptors, wherein the Insulin-Like-Growth Factor I Receptor and
Insulin Receptor mediated disease or disorder is cancer.
Accordingly, in an embodiment, the present invention relates to the crystalline form
of Compound I for use in the treatment of cancer.
The present invention also encompasses a method of treating or preventing cancer in
a mammal in need of such treatment which comprises administering to said mammal a
therapeutically effective amount of the crystalline form of the compound I .
In one embodiment, the present invention relates to a use of the crystalline form of
Compound I for the manufacture of a medicament for the treatment of diseases or disorders
mediated by Insulin-Like-Growth Factor I Receptor (IGF-IR) and Insulin Receptor (IR).
In another embodiment, the present invention relates to the use of the crystalline form
of Compound I for the manufacture of a medicament for the treatment of diseases or
disorders mediated by Insulin—Like-Growth Factor I Receptor and Insulin Receptor, wherein
the Insulin-Like-Growth Factor I Receptor and Insulin Receptor mediated disease or disorder
is cancer.
Accordingly, in an embodiment, the present invention relates to the use of the
crystalline form of Compound I for the manufacture of a medicament for the treatment of
cancer.
Types of cancers which may be treated using the crystalline form of the compound I
include, but are not limited to astrocytoma, basal or squamous cell carcinoma, brain cancer,
gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer,
chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer,
endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and
neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma,
non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer,
renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer,
testicular cancer or osteosarcoma. Preferably, the cancer being treated is selected from breast
cancer, colon carcinoma, colorectal cancer, Ewing's sarcoma or rhabdosarcoma.
The above-referenced PK-related disorder may be an IGFR-related disorder selected
from diabetes, an autoimmune disorder, Alzheimer's and other cognitive disorders, a
hyperproliferation disorder, aging, cancer, acromegaly, Crohn's disease, endometriosis,
diabetic retinopathy, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, an
inflammatory disorder and angiogenesis in yet another aspect of this invention.
A method of treating or preventing retinal vascularization which is comprised of
administering to a mammal in need of such treatment a therapeutically effective amount of
the crystalline form of the compound I is also encompassed by the present invention.
Methods of treating or preventing ocular diseases, such as diabetic retinopathy and agerelated
macular degeneration, are also part of the invention.
Also included within the scope of the present invention is a method of treating or
preventing inflammatory diseases, such as rheumatoid arthritis, psoriasis, contact dermatitis
and delayed hypersensitivity reactions, as well as treatment or prevention of bone associated
pathologies selected from osteosarcoma, osteoarthritis, and rickets.
Other disorders which might be treated with the compound of this invention include,
without limitation, immunological and cardiovascular disorders such as atherosclerosis.
Also included in the scope of the claims is a method of treating cancer that comprises
administering a therapeutically effective amount of the crystalline form of compound I in
combination with radiation therapy and/or in combination with a second compound which is
a therapeutically effective compound selected from: an estrogen receptor modulator, an
androgen receptor modulator, a retinoid receptor modulator, a cytotoxiccytostatic agent, an
antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase
inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis
inhibitor, PPAR-agonists, PPAR-agonists, an inhibitor of inherent multidrug resistance,
an anti-emetic agent, an agent useful in the treatment of anemia, an agent useful in the
treatment of neutropenia, an immunologic-enhancing drug, an inhibitor of cell proliferation
and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, -
secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and an agent
that interferes with a cell cycle checkpoint.
The instant invention also includes a pharmaceutical composition useful for treating
or preventing cancer that comprises a therapeutically effective amount of the crystalline form
of the compound I along with said second compound.
The PKs whose catalytic activity is modulated by the compound of this invention
include protein tyrosine kinases of which there are two types, receptor tyrosine kinases
(RTKs) and cellular tyrosine kinases (CTKs), and serine-threonine kinases (STKs). RTKmediated
signal transduction, is initiated by extracellular interaction with a specific growth
factor (ligand), followed by receptor dimerization (or conformational changes in the case of
IR, IGF-1R or IRR), transient stimulation of the intrinsic protein tyrosine kinase activity,
autophosphorylation and subsequent phosphorylation of other substrate proteins.
In another aspect, the protein kinase (PK), the catalytic activity of which is modulated
by contact with the crystalline form of the compound I, is a protein tyrosine kinase (PTK),
more particularly, a receptor protein tyrosine kinase (RTK). Among the RTKs whose
catalytic activity can be modulated with the compound of this invention, or salt thereof, are,
without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRa, PDGFRp, TrkA,
TrkB, TrkC, HGF, CSFIR, C-Kit, C-fms, Flk-IR, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-
2R, FGFR-3R and FGFR-4R. Preferably, the RTK is selected from IGF-1R.
The protein tyrosine kinase whose catalytic activity is modulated by contact with the
crystalline form of the compound I, can also be a non-receptor or cellular protein tyrosine
kinase (CTK). Thus, the catalytic activity of CTKs such as, without limitation, Src, Frk, Btk,
Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may
be modulated by contact with the crystalline form of the compound I .
Still another group of PKs which may have their catalytic activity modulated by
contact with crystalline form of the compound I are the serine-threonine protein kinases such
as, without limitation, CDK2 and Raf.
The present invention is directed to crystalline form of the compound I which
modulates RTK, CTK and/or STK mediated signal transduction pathways as a therapeutic
approach to cure many kinds of solid tumors, including, but not limited to, carcinomas,
sarcomas including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma,
astrocytoma, melonoma and myoblastoma. Treatment or prevention of non-solid tumor
cancers such as leukemia are also contemplated by this invention. Indications may include,
but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers,
pancreatic cancers, colon cancers, blood cancers, breast cancers, prostrate cancers, renal cell
carcinomas, lung cancer and bone cancers.
Further examples, without limitation, of the types of disorders related to inappropriate
PK activity that the compound described herein may be useful in preventing, treating and
studying, are cell proliferative disorders, fibrotic disorders and metabolic disorders.
These and other aspects of the invention will be apparent from the teachings
contained herein.
COMPOSITIONS AND FORMULATIONS
Pharmaceutical compositions of the compound of the present invention are a further
aspect of this invention.
As used herein, the term "composition" is intended to encompass a product
comprising the specified ingredients in the specified amounts, as well as any product which
results, directly or indirectly, from combination of the specified ingredients in the specified
amounts.
As used herein, the term "active ingredient" refers to any substance or mixture of
substances intended to be used in the manufacture of a drug (medicinal) product, which
substance(s) are intended to furnish pharmacological activity or other direct effect in the
diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or
function of the body. Reference: Manufacturing, Processing, or Holding Active
Pharmaceutical Ingredients FDA Guidance. In the context of the present invention, the term
"active ingredient" refers to Compound I .
The present invention also encompasses a pharmaceutical composition useful in the
treatment of cancer, comprising the administration of a therapeutically effective amount of
the crystalline form of the compound I, with or without pharmaceutically acceptable carriers
or diluents. Suitable compositions of this invention include aqueous solutions comprising the
crystalline form of the compound I and pharmacologically acceptable carriers, e.g., saline, at
a pH level, e.g., 7.4. The solutions may be introduced into a patient's bloodstream by local
bolus injection.
The crystalline form of the compound I may be administered to mammals, preferably
humans, either alone or, preferably, in combination with pharmaceutically acceptable
carriers, excipients or diluents, optionally with known adjuvants, such as alum, in a
pharmaceutical composition, according to standard pharmaceutical practice. The crystalline
form of the compound I can be administered orally or parenterally, including the intravenous,
intramuscular, intraperitoneal, subcutaneous, rectal and/or topical routes of administration.
