Patent Application
20250073358
The present disclosure relates to an aromatic vinyl compound, metal complex thereof, and preparation method therefor and use thereof.
Nuclear medicine which is mainly based on radionuclides and their labeled compounds is an important branch of modern medicine and applies nuclear technology to the research, diagnosis and treatment of diseases. Radiopharmaceuticals refer to a type of special drugs containing radionuclides for medical diagnosis and treatment, and they are radionuclide-labeled compounds or biological agents used for medical diagnosis or treatment in the body and are further divided into diagnostic radiopharmaceuticals and therapeutic radiopharmaceuticals. These two types of drugs include radioactive non-metal drugs and radioactive metal drugs. The former includes radioactive non-metal nuclide drugs represented by 131I, 18F, and so on, while the latter includes radioactive metal nuclide drugs represented by 68Ga, 177Lu, 186Re and so on.
PD-1 (programmed death 1) is an important immunosuppressive molecule. It is a member of CD28 superfamily and was originally cloned from the apoptotic mouse T-cell hybridoma 2B4.11. Immunomodulation targeting PD-1 is of great importance to anti-tumor, anti-infection, anti-autoimmune diseases and survival of organ transplantation. Its ligand PD-L1 can also be served as a target, and the corresponding antibodies and small molecular drugs can also inhibit the binding of PD-1 to PD-L1, thus serving to activate the immune response to destroy tumor cells.
Aromatic vinyl derived compounds containing radioactive metal elements have not been successfully marketed as small molecule nucleophiles targeting PD-L1 in the current state of the art, and there have been no technical reports of such compounds being used in PET tumor imaging and in tumor therapy.
The purpose of the present disclosure is to provide a novel aromatic vinyl compound, metal complex thereof, and preparation method therefor and use thereof.
The present disclosure provides a compound represented by general formula II or a metal complex thereof, or a tautomer or stereoisomer of any one of described above (namely, the compound represented by general formula II or the metal complex thereof), or a pharmaceutically acceptable salt of any one of described above (namely, the compound represented by general formula II or the metal complex thereof, the tautomer thereof, or the stereoisomer thereof), or a solvate of any one of described above (namely, the compound represented by general formula II or the metal complex thereof, the tautomer thereof, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof).
In some embodiments, q is 0, 1, or 2;
In some embodiments, q is 0.
In some embodiments, R1 is cyano or C1-C4 alkyl; X1, X2, and X3 are each independently CH or N.
In some embodiments, R1 is cyano or C1-C4 alkyl; X1 and X2 are CH, and X3 is N.
In some embodiments, R2 and R3 are hydrogen.
In some embodiments, R4 is C1-C4 alkyl or C1-C4 alkyl substituted by one or more Rb; each Rb is independently halogen; X4 and X5 are each independently CH or N.
In some embodiments, R4 is C1-C4 alkyl or C1-C4 alkyl substituted by one or more Rb; each Rb is independently halogen; X4 and X5 are each independently CH.
In some embodiments, R5, R6 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring substituted by one or more Rc; in the 5- to 7-membered heterocyclic ring, the number of heteroatoms is 1, 2, 3, or 4, and each heteroatom is independently selected from N, O, and S;
In some embodiments, L1 is
In some embodiments, the metal complex is a complex chelated by the compound represented by general formula II with the metal ion.
In some embodiments, X1, X2, X3, X4, and X5 are each independently CH or N;
each R8d-1-1 is independently 5- to 7-membered heterocyclic ring substituted by one carboxyl; in the 5- to 7-membered heterocyclic ring, the species of heteroatoms are independently selected from one or more of N, O, and S, and the number of heteroatoms is independently 1, 2, or 3;
In some embodiments, X1, X2, X4, and X5 are CH;
In some embodiments, the stereoisomer is chiral isomer.
In some embodiments, in R1, the C1-C4 alkyl is methyl or ethyl.
In some embodiments, in R4, the C1-C4 alkyl is methyl or ethyl.
In some embodiments, in R4, the C1-C4 alkyl in the C1-C4 alkyl substituted by one or more Rb is methyl or ethyl.
In some embodiments, in Rb, the halogen is fluorine or chlorine.
In some embodiments, in R4, the C1-C4 alkyl substituted by one or more Rb is trifluoromethyl.
In some embodiments, in the 5- to 7-membered heterocyclic ring substituted by one or more Rc, which is formed by R5, R6 together with the nitrogen atom they are attached, the 5- to 7-membered heterocyclic ring is a 6-membered saturated monocyclic heterocyclic ring.
In some embodiments, in the 5- to 7-membered heterocyclic ring substituted by one or more Rc, which is formed by R5, R6 together with the nitrogen atom they are attached, the number of heteroatoms is 1.
In some embodiments, in the 5- to 7-membered heterocyclic ring substituted by one or more Rc, which is formed by R5, R6 together with the nitrogen atom they are attached, the heteroatom is N.
In some embodiments, in the 5- to 7-membered heterocyclic ring substituted by one or more Rc, which is formed by R5, R6 together with the nitrogen atom they are attached, the 5- to 7-membered heterocyclic ring is piperidine ring.
In some embodiments, the 5- to 7-membered heterocyclic ring substituted by one or more Rc, which is formed by R5, R6 together with the nitrogen atom they are attached is
In some embodiments, the 5- to 7-membered heterocyclic ring substituted by one or more Rc, which is formed by R5, R6 together with the nitrogen atom they are attached is
(for example,
In some embodiments, the 5- to 7-membered heterocyclic ring substituted by one or more Rc, which is formed by R5, R6 together with the nitrogen atom to which they are attached is
In some embodiments, L1 is connected to ring A through —Y1—, and Y1 connected to ring A is —O—.
In some embodiments, L1 is connected to L2 via —CH2—.
In some embodiments, L1 is —O(CH2)n2—, —O(CH2)n2O(CH2)m2—, —O(CH2)n2O(CH2)m2O(CH2)m3—, —O(CH2)n2OC(O)(CH2)m2—, —O(CH2)n2NHC(O)(CH2)m2—, —O(CH2)n2NHC(O)—(CH2)n3—NHC(O)(CH2)m3—, —O(CH2)n2—O(CH2)n3—NHC(O)—(CH2)n4NHC(O)—(CH2)m3—, —O(CH2)n2NHC(O)—Y2—(CH2)n3NHC(O)—(CH2)m3—, —O(CH2)n2—Y2—C(O)—(CH2)m3—, —O(CH2)n2NHC(O)—Y2—C(O)—(CH2)m3—, or —O(CH2)n2NHC(O)—Y2—(CH2)n3NHC(O)—(CH2)n4NHC(O)—(CH2)m3—; the right end of L1 is connected to L2;
In some embodiments, m2 is 1 or 2.
In some embodiments, m3 is 1 or 2.
In some embodiments, m2 is 2 and m3 is 2.
In some embodiments, m2 is 1.
In some embodiments, m3 is 1.
In some embodiments, when Y2 is 5- to 7-membered carbocyclic ring, the 5- to 7-membered carbocyclic ring is
for example,
(for example,
In some embodiments, when Y2 is 5- to 7-membered heterocyclic ring, the 5- to 7-membered heterocyclic ring is
for example,
In some embodiments, R9 is phenyl or phenyl substituted by one or more Rd.
In some embodiments, in Rd, the C1-C4 alkyl is methyl or ethyl.
In some embodiments, R7 is
In some embodiments, L2 is R11 or -L4-(CH2)s—R11;
In some embodiments, when L2 is -L4-(CH2)s—R11, the -L4-(CH2)s—R11 is
In some embodiments, each Y5 is independently —NH— or —N(R11a)—.
In some embodiments, each R11a is independently C1-C4 alkyl substituted by one or more —COOH.
In some embodiments, L2 is R11, and R11 is defined as described herein.
In some embodiments, L2 is
and each R11b is independently H or R11a, and R11a is defined as described herein.
In some embodiments, in each structure of L2 above, at least 1, 2, or 3 R11b are R11a, and R11a is defined as described herein.
In some embodiments, L2 is
wherein the c-terminal is connected to L1.
In some embodiments, L2 is
wherein the c-terminal is connected to L1.
In some embodiments, the metal complex has a structure represented by the following general formula I:
In some embodiments, M is the metal ion.
In some embodiments, the metal ion is as defined in the remaining section.
In some embodiments, the metal complex has the following structure:
In some embodiments, in the metal complex, the molar ratio of the compound represented by general formula II to the metal atom or ion is 1:1.
In some embodiments, the metal ion is ion of the following metals: Al, Cu, Ga, Y, Zr, Tc, In, Lu, Re, At, Bi, or Tl.
In some embodiments, the metal ion is ion of the following metals: 27Al, 63Cu, 64Cu, 68Ga, 70Ga, 89Y, 90Y, 89Zr, 91Zr, 99mTc, 111In, 113In, 175Lu, 177Lu, 186Re, 188Re, 211At, 212Bi, 213Bi, 201Tl, or 203Tl.
In some embodiments, the valence state of the metal ion is monovalent, divalent, trivalent, or tetravalent.
In some embodiments, the valence state of the metal ion is trivalent.
In some embodiments, the metal ion is radioactive metal ion or non-radioactive metal ion.
In some embodiments, the metal ion may further bind to non-metal nuclide, for example, F. The non-metal nuclide may be radioactive non-metal nuclide or non-radioactive non-metal nuclide. The radioactive non-metal nuclide can be 18F.
In some embodiments, the metal ion may not bind to non-metal nuclide.
In some embodiments, the metal ion may be radioactive as a whole (for example, if the metal ion is combined with non-metal nuclide, then the non-metal nuclide is radioactive, which is also radioactive as a whole).
In some embodiments, the metal ion is [AlF]2+, [GaCl]2+, [LuCl]2+, Ga3+, or Lu3+.
In some embodiments, the metal ion is [Al18F]2+, [68GaCl]2+, [177LuCl]2+, 68Ga3+, or 177Lu3+.
In some embodiments,
In some embodiments, L1 is
wherein the upper end is connected to ring A, and the lower end is connected to L2.
In some embodiments, L2 is
In some embodiments, the L2 and the metal ion form any one of the following groups:
The present disclosure provides a compound represented by general formula II or a metal complex thereof, or a tautomer or stereoisomer of any one of described above (namely, the compound represented by general formula II or the metal complex thereof), or a pharmaceutically acceptable salt of any one of described above (namely, the compound represented by general formula II or the metal complex thereof, the tautomer thereof, or the stereoisomer thereof), or a solvate of any one of described above (namely, the compound represented by general formula II or the metal complex thereof, the tautomer thereof, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof):
or, two adjacent R8 together with the carbon atoms on the benzene ring to which they are attached form a 5- to 7-membered carbocyclic ring, a 5- to 7-membered heterocyclic ring, a 5- to 7-membered carbocyclic ring substituted by one or more C1-4 alkyl, or a 5- to 7-membered heterocyclic ring substituted by one or more C1-4 alkyl; in the 5- to 7-membered heterocyclic ring, the number of heteroatoms is 1, 2, 3, or 4, and each heteroatom is independently selected from N and 0;
In some embodiments, the metal complex is a complex chelated by the compound represented by general formula II with the metal ion.
In some embodiments, in the metal complex, the molar ratio of the compound represented by general formula II to the metal atom or ion is 1:1.
In some embodiments, the metal complex has the structure represented by the following general formula I:
In some embodiments, M is the metal ion.
In some embodiments, the metal is Cu, Ga, Y, Zr, Tc, In, Lu, Re, At, Bi, Tl, or their radioactive or non-radioactive isotopes thereof.
In some embodiments, the metal is Ga, Lu, or their radioactive or non-radioactive isotopes thereof.
In some embodiments, the metal is 63Cu, 64Cu, 68Ga, 70Ga, 89Y, 90Y, 89Zr, 91Zr, 99mTc, 111In, 113In, 175Lu, 177Lu, 186Re, 188Re, 211At 212Bi, 213Bi, 201Tl, or 203Tl.
In some embodiments, the valence state of the metal ion can be any valence state of the metal, for example, monovalent, divalent, trivalent, or tetravalent. In some embodiments, the valence state of the metal ion is trivalent.
In some embodiments, the metal ion is Ga3+ and Lu3+.
In some embodiments, X1 is CH.
In some embodiments, X2 is CH.
In some embodiments, X3 is CH.
In some embodiments, X4 is CH.
In some embodiments, X5 is CH.
In some embodiments, X1, X2, X3, X4, and X5 are CH.
In some embodiments, 1, 2, 3, or 4 of X1, X2, X3, X4, and X5 are N.
In some embodiments, q is 0.
In some embodiments, R1 is cyano or C1-C4 alkyl.
In some embodiments, R1 is cyano or methyl.
In some embodiments, R2 is hydrogen.
In some embodiments, R3 is hydrogen.
In some embodiments, R4 is C1-C4 alkyl or C1-C4 alkyl substituted by one or more Rb.
In some embodiments, each Rb is independently halogen, for example, fluorine.
In some embodiments, R4 is methyl or trifluoromethyl.
In some embodiments, R5, R6 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring or a 5- to 7-membered heterocyclic ring substituted by one or more Rc.
In some embodiments, when R5, R6 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring or a 5- to 7-membered heterocyclic ring substituted by one or more Rc, the heteroatom of the 5- to 7-membered heterocyclic ring is one N.
In some embodiments, when R5, R6 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic ring or a 5- to 7-membered heterocyclic ring substituted by one or more Rc, the 5- to 7-membered heterocyclic ring is saturated 5- to 7-membered heterocyclic ring, for example, piperidine.
In some embodiments, R5, R6 together with the nitrogen atom to which they are attached form
In some embodiments, each Rc is independently —COOH.
In some embodiments, R5, R6 together with the nitrogen atom to which they are attached form
(for example,
In some embodiments, R5, R6 together with the nitrogen atom to which they are attached form
In some embodiments, L1 is connected to ring A through —Y1—, and Y1 connected to ring A is —O—.
In some embodiments, L1 is connected to L2 via —CH2—.
In some embodiments, n, m, and p are each independently 5, 6, 7, 8, 9, 10, 11, or 12.
In some embodiments, L1 satisfies the definition of at least one of the foregoing embodiments, and L1 is a single bond, —(CH2)n1NH(CH2)m1—, —(CH2)n1O(CH2)m1—, —(CH2)n1NHC(O)NH(CH2)m1—, —O(CH2)n1NH—, —O(CH2)n1O—, —O(CH2)n1O(CH2)m1O—, —NH(CH2)n1NH(CH2)m1NH—, —NH(CH2)n1O(CH2)m1O—, —NH(CH2)n1NH(CH2)m1O—, —O(CH2)n1NH(CH2)m1O—, —O(CH2)n1O(CH2)m1NH—, —O(CH2)n1NH(CH2)m1NH—, —NH(CH2)n1O(CH2)m1NH—, —O—Y2—(CH2)n1NH—, —O(CH2)n2OC(O)(CH2)m2—, —O(CH2)n2O(CH2)m2—, —O(CH2)n2NHC(O)(CH2)m2—, —O(CH2)n2NHC(O)—(CH2)n3—NHC(O)(CH2)m3—, —O—(CH2)n2—Y2—C(O)—(CH2)m2—, —O(CH2)n2NHC(O)—Y2—(CH2)n3NHC(O)—(CH2)m3—, —O(CH2)n2NHC(O)—Y2—C(O)—(CH2)m3—, —O(CH2)n2—Y2—C(O)—(CH2)m3—, —O(CH2)n2NHC(O)—Y2—(CH2)n3NHC(O)—(CH2)n4NHC(O)—(CH2)m3, —O(CH2)n2NHC(O)—(CH2)n3NHC(O)—(CH2)m3—, or —O(CH2)n2—O(CH2)n3—NHC(O)—(CH2)n4NHC(O)—(CH2)m3—, and each n1, each n2, each n3, n4, each m1, each m2, and each m3 are each independently 1, 2, 3, 4, 5, or 6; L1 is unsubstituted or 1, 2, or 3 H contained in L1 are each independently substituted by R7.
In some embodiments, m2 is 1.
In some embodiments, m3 is 1.
In some embodiments, when Y2 is 5- to 7-membered carbocyclic ring, the 5- to 7-membered carbocyclic ring is
for example,
(for example,
In some embodiments, when Y2 is 5- to 7-membered heterocyclic ring, the 5- to 7-membered heterocyclic ring is
for example,
In some embodiments, L1 is unsubstituted or one H contained in L1 is substituted by R7.
In some embodiments, each R7 is independently -L3-R9.
In some embodiments, each Y4 is independently —C(O)NH—.
In some embodiments, L3 is —(CH2)k—, in which one CH2 is replaced by —Y4—, and Y4 is —C(O)—, —C(O)O—, —O—, —NH—, —C(O)NH—, or —NHC(O)NH—.
In some embodiments, each Y4 is independently —C(O)NH—.
In some embodiments, L3 is unsubstituted.
In some embodiments, j and k are each independently 7, 8, or 9.
In some embodiments, R9 is C6-C10 aryl or C6-C10 aryl substituted by one or more Rd.
In some embodiments, R9 is phenyl or phenyl substituted by one or more Rd.
In some embodiments, each Rd is independently C1-C4 alkyl.
In some embodiments, R7 is
In some embodiments, L1 is unsubstituted.
In some embodiments, L is
wherein the a-terminal is connected to ring A, and the b-terminal is connected to L2.
In some embodiments, L1 is
wherein the a-terminal is connected to ring A, and the b-terminal is connected to L2. Preferably, L1 is
wherein the a-terminal is connected to ring A, and the b-terminal is connected to L2.
