[ CAS No. 55750-53-3 ] {[proInfo.proName]}

Name: 55750-53-3|6-Maleimidohexanoic acid|98%
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Quality Control of [ 55750-53-3 ]

COA NMR CNMR HPLC LC-MS

Related Doc. of [ 55750-53-3 ]

SDS Specification

Alternatived Products of [ 55750-53-3 ]

Product Details of [ 55750-53-3 ]

CAS No. :	55750-53-3	MDL No. :	MFCD00043140
Formula :	C₁₀H₁₃NO₄	Boiling Point :	-
Linear Structure Formula :	-	InChI Key :	WOJKKJKETHYEAC-UHFFFAOYSA-N
M.W :	211.21	Pubchem ID :	573683
Synonyms :

Calculated chemistry of [ 55750-53-3 ]

Physicochemical Properties

Num. heavy atoms :	15
Num. arom. heavy atoms :	0
Fraction Csp3 :	0.5
Num. rotatable bonds :	6
Num. H-bond acceptors :	4.0
Num. H-bond donors :	1.0
Molar Refractivity :	56.58
TPSA :	74.68 Å²

Pharmacokinetics

GI absorption :	High
BBB permeant :	No
P-gp substrate :	No
CYP1A2 inhibitor :	Yes
CYP2C19 inhibitor :	No
CYP2C9 inhibitor :	No
CYP2D6 inhibitor :	No
CYP3A4 inhibitor :	No
Log Kp (skin permeation) :	-6.77 cm/s

Lipophilicity

Log Po/w (iLOGP) :	1.47
Log Po/w (XLOGP3) :	1.15
Log Po/w (WLOGP) :	0.18
Log Po/w (MLOGP) :	0.14
Log Po/w (SILICOS-IT) :	0.73
Consensus Log Po/w :	0.73

Druglikeness

Lipinski :	0.0
Ghose :	None
Veber :	0.0
Egan :	0.0
Muegge :	0.0
Bioavailability Score :	0.56

Water Solubility

Log S (ESOL) :	-1.48
Solubility :	7.03 mg/ml ; 0.0333 mol/l
Class :	Very soluble
Log S (Ali) :	-2.31
Solubility :	1.03 mg/ml ; 0.00487 mol/l
Class :	Soluble
Log S (SILICOS-IT) :	-1.04
Solubility :	19.3 mg/ml ; 0.0912 mol/l
Class :	Soluble

Medicinal Chemistry

PAINS :	0.0 alert
Brenk :	1.0 alert
Leadlikeness :	1.0
Synthetic accessibility :	2.17

Safety of [ 55750-53-3 ]

Signal Word:	Warning	Class:	N/A
Precautionary Statements:	P261-P305+P351+P338	UN#:	N/A
Hazard Statements:	H315-H319-H335	Packing Group:	N/A
GHS Pictogram:

Application In Synthesis of [ 55750-53-3 ]

* 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 [ 55750-53-3 ]
Downstream synthetic route of [ 55750-53-3 ]

[ 55750-53-3 ] Synthesis Path-Upstream 1~14

1
[ 6066-82-6 ]
[ 55750-53-3 ]
[ 55750-63-5 ]

Yield	Reaction Conditions	Operation in experiment
95%	With dicyclohexyl-carbodiimide In acetonitrile at 0 - 20℃; Inert atmosphere	Under nitrogen protection, 4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile.Another 4.5 g (22 mmol) of DCC was dissolved in 25 ml of acetonitrile,Keeping the internal temperature around 0°C,It was slowly dropped into the reaction solution.The reaction solution was reacted at 0°C for 2 hours and then reacted overnight at room temperature.filter,The filter cake was washed with acetonitrile 10ml×3.The filtrate was concentrated to dryness under reduced pressure.The resulting oil was dried under reduced pressure at room temperature for 6 h to give 6.4 g of light brown solid.Yield 95percent.
70%	With dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 3 h;	Dicyclohexyl-carbodiimide (2.68 g, 13.02 mmol) and N-hydroxysuccinimide (1 .36 g, 1 1 .84 mmol) are added at room temperature to a stirrer solution of compound [4] (2.5 g, 1 1 .84 mmol) in anhydrous dichloromethane (15 mL) and the mixture stirred at room temperature for 3 hours. The white solid is filtered with dichloromethane to remove the dicyclohexylurea, the organic phase is washed with HCI 0.1 N and water, then dried over anhydrous sodium sulfate and the solvent removed by rotatory evaporation. The resulting residue is subjected to flash column chromatography with a medium pressure system Sepacore® Buchi (silica gel; gradient A: petroleum ether/B: ethyl acetate; Bpercent 0-80 in 15 minutes) to give the activated acid [5] as a white solid, 2.4 g (70percent). MS: m/z 309 [M+H]⁺.¹H NMR (400 MHz, MeOD) δ 6.78 (s, 2H), 3.49 - 3.46 (m, 2H), 2.82 (s, 4H), 2.62 - 2.59 (m, 2H), 1 .78 - 1 .65 (m, 2H), 1 .64 - 1 .50 (m, 2H), 1 .46 - 1 .26 (m, 2H).¹³C NMR (100 MHz, MeOD) δ 169.8, 169.0, 167.3, 132.5, 35.4, 28.6, 26.1 , 23.8, 23.6, 22.3.
48%	With dicyclohexyl-carbodiimide In ethyl acetate at 0 - 20℃; for 20 h;	To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 °C, was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed to warm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48percent) as a clear oil that solidified slowly to a waxy white solid. NMR (400 MHz, Chloroform-i/) δ 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J= 7.4 Hz, 2H), 1.73 - 1.57 (m, 2H), 1.50 - 1.35 (m, 2H). m/z calcd. for C₁₄H₁₆N₂0₆ = 308.10. Found [M+H]⁺ = 309.13. Rf = 0.28 (50percent EtOAc/Hex).

48%

With dicyclohexyl-carbodiimide In ethyl acetate at 0 - 20℃; for 20 h;

To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 °C,was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed towarm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48percent) as a clear oil that solidified slowly to a waxy white solid. ‘H NMR (400 MHz, Chloroform-d) ö 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J = 7.4 Hz, 2H), 1.73 — 1.57 (m,2H), 1.50 1.35 (m, 2H). m/z calcd. for C,4H,6N206 = 308.10. Found [M+H] = 309.13. Rf= 0.28 (50percent EtOAc/Hex)

Reference： [1] Patent: CN107789630, 2018, A, . Location in patent: Paragraph 0064; 0065; 0066
[2] Patent: WO2018/178060, 2018, A1, . Location in patent: Page/Page column 58; 60; 61
[3] International Journal of Molecular Sciences, 2017, vol. 18, # 9,
[4] Patent: WO2015/95953, 2015, A1, . Location in patent: Page/Page column 118
[5] Patent: WO2016/41082, 2016, A1, . Location in patent: Page/Page column 97; 98
[6] Organic and Biomolecular Chemistry, 2014, vol. 12, # 34, p. 6624 - 6633
[7] Patent: CN108743968, 2018, A, . Location in patent: Paragraph 0007; 0008; 0009

2
[ 74124-79-1 ]
[ 55750-53-3 ]
[ 55750-63-5 ]

Yield	Reaction Conditions	Operation in experiment
70%	With sodium hydrogencarbonate In N,N-dimethyl-formamide at 0℃; for 1 h; Inert atmosphere	6-Maleimidohexanoic acid N-hydroxysuccinimide ester 4. Sodium bicarbonate (0.0512 g, 0.483 mmol ) was added to a solution of 6-maleimidocaproic acid (0.211 g, 1.000 mmol) in DI water (10 mL). After the reagents were dissolved in DI water, the DI water was evaporated by blowing with air. The resulting solid was dissolved in anhydrous DMF (3 mL) and the solution was cooled to 0 °C. Disuccinimide carbonate (0.282 g, 1.10 mmol) was added into the solution and the reaction was stirred at 0 °C for 1 h. CH₂C1₂ (25 mL) was added into reaction which was then washed with water (3 x 10 mL). The resulting organic layer was dried over Na₂S0₄, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0percentA / 100percentB (1 CV), 0percentA / 100percentB→ 2percentA / 98percentB (10 CV), 2percentA / 98percentB (2 CV); flow rate: 25 mL/min; monitored at 210 and 280 nm] to afford ester 4 (0.215 g, 0.697 mmol, 70percent yield) as a light yellow liquid. [000194] NMR (500 MHz, CDC1₃) 66.69 (2H, s), 3.53 (2H, t, J= 7.1 Hz), 2.83 (4H, s), 2.60 (2H, t, .7=7.4 Hz), 1.78 (2H,p, .7=7.5 Hz), 1.63 (2H,p, .7=7.4 Hz), 1.41 (2H,p,J=7.6 Hz). [000195] ¹³CNMR(125 MHz, CDC1₃) δ 170.9, 169.2, 168.5, 134.2,37.5,30.8,28.1,25.9,25.7,24.1.

Reference： [1] Journal of the American Chemical Society, 1994, vol. 116, # 14, p. 6101 - 6106
[2] Patent: WO2017/66668, 2017, A1, . Location in patent: Paragraph 000193-000195

3
[ 6066-82-6 ]
[ 55750-53-3 ]
[ 55750-63-5 ]
[ 57-13-6 ]

Reference： [1] Organic and Biomolecular Chemistry, 2009, vol. 7, # 17, p. 3400 - 3406

4
[ 108-31-6 ]
[ 60-32-2 ]
[ 55750-53-3 ]

Yield	Reaction Conditions	Operation in experiment
80%	at 120℃;	To 30 ml of glacial acetic acid was added 6-aminocaproic acid 3.9 g (0.03 mol) and 1.2 eq of maleic anhydride 3.5 g(0.036 mol).The reaction was stirred at 120° C. for 4-6 h.After the reaction is completed,Stop heating,Cool naturally to room temperature.It is concentrated under reduced pressure at 60°C to remove most of the acetic acidThe resulting brown-yellow viscous liquid is poured into water.Then add ethyl acetate 20ml×3 extraction,Combine the organic layers.The organic layer is watered in that orderSaturated saline wash,Drying with anhydrous sodium sulfate,filter,The filtrate was concentrated under reduced pressure to give a brown-yellow oil.Add 50ml water to stir,There are white solids precipitated,filter,50° C. vacuum drying target product 5.08g,Yield 80percent.
80%	at 120℃;	To 30 ml of glacial acetic acid, 3.9 g (0.03 mol) of 6-aminocaproic acid and 1.2 g of maleic anhydride 3.5 g (0.036 mol) were added. The reaction solution was stirred at 120 ° C for 4-6 h.After the reaction was completed, the heating was stopped and naturally cooled to room temperature.Most of the acetic acid was removed by concentration under reduced pressure at 60 °C. The resulting brownish yellow viscous liquid is poured into the water.Add ethyl acetate 20 ml × 3 for extraction.Combine the organic layers. The organic layer was washed successively with water and saturated brine.Dry over anhydrous sodium sulfate, filter,The filtrate was concentrated under reduced pressure to give a brown oil.An off-white solid precipitates, is filtered,The target product was dried under reduced pressure at 50 ° C, 5.08 g, yield 80percent.
77.4%	at 20℃; for 10 h; Reflux	Compound 1 (150 g, 1.53 mol) was added to a stirred solution of Compound 2 (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc(500 mL x 3), washed with H20. The combined organic layers was dried over Na2SO4 and concentrated to give the crude product. It was washed with petroleum ether to give compound 3 as white solid (250 g, 77.4 percent).

