JP2020532496A - High affinity CXCR4 selective binding conjugate and how to use it - Google Patents

Abstract

The present invention provides compounds that can be used for targeted drug delivery, patient imaging, or subject diagnosis for clinical conditions that may be associated with overexpression and / or upregulation of CXCR4. In particular, the present invention provides a high affinity CXCR4 selective binding ligand peptide conjugate (PC) of formula (I) or a pharmaceutically acceptable salt thereof, as well as methods of use and production thereof: the present invention. The high affinity CXCR4 selective binding ligand peptide conjugate (PC) of the invention is useful for patient diagnosis, treatment or imaging. In the compound of formula (I), n is an integer of the sum of 1 to (total number of amino acid residues in P and total number of side chain functional groups in P amino acid residues); each A is independent, A diagnostic, therapeutic, or imaging agent; L is a linker or absent; and P is a high-affinity CXCR4 selective binding peptidyl ligand. In particular, the present invention provides targeted drug delivery or patient imaging or patient diagnosis for diseases associated with CXCR4 overexpression and / or upregulation, such as cancer, HIV infection, and immune impairment. Compositions and kits comprising a peptide conjugate of formula (I), as well as methods of use and production of the peptide conjugate of formula (I), are disclosed herein. [Selection diagram] None

Description

[Cross-reference of related applications]
This application claims the priority benefit of US Provisional Application No. 62 / 554,354 filed on September 5, 2017, which is incorporated herein by reference in its entirety.

特に、本発明の高親和性CXCR4選択的結合リガンドペプチド抱合体は、患者の診断、治療またはイメージングに有用である。式（I）の化合物において、各Aは独立して、診断剤、治療剤、またはイメージング剤であり、Lはリンカーまたは不存在であり、およびPは高親和性CXCR4選択的結合ペプチジルリガンドである。特に、本発明は、癌、HIV感染、および免疫障害などのCXCR4の過剰発現および/または上方調節が関係する疾患について、標的化された薬物送達または患者のイメージングまたは患者の診断に関する。このような用途のための組成物、キットおよび方法が本明細書に開示される。 [Field of invention]
The present invention relates to a high affinity CXCR4 selective binding ligand peptide conjugate (“PC”) of formula (I) or a pharmaceutically acceptable salt thereof, and methods of use and production thereof:

In particular, the high affinity CXCR4 selective binding ligand peptide conjugate of the present invention is useful for diagnosis, treatment or imaging of patients. In the compounds of formula (I), each A is independently a diagnostic, therapeutic or imaging agent, L is a linker or absent, and P is a high affinity CXCR4 selective binding peptidyl ligand. .. In particular, the present invention relates to targeted drug delivery or patient imaging or patient diagnosis for diseases associated with CXCR4 overexpression and / or upregulation, such as cancer, HIV infection, and immune impairment. Compositions, kits and methods for such applications are disclosed herein.

[Background of invention]
CXCL12 (also called stromal cell-derived factor-1 or SDF-1) and CXCR4, chemokines and chemokine receptor pairs are important in hematopoiesis, multistage tumorigenesis, and embryogenesis Studies have been shown to play a role (Broxmeyer, HE et al., Int. J. Hematol. 2001, 74, 9-17; Horuk, R., Nat. Rev. Drug Discov. 2009, 8, 23- 33). For example, activation of CXCR4 by CXCL12 has been shown to be directed towards leukocyte chemotaxis in the immune system in response to inflammation and progenitor cell migration during embryogenesis. Activation of CXCR4 by CXCL12 has also been shown to mediate signaling pathways involved in breast cancer metastasis and memory T cell migration (Orimo, A., et al., Cell 2005, 121, 335-348).

CXCR4, a G protein-coupled receptor also known as fucin or CD184 (differentiation cluster 184), is constitutively or overexpressed in abundant human cancers and promotes local tumor cell proliferation, survival and angiogenesis (Huang, EH, et al., J. Surg.Res. 2009, 155, 231-236). CXCR4 is a co-receptor for HIV entry and infection in host cells and has also been reported to be evaluated as a potential HIV treatment (Tamamura, H., et al., Biochem. Biophys. Res. Commun. 1998, 253, 877-882; Oberlin, E. et al., Nature, 1996, 382, 833-835).

It has been confirmed that CXCR4 is overexpressed in many human cancers. CXCR4 antagonism has been shown to disrupt tumor-interstitial interactions, sensitize cancer cells to cytotoxic drugs, and reduce tumor growth and metastatic burden. Therefore, CXCR4 is a target not only for potential interventions in the treatment of cancer, but also for non-invasive monitoring of disease progression, treatment guidance, and other diagnostic purposes (Chatterjee, S. et al.,, Adv Cancer Res. 2014; 124: 31-82). Binding and interaction with CXCR4 has been suggested as a potential method of targeted drug delivery (Wang, Y. et al., Curr Pharmcol Rep (2016) 2: 1-10).

Thus, compounds with moieties capable of selectively binding to CXCR4 (ie, CXCR4 selective binding conjugates) can be used to treat a wide range of clinical conditions associated with activation or overexpression of CXCR4, to diagnose patients, and to diagnose patients. It is believed that it may have a wide range of applications, including, but not limited to, medical imaging.

Therefore, there is a need for conjugates that can selectively bind to CXCR4.

A first aspect of the invention provides a high affinity CXCR4 selective binding ligand peptide conjugate (“PC”). In some embodiments, the high affinity CXCR4 selective binding ligand peptide conjugate selectively binds (optionally via a linker) to CXCR4 that is linked or attached to the active ingredient. Contains a peptidyl moiety having a high affinity for binding). The active ingredient can be a diagnostic agent, a therapeutic agent, or an imaging agent. In this way, the peptidyl moiety selectively binds to the CXCR4 receptor and delivers the active ingredient.

式中、nは１から側鎖官能基を有するP内のアミノ酸残基の総数までの整数であり；Pは高親和性CXCR4選択的結合ペプチド部分であり；各Lは独立して、任意のリンカーであり（すなわち、それは不存在またはポリエチレングリコール部分などのリンカーまたは当業者に知られている他のリンカーであり得る）；および各Aは独立して、診断剤、治療剤、またはイメージング剤などの活性成分である。当業者には容易に明らかであるが、Lが不存在の場合、Aは、例えばアミド結合またはエステル結合などの化学結合を介して、Pに直接結合（attached）されていることに留意されたい。1つの特定の実施形態において、式（I）の化合物は、CXCR4の過剰発現または上方調節に関連する臨床状態の診断または治療において使用され、すなわち、Aは診断剤または薬剤である。典型的には、式（I）の化合物は、（1）CXCR4に対して高親和性を有するペプチジルリガンド（すなわち、ペプチド部分）、（2）任意にリンカー、および（3）活性成分、例えば診断剤、治療剤（例えば薬剤）、またはイメージング剤（例えば放射性部分、蛍光部分など）を含む。 In one particular embodiment, the high affinity CXCR4 selective binding ligand peptide conjugate is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

In the formula, n is an integer from 1 to the total number of amino acid residues in P with side chain functional groups; P is the high affinity CXCR4 selective binding peptide moiety; each L is independent and arbitrary. It is a linker (ie, it can be absent or a linker such as a polyethylene glycol moiety or another linker known to those skilled in the art); and each A is independently a diagnostic, therapeutic, or imaging agent, etc. Is the active ingredient of. It should be noted to those skilled in the art that in the absence of L, A is directly attached to P via a chemical bond, such as an amide or ester bond. .. In one particular embodiment, the compound of formula (I) is used in the diagnosis or treatment of a clinical condition associated with overexpression or upregulation of CXCR4, i.e. A is a diagnostic agent or agent. Typically, the compounds of formula (I) are (1) peptidyl ligands (ie, peptide moieties) having a high affinity for CXCR4, (2) optionally linkers, and (3) active ingredients such as diagnostics. Includes agents, therapeutic agents (eg, agents), or imaging agents (eg, radioactive moieties, fluorescent moieties, etc.).

式中、
aは0または1であり；
AA¹はそれに結合（attached）されているイオウ原子と共に、3−メルカプトプロピオン酸、任意に置換されたシステイン、または任意に置換されたホモシステインであり；
AA²はそれに結合（attached）されているイオウ原子と共に、システインまたはホモシステインであり；
Ar¹は任意に置換されたアリールであり；
X¹はArg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、またはLys（iPr）であり；
X²はArg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、Lys（iPr）、D-Arg、D-Dap、D-Dab、D-Orn、D-Lys、D-Dap（iPr）、D-Dab（iPr）、D-Orn（iPr）、D-Lys(iPr)、または不存在であり；
X³はLys、Glyまたは不存在であり；
X⁴はLys、Phe、2Nal、1Nal、それらのD異性体、Gly、または不存在であり；

X⁵はLys、Glyまたは不存在であり；および
R²は-OR⁴または-NHR⁵であり、ここでR⁴およびR⁵はH、アルキル、任意に置換されたアリールまたは任意に置換されたアラルキルである。
当然のことながら、式（I）の任意のリンカーおよび部分A（すなわち、−L−A部分）は、それらの側鎖に存在する官能基を介して任意のアミノ酸に結合（attached）され得る。 In one particular embodiment, the partial P (ie, the high affinity CXCR4 selective binding peptide moiety) is represented by the following formula (II):

During the ceremony
a is 0 or 1;
AA ¹ is 3-mercaptopropionic acid, optionally substituted cysteine, or optionally substituted homocysteine, along with the sulfur atom attached to it;
AA ² is cysteine or homocysteine, along with the sulfur atom attached to it;
Ar ¹ is an arbitrarily substituted aryl;
X ¹ is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), or Lys (iPr);
X ² is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), Lys (iPr), D-Arg, D-Dap, D-Dab, D-Orn, D -Lys, D-Dap (iPr), D-Dab (iPr), D-Orn (iPr), D-Lys (iPr), or absent;
X ³ is Lys, Gly or absent;
X ⁴ is Lys, Phe, 2Nal, 1Nal, their D isomers, Gly, or absent;

X ⁵ is Lys, Gly or absent; and
R ² is -OR ⁴ or -NHR ⁵ , where R ⁴ and R ⁵ are H, alkyl, optionally substituted aryl or optionally substituted aralkyl.
Of course, any linker and moiety A (ie, the -LA moiety) of formula (I) can be attached to any amino acid via a functional group present in their side chain.