The pharmaceutical compositions containing the active ingredient may be in a form
suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any method known to the
art for the manufacture of pharmaceutical compositions and such compositions may contain
one or more agents selected from the group consisting of sweetening agents, flavoring agents,
coloring agents and preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets contain the active ingredient in admixture with non-toxic
pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets.
These excipients may be for example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating
agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or
alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and
lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be
uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug
or delay disintegration and absorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a water soluble taste masking material
such as hydroxypropyl-methylcellulose or hydroxypropyl-cellulose, or a time delay material
such as ethyl cellulose, cellulose acetate buryrate may be employed.
Formulations for oral use may be presented as hard gelatin capsules wherein the
active ingredient is mixed with an inert solid diluent, for example, calcium carbonate,
calcium phosphate, kaolin, lactose or dried cornstarch, or as soft gelatin capsules wherein the
active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil
medium, for example peanut oil, liquid paraffin, or olive oil. For oral use of the compound
according to this invention, particularly for chemotherapy, the compound may be
administered, for example, in the form of a tablet or a capsule, or as an aqueous solution or
suspension. When aqueous suspensions are required for oral use, the active ingredient is
combined with emulsifying and suspending agents. If desired, certain sweetening and/or
flavoring agents may be added. For intramuscular, intraperitoneal, subcutaneous and
intravenous use, sterile solutions of the active ingredient are usually prepared, and the pH of
the solutions should be suitably adjusted and buffered. For intravenous use, the total
concentration of solutes should be controlled in order to render the preparation isotonic.
Aqueous suspensions contain the active material in admixture with excipients suitable
for the manufacture of aqueous suspensions. Such excipients are suspending agents, for
example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,
sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for example lecithin, or
condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene
stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for
example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with
partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol
monooleate, or condensation products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous
suspensions may also contain one or more preservatives, for example ethyl, or n-propyl phydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one or
more sweetening agents, such as sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the active ingredient in a
vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil
such as liquid paraffin. The oily suspensions may contain a thickening agent, for example
beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and
flavoring agents may be added to provide a palatable oral preparation. These compositions
may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or
alpha-tocopherol.
Dispersible powders and granules suitable for preparation of an aqueous suspension
by the addition of water provide the active ingredient in admixture with a dispersing or
wetting agent, suspending agent and one or more preservatives. Suitable dispersing or
wetting agents and suspending agents are exemplified by those already mentioned above.
Additional excipients, for example sweetening, flavoring and coloring agents, may also be
present. These compositions may be preserved by the addition of an anti-oxidant such as
ascorbic acid.
The pharmaceutical compositions of the invention may also be in the form ofan oilin-
water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis
oil, or a mineral oil, for example liquid paraffin phosphatides, for example soy bean lecithin,
and esters or partial esters derived from fatty acids and hexitol anhydrides, for example
sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide,
for example polyoxyethylene sorbitan monooleate. The emulsions may also contain
sweetening, flavoring agents, preservatives and antioxidants.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol,
propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a
preservative, flavoring and coloring agents and antioxidant.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous
solution. Among the acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution.
The sterile injectable preparation may also be a sterile injectable oil-in-water
microemulsion where the active ingredient is dissolved in the oily phase. For example, the
active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil
solution then introduced into a water and glycerol mixture and processed to form a
microemulation.
The injectable solutions or microemulsions may be introduced into a patient's
bloodstream by local bolus injection. Alternatively, it may be advantageous to administer the
solution or microemulsion in such a way as to maintain a constant circulating concentration
of the instant compound. In order to maintain such a constant concentration, a continuous
intravenous delivery device may be utilized. An example of such a device is the Deltec
CADD-PLUS™ model 5400 intravenous pump.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous
or oleagenous suspension for intramuscular and subcutaneous administration. This
suspension may be formulated according to the known art using those suitable dispersing or
wetting agents and suspending agents, which have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In
addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including synthetic mono- or
diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of
injectables.
The compound of the present invention may also be administered in the form of
suppositories for rectal administration of the drug. These compositions can be prepared by
mixing the drug with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will therefore melt in the rectum to
release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated
vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid
esters of polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing
the compound of the present invention are employed. (For purposes of this application,
topical application shall include mouth washes and gargles.)
The compound of the present invention can be administered in intranasal form via
topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes,
using those forms of transdermal skin patches well known to those of ordinary skill in the art.
To be administered in the form of a transdermal delivery system, the dosage administration
will, of course, be continuous rather than intermittent throughout the dosage regimen. The
compound of the present invention may also be delivered as a suppository employing bases
such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of
polyethylene glycols of various molecular weights and fatty acid esters of polyethylene
glycol.
When the compound of the present invention is administered into a human subject,
the daily dosage will normally be determined by the prescribing physician with the dosage
generally varying according to the age, weight, and response of the individual patient, as well
as the severity of the patient's symptoms.
In one exemplary application, a suitable amount of the compound is administered to a
mammal undergoing treatment for cancer. Administration occurs in an amount between
about 0.1 mg/kg of body weight to about 60 mg/kg of body weight per day, preferably of
between 0.5 mg/kg of body weight to about 40 mg/kg of body weight per day.
The use of all of these approaches in combination with the instant compound
described herein are within the scope of the present invention.
In respect of the schemes of preparation (Scheme(s) 1 and 2) depicted herein above, it
may be noted that in addition to other standard manipulations that are known in the literature
or exemplified in the experimental procedures. Substituent numbering, as shown in the
schemes, does not necessarily correlate to that used in the claims. It should also be noted that
any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples
and Tables herein is assumed to have the sufficient number of hydrogen atom(s) to satisfy the
valences. According to the present disclosure, the definition "substantially pure compound"
means compound comprising less than 10 , preferably less than 5 % of undesired chemical
impurities, which can be determined, for example, by HPLC.