77.4%	at 20℃; for 10 h; Reflux	Compound 1 (150 g, 1.53 mol) was added to a stirred solution of Compound 2 (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL × 3), and washed with H₂O. The combined organic layers were dried over Na₂SO₄ and concentrated to give the product. The resulting product was washed with petroleum ether to give compound 3 as white solid (250 g, 77.4 percent).
77.4%	at 20℃; for 10 h; Reflux	Maleic anhydride, furan-2,5-dione (150 g, 1.53 mol) was added to a stirred solution of 6-aminohexanoic acid (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL x 3), washed with 10. The combined organic layers was dried over Na2SC>4 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoic acid as white solid (250 g, 77.4 percent).
77.4%	at 20℃; for 10 h; Reflux	Maleic anhydride, furan-2,5 -dione (150 g, 1.53 mol) was added to a stirred solution of 6-aminohexanoic acid (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t.for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL x 3), washed with H20. The combined organic layers was dried over Na2504 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acidas white solid (250 g, 77.4 percent). DPPA (130 g, 473 mmol) and TEA (47.9 g, 473 mmol) was added to a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (100 g, 473 mmol) in t-BuOH (200 mL). The mixture was heated at reflux for 8 h under N2. The mixture was concentrated, and the residue was purified by column chromatography on silica gel (PE:EtOAc= 3:1) to give tert-butyl 5-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 -yl)pentylcarbamate (13 g, 10 percent).To a solution of tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamate (28 g, 992 mmol) in anhydrous EtOAc (30 mL) was added HC1/EtOAc (50 mL) dropwise. After the mixture was stirred at r.t. for 5 h, it was filtered and the solid was dried to give 1-(5- aminopentyl)-1H-pyrrole-2,5-dione hydrochloride lOa (16 g, 73.7 percent). ‘H NMR (400 MHz, DMSO-d6): 5 8.02 (s, 2H), 6.99 (s, 2H), 3.37-3.34 (m, 2H), 2.71-2.64 (m, 2H), 1.56-1.43 (m,4H), 1.23-1.20 (m, 2H)
77.4%	With trifluoroacetic acid In acetic acid at 20℃; for 10 h; Reflux	[00338] Maleic anhydride (furan-2,5-dione) la (150 g, 1.53 mol) was added to a stirred solution of 6-aminohexanoic acid lb (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. (room temperature) for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL ^χ 3), washed with H2O. The combined organic layers was dried over Na2S04 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoic acid lc as white solid (250 g, 77.4 percent).
77.4%	at 20℃; for 10 h; Reflux	Maleic anhydride, furan-2,5-dione (150 g, 1.53 mol) was added to a stirred solution of6-aminohexanoic acid (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred atr.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL x 3), washed with H20. The combined organic layers was dried over Na2504 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid as white solid (250 g, 77.4 percent). DPPA (130 g, 473 mmol) and TEA (47.9 g,473 mmol) was added to a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid(100 g, 473 mmol) in t-BuOH (200 mL). The mixture was heated at reflux for 8 h under N2.The mixture was concentrated, and the residue was purified by column chromatography onsilica gel (PE:EtOAc= 3:1) to give tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamate (13 g, 10 percent). To a solution of tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)pentylcarbamate (28 g, 992 mmol) in anhydrous EtOAc (30 mL) was added HC1/EtOAc (50 mL) dropwise. After the mixture was stirred at r.t. for 5 h, it was filtered and the solid was dried to give 1-(5-aminopentyl)-1H-pyrrole-2,5-dione hydrochloride lOa (16 g, 73.7 percent). ‘H NMR (400 MI-Tz, DMSO-d6): 5 8.02 (s, 2H), 6.99 (s, 2H), 3.37-3.34 (m, 2H),2.7 1-2.64 (m, 2H), 1.56-1.43 (m, 4H), 1.23-1.20 (m, 2H).
57%	With acetic anhydride In acetic acid for 1 h; Reflux	Maleic anhydride (2.94g, 30mmol) and 6-aminocaproic acid (3.94g, 30mmol) were refluxed in AcOH (70mL) overnight. Ac₂O (2.83mL, 30mmol) was added dropwise and reflux continued for a further 1h. The acetic acid was co-evaporated with toluene in vacuo to yield yellow syrup. The material was purified over SiO₂ (DCM:MeOH, 10:0.7) affording a cream crystalline solid in 57percent yield (3.58g): R_f (DCM/MeOH, 10:0.7)=0.50; ESI-MS (C₁₀H₁₃NO₄, 211.1): m/z=212.0 [M+H⁺] (calcd 212.1), 423.1 [M+2H⁺] (calcd 423.2); ¹H NMR: δ=6.95 (2H, s, CH=CH), 3.35 (2H, t, J 7.4Hz, NCH₂), 2.15 (2H, t, J 7.2Hz, CH₂COO⁻), 1.45 (4H, m, 2CH₂), 1.18 (2H, m, CH₂).
19%	Stage #2: With triethylamine In toluene for 5 h; Dean-Stark; Reflux	To a stirred solution of 6-aminocaproic acid (10.0 g, 76.2 mmol, 1.0 eq) in acetic acid (75 mL), maleic anhydride (7.85 g, 80.0 mmol, 1.05 eq) was added. The solids took a few minutes todissolve, then after ca. 5 mm, white solids began to crash out. After an hour, the suspension thickened to a white cake. This material was scooped onto a fitted funnel and washed with toluene and dried in vacuo with heating to remove all traces of acetic acid.The intermediate powder was taken up in toluene (250 mL), triethylamine (21.3 mL, 152mmol, 2.0 eq) was added, and the mixture heated to reflux with a Dean—Stark trap. After 5 h of reflux,the mixture was cooled and the clear toluene layer was decanted from the rest of the sticky residue in the flask. The toluene was removed in vacuo to yield the a triethylamine salt of 6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol- 1 -yl)hexanoate. The salt was redissolved in toluene, and a small amount of acetic acid was added, then concentrated. Next, the mixture was taken up in 50percent saturated sodiumbicarbonate, and 1 M HC1 was added to adjust the pH to 3, forming a milky precipitate. This wasextracted three times with EtOAc, combined organics dried over sodium sulfate, filtered, andconcentrated in vacuo to yield pure 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-l-yl)hexanoate (3.08 g, 19percent).

Reference： [1] Patent: CN107789630, 2018, A, . Location in patent: Paragraph 0061; 0062; 0063
[2] Patent: CN108743968, 2018, A, . Location in patent: Paragraph 0004; 0005; 0006
[3] Patent: WO2015/95223, 2015, A2, . Location in patent: Page/Page column 67
[4] Patent: WO2016/90050, 2016, A1, . Location in patent: Page/Page column 61-62
[5] Patent: WO2016/90038, 2016, A1, . Location in patent: Page/Page column 123
[6] Patent: WO2016/90040, 2016, A1, . Location in patent: Page/Page column 89
[7] Patent: WO2016/205176, 2016, A1, . Location in patent: Paragraph 00337; 00338
[8] Patent: WO2017/214024, 2017, A1, . Location in patent: Page/Page column 81
[9] Journal of Medicinal Chemistry, 2013, vol. 56, # 24, p. 9955 - 9968
[10] International Journal of Molecular Sciences, 2017, vol. 18, # 9,
[11] Drug Delivery and Translational Research, 2018, vol. 8, # 5, p. 1162 - 1170
[12] Synthesis, 2008, # 8, p. 1316 - 1318
[13] Bioorganic and Medicinal Chemistry, 2014, vol. 22, # 17, p. 4848 - 4854
[14] Patent: US6669951, 2003, B2, . Location in patent: Drawing sheet 23
[15] Organic and Biomolecular Chemistry, 2014, vol. 12, # 37, p. 7261 - 7269
[16] Patent: WO2016/41082, 2016, A1, . Location in patent: Page/Page column 97
[17] Patent: EP1382600, 2004, A1, . Location in patent: Page 5
[18] Organic and Biomolecular Chemistry, 2009, vol. 7, # 17, p. 3400 - 3406
[19] Australian Journal of Chemistry, 2011, vol. 64, # 6, p. 779 - 789
[20] Patent: US2017/106094, 2017, A1, . Location in patent: Paragraph 0066
[21] Patent: CN106632272, 2017, A, . Location in patent: Paragraph 0034; 0035; 0036
[22] Bioorganic and Medicinal Chemistry, 2018, vol. 26, # 9, p. 2599 - 2609
[23] Bioconjugate Chemistry, 2018, vol. 29, # 6, p. 1852 - 1858
[24] Patent: CN108164512, 2018, A, . Location in patent: Paragraph 0037; 0038; 0039
[25] Patent: CN108309938, 2018, A, . Location in patent: Paragraph 0018; 0021; 0056-0059; 0062-0064

5
[ 57079-14-8 ]
[ 55750-53-3 ]

Yield	Reaction Conditions	Operation in experiment
55%	Stage #1: With 10H-phenothiazine; acetic anhydride; triethylamine In tetrahydrofuran for 4 h; Heating / reflux Stage #2: With hydrogenchloride In tetrahydrofuran; water at 20 - 60℃; for 3 h;	(Z)-4-Oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles), acetic anhydride, 68 ml (73.5 g, 0.721 moles), and phenothiazine, 500 mg, were added to a 2 liter three-neck round bottom flask equipped with an overhead stirrer. Triethylamine (TEA), 91 ml (0.653 moles), and 600 ml of tetrahydrofuran (THF) were added and the mixture was heated to reflux while stirring. After a total of 4 hours of reflux, the dark mixture was cooled to <60° C. and poured into a solution of 250 ml of 12 N HCl in 3 liters of water. The mixture was stirred 3 hours at room temperature and then was filtered through a filtration pad (Celite 545, J. T. Baker, Jackson, Tenn.) to remove solids. The filtrate was extracted with 4.x.500 ml of chloroform and the combined extracts were dried over sodium sulfate. After adding 15 mg of phenothiazine to prevent polymerization, the solvent was removed under reduced pressure. The 6-maleimidohexanoic acid was recrystallized from 2:1 hexane:chloroform to give typical yields of 55-60percent with a melting point of 81-85° C.

Reference： [1] Dalton Transactions, 2005, # 24, p. 3886 - 3897
[2] Patent: US2008/63627, 2008, A1, . Location in patent: Page/Page column 16
[3] Journal of Medicinal Chemistry, 1996, vol. 39, # 8, p. 1692 - 1703
[4] Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 1996, vol. 35, # 3, p. 184 - 186
[5] Tetrahedron Letters, 1994, vol. 35, # 35, p. 6515 - 6516

6
[ 108-30-5 ]
[ 60-32-2 ]
[ 55750-53-3 ]

Yield	Reaction Conditions	Operation in experiment
77.4%	at 20℃; for 10 h; Reflux	Compound 1 (150 g, 1.53 mol) was added to a stirred solution of Compound 2 (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL x 3), washed with H₂0. The combined organic layers was dried over Na₂S0₄ and concentrated to give the crude product. It was washed with petroleum ether to give compound 3 as white solid (250 g, 77.4 percent).

Reference： [1] Patent: WO2015/95227, 2015, A2, . Location in patent: Page/Page column 261; 262

7
[ 57079-14-8 ]
[ 55750-53-3 ]

Yield	Reaction Conditions	Operation in experiment
33%	for 1.5 h; Reflux	[0076] A 1,4-Michael addition was applied to stepwise couple the fully functionalizedgeneration 1 and generation 3 half dendrons to a bis(maleicimido) linker. 6-Aminocaproicacid was treated with maleic anhydride in acetic acid to generate the ring-open product 15,followed by heating to reflux to form the cyclic condensation product 16. Then in the presence of N-methylmorpholine, the carboxylic acid group of 16 was activated by ethyl chloroformate to yield 17. To increase the solubility of the linker, and the length of the linker so that the first half dendron on the linker will not embed it and prevent a second Michael addition reaction from occuffing, 4,7,1O-trioxa-1,13-tridecanediamine was added to couple 2 eq of compound 16.

Reference： [1] Patent: WO2015/38493, 2015, A1, . Location in patent: Paragraph 0076
[2] Patent: WO2005/77923, 2005, A1, . Location in patent: Page/Page column 11
[3] Patent: US2014/249319, 2014, A1, . Location in patent: Paragraph 0214; 0218

8
[ 108-20-3 ]
[ 57079-14-8 ]
[ 55750-53-3 ]

Yield	Reaction Conditions	Operation in experiment
32%	With triethylamine In dichloromethane; acetone; acetonitrile	B. 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1 hexanoic acid To a solution of Part A compound (3 grams, 0.013 mole) in acetonitrile (90 ml) were added dropwise at RT chlorotrimethyl silane (8.3 ml, 0.065 mole) and triethylamine (9 ml, 0.06 m mol) sequentially. The homogenous mixture was then heated at reflux temperature for 7 hours. After cooling to RT, the precipitated solid (triethylamine hydrochloride salt) was filtered and the filtrate was concentrated under vacuum to give a brown solid. The residue was dissolved in dichloromethane (75 ml) and boiled with neutral Norit (1.5 grams) for 20 minutes. It was filtered through a bed of celite and washed with hot dichloromethane (25 ml). The combined filtrate was concentrated in vacuum to afford 1.4 grams (50percent) of the product. The crude product was crystallized using acetone and isopropyl ether (1:2) to give 0.91 grams of title compound in 32percent yield. m.p.: 83-85° C. TLC: R_f= 0.26, silica gel, MeOH-CHCl₃ 5:95, visualized by PMA spray.

Reference： [1] Patent: EP665020, 1995, A3,

9
[ 108-31-6 ]
[ 7440-44-0 ]
[ 60-32-2 ]
[ 55750-53-3 ]

Reference： [1] Patent: EP665020, 1995, A3,

10
[ 60-32-2 ]
[ 55750-53-3 ]

Reference： [1] Dalton Transactions, 2005, # 24, p. 3886 - 3897
[2] Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 1996, vol. 35, # 3, p. 184 - 186
[3] Journal of Medicinal Chemistry, 1996, vol. 39, # 8, p. 1692 - 1703
[4] Tetrahedron Letters, 1994, vol. 35, # 35, p. 6515 - 6516
[5] Patent: US2014/249319, 2014, A1,
[6] Patent: WO2015/38493, 2015, A1,
[7] Patent: WO2015/95953, 2015, A1,
[8] Patent: WO2018/178060, 2018, A1,

11
[ 108-31-6 ]
[ 55750-53-3 ]
[ 57079-14-8 ]

Reference： [1] Patent: US8241655, 2012, B2,

12
[ 108-31-6 ]
[ 660-88-8 ]
[ 55750-53-3 ]

Reference： [1] Organic and Biomolecular Chemistry, 2014, vol. 12, # 34, p. 6624 - 6633

13
[ 55750-53-3 ]
[ 159857-81-5 ]

Reference： [1] Patent: WO2014/191578, 2014, A1,
[2] Patent: WO2017/66668, 2017, A1,
[3] Patent: WO2017/66668, 2017, A1,
[4] International Journal of Molecular Sciences, 2017, vol. 18, # 9,
[5] Patent: CN107789630, 2018, A,
[6] Patent: CN108743968, 2018, A,

14
[ 55750-53-3 ]
[ 76-05-1 ]
[ 151038-94-7 ]

Reference： [1] Journal of Controlled Release, 2014, vol. 192, p. 29 - 39

[ 55750-53-3 ] Synthesis Path-Downstream 1~38

1
[ 57079-14-8 ]
[ 55750-53-3 ]

Yield	Reaction Conditions	Operation in experiment
55 - 60%		(Z)-4-Oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles), acetic anhydride, 68 ml (73.5 g, 0.721 moles), and phenothiazine, 500 mg, were added to a 2 liter three-neck round bottom flask equipped with an overhead stirrer. Triethylamine (TEA), 91 ml (0.653 moles), and 600 ml of tetrahydrofuran (THF) were added and the mixture was heated to reflux while stirring. After a total of 4 hours of reflux, the dark mixture was cooled to <60° C. and poured into a solution of 250 ml of 12 N HCl in 3 liters of water. The mixture was stirred 3 hours at room temperature and then was filtered through a filtration pad (Celite 545, J. T. Baker, Jackson, Tenn.) to remove solids. The filtrate was extracted with 4.x.500 ml of chloroform and the combined extracts were dried over sodium sulfate. After adding 15 mg of phenothiazine to prevent polymerization, the solvent was removed under reduced pressure. The 6-maleimidohexanoic acid was recrystallized from 2:1 hexane:chloroform to give typical yields of 55-60percent with a melting point of 81-85° C.

