TECHNICAL FIELD
The present invention belongs to the field of drug development and
particularly relates to a crystalline PD-1/PD-L1 inhibitor compound or basic salt,
5 preparation method therefor and use thereof.
BACKGROUND
The immune system plays a very important role in controlling and
eliminating diseases, such as cancer. However, tumor cells are often able to
10 develop a strategy for escaping or suppressing the monitoring of the immune
system to promote their malignant growth. One very important mechanism is to
change the expression of co-stimulatory and co-inhibitory immune checkpoint
molecules on immunocytes. Blocking the signal pathway of immune checkpoint
molecules, such as PD-1, has been shown to be an extremely promising and
15 effective therapeutic means.
Programmed cell death protein 1 (PD-1), also known as CD279, is a receptor
expressed on the surfaces of activated T cells, natural killer T cells, B cells and
macrophages. Its structure contains an extracellular domain similar to an
immunoglobulin variable region, a transmembrane domain and an intracellular
20 domain, where the intracellular domain contains two phosphorylation sites located
in an immunoreceptor tyrosine kinase-based inhibitory domain and an
immunoreceptor tyrosine kinase-based transduction domain, suggesting that PD-1
can down-regulate T cell receptor-mediated signal pathways.
PD-1 has two ligands: PD-L1 and PD-L2, and they are different in their
25 expression profile. The expression of PD-L1 will be up-regulated in macrophages
and dendritic cells after treatment with lipopolysaccharide (LPS) and granulocytemacrophage colony-stimulating factor (GM-CSF), and will also be up-regulated in
T cells and B cells after stimulation of T cell receptor and B cell receptor signal
pathways. PD-L1 is also highly expressed in almost all tumor cells, and the
30 expression will be up-regulated after stimulation of interferon (IFN) gamma. As a
3
matter of fact, the expression of PD-L1 in a variety of tumors is considered to
have prognostic relevance; in contrast, the expression of PD-L2 is relatively
concentrated, mainly on dendritic cells.
When T cells expressing PD-1 come into contact with cells expressing the
5 ligands of PD-1, those antigen-stimulated functional activities, such as cell
proliferation, cytokine release and cell lysis activity, are all inhibited. Therefore,
the interaction between PD-1 and ligands thereof can function as an intrinsic
negative feedback regulation mechanism to prevent T cell hyperactivation during
infection, immune tolerance or tumorigenesis, thus reducing the occurrence of
10 autoimmune diseases and promoting self-tolerance. Long-term antigenic
stimulation, e.g., in tumor or long-term infection, can cause T cells to express high
levels of PD-1 and make them inactive and nonfunctional in these long-term
reactions with antigens, which is also referred to as T cell exhaustion. For B cells,
there are also inhibitory effects caused by PD-1 and its ligands and corresponding
15 functional exhaustion.
Some evidence from preclinical animal studies indicates that PD-1 and
ligands thereof can down-regulate the immunoreaction. PD-1-deficient mice will
develop lupus erythematosus-like acute proliferative glomerulonephritis and
dilated cardiomyopathy. Utilizing the antibody of PD-L1 to block PD-1/PD-L1
20 interaction has been shown to be able to restore and enhance T cell activation in
many systems. The monoclonal antibody of PD-L1 can also benefit patients with
advanced cancers. In some preclinical animal tumor models, it was also shown
that blocking the signal pathway of PD-1/PD-L1 with a monoclonal antibody can
enhance immunoreaction and lead to immunoreactions to a series of histologically
25 significantly different tumors. With the long-term infection LCMV model, the
interaction between PD-1 and PD-L1 has been found to be able to inhibit the
activation and proliferation of virus-specific CD8 T cells and the acquisition of
effector cell functions. Besides being capable of enhancing the immunoreaction to
long-term antigens, blocking the pathway of PD-1/PD-L1 was also discovered to
4
be able to enhance response to vaccines, including response to a therapeutic
vaccine in long-term infection.
These results indicate that small-molecule inhibitors that target and block the
PD-1/PD-L1 interaction can be an effective therapeutic means for blocking the
5 PD-1/PD-L1-mediated inhibitory signal pathway to enhance or restore the
function of T cells, and will have very good efficacy in immunotherapy for a
variety of cancers and other immunity-related diseases.
During long-term research, Abbisko Therapeutics Co., Ltd. found a smallmolecule compound having the effect of inhibiting PD-1/PD-L1
10 (WO2019149183A1, international publication date: August 8, 2019), a
representative compound of which is shown below:
.
The name of the compound is (2S,2'S)-1,1'-(((((2,2'-dimethyl-[1,1'-biphenyl]-
3,3'-diyl)bis(azanediyl))bis(carbonyl))bis(4-cyclopropylpyridine-6,3-
15 diyl))bis(methylene))bis(piperidine-2-carboxylic acid) (the compound of formula
(I)). The compound has a strong inhibitory effect on the protein interaction of PD1/PD-L1, and the inhibitory effect can enhance or restore the activation of T cells
at the cellular level. The compound can satisfy the requirements of target and
immune therapy for tumors, immunity-related diseases and disorders,
20 communicable diseases, infectious diseases or metabolic diseases, etc., at this
stage in China and other countries.
During later pharmaceutical research, it was found that the compound of
formula (I) reported in WO2019149183A1 is a trifluoroacetate freeze-dried
amorphous compound that has poor solid-state properties, is very hygroscopic,
25 has poor solubility and is not suitable for clinical formulation development.
Therefore, in order to satisfy the needs of clinical research and the launch of drug
5
formulations, there is an urgent need to develop an aggregate form suitable for
drug development to overcome the defects in the prior art.
SUMMARY
5 To solve the problems in the prior art, the inventors intensively studied
different aggregate forms of the compound of formula (I) and developed several
crystalline free forms of the compound or basic salts thereof through a large
number of salt form and crystalline form screening experiments. The crystalline
free forms of the compound greatly improve physicochemical properties, such as
10 hygroscopicity, solubility and physicochemical stability, of the compound of
formula (I), and the crystalline basic salts of the compound greatly improve
physicochemical properties such as solubility and physicochemical stability. The
crystalline free forms of the compound or the basic salts thereof have improved
bioavailability, meet the requirements for industrial production and can satisfy the
15 need to develop clinical pharmaceutical formulations. The crystalline forms of the
compound or the basic salts thereof have very important clinical application value
and are expected to be developed into new-generation PD-1/PD-L1 smallmolecule inhibitors soon.
A first aspect of the present invention provides a crystalline basic salt of a
20 compound of formula (I):
,
wherein the basic salt is selected from the group consisting of lithium salt,
sodium salt, potassium salt, magnesium salt, calcium salt and ammonium salt.
As a preferred embodiment, the crystalline basic salt of the compound of
25 formula (I) is a sodium salt.
6
As a further preferred embodiment, each molecule of the crystalline sodium
salt of the compound of formula (I) comprises the compound of formula (I) in free
form and the sodium atom in a molar ratio of 1:1 or 1:2, wherein the sodium salt
is referred to as a monosodium salt when the molar ratio is 1:1, and the sodium
5 salt is referred to as a disodium salt when the molar ratio is 1:2.
As a more further preferred embodiment, each molecule of the crystalline
sodium salt of the compound of formula (I) comprises the compound of formula
(I) in free form and the sodium atom in a molar ratio of 1:2.
As a preferred embodiment, the crystalline sodium salt of the compound of
10 formula (I) is an anhydrate or a hydrate.
As a further preferred embodiment, each molecule of the crystalline sodium
salt hydrate of the compound of formula (I) comprises the compound of formula
(I) in free form and the water molecule in a molar ratio of 1.0:(0.1-10.0), wherein
the hydrate is referred to as a hemihydrate when the molar ratio is 1.0:0.5, the
15 hydrate is referred to as a monohydrate when the molar ratio is 1.0:1.0, and the
hydrate is referred to as a dihydrate when the molar ratio is 1.0:2.0; and following
this pattern, subsequent ratios are termed trihydrate, tetrahydrate, pentahydrate,
and so on.
As a more further preferred embodiment, each molecule of the crystalline
20 sodium salt hydrate of the compound of formula (I) comprises the compound of
formula (I) in free form and the water molecule in a molar ratio of 1.0:(1.0-5.0).
As a more further preferred embodiment, each molecule of the crystalline
sodium salt hydrate of the compound of formula (I) comprises the compound of
formula (I) in free form and the water molecule in a molar ratio of 1.0:4.0 or
25 1.0:2.0.
As a more further preferred embodiment, the crystalline basic salt of the
compound of formula (I) is a crystalline form A of disodium salt hydrate having
an X-ray powder diffraction (XRPD) pattern comprising five or more peaks at
angles of diffraction (2θ) of 8.60±0.2°, 9.97±0.2°, 12.92±0.2°, 15.03±0.2°,
7
17.62±0.2°, 17.93±0.2°, 20.04±0.2°, 22.07±0.2°, 23.18±0.2°, 23.60±0.2° and
27.06±0.2°.
As the most preferred embodiment, the X-ray powder diffraction (XRPD)
pattern of the crystalline form A of disodium salt hydrate comprises peaks
5 substantially identical (±0.2°) to those at angles of diffraction (2θ) shown in FIG.