In some embodiments, L2 is R11 or -L4-(CH2)s—R11;
In some embodiments, when L2 is -L4-(CH2)s—R11, the -L4-(CH2)s—R11 is
In some embodiments, each Y5 is independently —NH— or —N(R11a)—.
In some embodiments, each R11a is independently C1-C4 alkyl substituted by one or more —COOH.
In some embodiments, L2 is R11, and R11 is defined as described herein.
In some embodiments, L2 is
and each R11b is independently H or R11a, and R11a is defined as described herein. In some embodiments, in each structure of L2 above, at least 1, 2, or 3 R11b are R11a, and R11a is defined as described herein.
In some embodiments, L2 is
wherein the c-terminal is connected to L1.
In some embodiments, L2 is
wherein the c-terminal is connected to L1.
In some embodiments, the metal complex has the following structure:
In some embodiments, the compound represented by general formula II is any one of the following structures:
In some embodiments, the metal complex is a complex formed by any one of the following compounds with 177Lu3+:
In some embodiments, the metal complex is a complex formed by any one of the
following compounds with 68Ga3+:
In some embodiments, the metal complex is a complex formed by any one of the following compounds with [Al18F]2+.
In some embodiments, the metal complex is a complex formed by any one of the following compounds with [68GaCl]2+:
In some embodiments, the metal complex is a complex formed by any one of the following compounds with [177LuCl]2+:
In some embodiments, the metal complex has any one of the following structures:
The present disclosure also provides a method for preparing the compound represented by the general formula I as described above, which comprises the following steps: in a solvent (for example water, or a mixed solvent of water and methanol), reacting the compound represented by the general formula II with M metal halide (for example, chloride, another example, [AlF]2+) in the presence or absence of a buffer (for example, sodium acetate-acetic acid), to obtain the compound represented by the general formula I;
The present disclosure also provides a method for preparing the compound represented by the general formula II as described above, which comprises the following steps: in a solvent (for example, dichloromethane), removing the Boc protective group of the compound represented by the general formula III in the presence of acid (for example, trifluoroacetic acid), to obtain the compound represented by the general formula II;
The present disclosure also provides a compound represented by general formula III:
The present disclosure also provides a compound represented by any one of the following:
The present disclosure also provides a pharmaceutical composition comprising the compound represented by the general formula II or the metal complex thereof, or the tautomer or stereoisomer of any one of described above, or the pharmaceutically acceptable salts of any one of described above, or the solvate of any one of described above, and at least one pharmaceutical excipient.
The present disclosure also provides a use of the compound represented by the general formula II or the metal complex thereof, or the tautomer or stereoisomer of any one of described above, or the pharmaceutically acceptable salts of any one of described above, or the solvate of any one of described above in the manufacture of a medicament for the treatment of tumor.
In some embodiments, the metal complex is a complex chelated by the compound represented by general formula II with radioactive metal ion; the radioactive metal ion is radioactive metal ion for treatment.
In some embodiments, in the metal complex chelated by the compound represented by general formula II with radioactive metal ion, the metal ion is [177LuCl]2+ or 177Lu3+.
The present disclosure also provides a use of the metal complex as described above, or the tautomer or stereoisomer of any one of described above, or the pharmaceutically acceptable salts of any one of described above, or the solvate of any one of described above in the manufacture of a medicament for the radiodiagnosis of tumor.
In some embodiments, the metal complex is a complex chelated by the compound represented by general formula II with radioactive metal ion; the radioactive metal ion is radioactive metal ion for diagnosis.
In some embodiments, in the metal complex chelated by the compound represented by general formula II with radioactive metal ion, the metal ion is [Al18F]2+, [68GaCl]2+, or 68Ga3+.
The present disclosure also provides a method for treating tumor, which comprises administering to a tumor patient a therapeutically effective amount of the compound represented by the general formula II or the metal complex thereof, or the tautomer or stereoisomer of any one of described above, or the pharmaceutically acceptable salts of any one of described above, or the solvate of any one of described above.
In some embodiments, the metal complex is a complex chelated by the compound represented by general formula II with radioactive metal ion; the radioactive metal ion is radioactive metal ion for treatment.
In some embodiments, in the metal complex, the metal ion is [177LuCl]2+ or 177Lu3+.
In some embodiments, the tumor is lung cancer, gastric cancer, colorectal cancer, cervical cancer, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, liver cancer, bladder cancer, renal cancer, bone cancer, skin cancer, melanoma, glioma, glioblastoma, leukemia, or lymphoma.
The present disclosure also provides a method for diagnosing tumor, which comprises administering to a tumor patient a therapeutically effective amount of the metal complex as described above, or the tautomer or stereoisomer of any one of described above, or the pharmaceutically acceptable salts of any one of described above, or the solvate of any one of described above.
In some embodiments, the metal complex is a complex chelated by the compound represented by general formula II with radioactive metal ion; the radioactive metal ion is radioactive metal ion for diagnosis.
In some embodiments, in the metal complex chelated by the compound represented by general formula II with radioactive metal ion, the metal ion is [Al18F]2+, [68GaCl]2+, or 68Ga3+.
In some embodiments, the tumor is lung cancer, gastric cancer, colorectal cancer, cervical cancer, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, liver cancer, bladder cancer, renal cancer, bone cancer, skin cancer, melanoma, glioma, glioblastoma, leukemia, or lymphoma.
Unless otherwise indicated, the following terms and phrases used herein are intend to have the following meanings. A particular term or phrase should not be considered uncertain or unclear in the absence of a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.
In the present disclosure, the term “metal chelating group” refers to a group that forms a complex with a metal atom or ion. The metal chelating group may be any metal chelating group known in the art for complexing pharmaceutically useful metal atoms or ions.
In the present disclosure, the term “substitution” or “substituent” means that a hydrogen atom in a group is replaced by a specified group. When the position of substitution is not specified, substitution may be at any position, but only the formation of a stable or chemically viable chemical is allowed. An example is as follows: structure
indicates that the hydrogen atoms on the benzene ring are substituted by q R8, and each R8 is the same or different when there are multiple R8.
When any variable (for example R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted by 0-2 R, then the group may optionally be substituted by up to two R, and R in each case have independent options. In addition, the combination of substituent and/or variant thereof is allowed only if such a combination will result in a stable compound.
When the connecting group listed in the present disclosure does not specify its connecting direction, its connecting direction can be arbitrary and comprises both left to right connection and from right to left connection. An example is as follows, the linking group L in -A-L-B is —C-D-, and when the connection direction of L is not specified, -A-L-B comprises -A-C-D-B and -A-D-C—B.
When one of the variables is selected from a single bond, it means that the two groups connected to it are directly connected, for example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.
In the present disclosure, the term “alkyl” refers to a saturated linear or branched chain monovalent hydrocarbyl. C1-C4 alkyl refers to an alkyl with 1-4 carbon atoms, specifically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.
In the present disclosure, the term “alkylene” refers to a saturated linear or branched chain divalent hydrocarbyl. C1-C4 alkylene refers to an alkylene with 1-4 carbon atoms, specifically methylene, ethylene (for example, —CH2CH2—, —CH(CH3)—), propylene (for example, —CH2CH2CH2—, —C(CH3)2—, —CH2CH(CH3)—), butylene (for example, —CH2CH2CH2CH2—, —CH(CH3)CH(CH3)—, —CH2CH(CH3)CH2—).
In the present disclosure, halogen refers to F, Cl, Br, or I.
In the present disclosure, the term “halogenated alkyl” refers to a group formed by the substitution of one or more hydrogen atoms in an alkyl by halogen, wherein the alkyl is as defined above, and each halogen is independently F, Cl, Br, or I. Halogenated C1-C4 alkyl refers to C1-C4 alkyl substituted by one or more halogens, for example, fluorinated C1-C4 alkyl, wherein C1-C4 alkyl is as defined above. Examples of halogenated alkyl include, but are not limited to trifluoromethyl, pentafluoroethyl, 1-fluoro-2-chloroethyl.
In the present disclosure, the term “carbocyclic ring” refers to a saturated, partially unsaturated, or aromatic monocyclic or polycyclic (for example, paracyclic, spirocyclic, or bridged ring) cyclic group formed by carbon atoms. In the saturated carbocyclic ring, each carbon atom on the ring is saturated, and examples of saturated carbocyclic rings include, but are not limited to
In the aromatic carbocyclic ring, each ring is aromatic, and examples of aromatic carbocyclic rings include, but are not limited to
In the partially unsaturated carbocyclic ring, at least one carbon atom on the ring is saturated and at least one carbon atom is unsaturated or aromatic, and examples of partially unsaturated carbocyclic rings include, but are not limited to
The 5- to 7-membered carbocyclic ring may specifically be a 5, 6, or 7-membered carbocyclic ring. In some embodiments, the 5- to 7-membered carbocyclic ring may specifically be 5, 6, or 7-membered saturated carbocyclic ring. In some embodiments, the 5- to 7-membered carbocyclic ring may specifically be 5, 6, or 7-membered saturated monocyclic carbocyclic ring, including
In some embodiments, the 5- to 7-membered carbocyclic ring may specifically be benzene ring.
In the present disclosure, the term “heterocyclic ring” refers to a saturated, partially unsaturated, or aromatic monocyclic or polycyclic (for example paracyclic, spirocyclic or bridged ring) cyclic group formed by carbon atoms and at least one heteroatom, wherein the heteroatoms are independently selected from N, O, and S. In the saturated heterocyclic ring, the carbon atoms and heteroatoms on the ring are all saturated, and examples of saturated heterocyclic rings include, but are not limited to
In the aromatic heterocyclic ring, each ring is aromatic, and examples of the aromatic heterocyclic rings include, but are not limited to
In the partially unsaturated heterocyclic ring, at least one atom on the ring is saturated and at least one atom is unsaturated or aromatic, and examples of partially unsaturated heterocyclic rings include, but are not limited to
The 5- to 7-membered heterocyclic ring may specifically be 5, 6, or 7-membered heterocyclic ring. In some embodiments, the 5- to 7-membered heterocyclic ring may specifically be 5, 6, or 7-membered saturated heterocyclic ring. In some embodiments, the 5- to 7-membered heterocyclic ring may specifically be 5, 6, or 7-membered saturated monocyclic heterocyclic ring, including
In some embodiments, the 5- to 7-membered carbocyclic ring may specifically be a benzene ring.
In the present disclosure, the term “aryl” refers to an aromatic carbocyclic group, wherein each ring is aromatic. The C6-C10 aryl may specifically be phenyl or naphthyl. In some embodiments, the C6-C10 aryl may specifically be phenyl.
In the present disclosure, the term “stereoisomer” includes enantiomer, diastereomer, geometrical isomer. They may be defined as (R)-/(S)—, or (D-/(L)-, or (R, R)—/(R, S)—/(S, S)-according to absolute stereochemistry for amino acids. The present disclosure includes racemic, enantioenriched and optionally pure forms thereof. These isomers can be synthesized using chiral raw materials, prepared by chiral resolution, or can be resolved using conventional techniques such as, but not limited to, high performance liquid chromatography (HPLC) using chiral columns.
In the chemical structure, the wedge-shaped solid bond () and the wedge-shaped dashed bond (
) are used to represent absolute configuration of a stereocenter, and the straight solid bond (
) and straight dashed bond (
) are used to represent the relative configuration of a stereocenter. The bond “
” does not specify a configuration that is, if configurational isomerism exists in the chemical structure, the bond “
” can be “
” or “
”, or contain both “
” and “
” configurations (for example the ratio of “
” and “
” is 1:1).
In the present disclosure, the term “pharmaceutically acceptable” refers to a substance (for example, a carrier or diluent) that do not affect the biological activity or properties of the compound of the present disclosure and are relatively non-toxic, namely, the substance can be administered to an individual without causing undesirable biological reaction or interaction in an undesirable manner with any component contained in the composition.
In the present disclosure, the term “pharmaceutically acceptable salt” means a salt formed from a suitable non-toxic organic acid, inorganic acid, organic base, or inorganic base with a compound, which retains the biological activity of the compound. The organic acid may be a variety of conventional organic acids capable of forming salts in the art, preferably one or more of methanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, oxalic acid, butanedioic acid, benzoic acid, hydroxyethylsulfonic acid, naphthalenesulfonic acid, and salicylic acid. The inorganic acid may be a variety of conventional inorganic acids capable of forming salts in the art, preferably one or more of hydrochloric acid, sulfuric acid, and phosphoric acid. The organic base may be a variety of conventional organic bases capable of forming salts in the art, preferably one or more of pyridines, imidazoles, pyrazines, indoles, purines, tertiary amines, and anilines. The tertiary amine organic base is preferably triethylamine and/or N, N-diisopropylethylamine. The aniline organic base is preferably N, N-dimethylaniline. The pyridine organic base is preferably one or more of pyridine, methylpyridine, 4-dimethylaminopyridine, and 2-methyl-5-ethylpyridine. The inorganic base may be a variety of conventional inorganic bases capable of forming salts in the art, preferably one or more of alkali metal hydride, alkali metal hydroxide, alkali metal alkoxide, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, potassium bicarbonate, and sodium bicarbonate. The alkali metal hydride is preferably sodium hydride and/or potassium hydride. The alkali metal hydroxide is preferably one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide. The alkali metal alkoxide is preferably one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide, and sodium tert-butoxide.
In the present disclosure, the term “solvate” refers to a substance formed by a compound or a salt thereof with a suitable solvent. The solvent is preferably water or an organic solvent.
In the present disclosure, the term “therapeutically effective amount” refers to a sufficient amount of medicament or agent that is non-toxic but can achieve the desired effect. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, as well as on the specific active substance. The appropriate effective amount in individual cases can be determined by those skilled in the art based on routine experiments.
In the present disclosure, the term “patient” includes any animal, preferably a mammal, and more preferably a human.
In the present disclosure, the term “one or more” may be 1, 2, 3, 4, 5, or 6.
On the basis of not violating common sense in the art, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present disclosure.
The reagent and raw material used in the present disclosure are all commercially available.
The positive progress effect of the present disclosure lies in that the present disclosure provides a novel aromatic vinyl compound, metal complex thereof, and preparation method therefor and use thereof. The aromatic vinyl compounds and metal complexes thereof of the present disclosure have inhibitory activity on PD-1/PD-L1 binding, and thus can be used to treat related diseases such as tumor. In addition, the metal complexes of the present disclosure can also be used as imaging agents.
The present disclosure is further illustrated below by means of examples, but the present disclosure is not limited to the scope of the examples. The experimental methods for which specific conditions are not indicated in the following examples are selected according to conventional methods and conditions, or according to the product instructions.
Radioactivity meter (CRC-55tR type); electronic balance (YP30002); germanium-gallium generator (20 mCi); vortex mixer (MX-F); HPLC (1200); TLC (Scan-RAM); gamma radioimmunoassay counter (GC-2016); UV spectrophotometer (T6 New Century);
3-Bromo-4-methylphenol (3.74 g, 20 mmol), paraformaldehyde (4.41 g, 152 mmol), magnesium chloride (2.86 g, 30 mmol), and triethylamine (7.56 g, 75 mmol) were dissolved in acetonitrile (150 mL), and the reaction solution was heated and stirred at 80° C. for 4 hours. The reaction solution was cooled to room temperature, diluted with water (500 mL), and the pH was adjusted to 2-3 with 1 M hydrochloric acid. The mixture was extracted with ethyl acetate (500 mL×2). The organic phases were combined, washed with saturated brine (200 mL×1), and the organic phases were concentrated under reduced pressure. The obtained residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate=50:1) to obtain white solid 1-e (2.45 g, yield: 57%).
1-e (645 mg, 3.0 mmol), (2-bromoethoxy) (tert-butyl) dimethylsilane (1.08 g, 4.5 mmol), and potassium carbonate (829 mg, 6.0 mmol) were dissolved in N, ′N-dimethylformamide (5 mL), and the reaction solution was heated at 60° C. for 16 hours. The reaction solution was cooled to room temperature and diluted with water (50 mL). The obtained mixture was extracted with ethyl acetate (50 mL×2). The organic phase was washed sequentially with water (50 mL×2) and saturated brine (20 mL×2), dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate=20:1) to obtain white solid 1-d (767 mg, yield: 68%).
Compound 1-d (373 mg, 1.0 mmol), (E)-3-(2-(4,4,5,5-tetramethyl-1,3,2-dioxyboran-2-yl)vinyl)-[1,1′-biphenyl]-2-carbonitrile (397 mg, 1.2 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (73 mg, 0.1 mmol) and potassium carbonate (276 mg, 2.0 mmol) were dissolved in 1,4-dioxane (5 mL) and water (0.5 mL), and the reaction solution was heated and stirred at 90° C. for 16 hours. The reaction solution was cooled to room temperature, and the reaction solution was diluted with saturated brine (20 mL). The resulting mixture was extracted with dichloromethane (30 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain yellow solid 1-c (355 mg, yield: 71%).
LC-MS (ESI): m/z=498.5(M+H)+.
Compound 1-c (75 mg, 0.15 mmol) and (S)-piperidine-2-carboxylic acid (39 mg, 0.30 mmol) were dissolved in methanol (1 mL) and tetrahydrofuran (1 mL), and sodium cyanoborohydride (38 mg, 0.60 mmol) was added. The reaction solution was stirred at 60° C. for 1 hour. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and tetrahydrofuran (1 mL), water (0.2 mL), and trifluoroacetic acid (0.5 mL) were added to the reaction solution, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by pre-HPLC to obtain white solid 1-b (48.3 mg, yield: 53%).