式中、aは0または1であり；
AA¹はそれに結合（attached）されているイオウ原子と共に、3−メルカプトプロピオン酸、任意に置換されたシステイン、または任意に置換されたホモシステインであり；
AA²はそれに結合（attached）されているイオウ原子と共に、システインまたはホモシステインであり；
Ar¹は任意に置換されたアリールであり；
X¹はArg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、またはLys（iPr）であり；
X²はArg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、Lys（iPr）、D-Arg、D-Dap、D-Dab、D-Orn、D-Lys、D-Dap（iPr）、D-Dab（iPr）、D-Orn（iPr）、D-Lys 、または不存在であり；
LおよびAは、本明細書で定義されている。 In yet another embodiment, the compound of the invention is a compound of formula (III) or a pharmaceutically acceptable salt thereof:

In the formula, a is 0 or 1;
AA ¹ is 3-mercaptopropionic acid, optionally substituted cysteine, or optionally substituted homocysteine, along with the sulfur atom attached to it;
AA ² is cysteine or homocysteine, along with the sulfur atom attached to it;
Ar ¹ is an arbitrarily substituted aryl;
X ¹ is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), or Lys (iPr);
X ² is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), Lys (iPr), D-Arg, D-Dap, D-Dab, D-Orn, D -Lys, D-Dap (iPr), D-Dab (iPr), D-Orn (iPr), D-Lys, or absent;
L and A are defined herein.

Another aspect of the invention provides a diagnostic kit comprising a high affinity CXCR4 selective binding ligand peptide conjugate disclosed herein, eg, a compound of formula (I) where A is a diagnostic agent.

Yet another aspect of the invention is (i) a high affinity CXCR4 selective binding ligand peptide conjugate disclosed herein, and (ii) a pharmaceutically acceptable carrier, diluent, excipient. , Or a composition comprising a combination thereof.

Yet another aspect of the invention provides a method of imaging cancer cells in a patient. Such methods generally include administering to the patient an image-effective amount of a high affinity CXCR4 selective binding ligand peptide conjugate of the invention, eg, a compound of formula (I) where A is the imaging agent; And the step of imaging the cancer cells in the patient using an imaging device.

Yet another aspect of the invention is by administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (I) in which A in the formula is a therapeutic agent for cancer (ie, a cancer or oncological agent). , Provide a method of treating cancer in patients.

Of course, if compound A of formula (I) is a diagnostic or imaging agent, the compounds of the invention can be used in diagnostic or imaging kits, respectively.

In one particular embodiment of the invention, the compounds of formula (I) are used in the treatment of patients suffering from rheumatoid arthritis, pulmonary fibrosis, HIV infection, or cancer. The method treats a therapeutically effective amount of a compound of formula (I), in which A is a therapeutic agent for treating rheumatoid arthritis, pulmonary fibrosis, HIV infection, or cancer, respectively. Includes the step of administering to the patient in need. Typical cancers treated with the compound of formula (I) include breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colonic rectal cancer, and multiple cancers. Includes, but is not limited to, sex myeloma, multiple neuroblastoma, and chronic lymphocytic leukemia.

Another aspect of the invention provides a targeted drug delivery method for clinical conditions associated with overexpression and / or upregulation of CXCR4. Exemplary clinical conditions include, but are not limited to, rheumatoid arthritis, pulmonary fibrosis, HIV infection, and cancer. Specific examples of cancer are breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, polyglioblastoma , And chronic lymphocytic leukemia.

Another aspect of the invention provides a method of disease diagnosis and monitoring for clinical conditions associated with overexpression and / or upregulation of CXCR4. Exemplary clinical conditions include, but are not limited to, rheumatoid arthritis, pulmonary fibrosis, HIV infection, and cancer. Examples of specific cancers are breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, polyglioblastoma , And chronic lymphocytic leukemia.

Another aspect of the invention provides a disease diagnosis and monitoring kit for clinical conditions associated with overexpression and / or upregulation of CXCR4. Exemplary clinical conditions include, but are not limited to, rheumatoid arthritis, pulmonary fibrosis, HIV infection, and cancer. Examples of specific cancers are breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colorectal cancer, multiple myeloma, polyglioblastoma , And chronic lymphocytic leukemia.

[Detailed description of the invention]
CXCR4 plays an important role in the immune and inflammatory responses in a variety of diseases and disorders, including cancer, viral infections, and autoimmune pathologies such as rheumatoid arthritis. The present invention is at least partially based on reducing or preventing CXCR4 overexpression or activation in order to treat, diagnose or image the clinical conditions associated with CXCR4 overexpression and / or activation. As used herein, the term "overexpression and / or activation" refers to the expression of a gene above its normal (ie, control) or baseline level, and / or its normal, control or base, respectively. Activation of CXCR4 above the line level.

The terms "normal," "baseline level," and "control level" are used interchangeably herein, and overexpression and / or activation of CXCR4, such as those disclosed herein. Refers to the expression and / or activity level of CXCR4 in a subject who does not have a disease or clinical condition associated with. In some embodiments, baseline levels can be normal levels, which are levels in samples from normal subjects that do not have a clinical condition associated with overexpression and / or activation (or activity) of CXCR4. Means. This is a baseline-based determination of CXCR4 expression or its biological activity, i.e., a sample tested or evaluated for a disease or clinical condition is measurable in CXCR4 expression or activation as compared to baseline levels. Allows the determination of whether to increase, decrease, or have virtually no change.

Of course, overexpression and / or activation of CXCR4 can also be determined by comparing sample results with positive controls. As used herein, the term "positive control" refers to the level of CXCR4 expression and / or activation (or activity) established in a sample from a subject or a sample from a population of individuals, wherein the sample. Has a disease or clinical condition associated with overexpression and / or activation of CXCR4 (eg, cancer, autoimmune diseases such as rheumatoid arthritis, and viral infections such as HIV infection) based on data from that sample. it was thought.

In other embodiments, baseline levels can be established from previous samples from the subject being tested, so that the progression or regression of the subject's disease can be monitored over time and / or the efficacy of treatment is assessed. Can be done.

Some aspects of the invention provide a compound that has a high affinity for CXCR4, optionally bound via a linker, to a diagnostic, therapeutic or imaging agent. Such compounds include a CXCR4 binding moiety and an active ingredient. The invention also provides a method of using the compound in targeted delivery of a therapeutic agent, eg, for treating a clinical condition manifested by overexpression and / or activation of CXCR4 or associated therewith. To do. As used herein, the term "high affinity" refers to a compound or moiety that binds to CXCR4 of about 10 nM or less, typically about 3 nM or less, often less than 1 nM (K _b ). Means to have. Alternatively, the term "high affinity" means that the compound or moiety that binds to CXCR4 has a 50% binding inhibition concentration (IC ₅₀ ) of about 30 nM or less, typically about 10 nM or less, often about 3 nM or less. means. Methods of determining the coupling constant and the IC ₅₀ are well known to those of skill in the art. For example, US Provisional Patent Application No. 62 / 384,132 filed on September 6, 2016, and US Provisional Patent Application No. 62 / 505,064 filed on May 11, 2017, assigned to the assignee of the invention. See No. and PCT Patent Application No. PCT / US17 / 50106 filed on September 5, 2017, assigned to the assignee of the invention. All of these are incorporated herein by reference in their entirety. In particular, the values of K _b and IC ₅₀ are determined using the CXCR4 / ¹²⁵ I-SDF-1α binding experiment described in the provisional patent application referenced above. When referring to a number, the term "about" refers to ± 20%, typically ± 10%, and often ± 5% of that number.

式中、nは1から（P中のアミノ酸の総数および側鎖官能基の総数）の合計までの整数であり、典型的にはnは1からP中のアミノ酸の数であり、またはnは1から官能基を有する側鎖を有するP中のアミノ酸の数であり、しばしば、nは1から5の整数であり、より多くの場合、nは1から3の整数であり；
Aは、1以上の診断剤、治療剤、またはイメージング剤であり；
各Lは独立して二官能性リンカーまたは不存在であり；Lが不存在の場合、Aは例えば、アミド結合またはエステル結合などの化学結合を介してPに結合（attached）されていて；およびPは高親和性CXCR4選択的結合ペプチジルリガンド（すなわち、CXCR4に選択的に結合するペプチド部分）である。 In one particular aspect of the invention, the high affinity CXCR4 selective binding ligand peptide conjugate (“PC”) is the following formula (I) or a pharmaceutically acceptable salt thereof:

In the formula, n is an integer from 1 to the sum of (total number of amino acids in P and total number of side chain functional groups), typically n is the number of amino acids in 1 to P, or n is The number of amino acids in P having a side chain with a functional group from 1, often n is an integer of 1 to 5, and more often n is an integer of 1 to 3;
A is one or more diagnostic, therapeutic, or imaging agents;
Each L is independently a bifunctional linker or absent; in the absence of L, A is attached to P via a chemical bond, such as an amide or ester bond; P is a high affinity CXCR4 selective binding peptidyl ligand (ie, the peptide moiety that selectively binds to CXCR4).

The variable n is an integer from 1 to the sum of (total number of amino acids in P and total number of side chain functional groups). Typically, n is an integer of 1-7, often 1-5, more often 1-3, and most often 1 or 2. For example, if there are a total of 7 amino acid residues in P and 2 lysine groups (with side chain functional group-NH ₂ ), n is from 1 to 9 (7 amino acid residues of P + It can be an integer of two side chain functional groups). In this way, all functional groups of P can be attached to the -LA moiety.

Part A in the compound of formula (I) can be attached to any part of the P part. Typically, the A moiety is at any one of the N-terminus or C-terminus of the peptide (P moiety), the functional groups present on the side chain of the amino acid residue of the peptide, or their positions. It is attached. In some embodiments, the compound of formula (I) has multiple A moieties.

式中：
aは0または1であり；
AA¹はそれに結合（attached）されているイオウ原子と共に、3−メルカプトプロピオン酸、任意に置換されたシステイン、または任意に置換されたホモシステインであり；
AA²はそれに結合（attached）されているイオウ原子と共に、システインまたはホモシステインであり；
Ar¹は任意に置換されたアリールであり；
X¹はArg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、またはLys（iPr）であり；
X²はArg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、Lys（iPr）、D-Arg、D-Dap、D-Dab、D-Orn、D-Lys、D-Dap（iPr）、D-Dab（iPr）、D-Orn（iPr）、D-Lys（iPr）または不存在であり；
X³はLys、Glyまたは不存在であり；
X⁴はLys、Phe、2Nal、1Nal、そのD-異性体、Gly、または不存在であり；
X⁵はLys、Glyまたは不存在であり；および
R²は-OR⁴または-NHR⁵であり、ここでR⁴およびR⁵はH、アルキル、任意に置換されたアリール、または任意に置換されたアラルキルである。
式（I）の化合物の部分−L−Aは、AA¹（例えば、システインまたはホモシステインのα−アミノ基）および/またはR⁴および/またはR⁵に結合（attached）され得、またはR⁴およびR⁵は−L−Aであり得、ここでLは任意にリンカーであり、およびAは治療剤、治療剤、またはイメージング剤である。また更に、部分−L−Aは、ペプチジル部分のN末端アミノ酸のα−アミノ基または任意のアミノ酸の側鎖の官能基に結合（attached）され得る。 In one particular embodiment, P is a high affinity CXCR4-linked peptidyl of formula (II):

During the ceremony:
a is 0 or 1;
AA ¹ is 3-mercaptopropionic acid, optionally substituted cysteine, or optionally substituted homocysteine, along with the sulfur atom attached to it;
AA ² is cysteine or homocysteine, along with the sulfur atom attached to it;
Ar ¹ is an arbitrarily substituted aryl;
X ¹ is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), or Lys (iPr);
X ² is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), Lys (iPr), D-Arg, D-Dap, D-Dab, D-Orn, D -Lys, D-Dap (iPr), D-Dab (iPr), D-Orn (iPr), D-Lys (iPr) or absent;
X ³ is Lys, Gly or absent;
X ⁴ is Lys, Phe, 2Nal, 1Nal, its D-isomer, Gly, or absent;
X ⁵ is Lys, Gly or absent; and
R ² is -OR ⁴ or -NHR ⁵ , where R ⁴ and R ⁵ are H, alkyl, optionally substituted aryl, or optionally substituted aralkyl.
A portion of the compound of formula (I) -LA can be attached to AA ¹ (eg, the α-amino group of cysteine or homocysteine) and / or R ⁴ and / or R ⁵ , or R ⁴ And R ⁵ can be −LA, where L is optionally a linker, and A is a therapeutic, therapeutic, or imaging agent. Furthermore, the partial-LA can be attached to the α-amino group of the N-terminal amino acid of the peptidyl moiety or the functional group of the side chain of any amino acid.