1, and X-ray powder diffraction data thereof are shown in Table 1:
Table 1
2θ ( ° ) Intensity% 2θ ( ° ) Intensity%
8.60 100.0% 24.35 4.0%
9.38 1.3% 25.77 2.6%
9.97 6.9% 26.08 2.8%
11.76 0.7% 27.06 6.8%
12.92 11.1% 27.40 2.1%
15.03 7.5% 28.20 1.6%
15.49 1.2% 28.46 2.8%
16.01 2.7% 28.80 1.3%
17.62 17.6% 30.32 3.0%
17.93 15.5% 30.81 1.5%
18.78 1.7% 32.78 0.6%
19.83 5.0% 33.85 1.2%
20.04 11.9% 35.80 1.3%
20.46 5.9% 37.68 0.4%
21.24 1.3% 38.21 1.1%
22.07 8.9% 38.82 0.3%
22.64 0.4% 39.88 1.3%
23.18 7.2% 40.78 0.5%
23.60 7.0% 43.21 0.3%
24.02 1.0% 44.24 0.5%
8
The crystalline form is designated crystalline form A of disodium salt
hydrate.
As the most preferred embodiment, the crystalline basic salt of the compound
of formula (I) is a crystalline form A of disodium salt hydrate, wherein a unit cell
5 of the crystalline form A of disodium salt hydrate is a monoclinic crystal system
with the following unit cell parameters: a = 20.00(5) Å, b = 5.798(16) Å, c =
20.44(11) Å, α = 90°, β = 94.8(2)°, γ = 90° and a unit cell volume V of 2362(15)
Å3. Z' is 0.5, and an asymmetric unit consists of 0.5 API anions, 1 sodium ion and
2 water molecules. The structure of a unit cell of a single crystal thereof is shown
10 in FIG. 7.
As the most preferred embodiment, the crystalline basic salt of the compound
of formula (I) is a crystalline form A of disodium salt hydrate having DSC/TGA
graphs that are substantially those shown in FIG. 8.
As a more further preferred embodiment, the crystalline basic salt of the
15 compound of formula (I) is a crystalline form B of disodium salt anhydrate having
an X-ray powder diffraction (XRPD) pattern comprising five or more peaks at
angles of diffraction (2θ) of 6.60±0.2°, 7.46±0.2°, 12.43±0.2°, 13.32±0.2°,
13.63±0.2°, 15.54±0.2°, 17.32±0.2°, 18.68±0.2° and 21.73±0.2°.
As the most preferred embodiment, the X-ray powder diffraction (XRPD)
20 pattern of the crystalline form B of disodium salt anhydrate comprises peaks
substantially identical (±0.2°) to those at angles of diffraction (2θ) shown in FIG.
2, and X-ray powder diffraction data thereof are shown in Table 2:
Table 2
2θ ( ° ) Intensity% 2θ ( ° ) Intensity%
6.60 100.0% 18.41 6.8%
7.46 89.9% 18.68 8.4%
11.16 4.1% 20.51 3.1%
12.43 23.3% 21.73 13.7%
13.32 58.0% 23.57 3.5%
9
13.63 10.3% 27.26 5.6%
14.99 4.8% 28.64 2.1%
15.54 13.3% 31.83° 2.6%
17.32 66.6% 37.82 1.9%
The crystalline form is designated crystalline form B of disodium salt
anhydrate and has a melting point of 316.4 °C.
As the most preferred embodiment, the crystalline basic salt of the compound
of formula (I) is a crystalline form B of disodium salt anhydrate having DSC/TGA
5 graphs that are substantially those shown in FIG. 9.
A second aspect of the present invention provides a preparation method for
the aforementioned crystalline basic salt of the compound of formula (I),
comprising the following steps:
10 1) dissolving or dispersing the compound of formula (I) in free form in water or
a suitable organic solvent, and adding a basic solution to the above system;
or adding the compound of formula (I) in free form to a basic solution; and
2) collecting a solid product precipitated during the above salt-forming reaction,
or creating a degree of supersaturation in the salt-forming system to obtain a
15 crystalline product;
wherein the basic salt is selected from the group consisting of lithium salt,
sodium salt, potassium salt, magnesium salt, calcium salt and ammonium salt.
As a preferred embodiment, the basic solution is selected from the group
consisting of solutions of lithium hydroxide, sodium hydroxide, potassium
20 hydroxide, magnesium hydroxide, calcium hydroxide and ammonia in water or in
an organic solvent.
As a preferred embodiment, methods for creating the degree of
supersaturation in the salt-forming system in step 2) of the preparation method
include one or more of the following: adding a seed crystal, volatilizing the
25 solvent, adding an anti-solvent and cooling.
10
As a preferred embodiment, the organic solvent during salt formation in step
1) of the preparation method is selected from the group consisting of alcohols,
chloroalkanes, ketones, ethers, cyclic ethers, esters, alkanes, cycloalkanes,
benzenes, amides and sulfoxides, and mixtures thereof and aqueous solutions
5 thereof.
As a further preferred embodiment, the organic solvent during salt formation
in step 1) of the preparation method is selected from the group consisting of
methanol, ethanol, n-propanol, isopropanol, dichloromethane, heptane,
acetonitrile, acetone, methyl ethyl ketone, toluene, 1,4-dioxane, tetrahydrofuran,
10 N,N-dimethylformamide, ethyl acetate, isopropyl acetate, methyl tert-butyl ether
and 2-methoxyethyl ether, and mixtures thereof and aqueous solutions thereof.
A third aspect of the present invention provides a preparation method for the
aforementioned crystalline basic salt of the compound of formula (I), comprising
15 the following step: transforming one crystalline form of the basic salt of the
compound of formula (I) into another crystalline form of the salt by using a
crystalline form transformation method, wherein the crystalline form
transformation method includes heating or suspension crystalline form
transformation in a solvent.
20 As a preferred embodiment, the solvent is selected from the group consisting
of methanol, ethanol, n-propanol, isopropanol, dichloromethane, heptane,
acetonitrile, acetone, methyl ethyl ketone, toluene, 1,4-dioxane, tetrahydrofuran,
N,N-dimethylformamide, ethyl acetate, isopropyl acetate, methyl tert-butyl ether
and 2-methoxyethyl ether, and mixtures thereof and aqueous solutions thereof.
25
A fourth aspect of the present invention provides a crystalline form of the
compound of formula (I) in free form:
11
.
As a preferred embodiment, the crystalline form of the compound of formula
(I) in free form is crystalline form C, crystalline form D, crystalline form E or
crystalline form F.
5 As a further preferred embodiment, the crystalline form of the compound of
formula (I) in free form is a crystalline form C having an X-ray powder diffraction
(XRPD) pattern comprising five or more peaks at angles of diffraction (2θ) of
7.80±0.2°, 11.97±0.2°, 12.42±0.2°, 15.98±0.2°, 17.37±0.2°, 18.82±0.2°,
22.47±0.2°, 23.30±0.2°, 25.01±0.2° and 25.63±0.2°.
10 As a more further preferred embodiment, the X-ray powder diffraction
(XRPD) pattern of the crystalline form C comprises peaks substantially identical
(±0.2°) to those at angles of diffraction (2θ) shown in FIG. 3, and X-ray powder
diffraction data thereof are shown in Table 3:
12
Table 3
2θ ( ° ) Intensity% 2θ ( ° ) Intensity%
6.21 10.9% 24.35 3.6%
7.80 21.1% 25.01 15.6%
8.82 1.1% 25.63 25.5%
11.97 59.2% 26.48 2.6%
12.42 67.2% 27.48 1.3%
14.59 9.5% 28.41 4.7%
15.17 1.4% 28.58 2.4%
15.81 8.7% 29.38 2.9%
15.98 21.2% 30.09 2.0%
17.37 100.0% 31.47 6.3%
18.82 12.1% 33.05 1.1%
19.52 4.0% 35.36 1.9%
20.39 6.0% 38.00 1.0%
20.94 3.9% 38.63 1.8%
21.60 3.5% 39.90 1.0%
22.47 13.3% 42.72 0.8%
23.30 27.1% 43.95 0.7%
23.86 9.6%
The crystalline form is designated free-form crystalline form C and has a
melting point of 169.5 °C.
As the most preferred embodiment, the crystalline form of the compound of
5 formula (I) is a free-form crystalline form C having DSC/TGA graphs that are
substantially those shown in FIG. 10.
As a further preferred embodiment, the crystalline form of the compound of
formula (I) in free form is a crystalline form D having an X-ray powder
diffraction (XRPD) pattern comprising five or more peaks at angles of diffraction
13
(2θ) of 7.97±0.2°, 11.82±0.2°, 12.18±0.2°, 14.87±0.2°, 16.13±0.2°, 17.18±0.2°,
19.21±0.2°, 20.72±0.2° and 25.28±0.2°.
As a more further preferred embodiment, the X-ray powder diffraction
(XRPD) pattern of the crystalline form D comprises peaks substantially identical
5 (±0.2°) to those at angles of diffraction (2θ) shown in FIG. 4, and X-ray powder
diffraction data thereof are shown in Table 4:
Table 4
2θ ( ° ) Intensity% 2θ ( ° ) Intensity%
6.06 12.4% 18.81 14.5%
7.97 100.0% 19.21 19.4%
11.82 66.7% 20.72 58.4%
12.18 50.6% 21.55 11.7%
14.87 22.9% 24.48 13.2%
15.48 13.6% 25.28 37.9%
16.13 61.3% 27.88 10.9%
17.18 60.5%
The crystalline form is designated free-form crystalline form D and has a
melting point of 239.9 °C.