LC-MS (ESI): m/z=497.5 (M+H)+;
1H-NMR (400 MHz, DMSO) δ: 8.05 (d, J=8.0 Hz, 1H), 7.79 (t, J=7.9 Hz, 1H), 7.67 (d, J=16.0 Hz, 1H), 7.63-7.47 (m, 6H), 7.40 (d, J=16.0 Hz, 1H), 7.27 (s, 2H), 7.07 (s, 1H), 4.13-3.90 (m, 5H), 3.75 (t, J=4.6 Hz, 2H), 3.28-3.21 (m, 1H), 3.08-3.00 (m, 1H), 2.38 (s, 3H), 1.97-1.86 (m, 1H), 1.78-1.66 (m, 1H), 1.63-1.48 (m, 3H), 1.45-1.31 (m, 1H).
Compound 1-b (40 mg, 0.08 mmol) and triethylamine (65 mg, 0.64 mmol) were dissolved in dichloromethane (1 mL). The reaction solution was cooled to 0° C., and 2-bromoacetyl bromide (19 mg, dissolved in 1 mL of dichloromethane) and tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecan-1,4,7-triyl) triacetate (206 mg, 0.40 mmol) were added to the reaction solution sequentially. The reaction solution was raised to room temperature and stirred at room temperature for 5 hours. The reaction solution was concentrated under reduced pressure and the obtained residue was separated by reversed-phase preparative chromatography (column xBridge C18, 19*150 mm*5 um; mobile phase: water (0.1% trifluoroacetic acid), methanol; gradient: 65%-80% (initial mobile phase was 35% water/65% methanol, and ending mobile phase was 20% water/80% methanol, wherein % refers to volume percentage); 13 min; flow rate 15 mL/min) to obtain white solid 1-a (22 mg, yield: 26%).
LC-MS (ESI): m/z=1051.87(M+H)+.
Compound 1-a (22 mg, 0.021 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (2 mL) was added, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure and the obtained residue was separated by reversed-phase preparative chromatography (column xBridge C18, 19*150 mm*5 um; mobile phase: water (0.1% trifluoroacetic acid), methanol; gradient: 65%-80% (initial mobile phase was 35% water/65% methanol, and ending mobile phase was 20% water/80% methanol, wherein % refers to volume percentage); 13 min; flow rate 15 mL/min) to obtain white solid 1 (7 mg, yield: 38%). LC-MS (ESI): m/z=883.8(M+H)+;
1H-NMR (400 MHz, DMSO) δ: 8.05 (d, J=8.2 Hz, 1H), 7.79 (t, J=7.9 Hz, 1H), 7.66 (d, J=16.1 Hz, 1H), 7.62-7.47 (m, 6H), 7.39 (d, J=16.0 Hz, 1H), 7.28 (d, J=6.1 Hz, 2H), 4.56-4.41 (m, 1H), 4.39-4.21 (m, 3H), 4.08-3.96 (m, 1H), 3.82-3.40 (m, 15H), 3.11-2.73 (m, 16H), 2.38 (s, 3H), 2.36-2.28 (m, 1H), 1.94-1.62 (m, 3H), 1.59-1.33 (m, 3H).
(2S)-Hexahydropyridine-2-carboxylic acid (20 g, 154.847 mmol) was dissolved in 2-methylpropan-2-yl acetate (313.727 mL, 2322.701 mmol), and perchloric acid (18.630 mL, 309.693 mmol) was added. The reaction solution was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the obtained residue was diluted with ethyl acetate (50 mL), and the pH was adjusted to 9-10 with saturated sodium carbonate solution, and the mixture was washed sequentially with water (50 mL×2) and saturated brine (50 mL×1). The organic phase was dried and concentrated under reduced pressure to obtain 2-e (23.24 g, yield: 81.00%).
3-Bromo-4-(trifluoromethyl) phenol (10.96 g, 45.475 mmol) was dissolved in toluene (200 mL), 2-e (12.64 g, 68.213 mmol) and paraformaldehyde (2.73 g, 90.951 mmol) were added to the reaction solution, and the reaction was heated and stirred at 110° C. for 6 hours. The reaction solution was cooled to room temperature, and the reaction solution was diluted with ethyl acetate (50 mL), and washed sequentially with water (50 mL×2) and saturated brine (50 mL×1). The organic phase was dried, and concentrated under reduced pressure to obtain 2-d (15.26 g, yield: 76.57%).
4,4,5,5-Tetramethyl-2-[(1E)-2-(2-methyl-3-phenylphenyl)vinyl]-1,3,2-dioxyborane (9.00 g, 28.092 mmol) was dissolved in dioxane (160 mL) and water (4 mL), and compound 2-d (10.26 g, 23.410 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (2.02 g, 2.341 mmol), and sodium carbonate (6.20 g, 58.524 mmol) were added to the reaction solution, and the reaction solution was heated and stirred at 80° C. for 12 hours. The reaction solution was cooled to room temperature, and the reaction solution was diluted with ethyl acetate (50 mL), and washed sequentially with water (50 mL×2) and saturated brine (50 mL×1). The organic phase was dried and concentrated to obtain 2-c (10.78 g, yield: 83.50%).
Compound 2-c (551.65 mg, 1.0 mmol) and 1-bromo-2-[(2-bromoethyl)oxy]ethane (695.73 mg, 3.000 mmol) were dissolved in N, N-dimethylformamide (5 mL), and potassium carbonate (276.42 mg, 2.000 mmol) was added, and the reaction solution was stirred for at 70° C. for 3 hours. The reaction solution was cooled to room temperature, and the reaction solution was diluted with ethyl acetate (50 mL), washed sequentially with water (20 mL×2) and saturated brine (20 mL×2). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain product 2-b (548 mg, yield: 77.99%).
Compound 2-b (400 mg, 0.569 mmol) and tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecan-1,4,7-triyl) triacetate (292.87 mg, 0.569 mmol) were dissolved in acetonitrile (4 mL), and potassium carbonate (78.68 mg, 0.569 mmol) was added. The reaction solution was stirred at 60° C. for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (acetic acid in methanol:dichloromethane=1:10) to obtain 2-a (450 mg, yield: 69.56%).
Compound 2-a was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (4 mL, 0.510 mmol) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by Prep-HPLC to obtain 2 (153 mg, yield: 42.37%).
1H-NMR (400 MHz, MeOD) δ: 7.89 (s, 1H), 7.66 (d, J=15.8 Hz, 1H), 7.59 (s, 1H), 7.54 (d, J=7.5 Hz, 1H), 7.46-7.39 (m, 2H), 7.38-7.24 (m, 5H), 7.18 (d, J=6.9 Hz, 1H), 4.61 (d, J=13.0 Hz, 1H), 4.50-4.37 (m, 3H), 4.00-3.83 (m, 4H), 3.77-3.56 (m, 7H), 3.53-3.26 (m, 9H), 3.25-2.98 (m, 11H), 2.35-2.29 (m, 3H), 2.25 (d, J=15.2 Hz, 1H), 2.02-1.51 (m, 5H).
Compound 2-c (22.53 mg, 0.05 mmol), 2-(2-bromoethyl) isoindole-1,3-dione (508.16 mg, 2.000 mmol) and potassium carbonate (276.40 mg, 2.000 mmol) were dissolved in N, N-dimethylformamide (5 mL). The reaction solution was heated and stirred at 60° C. for 16 hours, and the reaction solution was cooled to room temperature, and the reaction solution was directly separated by C18 reversed-phase column chromatography to obtain a mixture of product and raw material 3-c (450 mg), which was directly put into the next step of reaction.
Compound 3-c (450 mg, 0.174 mmol) and hydrazine hydrate (10.89 mg, 0.174 mmol) were dissolved in methanol (5 mL). The reaction solution was heated and stirred at 65° C. for 2 hours. The reaction solution was cooled to room temperature, and the reaction solution was directly separated by prep-HPLC to obtain product 3-b (35 mg, yield: 33.82%).
Compound 3-b (35 mg, 0.059 mmol), (10-{1-[(methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (40.45 mg, 0.071 mmol) and HBTU reagent (0-(IH-benzotriazol-1-yl)-N,N,N′,N′-tetramethylisourea phosphorus hexafluoride, 33.48 mg, 0.088 mmol) was dissolved in N ′N-dimethylformamide (1 mL), and N, N-diisopropylethylamine (0.019 mL, 0.118 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was directly separated by reversed-phase preparative chromatography to obtain 3-a (76 mg, 0.051 mmol, 86.36%).
Compound 3-a (76 mg, 0.051 mmol) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (1.0 mL) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure and the residue was separated by reversed-phase preparative chromatography (column xBridge C18, 19*150 mm*5 um; mobile phase: water (0.1% trifluoroacetic acid), methanol; gradient: 65%-80% (initial mobile phase was 35% water/65% methanol, and ending mobile phase was 20% water/80% methanol, wherein % refers to volume percentage); 13 min; flow rate 15 mL/min) to obtain 3 (37 mg, yield: 57.26%). LC-MS (ESI): m/z=925.72(M+H)+;
1H-NMR (400 MHz, D2O) δ: 7.45 (s, 1H), 7.27-6.18 (m, 11H), 4.3-2.25 (m, 32H), 2.22-0.60 (m, 10H).
Compound 2-c (275.82 mg, 0.5 mmol) and 1,4-diodobutane (619.84 mg, 2.000 mmol) were dissolved in N, N-dimethylformamide (2 mL), and potassium carbonate (138.21 mg, 1.00 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (50 mL), and washed sequentially with water (20 mL×2) and saturated brine (20 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain product 4-c (310 mg, yield: 84.51% w).
Compound 4-c (410 mg, 0.559 mmol) was dissolved in ammonia-methanol solution (7 M, 11.975 mL), and the mixture was sealed in a microwave tube, and the reaction solution was stirred at 50° C. for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was dissolved in dichloromethane (20 mL) and methanol (2 mL), and washed with 10% potassium carbonate solution (5 mL). The organic phase was separated, dried over anhydrous sodium sulfate, and filtered to obtain yellow oil 4-b (280 mg, yield: 80.45%), which was used directly in the next step without purification.
Compound 4-b (180 mg, 0.289 mmol), 2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl) acetate (198.65 mg, 0.347 mmol), HBTU reagent (0-(IH-benzotriazol-1-yl)-N,N,N′,N′-tetramethylisourea phosphorus hexafluoride, 164.42 mg, 0.434 mmol) was dissolved in N, N-dimethylformamide (2 mL). N, N-Diisopropylethylamine (0.096 mL, 0.578 mmol) was added. The reaction solution was stirred at room temperature for 0.5 hour, and directly separated by reversed-phase preparative chromatography (column xBridge C18, 19*150 mm*5 um; mobile phase: water (0.1% trifluoroacetic acid), methanol; gradient: 65%-80% (initial mobile phase was 35% water/65% methanol, and ending mobile phase was 20% water/80% methanol, wherein % refers to volume percentage); 13 min; flow rate 15 mL/min) to obtain 4-a (268 mg, 0.176 mmol, yield: 61.03%). LC-MS (ESI): m/z=1178.18(M+H)+.
Compound 4-a (268 mg, 0.176 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by reversed-phase preparative chromatography (column xBridge C18, 19*150 mm*5 um; mobile phase: water (0.1% trifluoroacetic acid), methanol; gradient: 65%-80% (initial mobile phase was 35% water/65% methanol, and ending mobile phase was 20% water/80% methanol, wherein % refers to volume percentage); 13 min; flow rate 15 mL/min) to obtain 4 (156 mg, yield: 68.44%). LC-MS (ESI): m/z=953.64(M+H)+.
1H-NMR (400 MHz, D2O) δ: 7.48 (s, 1H), 7.19-6.19 (m, 11H), 4.41-2.32 (m, 33H), 2.19-0.83 (m, 13H).
Compound 4 (56 mg, 0.043 mmol) and lutetium trichloride (60.82 mg, 0.216 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (2.0 mL), and the reaction solution was stirred at 90° C. for 0.5 hour. The reaction solution was cooled to room temperature and purified by pre-HPLC to obtain 5 (33 mg, yield: 52.03%). LC-MS (ESI): m/z=1125.95(M+H)+.
Compound 2 (182.40 mg, 0.2 mmol) and lutetium trichloride (56.26 mg, 0.200 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (2.0 mL), and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was purified by pre-HPLC to obtain 6 (80 mg, 0.074 mmol, 36.90%).
LC-MS (ESI): m/z=1084.68(M+H)+;
1H-NMR (400 MHz, MeOD) δ: 7.85 (s, 1H), 7.62 (d, J=16.0 Hz, 1H), 7.58-7.46 (m, 2H), 7.46-7.38 (m, 2H), 7.38-7.21 (m, 5H), 7.17 (d, J=7.4 Hz, 1H), 4.66-1.40 (m, 44H).
Compound 2 and gallium trichloride (3.52 mg, 0.020 mmol) were dissolved in water (0.2 mL), and the reaction solution was stirred at 90° C. for 5 minutes. The reaction solution was purified directly by prep-HPLC (column XT C18, 19*150 mm*5 um; mobile phase: water (0.1% FA), acetonitrile; gradient: 25%-95% (initial mobile phase was 75% water: 25% acetonitrile, and ending mobile phase was 5% water/95% acetonitrile, wherein % refers to volume percentage); 10 min; flow rate 15 mL/min) to obtain white solid 7 (9.7 mg, yield: 99.08%).
LC-MS (ESI): m/z=978.61 (M+H)+.
1H-NMR (400 MHz, MeOD) δ: 7.88 (s, 1H), 7.63 (d, J=15.9 Hz, 1H), 7.58-7.51 (m, 2H), 7.47-7.39 (m, 2H), 7.38-7.24 (m, 5H), 7.18 (d, J=7.5 Hz, 1H), 4.62 (d, J=13.0 Hz, 1H), 4.50-4.42 (m, 2H), 4.40 (d, J=13.1 Hz, 1H), 4.06-3.86 (m, 6H), 3.85-3.45 (m, 10H), 3.44-3.32 (m, 9H), 3.26-3.10 (m, 5H), 3.09-2.97 (m, 1H), 2.30 (s, 3H), 2.28-2.17 (m, 1H), 2.05-1.68 (m, 4H), 1.66-1.52 (m, 1H).
Compound 4 (12.95 mg, 0.010 mmol), gallium trichloride (3.52 mg, 0.020 mmol) and sodium acetate (0.150 mL, 0.030 mmol) were dissolved in water (0.5 mL), and the reaction solution was stirred at 90° C. for 10 minutes. The reaction solution was cooled to room temperature, and the reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography to obtain 8 (8.76 mg, yield: 85.90%). LC-MS (ESI): m/z=1019.65 (M+H)+;
1H-NMR (400 MHz, MeOD) δ: 7.91 (s, 1H), 7.62 (d, J=15.9 Hz, 1H), 7.57-7.49 (m, 2H), 7.46-7.38 (m, 2H), 7.38-7.23 (m, 5H), 7.18 (d, J=6.8 Hz, 1H), 4.52 (dd, J=34.7, 12.7 Hz, 2H), 4.37-4.22 (m, 2H), 3.89-3.46 (m, 13H), 3.45-2.96 (m, 16H), 2.30 (s, 3H), 2.28-2.19 (m, 1H), 2.05-1.90 (m, 3H), 1.89-1.67 (m, 5H), 1.66-1.50 (m, 1H).
2-Methylpropan-2-yl (2S)-1-[(2-{[2-(1,3-dioxo-2,3-dihydro-1H-isoindole-2-yl)ethyl]oxy}-4-[(1E)-2-(2-methyl-3-phenylphenyl)vinyl]-5-(trifluoromethyl)phenyl)methyl]hexahydropyridine-2-carboxylate (610 mg, 0.842 mmol) and hydrazine hydrate (0.511 mL, 8.416 mmol) were dissolved in methanol (6 mL). The reaction solution was stirred at 60° C. for 2 hours. The reaction solution was cooled to room temperature, and concentrated under reduced pressure, and the residue was washed with dichloromethane (6 mL), filtered, and the filtrate was concentrated under reduced pressure to obtain 9-d (498 mg, yield: 99.50%). LC-MS (ESI): m/z=595.53(M+H)+.
N, N-Diisopropylethylamine (0.020 mL, 0.120 mmol), compound 9-d (59.47 mg, 0.1 mmol), (1r,4r)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)cyclohexane-1-carboxylic acid (37.95 mg, 0.100 mmol), and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (45.51 mg, 0.120 mmol) were dissolved in N, N-dimethylformamide (0.5 mL), and the reaction solution was stirred at room temperature for 30 minutes. The reaction solution was diluted with ethyl acetate (20 mL), and washed sequentially with water (20 mL×2) and saturated brine (10 mL×1). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate:ethanol=20:10:1) to obtain 9-c (72 mg, yield: 75.30%).
Piperidine (0.021 mL, 0.226 mmol) and compound 9-c (72 mg, 0.075 mmol) were dissolved in acetonitrile (1 mL). The reaction solution was stirred at room temperature for 5 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (dichloromethane:methanol:ammonia methanol=400:100:1) to obtain 9-b (49 mg, yield: 88.66%).
N, N-Diisopropylethylamine (0.017 mL, 0.100 mmol), compound 9-b (49 mg, 0.067 mmol), (10-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (42.06 mg, 0.073 mmol), and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (32.92 mg, 0.087 mmol) were dissolved in N, N-dimethylformamide (0.5 mL), and the reaction solution was stirred at room temperature for 1 hour, and the residue was separated by Prep-HPLC to obtain 9-a (72 mg, yield: 83.39%).