In yet another embodiment, A is an imaging agent. One particular example of a useful imaging agent of the present invention is a positron (positron) such as ³⁴ Cl, ⁴⁵ Ti, ⁵¹ Mn, ⁶¹ Cu, ⁶³ Zn, ⁶⁸ Ga, ¹¹ C, ¹³ N, ¹⁵ O, and ¹⁸ F. ) Includes released nuclides. Typically, the positron emitting nuclide is attached to or attached as part of the linker (L portion).

Another example of a useful imaging agent includes a radiometal isotope coordinated (ie, chelated) to a chelating group. Particularly useful radioactive metal isotopes include technetium, rhenium, gallium, gadolinium, indium, copper and combinations thereof. Suitable chelating groups for specific radioactive metal isotopes are well known to those of skill in the art. For example, ferrocene and its derivatives, ethylenediaminetetraacetic acid (“EDTA”), its derivatives, peptidyl moiety Dap-Asp-Cys and its derivatives (see US Pat. No. 7,128,893), and others known in the art. It is a thing.

Yet another example of a useful imaging agent comprises a contrast agent. Contrast agents are widely used, for example, in magnetic resonance imaging (MRI). A wide variety of contrast media are known to those skilled in the art, including gadobenate, gadobutrol, gadodiamide, gadophosbeset, gadopentate, gadoterate, gadoteridol, gadobersetamide, gadoxetate, and iron oxide.

Yet another example of a useful imaging agent is a fluorescent dye such as fluorenylmethyloxycarbonyl (FMOC) and its derivatives, AlexaFluor dye, Oregon Green dye, fluorescein, BODIPY (boron-dipyrromethene) dye, cyanine dye, rhodamine dye. , DyLight dyes, and Texas Red.

In other embodiments, A is a diagnostic agent. Exemplary diagnostic agents that can be used in the compounds of the invention include imaging agents, isotopic agents, or radioactive agents.

In yet another embodiment, the linker L comprises a functional group capable of releasing A in vivo. In this way, part A is released in vivo. The appropriate functional group capable of releasing A depends on the nature of the functional group on the moiety A linked to the linker. For example, when the functional group on A is a hydroxyl group (ie-OH) or an amino group (-NH ₂ ), the functional group on L is such that an ester bond or an amide bond is formed between A and L, respectively. It can be a carboxylate. If the functional group on A is a carboxylic acid, the corresponding functional group on L can be a hydroxyl or amino group for forming an ester or amide bond, respectively. Other suitable functional groups on L capable of releasing A in vivo include disulfide bond linkages, ester linkages, thiol-maleimide linkages, etc. It is well known to.

In yet another embodiment, A is a therapeutic agent. Suitable therapeutic agents include those known to those skilled in the art for the treatment of cancer, autoimmune diseases (eg, rheumatoid arthritis), viral infections (eg, HIV infection) and the like. Exemplary therapeutic agents useful in the compounds of the present invention are bleomycin, daunorubicin, doxorubicin, docetaxel, irinotecan, monomethyloristatin E, mertancin, paclitaxel, SN-38, tesillin, tubericin, binca alkaloids, and similar thereof. It includes, but is not limited to, body or derivatives, HIV protease inhibitors, HIV fusion inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV entry inhibitors, and therapeutic agents for autoimmune diseases.

、
ここでR^aまたはR^bの少なくとも1つがS-パクリタキセルを含む場合には、
R^aはアセチル-、アセチル-Cys（S-パクリタキセル）-、またはアセチル-Cys（S-パクリタキセル）-（mini-PEG6）-であり；および
R^bはグリシルアミド、グリシル-Cys（S-パクリタキセル）-アミド、または（mini-PEG6）-Cys（S-パクリタキセル）-アミドである。 Specific examples of the high affinity CXCR4 selective binding ligand peptide conjugates of the invention include, but are not limited to:
Cyclo [Phe-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly- (D-Glu)]-Lys (iPr)-(mini-PEG6) -Cys (S-paclitaxel) -Gly-NH ₂ , Where the cyclic structure is formed between α-amino of Phe linked to the side chain of D-Glu (SEQ ID NO: 3);
R ^a -Cyclo [Cys-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly-Cys] -Lys (iPr) -R ^b (SEQ ID NO: 4);
R ^a -cyclo [hCys-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly-Cys] -Lys (iPr) -R ^b (SEQ ID NO: 5);
R ^a -cyclo [Cys-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly-hCys] -Lys (iPr) -R ^b (SEQ ID NO: 6); and

,
Where at least one of R ^a or R ^b contains S-paclitaxel,
R ^a is acetyl-, acetyl-Cys (S-paclitaxel)-, or acetyl-Cys (S-paclitaxel)-(mini-PEG6)-; and
R ^b is glycylamide, glycyl-Cys (S-paclitaxel) -amide, or (mini-PEG6) -Cys (S-paclitaxel) -amide.

式中：
aは0または1であり；
AA¹は、それに結合（attached）されているイオウ原子と共に、3−メルカプトプロピオン酸、任意に置換されたシステイン、または任意に置換されたホモシステインであり、ここでAは、前記システインまたはホモシステインのα−アミノ基に任意に結合され；
AA²は、それに結合（attached）されているイオウ原子と共に、システインまたはホモシステインであり；
Ar¹は、任意に置換されたアリールであり；
X¹は、Arg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、またはLys（iPr）であり；
X²は、Arg、Dap、Dab、Orn、Lys、Dap（iPr）、Dab（iPr）、Orn（iPr）、Lys（iPr）、D-Arg、D-Dap、D-Dab、D-Orn、D-Lys、D-Dap（iPr）、D-Dab（iPr）、D-Orn（iPr）、D-Lys 、または不存在であり；
Lは任意にリンカーであり；および
Aは治療剤、診断剤またはイメージング剤である。 In some embodiments, the compound of the invention is a compound of formula (III) or a pharmaceutically acceptable salt thereof:

During the ceremony:
a is 0 or 1;
AA ¹ is 3-mercaptopropionic acid, optionally substituted cysteine, or optionally substituted homocysteine, along with the sulfur atom attached to it, where A is said cysteine or homocysteine. Arbitrarily attached to the α-amino group of
AA ² is cysteine or homocysteine, along with the sulfur atom attached to it;
Ar ¹ is an arbitrarily substituted aryl;
X ¹ is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), or Lys (iPr);
X ² is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), Lys (iPr), D-Arg, D-Dap, D-Dab, D-Orn, D-Lys, D-Dap (iPr), D-Dab (iPr), D-Orn (iPr), D-Lys, or absent;
L is optionally a linker; and
A is a therapeutic, diagnostic or imaging agent.

In these embodiments, a is 0 in some examples. In yet another example, a is 1. In yet another example, AA ¹ is 3-mercaptopropionic acid, along with the sulfur atom attached to it. In yet another example, AA ¹ is cysteine, along with the sulfur atom attached to it. In another example, AA ¹ is homocysteine, along with the sulfur atom attached to it.

In yet another embodiment, AA ² is a cysteine, along with a sulfur atom attached to it. In yet another embodiment, AA ² is homocysteine, along with the sulfur atom attached to it.

In yet another embodiment, A in the compound of formula (III) is an imaging agent.

In other embodiments, A in the compound of formula (III) is a therapeutic agent. Exemplary therapeutic agents within the compounds of formula (III) are bleomycin, calikeamycin, daunorubicin, docetaxel, doxorubicin, irinotecan, meltancin, monomethyloristatin E, paclitaxel, SN-38, tesillin, topotecan, tubericin, binca. Includes, but is not limited to, alkaloids, their analogs or derivatives, and combinations thereof.

L is, for example, H ₂ N-CH ₂ CH _2- (PEG) m-CH ₂ CH _2- COOH, HOOC-CH ₂ CH _2- (PEG) m-CH ₂ CH _2- COOH, or H ₂ N-CH. ₂ CH _2- (PEG) m-CH ₂ Can be any biocompatible bifunctional linker such as polyethylene glycol (PEG) in the form of CH _2- NH ₂ , natural and unnatural amino acids or polyamino acids (PAA). , Where m is an integer from 0 to 100, typically 1 to 50, often 1 to 25, and more often 1 to 10. In general, when L is a polymer (eg, PEG, PAA), the total number of monomers in the chain is about 1 (ie, monomer) to about 20, typically about 1 to about 10, and often about 1 to 6. Is.

In yet another embodiment, A in the compound of formula (III) is a diagnostic agent such as a radiopharmaceutical, a fluorescent agent or the like. Such imaging agents are well known to those of skill in the art. For example, magnetic resonance imaging agents, ultrasonic contrast agents, and contrast agents for radiocontrast agents. For example, see en.wikipedia.org/wiki/Contrast_agent.

Furthermore, the combinations of the various groups described herein can form other embodiments. In this way, various compounds are embodied within the scope of the present invention.

Another aspect of the invention provides a diagnostic kit comprising the high affinity CXCR4 selective binding ligand peptide conjugate described herein in which A of compound of formula (I) is a diagnostic agent.

Yet another aspect of the invention provides a composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can include diluents, excipients, flavors, adjuvants, binders, stabilizers, colorants, or combinations thereof. In general, "pharmaceutically acceptable carriers" are generally safe, non-toxic, and useful for preparing pharmaceutical compositions that are neither biologically nor otherwise desirable. Any excipient, including excipients acceptable for veterinary and human pharmaceutical applications.

The present invention comprises at least one pharmaceutically acceptable mixture of at least one compound of the invention, or individual isomers, racemic or non-racemic isomers, or pharmaceutically acceptable salts or solvates thereof. Carrier, and optionally other pharmaceutical compositions, including with other therapeutic and / or prophylactic ingredients.