10 As the most preferred embodiment, the crystalline form of the compound of
formula (I) is a free-form crystalline form D having DSC/TGA graphs that are
substantially those shown in FIG. 11.
As a further preferred embodiment, the crystalline form of the compound of
formula (I) in free form is a crystalline form E having an X-ray powder diffraction
15 (XRPD) pattern comprising five or more peaks at angles of diffraction (2θ) of
8.16±0.2°, 12.05±0.2°, 12.83±0.2°, 15.19±0.2°, 16.18±0.2°, 16.59±0.2°,
17.62±0.2°, 18.41±0.2°, 22.90±0.2° and 25.65±0.2°.
As a more further preferred embodiment, the X-ray powder diffraction
(XRPD) pattern of the crystalline form E comprises peaks substantially identical
14
(±0.2°) to those at angles of diffraction (2θ) shown in FIG. 5, and X-ray powder
diffraction data thereof are shown in Table 5:
Table 5
2θ ( ° ) Intensity% 2θ ( ° ) Intensity%
6.37 9.4% 17.62 100.0%
8.16 46.1% 18.41 25.3%
12.05 74.6% 19.59 18.8%
12.83 36.5% 20.57 6.6%
14.50 11.2% 21.33 7.7%
15.19 24.7% 22.90 50.8%
16.18 32.6% 25.65 24.7%
16.59 47.8%
The crystalline form is designated free-form crystalline form E and has a
5 melting point of 226 °C.
As the most preferred embodiment, the crystalline form of the compound of
formula (I) is a free-form crystalline form E having a DSC graph that is
substantially the one shown in FIG. 12.
As a further preferred embodiment, the crystalline form of the compound of
10 formula (I) in free form is a crystalline form F having an X-ray powder diffraction
(XRPD) pattern comprising five or more peaks at angles of diffraction (2θ) of
6.92±0.2°, 9.10±0.2°, 12.42±0.2°, 13.46±0.2°, 14.38±0.2°, 15.84±0.2°,
19.04±0.2°, 22.00±0.2° and 26.23±0.2°.
As a more further preferred embodiment, the X-ray powder diffraction
15 (XRPD) pattern of the crystalline form F comprises peaks substantially identical
(±0.2°) to those at angles of diffraction (2θ) shown in FIG. 6, and X-ray powder
diffraction data thereof are shown in Table 6:
20
15
Table 6
2θ ( ° ) Intensity% 2θ ( ° ) Intensity%
5.79 10.7% 14.38 42.9%
6.92 40.6% 15.84 59.3%
9.10 100.0% 19.04 11.8%
11.39 8.1% 22.00 12.0%
12.42 24.0% 26.23 13.7%
13.46 21.1%
The crystalline form is designated free-form crystalline form F and has a
melting point of 221 °C.
5 As the most preferred embodiment, the crystalline form of the compound of
formula (I) is a free-form crystalline form F having DSC/TGA graphs that are
substantially those shown in FIG. 13.
A fifth aspect of the present invention provides a use of the aforementioned
10 crystalline form of the compound of formula (I) in free form, wherein the
crystalline form of the compound of formula (I) in free form is used as a starting
material to prepare the crystalline basic salt of the compound of formula (I)
according to any one of the above.
A sixth aspect of the present invention provides a pharmaceutical
15 composition comprising a clinically effective amount of the aforementioned
crystalline basic salt of the compound of formula (I) or the crystalline form of the
compound of formula (I) in free form and a pharmaceutically acceptable carrier.
As a preferred embodiment, the clinically effective amount means that the
pharmaceutical composition comprises 0.01-99.0% W/W of the crystalline basic
20 salt of the compound of formula (I) or the crystalline form of the compound of
formula (I) in free form relative to a total content of the pharmaceutical
composition.
16
A seventh aspect of the present invention provides a use of the
aforementioned crystalline basic salt of the compound of formula (I) or the
aforementioned crystalline form of the compound of formula (I) in free form in
5 preparation of a medicament for treating a PD-1/PD-L1 signal pathway-mediated
tumor, immunity-related disease and disorder, communicable disease, infectious
disease or metabolic disease; preferably, the tumor is cancer.
As a preferred embodiment, the infectious disease is a bacterial infectious
disease, a viral infectious disease or a fungal infectious disease.
10 As a preferred embodiment, the tumor is lymphoma, sarcoma, melanoma,
glioblastoma, synovioma, meningioma, biliary tract tumor, neuroma, seminoma,
nephroblastoma, hepatocellular papilloma, papilloma, leiomyoma, rhabdomyoma,
hemangioma, lymphangioma, osteoma, lipoma, fibroma, central nervous system
tumor, spinal axis tumor, brain stem glioma, multiple myeloma, ovarian tumor,
15 myelodysplastic syndrome or mesothelioma, anal cancer, testicular cancer,
urethral carcinoma, penile cancer, bladder cancer, ureteral cancer, uterine cancer,
ovarian cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar
cancer, Merkel cell carcinoma, embryonal carcinoma, chronic or acute leukemia,
bronchial carcinoma, esophageal cancer, nasopharyngeal carcinoma,
20 hepatocellular carcinoma, basal cell carcinoma, lung cancer, adenocarcinoma,
papillary carcinoma, rectal cancer, colon cancer, gastric cancer, head and neck
cancer, bone cancer, skin cancer, small intestine cancer, endocrine cancer, renal
pelvic carcinoma, epidermoid carcinoma, transitional cell carcinoma or
choriocarcinoma;
25 the immunity-related disease and disorder is rheumatic arthritis, renal failure,
lupus erythematosus, asthma, psoriasis, ulcerative colitis, pancreatitis, allergy,
fibrosis, anemia, fibromyalgia, Alzheimer’s disease, congestive heart failure,
stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson’s disease,
17
Crohn’s disease, ulcerative colitis, allergic contact dermatitis and eczema,
systemic sclerosis or multiple sclerosis;
the communicable disease or infectious disease is sepsis, liver infection,
hepatitis A, hepatitis B, hepatitis C, hepatitis D, herpes virus, papillomavirus or
5 influenza; and
the metabolic disease is diabetes, diabetic ketoacidosis, hyperglycemic
hyperosmolar syndrome, hypoglycemia, gout, malnutrition, vitamin A deficiency,
scurvy, vitamin D deficiency or osteoporosis.
As a preferred embodiment, the lymphoma is lymphocytic lymphoma,
10 primary central nervous system lymphoma, T cell lymphoma, diffuse large B cell
lymphoma, follicle center lymphoma, Hodgkin lymphoma, non-Hodgkin
lymphoma or primary mediastinal large B cell lymphoma;
the sarcoma is Kaposi’s sarcoma, fibrosarcoma, liposarcoma,
chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, soft tissue
15 sarcoma, angiosarcoma or lymphangiosarcoma; and
the chronic or acute leukemia is acute myeloid leukemia, chronic myeloid
leukemia, acute lymphoblastic leukemia, chronic granulocytic leukemia or
chronic lymphoblastic leukemia.
As a preferred embodiment, the tumor is small cell lung cancer, squamous
20 non-small cell lung cancer, non-squamous non-small cell lung cancer, chondroma,
colorectal cancer, gastrointestinal cancer, endometrial cancer, head and neck
squamous cell carcinoma, abdominal wall carcinoma, renal cell carcinoma or
recurrent or existing drug-resistant prostate cancer.
As a preferred embodiment, the tumor is thymic tumor, pleomorphic
25 adenoma, renal tubule adenoma, cystadenoma, pituitary adenoma, prostate cancer,
thyroid cancer, parathyroid cancer, adrenal cancer, breast cancer,
cystadenocarcinoma or pancreatic cancer.
18
An eighth aspect of the present invention provides the aforementioned
crystalline basic salt of the compound of formula (I) or the aforementioned
crystalline form of the compound of formula (I) in free form for use as a
medicament for treating a PD-1/PD-L1 signal pathway-mediated tumor,
5 immunity-related disease and disorder, communicable disease, infectious disease
or metabolic disease.
The present invention also relates to a method for treating a PD-1/PD-L1
signal pathway-mediated tumor, immunity-related disease and disorder,
10 communicable disease, infectious disease or metabolic disease, comprising
administering to a patient in need thereof the aforementioned crystalline basic salt
of the compound of formula (I) or the aforementioned crystalline form of the
compound of formula (I) in free form.
15 BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an X-ray powder diffraction pattern of the crystalline form A
of disodium salt hydrate of the compound of formula (I) of the present invention.
The abscissa represents the value of 2θ (degrees), and the ordinate represents peak
intensity.
20 FIG. 2 shows an X-ray powder diffraction pattern of the crystalline form B of
disodium salt anhydrate of the compound of formula (I) of the present invention.
The abscissa represents the value of 2θ (degrees), and the ordinate represents peak
intensity.
FIG. 3 shows an X-ray powder diffraction pattern of the free-form crystalline
25 form C of the compound of formula (I) of the present invention. The abscissa
represents the value of 2θ (degrees), and the ordinate represents peak intensity.
FIG. 4 shows an X-ray powder diffraction pattern of the free-form crystalline
form D of the compound of formula (I) of the present invention. The abscissa
represents the value of 2θ (degrees), and the ordinate represents peak intensity.
19
FIG. 5 shows an X-ray powder diffraction pattern of the free-form crystalline
form E of the compound of formula (I) of the present invention. The abscissa
represents the value of 2θ (degrees), and the ordinate represents peak intensity.