Abbreviations, which may be used in the description of the chemistry and in the Examples
that follow, include:
ATCC American Type Culture Collection, USA;
ATP Adenosine triphosphate;
Ac20 Acetic anhydride;
AcOH Acetic acid;
A1C13 Aluminium chloride;
BF3 Boron trifluoride;
CDCI3 Deuterated chloroform;
co2 Carbon dioxide;
DCM Dichloromethane ;
DMF N,N-Dimethylf ormamide ;
DMSO Dimethyl sulfoxide;
DMSO-d Deuterated dimethyl sulfoxide;
D20 Deuterated water;
EDTA Ethylenediaminetetraacetic acid;
Et3N Triethylamine;
EtOAc Ethyl acetate;
EtOH Ethanol;
FBS Fetal bovine serum (Gibco, USA);
Fe Iron;
GC Gas chromatography;
HPLC High-performance liquid chromatography;
HC1 Hydrochloric acid;
LOD Loss on drying;
KOH Potassium hydroxide;
M.C. Moisture content;
MeOH Methanol;
MS Mass Spectroscopy;
NMT Not more than;
NLT Not less than;
NH3 Ammonia
NaOH Sodium hydroxide;
NaOEt Sodium ethoxide;
Na2S0 4 Sodium sulfate;
NaN0 2 Sodium nitrite;
NH4C Ammonium chloride;
NMR Nuclear Magnetic Resonance;
PBS Phosphate buffered saline (Sigma Aldrich, USA);
Pd/C Palladium over activated charcoal or Palladium-carbon;
p
2o5 Phosphorous pentoxide;
S11CI2 Stannous chloride;
RT Room Temperature;
TFA Trifluoroacetic acid;
THF Tetrahydrofuran ;
Zn Zinc;
ZnCl2 Zinc chloride;
Cell-lines (Source: ATCC. USA):
Colo205 : Human colon adenocarcinoma cell-line
MCF-7 : Human breast adenocarcinoma cell-line
A673 Ewing tumor cell-line
SK-ES Human Ewing' s sarcoma cell-line
RD-ES Human Ewing' s sarcoma cell-line
HCT-116 Colon cancer cell-line
RD Rhabdomyosarcoma cell-line
EXAMPLES
Example 1:
Ethyl 2-(2-(4-chloro-2-nitrophenyl)hydrazono)propanoate (compound 2)
To an ice-cold solution of NaOEt (Spectrochem, 49.3 g, 0.724 mol) in ethanol (Commercial
grade, 500 mL) at -15 °C to -10 °C, was added ethyl-2-methyl acetoacetate (Aldrich, 50.95 g,
0.353 mol) drop-wise maintaining temperature below -15 °C over a period of 0.45-1 h and the
resulting mixture was stirred for 45 min at -15 °C to -20 °C. Simultaneously a cold diazonium
salt solution was prepared by addition of sodium nitrite (Spectrochem, 27.9 g, 0.405 mol) to a
solution of 2-nitro-4-chloro aniline (Aldrich, 50 g, 0.289 mol) in a mixture of cone. HC1 (100
mL) and water (225 mL) at -10 °C to -5 °C. The diazonium salt mixture was then added into the
ethanol solution of ethyl-2-methyl acetoacetate with constant stirring, maintaining the
temperature below -10 °C. The reaction was stirred for another 30 to 40 min (pH = 2-3). The
solid was then filtered by suction filtration to yield crude compound 2, which is washed with
water (150 mL) and again filtered by suction filtration. The compound is dried at 12-16 h at 45-
50 °C to afford pure compound 2.
Yield range: 66-72 ; HPLC Purity: ~ 65 ; Moisture content: NMT 1 ; H NMR (300
MHz, DMSO-d ) : 10.87 (s, 1H), 8.19 (s, 1H), 8.01-7.99 (d, J = 8.4 Hz, 1H), 7.57-7.54 (d, J
= 7.8 Hz, 1H), 4.37-4.35 (q, 2H), 2.24 (s, 3H), 1.40 (t, 3H); MS: m/z 284 (M-H)Example 2 :
Ethyl 5-chloro-7-nitro-lH-indole-2-carboxylate (Compound 3)
A mixture of o-phosphoric acid (Spectrochem, 38.4 mL), polyphosphoric acid (PPA)
(Spectrochem, 384 g) and the compound 2 of example 1 (128 g, 0.4481 mol) were heated
with stirring at 80-85 °C for 2-3 h. On completion of the reaction, the temperature was
reduced to 55-60 °C and water was added to the reaction mixture slowly with stirred for
another 30 minutes. The solid precipitate was filtered and redissolved in EtOAc, treated with
charcoal (Norit CA1, 6.4 g) for 1 h and filtered through a celite bed. The organic layer was
washed using 5 % sodium bicarbonate (Merck, 2.56 L) and 10 % NaCl solution (1.2 L), dried
over anhydrous Na2S0 4 (Merck, 100 g) and evaporated to yield the crude compound 3, which
was treated with «-heptane (768 mL) and filtered to afford the title compound 3, which was
dried at - 50 °C for 15-16 h.
Yield range: 32-42 , HPLC Purity: ~ 85 , LOD: NMT 2 % w/w, H NMR (300 MHz,
DMSO-de): 10.31 (s, 1H), 8.27-8.26 (d, J = 1.5 Hz, 1H), 8.01- 8.01 (d, J = 1.2 Hz, 1H),
7.30-7.27 (s, 1H), 4.51-4.44 (q, 2H), 1.48-1.41 (t, 3H); MS: m/z 267 (M-H)Example 3:
5-Chloro-2-(ethoxycarbonyl)-7-nitro-lH-indole-3-sulfonic acid (Compound 4)
To compound 3 of example 2 (84 g, 0.3128 mol) was added acetic anhydride (S.D.
Finechem, 336 mL) at room temperature. The reaction mixture was subsequently cooled to
0-10 °C, and sulphuric acid (Rankem, 153.3 g, 83.31 mL) was added drop wise over a period
of 35-40 min. The reaction was stirred for 15-16 h at room temperature to ensure completion
of the reaction. The solid was then filtered by suction filtration to obtain crude compound 3,
which was washed with AcOH (Spectrochem, 84 mL), subsequently washed with EtOAc
(Commercial grade, 84 mL) and dried at 45-50 °C to afford the title compound 4.
Yield range: 54-62 ; HPLC Purity: -90 ; LOD: NMT 5 % w/w, M. C : NMT 5 % w/w;
H NMR (300 MHz, DMSO-d6) 12.28 (s, lH),s 8.357-8.351 (d, J = 1.8 Hz, 1H), 8.18- 8.17
(d, J = 1.8 Hz, 1H), 4.33-4.25 (q, 2H), 1.33-1.29 (t, 3H); MS: m/z 347 (M-H)Example 4 :
Ethyl 5-chloro-3-(chlorosulfonyl)-7-nitro-lH-indole-2-carboxylate (Compound 4A)
Compound 4 of example 3 (40 g, 0.115 mol) was suspended in dichloromethane
(Commercial grade, 550 mL) to which catalytic amount of DMF (A.R. Grade -20 mL) was
added and the mixture was stirred at room temperature till a clear solution was obtained.
Oxalyl chloride (Spectrochem, 72.9 g, 50 mL, 0.574 mol) in dichloromethane (250 mL) was
added to the reaction mixture drop wise over a period of 30-45 min at room temperature. The
reaction mixture was heated at 40 °C with stirring for 2-3 h. After evaporating a portion of
dichloromethane (3-5 vol), the reaction mixture is cooled to 0-5 °C and maintained under an
atmosphere of nitrogen for 1.5-2 h. The crude compound obtained was filtered, washed with
chilled dichloromethane (40 mL) and dried at 40-45 °C to afford the title compound 4A.
Yield Range: 76-85 ; HPLC: 95 ; LOD: NMT 3 % w/w.
Example 5 :
(S)-Ethyl 5-chloro-7-nitro-3-(2-(phenoxymethyl)morpholinosulfonyl)-lH-indole-2-
carboxylate (Compound 5)
To a solution of (S)-2-(phenoxymethyl)morpholine (Compound E, 17.3 g, 0.09 mol) in
dichloromethane (Commercial grade, 300 mL), triethylamine (L.R. grade, 16.5 g, 0.163 mol)
was added over a period of 30-45 min at -5 to -10 °C. At this temperature, compound 4a of
example 4 (30 g, 0.081 mol) was added in portions over a period of 1 h. The reaction was
then stirred for 2-3 h at 30 °C. On completion of the reaction, the reaction was quenched with
water (250 mL). The organic layer was dried over anhydrous sodium sulfate (10 g). The
organic layer was concentrated to a small volume and the residue was treated with methanol
(Commercial grade, 360 mL) at room temperature for 30-60 min. The solid obtained is
suction-filtered and washed with chilled methanol (60 mL). The solid is dried at 45-50 °C to
afford the title compound 5, which was purified using methanol.