In general, the compounds of the present invention are administered in therapeutically effective amounts by any acceptable mode of administration for agents of similar utility. Appropriate dose ranges include the severity of the disease being treated, the age and relative health of the subject, the efficacy of the compounds used, the route and form of administration, the indications for which administration is directed, and the preferences of the physician involved. And, depending on a number of factors such as experience, typically 1 to 500 mg / day, typically 1 to 100 mg / day, and often 1 to 30 mg / day. Those skilled in the art treating such diseases will be able to ascertain therapeutically effective amounts of the compounds of the invention, typically without undue experimentation and, relying on personal knowledge and disclosure of the present application.

Typically, the compounds of the invention are oral (including cheek and sublingual), rectal, nasal, topical, lung, vaginal, or parenteral (intramuscular, intraarterial, sublingual, subcutaneous and venous). It is administered as a pharmaceutical formulation, including those suitable for administration (including), or in a form suitable for administration by inhalation or infusion. A typical method of administration is generally oral administration using a convenient daily dose regimen that can be adjusted according to the degree of distress.

One or more compounds of the invention may be in the form of pharmaceutical compositions and unit doses, along with one or more conventional adjuvants, carriers, or diluents. Pharmaceutical compositions and unit dosage forms may contain conventional proportions of conventional ingredients with or without additional active compounds or principles, and unit dosage forms are commensurate with the intended daily dose range used. It may contain any suitable effective amount of active ingredient. Pharmaceutical compositions may be used as solids such as tablets or filled capsules, semi-solids, powders, sustained release formulations, or liquids such as liquids, suspensions, emulsions, elixirs, or filled capsules for oral use. Or can be used in the form of suppositories for rectal or vaginal administration; or can be used in the form of sterile injectable solutions for parenteral use. Formulations containing about 1 milligram of active ingredient per tablet, or more broadly, about 0.01 to about 100 milligrams of active ingredient, are suitable representative unit dosage forms.

The compounds of the present invention can be formulated in a wide variety of oral dosage forms. Pharmaceutical compositions and dosage forms can include as active ingredients one or more compounds of the invention or pharmaceutically acceptable salts thereof. The pharmaceutically acceptable carrier can be either solid or liquid. Solid formulations include powders, tablets, pills, capsules, cashiers, suppositories, and dispersible granules. The solid support can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrants, or encapsulating materials. In powders, the carrier is generally a pulverized solid that is a mixture with the pulverized active ingredient. In tablets, the active ingredient is generally mixed in an appropriate proportion with a carrier having the required binding capacity and compressed to the desired shape and size. Powders and tablets preferably contain from about 1 to about 70% active compound. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacant, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cacao butter and the like. The term "formulation" is intended to include a formulation of an active compound using an encapsulating material as a carrier, which provides a capsule in which the active ingredient with or without a carrier is enclosed by a carrier associated thereto. .. Similarly, cashiers and lozenges are included. Tablets, powders, capsules, pills, cashews, and lozenges can be in solid form suitable for oral administration.

Other forms suitable for oral administration are liquid forms containing emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid forms intended to be converted to liquid forms immediately prior to use. Includes formulations of. The emulsion can be prepared in a solution, such as in an aqueous propylene glycol solution, or may contain an emulsifier such as, for example, lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active ingredient in water and adding the appropriate colorants, flavors, stabilizers, and thickeners. Aqueous suspensions are prepared by dispersing the micronized active ingredient in water using viscous materials such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other well-known suspending agents. obtain. Solid formulations include solutions, suspensions, and emulsions, and in addition to active ingredients, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc. May include.

The compounds of the invention can also be formulated for parenteral administration (eg, by injection, eg, bolus injection or continuous infusion), with ampoules, prefilled syringes, small volume infusions, or preservatives added. Can be provided in unit dose form in multiple dose containers. The composition can take the form of suspensions, liquids, or emulsions in oily or aqueous vehicles, such as liquids in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (eg olive oil), and organic esters for injection (eg ethyl oleate), preservatives, wetting agents. , Emulsifiers or suspending agents, stabilizers and / or formulation agents such as dispersants. Alternatively, the active ingredient is in powder form obtained by aseptic isolation of a sterile solid or by lyophilization from solution to compose with a suitable vehicle, eg sterile water, pyrogen-free water, prior to use. obtain.

The compounds of the present invention can be formulated for topical administration to the epidermis as ointments, creams or lotions, or as transdermal patches. Ointments and creams can be formulated with aqueous or oily bases, for example, with the addition of appropriate thickeners and / or gelling agents. Lotions can be formulated with aqueous or oily bases, and generally also contain one or more emulsifiers, stabilizers, dispersants, suspensions, thickeners, or colorants. Let's go. Formulations suitable for topical oral administration are lozenges containing active agents in flavored bases, usually sucrose and gum arabic (acacia) or tragacant; gelatin and glycerin or sucrose and gum arabic. Includes troches containing the active ingredient in an inert base such as; and a mouthwash containing the active ingredient in a suitable liquid carrier.

The compounds of the invention can be formulated for administration as suppositories. A low melting point wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active ingredient is homogeneously dispersed, for example by stirring. The molten homogeneous mixture is then poured into a mold of a convenient size, cooled and solidified.

The compounds of the present invention can also be formulated for intravaginal administration. Pessaries, tampons, creams, gelling agents, pastes, foams or sprays contain, in addition to the active ingredient, carriers as known to be suitable in the art.

The compounds of the present invention can be formulated for nasal administration. The liquid or suspension is applied directly to the nasal cavity by conventional means, for example using an infusion device, pipette or spray. The formulation may be provided in a single dose form or a multiple dose form. In the latter case of drip or pipette, this can be achieved by the patient being administered an appropriate predetermined amount of liquid or suspension. In the case of sprays, this can be achieved, for example, with a measuring atomizing spray pump.

The compounds of the present invention can be formulated for administration of aerosols, especially to the respiratory tract, including intranasal administration. Compounds will generally have small particle sizes on the order of, for example, 5 microns or less. Such particle sizes can be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as chlorofluorocarbon (CFC), such as dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas. Aerosols can also conveniently include surfactants such as lecithin. The dose of drug can be controlled by a metering valve. Alternatively, the active ingredient may be provided in the form of a dry powder, for example a starch derivative such as lactose, starch, hydroxypropyl methylcellulose and a powder mixture of compounds in a suitable powder base such as polyvinylpyrrolidin (PVP). The powder carrier typically forms a gel in the nasal cavity. The powder composition may be provided in a unit dose form, eg, in a blister pack, eg capsule or cartridge, in which gelatin or powder can be administered by an inhaler, for example.

If desired, the formulation can be prepared with an enteric coating suitable for continuous or controlled release administration of the active ingredient. For example, the compounds of the invention can be formulated in a transdermal drug delivery device or a subcutaneous drug delivery device. These delivery systems are advantageous when sustained release of the compound is required or desired, and when patient compliance with the treatment regimen is important. Compounds in transdermal delivery systems are often attached to a skin-adhesive solid support. The compound of interest can also be combined with a penetration enhancer, such as Azone (1-dodecylazacycloheptane-2-one). The continuous release delivery system can be inserted subcutaneously into the subcutaneous layer by surgery or injection. Subcutaneous implants encapsulate compounds in lipophilic membranes such as silicone rubber or biodegradable polymers such as polylactic acid.

Pharmaceutical formulations are typically in unit dosage form. In such a form, the formulation is often subdivided into unit doses containing the appropriate amount of active ingredient. The unit dosage form can be a packaged formulation, which package comprises a separate amount of formulation, such as packaged tablets, capsules, and powder in vials or ampoules. Also, the unit dosage form can be either a capsule, a tablet, a cashier, or a lozenge itself, or any of the appropriate number of packaging forms.

Other suitable pharmaceutical carriers and their formulations are described in Remington: The Science and Practice of Pharmacy 1995, edited by E.W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.

Provide the active ingredient as a pharmaceutical composition where therapeutically effective amounts of the compounds of formula (I), as well as their pharmaceutically acceptable salts, can be administered as untreated chemicals for therapeutic use. Is possible. That is, the present disclosure describes therapeutically effective amounts of compounds of formula (I) or pharmaceutically acceptable salts thereof or prodrugs thereof, as well as one or more pharmaceutically acceptable carriers, diluents or excipients. Further provided are pharmaceutical compositions comprising. When a combination is applied, the term refers to the amount of active ingredient combination that provides a therapeutic effect, whether administered in combination, continuously or simultaneously. The compounds of formula (I) and their pharmaceutically acceptable salts are as described above. The carrier, diluent, or excipient must be acceptable in the sense that it is compatible with the other ingredients of the formulation and is not harmful to its recipient. According to another aspect of the present disclosure, one or more pharmaceutically acceptable carriers, diluents, or excipients of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a prodrug thereof. Also provided is a method of making a pharmaceutical formulation comprising mixing with an agent.

When the compositions of the present disclosure include a combination of the compounds of the present disclosure and one or more additional therapeutic or prophylactic agents, both the compounds and the additional agents are usually about 10 doses commonly administered in a monotherapy regimen. It is provided at dose levels between ~ 150%, more typically between about 10-80%.

Yet another aspect of the invention is to administer to a patient a high affinity CXCR4 selective binding ligand peptide conjugate of formula (I) where A is an imaging agent in an amount of formula effective for imaging, and imaging. Provided is a method of imaging a cancer cell in a patient, which comprises imaging the cancer cell in the patient using an apparatus. The imaging device used depends on the nature of the imaging agent A of the compound of formula (I). For example, if A is a positron emitting nuclide, the imaging device used is a PET scan, and if A is a contrast agent, the imaging device can be a computer topography device or an MRI device. If A is a radioisotope, the imaging device can be an X-ray machine or other similar device.

One particular aspect of the invention provides a method of treating cancer in a patient. The method comprises a therapeutically effective amount of a compound of formula (I) (in the formula, A is a cancer drug) or a compound of formula (I) (in the formula, A is a cancer drug). Includes administering to a cancer patient a pharmaceutical composition comprising.

Another particular aspect of the invention is a diagnostic or imaging kit comprising a high affinity CXCR4 selective binding ligand peptide conjugate (PC) of formula (I), wherein A is a diagnostic or imaging agent, respectively. I will provide a.

Yet another particular aspect of the invention provides a method of treating a patient suffering from rheumatoid arthritis, pulmonary fibrosis, HIV infection, or cancer. The method comprises administering a therapeutically effective amount of a compound of formula (I) to a patient in need of the treatment. In this method, compound A of formula (I) is a therapeutic agent that can be used to treat a particular clinical condition to be treated. Some of the cancers that can be treated with the compounds of the invention are breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colonic rectal cancer, multiple cancers. Includes, but is not limited to, myeloma, multiple neuroblastoma, and chronic lymphocytic leukemia.