FIG. 6 shows an X-ray powder diffraction pattern of the free-form crystalline
5 form F of the compound of formula (I) of the present invention. The abscissa
represents the value of 2θ (degrees), and the ordinate represents peak intensity.
FIG. 7 shows the structure of a unit cell of a single crystal of the crystalline
form A of disodium salt hydrate of the compound of formula (I) of the present
invention.
10 FIG. 8 shows DSC/TGA graphs of the crystalline form A of disodium salt
hydrate of the compound of formula (I) of the present invention. The abscissa
represents temperature (°C), the left ordinate represents heat flow (w/g), and the
right ordinate represents weight (%).
FIG. 9 shows DSC/TGA graphs of the crystalline form B of disodium salt
15 anhydrate of the compound of formula (I) of the present invention. The abscissa
represents temperature (°C), the left ordinate represents heat flow (w/g), and the
right ordinate represents weight (%).
FIG. 10 shows DSC/TGA graphs of the free-form crystalline form C of the
compound of formula (I) of the present invention. The abscissa represents
20 temperature (°C), the left ordinate represents weight (%), and the right ordinate
represents heat flow (w/g).
FIG. 11 shows DSC/TGA graphs of the free-form crystalline form D of the
compound of formula (I) of the present invention. The abscissa represents
temperature (°C), the left ordinate represents heat flow (w/g), and the right
25 ordinate represents weight (%).
FIG. 12 shows a DSC graph of the free-form crystalline form E of the
compound of formula (I) of the present invention. The abscissa represents
temperature (°C), and the ordinate represents heat flow (w/g).
20
FIG. 13 shows DSC/TGA graphs of the free-form crystalline form F of the
compound of formula (I) of the present invention. The abscissa represents
temperature (°C), the left ordinate represents heat flow (w/g), and the right
ordinate represents weight (%).
5 FIG. 14 shows a DVS graph of the free-form crystalline form C of the
compound of formula (I) of the present invention. The abscissa represents relative
humidity (%), and the ordinate represents weight change (%).
FIG. 15 shows a DVS graph of the free-form crystalline form D of the
compound of formula (I) of the present invention. The abscissa represents relative
10 humidity (%), and the ordinate represents weight change (%).
FIG. 16 shows a DVS graph of the free-form crystalline form F of the
compound of formula (I) of the present invention. The abscissa represents relative
humidity (%), and the ordinate represents weight change (%).
FIG. 17 shows a DVS graph of the crystalline form A of disodium salt
15 hydrate of the compound of formula (I) of the present invention. The abscissa
represents relative humidity (%), and the ordinate represents weight change (%).
FIG. 18 shows an X-ray powder diffraction pattern of an amorphous
potassium salt of the compound of formula (I) of the present invention. The
abscissa represents the value of 2θ (degrees), and the ordinate represents peak
20 intensity.
FIG. 19 shows an X-ray powder diffraction pattern of an amorphous arginine
salt of the compound of formula (I) of the present invention. The abscissa
represents the value of 2θ (degrees), and the ordinate represents peak intensity.
25 DETAILED DESCRIPTION OF THE INVENTION
The inventors of the present invention have intensively studied different
aggregate forms of the compound of formula (I) and provide a crystalline free
form of the PD-1/PD-L1 inhibitor or a basic salt thereof. The crystalline free form
of the compound greatly improves physicochemical properties, such as
21
hygroscopicity, solubility and physicochemical stability, of the compound of
formula (I), and the crystalline basic salt of the compound greatly improves
physicochemical properties such as solubility and physicochemical stability, so
that the crystalline free form of the compound of formula (I) or the basic salt
5 thereof can satisfy the need to develop clinical pharmaceutical formulations, has
very important clinical application value, can be widely applied to the preparation
of drugs for treating tumors, immunity-related diseases and disorders,
communicable diseases, infectious diseases or metabolic diseases, particularly
drugs for treating ovarian cancer, pancreatic cancer, prostate cancer, breast cancer,
10 cervical cancer, glioblastoma, multiple myeloma, metabolic diseases,
neurodegenerative diseases, primary tumor metastasis or metastatic cancer in
bones, and is expected to be developed into a new-generation PD-1/PD-L1
inhibitor drug soon. The present invention is achieved on this basis.
Detailed description: Unless otherwise stated, the following terms used in the
15 specification and claims have the following meanings.
“Pharmaceutical composition” refers to a mixture containing one or more of
the compounds described herein or a physiologically/pharmaceutically acceptable
salt or pro-drug thereof and other chemical components, as well as other
components such as physiologically/pharmaceutically acceptable carriers and
20 excipients. The purpose of the pharmaceutical composition is to promote
administration to an organism and facilitate the absorption of the active
ingredient, so that the active ingredient can exert biological activity.
The compound of formula (I) has a variety of separated free forms or basic
salts, showing polymorphism or monomorphism. For example, each of the sodium
25 salts is shown as a polymorph. These “polymorphs” differ in terms of their X-ray
powder diffraction patterns, physicochemical and pharmacokinetic properties and
thermodynamic stability.
22
As used herein, “salt” refers to a compound prepared by reacting an organic
acid or base medicament with a pharmaceutically acceptable inorganic or organic
acid or base.
5 Methods and Materials
Diffraction data of the crystalline free form of the compound of formula (I)
or the basic salt thereof were collected at room temperature using a Bruker D8
ADVANCE (Bruker, Germany) X-ray powder diffractometer with an X-ray
source of Cu K ( = 1.5406 Å, light tube voltage, current: 40 kV, 40 mA). When
10 a sample was prepared, an appropriate amount of the sample was placed on a
zero-background sample tray, and the surface was flattened by gently pressing.
The scan range for the sample was from 3° to 40° (2θ), the step size was 0.02°
(2θ), and the duration of each step of scanning was 0.02 s. Diffraction patterns
were analyzed using DIFFRAC.EVA (V4.3.1). Measurement differences
15 associated with such X-ray powder diffraction analyses result from a variety of
factors including: (a) errors in sample preparation (e.g., sample height), (b)
instrument errors, (c) calibration errors, (d) operator errors (including those errors
present when determining peak positions), and (e) the nature of the material (e.g.,
preferred orientation errors). Calibration errors and sample height errors often
20 result in a shift of all the peaks in the same direction. In general, this calibration
factor will cause the measured peak positions to be in agreement with the
expected peak positions and may be in the range of the expected 2θ value ± 0.2°.
The angle 2θ values (°) and intensity values (as a % of the value of the highest
peak) for each polymorph obtained in the examples of the present invention are
25 shown in Tables 1-6.
The experimental method of characterizing the crystalline free form of the
compound of formula (I) or the basic salt thereof using differential scanning
calorimetry (DSC) is as follows: a small amount of the crystalline sample was
placed in an aluminum pan that was compatible with the instrument and could be
23
covered with a lid; after the sample was loaded, the aluminum pan was covered
with a lid and then placed into the instrument for analysis. In this patent, all the
instruments used for differential scanning calorimetry were TA Discovery
DSC25, the scanning parameter was set to using a nitrogen atmosphere, and the
5 warming rate was 10 °C/min.
The experimental method of characterizing the crystalline free form of the
compound of formula (I) or the basic salt thereof using thermogravimetric
analysis (TGA) is as follows: a small amount of the crystalline sample was placed
in an aluminum pan that was compatible with the instrument; after the sample was
10 loaded, the aluminum pan was placed into the instrument for analysis. In this
patent, all the instruments used for thermogravimetric analysis were TA
Discovery TGA550, the scanning parameter was set to using a nitrogen
atmosphere, and the warming rate was 10 °C/min.
The experimental method of characterizing the crystalline free form of the
15 compound of formula (I) or the basic salt thereof using dynamic vapor sorption
(DVS) is as follows: a small amount of the crystalline sample was placed in a
precision sample tray that was compatible with the instrument; after the sample
was loaded, the sample tray was placed into the instrument for analysis. In this
patent, the instruments used for dynamic vapor sorption were all the DVS
20 Intrinsic plus model, and the following experimental parameters were set:
nitrogen was used as the carrier gas, a constant temperature of 25 °C was set, the
rate of mass percentage change over a unit of time (dm/dt) = 0.002%/min was
used as a criterion for determining if equilibrium is reached, and the program
humidity change cycle was set to 50-95-0-95-50% RH; within the range of 0-90%
25 RH, a change of 10% RH was taken as a step, and within the range of 90% RH95% RH, a change of 5% RH was taken as a step.
The stability and solubility of the crystalline free form of the compound of
formula (I) or the basic salt thereof of the present application were determined by
HPLC. The specific test method is shown in the table below.
24
Instrument Thermo HPLC
Chromatographic column Xbridge-C18
Chromatographic column
supplier
Waters
Particle size (μm) 3.5
Size (mm) 4.6 × 150
Column temperature (°C) 40
Flow rate (mL/minute) 0.8
Injection volume (μL) 10
Dilution solvent MeOH
Sample concentration
(μg/mL)
400
Wavelength (nm) 277
Mobile phase A 0.1% TFA in water
Mobile phase B 0.1% TFA in acetonitrile
Run time (minutes) 21.0
Elution gradient
% B Minutes
5 Initial
95 19
95 20
5 20.1
5 21
The reagents in the examples of the present invention are known and
commercially available, or can be synthesized using or according to methods
known in the art. The starting material used in the study was prepared according
to Patent No. WO2019149183A1, or was optionally subjected to conventional
5 treatment in the art to obtain a free form.