Reference： [1]Dalton Transactions,2005,p. 3886 - 3897
[2]Patent: US2008/63627,2008,A1 .Location in patent: Page/Page column 16
[3]Journal of Medicinal Chemistry,1996,vol. 39,p. 1692 - 1703
[4]Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry,1996,vol. 35,p. 184 - 186
[5]Tetrahedron Letters,1994,vol. 35,p. 6515 - 6516

2
[ 74124-79-1 ]
[ 55750-53-3 ]
[ 55750-63-5 ]

Yield	Reaction Conditions	Operation in experiment
70%	With sodium hydrogencarbonate; In N,N-dimethyl-formamide; at 0℃; for 1h;Inert atmosphere;	6-Maleimidohexanoic acid N-hydroxysuccinimide ester 4. Sodium bicarbonate (0.0512 g, 0.483 mmol ) was added to a solution of <strong>[55750-53-3]6-maleimidocaproic acid</strong> (0.211 g, 1.000 mmol) in DI water (10 mL). After the reagents were dissolved in DI water, the DI water was evaporated by blowing with air. The resulting solid was dissolved in anhydrous DMF (3 mL) and the solution was cooled to 0 °C. Disuccinimide carbonate (0.282 g, 1.10 mmol) was added into the solution and the reaction was stirred at 0 °C for 1 h. CH2C12 (25 mL) was added into reaction which was then washed with water (3 x 10 mL). The resulting organic layer was dried over Na2S04, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH2C12; gradient: 0percentA / 100percentB (1 CV), 0percentA / 100percentB? 2percentA / 98percentB (10 CV), 2percentA / 98percentB (2 CV); flow rate: 25 mL/min; monitored at 210 and 280 nm] to afford ester 4 (0.215 g, 0.697 mmol, 70percent yield) as a light yellow liquid. [000194] NMR (500 MHz, CDC13) 66.69 (2H, s), 3.53 (2H, t, J= 7.1 Hz), 2.83 (4H, s), 2.60 (2H, t, .7=7.4 Hz), 1.78 (2H,p, .7=7.5 Hz), 1.63 (2H,p, .7=7.4 Hz), 1.41 (2H,p,J=7.6 Hz). [000195] 13CNMR(125 MHz, CDC13) delta 170.9, 169.2, 168.5, 134.2,37.5,30.8,28.1,25.9,25.7,24.1.
	With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 6h;	A solution of compound 8 (0.860 g, 1.43 mmol,1.0 equiv) in DMF (0.2 M) was treated with triethylamine (4.00 mL, 28.6 mmol, 20.0 equiv) and stirred at room temperature for 24 hours. DMF and excess triethylamine were removed under reduced pressure to obtain the crude deprotected intermediate. Separately, to a solution of 6-maleimidohexanoic acid (0.452 g, 2.14 mmol, 1.5 equiv) and N,N'-disuccinimidyl carbonate (0.439 g, 1.72 mmol, 1.2 equiv) in DMF (0.2 M), triethylamine (0.40 mL, 3.0 mmol, 2.0 equiv) was added followed by stirring at room temperature for 6 hours. To this reaction mixture, the previously generated amine dissolved in DMF (0.2 M) was added and stirred at room temperature for 20 hours. DMF was removed under reduced pressure and the resulting residue was purified by flash column chromatography using 3-12percent MeOH-CH2Cl2 as solvent. The product was obtained as white solid. Yield: 91percent.

Reference： [1]Journal of the American Chemical Society,1994,vol. 116,p. 6101 - 6106
[2]Patent: WO2017/66668,2017,A1 .Location in patent: Paragraph 000193-000195
[3]Tetrahedron Letters,2018,vol. 59,p. 3594 - 3599

3
[ 55750-53-3 ]
[ 85-61-0 ]
C₃₁H₄₉N₈O₂₀P₃S [ No CAS ]

Reference： [1]Journal of the American Chemical Society,2004,vol. 126,p. 13570 - 13571

4
[ 67-56-1 ]
[ 55750-53-3 ]
[ 24163-93-7 ]
[ 5893-05-0 ]
[ 73724-43-3 ]
palmitoyl chloride [ No CAS ]
Fmoc-Lys(Aloc)-Cys(Trt)-NH-NH-(NovaGel resin) [ No CAS ]
MIC-Gly-Cys(palmitoyl)-Lys(Aloc)-Cys(farnesyl)-OMe [ No CAS ]

Reference： [1]Chemistry - A European Journal,2003,vol. 9,p. 3683 - 3691

5
[ 945528-81-4 ]
[ 55750-53-3 ]
{(2-(bis-tert-butoxycarbonylmethylamino)-3-{4-[7-(2,5-dioxopyrrolidin-1-yl)hexanoylamino]phenyl}propyl)-[2-(bis-tert-butoxycarbonylmethylamino)propyl]amino}acetic acid tert-butyl ester [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
84%		To solution of aniline 2 (0.22 g, 0.28 mmol) in CH2Cl2 (10 mL) is added l-[3- (dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.12 g, 0.63 mmol), N-epsilon- maleimidocaproic acid (0.06 g, 0.30 mmol). The mixture is stirred at room temperature for 18 h. Afterwards, the reaction solution is diluted with CH2Cl2 (50 mL), and then washed successively with H2O (2 x 50 mL), 5percent w/v NaHCO3 (2 x 50 mL), and H2O (2 x 50 mL). The organic layer is dried over anhydrous Na2SO4, evaporated, and chromatographed on silica gel eluting with EtOH-hexanes 1 :4 to 1 : 1 to yield 6 as a colorless oil (0.23 g, 84percent). [0059] 1H NMR (DMSO-d6) delta 9.82 (s, IH), 7.55 (d, J= 8.1Hz, 2H), 7.18 (d, J= 8.4 Hz, 2H), 7.09 (s, 2H), 3.45 (m, 12H), 3.20-2.40 (complicated m, 8 H), 2.32 (t, J= 6.9 Hz, 2H), 1.80-1.20 (m, 51H), 1.00 (d, J= 6.3 Hz 3H); ES-MS: calcd for C52H84N5Oi3 [M + H+]: 986.6, found 986.6.

Reference： [1]Journal of Medicinal Chemistry,2007,vol. 50,p. 3185 - 3193
[2]Patent: WO2008/70384,2008,A2 .Location in patent: Page/Page column 17; 1/4

6
[ 55750-53-3 ]
[ 175290-73-0 ]

Yield	Reaction Conditions	Operation in experiment
	Multi-step reaction with 2 steps 1: 1-hydroxybenzotriazole, 1-(dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride / N,N-dimethyl-acetamide / Ambient temperature 2: HCl / ethyl acetate / 0.5 h
	Multi-step reaction with 2 steps 1.1: diisopropyl-carbodiimide; benzotriazol-1-ol / N,N-dimethyl-formamide / 0.33 h / 0 °C / Inert atmosphere 1.2: 0 - 20 °C / Inert atmosphere 2.1: methoxybenzene; hydrogenchloride / 1,4-dioxane; ethyl acetate / 20 °C

Reference： [1]Ambekar, Sarvottam Y.; Gowda, D. Channe [Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 1996, vol. 35, # 3, p. 184 - 186]
[2]Current Patent Assignee: THE FIRST AFFILIATED HOSPITAL OF HAINAN MEDICAL UN - CN111346235, 2020, A

7
[ 108-31-6 ]
[ 60-32-2 ]
[ 55750-53-3 ]

Yield	Reaction Conditions	Operation in experiment
80%	With acetic acid; at 120℃;	To 30 ml of glacial acetic acid was added 6-aminocaproic acid 3.9 g (0.03 mol) and 1.2 eq of maleic anhydride 3.5 g(0.036 mol).The reaction was stirred at 120° C. for 4-6 h.After the reaction is completed,Stop heating,Cool naturally to room temperature.It is concentrated under reduced pressure at 60°C to remove most of the acetic acidThe resulting brown-yellow viscous liquid is poured into water.Then add ethyl acetate 20ml×3 extraction,Combine the organic layers.The organic layer is watered in that orderSaturated saline wash,Drying with anhydrous sodium sulfate,filter,The filtrate was concentrated under reduced pressure to give a brown-yellow oil.Add 50ml water to stir,There are white solids precipitated,filter,50° C. vacuum drying target product 5.08g,Yield 80percent.
80%	With acetic acid; at 120℃;	To 30 ml of glacial acetic acid, 3.9 g (0.03 mol) of 6-aminocaproic acid and 1.2 g of maleic anhydride 3.5 g (0.036 mol) were added. The reaction solution was stirred at 120 ° C for 4-6 h.After the reaction was completed, the heating was stopped and naturally cooled to room temperature.Most of the acetic acid was removed by concentration under reduced pressure at 60 °C. The resulting brownish yellow viscous liquid is poured into the water.Add ethyl acetate 20 ml × 3 for extraction.Combine the organic layers. The organic layer was washed successively with water and saturated brine.Dry over anhydrous sodium sulfate, filter,The filtrate was concentrated under reduced pressure to give a brown oil.An off-white solid precipitates, is filtered,The target product was dried under reduced pressure at 50 ° C, 5.08 g, yield 80percent.
80%	With acetic acid; at 120℃;	To 30 ml of glacial acetic acid, 3.9 g (0.03 mol) of 6-aminocaproic acid and 1.2 g of maleic anhydride 3.5 g (0.036 mol) were added. The reaction solution was stirred at 120 °C for 4-6 h. After the reaction is completed, stop heating. Cool naturally to room temperature. Most of the acetic acid was removed by concentration under reduced pressure at 60 °C.The resulting brownish yellow viscous liquid is poured into the water.Add ethyl acetate 20 ml × 3 for extraction.Combine the organic layers. The organic layer was washed with water and aq. There is a white solid precipitated, filtered, the target product was dried under reduced pressure at 50 °C, 5.08 g, yield 80percent.

