[ CAS No. 139504-50-0 ] {[proInfo.proName]}

Name: 139504-50-0|(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-Chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-mercaptopropanoyl)-N-methyl-L-alaninate|99%
Brand: Ambeed
SKU: A686559
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Quality Control of [ 139504-50-0 ]

COA NMR CNMR HPLC LC-MS

Related Doc. of [ 139504-50-0 ]

SDS Specification

Alternatived Products of [ 139504-50-0 ]

Product Details of [ 139504-50-0 ]

CAS No. :	139504-50-0	MDL No. :	MFCD28398157
Formula :	C₃₅H₄₈ClN₃O₁₀S	Boiling Point :	-
Linear Structure Formula :	-	InChI Key :	-
M.W :	738.29	Pubchem ID :	-
Synonyms :	Maytansinoid DM 1;Mertansine;emtansine;maytansine, Mertansine;UNII-DDZ29HGH0E;DM1 [Maytansinoid];DM1 Compound	Chemical Name :	(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-Chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-mercaptopropanoyl)-N-methyl-L-alaninate

Calculated chemistry of [ 139504-50-0 ]

Physicochemical Properties

Num. heavy atoms :	50
Num. arom. heavy atoms :	6
Fraction Csp3 :	0.6
Num. rotatable bonds :	9
Num. H-bond acceptors :	10.0
Num. H-bond donors :	2.0
Molar Refractivity :	197.25
TPSA :	195.27 Å²

Pharmacokinetics

GI absorption :	Low
BBB permeant :	No
P-gp substrate :	Yes
CYP1A2 inhibitor :	No
CYP2C19 inhibitor :	No
CYP2C9 inhibitor :	No
CYP2D6 inhibitor :	No
CYP3A4 inhibitor :	Yes
Log Kp (skin permeation) :	-9.23 cm/s

Lipophilicity

Log Po/w (iLOGP) :	4.81
Log Po/w (XLOGP3) :	2.21
Log Po/w (WLOGP) :	3.07
Log Po/w (MLOGP) :	1.69
Log Po/w (SILICOS-IT) :	2.81
Consensus Log Po/w :	2.92

Druglikeness

Lipinski :	2.0
Ghose :	None
Veber :	1.0
Egan :	1.0
Muegge :	2.0
Bioavailability Score :	0.17

Water Solubility

Log S (ESOL) :	-5.3
Solubility :	0.00366 mg/ml ; 0.00000496 mol/l
Class :	Moderately soluble
Log S (Ali) :	-5.95
Solubility :	0.000838 mg/ml ; 0.00000113 mol/l
Class :	Moderately soluble
Log S (SILICOS-IT) :	-5.66
Solubility :	0.00163 mg/ml ; 0.00000221 mol/l
Class :	Moderately soluble

Medicinal Chemistry

PAINS :	0.0 alert
Brenk :	3.0 alert
Leadlikeness :	2.0
Synthetic accessibility :	8.63

Safety of [ 139504-50-0 ]

Signal Word:	Danger	Class:	8,6.1
Precautionary Statements:	P201-P280-P303+P361+P353-P304+P340+P310-P305+P351+P338+P310-P308+P313	UN#:	2923
Hazard Statements:	H302-H311-H314-H340-H350	Packing Group:	Ⅲ
GHS Pictogram:

Application In Synthesis of [ 139504-50-0 ]

* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.

Upstream synthesis route of [ 139504-50-0 ]
Downstream synthetic route of [ 139504-50-0 ]

[ 139504-50-0 ] Synthesis Path-Upstream 1~4

1
[ 138148-68-2 ]
[ 139504-50-0 ]

Reference： [1] Journal of Medicinal Chemistry, 2006, vol. 49, # 14, p. 4392 - 4408
[2] Journal of Medicinal Chemistry, 2014, vol. 57, # 16, p. 6949 - 6964

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[ 138148-62-6 ]
[ 139504-50-0 ]

Reference： [1] Journal of Medicinal Chemistry, 2006, vol. 49, # 14, p. 4392 - 4408

3
[ 57103-68-1 ]
[ 139504-50-0 ]

Reference： [1] Journal of Medicinal Chemistry, 2006, vol. 49, # 14, p. 4392 - 4408