25
Unless otherwise stated, all reactions of the present invention are carried out
in a dry nitrogen or argon atmosphere with continuous magnetic stirring, the
solvent is a dry solvent, and the temperature is in degrees centigrade (°C).
Unless otherwise specified, each of the various crystalline forms referred to
5 in the present invention may be an anhydrous crystalline form or a hydrated
crystalline form. For example, it is a hydrated crystalline form; preferably, each
molecule of the crystal contains 1, 2, 3, 4 or 5 waters of crystallization; and more
preferably, each molecule of the crystal contains 2 or 4 waters of crystallization.
The present invention is further explained in detail below using the drawings
10 and specific examples, which are illustrative only of particular embodiments of the
present invention and should not be construed as limiting the scope of the present
invention in any way.
Preparation of Specific Examples
Example 1. Preparation of Crystalline Form A of Disodium Salt Hydrate
15 About 10 mg of the compound of formula (I) in free form was dissolved in
0.5 mL of ethanol/water (volume ratio: 10:1), and a solution of 2 equivalents of
sodium hydroxide in ethanol/water was added. The mixture was stirred at room
temperature for 3 days and filtered, and the filter cake was dried in a drying oven
at 50 °C. The crystalline form A of disodium salt hydrate was a solid with high
20 crystallinity and had a solid-solid crystalline form transformation peak at 237.3
°C.
Analysis showed that the crystalline form had the XRPD pattern shown in
FIG. 1 and the DSC/TGA graphs shown in FIG. 8.
Example 2. Preparation of Crystalline Form B of Disodium Salt Anhydrate
25 About 10 mg of the crystalline form A of disodium salt hydrate of the
compound of formula (I) was heated to 245 °C to obtain the crystalline form B of
disodium salt anhydrate, which had a melting point of 316.4 °C. The crystalline
form B of disodium salt anhydrate was a solid with high crystallinity.
26
Analysis showed that the crystalline form had the XRPD pattern shown in
FIG. 2 and the DSC/TGA graphs shown in FIG. 9.
Example 3. Preparation of Free-Form Crystalline Form C
About 25 mg of the compound of formula (I) in free form was added to 200
5 μL of ethyl acetate and suspended at 45 °C for 7 days. A solid sample was then
isolated by centrifugation and dried under vacuum at 40 °C overnight. The freeform crystalline form C was a solid with high crystallinity and had a melting peak
at 169.5 °C.
Analysis showed that the crystalline form had the XRPD pattern shown in
10 FIG. 3 and the DSC/TGA graphs shown in FIG. 10.
Example 4. Preparation of Free-Form Crystalline Form D
About 10 mg of the compound of formula (I) in free form was added to 200
μL of ethyl acetate and suspended at 45 °C for 7 days. The mixture was filtered,
and the filter cake was dried in a drying oven at 50 °C. The free-form crystalline
15 form D was a solid with moderate crystallinity, and DSC showed a melting peak
at 239.9 °C.
Analysis showed that the crystalline form had the XRPD pattern shown in
FIG. 4 and the DSC and TGA graphs shown in FIG. 11.
Example 5. Preparation of Free-Form Crystalline Form E
20 5 mg of the free-form crystalline form C of the compound of formula (I) was
heated to 166 °C to obtain a solid sample of the free-form crystalline form E. The
free-form crystalline form E was a solid with moderate crystallinity and had a
melting peak at 224.7 °C.
Analysis showed that the crystalline form had the XRPD pattern shown in
25 FIG. 5 and the DSC graph shown in FIG. 12.
Example 6. Preparation of Free-Form Crystalline Form F
5 mg of the compound of formula (I) in free form was added to 200 μL of
methanol and suspended at 50 °C for 7 days. Then, a solid sample was separated
by centrifugation and dried under vacuum at 40 °C overnight. The free-form
27
crystalline form F was a solid with moderate crystallinity and had a melting peak
at 221.1 °C.
Analysis showed that the crystalline form had the XRPD pattern shown in
FIG. 6 and the DSC and TGA graphs shown in FIG. 13.
5 Example 7. Preparation of Amorphous Potassium Salt
About 10 mg of the compound of formula (I) in free form was dissolved in
0.5 mL of trifluoroethanol, and a solution of 2 equivalents of potassium hydroxide
in trifluoroethanol was added. The mixture was stirred at room temperature for 3
days and filtered, and the filter cake was dried in a drying oven at 50 °C.
10 Analysis showed that the product had the XRPD pattern shown in FIG. 18.
The potassium salt obtained was an amorphous solid.
Example 8. Preparation of Amorphous Arginine Salt
About 10 mg of the compound of formula (I) in free form was dissolved in
0.5 mL of trifluoroethanol, and a solution of 2 equivalents of arginine in
15 trifluoroethanol was added. The mixture was stirred at room temperature for 3
days and filtered, and the filter cake was dried in a drying oven at 50 °C.
Analysis showed that the product had the XRPD pattern shown in FIG. 19.
The arginine salt obtained was an amorphous solid.
Example 9. Single Crystal Structure Analysis of Crystalline Form A of
20 Disodium Salt Hydrate
1. Experimental instruments
Transmission electron microscope: Thermo Scientific Glacios
Detector: Thermo Scientific Ceta-D
Sample rod: Autoloader
25 2. Experimental conditions
Voltage: 200 kV
Temperature: 83 K
Vacuum value: 10-7 Pa high vacuum
28
3. Application software
Data collection: Thermo Scientific EPU-D
Data processing: XDS, SHELXT and SHELXL
4. Sample preparation
5 A small amount of the powdered sample was directly transferred onto a TEM
grid, and the excess powder was blown away with a rubber bulb. The grid was
rapidly plunged into liquid ethane using a Thermo Scientific Vitrobot cryoelectron microscope sample preparation system. After being frozen, the sample
was transferred to liquid nitrogen for storage and then loaded into the electron
10 microscope for observation.
5. Image collection
Multiple particles of appropriate size with clear diffraction signals were
selected, and a series of diffraction data generated by rotation of the particles were
collected automatically using EPU-D software.
15 6. Data processing
The diffraction images were indexed and subjected to intensity integration to
obtain unit cell parameters and HKL files. Multiple sets of data were merged to
solve the crystal structure.
7. Analysis results
20 From 26 different particles of the sodium salt of the compound of formula
(I), 19 sets of diffraction images were collected. These images were each indexed
and integrated using XDS, and the following unit cell constants were obtained: a =
20.00(5) Å, b = 5.798(16) Å, c = 20.44(11) Å, α = 90°, β = 94.8(2)°, γ = 90° and
unit cell volume V = 2362(15) Å3
.
25 8. Structure determination
The 9 best-quality sets of data were merged using XSCALE. The total data
set contained a total of 16,135 diffraction points, of which 2821 were independent
diffraction points. From the above data, the crystal structure of the crystalline
form A of disodium salt hydrate was successfully determined. It is a monoclinic
29
crystal system with a space group of C2 (No. 5), a molecular weight of 450.48
g·mol-1 and Z' = 0.5. The asymmetric unit consists of 0.5 API anions, 1 sodium
ion and 2 water molecules. The structure of a unit cell of the single crystal is
shown in FIG. 7. Crystallographic data and refinement parameters are shown in
5 the table below.
Chemical formula C46H50N6O6
-
· 2Na+
· 4H2O
Molecular weight 900.97 g·mol-1
Temperature 83(2) K
Wavelength 0.02508 Å
Crystal system, space group Monoclinic, C2 (No. 5)
Unit cell parameters
a = 20.00(5) Å
b = 5.798(16) Å
c = 20.44(11) Å
α = 90°
β = 94.8(2)°
γ = 90°
Volume 2362(15) Å3
Z' 0.5
Density 1.267 g/cm3
Resolution 0.90 Å
All diffraction points 16135
Independent diffraction points 2821
Completeness 82.7 %
Rint 0.2522
Initial model generation method ab initio
Hydrogen atom refinement Geometric constraints
Goodness-of-fit
on F2
1.284
30
R1 [I ≥ 2sigma(I)] 0.1534
R1 [all data] 0.1736
wR2[I ≥ 2sigma(I)] 0.3589
wR2[all data] 0.3752
Example 10. Solubility Determination
The desired amount of the compound was weighed into a glass vial, and the
desired amount of a vehicle was added. A stir bar was added to the vial, and the
5 mixture was stirred at room temperature for 24 hrs. After 24 hrs, the appearance
of the sample was observed. An appropriate amount of the sample was filtered
through a PVDF 0.45 μm filter membrane, and the filtrate was collected. The pH
value of the filtrate was determined, and the sample was diluted with MeOH. The
diluted sample was injected into an HPLC system to determine the concentration.
10 The process of preparing pH buffers is shown in the table below:
pH value Preparation process
1.2
8.5 mL of a 0.2 M hydrochloric acid solution and 5 mL of a
0.2 M potassium chloride solution were added to a 20 mL
volumetric flask, and the mixture was brought to volume with
water and well mixed. The pH value was determined, and the
pH should be 1.2±0.03. The pH was adjusted to 1.2 with
hydrochloric acid or sodium hydroxide if necessary.