Yield range: 63-80 ; HPLC Purity: NLT 97 ; Chiral Purity: NLT 95 % ee; LOD: NMT
2.0 % w/w; H NMR (300 MHz, DMSO-d ) : 13.46 (s, 1H), 8.338-8.332 (d, J = 1.8 Hz 1H),
8.26-8.25 (d, J = 1.8 Hz, 1H), 7.29-7.24 (m, 2H), 6.95-6.88 (m, 3H), 4.41-4.34 (q, 2H), 3.98-
3.93 (m, 3H), 3.81-3.77 (m, 2H), 3.67-3.58 (m, 2H), 2.60-2.49 (m, 2H), 1.32-1.28 (t, 3H);
MS: m/z 524 (M+H)+.
Example 6:
(S)-Ethyl 7-amino-5-chloro-3-(2-(phenoxymethyl)morpholinosulfonyl)-lH-indole-2-
carboxylate (Compound 6)
Compound 5 of example 5 (27 g, 0.0516 mol), iron powder (Merck, 100 mesh electrolytic,
9.25 g, 0.1652 mol), ammonium chloride (Merck, 8.83 g, 0.1652 mol) was added to a
mixture of ethanol (Commercial grade, 151 mL), THF (Spectrochem, 75 mL) and water (37
mL). The reaction mixture was heated to 70-80 °C and maintained at that temperature for 3-
4 h. On completion of the reaction, the reaction mixture was cooled to 55-60 °C and filtered
hot through a bed of celite. The celite bed was further washed with EtOAc (Commercial
grade, 135 mL). The filtrate was concentrated to reduce the volume, which was chased with
EtOH (Commercial grade, 54 mL), water (540 mL) was added and stirred at room
temperature for 30-45 min. The solid obtained was suction-filtered, washed with water (54
mL) and dried at 45-50 °C for 12 - 16 h to afford the title compound 6. The compound 6
obtained may be optionally purified further by treatment with isopropyl alcohol (Commercial
grade, 130 mL) followed by filtration and drying.
Yield range: 63-72 ; HPLC Purity: NLT 95 ; LOD: NMT 3 % w/w; H NMR (300 MHz,
DMSO-d ) : 12.66 (s, 1H), 7.29-7.24 (m, 2H), 7.17 (s, 1H), 6.95-6.88 (m, 3H), 6.52 (s, 1H),
6.00 (bs, 2H), 4.41-4.34 (q, 2H), 3.99-3.90 (m, 3H), 3.81-3.78 (m, 2H), 3.61-3.52 (m, 2H),
2.59-2.50 (m, 2H), 1.34-1.22 (t, 3H); MS: m z 494.1 (M+H)+.
Example 7 :
(S)-7-Amino-5-chloro-3-(2-(phenoxymethyl)morpholinosulfonyl)-lH-indole-2-
carboxamide (Compound 7)
Compound 6 of example 6 (36 g, 0.0728 mol) was dissolved in isopropyl alcohol (IPA)
(Commercial grade, 720 mL) in a 2 L autoclave and the reaction mixture was cooled to -10 to
-5 °C for 30 min. Ammonia gas was purged for about 30 minutes at the same temperature and
maintaining a pressure of 2 kg/m2. The reaction mixture was heated to 105-110 °C for 14-16
h. The reaction was cooled to -10 °C and unloaded from the autoclave. The organic layer
was partially distilled below 50 °C and the residue was cooled to room temperature and
maintained at room temperature for 45-60 min. The solid was suction-filtered, washed with
IPA (2 x 36 mL) and dried at 45-50 °C to afford the title compound 7, which was purified
using isopropyl alcohol.
Yield range: 73-87 , HPLC Purity: NLT 96 , LOD: NMT 3 % w/w, H NMR (300 MHz,
DMSO-d ) : 12.59 (s, 1H), 8.30-8.23 (d, J = 21.0 Hz, 2H), 7.28-7.23 (m, 2H), 7.108-7.102
(d, J = 1.8 Hz, 1H), 6.94 -6.87 (m, 3H), 6.49-6.48 (d, J = 1.8 Hz, 1H), 6.01 (bs, 2H), 4.03-
3.94 (m, 2H), 3.90-3.79 (m, 2H), 3.68-3.46 (m, 3H), 2.50-2.31 (m, 2H). MS: m/z 465.1
(M+H)+.
Example 8:
(S)-Ethyl 4-((2-carbamoyl-5-chloro-3-((2-(phenoxymethyl)morpholino)sulfonyl)-lHindol-
7-yl)amino)piperidine-l-carboxylate (Compound I as free base)
Compound 7 of example 7 (18 g, 0.0387 mol) and ethyl 4-oxopiperidine-l-carboxylate
(Oakwood Corporation Inc., 9.96 g, 8.7 mL, 0.0581 mol) were taken in dichloromethane
(Commercial grade, 360 mL) and the turbid solution was stirred for 20 h at room
temperature. Trifluoroacetic acid (TFA) (Merck, 2.98 mL, 0.0387 mol) in dichloromethane
(18 mL) was added dropwise and stirred for 2 h. Following this, sodium triacetoxyborohydride
(Spectrochem, 24.55 g, 0.1163 mol) was added and the reaction mixture
was stirred for another 1.5 h at room temperature. The reaction mass was partially
concentrated and the residue was dissolved in ethyl acetate (Commercial grade, 360 mL).
After cooling to room temperature, the organic layer was washed with 5 % sodium
bicarbonate solution (Merck, 180 mL) and 20 % brine (180 mL). The organic phase was
filtered through a celite bed, which was washed with ethyl acetate (36 mL) and dried over
anhydrous sodium sulphate (Merck, 20 g). The organic solvent was distilled under 45 °C to
yield a crude solid, which was treated with dichloromethane (180 mL) at room temperature
for 30-40 min, suction-filtered and dried at 45-50 °C for 12-14 h to afford the title compound
I as a free base, which was purified using dichloromethane.
Yield range: 67-75 ; HPLC Purity: NLT 97 ; LOD: NMT 2 % w/w; H NMR (300 MHz,
DMSO-d ) : 12.66 (s, 1H), 8.31-8.31 (d, J = 12.6 Hz, 2H), 7.28-7.23 (t, J = 8.1 Hz, 2H),
7.14-7.13 (d, J = 1.2 Hz, 1H), 6.95-6.87 (m, 2H), 6.474-6.471 (d, J = 0.9 Hz, 1H), 6.38-6.36
(d, J = 7.2, 1H), 4.08-3.94 (m, 2H), 3.97-3.91 (m, 4H), 3.82-3.80 (m, 2H), 3.67-3.64 (d, J =
10.5 Hz, 2H), 3.58-3.43 (m, 2H), 3.07 (m, 2H), 2.45-2.30 (m, 3H), 2.02-1.98 (d, J = 9.9 Hz,
2H), 1.37-1.26 (m, 2H), 1.21-1.17 (t, J = 6.9 Hz, 3H); MS: m/z 620.2 (M+H)+.