4
[ 138148-62-6 ]
[ 57103-68-1 ]
[ 139504-50-0 ]

Reference： [1] Journal of Medicinal Chemistry, 2014, vol. 57, # 16, p. 6949 - 6964

[ 139504-50-0 ] Synthesis Path-Downstream 1~24

1
[ 452072-22-9 ]
[ 139504-50-0 ]
N^2'-deacetyl-N^2'-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]maytansine [ No CAS ]

Reference： [1]Journal of Medicinal Chemistry,2006,vol. 49,p. 4392 - 4408

2
[ 139504-50-0 ]
N^2'-deacetyl-N^2'-(3-mercapto-1-oxopropyl)maytansine dimer [ No CAS ]

Reference： [1]Journal of Medicinal Chemistry,2006,vol. 49,p. 4392 - 4408

3
[ 138148-68-2 ]
[ 139504-50-0 ]

Reference： [1]Journal of Medicinal Chemistry,2006,vol. 49,p. 4392 - 4408
[2]Journal of Medicinal Chemistry,2014,vol. 57,p. 6949 - 6964

4
[ 138148-62-6 ]
[ 139504-50-0 ]

Reference： [1]Journal of Medicinal Chemistry,2006,vol. 49,p. 4392 - 4408

5
[ 57103-68-1 ]
[ 139504-50-0 ]

Reference： [1]Journal of Medicinal Chemistry,2006,vol. 49,p. 4392 - 4408

Yield	Reaction Conditions	Operation in experiment
		EXAMPLE 2; This example demonstrates a process of preparing a conjugate comprising an antibody chemically coupled to a drug, which includes a holding step performed after modification of the antibody with a bifunctional crosslinking reagent and before conjugation of the antibody to the drug. A solution of huC242 antibody (16 mg at 8 mg/mL) in a buffer at pH 6.5, consisting of 50 mM potassium phosphate, 50 mM sodium chloride, and 2 mM ethylenediaminetetraacetic acid (EDTA) disodium salt, was treated at ambient temperature with a solution of SPP (10 mM stock in ethanol, 6.5 molar excess, final ethanol concentration was 5% v/v). The reaction mixture was incubated at ambient temperature for 90 minutes. A portion (0.4 mL) of the mixture was then passed through a gel filtration column packed with SEPHADEX G25 (NAP 10 column obtained from Amersham Biosciences) to remove any unreacted SPP and other low molecular weight material. Elution with the above buffer gave the modified antibody (?sample A?). A second portion of the reaction mixture (0.2 mL) was treated with a solution of lysine (0.05 mL of a stock solution of 250 mM lysine in 500 mM potassium phosphate buffer pH 7.5, containing 10 mM EDTA) to give a final lysine concentration of 50 mM. The reaction mixture was incubated at ambient temperature for two hours and then purified by passage through a gel filtration column as described above to give ?sample B?. The number of pyridyldithio groups (-SSPy) linked to the antibody was assayed by treatment with dithiothreitol to release pyridine-2-thione, which has an extinction coefficient of 8,080 M-1cm-1 at 343 nM. The parent sample A that had not been treated with lysine had 4.9 -SSPy linked per molecule of antibody. Sample B had 3.5 -SSPy linked per antibody molecule, indicating that lysine treatment removes unstably-bound linker, which in this sample was as much as 40% of the total initially bound linker. The modified antibody samples A and B (2.3 mgs of antibody) were each diluted to a final concentration of 2.5 mg/mL in buffer at pH 6.5, consisting of 50 mM potassium phosphate, 50 mM sodium chloride, and 2 mM EDTA. Both samples were treated with DM1 in dimethylacaetamide (final concentration of dimethylacetamide 3% v/v) using a 1.7-fold molar excess of DM1 over -SSPy that were linked. Thus, sample A was treated with 0.13 micromole of DM1, while the lysine-treated sample B required only 0.086 micromole of DM1. The reaction mixtures were incubated for 24 h at ambient temperature. The monomeric antibody-DM1 conjugates were separated by passage through gel filtration columns (20 mL) packed with SEPHACRYL S300, eluting with phosphate-buffered saline (PBS) at pH 6.5. The number of DM1 molecules linked per antibody molecule was determined as described in Example 1. Sample A, which originally contained 4.9 -SSPy groups, gave 3.5 DM1 per antibody, while sample B, which had 3.5 -SSPy resulted in 3.2 DM1 per antibody. Both antibody-DM1 conjugates were dialyzed into PBS for 48 hours, and re-assayed for DM1 content. In sample A, the number of DM1 molecules linked was reduced from 3.5 to 3.4, while the number of DM1 molecules linked in sample B remained unchanged at 3.2. This confirms the conclusion that treatments with lysine removes unstably bound linkers while having no effect on the level of stably bound DM1 drug that may be achieved.