4.7
359 mg of sodium acetate trihydrate solid was weighed into a
100 mL volumetric flask, and an appropriate amount of water
was added to dissolve the solid. Then, 1.18 mL of a 2 N acetic
acid solution was transferred into the volumetric flask, and the
mixture was brought to volume with water and well mixed.
The pH value was determined, and the pH should be 4.7±0.03.
31
6.8
169.96 mg of anhydrous potassium dihydrogen phosphate and
29.17 mg of sodium hydroxide were added to a 25 mL
volumetric flask, and the solution was brought to volume with
purified water. The pH was adjusted to 6.8 with phosphoric
acid.
The process of preparing biological media is shown in the table below:
Biological
medium
Preparation process
FaSSIF
fasted-state
simulated
intestinal
fluid
1.0471 g of concentrated FaSSIF solution was weighed into a
25 mL volumetric flask and diluted with an appropriate
amount of purified water, and 55.9 mg of
FaSSIF/FeSSIF/FaSSGF powder was added. The mixture was
brought to volume (25 mL) and well mixed (pH 6.5).
FeSSIF fedstate
simulated
intestinal
fluid
2.0365 g of concentrated FeSSIF solution was weighed into a
25 mL volumetric flask and diluted with an appropriate
amount of purified water, and 280.2 mg of
FaSSIF/FeSSIF/FaSSGF powder was added. The mixture was
brought to volume (25 mL) and well mixed (pH 5.0).
FaSSGF
simulated
gastric fluid
3.6790 g of concentrated FaSSGF solution was weighed into a
100 mL volumetric flask and diluted with an appropriate
amount of purified water, and 6.0 mg of
FaSSIF/FeSSIF/FaSSGF powder was added. The mixture was
brought to volume (100 mL) and well mixed (pH 1.6).
Note
The concentrated FaSSIF solution, concentrated FeSSIF
solution and concentrated FaSSGF solution and
FaSSIF/FeSSIF/FaSSGF powders used are all commercially
available products (supplier: Biorelevant).
32
The experimental results are shown below:
Equilibrium solubility data of free-form crystalline forms C, D and F
No
.
Vehicle
Target
concentrati
on
(μg/mL)
Equilibrium solubility (μg/mL, pH24 hrs)
Solubility of
crystalline form
C
(μg/mL, pH24
hrs)
Solubility of
crystalline
form D
(μg/mL, pH24
hrs)
Solubility of
crystalline
form F (μg/mL,
pH24 hrs)
1 Water
2000
3.84(6.78) 51.93(5.82) 69.47(5.52)
2
pH 1.2,
HCl 100
mM
18.92(1.30) 281.84(1.16) 244.49(1.15)
3
pH 4.7, 50
mM acetic
acid buffer
3.3(4.77) 47.41(4.73) 67.33(4.68)
4
pH6.8,
50 mM
phosphoric
acid buffer
2.31(6.89) 42.11(6.90) 61.01(6.93)
5 FaSSGF 8.9(1.56) 87.01(1.57) 117.15(1.58)
6 FaSSIF 3.21(6.41) 78.44(6.43) 306.56(6.42)
7 FeSSIF 5.34(4.90) 433.75(4.94) 1723.16(4.99)
As can be seen from the above data, the free-form crystalline form C
5 exhibited improved solubility in the buffer system having a pH of 1.2 compared to
the other test conditions. Compared to the free-form crystalline form C, the freeform crystalline form D and free-form crystalline form F had solubility values that
were increased by a factor of at least 10 or more under various test conditions,
such as buffer systems having a pH of 1.2-6.8, water and simulated biological
33
media (FaSSGF, FaSSIF and FeSSIF), indicating significantly improved
solubility. More surprisingly, compared to the free-form crystalline form C, the
free-form crystalline form F had a solubility value that was increased by a factor
of nearly one hundred in the simulated biological medium FaSSIF and a solubility
5 value that was increased by a factor of several hundreds in the simulated
biological medium FeSSIF.
Solubility data of crystalline form A of disodium salt hydrate and crystalline
form B of disodium salt anhydrate
No. Solvent/medium
Target
concentration
(μg/mL)
Equilibrium solubility (μg/mL,
pH24 hrs)
Solubility of
crystalline
form B
(μg/mL, pH24
hrs)
Solubility of
crystalline
form A
(μg/mL, pH24
hrs)
1 Water
2000
≥2000(9.96) >2000(9.61)
2
pH 1.2,
HCl 100 mM
≥2000(0.95) 1120(1.01)
3
pH 4.7,
50 mM acetic acid
buffer
135(4.92) 25(4.61)
4
pH 6.8, 50 mM
phosphoric acid
buffer
194(7.03) 34(6.88)
5 FaSSGF 485(1.72) 22(1.73)
6 FaSSIF 474(6.93) 303(6.93)
7 FeSSIF ≥2000(6.90) >2000(7.95)
34
As can be seen from the above data, the crystalline sodium salts of the
compound of formula (I) exhibited significantly improved solubility, which was at
least several tens of times higher than that of the free-form crystalline form C of
the compound of formula (I). In particular, the crystalline form B of disodium salt
5 anhydrate exhibited excellent solubility in various buffers and biological medium
solutions.
Example 11. Stability Determination
An appropriate amount of the sample was placed in a vial and left to stand at
50 °C, 80 °C or 50 °C & 75% RH (open) for 7 days to evaluate thermal stability.
10 An appropriate amount of the sample was placed in a transparent vial, an amber
vial or an aluminum foil transparent vial and left to stand in a light box for 10
days to obtain a photostability evaluation under conditions of overall nearultraviolet energy of 1.2 * 106
Lux·hr and no less than 200 W·hr/m2. The
experimental results are shown below.
15 Stability data of free-form crystalline form C
Condition Time Appearance Purity (%) XRPD
Initial 0 days
White
powder
98.42
Free-form crystalline
form C
50 ℃/75%RH 7 days
White
powder
98.43
Free-form crystalline
form C
50 ℃ 7 days
White
powder
98.31
Free-form crystalline
form C
80 ℃ 7 days
White
powder
97.90
Free-form crystalline
form C
Transparent light
exposure
9.2 days
White
powder
97.91
Free-form crystalline
form C
Aluminum foil light
exposure control
9.2 days
White
powder
98.51
Free-form crystalline
form C
35
Stability data of free-form crystalline form D
Condition Time Appearance Purity (%) XRPD
Initial 0 days
White
powder
98.17
Free-form crystalline
form D
50 ℃/75%RH 7 days
White
powder
98.24
Free-form crystalline
form D
50 ℃ 7 days
White
powder
98.13
Free-form crystalline
form D
80 ℃ 7 days
White
powder
97.22
Free-form crystalline
form D
Transparent light
exposure
9.2
days
White
powder
96.06
Free-form crystalline
form D
Aluminum foil light
exposure control
9.2
days
White
powder
98.26
Free-form crystalline
form D
Stability data of free-form crystalline form F
Condition Time Appearance
Purity
(%)
XRPD
Initial 0 days
White
powder
99.44
Free-form
crystalline form F
50 ℃/75%RH
11
days
White
powder
99.36
Free-form
crystalline form F
50 ℃
11
days
White
powder
99.36
Free-form
crystalline form F
80 ℃
11
days
White
powder
99.22
Free-form
crystalline form F
Transparent light
exposure
9.2
days
White
powder
99.43
Free-form
crystalline form F
36
Aluminum foil light
exposure control
9.2
days
White
powder
99.39
Free-form
crystalline form F
Stability data of crystalline form A of disodium salt hydrate
Condition Time Appearance Purity (%) XRPD
Initial 0 days White powder 99.38
Crystalline form A of
disodium salt
50 ℃ 7 days White powder 99.45
Crystalline form A of
disodium salt
80 ℃ 7 days White powder 99.34
Crystalline forms A +
B of disodium salt
50 ℃/75%RH 7 days White powder 99.46
Crystalline form A of
disodium salt
Transparent
light exposure
9.2 days White powder 99.22
Crystalline forms A +
B of disodium salt
Brown vial light
exposure control
9.2 days White powder 99.50
Crystalline form A of
disodium salt
Aluminum foil
light exposure
control
9.2 days White powder 99.51
Crystalline form A of
disodium salt
Stability data of crystalline form B of disodium salt anhydrate
Condition Time Appearance
Purity
(%)
XRPD
Initial 0 days White powder 91.23
Crystalline form B
of disodium salt
50 ℃ 7 days White powder 92.11
Crystalline form B
of disodium salt
80 ℃ 7 days White powder 90.77
Crystalline form B
of disodium salt
37
50 ℃/75%RH 7 days White powder 92.97
Crystalline form B
of disodium salt
Transparent
light exposure
9.2 days White powder 83.10
Crystalline form B
of disodium salt
Brown vial
light exposure
control
9.2 days White powder 88.10
Crystalline form B
of disodium salt
Aluminum foil
light exposure
control
9.2 days White powder 89.50
Crystalline form B
of disodium salt
As can be seen from the above results,
1) Under conditions of 50 °C, 50 °C/75% RH (open), 80 °C for 7 days and
photostress for 10 days and overall near-ultraviolet energy of 1.2 * 106
Lux·hr
5 and no less than 200 W·hr, the free-form crystalline form C, crystalline form
D and crystalline form F all exhibited extremely high physical and chemical
stability.
2) The crystalline form A of disodium salt hydrate exhibited good chemical
stability under various stability testing conditions, with no significant increase
10 in impurity content; however, the crystalline form underwent crystalline form
transformation under high temperature and light exposure. The crystalline
form B of disodium salt anhydrate exhibited good physical stability under the
stability testing conditions, and no crystalline form transformation was
observed; however, the crystalline form was sensitive to light exposure.