Trifluoroacetic acid (2.0 mL) and compound 9-a (72 mg, 0.056 mmol) were dissolved in dichloromethane (2.0 mL), and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by Prep-HPLC to obtain 9 (59.46 mg, yield: 67.27%). LC-MS (ESI): m/z=1065.21 (M+H)+.
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (191.70 mg, 1.000 mmol), 4-(4-methylphenyl) butyric acid (178.23 mg, 1.00 mmol) and 1-hydroxytetrahydro-1H-pyrrole-2,5-dione (115.09 mg, 1.000 mmol) were dissolved in dimethyl sulfoxide (2 mL). The reaction was stirred at room temperature for 48 hours. (2S)-6-Amino-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)hexanoic acid (368.43 mg, 1.000 mmol) was added, and the reaction solution was continuously stirred at room temperature for 1 hour. The reaction solution was directly separated by Prep-HPLC to obtain 10-d (400 mg, yield: 75.66%).
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (188.56 mg, 0.984 mmol), compound 10-d and 1-hydroxytetrahydro-1H-pyrrole-2,5-dione (95.79 mg, 0.832 mmol) were dissolved in dimethyl sulfoxide (2 mL). The reaction solution was stirred at room temperature for 2 hours. The reaction solution was directly separated by Prep-HPLC to obtain 10-c (400 mg, yield: 84.49%).
Compound 9-d (59.47 mg, 0.1 mmol) and compound 10-c (75.09 mg, 0.120 mmol) were dissolved in acetonitrile (1 mL). The reaction solution was stirred at room temperature for 1 hour. Piperidine (0.037 mL, 0.400 mmol) was added, and the reaction solution was continuously stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1, 6 CV, then dichloromethane:methanol=10:1, 8 CV, CV is the column volume) to obtain 10-b (78 mg, yield: 88.32%).
N, N-Diisopropylethylamine (0.029 mL, 0.177 mmol), compound 10-b (78 mg, 0.088 mmol), (10-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (55.65 mg, 0.097 mmol), and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (43.54 mg, 0.115 mmol) were dissolved in N, N-dimethylformamide (1.0 mL), and the reaction solution was stirred at room temperature for 1 hour, and directly separated by Prep-HPLC to obtain 10-a (108 mg, yield: 85.04%).
Trifluoroacetic acid (2.0 mL) and compound 10-a (72 mg, 0.056 mmol) were dissolved in dichloromethane (2.0 mL), and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by Prep-HPLC to obtain 10 (50 mg, yield: 54.86%). LC-MS (ESI): m/z=1214.47 (M+H)+.
Compound 4-b (124.55 mg, 0.20 mmol) and compound 10-c (150.17 mg, 0.240 mmol) were dissolved in acetonitrile (5.0 mL). The reaction solution was stirred at room temperature for 1 hour, and dichloromethane (2.0 mL) was added and the reaction solution was stirred for 1 hour. Piperidine (0.073 mL, 0.800 mmol) was added, and the reaction solution was continuously stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 11-b (177 mg, yield: 97.13%).
N, N-Diisopropylethylamine (0.064 mL, 0.389 mmol), 11-b (78 mg, 0.088 mmol), (10-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (122.39 mg, 0.214 mmol) and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (95.77 mg, 0.253 mmol) were dissolved in N, N-dimethylformamide (2.0 mL), and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (ethyl acetate, 6 CV, then dichloromethane:methanol=10:1, 8 CV, CV is column volume) to obtain 11-a (284 mg, yield: 99.73%).
Compound 11-a (284 mg, 0.194 mmol) was dissolved in dichloromethane (3 mL), and the reaction solution was cooled to 0° C., and trifluoroacetic acid (3 mL) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by reversed-phase preparative chromatography to obtain 11 (150 mg, 0.121 mmol, 62.37%).
1H-NMR (400 MHz, MeOD) δ: 8.00 (s, OH), 7.80 (s, 1H), 7.52 (d, J=15.8 Hz, 1H), 7.47-7.40 (m, 2H), 7.36-7.29 (m, 2H), 7.28-7.15 (m, 5H), 7.08 (d, J=7.0 Hz, 1H), 6.94 (s, 4H), 4.48 (d, J=12.7 Hz, 1H), 4.33 (d, J=12.9 Hz, 1H), 4.23-4.13 (m, 3H), 3.75-3.59 (m, 5H), 3.56-3.26 (m, 11H), 3.10-2.87 (m, 11H), 2.50-2.41 (m, 2H), 2.20 (s, 3H), 2.16 (s, 3H), 2.11-2.05 (m, 2H), 1.96-1.16 (m, 22H).
Compound 11 (30 mg, 0.024 mmol) and lutetium trichloride (13.60 mg, 0.048 mmol) were dissolved in methanol (1.0 mL) and sodium acetate-acetic acid buffer (pH=4.5) (0.5 mL). The reaction solution was stirred at room temperature for 20 minutes, then stirred at 90° C. for 5 minutes, and the reaction solution was cooled to room temperature. The reaction solution was directly separated by reversed-phase Pre-HPLC to obtain 12 (14 mg, yield: 48.08%). LC-MS (ESI): m/z=1414.3 (M+H)+.
Compound 10 (12.13 mg, 0.01 mmol) and lutetium trichloride (5.63 mg, 0.020 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (1.0 mL). The reaction solution was stirred at room temperature for 20 minutes, then stirred at 90° C. for 5 minutes, and the reaction solution was cooled to room temperature. The reaction solution was directly separated by reversed-phase Pre-HPLC to obtain 13 (10 mg, yield: 72.20%). LC-MS (ESI): m/z=1386.7 (M+H)+.
Compound 9 (10.64 mg, 0.01 mmol) and lutetium trichloride (5.63 mg, 0.020 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (1.0 mL). The reaction solution was stirred at room temperature for 20 minutes, then the reaction solution was stirred at 90° C. for 5 minutes, and the reaction solution was cooled to room temperature. The reaction solution was directly separated by reversed-phase Pre-HPLC to obtain 14 (6 mg, yield: 48.54%). LC-MS (ESI): m/z=1237.3 (M+H)+.
Compound 1 (15 mg, 0.017 mmol) and lutetium trichloride (23.89 mg, 0.085 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (1.0 mL), and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was directly separated by Prep-HPLC to obtain 15 (8.6 mg, yield: 36%). LC-MS (ESI): m/z=1055.79(M+H)+.
1H-NMR (400 MHz, D2O) δ: 7.20 (s, 1H), 7.15-6.87 (m, 6H), 6.81 (s, 1H), 6.75-6.22 (m, 4H), 4.50-3.82 (m, 3H), 3.78-2.89 (m, 17H), 2.86-2.18 (m, 13H), 2.08-0.96 (m, 9H).
Compound 2-c (500 mg, 0.910 mmol) was dissolved in N, N-dimethylformamide (15 mL), and 1,5-diiodopentane (294.66 mg, 0.910 mmol) and potassium carbonate (314.30 mg, 2.274 mmol) were added, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL), and washed sequentially with water (20 mL×2) and saturated brine (20 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography to obtain 16-c (171 mg, yield: 25.20%).
Compound 16-c (171 mg, 0.229 mmol) was dissolved in tetrahydrofuran (2 mL), and ammonia methanol solution (7 M, 10 mL) was added. The reaction solution was stirred at 50° C. for 12 hours. The reaction solution was cooled to room temperature, and the reaction solution was diluted with ethyl acetate (5 mL), and washed sequentially with water (2 mL×2) and saturated brine (2 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 16-b (145 mg, yield: 99.61%).
Compound 16-b (145 mg, 0.228 mmol) was dissolved in N, N-dimethylformamide (5 mL), and [4,7,10-tris(4,4-dimethyl-2-oxopentyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid (155.36 mg, 0.274 mmol), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (129.70 mg, 0.342 mmol), and N, N-diisopropylethylamine (0.075 mL, 0.456 mmol) were added. The reaction solution was stirred at room temperature for 1 hour, then the reaction solution was diluted with ethyl acetate (5 mL), and washed sequentially with water (2 mL×2) and saturated brine (2 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 16-a (243 mg, yield: 98.10%).
Compound 16-a (243 mg, 0.224 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10 mL) was added. The reaction solution was stirred at room temperature for 18 hours, and the reaction solution was concentrated under reduced pressure, and the residue was directly separated by prep-HPLC to obtain 16 (104.77 mg, yield: 48.35%). LC-MS (ESI): m/z=968.4 (M+H)+.
Compound 16 (49 mg, 0.051 mmol) was dissolved in sodium acetate-acetic acid buffer (pH=4.5) (10 mL), and lutetium trichloride (28.69 mg, 0.102 mmol) was added. The reaction solution was stirred at room temperature for 10 minutes, and directly separated by prep-HPLC to obtain 17 (29.67 mg, yield: 51.41%). LC-MS (ESI): m/z=1140.7 (M+H)+.
1-{[(9H-Fluoren-9-ylmethyl)oxy]carbonyl}hexahydropyridine-4-carboxylic acid (702.80 mg, 2.00 mmol), 1-hydroxytetrahydro-1H-pyrrole-2,5-dione (230.18 mg, 2.000 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (383.40 mg, 2.000 mmol) were dissolved in acetonitrile (7.0 mL) and dichloromethane (7.0 mL), and the reaction solution was stirred at room temperature for 5 hours, and directly separated by reversed-phase chromatography to obtain 18-c (800 mg, yield: 89.19%).
Compound 9-d (59.47 mg, 0.100 mmol) and compound 18-c (57.41 mg, 0.128 mmol) were dissolved in acetonitrile (1.0 mL), and the reaction solution was stirred at room temperature for 16 hours. Piperidine (85.15 mg, 1.000 mmol) was added, and the stirring was continued for 1 hour, and the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column (petroleum ether:ethyl acetate=5:1, 6 CV, dichloromethane:methanol=7:3, 6 CV) to obtain 18-b (50 mg, yield: 70.83%).
N, N-Diisopropylethylamine (0.023 mL, 0.142 mmol), compound 18-b (50 mg, 0.071 mmol), (10-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (44.63 mg, 0.078 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (34.92 mg, 0.092 mmol) were dissolved in N, N-dimethylformamide (1.0 mL), and the reaction solution was stirred at room temperature for 1 hour, then directly purified by silica gel column (petroleum ether:ethyl acetate=5:1, 6 CV, dichloromethane:methanol=10:1, 6 CV, CV is column volume) to obtain 18-a (80 mg, yield: 89.59%).
Compound 18-a (80 mg, 0.063 mmol) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (2.0 mL) was added. The reaction solution was stirred at room temperature for 24 hours, and the reaction solution was concentrated under reduced pressure to obtain compound 18 (30 mg, yield: 45.62%). LC-MS (ESI): m/z=1037.3 (M+H)+.
Compound 19-c (284 mg, 0.379 mmol) was dissolved in tetrahydrofuran (2 mL) and hydrochloric acid in 1,4-dioxane (4 M in dioxane) (1.5 mL) was added. The reaction solution was stirred at room temperature for 5 hours. The reaction solution was quenched with potassium carbonate aqueous solution (20% aqueous, 5 mL). The mixture was extracted with dichloromethane (20 mL×2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 19-b (246 mg, yield: 99.990%), which was directly carried out in the next step of reaction without purification.
Compound 19-b (246 mg, 0.379 mmol), (10-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (238.87 mg, 0.417 mmol), and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (186.93 mg, 0.493 mmol) were dissolved in N, N-dimethylformamide (2.0 mL), and N, N-diisopropylethylamine (0.125 mL, 0.758 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate:ethanol=10:20:1) to obtain 19-a (360 mg, yield: 78.89%).
Compound 19-a (360 mg, 0.299 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (3 mL) was added. The reaction solution was stirred at room temperature for 16 hours, and the reaction solution was concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 19 (194 mg, yield: 66.24%). LC-MS (ESI): m/z=980.7 (M+H)+.
Compound 19 (23.50 mg, 0.024 mmol) and lutetium trichloride (13.50 mg, 0.048 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (1 mL), and the reaction solution was stirred at 90° C. for 5 minutes. The reaction solution was cooled to room temperature, and directly separated by pre-HPLC to obtain 20 (23 mg, yield: 83.24%). LC-MS (ESI): m/z=1152.1 (M+H)+.
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (383.40 mg, 2.000 mmol), 4-(4-methylphenyl) butyric acid (356.46 mg, 2.00 mmol), and 1-hydroxytetrahydro-1H-pyrrole-2,5-dione (230.18 mg, 2.000 mmol) were added to dimethyl sulfoxide (4 mL). The reaction was stirred at room temperature for 48 hours. (2S)-6-Amino-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)hexanoic acid (736.86 mg, 2.00 mmol) was added, and the reaction solution was continuously stirred at room temperature for 1 hour. The reaction solution was directly separated by reversed-phase chromatography (120 g of C18, water (0.1% formic acid), 0-95% acetonitrile, 6 CV, CV is column volume) to obtain 21-f (860 mg, yield: 81.34%).
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (498.97 mg, 2.603 mmol), compound 21-f (860 mg, 1.627 mmol) and 1-hydroxytetrahydro-1H-pyrrole-2,5-dione (252.76 mg, 2.196 mmol) were added to dimethyl sulfoxide (2 mL). The reaction solution was stirred at room temperature for 2 hours. The reaction solution was directly separated by reversed-phase chromatography (120 g of C18, water (0.1% formic acid), 0-95% acetonitrile, 6 CV, CV is column volume) to obtain 21-e (900 mg, yield: 88.42%).
N, N-Diisopropylethylamine (0.040 mL, 0.240 mmol), compound 9-d (118.94 mg, 0.20 mmol), (1r,4r)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)cyclohexane-1-carboxylic acid (75.89 mg, 0.200 mmol), and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (91.02 mg, 0.240 mmol) were dissolved in N, N-dimethylformamide (1.0 mL). The reaction was stirred at room temperature for 30 minutes. The reaction solution was diluted with ethyl acetate (20 mL), and washed sequentially with water (20 mL×2) and saturated brine (10 mL×1). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate:ethanol=20:10:1) to obtain 21-d (164 mg, yield: 85.76%).
Piperidine (0.047 mL, 0.515 mmol) and compound 21-d (164 mg, 0.172 mmol) were dissolved in acetonitrile (2 mL). The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column (dichloromethane:methanol:ammonia methanol (7 M in MeOH)=400:100:1) to obtain 21-c (110 mg, yield: 87.38%).
Compound 21-c (110 mg, 0.150 mmol) and compound 21-e (112.63 mg, 0.180 mmol) were dissolved in acetonitrile (2 mL). The reaction solution was stirred at room temperature for 1 hour, and dichloromethane (2.0 mL) was added to continue stirring for 1 hour. Piperidine (0.069 mL, 0.749 mmol) was added, and the reaction solution was continuously stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1, then, dichloromethane:methanol:ammonia methanol (7 M in MeOH)=100:10:2) to obtain 21-b (127 mg, yield: 82.82%).
Compound 21-b (127 mg, 0.124 mmol), (4,7,10-tris{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (92.33 mg, 0.161 mmol) and O-benzotriazole-tetramethyluronium hexafluorophosphate (70.54 mg, 0.186 mmol) were dissolved in N, N-dimethylformamide (1.0 mL), and N, N-diisopropylethylamine (0.041 mL, 0.248 mmol) was added. The reaction solution was stirred at room temperature for 1 hour, and the reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column (petroleum ether:ethyl acetate=4:1, 6 CV, then 100% PE, 5 CV, then dichloromethane:methanol=10:1, 8 CV, CV is column volume) to obtain 21-a (195 mg, yield: 99.54%).
Compound 21-b (195 mg, 0.124 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added. The reaction solution was stirred at room temperature for 16 hours, and the reaction solution was concentrated under reduced pressure, and directly separated by pre-HPLC to obtain 21 (100 mg, yield: 59.79%). LC-MS (ESI): m/z=1353.61 (M+H)+.
Compound 21 (13.53 mg, 0.01 mmol) was dissolved in sodium acetate-acetic acid buffer (pH=4.5) (1 mL), and lutetium trichloride (5.63 mg, 0.020 mmol) was added, and the reaction solution was heated and stirred at 90° C. for 10 minutes. The reaction solution was directly separated by Prep-HPLC to obtain 22 (10 mg, yield: 65.59%). LC-MS (ESI): m/z=1525.55 (M+H)+.
Compound 2-c (500 mg, 0.910 mmol) was dissolved in N, N-dimethylformamide (15 mL), and 1,6-diiodohexane (307.55 mg, 0.910 mmol) and potassium carbonate (314.43 mg, 2.275 mmol) were added, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL), and washed sequentially with water (20 mL×2) and saturated saline (20 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography to obtain 23-c (462 mg, yield: 66.82%).
Compound 23-c (462 mg, 0.608 mmol) was dissolved in tetrahydrofuran (2 mL), and ammonia methanol solution (7 M, 15 mL) was added. The reaction solution was stirred at 50° C. for 16 hours. The reaction solution was cooled to room temperature, and the reaction solution was diluted with ethyl acetate (5 mL), and washed sequentially with water (2 mL×2) and saturated saline (2 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 23-b (483 mg, yield: 122.41%), which was directly carried out in the next step of reaction without purification.
Compound 23-b (145 mg, 0.228 mmol) was dissolved in N, N-dimethylformamide (5 mL), and [4,7,10-tris(4,4-dimethyl-2-oxopentyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid (104.83 mg, 0.185 mmol), benzotriazole-N,N,N′, N′-tetramethyluronium hexafluorophosphate (87.67 mg, 0.231 mmol), and N, N-diisopropylethylamine (0.051 mL, 0.308 mmol) were added. The reaction solution was stirred at room temperature for 1 hour, and the reaction solution was diluted with ethyl acetate (5 mL) and washed sequentially with water (2 mL×2) and saturated brine (2 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 23-a (91 mg, yield: 49.10%).