Yield	Reaction Conditions	Operation in experiment
		EXAMPLE 3; This example demonstrates a process of preparing a conjugate comprising an antibody chemically coupled to a drug, which comprises a holding step after purification of the conjugate reaction mixture and diafiltration (using membrane-based TFF) of the conjugate after the holding step. The BIWA 4 antibody was modified with SPP (6.6-fold molar excess of SPP over antibody) for 105 minutes at room temperature in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. Modified antibody was then purified by chromatography on SEPHADEX G25F equilibrated in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl and 2 mM EDTA. The purified modified antibody was then conjugated with DM1 (1.7 fold molar excess over linker) in dimethylacetamide (DMA) (final concentration 3%). After incubation overnight at room temperature, the conjugate was purified by chromatography on SEPHADEX G25F equilibrated in PBS, pH 6.5. One portion of the conjugate (Sample A) was placed at 4 C. without further treatment. A second portion (Sample B) was held for four days at 4 C., then diafiltered against PBS, pH 6.5, and also placed at 4 C. Free drug was measured at intervals as described in Example 1. The results are set forth in Table 2; As shown by the data set forth in Table 2, release of free drug from the diafiltered sample was significantly reduced relative to release from the untreated sample; EXAMPLE 4A; This example demonstrates a process for preparing a conjugate comprising an antibody chemically coupled to a drug, which process comprises a prolonged hold step prior to diafiltration by membrane-based TFF. The BIWA 4 monoclonal antibody (final concentration of 20 mg/mL) was reacted with SPP (6.6 fold molar excess of SPP) for approximately 130 minutes at room temperature in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. The modified antibody was purified using a column of SEPHADEX G25F equilibrated and eluted in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl and 2 mM EDTA. Modified antibody was conjugated with DM1 (1.7 fold molar excess over linker) dissolved in dimethylacetamide (DMA, final concentration 3%). After overnight incubation at room temperature, the conjugated antibody samples were purified by chromatography on SEPHADEX G25F equilibrated in phosphate buffered saline (PBS), pH 6.5. One portion of the conjugate was held for one day at 4 C., diafiltered by membrane-based TFF against 10 mM sodium succinate, pH 5.5, and placed at 4 C. A second portion of the conjugate was held for thirty days, diafiltered by membrane-based TFF against 10 mM sodium succinate, pH 5.5, and placed at 4 C. Free drug was measured as described in Example 1. After eight weeks of storage at 4 C., the sample that had been diafiltered after a one day hold had 1.7% free drug, whereas the sample diafiltered after a thirty day hold had 0.8% free drug. This result demonstrates that a prolonged hold step lowers the level of free drug in the conjugate; EXAMPLE 4B; This example demonstrates a process for preparing a conjugate comprising an antibody chemically coupled to a drug, which process comprises a prolonged hold step prior to diafiltration by membrane-based TFF and shows that holding at a higher pH (6.5) is more beneficial. The BIWA 4 monoclonal antibody (final concentration of 20 mg/mL) was reacted with SPP (4.4-4.6 fold molar excess of SPP) for approximately 120 minutes at room temperature in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. The modified antibody was purified using a column of SEPHADEX G25F equilibrated and eluted in 35 mM sodium citrate buffer, containing 150 mM NaCl and 2 mM EDTA, at a pH 5.0. Modified antibody was conjugated with DM1 (1.7 fold molar excess over linker) dissolved in dimethylacetamide (DMA, final concentration 3%). After overnight incubation at room temperature, the conjugated antibody samples were purified by chromatography on SEPHADEX G25F equilibrated in either phosphate buffered saline (PBS), pH 6.5 (Sample A) or 10 mM sodium succinate, pH 5.5 (Sample B). Both samples were held at a conjugate concentration of 2 mg/mL at 2-8 C. for 29 days. Sample A was then diafiltered against PBS, pH 6.5. Sample B was diafiltered against 10 mM sodium succinate, pH 5.5. Both samples were stored at 2-8 C. and analyzed for free drug at intervals as described in Example 1A. The results of this analysis are shown in Table 3. As shown by the data set forth in Table 3, stability (with respect to free drug release) of the conjugate that had been held at pH 6.5 (Sample A) was significantly greater than that of the conjugate sample (Sample B) that had been held at pH 5.5. It should be noted that the final formulation of Sample A was at pH 6.5, compared to pH 5.5 for Sample B. However, as disclosed in U.S. Patent Application Publication 2004/0241174 A1, formulating a conjugate at pH 5.5 ...
		EXAMPLE 11; This example demonstrates a process for preparing a conjugate comprising an antibody chemically coupled to a drug, wherein the conjugation reaction mixture contains sucrose. The BIWA 4 antibody was modified with SPP (6.6-fold molar excess of SPP over antibody) for 120 minutes at room temperature in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. Modified antibody was purified by chromatography on SEPHADEX G25F equilibrated in 50 mM potassium phosphate buffer pH 6.5, containing 50 mM NaCl and 2 mM EDTA. Purified modified antibody was then conjugated with DM1 (1.7 fold molar excess over linker) in dimethylacetamide (DMA, final concentration 3%), in either the presence or absence of 10% (w/v) sucrose. After incubation overnight at room temperature, the sample conjugated without sucrose was purified by chromatography on SEPHADEX G25F equilibrated in PBS, pH 6.5. The sample conjugated in the presence of sucrose was divided into two portions. One portion was purified by chromatography on SEPHADEX G25F equilibrated in PBS, pH 6.5. The second portion was purified by chromatography on SEPHADEX G25F equilibrated in PBS, pH 6.5 containing 10% (w/v) sucrose. The conjugation step yield (determined as described in Example 7) for the process run with sucrose in both the conjugation reaction and the subsequent G25 column was 80%, whereas the yield for the process with sucrose only in the conjugation reaction was 68%, and the yield for the process run without sucrose was 62%. Thus, the results of this example demonstrate that sucrose improves the yield of the conjugation reaction and subsequent purification step.