77.4%	With acetic acid; at 20℃; for 10h;Reflux;	Compound 1 (150 g, 1.53 mol) was added to a stirred solution of Compound 2 (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc(500 mL x 3), washed with H20. The combined organic layers was dried over Na2SO4 and concentrated to give the crude product. It was washed with petroleum ether to give compound 3 as white solid (250 g, 77.4 percent).
77.4%	With acetic acid; at 20℃; for 10h;Reflux;	Compound 1 (150 g, 1.53 mol) was added to a stirred solution of Compound 2 (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL × 3), and washed with H2O. The combined organic layers were dried over Na2SO4 and concentrated to give the product. The resulting product was washed with petroleum ether to give compound 3 as white solid (250 g, 77.4 percent).
77.4%	With acetic acid; at 20℃; for 10h;Reflux;	Maleic anhydride, furan-2,5-dione (150 g, 1.53 mol) was added to a stirred solution of 6-aminohexanoic acid (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL x 3), washed with 10. The combined organic layers was dried over Na2SC>4 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoic acid as white solid (250 g, 77.4 percent).
77.4%	In acetic acid; at 20℃; for 10h;Reflux;	Maleic anhydride, furan-2,5 -dione (150 g, 1.53 mol) was added to a stirred solution of 6-aminohexanoic acid (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t.for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL x 3), washed with H20. The combined organic layers was dried over Na2504 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acidas white solid (250 g, 77.4 percent). DPPA (130 g, 473 mmol) and TEA (47.9 g, 473 mmol) was added to a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (100 g, 473 mmol) in t-BuOH (200 mL). The mixture was heated at reflux for 8 h under N2. The mixture was concentrated, and the residue was purified by column chromatography on silica gel (PE:EtOAc= 3:1) to give tert-butyl 5-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 -yl)pentylcarbamate (13 g, 10 percent).To a solution of tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamate (28 g, 992 mmol) in anhydrous EtOAc (30 mL) was added HC1/EtOAc (50 mL) dropwise. After the mixture was stirred at r.t. for 5 h, it was filtered and the solid was dried to give 1-(5- aminopentyl)-1H-pyrrole-2,5-dione hydrochloride lOa (16 g, 73.7 percent). ?H NMR (400 MHz, DMSO-d6): 5 8.02 (s, 2H), 6.99 (s, 2H), 3.37-3.34 (m, 2H), 2.71-2.64 (m, 2H), 1.56-1.43 (m,4H), 1.23-1.20 (m, 2H)
77.4%	With trifluoroacetic acid; In acetic acid; at 20℃; for 10h;Reflux;	[00338] Maleic anhydride (furan-2,5-dione) la (150 g, 1.53 mol) was added to a stirred solution of 6-aminohexanoic acid lb (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred at r.t. (room temperature) for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL chi 3), washed with H2O. The combined organic layers was dried over Na2S04 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoic acid lc as white solid (250 g, 77.4 percent).
77.4%	With acetic acid; at 20℃; for 10h;Reflux;	Maleic anhydride, furan-2,5-dione (150 g, 1.53 mol) was added to a stirred solution of6-aminohexanoic acid (201 g, 1.53 mol) in HOAc (1000 mL). After the mixture was stirred atr.t. for 2 h, it was heated at reflux for 8 h. The organic solvents were removed under reduced pressure and the residue was extracted with EtOAc (500 mL x 3), washed with H20. The combined organic layers was dried over Na2504 and concentrated to give the crude product. It was washed with petroleum ether to give 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid as white solid (250 g, 77.4 percent). DPPA (130 g, 473 mmol) and TEA (47.9 g,473 mmol) was added to a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid(100 g, 473 mmol) in t-BuOH (200 mL). The mixture was heated at reflux for 8 h under N2.The mixture was concentrated, and the residue was purified by column chromatography onsilica gel (PE:EtOAc= 3:1) to give tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentylcarbamate (13 g, 10 percent). To a solution of tert-butyl 5-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)pentylcarbamate (28 g, 992 mmol) in anhydrous EtOAc (30 mL) was added HC1/EtOAc (50 mL) dropwise. After the mixture was stirred at r.t. for 5 h, it was filtered and the solid was dried to give 1-(5-aminopentyl)-1H-pyrrole-2,5-dione hydrochloride lOa (16 g, 73.7 percent). ?H NMR (400 MI-Tz, DMSO-d6): 5 8.02 (s, 2H), 6.99 (s, 2H), 3.37-3.34 (m, 2H),2.7 1-2.64 (m, 2H), 1.56-1.43 (m, 4H), 1.23-1.20 (m, 2H).
57%	With acetic anhydride; In acetic acid; for 1h;Reflux;	Maleic anhydride (2.94g, 30mmol) and 6-aminocaproic acid (3.94g, 30mmol) were refluxed in AcOH (70mL) overnight. Ac2O (2.83mL, 30mmol) was added dropwise and reflux continued for a further 1h. The acetic acid was co-evaporated with toluene in vacuo to yield yellow syrup. The material was purified over SiO2 (DCM:MeOH, 10:0.7) affording a cream crystalline solid in 57percent yield (3.58g): Rf (DCM/MeOH, 10:0.7)=0.50; ESI-MS (C10H13NO4, 211.1): m/z=212.0 [M+H+] (calcd 212.1), 423.1 [M+2H+] (calcd 423.2); 1H NMR: delta=6.95 (2H, s, CH=CH), 3.35 (2H, t, J 7.4Hz, NCH2), 2.15 (2H, t, J 7.2Hz, CH2COO?), 1.45 (4H, m, 2CH2), 1.18 (2H, m, CH2).
19%		To a stirred solution of 6-aminocaproic acid (10.0 g, 76.2 mmol, 1.0 eq) in acetic acid (75 mL), maleic anhydride (7.85 g, 80.0 mmol, 1.05 eq) was added. The solids took a few minutes todissolve, then after ca. 5 mm, white solids began to crash out. After an hour, the suspension thickened to a white cake. This material was scooped onto a fitted funnel and washed with toluene and dried in vacuo with heating to remove all traces of acetic acid.The intermediate powder was taken up in toluene (250 mL), triethylamine (21.3 mL, 152mmol, 2.0 eq) was added, and the mixture heated to reflux with a Dean?Stark trap. After 5 h of reflux,the mixture was cooled and the clear toluene layer was decanted from the rest of the sticky residue in the flask. The toluene was removed in vacuo to yield the a triethylamine salt of 6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol- 1 -yl)hexanoate. The salt was redissolved in toluene, and a small amount of acetic acid was added, then concentrated. Next, the mixture was taken up in 50percent saturated sodiumbicarbonate, and 1 M HC1 was added to adjust the pH to 3, forming a milky precipitate. This wasextracted three times with EtOAc, combined organics dried over sodium sulfate, filtered, andconcentrated in vacuo to yield pure 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-l-yl)hexanoate (3.08 g, 19percent).
		A solution of one mole equivalent of maleic anhydride in acetonitrile is added to a one mole equivalent of 6-aminocaprioc acid in acetic acid. The mixture is allowed to react for three hours at room temperature. The formed white crystals are filtered off, washed with cold acetonitrile and dried to produce the amic acid adduct. Amic acid is mixed with triethylamine in toluene. The mixture is heated to 130°C for two hours and water is collected in Dean-Stark trap. The organic solvent is evaporated and the 2M HCL added to reach pH 2. Extraction with ethyl acetate and drying over MgSO4 followed by evaporating the solvent gave 6-maleimidocaproic acid (MCA).
	With acetic acid; at 140℃; for 2.5h;	Take maleic anhydride7.84 g (80 mmol) and 10.494 g (80 mmol) of 6-aminocaproic acid were dissolved in 70 mLGlacial acetic acid, refluxed in an oil bath at 140 ° C for about 2.5 hours,(2 × 30mL), spin-dried ethyl acetate extraction, organic layer combined with anhydrous sodium sulfate dried overnight, spin dry, silica gel column separation and purification, the product ().
	With acetic acid; at 120℃;	(a) 18.06 g (100 mmol) of maleic anhydride and 13.118 g (100 mmol) of aminocaproic acid were dissolved in 100 mL of glacial acetic acid, and reacted at 120 ° C for 3-4 h, and the organic reagent was removed by rotary evaporation, and water was taken with toluene and ethyl acetate. After the extraction, the organic layers were combined, and anhydrous sodium sulfate was added and stirred to remove water, and the column was obtained to afford white intermediate (I).
		6-aminocaproic acid and maleic anhydride were placed in a three-necked flask, dissolved in glacial acetic acid, refluxed for 6 hours in an oil bath at 135 ° C, and a small amount of acetic anhydride was measured and added dropwise to the reaction solution at a rate of 1 drop per second. In the middle, reflux for 2h, using a silica gel column to obtain 6-Maleimide caproic acid. The 6-maleimidocaproic acid was weighed and added to a 100 mL eggplant type bottle, and the sample was stirred and stirred by adding oxalyl chloride. In a 70 ° C oil bath, stirring conditions,After refluxing for 2 h, 6-maleimidohexanoyl chloride was obtained. The reaction solution was spin-dried using a rotary evaporator to remove oxalyl chloride. The 6-maleimidohexanoyl chloride was dissolved in re-distilled dichloromethane and magnetically stirred under a 40 ° C oil bath for 15 min. At the same time, PLGA was dissolved in re-distilled dichloromethane, and the liquid was added dropwise at a rate of 1 drop/second using a constant pressure dropping funnel, and then reacted for 24 hours to further separate PLGA-Mal. Its reaction formula is as follows:

Reference： [1]Patent: CN107789630,2018,A .Location in patent: Paragraph 0061; 0062; 0063
[2]Patent: CN108743968,2018,A .Location in patent: Paragraph 0004; 0005; 0006
[3]Patent: CN108714220,2018,A .Location in patent: Paragraph 0008-0010
[4]Patent: WO2015/95223,2015,A2 .Location in patent: Page/Page column 67
[5]Patent: WO2016/90050,2016,A1 .Location in patent: Page/Page column 61-62
[6]Patent: WO2016/90038,2016,A1 .Location in patent: Page/Page column 123
[7]Patent: WO2016/90040,2016,A1 .Location in patent: Page/Page column 89
[8]Patent: WO2016/205176,2016,A1 .Location in patent: Paragraph 00337; 00338
[9]Patent: WO2017/214024,2017,A1 .Location in patent: Page/Page column 81
[10]Journal of Medicinal Chemistry,2013,vol. 56,p. 9955 - 9968
[11]International Journal of Molecular Sciences,2017,vol. 18
[12]Drug Delivery and Translational Research,2018,vol. 8,p. 1162 - 1170
[13]Synthesis,2008,p. 1316 - 1318
[14]Bioorganic and Medicinal Chemistry,2014,vol. 22,p. 4848 - 4854
[15]Patent: US6669951,2003,B2 .Location in patent: Drawing sheet 23
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[18]Patent: WO2016/41082,2016,A1 .Location in patent: Page/Page column 97
[19]Patent: EP1382600,2004,A1 .Location in patent: Page 5
[20]Organic and Biomolecular Chemistry,2009,vol. 7,p. 3400 - 3406
[21]Australian Journal of Chemistry,2011,vol. 64,p. 779 - 789
[22]Patent: US2017/106094,2017,A1 .Location in patent: Paragraph 0066
[23]Patent: CN106632272,2017,A .Location in patent: Paragraph 0034; 0035; 0036
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[27]Patent: CN108309938,2018,A .Location in patent: Paragraph 0018; 0021; 0056-0059; 0062-0064

8
[ 137-00-8 ]
[ 55750-53-3 ]
[ 644985-61-5 ]

Yield	Reaction Conditions	Operation in experiment
62%	With sulfuric acid; In toluene; at 80 - 140℃; for 11h;	6-Maleimidocaproic acid (MCA, 45.13 g, 0.214 mole), 5-(2-hydroxyethyl)-4-methylthiazole (30.41 g, 0.214 mole) and 250 mL toluene were added to a 500 mL three-necked flask and heated to 80C in an oil bath until dissolved. Sulfuric acid catalyst (0.384 g) was added and heat was increased to 140C. After 11 hours of heating, theoretical water (3.9 mL) was dropped along with 25 mL of toluene from Dean-Stark apparatus. Toluene, 25 mL, was replaced into the flask and the reaction was continued. This was done three additional times. Triethyl amine (10.80 mL) was added and the mixture was allowed to stir for one hour at room temperature. NaCI (20%) was added to the mixture and the mixture was transferred to a separatory funnel. The organic layer was isolated and dried over MgSO4, followed by evaporating the solvent to give the product in 62% yield.

Reference： [1]Patent: EP1382600,2004,A1 .Location in patent: Page 5

9
[ 55750-53-3 ]
[ 157503-18-9 ]

Yield	Reaction Conditions	Operation in experiment
54%	With borane-THF In tetrahydrofuran at 23℃; for 3h;	L18 26 ml of a [1,] OM solution of borane-tetrahydrofurane-complex in tetrahydrofurane is added to a solution of 5.0 g (23.7 mmol) of the acid prepared according to Example L5 in 50 ml of anhydrous tetrahydrofurane and the mixture is stirred for 3 hours at [23XB0;C.] The mixture is poured into a saturated solution of sodium bicarbonate, extracted several times with ethyl acetate, and the combined organic extracts are dried over sodium sulfate. After filtration and removal of the solvent, the residue is purified by chromatography. 2.53 g (12.8 mmol, 54%) of the title compound are isolated. 'H-NMR [(CDC13)] : [8= 1.] 24-1.65 (9H), 3.52 (2H), 3.63 (2H), 6.68 (2H) ppm.

Reference： [1]Current Patent Assignee: BAYER AG - WO2004/12735, 2004, A2 Location in patent: Page/Page column 51

10
[ 55750-53-3 ]
[ 839-90-7 ]
C₃₉H₄₈N₆O₁₅ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
50%	Stage #1: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid; 1,3,5-tris(2-hydroxyethyl)-S-triazine-2,4,6-trione With sulfuric acid In toluene at 115℃; Heating / reflux; Stage #2: With triethylamine for 1h;	1 A one liter flask was charged with 1,3, 5-tris (2-hydroxyethyl) cyanuric acid (25.00 g, 96 mmol), 6- maleimidocaproic acid (60.65 g, 287 mmol), sulfuric acid (1.00 g, 10 mmol), and toluene (400 mL). The reaction vessel was equipped with an overhead stirrer, Dean-Stark trap, and condenser. The contents were heated to 115°C and allowed to reflux. The reaction was continued until the calculated amount of water was achieved. After the reaction flask cooled to room temperature, the mixture was filtered. Triethylamine (12.4 g, 123 mmol) was added to the flask and stirred for one hour. After this interval, the mixture was washed with 20% NaCI solution (3 x 400 mL). The organics were collected and silica gel (50 g) was added, stirred for one hour, filtered, and the solvent was removed in vacuo to afford a clear, but slightly yellow liquid. The yield was approximately 50%. The viscosity of this trifunctional maieimide resin was 26,000 cPs at 50 °C while the volatility was determined to be 0.12% at 200 °C, based on TGA analysis. The NMR is attached as Figure 1.

Reference： [1]Current Patent Assignee: HENKEL AG & CO. KGAA - WO2005/77923, 2005, A1 Location in patent: Page/Page column 11-12

11
C₁₆H₂₅F₃N₆O₄S [ No CAS ]
[ 55750-53-3 ]
C₂₆H₃₆F₃N₇O₇S [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
89%		Compound 5A (1.1 grams), 0.8 grams of maleimidocaproic acid, 1.1 ml of DIEA, and 2.5 grams of HBTU were dissolved in 33 ml DMF. After stirring at RT for 18 hours, the reaction was quenched by addition of 5 ml of water and diluted with 400 ml of EtOAc. The organic layer was washed with brine and worked up as described in General Procedure. Pure title compound (6A) was obtained by purification on a fresh silica gel column eluted with 10percent MeOH in DCM (1.4 grams, yield 89percent).