Further objects, advantages, and novel features of the present invention will become apparent to those skilled in the art by examining the following examples not intended to be limiting. In the examples, the structurally simplified procedures to be performed are described in the present tense, and the procedures performed in the laboratory are described in the past tense.

The following abbreviations are used: Ac: acetyl; Boc: tert-butyloxycarbonyl; BOP: (benzotriazole-1-yloxy) -tris (dimethylamino) phosphonium hexafluorophosphate; Bz: benzoyl; Bzl: benzyl; Dab: 1,4-diaminobutyric acid; Dap: 1,3-diaminopropionic acid; DCC: dicyclohexylcarbodiimide; DCM: dichloromethane; DIC: diisopropylcarbodiimide; DIEA: diisopropyl-ethylamine; DMAP: 4- (N, N-dimethylamino) ) Pyridine; DMF: N, N-dimethylformamide; DMSO: dimethyl sulfoxide; EDT: 1,2-ethanedithiol; Et: ethyl; Fmoc: 9-fluoro-enylmethoxycarbonyl; HATU: N-[(dimethylamino)- 1H-1,2,3-triazolo [4,5-b] pyridine-1-ylmethylene] -N-methylmethaneaminium hexafluorophosphate N-oxide; HBTU: O-benzo-triazolyl-N, N, N' , N'-tetramethyluronium hexafluorophosphate; HCTU: 1H-benzotriazolium 1- [bis (dimethylamino) methylene] -5-chloro-3-oxide hexafluorophosphate; HOBt: hydroxybenzotriazole; hCys: Homocysteine; iPr: isopropyl; IPA: isopropyl alcohol; Me: methyl; Mmt: 4-methoxytrityl; Mpa: 3-mercaptopropionic acid; 2Nal: 2-naphthylalanine; 1Nal: 1-naphthylalanine; NMM: N-methyl Morphorin; NMP: N-methylpyrrolidone; Orn: Ornitine; Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl; PBS: phosphate buffered saline; PyBOP: (benzotriazole-1- Iloxy) -tris (pyrrolidino) -phosphonium hexafluoro-phosphate; PyBrOP: bromotris (pyrrolidino) phosphonium hexafluorophosphate; tBu: tert-butyl; TFA: trifluoroacetic acid; TFE: trifluoroethanol; THF: tetrahydrofuran; TIS: tri Isopropylsilane; Trt: Trityl; mini-PEG6: Ethylene glycol 6 Dimer (6-mer); All common amino acids are expressed as three-letter symbols or otherwise specified.

Mass Spectrometry (MS) Analysis: The preparation of the compounds of the invention described in the Examples below is meant to be exemplary, but not limiting. In each of these examples, the observed molecular weight is reported as a de-convoluted value. The deconvolution value is derived from the formula MW (measured value) = n (m / z) -n, in which m / z represents a charged ion (positive mode) and n is the number of charges of a specific species. is there. If there are multiple charged species in the mass spectrum, the measured molecular weights are reported as average.

Common Methods for Peptide Synthesis, Cyclic Structure Formation, and Salt Exchange: Peptides were synthesized using solid phase peptide synthesis chemistry known in the art. The cyclic structure of these peptides, for disulfides, by using air oxidation, or by using iodine oxidation in the presence of acidic acids, or for bisthioether rings, such as a 15 mM ammonium bicarbonate solution. It was established by nucleophilic substitution with a bis (halomethyl) allyl compound, typically 1.3 equivalents of a bis (bromomethyl) allyl compound, in the presence of the base.

Isotope or radiolabeled acetone are commercially available from various manufacturers. If there is a need for custom preparation of isotopes or radiolabeled acetone, methods can be found in known techniques such as Rolf Voges, et al., Preparation of Compounds Labeled with Tritium and Carbon-14 (John Wiley & Sons (2009)). Can be issued.

Peptides with different linker - preparation of a medicament conjugates are known in the art (. GTHermanson, Bioconjugate Techniques, 2 nd Ed, Academic PressElsevier, 2008). For example, procedures for thiol-mediated cysteine side chains (ie, peptide linkage or attachment) have been reported by Backer and collaborators (MV Backer, et al., Pp 275). -294 in Methods in Molecular Biology, vol. 494: Peptide-Based Drug Design, edited by L. Otvos, Humana Press, NewYork, NY, 2008).

Paclitaxel Activation-2'-Maleimide-Preparation of Paclitaxel: Dissolve 1 gram of paclitaxel (1.2 mmol) in 160 mL DCM, add 0.12 mmol DMAP, and cool the reaction mixture to 0 ° C. 2.4 mmol of 3-maleimide propionic acid followed by 1.2 mmol of DIC was added to the cooled reaction mixture under stirring. The reaction mixture was then slowly warmed to room temperature and the coupling reaction was allowed to proceed at room temperature for 18 hours under continuous stirring. The crude product of 2'-maleimide-paclitaxel was purified to purity> 90% and used for conjugation to the cyclic CXCR4 antagonist peptide.

Most of the drugs disclosed herein as cancer treatments conjugated to a high affinity CXCR4 binding ligand peptide conjugate can be activated and incorporated in a similar manner known to those of skill in the art.

Purification, salt morphology conversion, and final product characterization: The final product was purified by reverse phase HPLC and further characterized by analytical HPLC and mass spectrometry. Peptides purified from reverse phase HPLC were usually in the form of trifluoroacetic acid (TFA). This salt was typically converted to a more pharmaceutically convenient salt form, such as acetate or hydrochloride form. Conversion of the peptides in the TFA salt to the hydrochloride salt could be achieved by repeated lyophilization of the peptides in the TFA salt in dilute hydrochloric acid solution. The following methods were typically used to convert the peptides in the TFA salt to acetate. The strong anion exchange resin (chloride form, substitution 3 mmol / g, water content 50%, using 2 grams of resin per gram of peptide) was first washed 3 times with milliQ water and then 1 N NaOH solution. Was washed 3 times, 5 minutes / 1 time, and then 5 times, 5 minutes / 1 time with milliQ water. The resin was further washed with 75% ethanol water until the pH reached about 7.4. The resin was treated with 10% acetic acid solution 3 times, 5 minutes each time. The resin was then washed 3 times with 1% acetic acid solution for 5 minutes each time. The resin is ready for salt conversion of the purified peptide.

The purified lyophilized peptide was dissolved in 1% acetic acid solution and added to the prepared resin described above. The mixture was stirred at room temperature for 1 hour or magnetically. The supernatant was separated. The resin was washed 3 times with 1% acetic acid solution. The supernatant and wash solution were mixed, filtered through a 0.22 mm membrane and lyophilized to give the peptide in acetate.

Example 1: Synthesis of (MLB-1707)

Peptide chain construction: Cys (Mmt) -Tyr (tBu) -Lys (iPr, Boc)-(D-Arg (Pbf))-2Nal-Gly-Cys (Mmt) -Lys (iPr, Boc)-(mini-PEG6 ) -The peptide chain of Cys (Trt) (SEQ ID NO: 8) was constructed by standard Fmoc chemistry using Lysine AM resin. Briefly, 0.8 g of Rink AM resin was swollen in DCM for 14 hours and then washed 4 times with DMF. Removal of Fmoc was performed in 20% piperidine in DMF for 20 minutes at room temperature and washed several times with DMF. The ninhydrin test was negative. Stepwise chain construction was initiated from the C-terminus of the linear peptide using Fmoc-Cys (Trt) -OH. Three equivalents of the protected amino acid Fmoc-Cys (Trt) -OH were activated with DIC / HOBt in DMF and coupled to the Fmoc-removed Rink AM resin prepared above for 2 hours at room temperature. The ninhydrin test was negative. Capping of unreacted amino groups was performed for 30 minutes using a 1: 1: 2 volume ratio mixture of 5 mL acetic anhydride / DIEA / DCM. This was followed by removal of Fmoc using 20% piperidine in DMF for 20 minutes. The following residues were continuously coupled without capping: Fmoc-mini-PEG6-OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Gly- OH, Fmoc-2Nal-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Tyr (tBu) -OH, and Fmoc-Cys (Mmt) -OH. Fmoc protection was removed again using 20% piperidine in DMF for 20 minutes after coupling of the last residue Fmoc-Cys (Mmt). N-terminal acetylation was performed at room temperature for 30 minutes using a mixture of 5 mL acetic anhydride / DIEA / DMF (1: 1: 4, v / v / v). The resin was then washed 3 times with DMF, then 2 times with DCM and dried under vacuum.

Removal of Mmt protection on Cys residues and cyclization on solid phase: Mmt protection of Cys side chains is 30 mL of cleavage cocktail per gram of resin (TFA / EDT / TIS / DCM, 3: 1.5: 1.5: Removed using 100, v / v). This deprotection procedure was repeated 3 times, 10 minutes each, at room temperature. The resin was then washed 3 times with DCM and 10 times with DMF to ensure complete removal of residual TFA. To the well-washed resin, 10 mL of DMF and 2 mL of DIEA per gram of resin were added, followed by a slow instillation of 1.2 equivalents of 1,2-bis (bromomethyl) benzene. The cyclization reaction was allowed to proceed for 1 hour at room temperature. Test cleavage and MS confirmed the completion of cyclization. The reaction mixture was then drained from the resin, and the resin was further washed 3 times with DMF and 2 times with DCM. The resin was then dried under vacuum prior to cutting.

Peptide cleavage and side chain deprotection from solid support: The finished peptide is cleaved with 10 mL / g resin for 70 minutes at room temperature Cocktail (TFA / EDT / TIS / H ₂ O / thioanisol / phenol, solution 100 mL) Deprotected with 81.5 mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mL H ₂ O, 5.0 mL thioanisol, and 5.0 g of phenol), and dry resin Separated from. The resin was removed by filtration and washed with a few milliliters of cutting cocktail. Eight volumes of methyl t-butyl ether was added to the cleavage mixture. The crude peptide precipitate was separated by centrifugation at 3000 rpm for 3 minutes. The crude peptide precipitate was washed 3 times with methyl t-butyl ether. The crude peptide was purified by preparative HPLC to a purity> 90% and lyophilized.

Paclitaxel conjugation: The purified cyclic peptide is mixed with the previously prepared 2'-maleimide-paclitaxel in a molar ratio of 1: 1.2, and a 30% aqueous acetonitrile solution is added to give a final peptide concentration of 10 mg / mL. It was. A 0.5 mol / L NH ₄ HCO ₃ solution was used to adjust the reaction mixture to pH 7.5. The conjugation reaction was completed in about 30 minutes, as confirmed by MS. The final product was purified using a reverse phase preparative column Daisogel (50 x 250 mm, 8 mm); mobile phase-solvent A: 0.1% TFA water; solvent B: 0.1% TFA acetonitrile. Fractions containing the target product were mixed and lyophilized (TFA salt).

Peptides in acetate were obtained by salt exchange as described above. Analysis of final peptide product HPLC purity 95.14%; MW calculated (cal.): 2725.56; MW observed (obs.): 2274.75.