Yield	Reaction Conditions	Operation in experiment
		EXAMPLE 1A; This example demonstrates a process of preparing a conjugate comprising an antibody chemically coupled to a drug, which includes a holding step performed after modification of the antibody with a bifunctional crosslinking reagent and before conjugation of the antibody to the drug. The huN901 monoclonal antibody (final concentration 8 mg/mL) was incubated with N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (7 fold molar excess of SPP) for approximately 100 minutes at room temperature in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. The reaction mixture was purified using a column of SEPHADEX G25F equilibrated and eluted in the aforementioned potassium phosphate buffer lacking ethanol. Samples of modified antibody were held for up to two weeks at 4 C., then conjugated with the <strong>[139504-50-0]maytansinoid DM1</strong> (1.7 fold molar excess over linker) dissolved in dimethylacetamide (DMA, final concentration is 3%). After overnight incubation at room temperature, the conjugated antibody samples were purified by chromatography on SEPHADEX G25F equilibrated in phosphate buffered saline (PBS), pH 6.5. Samples were stored at 4 C. for one week. The number of DM1 molecules linked per antibody molecule was determined using the previously reported extinction coefficients for antibody and drug (Liu et al., Proc. Natl. Acad. Sci. USA, 93, 8618-8623 (1996)). The amount of free drug present after the conjugation reaction was determined by injecting 20-50 mug conjugate onto a HiSep column equilibrated in 25% acetonitrile in 100 mM ammonium acetate buffer, pH 7.0, and eluting in acetonitrile. The peak area of total free drug species (eluted in the gradient and identified by comparison of elution time with known standards), which was measured using an absorbance detector set to a wavelength of 252 nm, was compared with the peak area related to bound drug (eluted in the conjugate peak in the column flow-through fractions) to calculate the proportion of total drug species that was free. The results of this analysis are shown in Table 1; As shown by the data set forth in Table 1, holding the modified antibody at 4 C., after modification but prior to conjugation, significantly reduced the amount of free drug in the conjugate sample after storage for one week, without significantly affecting the amount of drug that was bound (as reflected in the drug/antibody ratio); EXAMPLE 5; This example demonstrates a process for preparing a conjugate comprising an antibody chemically coupled to a drug at pH less than 6. The huN901 antibody was modified with SPP (molar excess of SPP as shown in Table 3) for 90-120 minutes at room temperature in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. Aliquots of modified antibody were then purified on separate columns of G25F equilibrated in either 35 mM sodium citrate buffer, pH 5.0, containing 150 mM NaCl, or 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl and 2 mM EDTA. Modified antibody from each solution was conjugated with DM1 and purified on SEPHADEX G25 as described in Example 1. Drug/antibody ratios were also determined as described in Example 1. Linker/antibody ratios were determined by measuring releasable pyridyldithio groups as described in Example 2. The results of the analysis of the samples conjugated in the solutions with different pH values are shown in Table 4; As shown by the data set forth in Table 4, when conjugation is performed at a lower pH, a smaller excess of SPP is required to achieve the same final drug/antibody level observed at higher pH. As a result, less DM1 is required to achieve the same drug/antibody level, since the amount of drug added is based on the measured linker/antibody ratio. The more efficient use of linker and drug at lower pH not only reduces material costs but also is indicative of reduced side reactions during the conjugation reaction; EXAMPLE 6; This example further demonstrates the beneficial effects of conjugating a modified antibody with a drug at a pH of below 6.0. The huN901 monoclonal antibody (final concentration of 8 mg/ml) was incubated with N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP, 5.6-fold molar excess) for approximately 180 minutes at 20 C. in 50 mM potassium phosphate buffer (pH 7.5) containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. In a first group, the reaction mixture was purified using a column of SEPHADEX G25F resin equilibrated and eluted in 50 mM sodium citrate buffer (pH 5.0) containing 50 mM NaCl and 2 mM EDTA. In a second group, the reaction mixture was purified using a column of SEPHADEX G25F resin equilibrated and eluted in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl and 2 mM EDTA. Both samples were conjugated with DM1 (1.7-fold molar excess over bound linker) for 3, 19, 25, 48, and 120 hours at room temperature in a final concentration of dimethylacetamide (DMA) of 3%. Thus the first group of samples...