Example 9 :
Methanesulfonic acid salt of (S)-ethyl 4-((2-carbamoyl-5-chloro-3-((2-
(phenoxymethyl)morpholino)sulfonyl)-lH-indol-7-yl)amino)piperidine-l-carboxylate
(Compound I as mesylate)
To isopropyl acetate (Commercial grade, 200 mL), compound I of example 8 (25 g, 0.0403
mol) was added and stirred at room temperature for 15-30 min. To the clear solution
obtained, 10 % activated charcoal (Norit (CA1 0155-9), 2.5 g) and 5 % SiThiol (Silicycle,
1.25 g) were added and the reaction mixture was stirred for 30-35 min. The compound was
then filtered through a celite bed (Commercial grade, 75 g), which was washed with
isopropyl acetate (Commercial grade, 25 mL). After complete removal of solvent by
distillation, the residue was dissolved in isopropyl acetate (Commercial grade, 200 mL) at
room temperature. To this methane sulfonic acid (Avra Labs, 4.27 g, 0.0444 mol) was added
over a period of 15-30 min at room temperature. The reaction mixture was heated to 75-80
°C and maintained at that temperature for 4.5-5 h to complete crystallization. The reaction
mixture was cooled and maintained at room temperature for 1 h followed by at 0-5 °C for 2
h. The solid obtained as crystals were suction-filtered, washed with chilled isopropyl acetate
(25 mL) and dried at 50-55 °C to afford the crystals of the title compound I as mesylate salt.
Yield range: 78-86 ; HPLC Purity: NLT 97 ; Chiral Purity: NLT 95 % ee; LOD: ~ 1.0 ;
H NMR (300 MHz, DMSO-d ) : 12.66 (s, 1H), 8.30-8.26 (d, J = 13.2 Hz, 2H), 7.28-7.23 (t,
J = 7.5 Hz, 2H), 7.14 (s, 1H), 6.94-6.87 (m, 3H), 6.47 (s, 1H), 4.06-4.01 (m, 2H), 3.95-3.90
(m, 4H), 3.81 (m, 1H), 3.67-3.59 (m, 2H), 3.50-3.46 (m, 2H), 3.07 (m, 2H), 2.44 (s, 3H),
2.37-2.30 (m, 2H), 2.02-1.98 (d, J = 10.5 Hz, 2H), 1.75 (m, 1H), 1.34-1.31 (m, 2H), 1.21-
1.17 (t, J = 7.2 Hz, 3H); IR (Perkin Elmer, KBr): cm 1 3400, 1698, 1687, 1338, 1155.
Characterisation of the crystalline form of Compound I :
The crystalline form was characterised using following procedures:
1. X-Ray powder diffraction XRPD pattern analysis:
X-Ray diffractograms of the crystalline form of Compound I was recorded on a X-Ray
difractometer, Bruker, D8 Advance, LynxEye detector, X-Ray tube with Cu target anode,
slit 0.3, antiscatter slit 1°, Power 40 kV, 40 mA, Scanning speed 0.25 sec/step, 0.02 deg,
Wave length: 1.5406 A
The X-Ray diffractograms were recorded for the crystalline form of Compound I, obtained
using solvent of crystallisation selected from THF, 2-methyl tetrahydrofuran, a mixture of 2-
methyl tetrahydrofuran and toluene, a mixture of 2-methyl tetrahydrofuran and heptane,
methylethylketone, ethyl acetate or isopropyl acetate and were found to be identical,
indicating that an identical crystalline form of Compound I was obtained with each of the
above-mentioned solvents.
Table 1 indicates the main peaks of % intensity greater than 10, at an angle of refraction 2-
theta of 9.22, 11.92, 13.58, 15.74, 18.37, 18.65, 18.95, 19.37, 19.59, 20.33, 20.92, 22.48,
22.79, 23.97, 24.19, 24.59, 28.48 ± 0.2°, obtained for the crystalline form of Compound I,
obtained using isopropyl acetate as crystallization solvent.
Table 1:
Angle value Intensity
2-T a , Angstrom %
7.662 1 .52853 7.4
8.93 .895 3.8
9.22 9.534 18 45
f . 23 7.74044 5. 1
.916 7.421 3 3.5
12.888 6,85822 5.6
13. 151 6.72658 3.5
13.577 6.5169 1 .
14.322 6.1793 5.2
15.378 5.75792 .5
15.745 5.62408 20.7
19.275 5.44207 12.5
17.478 5.07012 5.4
17.767 4.98804 4.8
18.369 4,82593 43.
18.649 4.75422 16.8
13.953 4.6787 1 .9
19.374 4.57784 12.9
19.589 4 .52 7 25. 1
19.855 4.46808 5.7
20.331 4.36445 34. 1
20.918 4.24339 100
22.484 3.951 2 1 46.
22.794 3.89 18.8
23.97 f 3.70928 30.1
24.189 3.67S45 1 .5
24.587 3.61 779 .7
25. 129 3.54094 9.4
25.826 3.44703 5.4
26.027 3.42076 5.3
26.459 3.3659 7.5
28.718 3.33408 9.4
27.222 3.27326 8.5
27.433 3.24856 8.4
27.827 3.20351 8
28. 85 3.1 6245 5.8
28.475 3.1 204 12. !
28.724 3.1 0542 5.7
29.041 3.07228 3.9
29.352 3.04039 5.8
30.285 2.94887 5.1
30.831 2.89788 8.7
3 1.236 2.861 8 7.9
3 1.585 2.83041 5.1
3 1.959 2.7S81 5.1
32.6! 2.7437 3.6
32.994 2.71 264 4.5
33.839 2.64S82 4.6
34.056 2.63048 5.S
34.517 2.59639 7.S
35.351 2.537 4
35.7S8 2.50638 3.8
36. 128 2 .48 3.4
38.455 2.4627 4.7
38.389 2.43469 3.
38.094 2.36042 3.6
38.42 2.341 8 4.7
38.759 2.321 1 8.5
2. Differential Scanning Calorimetry (DSC):
Melting point was measured by differential scanning calorimetry (DSC) using a
Parkin Elmer, Diamond DSC, the temperature gradient program is 50 °C to 260 °C at a ramp
of 20 °C per min and sample mass of 1-2 mg.
The melting points were recorded for the crystalline form of Compound I, obtained
using solvent of crystallisation selected from THF, 2-methyl tetrahydrofuran, a mixture of 2-
methyl tetrahydrofuran and toluene, a mixture of 2-methyl tetrahydrofuran and heptane,
methylethylketone, ethyl acetate or isopropyl acetate, and the melting points recorded were
found to be identical, indicating that an identical crystalline form of Compound I was
obtained with each of the above-mentioned solvents.
The melting temperature onset of the crystalline form of Compound I obtained using
isopropyl acetate as solvent of crystallisation was determined to be 224.08 ± 0.5 °C at 20
deg/min under nitrogen. The peak melting temperature was determined to be 226.83+ 0.5 °C.
Example 10:
(S)-2-Phenoxymethyloxirane (Compound A)
To a solution of NaOH (Merck, 159.99 g) in water (3.2 L), phenol (Spectrochem, 400 g,
4.211 mol) was added at room temperature and stirred for 10-15 min. To this reaction
mixture was added R-epichlorohydrin (Frapps, 467.3 g, 5.053 mol) and tetrabutylammonium
hydrogensulphate (Sisco, 8 g, 0.5797 mol) over a period of 10 -15 minutes along with
vigorous stirring by maintaining the temperature at room temperature (25-30 °C). The
mixture was stirred for 3-3.5 h and on completion of the reaction, it was extracted with 1:1
ethyl acetate: petroleum ether (Commercial grade, 800 mL). The combined organic layer was
dried over anhydrous sodium sulfate (Commercial grade, 200 g) and concentrated completely
below 40 °C to afford the title compound A.