Reference： [1]Patent: US2005/256030,2005,A1 .Location in patent: Page/Page column 31-32

12
[ 55750-53-3 ]
[ 678-39-7 ]
C₂₀H₁₆F₁₇NO₄ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With hydroquinone;sulfuric acid; In toluene; at 95 - 130℃; for 5 - 10h;	By the similar procedure as in Example 1, 464.1 g of 2-Perfluorooctylethanol, 211 g of maleimide caproic acid, 10 g of sulfuric acid, 5 g of hydroquinone and 400 g toluene were charged, and the solution was heated while agitating. The dehydration reaction was continued at 95 to 130 C. for about 5 to 10 hours and terminated when 18 g of water formed were obtained. The reaction mixture was transferred to a separating funnel, dissolved in 1,000 g of toluene, neutralized with 100 g of a 20% aqueous solution of NaOH and then washed three times with 300 g of a 15% aqueous solution of NaCl. The solvent was distilled off under a reduced pressure to obtain 580 g of a reaction product. The refractive index (25 C.) of the reaction product was 1.390. The reaction product was a solid of which the melting point was 50 C. The main component of the reaction product was confirmed to be the compound represented by the following structural formula from the results of NMR and LCMS analysis.

Reference： [1]Patent: US7169829,2007,B2 .Location in patent: Page/Page column 21-22

13
[ 55750-53-3 ]
[ 82333-93-5 ]

Yield	Reaction Conditions	Operation in experiment
100%	With oxalyl dichloride; N,N-dimethyl-formamide; In dichloromethane; at 20℃; for 3h;	To a stirring solution of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoic acid (3.15 g, 14.9 mM) in 15 mL of dichloromethane, oxalyl chloride (1.61 mL, 17.9 mM) was added followed by one drop of DMF. The reaction was allowed to stir at room temperature for three hours. The reaction was concentrated in vacuo. The residue was dissolved in a one to one solution of heptane and dichloromethane and then concentrated in vacuo. This process was repeated two more times producing a solid 248 (3.43 g, 100%). . NMR (400 MHz, DMSO-i): delta [7.02 (s, 2H), 3.43 (m, 2H), 2.53 (m, 1H), CH2.18 (m, 1H), 1.54 (m, 4H), 1.26 (m, 2H).]
100%	With oxalyl dichloride; at 20℃; for 5h;	Compound G- 12-1: benzyl N-(4-aminophenethyl)-N-methyl-L-valinatedihydrochioride Into oxalyl chloride (3 mL) was dissolved 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)hexanoic acid (200 mg, 0.947 mmol, 1 eq.). The solution was stirred at room temperature for 5 hours before evaporating to dryness under reduced pressure.Compound G-12-1 was obtained as a beige solid (217 mg, 100 %) and used in the next step without purification.
100%	With oxalyl dichloride; at 20℃; for 5h;	Into oxalyl chloride (3 mL) was dissolved 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)hexanoic acid (200 mg, 0.947 mmol, 1 eq.). The solution was stirred at room temperature for 5 hours before evaporating to dryness under reduced pressure. Compound G-12-1 was obtained as a beige solid (217 mg, 100 %) and used in the next step without purification.