Example 2: Synthesis of (MLB-1708)

Peptide chain construction: Cys (Trt) -Cys (Mmt) -Tyr (tBu) -Lys (iPr, Boc)-(D-Arg (Pbf))-2Nal-Gly-Cys (Mmt) -Lys (iPr, Boc) The -Gly (SEQ ID NO: 9) peptide chain was constructed using standard Fmoc chemistry using Rink AM resin. Briefly, 3.6 g of Rink AM resin was swollen in DCM for 14 hours and then washed 4 times with DMF. Removal of Fmoc was performed in 20% piperidine in DMF for 20 minutes at room temperature and washed several times with DMF. The ninhydrin test was negative. Stepwise chain construction was initiated from the C-terminus of the linear peptide using Fmoc-Gly-OH. Three equivalents of the protected amino acid Fmoc-Gly-OH were activated with DIC / HOBt in DMF and coupled to the Fmoc-removed Rink AM resin prepared above for 2 hours at room temperature. The ninhydrin test was negative. Capping of unreacted amino groups was performed for 30 minutes using a mixture of 20 mL acetic anhydride / DIEA / DCM in a volume ratio of 1: 1: 2. This was followed by removal of Fmoc using 20% piperidine in DMF for 20 minutes. The following residues were continuously coupled without capping: Fmoc-Lys (iPr, Boc) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Mmt) -OH, and Fmoc-Cys (Trt) -OH. Fmoc protection was removed again using 20% piperidine in DMF for 20 minutes after coupling of the last residue Fmoc-Cys (Trt) -OH. N-terminal acetylation was performed at room temperature for 30 minutes using a mixture of 20 mL acetic anhydride / DIEA / DMF (1: 1: 4, v / v / v). The resin was then washed 3 times with DMF, then 2 times with DCM and dried under vacuum.

Removal of Mmt protection on Cys residues and cyclization on solid phase: Mmt protection of Cys side chains is 30 mL of cleavage cocktail per gram of resin (TFA / EDT / TIS / DCM, 3: 1.5: 1.5: Removed using 100, v / v). The deprotection procedure was repeated 3 times, 10 minutes each, at room temperature. The resin was then washed 3 times with DCM and 10 times with DMF to ensure complete removal of residual TFA. To the thoroughly washed resin was added 10 mL DMF and 2 mL DIEA per gram of resin. The cyclization reaction was allowed to proceed for 1 hour at room temperature. Test cleavage and MS confirmed the completion of cyclization. The reaction mixture was then drained from the resin, and the resin was further washed 3 times with DMF and 2 times with DCM. The resin was then dried under vacuum prior to cutting.

Peptide cleavage and side chain deprotection from solid support: The finished peptide is cleaved with 10 mL / g resin for 70 minutes at room temperature Cocktail (TFA / EDT / TIS / H ₂ O / thioanisol / phenol, solution 100 mL) Deprotected with 81.5 mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mL H ₂ O, 5.0 mL thioanisol, and 5.0 g of phenol), and dry resin Separated from. The resin was removed by filtration and washed with a few milliliters of cutting cocktail. Eight volumes of methyl t-butyl ether was added to the cleavage mixture. The crude peptide precipitate was separated by centrifugation at 3000 rpm for 3 minutes. The crude peptide precipitate was washed 3 times with methyl t-butyl ether. The crude peptide was purified by preparative HPLC to a purity> 90% and lyophilized.

Paclitaxel conjugation: The purified cyclic peptide is mixed with the previously prepared 2'-maleimide-paclitaxel in a molar ratio of 1: 1.2, and a 30% aqueous acetonitrile solution is added to give a final peptide concentration of 10 mg / mL. It was. A 0.5 mol / L NH ₄ HCO ₃ solution was used to adjust the reaction mixture to pH 7.5. The conjugation reaction was completed in about 30 minutes, as confirmed by MS. The final product was purified using a reverse phase preparative column Daisogel (50 x 250 mm, 8 mm); mobile phase-solvent A: 0.1% TFA water; solvent B: 0.1% TFA acetonitrile. Fractions containing the target product were mixed and lyophilized (TFA salt).

Peptides in acetate were obtained by salt exchange as described above. Analysis of final peptide product HPLC purity 95.71%; MW calculated value: 2343.70; MW observed value: 2342.85.

Example 3: Synthesis of (MLB-1710)

Peptide chain construction: Cys (Trt) -Cys (Mmt) -Tyr (tBu) -Lys (iPr, Boc)-(D-Arg (Pbf))-2Nal-Gly-Cys (Mmt) -Lys (iPr, Boc) The -Gly (SEQ ID NO: 9) peptide chain was constructed using standard Fmoc chemistry using Rink AM resin. Briefly, 3.6 g of Rink AM resin was swollen in DCM for 14 hours and then washed 4 times with DMF. Removal of Fmoc was performed in 20% piperidine in DMF for 20 minutes at room temperature and washed several times with DMF. The ninhydrin test was negative. Stepwise chain construction was initiated from the C-terminus of the linear peptide using Fmoc-Gly-OH. Three equivalents of the protected amino acid Fmoc-Gly-OH were activated with DIC / HOBt in DMF and coupled to the Fmoc-removed Rink AM resin prepared above for 2 hours at room temperature. The ninhydrin test was negative. Capping of unreacted amino groups was performed for 30 minutes using a mixture of 20 mL acetic anhydride / DIEA / DCM in a volume ratio of 1: 1: 2. This was followed by removal of Fmoc using 20% piperidine in DMF for 20 minutes. The following residues were continuously coupled without capping: Fmoc-Lys (iPr, Boc) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Mmt) -OH, and Fmoc-Cys (Trt) -OH. Fmoc protection was removed again using 20% piperidine in DMF for 20 minutes after coupling of the last residue Fmoc-Cys (Trt) -OH. N-terminal acetylation was performed at room temperature for 30 minutes using a mixture of 20 mL acetic anhydride / DIEA / DMF (1: 1: 4, v / v / v). The resin was then washed 3 times with DMF, then 2 times with DCM and dried under vacuum.

Removal of Mmt protection on Cys residues and cyclization on solid phase: Mmt protection of Cys side chains is 30 mL of cleavage cocktail per gram of resin (TFA / EDT / TIS / DCM, 3: 1.5: 1.5: Removed using 100, v / v). The deprotection procedure was repeated 3 times, 10 minutes each, at room temperature. The resin was then washed 3 times with DCM and 10 times with DMF to ensure complete removal of residual TFA. To the well-washed resin, 10 mL of DMF and 2 mL of DIEA per gram of resin were added, followed by a slow instillation of 1.2 equivalents of 1,2-bis (bromomethyl) benzene. The cyclization reaction was allowed to proceed for 1 hour at room temperature. Test cleavage and MS confirmed the completion of cyclization. The reaction mixture was then drained from the resin, and the resin was further washed 3 times with DMF and 2 times with DCM. The resin was then dried under vacuum prior to cutting.

Peptide cleavage and side chain deprotection from solid support: The finished peptide is cleaved with 10 mL / g resin for 70 minutes at room temperature Cocktail (TFA / EDT / TIS / H ₂ O / thioanisol / phenol, solution 100 mL) Deprotected with 81.5 mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mL H ₂ O, 5.0 mL thioanisol, and 5.0 g of phenol), and dry resin Separated from. The resin was removed by filtration and washed with a few milliliters of cutting cocktail. Eight volumes of methyl t-butyl ether was added to the cleavage mixture. The crude peptide precipitate was separated by centrifugation at 3000 rpm for 3 minutes. The crude peptide precipitate was washed 3 times with methyl t-butyl ether. The crude peptide was purified by preparative HPLC to a purity> 90% and lyophilized.

Paclitaxel conjugation: The purified cyclic peptide is mixed with the previously prepared 2'-maleimide-paclitaxel in a molar ratio of 1: 1.2, and a 30% aqueous acetonitrile solution is added to give a final peptide concentration of 10 mg / mL. It was. A 0.5 mol / L NH ₄ HCO ₃ solution was used to adjust the reaction mixture to pH 7.5. The conjugation reaction was completed in about 30 minutes, as confirmed by MS. The final product was purified using a reverse phase preparative column Daisogel (50 x 250 mm, 8 mm); mobile phase-solvent A: 0.1% TFA water; solvent B: 0.1% TFA acetonitrile. Fractions containing the target product were mixed and lyophilized (TFA salt).

Peptides in acetate were obtained by salt exchange as described above. Analysis of final peptide product HPLC purity 95.07%; MW calculated value: 2446.96; MW observed value: 2446.50.

Example 4: Synthesis of (MLB-1711)

Peptide chain construction: Cys (Trt)-(mini-PEG6) -Cys (Mmt) -Tyr (tBu) -Lys (iPr, Boc)-(D-Arg (Pbf))-2Nal-Gly-Cys (Mmt)- The Lys (iPr, Boc) -Gly (SEQ ID NO: 10) peptide chain was constructed by standard Fmoc chemistry using Rink AM resin. Briefly, 3.6 g of Rink AM resin was swollen in DCM for 14 hours and then washed 4 times with DMF. Removal of Fmoc was performed in 20% piperidine in DMF for 20 minutes at room temperature and washed several times with DMF. The ninhydrin test was negative. Stepwise chain construction was initiated from the C-terminus of the linear peptide using Fmoc-Gly-OH. Three equivalents of the protected amino acid Fmoc-Gly-OH were activated with DIC / HOBt in DMF and coupled to the Fmoc-removed Rink AM resin prepared above for 2 hours at room temperature. The ninhydrin test was negative. Capping of unreacted amino groups was performed for 30 minutes using a mixture of 20 mL acetic anhydride / DIEA / DCM in a volume ratio of 1: 1: 2. This was followed by removal of Fmoc using 20% piperidine in DMF for 20 minutes. The following residues were continuously coupled without capping: Fmoc-Lys (iPr, Boc) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Mmt) -OH, Fmoc- (mini-PEG6) -OH, And Fmoc-Cys (Trt) -OH. Fmoc protection was removed again using 20% piperidine in DMF for 20 minutes after coupling of the last residue Fmoc-Cys (Trt) -OH. N-terminal acetylation was performed at room temperature for 30 minutes using a mixture of 20 mL acetic anhydride / DIEA / DMF (1: 1: 4, v / v / v). The resin was then washed 3 times with DMF, then 2 times with DCM and dried under vacuum.

Removal of Mmt protection on Cys residues and cyclization on solid phase: Mmt protection of Cys side chains is 30 mL of cleavage cocktail per gram of resin (TFA / EDT / TIS / DCM, 3: 1.5: 1.5: Removed using 100, v / v). The deprotection procedure was repeated 3 times, 10 minutes each, at room temperature. The resin was then washed 3 times with DCM and 10 times with DMF to ensure complete removal of residual TFA. To the well-washed resin, 10 mL DMF and 2 mL DIEA per gram of resin were added, followed by a slow instillation of 1.2 equivalents of 1,2-bis (bromomethyl) benzene. The cyclization reaction was allowed to proceed for 1 hour at room temperature. Test cleavage and MS confirmed the completion of cyclization. The reaction mixture was then drained from the resin, and the resin was further washed 3 times with DMF and 2 times with DCM. The resin was then dried under vacuum prior to cutting.