Yield	Reaction Conditions	Operation in experiment
		EXAMPLE 10; This example demonstrates a process for preparing a conjugate comprising an antibody chemically coupled to a drug, wherein the conjugation reaction mixture contains sucrose. The J591 antibody was modified with SPP (7.0-fold molar excess of SPP over antibody) for 130 minutes at room temperature in 50 mM potassium phosphate buffer, pH 6.0, containing 2 mM EDTA and 5% ethanol. Modified antibody was then purified by chromatography on SEPHADEX G25F equilibrated in 50 mM potassium phosphate buffer, pH 6.0, containing 2 mM EDTA. Modified antibody was conjugated with DM1 (1.7 fold molar excess over linker) in dimethylacetamide (DMA, final concentration 3%), in either the presence or absence of 10% (w/v) sucrose. After incubation overnight at room temperature, the samples conjugated without sucrose were purified by chromatography on SEPHADEX G25F equilibrated in 20 mM sodium succinate buffer, pH 5.5. The samples conjugated in the presence of sucrose were purified by chromatography on SEPHADEX G25F equilibrated with 20 mM sodium succinate buffer, pH 5.5, containing 5% (w/v) sucrose. The conjugation step yield (determined as described in Example 7) for the process run with added sucrose was 62% (average of two runs), whereas the yield for the process without sucrose was 49% (average of two runs). Thus, the results of this example demonstrate that sucrose improves the yield of the conjugation reaction.
		EXAMPLE 10; This example demonstrates a process for preparing a conjugate comprising an antibody chemically coupled to a drug, wherein the conjugation reaction mixture contains sucrose. The J591 antibody was modified with SPP (7.0-fold molar excess of SPP over antibody) for 130 minutes at room temperature in 50 mM potassium phosphate buffer, pH 6.0, containing 2 mM EDTA and 5% ethanol. Modified antibody was then purified by chromatography on SEPHADEX G25F equilibrated in 50 mM potassium phosphate buffer, pH 6.0, containing 2 mM EDTA. Modified antibody was conjugated with DM1 (1.7 fold molar excess over linker) in dimethylacetamide (DMA, final concentration 3%), in either the presence or absence of 10% (w/v) sucrose. After incubation overnight at room temperature, the samples conjugated without sucrose were purified by chromatography on SEPHADEX G25F equilibrated in 20 mM sodium succinate buffer, pH 5.5. The samples conjugated in the presence of sucrose were purified by chromatography on SEPHADEX G25F equilibrated with 20 mM sodium succinate buffer, pH 5.5, containing 5% (w/v) sucrose. The conjugation step yield (determined as described in Example 7) for the process run with added sucrose was 62% (average of two runs), whereas the yield for the process without sucrose was 49% (average of two runs). Thus, the results of this example demonstrate that sucrose improves the yield of the conjugation reaction.