15 Therefore, the development of the crystalline forms of disodium salt requires
attention to storage conditions, and high temperatures and exposure to light
should be avoided.
38
Example 12. Hygroscopic Behavior Test
In the present invention, the hygroscopic weight increases (hygroscopic
weight increase/weight before hygroscopy * 100%) of various crystalline forms
under various relative humidity conditions were determined using the dynamic
5 vapor sorption method, and the hygroscopicity of different crystalline compounds
was evaluated. The results are shown in FIGs. 14-17 and the table below:
Hygroscopic
weight
increase (%)

Crystalline form
Relative humidity (%) at 25 °C
0.0 10.0
20.
0
30.0 40.0 50.0 60.0 65.0 70.0 80.0 90.0
Free-form
crystalline form
C
0.0
0
0.50
0.7
5
0.80 1.20 1.35 1.50 / 1.75 2.21 2.95
Free-form
crystalline form
D
0.0
0
0.40
0.8
5
1.34 1.85 2.30 2.76 / 3.25 3.79 4.58
Free-form
crystalline form
F
0.0
0
0.18
0.3
8
0.60 0.84 1.14 1.54 / 2.04 2.55 3.28
Crystalline form
A of disodium
salt hydrate
0.0
0
2.40
7.4
5
9.28
10.6
2
11.6
4
12.4
3
13.2
5
14.5
9
/
Amorphous
potassium salt
/ / / / / / / 5.12 /
12.9
8
26.6
4
Note: “/” means “no detection”.
39
As can be seen from the above data, the free-form crystalline form C,
crystalline form D and crystalline form F were all moderately hygroscopic, and
showed no crystalline form changes after DVS testing.
The crystalline form A of disodium salt hydrate and the amorphous
5 potassium salt exhibited comparable hygroscopicity, their weight increased by
more than 10% at 80% RH, and they showed no crystalline form changes after
DVS testing.
As can be seen from the above experimental results, the free-form crystalline
form C has high crystallinity, allows for scale-up preparation, has moderate
10 hygroscopicity, and exhibits extremely high physical and chemical stability under
various stability testing conditions. However, the free-form crystalline form C
exhibits improved solubility only in a buffer system having a pH of 1.2.
The free-form crystalline form D has moderate crystallinity, allows for scaleup preparation, has moderate hygroscopicity, and exhibits extremely high physical
15 and chemical stability under various stability testing conditions. In addition, the
free-form crystalline form D exhibits significantly improved solubility under
various pH conditions.
The free-form crystalline form E has moderate crystallinity, and its scale-up
preparation is not easy at present.
20 The free-form crystalline form F has moderate crystallinity, allows for scaleup preparation, has moderate hygroscopicity, and exhibits extremely high physical
and chemical stability under various stability testing conditions. In addition, the
free-form crystalline form F exhibits extremely significantly improved solubility
under various pH conditions.
25 After salt-forming screening of a large number of basic salts, only crystalline
salt forms of the sodium salt, specifically the crystalline form A of disodium salt
hydrate and the crystalline form B of anhydrate, were obtained. The sodium salt
can be used to prepare salt forms with high crystallinity and allows for scale-up
preparation. In addition, under different pH conditions or in various biological
40
media, various crystalline forms of the sodium salt all exhibit significantly higher
solubility than the free-form crystalline form C.
Specifically, the crystalline form A of disodium salt hydrate exhibits
relatively high physical and chemical stability under various conditions, such as
5 50 °C, 50 °C/75% RH or brown light exposure, and under high temperature and
light exposure, it exhibits relatively high chemical stability but undergoes
crystalline form transformation. In further development of formulations or storage
and transportation, it is necessary to avoid exposure to light, and the ambient
temperature should not be kept too high.
10 The crystalline form B of disodium salt anhydrate exhibits relatively high
physical and chemical stability under various conditions, such as 50 °C, 50
°C/75% RH or brown light exposure; and it exhibits relatively high physical
stability under high temperature and high humidity conditions but is sensitive to
light. In further development of formulations or storage and transportation, it is
15 necessary to avoid exposure to light.
All documents mentioned in the present invention are incorporated as references,
just as each document is individually cited as a reference. In addition, it should be
understood that various modifications or changes may be made by those skilled in
the art after reading the above teachings of the present invention, and these
20 equivalent forms also fall within the scope to which the present invention relates.
25
30
41
WE CLAIM :
1. A crystalline basic salt of a compound of formula (I):
5 ,
wherein the basic salt is selected from the group consisting of lithium salt,
sodium salt, potassium salt, magnesium salt, calcium salt and ammonium salt.
2. The crystalline basic salt of the compound of formula (I) according to
10 claim 1, wherein the crystalline basic salt of the compound of formula (I) is a
sodium salt; and each molecule of the crystalline sodium salt of the compound of
formula (I) comprises the compound of formula (I) in free form and the sodium
atom in a molar ratio of 1:1 or 1:2.
15 3. The crystalline basic salt of the compound of formula (I) according to
claim 2, wherein each molecule of the crystalline sodium salt of the compound of
formula (I) comprises the compound of formula (I) in free form and the sodium
atom in a molar ratio of 1:2.
20 4. The crystalline basic salt of the compound of formula (I) according to
claim 2, wherein the crystalline sodium salt of the compound of formula (I) is an
anhydrate or a hydrate;
preferably, each molecule of the crystalline sodium salt hydrate of the
compound of formula (I) comprises the compound of formula (I) in free form and
25 the water molecule in a molar ratio of 1.0:(0.1-10.0);
42
more preferably, each molecule of the crystalline sodium salt hydrate of the
compound of formula (I) comprises the compound of formula (I) in free form and
the water molecule in a molar ratio of 1.0:(1.0-5.0);
most preferably, each molecule of the crystalline sodium salt hydrate of the
5 compound of formula (I) comprises the compound of formula (I) in free form and
the water molecule in a molar ratio of 1.0:4.0 or 1.0:2.0.
5. The crystalline basic salt of the compound of formula (I) according to
claim 4, wherein the crystalline basic salt of the compound of formula (I) is a
10 crystalline form A of disodium salt hydrate having an X-ray powder diffraction
(XRPD) pattern comprising five or more peaks at angles of diffraction (2θ) of
8.60±0.2°, 9.97±0.2°, 12.92±0.2°, 15.03±0.2°, 17.62±0.2°, 17.93±0.2°,
20.04±0.2°, 22.07±0.2°, 23.18±0.2°, 23.60±0.2° and 27.06±0.2°;
preferably, the X-ray powder diffraction (XRPD) pattern of the crystalline
15 form A of disodium salt hydrate comprises peaks substantially identical to those at
angles of diffraction (2θ) shown in FIG. 1.
6. The crystalline basic salt of the compound of formula (I) according to
claim 5, wherein a unit cell of the crystalline form A of disodium salt hydrate is a
20 monoclinic crystal system with the following unit cell parameters: a = 20.00(5) Å,
b = 5.798(16) Å, c = 20.44(11) Å, α = 90°, β = 94.8(2)°, γ = 90°, a unit cell
volume V of 2362(15) Å3 and space group of C2 (No. 5).
7. The crystalline basic salt of the compound of formula (I) according to
25 claim 4, wherein the crystalline basic salt of the compound of formula (I) is a
crystalline form B of disodium salt anhydrate having an X-ray powder diffraction
(XRPD) pattern comprising five or more peaks at angles of diffraction (2θ) of
6.60±0.2°, 7.46±0.2°, 12.43±0.2°, 13.32±0.2°, 13.63±0.2°, 15.54±0.2°,
17.32±0.2°, 18.68±0.2° and 21.73±0.2°;
43
preferably, the X-ray powder diffraction (XRPD) pattern of the crystalline
form B of disodium salt anhydrate comprises peaks substantially identical to those
at angles of diffraction (2θ) shown in FIG. 2.
5 8. A preparation method for the crystalline basic salt of the compound of
formula (I) according to any one of claims 1-7, comprising the following steps:
1) dissolving or dispersing the compound of formula (I) in free form in water or
a suitable organic solvent, and adding a basic solution to the above system to
conduct a salt-forming reaction; or adding the compound of formula (I) in
10 free form to a basic solution to conduct a salt-forming reaction; and
2) collecting a solid product precipitated during the above salt-forming reaction,
or creating a degree of supersaturation in the salt-forming system to obtain
the crystalline basic salt of the compound of formula (I);
wherein the basic salt is selected from the group consisting of lithium salt,
15 sodium salt, potassium salt, magnesium salt, calcium salt and ammonium salt; and
the basic solution is selected from the group consisting of solutions of
lithium hydroxide, sodium hydroxide, potassium hydroxide, magnesium
hydroxide, calcium hydroxide and ammonia in water or in an organic solvent.
20 9. The preparation method according to claim 8, wherein methods for
creating the degree of supersaturation in the salt-forming system in step 2) include
one or more of the following: adding a seed crystal, volatilizing the solvent,
adding an anti-solvent and cooling.