Peptide cleavage and side chain deprotection from solid support: The finished peptide is cleaved with 10 mL / g resin for 70 minutes at room temperature Cocktail (TFA / EDT / TIS / H ₂ O / thioanisol / phenol, solution 100 mL) Deprotected with 81.5 mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mL H ₂ O, 5.0 mL thioanisol, and 5.0 g of phenol), and dry resin Separated from. The resin was removed by filtration and washed with a few milliliters of cutting cocktail. Eight volumes of methyl t-butyl ether was added to the cleavage mixture. The crude peptide precipitate was separated by centrifugation at 3000 rpm for 3 minutes. The crude peptide precipitate was washed 3 times with methyl t-butyl ether. The crude peptide was purified by preparative HPLC to a purity> 90% and lyophilized.

Paclitaxel conjugation: The purified cyclic peptide is mixed with the previously prepared 2'-maleimide-paclitaxel in a molar ratio of 1: 1.2, and a 30% aqueous acetonitrile solution is added to give a final peptide concentration of 10 mg / mL. It was. A 0.5 mol / L NH ₄ HCO ₃ solution was used to adjust the reaction mixture to pH 7.5. The conjugation reaction was completed in about 30 minutes, as confirmed by MS. The final product was purified using a reverse phase preparative column Daisogel (50 x 250 mm, 8 mm); mobile phase-solvent A: 0.1% TFA water; solvent B: 0.1% TFA acetonitrile. Fractions containing the target product were mixed and lyophilized (TFA salt).

Peptides in acetate were obtained by salt exchange as described above. Analysis of final peptide product HPLC purity 95.10%; MW calculated value: 2781.25; MW observed value: 2781.75.

Example 5: Synthesis of (MLB-1713)

Peptide chain construction: Phe-Tyr (tBu) -Lys (iPr, Boc)-(D-Arg (Pbf)) -2Nal-Gly- (D-Glu (OAll) -Lys (iPr, Boc)-(mini-PEG6) The peptide chain of) -Cys (Trt) -Gly (SEQ ID NO: 11) was constructed by standard Fmoc chemistry using Rink AM resin. Briefly, 1.0 g of Rink AM resin is in DCM. It was swollen for 14 hours and then washed 4 times with DMF. Fmoc removal was performed in 20% piperidin in DMF for 20 minutes at room temperature and washed several times with DMF. The ninhydrin test was negative. Stepwise chain construction was initiated from the C-terminal of the linear peptide with Fmoc-Gly-OH. 3 equivalents of the protected amino acid Fmoc-Gly-OH were activated by DIC / HOBt in DMF, and Coupled to the Fmoc-removed Rink AM resin prepared above for 2 hours at room temperature. Ninhydrin test was negative. Capping of unreacted amino groups was 1: 1: 1 with 6 mL anhydrous acetic acid / DIEA / DCM. A mixture of 2 volumes was used for 30 minutes, followed by 20 minutes of removal of Fmoc using 20% piperidine in DMF. The following residues were capped. Coupled continuously without: Fmoc-Cys (Trt) -OH, Fmoc- (mini-PEG6) -OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-D-Glu (OAll) -OH , Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Tyr (tBu) -OH, and Fmoc-Phe-OH The resin was washed 3 times with DMF after coupling the last residue Fmoc-Phe-OH. The Fmoc protective group of Phe was not removed at this stage.

Removal of OAll protection, removal of Fmoc protection, and cyclization on solid phase: Allyl ester side chain protection of D-Glu is 0.1 equivalent Pd (Ph ₃ P) in dichloromethane in the presence of 24 equivalents of phenylsilane. ) Removed using ₄ . This procedure was repeated once for complete removal of allyl side chain deprotection. The N-terminal Fmoc protecting group was then removed with 20% piperidine in DMF for 20 minutes. The deprotected side chain carboxylic acid of D-Glu is then activated with PyBOP ((benzotriazole-1-yloxy) -tris (pyrrolidino) -phosphonium hexafluorophosphate) / DIEA, and the α of the Phe residue on the resin. It was cyclized to an amino group. Cyclization was completed within 2 hours, as confirmed by MS after test cleavage.

Peptide cleavage and side chain deprotection from solid support: The finished peptide is cleaved with 10 mL / g resin for 70 minutes at room temperature Cocktail (TFA / EDT / TIS / H ₂ O / thioanisol / phenol, solution 100 mL) Deprotected with 81.5 mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mL H ₂ O, 5.0 mL thioanisol, and 5.0 g of phenol), and dry resin Separated from. The resin was removed by filtration and washed with a few milliliters of cutting cocktail. Eight volumes of methyl t-butyl ether was added to the cleavage mixture. The crude peptide precipitate was separated by centrifugation at 3000 rpm for 3 minutes. The crude peptide precipitate was washed 3 times with methyl t-butyl ether. The crude peptide was purified by preparative HPLC to a purity> 90% and lyophilized.

Paclitaxel conjugation: The purified cyclic peptide is mixed with the previously prepared 2'-maleimide-paclitaxel in a molar ratio of 1: 1.2, and a 30% aqueous acetonitrile solution is added to give a final peptide concentration of 10 mg / mL. It was. A 0.5 mol / L NH ₄ HCO ₃ solution was used to adjust the reaction mixture to pH 7.5. The conjugation reaction was completed in about 30 minutes, as confirmed by MS. The final product was purified using a reverse phase preparative column Daisogel (50 x 250 mm, 8 mm); mobile phase-solvent A: 0.1% TFA water; solvent B: 0.1% TFA acetonitrile. Fractions containing the target product were mixed and lyophilized (TFA salt).

Peptides in acetate were obtained by salt exchange as described above. Analysis of final peptide product HPLC purity 95.03%; MW calculated value: 2690.40; MW observed value: 2690.25.

Example 6: Synthesis of (MLB-1703)

Peptide chain construction: Cys (Mmt) -Tyr (tBu) -Lys (iPr, Boc)-(D-Arg (Pbf))-2Nal-Gly-Cys (Mmt) -Lys (iPr, Boc) -Gly-Cys ( The peptide chain of Trt) (SEQ ID NO: 12) was constructed by standard Fmoc chemistry using Rink AM resin. Briefly, 0.8 g of Rink AM resin was swollen in DCM for 14 hours and then washed 4 times with DMF. Removal of Fmoc was performed in 20% piperidine in DMF for 20 minutes at room temperature and washed several times with DMF. The ninhydrin test was negative. Stepwise chain construction was initiated from the C-terminus of the linear peptide using Fmoc-Cys (Trt) -OH. Three equivalents of the protected amino acid Fmoc-Cys (Trt) -OH were activated with DIC / HOBt in DMF and coupled to the Fmoc-removed Rink AM resin prepared above for 2 hours at room temperature. The ninhydrin test was negative. Capping of unreacted amino groups was performed for 30 minutes using a 1: 1: 2 volume ratio mixture of 5 mL acetic anhydride / DIEA / DCM. This was followed by removal of Fmoc using 20% piperidine in DMF for 20 minutes. The following residues were continuously coupled without capping: Fmoc-Gly-OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Cys (Mmt) -OH, Fmoc-Gly-OH, Fmoc-2Nal-OH, Fmoc-D-Arg (Pbf) -OH, Fmoc-Lys (iPr, Boc) -OH, Fmoc-Tyr (tBu) -OH, and Fmoc-Cys (Mmt) -OH. Fmoc protection was removed again using 20% piperidine in DMF for 20 minutes after coupling of the last residue Fmoc-Cys (Mmt) -OH. N-terminal acetylation was performed at room temperature for 30 minutes using a mixture of 5 mL acetic anhydride / DIEA / DMF (1: 1: 4, v / v / v). The resin was then washed 3 times with DMF, then 2 times with DCM and dried under vacuum.

Removal of Mmt protection on Cys residues and cyclization on solid phase: Mmt protection of Cys side chains is 30 mL of cleavage cocktail per gram of resin (TFA / EDT / TIS / DCM, 3: 1.5: 1.5: Removed using 100, v / v). The deprotection procedure was repeated 3 times, 10 minutes each, at room temperature. The resin was then washed 3 times with DCM and 10 times with DMF to ensure complete removal of residual TFA. To the well-washed resin, 10 mL of DMF and 2 mL of DIEA per gram of resin were added, followed by a slow instillation of 1.2 equivalents of 1,2-bis (bromomethyl) benzene. The cyclization reaction was allowed to proceed for 1 hour at room temperature. Test cleavage and MS confirmed the completion of cyclization. The reaction mixture was then drained from the resin, and the resin was further washed 3 times with DMF and 2 times with DCM. The resin was then dried under vacuum prior to cutting.

Peptide cleavage from solid support and side chain deprotection: The finished peptide is cleaved with 10 mL / g resin for 70 minutes at room temperature Cocktail (TFA / EDT / TIS / H ₂ O / thioanisol / phenol, solution 100 mL) Deprotected with 81.5 mL TFA, 2.5 mL EDT, 1.0 mL TIS, 5.0 mL H ₂ O, 5.0 mL thioanisol, and 5.0 g of phenol), and dry resin Separated from. The resin was removed by filtration and washed with a few milliliters of cutting cocktail. Eight volumes of methyl t-butyl ether was added to the cleavage mixture. The crude peptide precipitate was separated by centrifugation at 3000 rpm for 3 minutes. The crude peptide precipitate was washed 3 times with methyl t-butyl ether. The crude peptide was purified by preparative HPLC to a purity> 90% and lyophilized.

Paclitaxel conjugation: The purified cyclic peptide is mixed with the previously prepared 2'-maleimide-paclitaxel in a molar ratio of 1: 1.2, and a 30% aqueous acetonitrile solution is added to give a final peptide concentration of 10 mg / mL. It was. A 0.5 mol / L NH ₄ HCO ₃ solution was used to adjust the reaction mixture to pH 7.5. The conjugation reaction was completed in about 30 minutes, as confirmed by MS. The final product was purified using a reverse phase preparative column Daisogel (50 x 250 mm, 8 mm); mobile phase-solvent A: 0.1% TFA water; solvent B: 0.1% TFA acetonitrile. Fractions containing the target product were mixed and lyophilized (TFA salt).

Peptides in acetate were obtained by salt exchange as described above. Analysis of final peptide product HPLC purity 95.13%; MW calculated value: 2447.86; MW observed value: 2446.95.