10
[ 139504-50-0 ]
[ 74-88-4 ]
[ 912569-84-7 ]

Yield	Reaction Conditions	Operation in experiment
65%	With N-ethyl-N,N-diisopropylamine; In ISOPROPYLAMIDE; for 3h;	N2'-deacetyl-N2'-(3-methylthio-1-oxopropyl)-maytansine (S-Methyl-DM1, DM1Me): To a solution of N2'-deacetyl-N-2'(3-mercapto-1-oxopropyl)-maytansine (DM1, 30 mg, 0.041 mmol) in dimethylacetamide (0.25 mL) was added a solution of iodomethane (5.8 mg, 0.041 mmol) in 0.2 mL of dimethylacetamide under an argon atmosphere, with magnetic stirring. Diisopropylethylamine (5.0 mg, 0.041 mmol) was then added and the reaction was stirred for 3 hours. Purification by high pressure liquid chromatography using a reverse phase C 18 column and a gradient of deionized water and acetonitrile gave 20 mg (65% yield) of the desired product DM1Me. Mass spectrum: found 774.5 (M+Na+), calculated 774.3 (M+Na+); 1H NMR (CDCl3) delta 0.84 (3H, s), 1.33 (3H, d, J=5 Hz), 1.35 (3H, d, J=5 Hz), 1.60 (3H, s), 1.68 (3H, s), 2.05 (3H, s) 2.22 (1H, dd, J=3 Hz, 14 Hz, 2.60-2.82 (2H, m), 2.88 (3H, s), 3.08-3.20 (2H, m), 3.25 (3H, s), 3.39 (3H, s), 3.55 (1H, d, J=9 Hz), 3.71 (1H, d, J=12 Hz), 4.02 (3H, s), 4.32 (1H, t, J=10 Hz), 4.81 (1H, dd, J=3 Hz, 12 Hz), 5.45 (1H, q, J=7 Hz), 5.67 (1H, dd, J=9 Hz, 15 Hz), 6.25 (1H, s), 6.47 (1H, dd, J=11 Hz, 15 Hz), 6.70 (1H, d, J=1.5 Hz), 6.75 (1H, d, J=11 Hz), 6.86 (1H, d, J=1.5 Hz).

Reference： [1]Molecular Pharmaceutics,2015,vol. 12,p. 1762 - 1773
[2]Patent: US2006/233814,2006,A1 .Location in patent: Page/Page column 21; Sheet 20/20