Yield range: 82-100 ; G.C Purity: 70 ; H NMR (300 MHz, CDC13) : 7.28-7.34 (m, 2H),
6.93-7.03 (m, 3H), 4.255 (m, 1H), 4.00 (m, 1H), 3.390 (t, 1H), 2.95 (m, 1H), 2.785 (m, 1H);
MS: m/z 151 (M+H)+.
Example 11:
N-Benzyl ethanolamine hydrogen sulphate (Compound C)
A solution of N-benzylethanolamine (A.K. Scientific, 1000 g, 6.6225 mol) in
dichloromethane (Commercial grade, 6 L) was cooled to -5 to 0 °C. Chlorosulphonic acid
(Spectrochem, 771.5 g, 440.5 mL, 6.6255 mol) was added dropwise to the solution while
maintaining the reaction temperature below 10 °C. After addition was complete, the reaction
mixture was then stirred at room temperature for 15-16 h. On completion of the reaction,
ethanol (Commercial grade, 3 L) was added along with dichlorome thane (3 L) and the
reaction mixture was stirred at room temperature for 3-3.5 h. The solid obtained was filtered,
washed with 1:1 EtOH: dichloromethane (Commercial grade, 2 L) and dried at 45-50 °C to
afford the title compound C.
Yield range: 65-78 ; HPLC purity: NLT 97 ; LOD: NMT 2 % w/w; H NMR (300 MHz,
D20): 7.388(s, 5H), 4.214 (m, 4H), 3.32 (t, 2H); MS: m/z 232 (M+H)+.
Example 12:
(S)-l-Benzyl-2-phenoxymethylmorpholine (Compound D)
To a solution of NaOH (Merck, 933 g, 23, .33 mol) in water (1.75 L) which was cooled to 10-
15 °C, was added Compound C of example 11 (592.8 g, 2.566 mol) in portions while
maintaining the reaction temperature at room temperature. A solution of Compound A of
example 10 (350 g, 2.333 mol) in toluene (Commercial grade, 3.5 L) was added to the
reaction mixture over 10-15 min, tetrabutylammonium hydrogensulphate (Sisco, 17.5 g) was
added to the reaction mixture and the mixture was stirred at 45-50 °C for 15-16 h. On
completion of the reaction, water (2.45 L) was added and the organic layer was separated.
The organic layer was extracted with 10 % aqueous HCl (3.5 L) twice. The combined
aqueous layers were basified to pH of 9-10 with 10 % NaOH solution (Merck, 3 L) and
extracted with EtOAc (Commercial grade, 5.25 + 3.5 L). The combined organic layers were
washed with water (3.5 L), 10 % brine (3.5 L) and dried over anhydrous Na2S0 4 (100 g). The
solvent was removed completely by distillation below 50 °C to afford the title compound D
as an oil.
Yield range: 68-90 ; GC Purity: NLT 85 ; H NMR (300 MHz, CDC13) : 7.33-7.23 (m,
7H), 6.96-6.93 (d, J = 7.5 Hz, 1H), 6.90-6.88 (d, J = 8.1 Hz , 2H), 4.05-3.90 (m, 4H), 3.77-
3.66 (t, J = 11.1 Hz, 1H), 3.55 (s, 2H), 3.49-2.86 (d, J = 11.1 Hz, 1H), 2.70-2.66 (d, J = 11.1
Hz, 1H), 2.274-2.187 (t, J = 11.4 Hz, 1H), 2.131-2.063 (t, J = 9.6 Hz, 1H), MS: m/z 284
(M+H)+.
Example 13:
(S)- 2-(Phenoxymethyl)morpholine (Compound E)
To a solution of compound D of example 12 (560 g, 1.978 mol) in methanol (Commercial
grade, 5.6 L) in an atmosphere of CO2 (obtained by adding small pieces of dry ice to the
mixture) was added 10 % Pd/C (Johnson M, 112 g). To the above reaction mixture was
added ammonium formate (Avra, 560 g) at room temperature and the reaction mixture was
heated at 60-65 °C for 1-2 h. On completion of the reaction, the reaction mixture is cooled to
room temperature and was filtered through a celite bed, which was washed with MeOH (560
mL). The filtrate was concentrated completely below 45 °C. The residue was dissolved in
EtOAc (Commercial grade, 8.4 L) and the organic layer was washed with 10 % brine (5.6 L)
and dried over anhydrous Na2S0 4. The organic layer was concentrated completely below 45
°C and the residue degassed below 45 °C to afford the title compound E.
Yield range: 74-90 ; GC Purity: NLT 85 ; Chiral HPLC: NLT 90 % ee; H NMR (300
MHz, CDCI3) 7.31-7.26 (m, 2H), 6.99-6.91 (m, 3H), 4.11-4.09 (m, 2H), d 4.047-3.990 (m,
2H), 3.977-3.656 (t, 1H), 3.091-2.740 (m, 4H). MS: m/z 194 (M+H)+.
Example 14:
In vitro IGF-1R Kinase Assay:
The in vitro kinase assays using IGF-1R kinase GST fusion proteins were conducted
using a homogeneous time-resolved fluorescence (HTRF) format. Kinase reactions were
carried out in a 384-well plate format in a final volume of 20 . The standard enzyme
reaction buffer consisted of 50mM Tris HCL (pH: 7.4), 1 mM EGTA, 10 mM MgCl2, 2 mM
DTT, 0.01 % Tween-20, IGF-1R/ IR kinase enzyme, poly GT peptide substrate (Perkin
Elmer [Ulight Glu-Tyr (4:l)]n) and ATP [concentration equivalent to KmJ. Compound I
(methane sulfonate salt of (S)-ethyl 4-(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl)
morpholinosulfonyl) -lH-indol-7-ylamino) piperidine-l-carboxylate) in its amorphous and
crystalline forms respectively in DMSO (<1%), were added to give a final inhibitor
concentration ranging from 40 to 40 pM. Briefly, 2.5 enzyme and 2.5 inhibitor
was pre-incubated for 10 minutes at 23 °C followed by the addition of 2.5 of poly GT
substrate (final concentration of 50 nM). Reaction was initiated with the addition of 2.5 
of ATP (final concentration of 20 for IGF-1R assay). After 1 hour incubation at 23 °C,
the kinase reaction was stopped with the addition of 5 EDTA (final concentration of 10
mM in 20 ) . Europium cryptate - labeled antiphosphotyrosine antibody PY20 (5 ) was
added (final concentration of 2 nM) and the mixture was allowed to equilibrate for 1 hour at
23 °C followed by reading the plate in an Envision plate reader. The intensity of light
emission at 665 nm was directly proportional to the level of substrate phosphorylation. The
IC50 values for Compound I in both, the crystalline form and the amorphous form were
determined by a four-parameter sigmoidal curve fit (Sigma plot or Graph pad) as represented
in Table 2.
IGFRK enzyme used for the assay was intracellular kinase domain of human IGF-1R
and expressed as GST fusion proteins using the baculovirus expression system and purified
using glutathione - Sepharose column. IGFRK was used at a final concentration of 0.25 nM.