25 10. The preparation method according to claim 8, wherein the organic
solvent is selected from the group consisting of alcohols, chloroalkanes, ketones,
ethers, cyclic ethers, esters, alkanes, cycloalkanes, benzenes, amides and
sulfoxides, and mixtures thereof and aqueous solutions thereof;
44
preferably, the organic solvent is selected from the group consisting of
methanol, ethanol, n-propanol, isopropanol, dichloromethane, heptane,
acetonitrile, acetone, methyl ethyl ketone, toluene, 1,4-dioxane, tetrahydrofuran,
N,N-dimethylformamide, ethyl acetate, isopropyl acetate, methyl tert-butyl ether
5 and 2-methoxyethyl ether, and mixtures thereof and aqueous solutions thereof.
11. A preparation method for the crystalline basic salt of the compound of
formula (I) according to any one of claims 1-7, comprising the following step:
transforming one crystalline form of the basic salt of the compound of formula (I)
10 into another crystalline form of the salt by using a crystalline form transformation
method, wherein the crystalline form transformation method includes heating or
suspension crystalline form transformation in a solvent selected from the group
consisting of methanol, ethanol, n-propanol, isopropanol, dichloromethane,
heptane, acetonitrile, acetone, methyl ethyl ketone, toluene, 1,4-dioxane,
15 tetrahydrofuran, N,N-dimethylformamide, ethyl acetate, isopropyl acetate, methyl
tert-butyl ether and 2-methoxyethyl ether, and mixtures thereof and aqueous
solutions thereof.
12. A crystalline form of a compound of formula (I) in free form:
20 ;
preferably, the crystalline form of the compound of formula (I) in free form
is crystalline form C, crystalline form D, crystalline form E or crystalline form F.
13. The crystalline form of the compound of formula (I) in free form
25 according to claim 12, wherein the crystalline form of the compound of formula
45
(I) in free form is the crystalline form C, an X-ray powder diffraction (XRPD)
pattern of which comprises five or more peaks at angles of diffraction (2θ) of
7.80±0.2°, 11.97±0.2°, 12.42±0.2°, 15.98±0.2°, 17.37±0.2°, 18.82±0.2°,
22.47±0.2°, 23.30±0.2°, 25.01±0.2° and 25.63±0.2°;
5 preferably, the X-ray powder diffraction (XRPD) pattern of the crystalline
form C comprises peaks substantially identical to those at angles of diffraction
(2θ) shown in FIG. 3.
14. The crystalline form of the compound of formula (I) in free form
10 according to claim 12, wherein the crystalline form of the compound of formula
(I) in free form is the crystalline form D, an X-ray powder diffraction (XRPD)
pattern of which comprises five or more peaks at angles of diffraction (2θ) of
7.97±0.2°, 11.82±0.2°, 12.18±0.2°, 14.87±0.2°, 16.13±0.2°, 17.18±0.2°,
19.21±0.2°, 20.72±0.2° and 25.28±0.2°;
15 preferably, the X-ray powder diffraction (XRPD) pattern of the crystalline
form D comprises peaks substantially identical to those at angles of diffraction
(2θ) shown in FIG. 4.
15. The crystalline form of the compound of formula (I) in free form
20 according to claim 12, wherein the crystalline form of the compound of formula
(I) in free form is the crystalline form E, an X-ray powder diffraction (XRPD)
pattern of which comprises five or more peaks at angles of diffraction (2θ) of
8.16±0.2°, 12.05±0.2°, 12.83±0.2°, 15.19±0.2°, 16.18±0.2°, 16.59±0.2°,
17.62±0.2°, 18.41±0.2°, 22.90±0.2° and 25.65±0.2°;
25 preferably, the X-ray powder diffraction (XRPD) pattern of the crystalline
form E comprises peaks substantially identical to those at angles of diffraction
(2θ) shown in FIG. 5.
46
16. The crystalline form of the compound of formula (I) in free form
according to claim 12, wherein the crystalline form of the compound of formula
(I) in free form is the crystalline form F, an X-ray powder diffraction (XRPD)
pattern of which comprises five or more peaks at angles of diffraction (2θ) of
5 6.92±0.2°, 9.10±0.2°, 12.42±0.2°, 13.46±0.2°, 14.38±0.2°, 15.84±0.2°,
19.04±0.2°, 22.00±0.2° and 26.23±0.2°;
preferably, the X-ray powder diffraction (XRPD) pattern of the crystalline
form F comprises peaks substantially identical to those at angles of diffraction
(2θ) shown in FIG. 6.
10
17. Use of the crystalline form of the compound of formula (I) in free form
according to any one of claims 12-16, wherein the crystalline form of the
compound of formula (I) in free form is used as a starting material to prepare the
crystalline basic salt of the compound of formula (I) according to any one of
15 claims 1-7.
18. A pharmaceutical composition, comprising a clinically effective amount
of the crystalline basic salt of the compound of formula (I) according to any one
of claims 1-7 or the crystalline form of the compound of formula (I) in free form
20 according to any one of claims 12-16 and a pharmaceutically acceptable carrier;
preferably, the pharmaceutical composition comprises 0.01-99.0% W/W of
the crystalline basic salt of the compound of formula (I) or the crystalline form of
the compound of formula (I) in free form relative to a total content of the
pharmaceutical composition.
25
19. Use of the crystalline basic salt of the compound of formula (I) according
to any one of claims 1-7 or the crystalline form of the compound of formula (I) in
free form according to any one of claims 12-16 in preparation of a medicament for
47
treating a PD-1/PD-L1 signal pathway-mediated tumor, immunity-related disease
and disorder, communicable disease, infectious disease or metabolic disease;
preferably, the tumor is cancer.
5 20. The use according to claim 19, wherein the infectious disease is a
bacterial infectious disease, a viral infectious disease or a fungal infectious
disease.
21. The use according to claim 19, wherein the tumor is lymphoma, sarcoma,
10 melanoma, glioblastoma, synovioma, meningioma, biliary tract tumor, neuroma,
seminoma, nephroblastoma, hepatocellular papilloma, papilloma, leiomyoma,
rhabdomyoma, hemangioma, lymphangioma, osteoma, lipoma, fibroma, central
nervous system tumor, spinal axis tumor, brain stem glioma, multiple myeloma,
ovarian tumor, myelodysplastic syndrome or mesothelioma, anal cancer, testicular
15 cancer, urethral carcinoma, penile cancer, bladder cancer, ureteral cancer, uterine
cancer, ovarian cancer, fallopian tube cancer, cervical cancer, vaginal cancer,
vulvar cancer, Merkel cell carcinoma, embryonal carcinoma, chronic or acute
leukemia, bronchial carcinoma, esophageal cancer, nasopharyngeal carcinoma,
hepatocellular carcinoma, basal cell carcinoma, lung cancer, adenocarcinoma,
20 papillary carcinoma, rectal cancer, colon cancer, gastric cancer, head and neck
cancer, bone cancer, skin cancer, small intestine cancer, endocrine cancer, renal
pelvic carcinoma, epidermoid carcinoma, transitional cell carcinoma or
choriocarcinoma;
the immunity-related disease and disorder is rheumatic arthritis, renal failure,
25 lupus erythematosus, asthma, psoriasis, ulcerative colitis, pancreatitis, allergy,
fibrosis, anemia, fibromyalgia, Alzheimer’s disease, congestive heart failure,
stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson’s disease,
Crohn’s disease, ulcerative colitis, allergic contact dermatitis and eczema,
systemic sclerosis or multiple sclerosis;
48
the communicable disease or infectious disease is sepsis, liver infection,
hepatitis A, hepatitis B, hepatitis C, hepatitis D, herpes virus, papillomavirus or
influenza; and
the metabolic disease is diabetes, diabetic ketoacidosis, hyperglycemic
5 hyperosmolar syndrome, hypoglycemia, gout, malnutrition, vitamin A deficiency,
scurvy, vitamin D deficiency or osteoporosis.
22. The use according to claim 21, wherein the lymphoma is lymphocytic
lymphoma, primary central nervous system lymphoma, T cell lymphoma, diffuse
10 large B cell lymphoma, follicle center lymphoma, Hodgkin lymphoma, nonHodgkin lymphoma or primary mediastinal large B cell lymphoma;
the sarcoma is Kaposi’s sarcoma, fibrosarcoma, liposarcoma,
chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, soft tissue
sarcoma, angiosarcoma or lymphangiosarcoma; and
15 the chronic or acute leukemia is acute myeloid leukemia, chronic myeloid
leukemia, acute lymphoblastic leukemia, chronic granulocytic leukemia or
chronic lymphoblastic leukemia.
23. The use according to claim 19, wherein the tumor is small cell lung
20 cancer, squamous non-small cell lung cancer, non-squamous non-small cell lung
cancer, chondroma, colorectal cancer, gastrointestinal cancer, endometrial cancer,
head and neck squamous cell carcinoma, abdominal wall carcinoma, renal cell
carcinoma or recurrent or existing drug-resistant prostate cancer.
25 24. The use according to claim 19, wherein the tumor is thymic tumor,
pleomorphic adenoma, renal tubule adenoma, cystadenoma, pituitary adenoma,
prostate cancer, thyroid cancer, parathyroid cancer, adrenal cancer, breast cancer,
cystadenocarcinoma or pancreatic cancer.
49
25.The crystalline basic salt of the compound of formula (I) according to any one
of claims 1-7 or the crystalline form of the compound of formula (I) in free form
according to any one of claims 12-16 for use as a medicament for treating a PD1/PD-L1 signal pathway-mediated tumor, immunity-related disease and disorder,
5 communicable disease, infectious disease or metabolic disease.