Compound 23-a (91 mg, 0.076 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10 mL) was added. The reaction solution was stirred at room temperature for 18 hours, and the reaction solution was concentrated under reduced pressure, and the residue was directly separated by prep-HPLC to obtain 23 (55.42 mg, yield: 74.34%). LC-MS (ESI): m/z=982.12 (M+H)+.
Compound 23 (30 mg, 0.031 mmol) was dissolved in sodium acetate-acetic acid buffer (pH=4.5) (10 mL), and lutetium trichloride (17.20 mg, 0.061 mmol) was added. The reaction solution was stirred at room temperature for 10 minutes, and directly separated by prep-HPLC to obtain 24 (28.12 mg, yield: 78.67%). LC-MS (ESI): m/z=1154.3 (M+H)+.
Compound 23-b (200 mg, 0.308 mmol) was dissolved in acetonitrile (5 mL) and dichloromethane (5 mL), and 9H-fluoren-9-ylmethyl {[(2S)-125-b-[(2,5-dioxytetrahydro-1H-pyrrole-1-yl)oxy]-6-{[4-(4-methylphenyl)-1-oxybutyl]amino}-1-oxohex-2-yl]amino}formate (231.44 mg, 0.370 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. Piperidine (0.102 mL, 0.616 mmol) was added to continue stirring for 1 hour, and the reaction solution was diluted with ethyl acetate (5 mL), and washed sequentially with water (2 mL×2) and saturated brine (2 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 25-b (184 mg, yield: 63.69%).
Compound 25-b (184 mg, 0.196 mmol) was dissolved in N, N-dimethylformamide (5 mL), and (4,7,10-tris{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (112.44 mg, 0.196 mmol), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (111.68 mg, 0.294 mmol), and N, N-diisopropylethylamine (0.065 mL, 0.393 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (5 mL), and washed sequentially with water (2 mL×2) and saturated brine (2 mL×1). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 25-a (158.85 mg, yield: 54.16%).
Compound 25-a (158.85 mg, 0.106 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10 mL) was added. The reaction solution was stirred at room temperature for 18 hours, and the reaction solution was concentrated under reduced pressure, and the residue was directly separated by prep-HPLC to obtain 25 (84.5 mg, yield: 62.60%). LC-MS (ESI): m/z=1270.5 (M+H).
Compound 25 (20 mg, 0.016 mmol) was dissolved in sodium acetate-acetic acid buffer (pH=4.5) (3 mL), and lutetium trichloride (8.86 mg, 0.032 mmol) was added. The reaction solution was stirred at room temperature for 10 minutes, and directly separated by prep-HPLC to obtain 26 (19.02 mg, yield: 83.75%). LC-MS (ESI): m/z=1142.46(M+H)+.
Compound 25 (20 mg, 0.016 mmol) was added to water (2 mL), and gallium trichloride (0.640 mL, 0.032 mmol) was added. The reaction solution was stirred at 90° C. for 10 minutes, cooled to room temperature, and directly separated by prep-HPLC to obtain 27 (17.37 mg, yield: 81.24%). LC-MS (ESI): m/z=1337.21(M+H)+.
Compound 23 (8 mg, 0.008 mmol) was added to water (2 mL), and sodium acetate (0.164 mL, 0.024 mmol) was added, and the reaction solution was stirred for 1 minute, and gallium trichloride (0.319 mL, 0.016 mmol) was added. The reaction solution was stirred at 90° C. for 10 minutes, and the reaction solution was cooled to room temperature, and directly separated by prep-HPLC to obtain 28 (4.92 mg, yield: 57.61%). LC-MS (ESI): m/z=1048.82(M+H)+.
Compound 16 (20 mg, 0.021 mmol) was added to water (2 mL), and gallium trichloride (0.809 mL, 0.041 mmol) was added. The reaction solution was stirred at 90° C. for 10 minutes, and the reaction solution was cooled to room temperature, and directly separated by prep-HPLC to obtain 29 (19.64 mg, yield: 90.47%). LC-MS (ESI): m/z=1334.8(M+H)+.
Compound 2-c (551.65 mg, 1.00 mmol), 2-methylpropan-2-yl ({2-[(2-bromoethyl)oxy]ethyl}amino) formate (321.78 mg, 1.200 mmol), and potassium carbonate (179.66 mg, 1.300 mmol) were dissolved in N, N-dimethylformamide (3.0 mL). The reaction solution was stirred at 80° C. for 60 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (40 mL), and washed sequentially with water (20 mL×2) and saturated brine (20 mL×1). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate=10:3) to obtain 30-d (438 mg, yield: 59.28%).
Compound 30-d (438 mg, 0.593 mmol) was dissolved in tetrahydrofuran (4 mL), and hydrochloric acid in 1,4-dioxane (4 M) (4 mL) was added, and the reaction solution was stirred at room temperature for 1 hour, and the reaction solution was concentrated under reduced pressure to obtain 30-c (260 mg, yield: 68.64%).
Compound 30-c (127.75 mg, 0.2 mmol), compound 21-e (150.17 mg, 0.240 mmol) were dissolved in acetonitrile (2.0 mL) and dichloromethane (2.0 mL). The reaction solution was stirred at room temperature for 1 hour, and piperidine (0.183 mL, 2.00 mmol) was added, and the reaction solution was continuously stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column (petroleum ether:ethyl acetate=5:1, 6 CV, then, dichloromethane:methanol:ammonia methanol (7 M in MeOH)=100:10:2, 8 CV, CV is column volume) to obtain 30-b (160 mg, yield: 86.29%).
N, N-Diisopropylethylamine (0.057 mL, 0.346 mmol), compound 30-b (160 mg, 0.173 mmol), (4,7,10-tris{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (118.61 mg, 0.207 mmol), and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (85.29 mg, 0.225 mmol) were dissolved in N, N-dimethylformamide (2.0 mL), and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The residue was separated by column chromatography (petroleum ether:ethyl acetate=4:1, 6 CV, then 100% PE, 5 CV, then dichloromethane:methanol=10:1, 8 CV, CV is the column volume) to obtain 30-a (255 mg, 0.172 mmol, 99.71%).
Trifluoroacetic acid (3 mL) and compound 30-a (255 mg, 0.172 mmol) were dissolved in dichloromethane (3 mL). The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by Pre-HPLC to obtain 30 (121 mg, yield: 55.92%). LC-MS (ESI): m/z=1258.46(M+H)+.
N, N-Diisopropylethylamine (0.033 mL, 0.200 mmol), compound 30-c (63.88 mg, 0.1 mmol), (4,7,10-tris{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (68.73 mg, 0.120 mmol) and benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (49.30 mg, 0.130 mmol) were dissolved in acetonitrile (1.0 mL), and the reaction solution was stirred at room temperature for 0.5 hour. The reaction solution was concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1, then, dichloromethane:methanol:ammonia methanol (7 M in MeOH)=200:20:1) to obtain 31-a (119 mg, yield: 99.71%).
Trifluoroacetic acid (2.0 mL) and compound 31-a (119 mg, 0.100 mmol) were dissolved in dichloromethane (2.0 mL). The reaction solution was reacted at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by Pre-HPLC to obtain 31 (47 mg, yield: 48.64%). LC-MS (ESI): m/z=970.13(M+H)+.
Compound 30 (12.57 mg, 0.01 mmol) and lutetium trichloride (5.63 mg, 0.020 mmol) were added to sodium acetate-acetic acid buffer (pH=4.5) (1.0 mL), and the reaction solution was stirred at 90° C. for 10 minutes. The reaction solution was cooled to room temperature, and directly separated by reversed-phase chromatography to obtain 32 (7.2 mg, yield: 50.38%). LC-MS (ESI): m/z=1430.4(M+H)+.
Lutetium trichloride (5.63 mg, 0.020 mmol) and compound 31 (9.69 mg, 0.01 mmol) were added to sodium acetate-acetic acid buffer (pH=4.5) (1.0 mL), and the reaction solution was stirred at 90° C. for 5 minutes, and the reaction solution was cooled to room temperature. The reaction solution was directly separated by Pre-HPLC to obtain 33 (8.7 mg yield: 76.25%). LC-MS (ESI): m/z=1142.01(M+H)+.
Compound 31 (4.85 mg, 0.005 mmol) and gallium trichloride (1.76 mg, 0.010 mmol) were added to water (0.2 mL), and the reaction solution was heated and stirred at 90° C. for 5 minutes. The reaction solution was cooled to room temperature, and the reaction solution was directly separated by Pre-HPLC to obtain 34 (3.5 mg, yield: 67.57%). LC-MS (ESI): m/z=1036.77(M+H)+.
Compound 9 (5.32 mg, 0.005 mmol) and gallium trichloride (1.76 mg, 0.010 mmol) were added to water (0.2 mL), and the reaction solution was heated and stirred at 90° C. for 10 minutes. The reaction solution was cooled to room temperature, and directly separated by Pre-HPLC to obtain 35 (2.3 mg, yield: 40.71%). LC-MS (ESI): m/z=1131.91(M+H)+.
Compound 18 (5.18 mg, 0.005 mmol) and gallium trichloride (1.76 mg, 0.010 mmol) were added to water (0.2 mL), and the reaction solution was stirred at 90° C. for 10 minutes. The reaction solution was cooled to room temperature, and directly separated by Pre-HPLC to obtain 36 (2.7 mg, yield: 49.00%). LC-MS (ESI): m/z=1103.86(M+H)+.
Compound 19 (9.79 mg, 0.01 mmol) and gallium trichloride (3.52, 0.020 mmol) were added to water (0.5 mL), and the reaction solution was heated and stirred at 90° C. for 10 minutes, and the reaction solution was cooled to room temperature. The reaction solution was directly separated by Pre-HPLC to obtain 37 (8.5 mg, yield: 81.26%). LC-MS (ESI): m/z=1146.81(M+H)+.
Compound 18 (15.54 mg, 0.015 mmol) and lutetium trichloride (8.44 mg, 0.030 mmol) were added to sodium acetate-acetic acid buffer (pH=4.5) (1.0 mL), and the reaction solution was stirred at room temperature for 10 minutes, and directly separated by Pre-HPLC to obtain 38 (8 mg, yield: 44.15%). LC-MS (ESI): m/z=1209.1(M+H)+.
Compound 2-c (441.32 mg, 0.80 mmol) and 1,8-dibromo-3,6-dioxoctane (242.85 mg, 0.880 mmol) were dissolved in N, N-dimethylformamide (3.0 mL), and potassium carbonate (143.73 mg, 1.040 mmol) was added. The reaction solution was stirred at 80° C. for 60 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (40 mL), and the reaction solution was washed sequentially with water (20 mL×2) and saturated brine (20 mL×1). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to obtain 39-b (240 mg, yield: 40.18%).
Compound 39-b (140 mg, 0.187 mmol) and 2-methylpropan-2-yl (4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl) acetate (96.50 mg, 0.187 mmol) were dissolved in acetonitrile (2 mL), and potassium carbonate (25.91 mg, 0.187 mmol) was added, and the reaction solution was stirred at 60° C. for 16 hours. The reaction solution was cooled to room temperature, and the reaction solution was concentrated under reduced pressure, and the residue was purified by Pre-HPLC to obtain 39-a (240 mg, yield: 84.29%).
Compound 39-a (240 mg, 0.158 mmol) and trifluoroacetic acid (3.0 mL) were dissolved in dichloromethane (3.0 mL), and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure. The residue was purified by Pre-HPLC to obtain 39 (78 mg, yield: 51.76%). LC-MS (ESI): m/z=956.24(M+H)+.
3-Bromo-1-chloro-2-toluene (3082.20 mg, 15.00 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxyborane (2772.36 mg, 18.000 mmol), and triethylamine (20.850 mL, 150.000 mmol) were dissolved in toluene (30 mL), and bis(tri-tert-butylphosphine) palladium (383.29 mg, 0.750 mmol) was added, and the reaction solution was replaced with nitrogen for three times, and the reaction solution was heated at 80° C. for 16 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 40-f (1000 mg, yield: 23.93%).
Compound 2-d (1200 mg, 2.738 mmol) and compound 40-f (991.60 mg, 3.559 mmol) were dissolved in dioxane (10.0 mL) and water (2.0 mL), and (1,1′-bis(diphenylphosphino)ferrocene) dichloropalladium (100.17 mg, 0.137 mmol) and potassium carbonate (756.83 mg, 5.476 mmol) were added, and the reaction solution was heated and stirred at 80° C. for 16 hours under nitrogen protection. The reaction solution was cooled to room temperature, and the reaction solution was diluted with water (10 mL), and the mixture was extracted with dichloromethane (30 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain 40-e (220 mg, yield: 15.76%). LC-MS (ESI): m/z=510.39 (M+H)+.
Compound 40-e (220 mg, 0.431 mmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (219.09 mg, 0.863 mmol) were dissolved in toluene (3 mL), and palladium acetate (9.68 mg, 0.043 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (41.13 mg, 0.086 mmol), and potassium acetate (84.60 mg, 0.862 mmol) were added, and the reaction solution was heated at 100° C. for 16 hours under nitrogen protection. The reaction solution was cooled to room temperature, and the reaction solution was diluted with dichloromethane (40 mL), and the mixture was washed with water (10 mL×1). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 40-d (220 mg, yield: 84.86%).
Compound 40-d (220 mg, 0.366 mmol) and 4-{3-[(3-bromo-2-methylphenyl)oxy]propyl}-1,4-oxazinane (137.91 mg, 0.439 mmol) were dissolved in dioxane (2.0 mL) and water (0.4 mL), and (1,1′-bis(diphenylphosphino)ferrocene) dichloropalladium (26.76 mg, 0.037 mmol) and potassium carbonate (101.10 mg, 0.731 mmol) were added, and the reaction solution was heated and stirred at 90° C. for 16 hours under nitrogen protection. The reaction solution was cooled to room temperature, and the reaction solution was diluted with water (10 mL), and the obtained mixture was extracted with dichloromethane (40 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated by column chromatography (100% ethyl acetate) to obtain 40-c (195 mg, yield: 75.21%).
Compound 40-c (195 mg, 0.275 mmol) and 1-bromo-2-[(2-bromoethyl)oxy]ethane (191.39 mg, 0.825 mmol) were dissolved in N, N-dimethylformamide (2.0 mL), and potassium carbonate (76.02 mg, 0.550 mmol) was added, and the reaction solution was heated and stirred at 70° C. for 20 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (ethyl acetate:ethanol=20:1) to obtain 40-b (150 mg, yield: 63.44%).
Compound 40-b (150 mg, 0.174 mmol) and 2-methylpropan-2-yl (4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl) acetate (134.68 mg, 0.262 mmol) were dissolved in acetonitrile (2.0 mL), and potassium carbonate (36.17 mg, 0.262 mmol) was added, and the reaction solution was stirred at 60° C. for 16 hours, and 13% of 40-b was remained and the mixture was continuously heated and stirred for 8 hours. The reaction solution was evaporated to dryness, and the residue was separated by column chromatography (methanol:ammonia methanol (7 M)=5:1) to obtain 40-a (170 mg, yield: 75.33%).
Compound 40-a (170 mg, 0.131 mmol) was dissolved in dichloromethane (5.0 mL), and trifluoroacetic acid (5.0 mL) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by Pre-HPLC to obtain 40 (40 mg, yield: 21.64%).
Compound 39 (11.2 mg, 0.012 mmol) and lutetium trichloride (7.25 mg, 0.026 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (0.5 mL), and the reaction solution was heated at 90° C. for 20 minutes. The reaction solution was cooled to room temperature, and purified directly by Pre-HPLC to obtain 41 (11 mg, yield: 83.27%).
2-Chloro-4-phenylpyridine-3-carbonitrile (4293.20 mg, 20.00 mmol) and 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxyborane (7701.00 mg, 50.00 mmol) were dissolved in dioxane (50.0 mL) and water (10.0 mL), and potassium carbonate (8292.60 mg, 60.00 mmol) and (1,1′-bis(diphenylphosphino)ferrocene) dichloropalladium (1463.40 mg, 2.00 mmol) were mixed in a microwave tube, and the microwave tube was sealed after 1 minute of nitrogen substitution. The reaction solution was stirred at 100° C. for 16 hours, and the reaction solution was cooled to room temperature. The reaction solution was directly diluted with water (50 mL), and extracted with dichloromethane (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=85:15) to obtain 42-e (3150 mg, yield: 76.36%). LC-MS (ESI): m/z=207.29(M+H)+.
Compound 42-e (412.50 mg, 2.0 mmol) and compound 2-d (876.56 mg, 2.00 mmol) were dissolved in N, N-dimethylacetamide (6.0 mL), and palladium acetate (44.90 mg, 0.20 mmol), tri(o-tolyl)phosphine (121.75 mg, 0.40 mmol), and triethylamine (2.0 mL, 14.389 mmol) were added, and the mixture was heated via microwave at 160° C. for 0.5 hour under nitrogen protection. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 42-d (550 mg, yield: 48.79%).
Compound 42-d (225.45 mg, 0.4 mmol) and 2-methylpropan-2-yl-4-(iodomethyl) hexahydropyridine-1-carboxylate (650.38 mg, 2.00 mmol) were dissolved in N, N-dimethylformamide (2 mL), and potassium carbonate (276.42 mg, 2.00 mmol) was added, and the reaction solution was heated and stirred at 80° C. for 60 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (40 mL), and washed sequentially with water (20 mL×2) and saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 42-c (248 mg, yield: 81.48%).