*n/a：利用できない。 Human CXCR4 / ¹²⁵ I-SDF-1α binding inhibition assay: (performed by EUROFINS CEREP SA, Le Bois l'Eveque, 86600 Celle L'Evescault, France): Human chemokine receptor CXCR4 expressed in Chem-1 cells Was used in modified HEPES buffer pH 7.4. 0.5 μg (membrane protein may vary from lot to lot and concentration used will be adjusted as needed), aliquots 25 ° C with 0.03 nM [ ¹²⁵ I] SDF-1α Was incubated for 90 minutes. Non-specific binding was estimated in the presence of 30 nM SDF-1α. Membranes were filtered and washed, and filters were then counted to determine specifically bound [ ¹²⁵ I] SDF-1α. Compounds were screened starting from 10 μM at 11 point dilution (Valenzuela-Fernandez A, et al.J Biol Chem. 277 (18): 15677, 2002). CXCR4 binding data are shown in the table below along with their physical characterization.
Table 1: characterization and binding activity of exemplary peptides

* n / a: Not available.

In addition to the high-affinity CXCR4-binding ligand peptide drug conjugates disclosed herein, the preparation and characterization of other high-affinity CXCR4-binding ligand peptides can be found in the following US provisional patent application: No. 62 / 384,132 filed September 6, 2016, and No. 62 / 505,064 filed May 11, 2017 (see Table 2 below).

Isotope or radiolabeled acetone are commercially available from various manufacturers. If there is a need for custom preparation of isotopes or radiolabeled acetone, methods are used in known techniques such as Rolf Voges, et al., Preparation of Compounds Labeled with Tritium and Carbon-14 (John Wiley & Sons (2009)). Can be found.

*n/a：利用できない。 Peptides with different linker - preparation of a medicament conjugates are known in the art (. GTHermanson, Bioconjugate Techniques, 2 nd Ed, Academic PressElsevier, 2008). Examples of procedures for preparing peptide conjugates mediated by thiols in the cysteine side chain (eg, linkage or attachment of the active ingredient to the peptide) are backer, et al., Pp 275-294 in Methods. In Molecular Biology, vol. 494: Peptide-Based Drug Design, edited by L. Otvos, Humana Press, New York, NY, 2008.
Table 2: Other high affinity CXCR4 binding ligand peptides

* n / a: Not available.

The aforementioned description of the invention is presented for purposes of illustration and explanation. The above is not intended to limit the invention to the forms disclosed herein. Descriptions of the invention include description of one or more embodiments as well as specific modified and modified embodiments, eg, within the skill and knowledge of one of ordinary skill in the art after understanding the present disclosure. Other modified and modified embodiments may be within the scope of the present invention. It is intended to obtain rights to include an authorized range of alternative embodiments, including interchangeable and / or equivalent structures, functions, scopes or processes of the claims. To publicly dedicate a patentable subject matter, whether such alternative, interchangeable, and / or equivalent structures, functions, scopes, or processes are disclosed herein. Not intended. All references cited herein are incorporated by reference in their entirety.

Claims (35)

High affinity CXCR4 selective binding ligand peptide conjugate (PC) of formula (I) or a pharmaceutically acceptable salt thereof:

During the ceremony
n is an integer from 1 to about 5 or the sum of (total number of side chain functional groups in P)
A is one or more diagnostic, therapeutic, or imaging agents;
L is a bifunctional linker or absent; and
P is a high affinity CXCR4 selective binding peptidyl ligand. The first aspect of claim 1, wherein A is attached to any one of the N-terminus or C-terminus of the peptide, the functional groups present on the side chain of the amino acid residue of the peptide, or their positions. High affinity CXCR4 selective binding ligand peptide conjugate. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1, wherein the high affinity CXCR4 selective binding ligand peptide conjugate comprises a plurality of A's. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1, wherein P is a high affinity CXCR4 binding peptidyl of the following formula (II):

During the ceremony
a is 0 or 1;
AA ¹ is 3-mercaptopropionic acid, optionally substituted cysteine, or optionally substituted homocysteine, along with the sulfur atom attached to it;
AA ² is cysteine or homocysteine, along with the sulfur atom attached to it;
Ar ¹ is an arbitrarily substituted aryl;
X ¹ is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), or Lys (iPr);
X ² is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), Lys (iPr), D-Arg, D-Dap, D-Dab, D-Orn, D -Lys, D-Dap (iPr), D-Dab (iPr), D-Orn (iPr), D-Lys (iPr), or absent;
X ³ is Lys, Gly or absent;
X ⁴ is Lys, Phe, 2Nal, 1Nal, their D isomers, Gly, or absent;
X ⁵ is Lys, Gly or absent; and
R ² is -OR ⁴ or -NHR ⁵ , where R ⁴ and R ⁵ are H, alkyl, optionally substituted aryl or optionally substituted aralkyl. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1, wherein A is an imaging agent. The imaging agent is a positron emitting nuclide bound to the linker, and the positron emitting nuclide is ³⁴ Cl, ⁴⁵ Ti, ⁵¹ Mn, ⁶¹ Cu, ⁶³ Zn, ⁶⁸ Ga, ¹¹ C, ¹³ N, ¹⁵ O, and. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 5, selected from the group consisting of ¹⁸ F. The imaging agent comprises a chelating group and a radioactive metal isotope coordinated to the chelating group, and the radioactive metal isotope is selected from the group consisting of technetium, renium, gallium, gadolinium, indium, copper and combinations thereof. , The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 5. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 5, wherein the imaging agent is a fluorescent dye. The high affinity according to claim 8, wherein the fluorescent dye is selected from the group consisting of Alexa Fluor dye, Oregon Green dye, fluorescein, BODIPY (boron-dipyrromethene) dye, cyanine dye, rhodamine dye, DyLight dye, and Texas Red. CXCR4 selective binding ligand peptide conjugate. A is a diagnostic agent, the high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 10, wherein the diagnostic agent is an imaging agent, an isotope agent, or a radiopharmaceutical. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1, wherein the linker comprises a functional group capable of releasing A in vivo. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1, wherein A is a therapeutic agent. The therapeutic agents include bleomycin, calikeamycin, daunorubicin, docetaxel, doxorubicin, irinotecan, meltancin, monomethyloristatin E, paclitaxel, SN-38, tecillin, topotecan, tubericin, vinca alkaloids, and their analogs or derivatives. The high-affinity CXCR4 selective binding ligand peptide conjugate according to claim 13, selected from the group consisting of combinations thereof. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1, wherein the high affinity CXCR4 selective binding ligand peptide conjugate is:
Cyclo [Phe-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly- (D-Glu)]-Lys (iPr)-(mini-PEG6) -Cys (S-paclitaxel) -Gly-NH ₂ (SEQ ID NO: 3), where the cyclic structure is formed between the α-amino of Phe bound to the side chain of D-Glu;
R ^a -Cyclo [Cys-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly-Cys] -Lys (iPr) -R ^b (SEQ ID NO: 4);
R ^a -cyclo [hCys-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly-Cys] -Lys (iPr) -R ^b (SEQ ID NO: 5);
R ^a -cyclo [Cys-Tyr-Lys (iPr)-(D-Arg) -2Nal-Gly-hCys] -Lys (iPr) -R ^b (SEQ ID NO: 6); or

,
Where at least one of R ^a or R ^b contains S-paclitaxel,
R ^a is acetyl-, acetyl-Cys (S-paclitaxel)-, or acetyl-Cys (S-paclitaxel)-(mini-PEG6)-; and R ^b is glycylamide, glycyl-Cys (S-paclitaxel)-. Amide, or (mini-PEG6) -Cys (S-paclitaxel) -amide. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 1 of the following formula (III) or a pharmaceutically acceptable salt thereof:

During the ceremony:
a is 0 or 1;
AA ¹ is 3-mercaptopropionic acid, optionally substituted cysteine, or optionally substituted homocysteine, along with the sulfur atom attached to it, where A is optionally of said cysteine or homocysteine. Bonded to α-amino group;
AA ² is cysteine or homocysteine, along with the sulfur atom attached to it;
Ar ¹ is an arbitrarily substituted aryl;
X ¹ is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), or Lys (iPr);
X ² is Arg, Dap, Dab, Orn, Lys, Dap (iPr), Dab (iPr), Orn (iPr), Lys (iPr), D-Arg, D-Dap, D-Dab, D-Orn, D -Lys, D-Dap (iPr), D-Dab (iPr), D-Orn (iPr), D-Lys (iPr), or absent;
L is any linker; and
A is as defined in claim 1. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein a is 0. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein a is 1. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein AA ¹ is a 3-mercaptopropionic acid with a sulfur atom attached to it. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein AA ¹ is a cysteine with a sulfur atom attached to it. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein AA ¹ is homocysteine, along with the sulfur atom attached to it. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein AA ² is a cysteine with a sulfur atom attached to it. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein AA ² is homocysteine, along with the sulfur atom attached to it. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein A is an imaging agent. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein A is a therapeutic agent. The therapeutic agents include bleomycin, calikeamycin, daunorubicin, docetaxel, doxorubicin, irinotecan, mertancin, monomethyloristatin E, paclitaxel, SN-38, tecillin, topotecan, tubericin, vinca alkaloids, and their analogs or derivatives. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 25, selected from the group consisting of combinations thereof. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16, wherein A is a diagnostic agent. A diagnostic kit comprising the high affinity CXCR4 selective binding ligand peptide conjugate according to claim 27. A composition comprising the high affinity CXCR4 selective binding ligand peptide conjugate according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier, diluent, excipient or combination thereof. Administering to a patient an amount effective for imaging the high affinity CXCR4 selective binding ligand peptide conjugate according to claim 5, and imaging the cancer cells in the patient using an imaging apparatus. Methods of imaging cancer cells in a patient, including. A method of treating cancer in a patient, the method comprising administering to the cancer patient a therapeutically effective amount of the high affinity CXCR4 selective binding ligand peptide conjugate of claim 16. A diagnostic or imaging kit comprising the high affinity CXCR4 selective binding ligand peptide conjugate (PC) according to claim 1, wherein A of formula (I) is a diagnostic or imaging agent. A method of treating a patient suffering from rheumatoid arthritis, pulmonary fibrosis, HIV infection, or cancer, wherein the method comprises a therapeutically effective amount of the high affinity CXCR4 selective binding ligand peptide conjugate according to claim 16. The cancers include breast cancer, pancreatic cancer, melanoma, prostate cancer, kidney cancer, neuroblastoma, non-Hodgkin's lymphoma, lung cancer, ovarian cancer, colonic rectal cancer, including administration to patients in need of the treatment. A method selected from the group consisting of cancer, multiple myeloma, polyglioblastoma, and chronic lymphocytic leukemia. The therapeutic agent is selected from the group consisting of HIV protease inhibitors, HIV fusion inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV entry inhibitors, and combinations thereof, according to claim 13. High affinity CXCR4 selective binding ligand peptide conjugate. The high affinity CXCR4 selective binding ligand peptide conjugate according to claim 34, wherein the therapeutic agent is the HIV fusion inhibitor enfuvirtide.