100%	With oxalyl dichloride; at 20℃; for 5h;	Into oxalyl chloride (3 mL) was dissolved 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanoic acid (200 mg, 0.947 mmol, 1 eq.). The solution was stirred at room temperature for 5 hours before evaporating to dryness under reduced pressure. Compound G-12-1 was obtained as a beige solid (217 mg, 100 %) and used in the next step without purification.
98%	With oxalyl dichloride; In dichloromethane; at 20℃; for 7.5h;	Synthesis of 6-maleimidocaproic Acid Chloride Dry dichloromethane (250 mL) was added at room temperature under stirring in one portion to a one-liter one-neck round-bottom flask containing <strong>[55750-53-3]6-maleimidocaproic acid</strong> (1.0 equiv., 236.6 mmol, 49.97 g) resulting in a yellow solution. A small amount of insoluble impurities were removed by filtration through a filter paper (MN 617 1/4, Ø185 mm) followed by rinsing with dry DCM (25 mL). To the solution at room temperature was added oxalic acid chloride (1.1 equiv., 259.6 mmol, 22.50 mL) dropwise via a dropping funnel (over the course of 2 h) while stirring the reaction solution. Caution: Gas evolution was observed during the addition process. The reaction mixture was stirred at room temperature and allowed to react until HPLC analysis indicated complete consumption of <strong>[55750-53-3]6-maleimidocaproic acid</strong> after 7 h 30 min. The color of the reaction solution changed to dark yellow during the reaction time. 5 h 30 min after the complete addition of oxalic acid chloride the solvent was removed in vacuo at 30 C. The resulting dark yellow oil was then dried in high vacuum for 20 hours. The obtained light brownish solid was crushed with a spatula and dried for further 20 h under high vacuum to yield (53.35 g, 98%) as an light brownish solid (HPLC (220 nm) >97.6% as the methyl ester).
95%	With oxalyl dichloride; In dichloromethane; at 20℃; for 5h;Inert atmosphere;	To a stirring yellow solution of 6-maleimido caproic acid (EMC) (33 g, 156 mmol, 1.0 eq) in dry DCM (150 mL) at room temperature and under N2 atmosphere, was added within 30 min (~0.5 mL/ min) oxalyl chloride (15 mL, 171 mmol, 1.1 eq) using a dropping funnel. The reaction was stirred at room temperature for 5 h. The color of the reaction solution changed to dark yellow during the reaction time and HPLC analysis (PDA 220 nm) confirmed that the reaction was finished after 5 h (> 95% conversion). Solvent was removed with a rotary evaporator at 40 C to obtain an oil. This residual oil was dried under high vacuum overnight (solidified overnight). The obtained light brownish solid was crushed and dried for further 20 h under high vacuum to give 6-maleimidohexanoyl chloride as a yellow microcrystalline solid. The compound was used in the next reaction without further purification. Yield: 34 g (95%). Purity by RP-HPLC, 220 nm, > 95% as the methyl ester. LRMS-ESI (m/z) calcd. for CnHi6N04 (as methyl ester) [M+H]+: 226.10. Found: 225.97.
95%	With oxalyl dichloride; In dichloromethane; at 20℃; for 5.5h;Inert atmosphere;	To a stirring solution of <strong>[55750-53-3]6-maleimidocaproic acid</strong> (EMC) (33 g, 156 mmol, 1.0 equiv., Alfa Aesar) in dry dichloromethane (150 mL) at room temperature and under nitrogen atmosphere was added within 30 min (~l mL/2 min) oxalyl chloride (15 mL, 171 mmol, 1.1 equiv., Sigma Aldrich) using a dropping funnel. Caution: Gas evolution was observed during the addition process. The reaction was stirred at room temperature for 5 h. The color of the reaction solution changed to dark yellow during the reaction time and HPLC analysis (method 1, 220 nm) confirmed that the reaction was completed after 5 h. The solvent was removed under reduced pressure at 40 C to give an oil that was dried under high vacuum overnight resulting in a solidified compound. The obtained light brownish solid was crushed with a spatula and dried for further 20 h under high vacuum to give C as a yellow microcrystalline solid. The compound was used in the next reaction without further purification. Yield: 34 g, 95%. Purity by HPLC (method 1, 220 nm) >95% as the methyl ester. LRMS-ESI (m/z) calcd. for C11H16NO4 (as methyl ester) [M+H]+: 226.10. Found: 225.97.
		General Procedure S: Amide bond formation between the Linker and the Drug[0365] A Linker containing carboxylic acid (30 mg), and anhydrous DMF (10 mul) are placed under an inert atmosphere, preferably argon, and cooled on dry ice for about 5 min. To this mixture oxalyl chloride (1 mL) was added dropwise by syringe. Typically, after few minutes, the mixture is allowed to warm up to room temperature and left for 30 min with occasional manual stirring. Volatiles are removed under reduced pressure. The residue is re-suspended in anhydrous CH2Cl2 (1 mL) and the solvent is removed in vacuo. The residue is dried at vacuum pump overnight to produce Linker AN. [0366] The acylchloride AN is suspended in anhydrous CH2Cl2 (3 mL). A Drug Ib (0.006 mmol) and N,N-diisopropylethylamine (4 mul, ~4 eq.) are suspended in anhydrous CH2Cl2 (100 mul) and the mixtures is cooled on the ice bath typically for about 10 min. To this mixture, 150 mul of the acylchloride in CH2Cl2 (-1.1 eq.) are added via syringe. After 15 min on ice, reaction mixture is allowed to warm up to room temperature and stirring continued for about 2 more hours. Reaction progress can be monitored by RP-HPLC. Solvent then is removed in vacuo. The residue is suspended in DMSO (0.5 mL). Water (100 mul) was added and after 0.5 h the mixture is purified, for example, using preparative HPLC to yield Drug-Linker AO.; [0605] Maleimidocaproyl-MeVal-Val-Dil-Dap-Z can be prepared following General Procedure S.[0606] Briefly, maleimidocaproic acid (30 mg, Molecular Biosciences) and anhydrous DMF (10 mul) in 10 mL glass flask under Ar (balloon) are cooled on dry ice for 5 min. To this mixture oxalyl chloride (1 mL) is added with a syringe. (A vigorous reaction and pressure increase occurred.) After 5 min, the mixture is allowed to warm up to room temperature and left for 30 min with occasional manual stirring. Volatiles are removed on Rotavap, residue is co-evaporated with anhydrous CH2Cl2 (1 mL) and dried at vacuum pump overnight. Product is initially generated as white solid, progressively turning into off-white to brownish solid. 1H-NMR in CDCl3: 1.26-1.32 (2H, m), 1.51-1.59 (2H, m), 1.63-1.70 (2H, m), 2.82 (2H, t), 3.46 (2H, t), 6.70 (2H, s) ppm. Hydrolyzed material can be detected by triplet at 2.35 ppm. Product is used if the integral of the triplet at 2.42 ppm does not exceed 20% of the triplet at 3.46 ppm.
		General Procedure I: Amide bond formation between the Linker and the Drug.[0381] A Linker containing carboxylic acid (30 mg), and anhydrous DMF (10 mul) are placed under an inert atmosphere, preferably argon, and cooled on dry ice for about 5 min. To this mixture oxalyl chloride (1 niL) was added dropwise by syringe. Typically, after few minutes, the mixture is allowed to warm up to room temperature and left for 30 min with occasional manual stirring. Volatiles are removed under reduced pressure. The residue is re-suspended in anhydrous CH2Cl2 (1 mL) and the solvent is removed in vacuo. The residue is dried at vacuum pump overnight to produce Linker AN. [0382] The acylchloride AN is suspended in anhydrous CH2Cl2 (3 mL). The Drug Ib (0.006 mmol) and N,N-diisopropylethylamine (4 mul, ~4 eq.) are suspended in anhydrous CH2Cl2 (100 mul) and the mixtures is cooled on the ice bath typically for about 10 min. To this mixture, 150 mul of the acylchloride in CH2Cl2 (-1.1 eq.) are added via syringe. After 15 min on ice, reaction mixture is allowed to warm up to room temperature and stirring continued for about 2 more hours. Reaction progress can be monitored by RP-HPLC. Solvent then is removed in vacuo. The residue is suspended in DMSO (0.5 mL). Water (100 mul) was added and after 0.5 h the mixture is purified, for example, using preparative HPLC to yield Drag-Linker AO.; [0473] To an ice-bath cooled suspension of PP7 (177 mg, 0.23 mol) in 2 mL CH2Cl2 was added DIEA (145 muL, 0.83 mmol), which effected a clear solution, and then maleimidocaproyl chloride (0.3 mmol, prepared by the addition to maleimidocaproic acid, 63.2 mg, 0.3 mmol, of 1.5 mL oxalyl chloride, and 1 drop of DMF at 0 0C. The reaction was allowed to stir at ambient temperature, the volatiles removed in vacuo overnight, and the reagent checked by HPLC and 1H NMR, then used directly in the acylation.) The cooling bath was removed, and the reaction stirred at ambient temperature for 1.75 h, then concentrated in vacuo briefly before quenching the excess maleimidocaproyl chloride with water. The pH was adjusted to 3 with 0.1 N HCl, whereupon a white precipitate was formed. This was extracted into CHCl3, and the aqueous phase extracted twice more with CHCl3. The combined organic extracts were EPO <DP n="145"/>concentrated in vacuo to give 192 mg of the crude product. The product was purified via RP-HPLC in several batches. The best conditions were: 21.2 x 350 mm 4A, 80mu synergi MAX RP column using a method called 'full prep 10, 50, 60, 90', a series of connected linear gradients ranging from 10% to 90% % B in A over 56 min. where A = 0.05% TFA in H2O and B = CH3CN, at 25 mL/min, and monitoring at 215 and 254 nM. Fractions eluting at 21.6 to 22.3 min. were pooled and lyophilized as 35.7 mg (16%) isomer A (mc- MMApF-A, PP8-A). The fractions eluting between 23.0 and 23.6 min. were pooled and lyophilized as 11.2 mg isomer B (mc-MMApF-A, PP8-A).; [0493] Maleimidocaproic acid (30 mg, Molecular Biosciences), and anhydrous DMF (10 mul) in 10 mL glass flask under Ar (balloon) were cooled on dry ice for 5 min. To this mixture oxalyl chloride (1 mL) was added by syringe (noting a vigorous reaction and pressure increase). After 5 min, the mixture was allowed to warm up to room temperature and left for 30 min. with occasional manual stirring. Volatiles were removed on a Rotavap. The residue was co-evaporated with anhydrous CH2Cl2 (1 mL) and dried at vacuum pump overnight. Product is initially generated as a white solid, progressively turning into off-white to brownish solid. 1H-NMR in CDCl3: 1.26-1.32 (2H, m), 1.51-1.59 (2H, m), 1.63-1.70 (2H, m), 2.82 (2H, t), 3.46 (2H, t), 6.70 (2H, s) ppm. Hydrolyzed material can be detected by triplet at 2.35 ppm. Product is used if the integral of the triplet at 2.42 ppm does not exceed 20% of the triplet at 3.46 ppm. (Note: when the product was obtained as oil instead of solid, subsequent acylation was more difficult.); [0552] Maleimidocaproic acid (30 mg, Molecular Biosciences), and anhydrous DMF (10 mul) in 10 mL glass flask under Ar (balloon) were cooled on dry ice for 5 min. To this mixture oxalyl chloride (1 mL) was added by syringe (Attention: vigorous reaction, pressure increase). After 5 min, the mixture was allowed to warm up to room temperature and left for 30 min with occasional manual stirring. Volatiles were removed on Rotavap, residue was co-evaporated with anhydrous CH2Cl2 (1 mL) and dried at vacuum pump overnight. The product is initially generated as white solid, progressively turning into off-white to brownish solid. 1H-NMR in CDCl3: 1.26-1.32 (2H, m), 1.51-1.59 (2H, m), 1.63-1.70 (2H, m), 2.82 (2H, t), 3.46 (2H, t), 6.70 (2H, s) ppm. Hydrolyzed material can be detected by triplet at 2.35 ppm. Product can only be used if integral of the triplet at 2.42 ppm does not exceed 20% of the triplet at 3.46 ppm.
	With oxalyl dichloride; In chloroform; at 20℃;	EXAMPLE 6 Preparation of N-(5-isocyanatopentyl)maleimide (Mal-C5-NCO) Mal-EACA from Example 5 (5.0 g; 23.5 mmole) and CHCl3 (25 mL) were placed in a 100 mL round bottom flask and stirred using a magnetic bar with cooling in an ice bath. Oxalyl chloride (10.3 mL; ~15 g; 118 mmole) was added and the reaction was brought to room temperature with stirring overnight. The volatiles were removed on a rotary evaporator, and the residue was azetroped with three times with 10 mL CHCl3 each time. The intermediate Mal-EAC-Cl[N-(6-oxo-6-chlorohexyl)maleimide] (Compound 3) was dissolved in acetone (10 mL) and added to a cold (ice bath) stirred solution of sodium azide (2.23 g; 34.3 mmole) in water (10 mL). The mixture was stirred one hour using an ice bath. The organic layer was set aside in an ice bath, and the aqueous layer was extracted three times with 10 mL CHCl3. All operations of the acylazide were done at ice bath temperatures. The combined organic solutions of the azide reaction were dried for an hour over anhydrous sodium sulfate. The N-(6-oxo-6-azidohexyl)maleimide (Compound 4) solution was further dried by gentle swirling over molecular sieves over night. The cold azide solution was filtered and added to refluxing CHCl3, 5 mL over a 10 minute period. The azide solution was refluxed for 2 hours. The weight of Mal-C5-NCO (Compound 5) solution obtained was 55.5 g, which was protected from moisture. A sample of the isocyanate solution, 136 mg was evaporated and treated with DBB (1,4-dibromobenzene), 7.54 mg and chloroform-d, 0.9 mL: 1H NMR (CDCl3, 400 MHz) delta 6.72 (s,2H), 3.55 (t, 2H, J=7.2 Hz), 3.32 (t, 2H, J=6.6 Hz), 1.70-1.59 (m, 4H), 1.44-1.35 (m, 2H). The NMR spectra was consistent with desired product. The DBB internal standard delta at 7.38 (integral value was 2.0, 4H; per mole of product) was used to estimate the moles of Mal-C5-NCO in solution. The calculated amount of product in solution was 23.2 mmole for a yield of 98% of theory. NCO reagent (concentration was 0.42 mmole/g) was used to prepare a macromer in Example 12.
	With oxalyl dichloride; In dichloromethane;	DMAP (0.1 equiv, 14.8 mmol, 1.81 g) and DIPC (1.5 equiv., 222.3 mmol, 34.5 ml_) were added under nitrogen atmosphere slowly at 00C to a solution of Cbz-Glu-OtBu (1 equiv., 148.2 mmol, 50 g) and tetrt.butyl-carbazate (1.5 equiv., 222.3 mmol, 29.4 g) in dry DCM (100 mL). The mixture was stirred then at RT for 16 h. The solid was removed by suction filtration and washed with DCM. The organic layer was extracted with 0.1 M HCI (10 x 100 mL). The organic phase was then extracted with brine, water, dried and the solvent was removed under reduced pressure to give Cbz-Glu-NH-NH-BOC as a white solid (66.6 g) 60 g Cbz-Glu-NH-NH-BOC was dissolved under nitrogen in dry methanol (200 mL). Pd/C (10%, 2.5 g) was added and a hydrogen balloon was applied to the flask. The reaction mixture was stirred au RT for 4 d during which the balloons were exchanged with new ones. The suspension was then diluted with methanol, filtered over celite and the solvent was removed under reduced pressure. The oily rest was purified by FC on silica eluting with CHCI3ZMeOH 20:1 to give H-GIu-NH-NH- BOC as a white solid (42 g). The <strong>[55750-53-3]6-maleimidocaproic acid</strong> chloride was prepared from maleimidocaproic acid (82.78 mmol, 17.35 g) and oxalic acid chloride (1.0 equiv, 82.78 mmol, 9.93 mL) in dry DCM (180 mL). Then dry trietylamine (82.8 mmol, 1 1.47 mL) was added at RT under nitrogen atmosphere to a solution of H- GIu-NH-NH-BOC (82.8 mmol, 26.36 g) in dry DCM (300 mL) followed by slowly adding the solution of the <strong>[55750-53-3]6-maleimidocaproic acid</strong> chloride. The reaction solution was stirred at RT for 18 h. The solvent was removed under reduced pressure. The oily residue was purified by flash chromatography on silica eluting with EE/hexane (i) 1 :1 , (i) 2:1 , (i) 4:1 to give EMC-Glu-NH-NH-BOC as a white solid (36 g, 76 %).
	With oxalyl dichloride; In chloroform; at 0 - 20℃;	MaI-EACA from Example 5 (5.0 g; 23.5 mmole) and CHCl3 (25 mL) were placed in a 100 mL round bottom flask and stirred using a magnetic bar with cooling in an ice bath. Oxalyl chloride (10.3 mL; -15 g; 1 18 mmole) was added and the reaction was brought to room temperature with stirring overnight. The volatiles were removed on a rotary evaporator, and the residue was azetroped with three times with 10 mL CHCl3 each time. The intermediate MaI-EAC-Cl [N-(6-oxo-6- chlorohexyl)maleimide] (Compound 3) was dissolved in acetone (10 mL) and added to a cold (ice bath) stirred solution of sodium azide (2.23 g; 34.3 mmole) in <n="48"/>water (10 mL). The mixture was stirred one hour using an ice bath. The organic layer was set aside in an ice bath, and the aqueous layer was extracted three times with 10 mL CHCl3. All operations of the acylazide were done at ice bath temperatures. The combined organic solutions of the azide reaction were dried for an hour over anhydrous sodium sulfate. The N-(6-oxo-6-azidohexyl)maleimide (Compound 4) solution was further dried by gentle swirling over molecular sieves over night. The cold azide solution was filtered and added to refluxing CHCl3, 5 mL over a 10 minute period. The azide solution was refluxed for 2 hours. The weight of Mal-C5-NCO (Compound 5) solution obtained was 55.5 g, which was protected from moisture. A sample of the isocyanate solution, 136 mg was evaporated and treated with DBB (1 ,4-dibromobenzene), 7.54 mg and chloroform-d, 0.9 mL: 1H NMR (CDCl3, 400MHz) delta 6.72 (s,2H), 3.55 (t, 2H, J - 7.2 Hz), 3.32 (t, 2H, J = 6.6 Hz), 1.70-1.59 (m, 4H), 1.44-1.35 (m, 2H). The NMR spectra was consistent with desired product. The DBB internal standard delta at 7.38 (integral value was 2.0, 4H; per mole of product) was used to estimate the moles of Mal-C5-NCO in solution. The calculated amount of product in solution was 23.2 mmole for a yield of 98% of theory. NCO reagent (concentration was 0.42 mmole/g) was used to prepare a macromer in Example 12.
	With oxalyl dichloride; at 20℃; for 4h;Neat (no solvent);	The 6-Maleimidohexanoic acid, 2.24 g (10.6 mmol) was dissolved in 10.76 g (84.8 mmol) of oxalyl chloride and stirred as a neat solution for 4 hours at room temperature. The excess oxalyl chloride was then removed under reduced pressure and the resulting acid chloride was dissolved in 25 ml of methylene chloride. This solution was added with stirring to a solution of 3.60 g (10.6 mmol) N-epsilon-t-BOC lysine t-butyl ester hydrochloride (Bachem California) in 25 ml of methylene chloride and 3.21 g (31.7 mmol) of TEA. The resulting mixture was stirred overnight under nitrogen. After this time, the mixture was treated with water and the organic layer was separated and dried over sodium sulfate. The solvent was removed and the product was purified on a silica gel flash chromatography column using a 0-5% methanol in chloroform solvent gradient. Pooling of the desired fractions and evaporation of solvent gave 5.20 g of product (98% yield). Analysis on an NMR spectrometer was consistent with the desired product.
	In n-heptane; dichloromethane; N,N-dimethyl-formamide;	Step 1. Synthesis of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride (#248). To a stirring solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (3.15 g, 14.9 mM) in 15 mL of dichloromethane, oxalyl chloride (1.61 mL, 17.9 mM) was added followed by one drop of DMF. The reaction was allowed to stir at room temperature for three hours. The reaction was concentrated in vacuo. The residue was dissolved in a one to one solution of heptane and dichloromethane and then concentrated in vacuo. This process was repeated two more times producing a solid #248 (3.43 g, 100%). 1H NMR (400 MHz, DMSO-d6): delta [7.02 (s, 2H), 3.43 (m, 2H), 2.53 (m, 1H), CH2.18 (m, 1H), 1.54 (m, 4H), 1.26 (m, 2H).]
	With oxalyl dichloride; N,N-dimethyl-formamide; In dichloromethane; at 20℃;Cooling with ice;	To a suspension of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoic acid (1.00 g, 4.73 mmol) in DCM (20 mL) cooled in an ice bath was added a drop of DMF and then oxalyl chloride (2.03 mL, 23.67 mmol) dropwise. See Figure 10. The mixture was allowed to warm up to room temperature and stirred overnight giving a dark brown solution. All volatile components were removed by rotary evaporator and then high vacuum pump. The resultant residue was dissolved in DCM (5 mL) and the solvent was removed by rotary evaporator and then high vacuum pump. The above dissolving and removal procedure was repeated once more to give crude 6-maleimidocaproyl chloride as dark brown oil.
	With oxalyl dichloride; at 20℃; for 5h;	Into oxalyl chloride (3 mL) was dissolved 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)hexanoic acid (200 mg, 0.947 mmol, 1 eq.). The solution was stirred at room temperature for 5 hours before evaporating to dryness under reduced pressure. Compound G-12-1 was obtained as a beige solid (217 mg, 100 %) and used in the next step without purification.
	With oxalyl dichloride; In dichloromethane; N,N-dimethyl-formamide; at 20℃; for 2h;Inert atmosphere;	FIG. 1 1 illustrates a synthesis scheme for obtaining MC-DMED-KGP 18 Conjugates. FIG. 1 1 shows that to a solution of 6-Maleimidohexanoic acid (1 .0 equiv) and oxalyl chloride (3.0 equiv) in CH2C12, DMF was added using a syringe. It was stirred for 2h. Excess oxalyl chloride was removed under vacuum.
	With oxalyl dichloride; at 20℃; for 5h;	Compound G-12-1: benzyl N-(4-aminophenethyl)-N-methyl-L-valinate dihydrochloride Into oxalyl chloride (3 mL) was dissolved 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (200 mg, 0.947 mmol, 1 eq.). The solution was stirred at room temperature for 5 hours before evaporating to dryness under reduced pressure. Compound G-12-1 was obtained as a beige solid (217 mg, 100%) and used in the next step without purification.
	With oxalyl dichloride; N,N-dimethyl-formamide; at 20 - 30℃; for 1h;Inert atmosphere; Cooling with ice;	6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 4a (1.5 g, 7.10 mmol, prepared according to the known method of "") was added with a drop of N, N-dimethylformamide, and then added with 15 mL of oxalyl chloride dropwise with vigorous stirring under an argon atmosphere after cooling in a dry ice bath. Then the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residues were dissolved in methylene chloride and concentrated under reduced pressure to give the crude title product of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl chloride 4b. The product was used in the next step without further purification.
	With thionyl chloride;	6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl Chloride 200.0 mg (0.95 mmol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid were dissolved in 4.0 ml of dichloromethane, and 338.0 mg (2.84 mmol) of thionyl chloride were added. The reaction mixture was stirred at RT for 3 h, and 1 drop of DMF was then added. The mixture was stirred for another 1 h. The solvent was evaporated under reduced pressure and the residue was co-distilled three times with dichloromethane. The crude product was used without further purification in the next step of the synthesis.
	With oxalyl dichloride; at 70℃; for 2h;	6-aminocaproic acid and maleic anhydride were placed in a three-necked flask, dissolved in glacial acetic acid, refluxed for 6 hours in an oil bath at 135 C, and a small amount of acetic anhydride was measured and added dropwise to the reaction solution at a rate of 1 drop per second. In the middle, reflux for 2h, using a silica gel column to obtain 6-Maleimide caproic acid. The <strong>[55750-53-3]6-maleimidocaproic acid</strong> was weighed and added to a 100 mL eggplant type bottle, and the sample was stirred and stirred by adding oxalyl chloride. In a 70 C oil bath, stirring conditions,After refluxing for 2 h, 6-maleimidohexanoyl chloride was obtained. The reaction solution was spin-dried using a rotary evaporator to remove oxalyl chloride. The 6-maleimidohexanoyl chloride was dissolved in re-distilled dichloromethane and magnetically stirred under a 40 C oil bath for 15 min. At the same time, PLGA was dissolved in re-distilled dichloromethane, and the liquid was added dropwise at a rate of 1 drop/second using a constant pressure dropping funnel, and then reacted for 24 hours to further separate PLGA-Mal. Its reaction formula is as follows:
	With thionyl chloride; In dichloromethane; at 0℃; for 1h;	General procedure for preparation of compound 3 has beenreported in previous research [39]. Then, compound 3 (422 mg,2 mmol) was dissolved in 20 mL of anhydrous DCM which was thenreacted with 10 equiv of SOCl2 at 0 C for 1 h to produce the desiredintermediate compound 4. PTX (854 mg, 1 mmol) and 350 mul oftrimethylamine (TEA) were dissolved in 10 ml of anhydrous DCM,and then compound 4 was added at 0 C under Ar atmosphere. The resulting solution was stirred for 2 h at the dark. The reactionmixture with Ar atmosphere protection was evaporated undervacuum. Compound 5 was obtained by column chromatography(DCM: Methanol, 20:1) with a yield of 58% (Scheme 1).
	With thionyl chloride; N,N-dimethyl-formamide; In ethyl acetate; for 3h;Reflux;	S1, the maleimido hexanoic acid 21.1g of ethyl acetate were added to the 100ml,Then add 16.2 g of thionyl chloride,Add a drop of DMF and connect to the bubbler to observe the reaction gas.The reaction solution was heated to reflux for 3 h until the bubbler had no gas, and the reaction was stopped.The reaction solution was concentrated to dryness.Obtaining a yellow solid without post-treatment, directly proceeding to the next reaction;