Compound 42-c (248 mg, 0.326 mmol) was dissolved in tetrahydrofuran (3 mL), and dioxane solution of hydrochloric acid (1 mL) was added. The reaction solution was stirred at room temperature for 6 hours. 1 M K2CO3 solution (10 mL) was added. The mixture was extracted with dichloromethane (30 mL). The separated organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 42-b (215 mg, yield: 99.83%). The product 42-b was directly used in the next step of reaction without purification.
Compound 42-b (100 mg, 0.151 mmol), (10-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy)-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl) acetic acid (104.01 mg, 0.182 mmol) and O-benzotriazole-tetramethyluronium hexafluorophosphate (74.61 mg, 0.197 mmol) were dissolved in N, N-dimethylformamide (1.0 mL), and diisopropylethylamine (0.050 mL, 0.303 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether:ethyl acetate:ethanol=10:20:1) to obtain 42-a (360 mg, yield: 78.89%).
Compound 42-a (150 mg, 0.096 mmol) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (2.0 mL) was added. The reaction solution was stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by Pre-HPLC to obtain 42 (63 mg, yield: 66.01%).
Compound 42-d (112.72 mg, 0.2 mmol), 1,4-diiodobutane (247.94 mg, 0.80 mmol) was dissolved in N, N-dimethylformamide (1.0 mL), and potassium carbonate (55.28 mg, 0.40 mmol) was added, and the reaction solution was stirred at 50° C. for 1 hour. The reaction solution was cooled to room temperature, and the reaction solution was diluted with ethyl acetate (50 mL), washed sequentially with water (20 mL×2) and saturated brine (20 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 43-c (124 mg, yield: 83.15%).
Compound 43-c (124 mg, 0.166 mmol) was dissolved in tetrahydrofuran (3 mL), and ammonia methanol solution (10 mL, 70.00 mmol) was added. The reaction solution was heated and stirred at 50° C. for 16 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was dissolved in dichloromethane (30 mL) and washed with 1 M K2CO3 solution (10 mL). The separated organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 43-b (105 mg, yield: 99.47%), which was directly used in the next step of reaction without purification.
Compound 43-b (105 mg, 0.165 mmol), (4,7,10-tris{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (113.69 mg, 0.199 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (81.56 mg, 0.215 mmol) were dissolved in acetonitrile (1.0 mL), and diisopropylethylamine (0.055 mL, 0.331 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was separated by column chromatography (dichloromethane:methanol=94:6) to obtain 43-a (196 mg, yield: 99.61%).
Compound 43-a (196 mg, 0.165 mmol) was dissolved in dichloromethane (3.0 mL). Trifluoroacetic acid (3.0 mL) was added. The reaction solution was stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated by Pre-HPLC to obtain compound 43 (80 mg, yield: 50.31%). LC-MS (ESI): m/z=965.45 (M+H)+.
Compound 42 (9.91 mg, 0.01 mmol) was dissolved in sodium acetate-acetic acid buffer (pH=4.5) (0.5 mL), and lutetium trichloride (5.63 mg, 0.020 mmol) was added, and the reaction solution was stirred at 90° C. for 10 minutes. The reaction solution was cooled to room temperature, and separated directly by Pre-HPLC to obtain 44 (10.6 mg, yield: 91.14%). LC-MS (ESI): m/z=1163.12 (M+H)+.
Compound 43 (11 mg, 0.011 mmol) and lutetium trichloride (9.28 mg, 0.033 mmol) were dissolved in sodium acetate-acetic acid buffer (pH=4.5) (0.5 mL), and the reaction solution was stirred at 90° C. for 10 minutes. The reaction solution was directly separated by Pre-HPLC to obtain 45 (9 mg, yield: 71.94%). LC-MS (ESI): m/z=1137.80 (M+H)−.
Compound 40 (15 mg, 0.011 mmol) was dissolved in sodium acetate-acetic acid buffer (pH=4.5) (0.5 mL), and lutetium trichloride (5.98 mg, 0.021 mmol) was added. The reaction solution was heated and stirred at 90° C. for 10 minutes. The reaction solution was cooled to room temperature, and purified directly by Pre-HPLC to obtain 46 (5.5 mg, yield: 41.70%).
A solution of 2-methylpropan-2-yl bromoacetate (3019.14 mg, 15.478 mmol) dissolved in acetonitrile (40 mL) was added dropwise into a solution of 1,4,7-triazane (1000 mg, 7.739 mmol) dissolved in acetonitrile (10 mL) at 0° C. After dropwise addition, the reaction solution was stirred at room temperature for 24 hours, and the reaction solution was concentrated under reduced pressure, and diluted with pure water (5 mL). The mixture was adjusted to pH=3 with 1 M hydrochloric acid. The reaction solution was extracted with methyl tert-butyl ether (15 mL×3), and the mixture was adjusted to PH=8 with 1 M sodium hydroxide solution, extracted with dichloromethane (20 mL×2), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 47-b (1200 mg, yield: 43.37%).
Compound 2-b (140.53 mg, 0.20 mmol) and compound 47-b were dissolved in acetonitrile (1.0 mL), and potassium carbonate (55.28 mg, 0.400 mmol) was added, and the reaction solution was stirred at 60° C. overnight. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was separated by column chromatography (dichloromethane:methanol=5:1) to obtain 47-a (153 mg, yield: 78.12%).
Compound 47-a (153 mg, 0.156 mmol) was dissolved in dichloromethane (2.5 mL), and trifluoroacetic acid (2.5 mL) was added. The reaction solution was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was separated by Pre-HPLC to obtain 47 (75 mg, yield: 59.19%).
Aluminum chloride (0.80 mg, 0.006 mmol) was dissolved in 0.2 M sodium acetate-acetic acid buffer (pH=4.5) (0.60 mL), and sodium fluoride (0.25 mg, 0.006 mmol) was added, and the reaction solution was stirred at room temperature for 5 minutes. Compound 47 (4.05 mg, 0.005 mmol) was added, and the mixture was heated at 100° C. for 15 minutes. The reaction solution was cooled to room temperature, and the reaction solution was purified directly by Pre-HPLC to obtain 48 (3.0 mg, yield: 70.26%).
4,4,5,5-Tetramethyl-2-[(1E)-2-(2-methyl-3-phenylphenyl)vinyl]-1,3,2-dioxyborane (960.7 mg, 3.0 mmol) and 4-bromo-2-hydroxy-5-methylphenyl-1-formaldehyde (645.1 mg, 3.00 mmol) were dissolved in dioxane (100.0 mL) and water (20.0 mL), and (1,1′-bis(diphenylphosphino)ferrocene)dichloropalladium (219.3 mg, 0.3 mmol) and potassium carbonate (1243.8 mg, 9.00 mmol) were added. The reaction solution was heated and stirred at 90° C. for 12 hours, and the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 49-d (950 mg, yield: 86.78). LC-MS (ESI): m/z=329.1(M+Na)*.
Compound 49-d (620 mg, 2.89 mmol) and 1-bromo-2-[(2-bromoethyl)oxy]ethane (875.6 mg, 3.77 mmol) were dissolved in N, N-dimethylformamide (50 m1), and potassium carbonate (782.7 mg, 5.66 mmol) was added, and the reaction solution was stirred at 70° C. for 12 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was dissolved with dichloromethane (100 mL), and the mixture was washed sequentially with water (50 ml×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain 49-c (220 mg, yield: 23.09%).
Compound 49-c (280.0 mg, 0.58 mmol) and compound 2-e (216.41 mg, 1.16 mmol) were dissolved in methanol (40 mL), and sodium cyanoborohydride (110.39 mg, 1.752 mmol) and acetic acid (35.0 mg, 0.584 mmol) were added, and the reaction solution was stirred at 60° C. for 6 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the concentrate was separated by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 49-b (280 mg, yield: 70.21%). LC-MS (ESI): m/z=648.3 (M+H)+.
Compound 49-b (380 mg, 0.586 mmol) and compound 47-b (418.8 mg, 1.172 mmol) were dissolved in acetonitrile (30.0 mL), and potassium carbonate (323.36 mg, 2.343 mmol) was added. The reaction solution was stirred at 60° C. for 12 hours, and the reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was dissolved in dichloromethane (100 mL). The mixture was washed sequentially with water (50 ml×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 49-a (320 mg, yield: 56.09%).
Compound 49-a (320 mg, 0.346 mmol) was dissolved in dichloromethane (20.0 mL) and trifluoroacetic acid (20 mL) was added. The reaction solution was stirred at room temperature for 12 hours and concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 49 (52 mg, yield: 19.86%). LC-MS (ESI): m/z=757.5(M+H)+.
AlCl3 (100 mg) was dissolved in 0.2 M AcONa/AcoH buffer (50 mL), and the solution was shaken well and set aside. NaF (50 mg) and 0.2 M AcONa/AcoH buffer (40 mL) were added to a plastic (centrifuge) tube with cover, and the solution was shaken well and set aside. The above AlCl3 solution (10.0 mL) was added to the plastic (centrifuge) tube, then NaF solution (5.0 mL) was added. The mixture was shaken for 2 minutes, and set aside.
2. Compound 49 (1.0 mg, 0.13 mmol) was dissolved in anhydrous ethanol (0.6 mL), and [AlF]2+ solution (0.016 mmol, 1.6 mL) prepared above was added, and the reaction solution was stirred at 100° C. for 30 minutes. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was directly separated by prep-HPLC to obtain 50 (6.2 mg, yield: 58.6%). LC-MS (ESI): m/z=801.2(M+H)+.
The compound butane-1,4-diol (901.2 mg, 10.0 mmol) was dissolved in dichloromethane (50.0 mL), and pyridine (3160.0 mg, 40.0 mmol) was added, and the reaction solution was cooled to 0° C., then 4-methylbenzenesulfonyl chloride (5719.5 mg, 30.00 mmol) was added. The reaction solution was stirred at room temperature overnight, concentrated under reduced pressure, and the residue was dissolved with ethyl acetate (100 mL), and the mixture was washed with water (50.0 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=15:1) to obtain 51-d (670 mg, yield: 16.8%). LC-MS (ESI): m/z=329.1(M+Na)*.
Compound 49-d (656.8 mg, 2.0 mmol) and compound 51-d (1594.0 mg, 4.0 mmol) were dissolved in N, N-dimethylformamide (50.0 mL), and potassium carbonate (1382.0 mg, 10.0 mmol) was added. The reaction solution was heated and stirred at 50° C. for 6 hours, and the reaction solution was cooled to room temperature. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (100 mL), and the mixture was washed sequentially with water (50 ml×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain 51-c (610 mg, yield: 54.98%). LC-MS (ESI): m/z=555.2(M+H)+.
Compound 51-c (554.7 mg, 1.0 mmol) and compound 2-e (555.8 mg, 3.0 mmol) were dissolved in methanol (40 mL), and sodium cyanoborohydride (126.0 mg, 2.0 mmol) and acetic acid (120.0 mg, 2.0 mmol) were added. The reaction solution was stirred at 60° C. for 6 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 51-b (370 mg, yield: 51.1%). LC-MS (ESI): m/z=724.6(M+H)+.
Compound 57-b (370 mg, 0.511 mmol) and compound 47-b (365.4 mg, 1.02 mmol) were dissolved in acetonitrile (30.0 mL), and potassium carbonate (282.1 mg, 2.04 mmol) was added. The reaction solution was heated and stirred at 60° C. for 12 hours, and the reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in dichloromethane (100 mL), and the mixture was washed sequentially with water (50 ml×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol=10:1) to obtain 51-a (350 mg, yield: 75.32%). LC-MS (ESI): m/z=926.2(M+H)+.
Compound 51-a (350 mg, 0.385 mmol) was dissolved in dichloromethane (20.0 mL), and trifluoroacetic acid (20 mL) was added, and the reaction solution was stirred at room temperature for 12 hours and concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 51 (158 mg, yield: 55.4%). LC-MS (ESI): m/z=741.5(M+H)+.
Compound 51 (20 mmol, 0.027 mg) was dissolved in anhydrous ethanol (0.5 mL), and the [AlF]2+ solution (0.016 mmol, 1.6 mL) prepared in Embodiment 50 was added, and the reaction solution was stirred at 100° C. for 30 minutes. The reaction solution was cooled to room temperature, and directly separated by prep-HPLC to obtain 52 (15 mg, yield: 70.79%). LC-MS (ESI): m/z=785.2(M+H)+.
Compound 49-d (328.4 mg, 1.0 mmol) and 1,5-diiodopentane (647.8 mg, 2.0 mmol) were dissolved in N, N-dimethylformamide (20.0 mL), and potassium carbonate (414.6 mg, 3.0 mmol) was added. The reaction solution was stirred at room temperature for 12 hours, and water (60 mL) was added, and the mixture was extracted with dichloromethane (150 mL). The organic phases were combined, washed sequentially with water (50 m1×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 53-c (55 mg, yield: 9.96%). LC-MS (ESI): m/z=525.2 (M+H)+.
Compound 53-c (340 mg, 0.648 mmol) and compound 2-e (240.23 mg, 1.297 mmol) were dissolved in methanol (40 mL), and sodium cyanoborohydride (81.69 mg, 1.29 mmol) and acetic acid (77.8 mg, 1.29 mmol) were added. The reaction solution was heated and stirred at 60° C. for 5 hours, and the reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 53-b (158 mg, yield: 55.4%). LC-MS (ESI): m/z=694.8 (M+H)+.
Compound 53-b (230 mg, 0.332 mmol) and compound 47-b (237.0 mg, 0.663 mmol) were dissolved in acetonitrile (30.0 mL), and potassium carbonate (137.2 mg, 0.995 mol) was added. The reaction solution was heated and stirred at 65° C. for 12 hours, and the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was dissolved in dichloromethane (100 mL). The mixture was washed sequentially with water (50 ml×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 53-a (230 mg, yield: 75.13%). LC-MS (ESI): m/z=923.7 (M+H)+.
Compound 53-a (230 mg, 0.249 mmol) was dissolved in dichloromethane (20.0 mL), and trifluoroacetic acid (10.0 mL) was added. The reaction solution was stirred at room temperature for 18 hours, and the reaction solution was concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 53 (110.3 mg, yield: 58.65%). LC-MS (ESI): m/z=755.7 (M+H)+.
Compound 53 (0.033 mmol, 25.0 mg) was dissolved in anhydrous ethanol (0.5 mL), and the [AlF]2+ solution (0.016 mmol, 1.6 mL) prepared in Embodiment 50 was added, and the reaction solution was heated and stirred at 100° C. for 30 minutes. The reaction solution was cooled to room temperature, and directly separated by prep-HPLC to obtain 54 (14.1 mg, yield: 53.29%). LC-MS (ESI): m/z=800.6 (M+H)+.
Compound 49-d (328.41 mg, 1.0 mmol) and 1,6-diiodohexane (675.94 mg, 2.0 mmol) were dissolved in N, N-dimethylformamide (30.0 mL), and potassium carbonate (414.60 mg, 3.0 mmol) was added. The reaction solution was stirred at room temperature for 12 hours and concentrated under reduced pressure, and the residue was dissolved in dichloromethane (100 mL). The mixture was washed sequentially with water (50 m1×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 55-c (380 mg, yield: 67.04%). LC-MS (ESI): m/z=539.2 (M+H)+.
Compound 55-c (340 mg, 0.631 mmol) and compound 2-e (240.0 mg, 1.263 mmol) were dissolved in methanol (40 mL), and sodium cyanoborohydride (79.56 mg, 1.26 mmol) and acetic acid (75.8 mg, 1.26 mmol) were added. The reaction solution was heated and stirred at 60° C. for 5 hours, and the reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 55-b (230 mg, yield: 51.47%). LC-MS (ESI): m/z=709.8 (M+H)+.
Compound 55-b (380 mg, 0.586 mmol) and compound 47-b (418.8 mg, 1.172 mmol) were dissolved in acetonitrile (30.0 mL), and potassium carbonate (323.36 mg, 2.343 mmol) was added, and the reaction solution was heated and stirred at 60° C. for 12 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was dissolved in dichloromethane (100 mL). The mixture was washed sequentially with water (50 ml×2) and saturated brine (50 mlxi), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 55-a (180 mg, yield: 61.78%). LC-MS (ESI): m/z=926.2 (M+H)+.
Compound 55-a (220 mg, 0.235 mmol) was dissolved in dichloromethane (20.0 mL), and trifluoroacetic acid (10.0 mL) was added, and the reaction solution was stirred at room temperature for 12 hours and concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 55 (160 mg, yield: 88.65%). LC-MS (ESI): m/z=769.6 (M+H)+.
Compound 55 (0.039 mmol, 6.0 mg) was dissolved in anhydrous ethanol (1.0 mL), and the [AlF]2+ solution (0.05 mmol, 5.0 mL) prepared in Embodiment 50 was added, and the reaction solution was heated and stirred at 100° C. for 30 minutes. The reaction solution was cooled to room temperature, and directly separated by prep-HPLC to obtain 56 (6 mg, yield: 18.92%). LC-MS (ESI): m/z=814.6 (M+H)+.
Compound 42-d (195 mg, 0.346 mmol) and 1,5-diiodopentane (224.15 mg, 0.692 mmol) were dissolved in N, N-dimethylformamide (5 mL), and potassium carbonate (239.09 mg, 1.730 mmol) was added, and the reaction solution was stirred at 60° C. for 1 hour. The reaction solution was cooled to room temperature, and the reaction solution was extracted with ethyl acetate (10 mL), washed sequentially with water (10 mL×3) and saturated brine (10 mL×1), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 57-b (211 mg, yield: 80.49%).