Table 2 :
Example 15:
IGF-1R autophosphorylation assay:
Cells were grown and maintained in a medium containing 10 % FBS. Cells grown as
subconfluent monolayer, were subjected to serum starvation by replacing the respective
culture medium with plain medium (containing no serum) and incubated for about 16 h at 37
°C in 5 % CO2 incubator. Serum starved cells were treated with compound I at different
concentrations for 1 h at 37 °C in 5 % CO2 incubator and stimulated with IGF-1 (50 ng/mL)
for the last 5 minutes of treatment with Compound I . After stimulation cells were washed
twice with cold l x PBS, pH 7.2 and cell lysates were prepared using CelLytic M cell lysis
reagent (Sigma) containing protease and phosphatase inhibitors. Estimation of the total
protein content in each cell lysate was carried out using Bradford reagent. Equal amount of
protein from each lysate was subjected to Sodium Dodecyl Sulphate-Polyacrylamide Gel
Electrophoresis (SDS-PAGE) followed by Western blotting protocol using specific antibody
to pIGF-lR, IGF-IRp, pAkt, Akt, Pp70S6, p70S6, Pp44/42, p44/42 and loading control, -
actin. Imaging was done with BIO-RAD Image-Lab Software. The band density was then
estimated using the software Image J . From the band densities of the various proteins at
different concentrations of Compound I, the IC50 concentrations for various proteins were
calculated, and are enumerated in Table 3.
Table 3 :
Example 16:
Anti-proliferative assay
Method for measuring cell proliferation:
The cancer cell lines were seeded in triplicate (at density, from 3000-5000 cells/well
depending on cell type) with 10 % FCS in 180 of culture medium in tissue culture grade
96 well plates and allowed to recover for 24 h in humidified 5 % CO2 incubator at 37 ± 1 °C.
After 24 h, media was replaced from the plate completely and 180 of fresh media
containing 100 ng/mL IGF-1 without FCS was added followed with addition of 20 of
10X crystalline form of Compound I (dissolved first in DMSO and then in cell medium, final
DMSO concentration did not exceed 0.5 ) in wells. Compound I in crystalline form was
used at concentration range of 0.1, 1, 3 and 10 and the plates were incubated for 72 h in
humidified 5 % CO2 incubator at 37 ± 1 °C. Control wells were treated with vehicle
(DMSO). At the end of the incubation periods, the plates were assayed by the CellTiter-Glo®
Luminescent Cell Viability assay protocol. Percent cytoxicity was calculated at the various
drug concentrations. Graph for cytotoxicity vs. concentration of Compound I was plotted,
and the IC50 values were determined.
CellTiter-Glo® Luminescent Cell Viability Assay
The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to
determine the number of viable cells in culture based on quantitation of the ATP present,
which signals the presence of metabolically active cells. The amount of ATP is directly
proportional to the number of cells present in culture
Protocol
1. After 72 h incubation, the plate is equilibrated and its contents are maintained at room
temperature for approximately 30 minutes.
2. A volume of CellTiter-Glo® Reagent was added in a volume equal to the volume of
cell culture medium present in each well (e.g., 100 of reagent to 100 of
medium containing cells for a 96-well plate).
3. The contents are mixed for 2 minutes on an orbital shaker to induce cell lysis. The
plate is allowed to incubate at room temperature for 10 minutes to stabilize the
luminescent signal. The luminescence is recorded using the POLARstar optima plate
reader at excitation 536 nm and emission 590 nm.
Values are IC50 in nM for different cell lines are provided for the crystalline form of
Compound I in Table 4.
Table 4 :
It should be noted that, as used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural referents unless the content clearly dictates
otherwise. Thus, for example, reference to a composition containing "a compound" includes
a mixture of two or more compounds. It should also be noted that the term "or" is generally
employed in its sense including "and/or" unless the content clearly dictates otherwise.
All publications and patent applications in this specification are indicative of the level
of ordinary skill in the art to which this invention pertains.
The invention has been described with reference to various specific and preferred
aspects and techniques. However, it should be understood that many variations and
modifications may be made while remaining within the spirit and scope of the invention.
CLAIMS
We claim:
1. A stable crystalline form of (S)-ethyl 4-(2-carbamoyl-5-chloro-3-(2-(phenoxy
methyl)morpholinosulfonyl)- 1H-indol-7-ylamino) piperidine- 1-carboxylate methane
sulfonate (Compound I).
2. The crystalline form of Compound I according to claim 1, wherein said crystalline
form is characterized by X-Ray diffraction peaks at an angle of refraction 2-theta of 9.22,
11.92, 13.58, 15.74, 18.37, 18.65, 18.95, 19.37, 19.59, 20.33, 20.92, 22.48, 22.79, 23.97,
24.19, 24.59, 28.48 + 0.2°.
3. The crystalline form of Compound I according to claim 1 or claim 2, wherein said
crystalline form is characterized by a melting temperature onset determined to be 224.08 ±
0.5 °C at 20 deg/min under nitrogen, with a peak melting temperature of 226.83 ± 0.5 °C.
4. A process for the preparation of the crystalline form of Compound I as defined in
preceding claims 1 to 3, wherein said process comprises the steps of:
Step 1) purifying the free base of Compound I by treating a solution of said free base in a
solvent selected from isopropyl acetate, THF, 2-methyl tetrahydrofuran, toluene, heptane,
methylethylketone, ethyl acetate, isopropyl acetate or combinations thereof; with activated
charcoal and Si-thiol (silicycle), filtering the resulting mixture through a celite bed,
repeatedly washing the celite bed with the same solvent and evaporation of the filtrate; and
Step 2) reacting the purified free base of compound I with methanesulfonic acid in said
solvent at a temperature range of 70-80 °C for about 4-5 h to initiate crystallization of
Compound I as methane sulfonate salt, followed by cooling the resulting reaction mixture to
room temperature and further to a temperature range of 0-5 °C to afford a crystalline mass,
which is washed with the same chilled solvent to obtain the required crystalline form of
Compound I .
5. A process for the preparation of the crystalline form of Compound I as defined in
preceding claims 1 to 3, comprising crystallizing an amorphous form of the compound I, (S)-
ethyl 4-(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl) mo holinosulfonyl)-lH-indol-7-
ylamino)piperidine-l -carboxylate methane sulfonate) with a solvent selected from isopropyl
acetate, tetrahydrofuran (THF), 2-methyl tetrahydrofuran, toluene, heptane,
methylethylketone, ethyl acetate, isopropyl acetate or combinations thereof; wherein the
amorphous form of compound I is obtained by reacting the free base of Compound I with
methanesulphonic acid in tetrahydrofuran (THF) as solvent at room temperature (25-30 °C)
for 30 min to 2 h.