Reference： [1]Patent: WO2013/72813,2013,A2 .Location in patent: Page/Page column 224
[2]Patent: WO2015/162291,2015,A1 .Location in patent: Page/Page column 179
[3]Patent: WO2016/173682,2016,A1 .Location in patent: Page/Page column 109
[4]Patent: WO2017/72196,2017,A1 .Location in patent: Page/Page column 179
[5]Journal of Polymer Science, Part A: Polymer Chemistry,2019,vol. 57,p. 699 - 707
[6]Patent: US2019/2484,2019,A1 .Location in patent: Paragraph 0405; 0407
[7]Biomacromolecules,2018,vol. 19,p. 2156 - 2165
[8]Patent: WO2019/108975,2019,A1 .Location in patent: Paragraph 00130; 00133
[9]Patent: WO2019/108974,2019,A1 .Location in patent: Paragraph 00165
[10]Patent: WO2007/8603,2007,A1 .Location in patent: Page/Page column 140; 223
[11]Patent: WO2007/8848,2007,A2 .Location in patent: Page/Page column 111; 143; 144; 152; 167
[12]Patent: US2008/154241,2008,A1 .Location in patent: Page/Page column 18
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[14]Patent: WO2008/60359,2008,A2 .Location in patent: Page/Page column 46-47
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[16]Organic and Biomolecular Chemistry,2011,vol. 9,p. 5535 - 5540
[17]Patent: US2013/129753,2013,A1 .Location in patent: Page/Page column
[18]Patent: WO2015/23355,2015,A1 .Location in patent: Page/Page column 113
[19]Green Chemistry,2015,vol. 17,p. 4991 - 5000
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[28]Patent: CN109535058,2019,A .Location in patent: Paragraph 0014

14
[ 7299-86-7 ]
[ 55750-53-3 ]
[ 558483-53-7 ]

Yield	Reaction Conditions	Operation in experiment
	With hydroquinone In toluene at 95 - 130℃; for 5h;	7 Subsequently, the reaction mixture was added with 211 g of maleimide caproic acid, 3 g of sulfuric acid and 3 g of hydroquinone and heated while agitating. The dehydration reaction was continued at 95 to 130° C. for about 5 hours and terminated when 18 g of water formed were obtained. The reaction mixture was transferred to a 2-liter separating funnel, dissolved in 500 g of toluene, neutralized with 100 g of a 20% aqueous solution of NaOH and then washed three times with 200 g of a 15% aqueous solution of NaCl. The solvent was distilled off under a reduced pressure to obtain 360 g of a reaction product. The refractive index (25° C.) of the reaction product was 1.493 and the viscosity thereof was 1.3 Pa.s at 25° C. The main component of the reaction product was confirmed to be the compound represented by the following structural formula from the results of NMR and LCMS analysis.

Reference： [1]Current Patent Assignee: NIPPON KAYAKU CO LTD - US7169829, 2007, B2 Location in patent: Page/Page column 20-21

15
[ 55750-53-3 ]
[ 84624-28-2 ]
[ 1137667-22-1 ]

Yield	Reaction Conditions	Operation in experiment
	With benzotriazol-1-yloxyl-tris-(pyrrolidino)-phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20 - 50℃;	To a solution of 6-maleimido-hexanoic acid (88 mg, 0.42 mmol) in DMF (3 ml_) PYBOP (109 mg, 0.42 mmol) is added at rt. The solution is stirred at rt for 20 min. Fmoc-Lys-OH (155 mg, 0.42 mmol) and DIPEA (70 mul_, 0.42 mmol) are added and the solution is heated at 500C for 5 min. The solution is stirred at rt overnight. The crude mixture is poured into water (60 ml_). The aqueous phase is basified (pH = 9) with NaOH (1 M). The aqueous phase is washed with ethyl acetate. Upon acidification of the aqueous phase with acetic acid, a yellowish precipitate is formed. The solid is collected via filtration to afford compound (26). ESI-MS: m/z 562.63 [M+H]+.

Reference： [1]Patent: WO2009/43900,2009,A1 .Location in patent: Page/Page column 28

16
[ 55750-53-3 ]
[ 764-99-8 ]
[ 1139575-54-4 ]

Yield	Reaction Conditions	Operation in experiment
55%	With 4-methoxy-phenol at 80 - 90℃; for 19h;	3 To a pre-heated (90°C) three-neck round bottom flask was added 4- MEHQ (223mg, 1.8 mmoi) and diethyleneglycol divinyl ether (14.9 g, 94 mmol). MCA (50 g, 267 mmol) was crushed into small pieces and added into the reaction mixture in ten portions over two hours. The reaction temperature was maintained at 80°-90°C. Addition of MCA generally causes a decrease of the reaction temperature and increase of viscosity. The reaction mixture was stirred for another 17 hours at 85°C, after which the reaction mixture was cooled to ambient temperature. A very small amount of gel was found in the reaction mixture. Toluene (200 ml.) was added to dissolve the product mixture; Amberlyst A 21 (50 g) was added and the product mixture stirred for one hour. The Amberlyst resin was filtered and the solvent stripped under vacuum to give 30 g (55%) of a yellow viscous liquid with a viscosity of 3,800 mPa.s (25 °C). 1H-NMR (DMSO-d6, in ppm): δ 7.0 (s, 4H), 5.9 (q, 2H), 3.4-3.6 (m, 12H), 2.3 (t, 4H), 1.5 (m, 8H), 1.2 (m, 10H).

Reference： [1]Current Patent Assignee: HENKEL AG & CO. KGAA - WO2009/70172, 2009, A1 Location in patent: Page/Page column 30-31

17
[ 55750-53-3 ]
[ 125741-64-2 ]
[ 29022-11-5 ]
[ 35661-60-0 ]
[ 71989-31-6 ]
[ 71989-28-1 ]
[ 67436-13-9 ]
MIC-Gly-Cys(StBu)-Met-Gly-Leu-Pro-Cys(Far)OMe [ No CAS ]

Reference： [1]Journal of the American Chemical Society,2009,vol. 131,p. 1557 - 1564

18
[ 55750-53-3 ]
[ 1343407-40-8 ]
[ 1343407-41-9 ]

Yield	Reaction Conditions	Operation in experiment
68%	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; dichloromethane; at 20℃; for 24h;	Solid EEDQ (108 mg, 0.436 mmol, 2eq) was added to a solution of amine 10 (199 mg, 0.219 mmol, 1eq) and maleimido hexanoic acid (57 mg, 0.269, 1.23 eq) in a mixture of DCM (6 mL) and methanol (3 mL). The solution was allowed to stir at room temperature for 24 hours. The reaction was found complete by LC/MS (3.45 min (ES+) m/z (relative intensity) 1 101.78 ([M + Hf , 100)). The volatiles were removed by evaporation under reduced pressure. The residue was partitioned between DCM and saturated aqueous NaHC03, washed with brine, dried over MgS04, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient from 1/99 up to 2.5/97.5 methanol/chloroform) to yield 165 mg (68 percent) of pure product 11.Analytical Data: [a]24D = +94° (c = 0.09, CHCI3); 1H NMR (400 MHz, CDCI3) delta 8.62 (s, 1 H), 7.46-7.39 (m, 2H), 7.27 (s, 1 H), 7.22-7.06 (m, 9H), 6.79 (d, 2H, J = 8.65 Hz), 6.75 (bs, 1 H), 6.57 (s, 2H), 6.52 (s, 1 H), 6.28 (s, 1 H), 5.77 (bs, 1 H), 5.21 (s, 1 H), 4.75-4.60 (m, 2H), 4.40 (q, 1 H, J = 5.54 Hz), 3.93-3.86 (m, 1 H), 3.83 (s, 3H), 3.73 (s, 3H), 3.65 (s, 2H), 3.36 (t, 2H, J = 7.16 Hz), 3.30-3.23 (m, 1 H), 3.08-2.90 (m, 5H), 2.09 (t, 2H, J = 7.14 Hz), 1.93-1.75 (m, 1 H), 1.62-1 .31 (m, 17H), 1.30-1.04 (m, 6H). 13C NMR (100 MHz, CDCI3) delta 173.5, 170.9, 170.3, 169.6, 163.2, 159.1 , 156.3, 151.1 , 148.6, 136.1 , 134.0, 129.1 , 128.7, 127.2, 126.3, 126.2, 124.9, 123.3, 121.7, 120.0, 114.2, 1 12.7, 1 10.7, 86.1 , 79.3, 67.6, 59.5, 56.2, 56.1 , 55.3, 54.6, 53.9, 53.4, 37.8, 37.5, 36.7, 35.2, 31 .0, 29.4, 28.5, 28.2, 26.2, 24.8, 22.7; MS (ES+) m/z (relative intensity) 1 101.78 ([M + H]+ , 100).
68%	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; dichloromethane; at 20℃; for 24h;	Solid EEDQ (108 mg, 0.436 mmol, 2 eq) was added to a solution of amine 10 (199 mg, 0.219 mmol, 1 eq) and maleimido hexanoic acid (57 mg, 0.269, 1.23 eq) in a mixture of DCM (6 mL) and methanol (3 mL). The solution was allowed to stir at room temperature for 24 hours. The reaction was found complete by LC/MS (3.45 min (ES+) m/z (relative intensity) 1101.78 ([M+H]+., 100)). The volatiles were removed by evaporation under reduced pressure. The residue was partitioned between DCM and saturated aqueous NaHCO3, washed with brine, dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient from 1/99 up to 2.5/97.5 methanol/chloroform) to yield 165 mg (68percent) of pure product 11.[0434]Analytical Data: [alpha]24D=+94° (c=0.09, CHCl3); 1H NMR (400 MHz, CDCl3) delta 8.62 (s, 1H), 7.46-7.39 (m, 2H), 7.27 (s, 1H), 7.22-7.06 (m, 9H), 6.79 (d, 2H, J=8.65 Hz), 6.75 (bs, 1H), 6.57 (s, 2H), 6.52 (s, 1H), 6.28 (s, 1H), 5.77 (bs, 1H), 5.21 (s, 1H), 4.75-4.60 (m, 2H), 4.40 (q, 1H, J=5.54 Hz), 3.93-3.86 (m, 1H), 3.83 (s, 3H), 3.73 (s, 3H), 3.65 (s, 2H), 3.36 (t, 2H, J=7.16 Hz), 3.30-3.23 (m, 1H), 3.08-2.90 (m, 5H), 2.09 (t, 2H, J=7.14 Hz), 1.93-1.75 (m, 1H), 1.62-1.31 (m, 17H), 1.30-1.04 (m, 6H). 13C NMR (100 Hz, CDCl3) delta 173.5, 170.9, 170.3, 169.6, 163.2, 159.1, 156.3, 151.1, 148.6, 136.1, 134.0, 129.1, 128.7, 127.2, 126.3, 126.2, 124.9, 123.3, 121.7, 120.0, 114.2, 112.7, 110.7, 86.1, 79.3, 67.6, 59.5, 56.2, 56.1, 55.3, 54.6, 53.9, 53.4, 37.8, 37.5, 36.7, 35.2, 31.0, 29.4, 28.5, 28.2, 26.2, 24.8, 22.7; MS (ES+) m/z (relative intensity) 1101.78 ([M+H]+., 100).
68%	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; dichloromethane; at 20℃; for 24h;	Solid EEDQ (108 mg, 0.436 mmol, 2 eq.) was added to a solution of amine 32 (199 mg, 0.219 mmol, 1 eq.) and maleimido hexanoic acid (57 mg, 0.269, 1.23 eq.) in a mixture of DCM (6 mE) and methanol (3 mE). The solution was allowed to stir at room temperature for 24 hours. The reaction was found to be complete by EC/MS (3.45 mm (ES) mlz (relative intensity) 1101.78 ([M+H], 100)). The volatiles were removed by evaporation under reduced pressure. The residue was partitioned between DCM and saturated aqueous NaHCO3, washed with brine, dried over Mg504, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography (gradient from 1/99 up to 2.5/97.5 methanol/chloroform) to yield 165 mg (68percent) of pure product 33. Analytical Data: [a]24D=+94° (c=0.09, CHC13); ?H NMR (400 MHz, CDC13) oe 8.62 (s, 1H), 7.46-7.39 (m, 2H), 7.27 (s, 1H), 7.22-7.06 (m, 9H),6.79 (d, 2H, J=8.65 Hz), 6.75 (bs, 1H), 6.57 (s, 2H), 6.52 (s, 1H), 6.28 (s, 1H), 5.77 (bs, 1H), 5.21 (s, 1H), 4.75-4.60 (m, 2H), 4.40 (q, 1H, J=5.54 Hz), 3.93-3.86 (m, 1H), 3.83 (s, 3H), 3.73 (s, 3H), 3.65 (s, 2H), 3.36 (t, 2H, J=7.16 Hz),3.30-3.23 (m, 1H), 3.08-2.90 (m, 5H), 2.09 (t, 2H, J=7.14 Hz), 1.93-1.75 (m, 1H), 1.62-1.31 (m, 17H), 1.30-1.04 (m, 6H). ?3C NMR (100 MHz, CDC13) oe 173.5, 170.9, 170.3,169.6, 163.2, 159.1, 156.3, 151.1, 148.6, 136.1, 134.0,129.1, 128.7, 127.2, 126.3, 126.2, 124.9, 123.3, 121.7,120.0, 114.2, 112.7, 110.7, 86.1, 79.3, 67.6, 59.5, 56.2, 56.1,55.3, 54.6, 53.9, 53.4,37.8,37.5,36.7,35.2,31.0,29.4,28.5,28.2, 26.2, 24.8, 22.7; MS (ES) mlz (relative intensity)1101.78 ([M+H], 100).