Compound 57-b (211 mg, 0.278 mmol) and compound 47-b were dissolved in acetonitrile (10 mL), and potassium carbonate (76.98 mg, 0.557 mmol) was added, and the reaction solution was stirred at 60° C. overnight. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was dissolved with ethyl acetate (10 mL), and the mixture was washed sequentially with water (10 mL×3) and saturated brine (10 mL×1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 57-a (242 mg, yield: 87.85%).
Compound 57-a (242 mg, 0.245 mmol) was dissolved in dichloromethane (9 mL), and trifluoroacetic acid (9 mL, 1.223 mmol) was added. The reaction solution was stirred at room temperature for 30 minutes, and the reaction solution was concentrated under reduced pressure, and the residue was separated by prep-HPLC to obtain 57 (93.61 mg, yield: 46.61%).
Compound 57 (20 mg, 0.024 mmol) was dissolved in ethanol (0.50 mL), and 0.2 M sodium acetate-acetic acid buffer (pH=4.6) (1.0 mL), aluminum chloride (2 mg, 0.029 mmol), and sodium fluoride (1 mg, 0.029 mmol) were added, and the reaction solution was stirred at 100° C. for 30 minutes. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by prep-HPLC to obtain 58 (11.68 mg, yield: 55.43%).
Compound 47-b (715.00 mg, 2.0 mmol) and potassium carbonate (552.84 mg, 4.00 mmol) were dissolved in methanol (10 mL) and water (10 mL), and bromoacetic acid (383.48 mg, 2.76 mmol) was added. The reaction solution was stirred at room temperature overnight, and the pH was adjusted to 6 with hydrochloric acid (1 mol/L), and the reaction solution was extracted with dichloromethane (50 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 59-b (610 mg, yield: 73.40%).
Compound 42-b (115 mg, 0.174 mmol) was dissolved in N, N-dimethylformamide (5 mL), and 59-b (86.78 mg, 0.209 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (85.80 mg, 0.226 mmol), and diisopropylethylamine (0.058 mL, 0.348 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was extracted with ethyl acetate (10 mL), washed sequentially with water (10 mL×3) and saturated brine (10 mL×1), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 59-a (45.66 mg, yield: 24.79%).
Compound 59-a (45.66 mg, 0.043 mmol) was dissolved in dichloromethane (9 mL), and trifluoroacetic acid (9 mL, 1.223 mmol) was added, and the reaction solution was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and directly separated by pre-HPLC to obtain 59 (22.57 mg, yield: 58.78%). LC-MS (ESI): m/z=890 (M+H)+.
Compound 59 (5 mg, 0.006 mmol) was dissolved in 0.2 M sodium acetate-acetic acid buffer (pH=4.6) (1.0 mL), and aluminum chloride (AlCl3, 0.6 mL, 0.002 mmol) and sodium fluoride (0.3 mL, 0.002 mmol) were added, and the reaction solution was stirred at 100° C. for 30 minutes. The reaction solution was cooled to room temperature, and separated directly by prer-HPLC to obtain 60 (1.96 mg, yield: 37.33%). LC-MS (ESI): m/z=934 (M+H)+.
Compound 4-b (124.5 mg, 0.20 mmol) and compound 59-b (108.0 mg, 0.26 mmol) were dissolved in N, N-dimethylformamide (15.0 mL), and O-benzotriazole-tetramethyluronium hexafluorophosphate (113.7 mg, 0.30 mmol) and diisopropylethylamine (77.4 mg, 0.60 mmol) were added, and the reaction solution was stirred at room temperature for 18 hours, diluted with dichloromethane (100 mL), washed sequentially with water (50 ml×3) and saturated brine (50 ml×1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol=10:1) to obtain 61-a (60 mg, yield: 29.81%). LC-MS (ESI): m/z=1020.7 (M+H)+.
Compound 61-a (60 mg, 0.059 mmol) was dissolved in dichloromethane (12.0 mL), and trifluoroacetic acid (6.0 mL) was added, and the reaction solution was stirred at room temperature for 6 hours and concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 61 (15 mg, yield: 29.94%). LC-MS (ESI): m/z=852.8 (M+H)+.
Compound 61 (0.008 mmol, 7.0 mg) was dissolved in anhydrous ethanol (1.0 mL), and the [AlF]2+ solution (0.016 mmol, 1.6 mL) prepared in Embodiment 50 was added, and the reaction solution was heated and stirred at 100° C. for 2 hours. The reaction solution was cooled to room temperature, and directly separated by prep-HPLC to obtain 62 (1.6 mg, yield: 21.74%). LC-MS (ESI): m/z=897.4 (M+H)+.
Compound 2-c (551.65 mg, 1.0 mmol) and 1,3-dibromopropane (0.305 mL, 3.00 mmol) were dissolved in DMF (5.0 mL), and potassium carbonate (276.42 mg, 2.00 mmol) was added, and the reaction solution was heated and stirred at 50° C. for 7 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (50 mL), and washed sequentially with water (20 mL×2) and saturated brine (20 mL×1). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain 63-c (461 mg, yield: 68.54%).
Compound 63-c (461 mg, 0.807 mmol) and ammonia methanol solution (7 M) (23 mL) were sealed in a microwave tube, and the reaction solution was heated and stirred at 50° C. for 60 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was dissolved in dichloromethane (50 mL) and washed with 10% K2CO3 solution (10 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 63-b (409 mg, yield: 99.89%), which was directly used in the next step of reaction without purification.
Compound 63-b (121.7 mg, 0.20 mmol) and compound 59-b (108.0 mg, 0.26 mmol) were dissolved in N, N-dimethylformamide (15.0 mL), and O-benzotriazole-tetramethyluronium hexafluorophosphate (113.7 mg, 0.30 mmol) and diisopropylethylamine (77.4 mg, 0.60 mmol) were added, and the reaction solution was stirred at room temperature for 18 hours, diluted with dichloromethane (100 mL), washed sequentially with water (50 ml×3) and saturated brine (50 ml×1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (dichloromethane:methanol=10:1) to obtain 63-a (90 mg, yield: 44.72%). LC-MS (ESI): m/z=1006.7 (M+H)+.
Compound 63-a (90 mg, 0.089 mmol) was dissolved in dichloromethane (20.0 mL), and trifluoroacetic acid (10.0 mL) was added, and the reaction solution was stirred at room temperature for 12 hours and concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 63 (25 mg, yield: 33.36%). LC-MS (ESI): m/z=838.4 (M+H)+.
Compound 63 (0.008 mmol, 7.0 mg) was dissolved in anhydrous ethanol (1.0 mL), and the [AlF]2+ solution (0.016 mmol, 1.6 mL) prepared in Embodiment 50 was added, and the reaction solution was heated and stirred at 100° C. for 2 hours. The reaction solution was cooled to room temperature, and directly separated by prep-HPLC to obtain 64 (6.67 mg, yield: 70.42%). LC-MS (ESI): m/z=883.4 (M+H)+.
Compound 16-c (252 mg, 0.337 mmol) and compound 47-b (156.64 mg, 0.438 mmol) were dissolved in acetonitrile (3.0 mL), and potassium carbonate (72 mg, 0.521 mmol) was added, and the reaction solution was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (dichloromethane:methanol=10:1) to obtain 65-a (190 mg, yield: 57.68%).
Compound 65-a (190 mg, 0.194 mmol) was dissolved in dichloromethane (3.0 mL), and trifluoroacetic acid (3.0 mL) was added. The reaction solution was stirred at room temperature overnight, and the reaction solution was concentrated under reduced pressure, and directly separated by pre-HPLC to obtain 65 (74 mg, yield: 47.05%). LC-MS (ESI): m/z=809.5(M+H)+.
1H NMR (400 MHz, MeOD) δ 7.90 (s, 1H), 7.62 (d, J=15.9 Hz, 1H), 7.57-7.49 (m, 2H), 7.46-7.38 (m, 2H), 7.38-7.23 (m, 5H), 7.18 (d, J=7.5 Hz, 1H), 4.59 (d, J=13.0 Hz, 1H), 4.42 (d, J=13.0 Hz, 1H), 4.32 (t, J=6.3 Hz, 2H), 3.59 (dd, J=9.9, 3.6 Hz, 1H), 3.56-3.35 (m, 5H), 3.24-2.78 (m, 15H), 2.30 (s, 3H), 2.28-2.16 (m, 1H), 2.06-1.67 (m, 8H), 1.67-1.51 (m, 3H).
Aluminum chloride (1.60 mg, 0.012 mmol) was dissolved in 0.2 M sodium acetate-acetic acid buffer (pH=4.6) (0.8 mL), and sodium fluoride (0.50 mg, 0.012 mmol) was added, and the reaction solution was stirred at room temperature for 5 minutes. Ethanol (0.60 mL) and compound 65 (8.09 mg, 0.01 mmol) were added, and the reaction solution was heated at 100° C. for 30 minutes. The reaction solution was cooled to room temperature, and directly purified by Pre-HPLC to obtain 66 (4.6 mg, yield: 53.93%).
Compound 19-b (64.88 mg, 0.1 mmol), compound 59-b (54.02 mg, 0.130 mmol), and O-benzotriazole-tetramethyluronium hexafluorophosphate (75.85 mg, 0.200 mmol) were dissolved in N, N-dimethylformamide (1.0 mL), and diisopropylethylamine (0.050 mL, 0.30 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was directly purified by Pre-HPLC to obtain 67-a (58 mg, yield: 55.43%).
Compound 67-a (58 mg, 0.055 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added, and the reaction solution was stirred at room temperature for 24 hours. The reaction solution was directly purified by Pre-HPLC to obtain 67 (25 mg, yield: 41.10%).
1H NMR (400 MHz, MeOD) δ: 7.94 (s, 1H), 7.62 (d, J=15.8 Hz, 1H), 7.54 (t, J=3.4 Hz, 2H), 7.47-7.39 (m, 2H), 7.39-7.23 (m, 5H), 7.18 (d, J=7.5 Hz, 1H), 4.63-4.49 (m, 2H), 4.40 (d, J=13.3 Hz, 1H), 4.16 (d, J=6.3 Hz, 2H), 3.95 (d, J=12.7 Hz, 1H), 3.84-3.53 (m, 7H), 3.44-3.33 (m, 1H), 3.26-2.61 (m, 15H), 2.30 (s, 3H), 2.29-2.17 (m, 2H), 2.06-1.49 (m, 7H), 1.49-1.23 (m, 2H).
Aluminum chloride (1.60 mg, 0.012 mmol) was dissolved in 0.2 M sodium acetate-acetic acid buffer (pH=4.6) (0.8 mL), and sodium fluoride (0.50 mg, 0.012 mmol) was added, and the reaction solution was stirred at room temperature for 5 minutes. Ethanol (0.60 mL) and compound 67 (8.78 mg, 0.01 mmol) were added, and the reaction solution was heated at 100° C. for 30 minutes. The reaction solution was cooled to room temperature, and directly purified by Pre-HPLC to obtain 68 (4.74 mg, yield: 51.41%).
2-methylpropan-2-yl (2R)-1-{[2-({2-[(2-bromoethyl)oxy]ethyl}oxy)-4-[(1E)-2-(2-methyl-3-phenylphenyl)vinyl]-5-(trifluoromethyl)phenyl]methyl}hexahydropyridine-2-carboxylate (140.53 mg, 0.2 mmol) and compound tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecan-1,4,7-triyl)triacetate (154.41 mg, 0.300 mmol) were dissolved in acetonitrile (5.0 mL), and potassium carbonate (55.28 mg, 0.400 mmol) was added. The reactant was heated and stirred at 60° C. for 16 hours. The reaction solution was cooled to room temperature, filtered, and concentrated under reduced pressure to obtain 69-a (226 mg, yield: 99.43%), which was directly used in the next step of reaction without purification.
Compound 69-a (226 mg, 0.199 mmol) was dissolved in dichloromethane (3.0 mL), and trifluoroacetic acid (3.0 mL) was added, and the reaction solution was stirred at room temperature overnight, and directly separated by Pre-HPLC to obtain 69 (127 mg, yield: 70.02%).
Compound 69 (13.68 mg, 0.015 mmol) and lutetium trichloride (8.44 mg, 0.030 mmol) were dissolved in 0.2 M AcONa/AcOH buffer (pH=4.5, 1.0 mL), and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was directly purified by Prep-HPLC to obtain 70 (9.77 mg, yield: 60.09%). LC-MS (ESI): m/z=1084.68(M+H)+.
Compound 53-b (180 mg, 0.259 mmol) and tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecan-1,4,7-triyl) triacetate (237.0 mg, 0.519 mmol) were dissolved in acetonitrile (30.0 mL), and potassium carbonate (35.81 mg, 0.259 mmol) was added. The reaction solution was heated and stirred at 60° C. for 3 hours, and the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the residue was dissolved in dichloromethane (100 mL). The mixture was washed sequentially with water (50 ml×2) and saturated brine (50 m1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 71-a (230 mg, yield: 75.13%). LC-MS (ESI): m/z=1080.5 (M+H)+.
Compound 71-a (170 mg, 0.157 mmol) was dissolved in dichloromethane (15.0 mL), and trifluoroacetic acid (15.0 mL) was added, and the reaction solution was stirred at room temperature for 16 hours and concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 71 (85.7 mg, yield: 63.6%). LC-MS (ESI): m/z=856.0(M+H)+.
Compound 71 (13.68 mg, 0.015 mmol) and lutetium trichloride (8.44 mg, 0.030 mmol) were dissolved in 0.2 M AcONa/AcOH buffer (pH=4.5, 1.0 mL), and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was directly purified by prep-HPLC to obtain 72 (9.60 mg, yield: 53.3%). LC-MS (ESI): m/z=1028.02(M+H)+.
Compound 71 (11.0 mg, 0.013 mmol) and gallium trichloride (1.76 mg, 0.010 mmol) were added to water (0.2 mL), and the reaction solution was heated and stirred at 90° C. for 5 minutes. The reaction solution was cooled to room temperature, and the reaction solution was directly separated by Pre-HPLC to obtain 73 (11.1 mg, yield: 92.5%). LC-MS (ESI): m/z=922.77(M+H)+.
Compound 4 was dissolved in DMSO to a 10 mg/mL solution, and the solution was taken out a portion to dilute to 0.1 mg/mL with sodium acetate buffer which is metal-free and pH=5.5. The germanium-gallium generator was eluted with 5 mL of 0.1 M HCl in segments, and the highest activity portion (0.5 mL) was taken and 0.5 mL of sodium acetate buffer was added thereto, then 20-fold equivalent of compound 4 (0.1 mg/mL) was added. The mixture was vortexed and mixed for 10 s, and heated and shaken at 95° C. and 800 rpm for 30 minutes. The C18 cartridge was activated with ethanol, then eluted clean with pure water and eluted dry, and the solution after the reaction completed was passed through the C18 cartridge, and the C18 cartridge was eluted with pure water and eluted dry and then eluted with ethanol. TLC purity detection was carried out using 1% EDTA as a developing agent, and compound 4 labeled with 68Ga was detected by TLC scanning, and its radiochemical purity was 99.67%.
Appropriate amounts of 68Ga-labeled compound 4 and compound 8 were taken for HPLC characterization according to the following conditions, and chromatographic conditions were as follows:
After HPLC characterization, the UV peak time of compound 8 and the radioactive peak time of compound 4 labeled with 68Ga are both around 7:07 (mm:ss), indicating that the labeling of compound 4 by 68Ga was successful (namely, compound 74), which was consistent with the results of TLC scanning.
Compound 2 was dissolved in DMSO to a 10 mg/mL solution, and the solution was taken out a portion to dilute to 0.1 mg/mL with sodium acetate buffer which is metal-free and pH=5.5. The germanium-gallium generator was eluted with 5 mL of 0.1 M HCl in segments, and the highest activity portion (0.5 mL) was taken and 0.5 mL of sodium acetate buffer was added thereto, then 20-fold equivalent of compound 2 (0.1 mg/mL) was added. The mixture was vortexed and mixed for 10 s, and heated and shaken at 95° C. and 800 rpm for 30 minutes. The C18 cartridge was activated with ethanol, then eluted clean with pure water and eluted dry, and the solution after the reaction completed was passed through the C18 cartridge, and the C18 cartridge was eluted with pure water and eluted dry, and then eluted with ethanol. The compound 2 labeled with 68Ga was detected by TLC scanning, and its radiochemical purity was 99.74%.
Appropriate amounts of 68Ga-labeled compound 2 and compound 7 were taken for HPLC characterization according to the following conditions, and chromatographic conditions were as follows:
Method: Time: 0 min 25% B, 10 min 95% B
After HPLC characterization, the UV peak time of compound 7 and the radioactive peak time of compound 2 labeled with 68Ga are both around 7:03 (mm:ss), indicating that the labeling of compound 2 by 68Ga was successful (namely, compound 75), which was consistent with the resluts of TLC scanning.
Compound 1 was dissolved in DMSO to a 10 mg/mL solution, and the solution was taken out a portion to dilute to 0.1 mg/mL with sodium acetate buffer which is metal-free and pH=5.5. The metal bath reactor was turned on and preheated to 25° C. A solution of 1 mCi (about 50 pmol) 177LuCl3 was taken, and then a solution of compound 1 with 20-fold equivalent was added, and the solution was supplemented to 50 μL via sodium acetate buffer, and the mixture was reacted at 25° C. and 800 rpm for 120 minutes. TLC purity detection was carried out using 1% EDTA as a developing agent, and compound 1 labeled with 177Lu was detected by TLC scanning, and its radiochemical purity was 96.90%.