6. The process according to claim 4, wherein said free base of Compound I is prepared
by the steps comprising of:
step a : diazotising compound 1;
1
by reacting it with sodium nitrite (NaNC^) and hydrochloric acid (HC1) at a temperature
range of -10 to 5 °C, followed by a dropwise addition of the diazotized mixture to an alkaline
solution of the reagent, ethyl 2-methyl-3-oxobutanoate in a base selected from sodium
ethoxide (NaOEt), potassium hydroxide (KOH) or sodium hydroxide (NaOH) in a solvent
selected from methanol or ethanol at a temperature range of -20 °C to -15 °C to obtain
compound 2;
2
step b : cyclising the compound 2 by reaction with a Lewis acid selected from zinc chloride
(ZnCl2), aluminium chloride (AICI 3) , boron trifluoride (BF3), phosphorus pentoxide (P2O5) or
polyphosphoric acid at a temperatur - 120 °C for 5-12 h to obtain compound 3;
3
step c : sulphonating the compound 3 by reaction with sulphuric acid and acetic anhydride at
a temperature range of 0-30 °C for 10-20 h to obtain compound 4;
4
step d : reacting the compound 4 with oxalyl chloride or thionyl chloride in the presence of
an organic base selected from triethylamine or pyridine in a solvent selected from N,Ndimethylformamide
(DMF), methylene dichloride or a mixture thereof at a temperature range
of 25 - 50 °C for 2-4 h to obtain the corresponding sulphonyl chloride 4A;
4A
which is optionally isolated; and is then reacted with compound E:
in the presence of an organic base selected from pyridine or triethylamine in a solvent
selected from dichloromethane or chloroform at room temperature (25-30 °C) for 1-4 h to
obtain compound 5;
5
step e : reducing the compound 5 by reaction with a reducing agent selected from iron and
ammonium chloride (Fe and NH4C1), zinc and hydrochloric acid (Zn and HC1) or stannous
chloride (SnC^), for 2-8 h in a solvent selected from methanol, ethanol, THF, water or a
mixture thereof, to obtain compound 6, which is purified using alcohol;
6
step f : reacting the compound 6 with isopropyl alcohol and ammonia at a temperature range
of 80 - 120 °C at a pressure of 0.5 - 10 kg/cm2 for 10-18 h in an autoclave or in a microwave
for 10-15 min to obtain the compoun is purified using alcohol; and
step g : reacting the compound 7 with the compound F:
in the presence of trifluoroacetic acid in sodium triacetoxy borohydride as a base in a solvent
selected from dichloromethane or ethyl acetate at room temperature (25-30 °C) for 0.5 - 2 h
to obtain compound I as a free base, which is purified;
Compound I (free base)
7. The process according to claim 6, wherein the preparation of compound E used in step
d comprises the steps:
step a : reacting phenol with (R)-2-(chloromethyl)oxirane in the presence of a base selected
from aqueous sodium hydroxide (NaOH) or aqueous potassium hydroxide (KOH) and
tetrabutylammonium hydrogen sulphate as a phase transfer catalyst, at a temperature range of
80-120 °C for 1-4 h to obtain Co
step b : reacting Compound B;
OH
HN
Bn
B
with chlorosulfonic acid in a solvent selected from chloroform, carbon tetrachloride, or
dichlorome thane, at 0-10 °C during addition of the acid over a period of 15-30 min, followed
by at room temperature (25-30 °C) for 10-16 h to obtain Compound C;
C
step c : reacting the Compound A obtained in step a with the Compound C obtained in step b
in the presence of base selected from aqueous sodium hydroxide (NaOH) or aqueous
potassium hydroxide (KOH) in a solvent selected from toluene, dioxane or tetrahydrofuran
(THF) in the presence of tetrabutylammoniun hydrogen sulfate as a phase transfer catalyst, at
a temperature range of 30-50 °C for 10-16 h to obtain Compound D; and
step d : carrying out debenzylation of Compound D by refluxing said Compound D with
ammonium formate and 10 % palladium over carbon (Pd/C) in an atmosphere of carbon
dioxide in a solvent selected from ethanol or methanol at 50-70 °C for 1-3 h to obtain
Compound E.
8. The process according to claim 4, wherein the solvent used in steps 1 and 2 is
selected from tetrahydrofuran (THF), 2-methyl tetrahydrofuran, a mixture of 2-methyl
tetrahydrofuran and toluene, a mixture of 2-methyl tetrahydrofuran and heptane,
methylethylketone, ethyl acetate or isopropyl acetate.
9. The process according to any one of the claims 4 or 8, wherein the solvent is
isopropyl acetate.
10. The process according to claim 6, wherein the base used in step a is sodium ethoxide
(NaOEt).
11. The process according to claim 6, wherein in step b the cyclisation of the compound 2
is carried out using polyphosphoric acid in o-phosphoric acid as the Lewis acid at a
temperature range of 80 - 85 °C for 2-3 h.
12. The process according to claim 6, wherein compound 4A of step d is isolated prior to
reaction with the compound E.
13. The process according to claim 6, wherein the alcohol used for purification of the
compound 5 in step d is selected from methanol, ethanol, n-propanol, isopropanol or nbutanol.
14. The process according to claim 6 or claim 13, wherein the alcohol used for
purification of compound 5 in step d is methanol.
15. The process according to claim 6, wherein in step e the reduction of compound 5 is
carried out using iron and ammonium chloride (Fe and NH4C1) as the reducing agent in a
mixture of tetrahydrofuran (THF), water and ethanol as solvent at a temperature range of 70-
80 °C for 2-4 h.
16. The process according to claim 15, wherein the reduction using iron and ammonium
chloride (Fe and NH4C1) in step e affords compound 6 along with residual iron and iron
oxides, which are removed by using ethylenediaminetetraaceticacid (EDTA) and chloroform.
17. The process according to claim 15, wherein the reduction using iron and ammonium
chloride (Fe and NH4C1) in step e provides compound 6 along with residual iron and iron
oxides, which are removed by filtration.
18. The process according to claim 6, wherein the alcohol used for purification of the
compound 6 in step e is selected from methanol, ethanol, n-propanol, isopropanol or nbutanol.
19. The process according to claim 6 or claim 18, wherein the alcohol used for
purification of the compound 6 in step e of claim 6 is isopropanol.
20. The process according to claim 6, wherein the alcohol used for purification of the
compound 7 in step f is selected from methanol, ethanol, n-propanol, isopropanol or nbutanol.
21. The process according to claim 6 or claim 20, wherein the alcohol used for
purification of the compound 7 in step f of claim 6 is isopropanol.
22. A pharmaceutical composition comprising a therapeutically effective amount of
crystalline form of Compound I according to claim 1 and a pharmaceutically acceptable
excipient or a carrier.
23. A pharmaceutical composition comprising a therapeutically effective amount of
crystalline form of Compound I according to claim 1 and a pharmaceutically acceptable
carrier and optionally, other therapeutic agents.
24. The crystalline form of Compound I according to any one of the claims 1 to 3 for
use in the treatment of diseases or disorders mediated by Insulin-Like-Growth Factor I
Receptors or Insulin Receptors comprising administering to a subject in need thereof, a
therapeutically effective amount of the crystalline form of Compound I .
25. The crystalline form of Compound I for use according to claim 24, wherein the
Insulin-Like-Growth Factor I Receptor and Insulin Receptor mediated disease or disorder is
cancer.
26. The crystalline form of Compound I for use according to claim 25, wherein the
cancer is selected from astrocytoma, basal or squamous cell carcinoma, brain cancer,
gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer,
chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer,
endometrial carcinoma, erythroleukemia, Ewing' s sarcoma, gastrointestinal cancer, head and
neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma,
non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer,
renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer,
testicular cancer or osteosarcoma.
27. The crystalline form of Compound I for use according to claim 26, wherein the
cancer is selected from breast cancer, colon carcinoma, colorectal cancer, Ewing' s sarcoma
or rhabdosarcoma.
28. Use of the crystalline form of Compound I of any one of the claims 1 to 3 for the
manufacture of a medicament for the treatment of diseases or disorders mediated by Insulin-
Like-Growth Factor I Receptor and Insulin Receptor.
29. The use according to claim 28, wherein the Insulin-Like-Growth Factor I Receptor
and Insulin Receptor mediated disease or disorder is cancer.
30. The use according to claim 29, wherein the cancer is selected from astrocytoma,
basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer,
colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer,
choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing' s
sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular
carcinoma, leukemia, leiomyona, melanoma, non-small cell lung cancer, neural cancer,
ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma,
small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.
31. The use according to claim 30, wherein the cancer is selected from breast cancer,
colon carcinoma, colorectal cancer, Ewing' s sarcoma or rhabdosarcoma.