11
[ 139504-50-0 ]
murine anti-CA₆ glycotope DS₆ monoclonal antibody [ No CAS ]
[ 341498-08-6 ]
murine anti-CA₆ glycotope DS₆ monoclonal antibody-N₂'-deacetyl-N₂'-(3-mercapto-1-oxopropyl)-maytansine conjugate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
		Example 14: Preparation of muDS6-DMl cytotoxic conjugate; [257] The muDS6 antibody (8 mg/ml) was modified using 8-fold molar excess of N- succinimidyl-4-(2-pyridyldithio) pentanoate (SPP) to introduce dithiopyridyl groups. The reaction was carried out in 95% v/v Buffer A (50 mM KPi , 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v/v DMA for 2 h at room temperature. The slightly turgid reaction mixture was gel-filtered through a NAP or Sephadex G25 column (equilibrated in Buffer A). The degree of modification was determined by measuring the absorbance of the antibody at 280 nm and the DTT released 2-mercaptopyridine (Spy) at 280 and 343 nm. Modified muDS6 was then conjugated at 2.5 mg Ab/mL using a 1.7-fold molar excess of N2 -deacetyl-iV-2 (3-mercapto-l-oxopropyl)- maytansine (L-DMl) over SPy. The reaction was carried out in Buffer A (97% v/v) with DMA (3% v/v). The reaction was incubated at room temperature overnight for~20 h. The opaque reaction mixture was centrifuged (1162 x g, 10 min) and the supernatant was then gel-filtered through a NAP-25 or S300 (Tandem 3, 3x 26/10 desalting columns, G25 medium) column equilibrated in Buffer B (IX PBS pH 6.5).The pellet was discarded. The conjugate was sterile- filtered using a 0.22 mum Millex-GV filter and was dialyzed in Buffer B with a Slide-A-Lyzer. The number of DMl molecules linked per molecule of muDS6 was determined by measuring the absorbance at both 252 nm and 280 nm of the filtered material. The DMl /Ab ratio was found to be 4.36 and the step yield of conjugated MUDS6 was 55%. The conjugated antibody concentration was 1.32 mg/mL. The purified conjugate was biochemically characterized by size exclusion chromography (SEC) and found to be EPO <DP n="89"/>92% monomer. Analysis of DMl in the purified conjugated indicated that 99% was covalently bound to antibody. In Figure 20, flow cytometric binding of the muDS6- DMl conjugate and unmodified muDS6 to Caov-3 cells shows that conjugation of muDS results in only a slight loss of affinity

Reference： [1]Patent: WO2007/24222,2007,A1 .Location in patent: Page/Page column 87; 88

12
[ 138148-62-6 ]
[ 57103-68-1 ]
[ 139504-50-0 ]

Reference： [1]Journal of Medicinal Chemistry,2014,vol. 57,p. 6949 - 6964

13
[ 139504-50-0 ]
[ 2127-03-9 ]
DM₁-SSPy [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
87%	With acetic acid; In 1,2-dimethoxyethane; for 2h;	DM1 (100 mg, 0.14 mmol) was dissolved in dimethoxyethane (4 mL) and added to a magnetically stirred solution of 2,2?-dithiodipyridine (100 mg, 0.45 mmol) in dimethoxyethane (4 mL) containing acetic acid (0.4 mL). After 2 hours the reaction was purified by silica chromatography on a 20 g cartridge eluting with dichloromethane and a linear gradient from 3% to 8% methanol over 25 mm. Desired product was collected and solvent evaporated under vacuum to give 104 mg (87 yield) of desired product. MS (M + H) found 847.6; calcd. 847.3.
60%	With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 0.583333h;	[00346] To a solution of 2,2'-dithiodipyridine (1.24 g, 5.65 mmol) in DMF (8 mL) and diisopropylethylamine (1 mL) was added a solution of DM1 (417 mg, 0.565 mmol) in 2 mL DMF, dropwise over 5 min. The reaction was stirred at room temperature for another 30 min, and the reaction mixture loaded onto a CI 8 Isco gold column. Eluting with 25% to 85% acetonitrile in water provided DMl -SSPy (287 mg, 0.339 mmol, 60% yield). LCMS M/Z: 847.3 [M + 1] .

Reference： [1]Patent: WO2014/134483,2014,A2 .Location in patent: Page/Page column 55
[2]Patent: WO2017/3940,2017,A1 .Location in patent: Paragraph 00346

14
N-succinimidyl 4-(N-maleimidomethyl) trans-cyclohexane-1-carboxylate [ No CAS ]
[ 139504-50-0 ]
[ 56-87-1 ]
N²'-deacetyl-N²'-[3-[[1-[[4-[[[(1S)-5-amino-1-carboxypentyl]amino]carbonyl]cyclohexyl]methyl]-2,5-dioxo-3-pyrrolidinyl]thio]-1-oxopropyl]maytansine [ No CAS ]