Appropriate amounts of compound 15 and 177Lu-labeled compound 1 were taken for HPLC characterization according to the following conditions, and chromatographic conditions were as follows:
The 175Lu-labeled compound 1 was characterized by HPLC and the peak time was confirmed to be 5.731 minutes.
The amount of 177Lu-labeled compound 1 was so low that UV light could not be seen, so a tube of mobile phase was collected every 0.5 minute, and a total of 20 tubes were collected. The radioactivity count of each tube of mobile phase was detected, and the radioactivity count-time curve was drawn. Compared with the HPLC of compound 15, it could be found that the peak time of 177Lu-labeled compound 1 was consistent with compound 15, indicating that 177Lu-labeled compound 1 (namely, compound 76) was successfully prepared.
Compound 2 was dissolved in DMSO to a 10 mg/mL solution, and the solution was taken out a portion to dilute to 0.1 mg/mL with sodium acetate buffer which is metal-free and pH=5.5. The metal bath reactor was turned on and preheated to 95° C. A solution of 1 mCi (about 50 pmol) 177LuCl3 was taken, and then a solution of compound 2 with 20-fold equivalent was added, and the solution was supplemented to 50 μL via sodium acetate buffer, and the mixture was reacted at 95° C. and 800 rpm for 30 minutes. TLC purity detection was carried out using 1% EDTA as a developing agent, and compound 2 labeled with 177Lu was detected by TLC scanning, and its radiochemical purity reached 100%, which could meet the requirements for animal administration and no further purification was needed.
Appropriate amounts of compound 6 and 177Lu-labeled compound 2 were taken for HPLC characterization according to the following conditions, and chromatographic conditions were as follows:
The 175Lu-labeled compound 2 was characterized by HPLC and the peak time was confirmed to be 6.457 minutes.
The amount of 177Lu-labeled compound 2 was so low that UV light could not be seen, so a tube of mobile phase was collected every 0.5 minute, and a total of 20 tubes were collected. The radioactivity count of each tube of mobile phase was detected, and the radioactivity count-time curve was drawn. Compared with the HPLC of 175Lu-labeled compound 2, it could be found that the peak time of 177Lu-labeled compound 2 was consistent with compound 6, indicating that 177Lu-labeled compound 2 (namely, compound 77) was successfully prepared.
Compound 4 was dissolved in DMSO to a 10 mg/mL solution, and the solution was taken out a portion to dilute to 0.1 mg/mL with sodium acetate buffer which is metal-free and pH=5.5. The metal bath reactor was turned on and preheated to 95° C. A solution of 1 mCi (about 50 pmol)177LuCl3 was taken, and then compound 4 with 20-fold equivalent was added, and the solution was supplemented to 50 μL via sodium acetate buffer, and the mixture was reacted at 95° C. and 800 rpm for 30 minutes. TLC purity detection was carried out using 1% EDTA as a developing agent, and compound 4 labeled with 177Lu was detected by TLC scanning, and its radiochemical purity reached 100%, which could meet the requirements for animal administration and no further purification was needed.
Appropriate amounts of compound 5 and 177Lu-labeled compound 4 were taken for HPLC characterization according to the following conditions, and chromatographic conditions were as follows:
The 175Lu-labeled compound 4 was characterized by HPLC and the peak time was confirmed to be 6.690 minutes.
The amount of 177Lu-labeled compound 4 was so low that UV light could not be seen, so a tube of mobile phase was collected every 0.5 minute, and a total of 20 tubes were collected. The radioactivity count of each tube of mobile phase was detected, and the radioactivity count-time curve was drawn. Compared with the HPLC of compound 5, it can be found that the peak time of 177Lu-labeled compound 4 was consistent with compound 5, indicating that 177Lu-labeled compound 4 (namely, compound 78) was successfully prepared.
Buffer system: sodium acetate-acetic acid buffer solution with a pH of around 4.0 and a concentration of 0.5 M (freshly prepared).
QMA column activation: 5 mL of water, 5 mL of freshly prepared sodium acetate-acetic acid buffer solution with a pH of around 4.0 and a concentration of 0.5 M.
F-18 ion purification: the QMA column was passed through target water and eluted sequentially with 0.3 mL buffer.
Precursor solution: 100 μL buffer, 6 μL AlCl3 buffer solution with a concentration of 10 mM, 300 μL acetonitrile (reaction-promoting solvent), 20 μL precursor solution (3 mg/mL).
The precursor solution was mixed evenly and let stand to equilibrate for 5 minutes.
300 μL of QMA column eluent containing F-18 was added to the reaction solution.
The plastic centrifuge tube was sealed and heated for 15 minutes, and the heating temperature was 100° C.
C18 Light cartridge purification: the reaction solution was diluted with 5 mL of water, then the sample was loaded to the C18 Light cartridge, and the product was eluted with 1 mL of ethanol-water solution at a concentration of 50% (v/v). The eluent was injected into HPLC for analysis.
The chromatographic conditions were as follows
Quality control analysis of compound 79
The radiochemical purity of compound 79 is 95.28%.
Buffer system: sodium acetate-acetic acid buffer solution with a pH of around 4.0 and a concentration of 0.5 M (freshly prepared).
Compound 67 was dissolved in buffer solution, and 300 μL of 68Ga3+ solution was added. The plastic centrifuge tube was sealed and heated for 15 minutes, and the heating temperature was 100° C.
C18 Light cartridge purification: the reaction solution was diluted with 5 mL of water, then the sample was loaded to the C18 Light cartridge, and the product was eluted with 1 mL of ethanol-water solution at a concentration of 50% (v/v) to obtain compound 80.
Methyl 4-methoxypyridinecarboxylate (5000 mg, 29.94 mmol) was added to concentrated sulfuric acid (100 mL), and N-bromosuccinimide (7993.98 mg, 44.91 mmol) was added in batches at room temperature, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was cooled to 0° C., and then the reaction solution was slowly added into ice water (1.0 L), and the pH of the mixture was adjusted to 7-9 with saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (1.0 L), and the organic phase was collected, washed sequentially with water (500 mL×3) and saturated brine (500 mL×1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain 81-h (7 g, yield: 95.04%). LC-MS (ESI): m/z=248.3(M+H)+.
Compound 81-h (7.0 g, 28.448 mmol) and potassium trifluoro(vinyl)borate (5675.45 mg, 42.673 mmol) were dissolved in dioxane (60.0 mL) and water (6.0 mL), and 1,1′-bis(diphenylphosphino)ferrocene dichloropalladium (2081.57 mg, 2.845 mmol) and potassium carbonate (11795.5 mg, 85.3 mmol) were added. The reaction solution was heated and stirred at 90° C. for 16 hours, and the reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 81-g (3.4 g, yield: 61.86%). LC-MS (ESI): m/z=194.22(M+H)+.
Compound 81-g (966.0 mg, 5.0 mmol) and 3-bromo-2-methylphenyl-1-amine (1023.2 mg, 5.50 mmol) were dissolved in tetrahydrofuran (30 mL), and the reaction solution was cooled to 0° C., and sodium bis(trimethylsilyl)amide (6.5 mL, 1.0 M) was added dropwise to the reaction solution. After dropwise addition, the reaction solution was stirred at room temperature for 1 hour, and ammonium chloride aqueous solution (100 mL) was slowly added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (150 mL), and the organic phase was collected, washed sequentially with water (100 mL×3) and saturated brine (100 mL×1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=10:1) to obtain 81-f (1.4 g, yield: 80.64%). LC-MS (ESI): m/z=349.3(M+H)+.
Compound 81-f (850 mg, 2.448 mmol) and potassium osmate (90.09 mg, 0.245 mmol) were dissolved in dioxane (60 mL) and water (20 mL), and sodium periodate (1570.86 mg, 7.344 mmol) was added in batches, and the reaction solution was stirred at room temperature for 6 hours. The reaction solution was filtered, and the filter cake was washed with dioxane (50 mL), and the filter cake was purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 81-e (360 mg, yield: 42.11%). LC-MS (ESI): m/z=351.2(M+H)+.
Compound 81-e (360 mg, 1.03 mmol) and compound 2-e (382.02 mg, 2.06 mmol) were dissolved in methanol (60.0 mL), and sodium cyanoborohydride (129.91 mg, 2.06 mmol) and acetic acid (129.90 mg, 2.16 mmol) were added, and the reaction solution was heated and stirred at 60° C. for 6 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 81-d (280 mg, yield: 52.38%). LC-MS (ESI): m/z=520.3(M+H)+.
Compound 81-d (280.0 mg, 0.54 mmol) and compound 40-d (487.2 mg, 0.81 mmol) were dissolved in dioxane (50.0 mL) and water (5.0 mL), and 1,1′-bis(diphenylphosphino)ferrocene dichloropalladium (39.52 mg, 0.054 mmol) and potassium carbonate (223.9 mg, 1.62 mmol) were added, and the reaction solution was heated and stirred at 110° C. for 12 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 81-c (210 mg, yield: 38.46%). LC-MS (ESI): m/z=913.53 (M+H)+.
Compound 81-c (198 mg, 0.217 mmol) and 1-bromo-2-[(2-bromoethyl)oxy]ethane (150.8 mg, 0.651 mmol) were dissolved in N, N-dimethylformamide (50.0 mL), and potassium carbonate (89.77 mg, 0.651 mmol) was added, and the reaction solution was heated and stirred at 80° C. for 12 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was dissolved with dioxane (60 mL), and the mixture was washed sequentially with water (40 mL×3) and saturated brine (40 mL×1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain 81-b (70 mg, yield: 28.6%). LC-MS (ESI): m/z=1065.14(M+H)+.
Compound 81-b (70 mg, 0.066 mmol) and compound 47-b (47.07 mg, 0.132 mmol) were dissolved in acetonitrile (10.0 mL), and potassium carbonate (36.37 mg, 0.263 mmol) was added, and the reaction solution was heated and stirred at 60° C. for 3 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure, and the residue was separated by column chromatography (dichloromethane:methanol=10:1) to obtain 81-a (80 mg, yield: 90.7%). LC-MS (ESI): m/z=1341.0(M+H)+.
Compound 81-a (80 mg, 0.060 mmol) was dissolved in dichloromethane (10.0 mL), and trifluoroacetic acid (5.0 mL) was added, and the reaction solution was stirred at room temperature for 6 hours and concentrated under reduced pressure, and the residue was directly separated by pre-HPLC to obtain 81 (28 mg, yield: 42.04%). LC-MS (ESI): m/z=1116.64(M+H)+.
Compound 81 (8.0 mg, 0.007 mmol) was dissolved in ethanol (0.5 mL), and the [AlF]2+ solution (0.016 mmol, 1.6 mL) was added, and the reaction solution was stirred at 100° C. for 1 hour. The reaction solution was cooled to room temperature, and directly separated by pre-HPLC to obtain 82 (4 mg, yield: 48.08%). LC-MS (ESI): m/z=1160.8(M+H)+.
Homogenouse Time-Resolved Fluorescence (HTRF) binding assay was used to detect the binding ability of the compound of the present disclosure to PD-1/PD-L1.
The purchased kit (CisBio, #64CUS000C-1) contains reagents required for experiments such as PD-1, PD-L1, anti-tag1-Eu, Anti-tag2-XL665, Dilute Buffer, and Detection Buffer.
The biological activity of the compounds of the present disclosure were determined by the above tests, and the results obtained are as follows (Table 1):
Ten tumor-bearing mice with tumor volumes of 10-100 mm3 were systematically and randomly divided into 2 groups according to tumor size, with 5 mice in each group, corresponding to PBS and compound 78 respectively. After anesthesia with isoflurane, tumor-bearing mice were injected into the tail vein with compound 78 with 45 MBq (approximately 1.2 mCi) per mouse and a volume of 0.2 mL. After administration, the tumor size and weight of the animals were measured periodically, and the tumor growth curve was drawn to understand the inhibitory effect of the drug on tumor.
Experimental results show that compared with the control group, compound 78 has a very good therapeutic effects on inhibiting tumor growth. (see
The experiments were operated according to the above, and the experimental results show that: the injection of compound 5, compound 6, compound 12, compound 13, compound 14, compound 15, compound 17, compound 20, compound 22, compound 24, compound 26, compound 32, compound 33, compound 38, compound 41, compound 44, compound 45, compound 46, compound 70, compound 72, compound 76, and compound 77 respectively also has very good therapeutic effects on inhibiting tumor growth.
Tumor targeting study of compound 79 in Micro-PET/CT scanning 1. Experimental animal information: C57BL/6J mice, SPF grade; 15-21 g; model mouse construction: bilateral tumor
Human-derived PD-L1 gene knock-in MC-38 cells (MC-38-hPD-L1 cells) were adherently cultured in vitro, and the culture conditions were DMEM medium plus 10% heat-inactivated fetal bovine serum and hygromycinB (final concentration was 100 L/mL), and the cells were cultured at 37° C. with 5% CO2. Subculture of 2-3 times a week. When the cells were in the exponential growth phase, the cells were harvested, counted, and the left side of the mice was inoculated subcutaneously.
Mouse-derived MC-38 cells were adherently cultured in vitro, and the culture conditions were DMEM medium plus 10% heat-inactivated fetal bovine serum, and the cells were cultured at 37° C. with 5% CO2. Subculture of 2-3 times a week. When the cells were in the exponential growth phase, the cells were collected, counted, and the right side of the mice was inoculated subcutaneously.
100 μL of 1×106 MC-38-hPD-L1 cell suspension was inoculated subcutaneously on the left dorsal side of C57BL/6J mice. The same mice were subcutaneously inoculated with 100 μL of 1×106 MC-38 cell suspension on the right dorsal side on the second day. After inoculation, the mice were fed normally, and after a certain number of days, tumor-bearing mice with the volume of bilateral transplanted tumor in the range of 150 mm3−350 mm3 were selected for test.
After quality control, compound 79 was diluted with 10% ethanol in physiological saline, and the drug was extracted and injected into each animal through tail vein. The administration volume was 100 μL/animal, and the administration dose was 100-200 μCi/animal. The study of Micro-PET/CT imaging was performed at 0.5 h, 1.5 h, 2.5 h, 3.5 h after injection of compound 79.
The experimental results show that the left side of bilateral tumor mice scanned by compound 79 was MC38-PDL1 and the right side was MC38. The results show that the tumor uptake value on the left side is significantly higher than that on the right side, as shown in Table 2.
The experiments were operated according to the above, and the experimental results show that: compound 48, compound 50, compound 52, compound 54, compound 56, compound 58, compound 60, compound 62, compound 64, compound 66, compound 68, and compound 82 were injected respectively for Micro-PET/CT imaging, and it can be found that the uptake value of the mouse left tumor (MC38-PDL1) is significantly higher than that of the mouse right tumor (MC38).
Human-derived PD-L1 gene knock-in MC-38 cells (MC-38-hPD-L1 cells) were adherently cultured in vitro, and the culture conditions were DMEM medium plus 10% heat-inactivated fetal bovine serum and hygromycinB (final concentration was 100 L/mL), and the cells were cultured at 37° C. with 5% CO2. Subculture of 2-3 times a week. When the cells were in the exponential growth phase, the cells were harvested, counted, and the left side of the mice was inoculated subcutaneously.
Mouse-derived MC-38 cells were adherently cultured in vitro, and the culture conditions were DMEM medium plus 10% heat-inactivated fetal bovine serum, and the cells were cultured at 37° C. with 5% CO2. Subculture of 2-3 times a week. When the cells were in the exponential growth phase, the cells were collected, counted, and the right side of the mice was inoculated subcutaneously.
100 μL of 1×106 MC-38-hPD-L1 cell suspension was inoculated subcutaneously on the left dorsal side of C57BL/6J mice. The same mice were subcutaneously inoculated with 100 μL of 1×106 MC-38 cell suspension on the right dorsal side on the second day. After inoculation, the mice were fed normally, and after a certain number of days, tumor-bearing mice with the volume of bilateral transplanted tumor in the range of 150 mm3−350 mm3 were selected for test.
After quality control, compound 80 was diluted with 10% ethanol in physiological saline, and the drug was extracted and injected into each animal through tail vein. The administration volume was 100 μL/animal, and the administration dose was 100-200 μCi/animal. The study of Micro-PET/CT imaging was performed at 0.5 h, 1.5 h, 2.5 h, 3.5 h after injection of compound 80.
The experimental results show that the uptake value of the left side tumor (MC38-PDL1) in the model mice scanned with compound 80 is significantly higher than that of the right side (MC-38), as shown in Table 3.
The experiments were operated according to the above, and the experimental results show that: compound 7, compound 8, compound 27, compound 28, compound 29, compound 34, compound 35, compound 36, compound 37, compound 73, compound 74, and compound 75 were injected respectively for Micro-PET/CT imaging, and it can be found that the uptake value of the mouse left tumor (MC38-PDL1) is significantly higher than that of the mouse right tumor (MC38).
Although specific embodiments of the present disclosure are described above, it should be understood by those skilled in the art that these are merely illustrative and that a variety of changes or modifications can be made to these embodiments without departing from the principles and substance of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/141330 | Dec 2021 | WO | international |
| 202211627117.3 | Dec 2022 | CN | national |
The present application claims the priority of PCT patent application PCT/CN2021/141330 with the filing date of Dec. 24, 2021, and the present application claims the priority of Chinese patent application CN202211627117.3 with the filing date of Dec. 16, 2022. The contents of the above patent applications are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/141252 | 12/23/2022 | WO |