Reference： [1]Patent: WO2011/128650,2011,A1 .Location in patent: Page/Page column 60-61
[2]Patent: US2013/35484,2013,A1 .Location in patent: Paragraph 0432; 0433; 0434
[3]Patent: US2018/134717,2018,A1 .Location in patent: Paragraph 1210; 1211

19
N-Me-D/L-Ala-maytansinol [ No CAS ]
[ 55750-53-3 ]
[ 1375089-56-7 ]
[ 1452404-51-1 ]

Yield	Reaction Conditions	Operation in experiment
30.5%; 25.2%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Example 4 Condensation of N-Me-D/L-Ala-MDC with MA-ACP (D-3AA-MDC and L-3AA-MDC) [0181] [0182] Into above prepared N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, and dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250×20 mm, S 10 mum) gave two fractions (Rt=6.59 min and 6.98 min) as white solid. The higher Rt fraction was determined to be the D-aminoacyl ester diastereomer (D-3AA-MDC, 45.2percent), while the lower Rt fraction was the desired L-aminoacyl ester (L-3AA-MDC, 54.8percent). L-3AA-MDC: white solid (0.1364 g, 30.5percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.86 (3H, s), 1.22-1.38 (4H, m), 1.25 (3H, d, J=9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J=7.6 Hz), 1.56-1.70 (4H, m), 1.68 (3H, s), 1.75 (1H, d, J=13.6 Hz), 2.19 (1H, dd, J=14.4, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J=14.2, 12.0 Hz), 2.80 (1H, d, J=9.6 Hz), 3.01 (3H, s), 3.19 (1H, d, J=13.2 Hz), 3.32 (3H, s), 3.42 (1H, d, J=9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H, t, J=10.4 Hz), 4.88 (1H, dd, J=11.8, 3.2 Hz), 5.07 (1H, q, J=7.6 Hz), 5.84 (1H, dd, J=15.2, 9.2 Hz), 6.23 (1H, d, J=11.2 Hz), 6.27 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J=1.2 Hz), 6.84 (1H, d, J=1.2 Hz). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.98 min.
30.5%; 25.2%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Example 4 Condensation of N-Me-D/L-Ala-MDC with MA-ACP (D-3AA-MDC and L-3AA-MDC) [0181] [0182] Into above prepared N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, and dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250×20 mm, S 10 mum) gave two fractions (Rt=6.59 min and 6.98 min) as white solid. The higher Rt fraction was determined to be the D-aminoacyl ester diastereomer (D-3AA-MDC, 45.2percent), while the lower Rt fraction was the desired L-aminoacyl ester (L-3AA-MDC, 54.8percent). L-3AA-MDC: white solid (0.1364 g, 30.5percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.86 (3H, s), 1.22-1.38 (4H, m), 1.25 (3H, d, J=9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J=7.6 Hz), 1.56-1.70 (4H, m), 1.68 (3H, s), 1.75 (1H, d, J=13.6 Hz), 2.19 (1H, dd, J=14.4, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J=14.2, 12.0 Hz), 2.80 (1H, d, J=9.6 Hz), 3.01 (3H, s), 3.19 (1H, d, J=13.2 Hz), 3.32 (3H, s), 3.42 (1H, d, J=9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H, t, J=10.4 Hz), 4.88 (1H, dd, J=11.8, 3.2 Hz), 5.07 (1H, q, J=7.6 Hz), 5.84 (1H, dd, J=15.2, 9.2 Hz), 6.23 (1H, d, J=11.2 Hz), 6.27 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J=1.2 Hz), 6.84 (1H, d, J=1.2 Hz). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.98 min.
30.5%; 25.2%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Example 6 Condensation of N-Me-D/L-Ala-MDC with MA-ACP (D-3AA-MDC and L-3AA-MDC) [0167] [0168] Into above prepared N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, and dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250×20 mm, S 10 mum) gave two fractions (Rt=6.59 min and 6.98 min) as white solid. The higher Rt fraction was determined to be the D-aminoacyl ester diastereomer (D-3AA-MDC, 45.2percent), while the lower Rt fraction was the desired L-aminoacyl ester (L-3AA-MDC, 54.8percent). L-3AA-MDC: white solid (0.1364 g, 30.5percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.86 (3H, s), 1.22-1.38 (4H, m), 1.25 (3H, d, J=9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J=7.6 Hz), 1.56-1.70 (4H, m), 1.68 (3H, s), 1.75 (1H, d, J=13.6 Hz), 2.19 (1H, dd, J=14.4, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J=14.2, 12.0 Hz), 2.80 (1H, d, J=9.6 Hz), 3.01 (3H, s), 3.19 (1H, d, J=13.2 Hz), 3.32 (3H, s), 3.42 (1H, d, J=9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H, t, J=10.4 Hz), 4.88 (1H, dd, J=11.8, 3.2 Hz), 5.07 (1H, q, J=7.6 Hz), 5.84 (1H, dd, J=15.2, 9.2 Hz), 6.23 (1H, d, J=11.2 Hz), 6.27 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J=1.2 Hz), 6.84 (1H, d, J=1.2 Hz). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.98 min.

30.5%; 25.2%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃;Cooling with ice;	The crude N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) prepared in the previous step were dissolved in DMF (25 mL), cooled in an ice water bath, and EDC (0.407 g, 2.123 Mmol)was added. The reaction mixture was stirred overnight at room temperature, quenched with water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation. The, subjected to Column chromatography (silica gel, CH2C12/Me0H 30:1) to give crude product. Preparative HPLC (YMC 018 column, 250x20mm, S 10 mum) was further purified to obtain two pure diastereoisomers (Rt = 6.59 min and 6.98 min). The larger Rt component was identified as the D-aminoacyl ester diastereomer (D-3AA-MDC, 45.2percent), and the smaller Rt component was identified as the L-aminoacyl ester diastereomer. (L-3AA-MDC, 54.8percent). L-3AlphaAlpha-MuDC white solid (0.1364g, two-step total yield 30. 5percent).
0.1364 g; 0.1128 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Into above prepared N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EA, washed with brine, dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column, 250×20 mm, S 10 mum) gave two fractions (Rt=6.59 min and 6.98 min) as white solid. The higher Rt fraction was determined to be the D-aminoacyl ester diastereomer (D-3AA-MDC, 45.2percent), while the lower Rt fraction was the desired L-aminoacyl ester (L-3AA-MDC, 54.8percent). L-3AA-MDC: white solid (0.1364 g, 30.5percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.86 (3H, s), 1.22-1.38 (4H, m), 1.25 (3H, d, J=9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J=7.6 Hz), 1.56-1.70 (4H, m), 1.68 (3H, s), 1.75 (1H, d, J=13.6 Hz), 2.19 (1H, dd, J=14.4, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J=14.2, 12.0 Hz), 2.80 (1H, d, J=9.6 Hz), 3.01 (3H, s), 3.19 (1H, d, J=13.2 Hz), 3.32 (3H, s), 3.42 (1H, d, J=9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H, t, J=10.4 Hz), 4.88 (1H, dd, J=11.8, 3.2 Hz), 5.07 (1H, q, J=7.6 Hz), 5.84 (1H, dd, J=15.2, 9.2 Hz), 6.23 (1H, d, J=11.2 Hz), 6.27 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J=1.2 Hz), 6.84 (1H, d, J=1.2 Hz). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.98 min.
0.1364 g; 0.1128 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Into above prepared N-Me-D/L- Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0 °C was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, and dried over Na2S04. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2 MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250x20 mm, S 10 muiotatauiota) gave two fractions (Rt = 6.59 min and 6.98 min) as white solid. The higher Rt fraction was determined to be the D-aminoacyl ester diastereomer (D- 3AA-MDC, 45.2percent), while the lower Rt fraction was the desired L-aminoacyl ester (L-3AA- MDC, 54.8percent). L-3AA-MDC: white solid (0.1364 g, 30.5percent overall yield over two steps), 1H NMR (400 MHz, CDC13): 50.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s),1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J= 12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J = 9.6 Hz), 3.10 (1H, d, J = 8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J = 6.8Hz), 3.48 (2H, d, J = 6.8 Hz), 3.62 (1H, d, J = 12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J = 11.2 Hz), 4.76 (lH,d, J = 11.6 Hz), 5.36 (1H, q, J= 6.8 Hz), 5.65 ( 1H, dd, J = 15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J = 15.2 , 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J = 11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc: 865.3, found: 865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2percent overall yield over two steps), 1H NMR (400 MHz, CDC13): 50.86 (3H, s), 1.22-1.38 (4H, m), I .25 (3H, d, J= 9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J = 7.6 Hz), 1.56-1.70 (4H, m),1.68 (3H, s), 1.75 (1H, d, J = 13.6 Hz), 2.19 (1H, dd, J =14 A, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J = 14.2, 12.0 Hz), 2.80 (1H, d, J = 9.6Hz), 3.01 (3H, s), 3.19 (1H, d, J= 13.2 Hz), 3.32 (3H, s), 3.42 (1H, d, J= 9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H, t, J = 10.4 Hz), 4.88 (lH,dd, J = I I .8, 3.2 Hz), 5.07 (1H, q, J = 7.6 Hz), 5.84 ( 1H, dd, J = 15.2, 9.2 Hz), 6.23 (1H, d, J = 11.2 Hz), 6.27 (1H, s), 6.41 (1H, dd, J= 15.2 , 1 1.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J= 1.2 Hz), 6.84 (1H, d, J= 1.2 Hz). LC-MS (M+Na+) calc: 865.3, found: 865.3. Rt: 6.98 min.
0.1364 g; 0.1128 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Into above prepared N-Me-D/L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0° C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, and dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250×20 mm, S 10 mum) gave two fractions (Rt=6.59 min and 6.98 min) as white solid. The higher Rt fraction was determined to be the D-aminoacyl ester diastereomer (D-3AA-MDC, 45.2percent), while the lower Rt fraction was the desired L-aminoacyl ester (L-3AA-MDC, 54.8percent). L-3AA-MDC: white solid (0.1364 g, 30.5percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min. D-3AA-MDC: white solid (0.1128 g, 25.2percent overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.86 (3H, s), 1.22-1.38 (4H, m), 1.25 (3H, d, J=9.2 Hz), 1.38-1.45 (1H, m), 1.48 (3H, d, J=7.6 Hz), 1.56-1.70 (4H, m), 1.68 (3H, s), 1.75 (1H, d, J=13.6 Hz), 2.19 (1H, dd, J=14.4, 2.8 Hz), 2.28-2.36 (2H, m), 2.65 (1H, dd, J=14.2, 12.0 Hz), 2.80 (1H, d, J=9.6 Hz), 3.01 (3H, s), 3.19 (1H, d, J=13.2 Hz), 3.32 (3H, s), 3.42 (1H, d, J=9.6 Hz), 3.47-3.54 (3H, m), 3.98 (3H, s), 4.29 (1H, t, J=10.4 Hz), 4.88 (1H, dd, J=11.8, 3.2 Hz), 5.07 (1H, q, J=7.6 Hz), 5.84 (1H, dd, J=15.2, 9.2 Hz), 6.23 (1H, d, J=11.2 Hz), 6.27 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.69 (2H, s), 6.79 (1H, d, J=1.2 Hz), 6.84 (1H, d, J=1.2 Hz). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.98 min.

Reference： [1]Patent: US2014/178413,2014,A1 .Location in patent: Paragraph 150; 151
[2]Patent: US2014/178412,2014,A1 .Location in patent: Paragraph 181; 182
[3]Patent: US2014/178411,2014,A1 .Location in patent: Paragraph 167; 168
[4]Patent: CN107899020,2018,A .Location in patent: Paragraph 0193-0196
[5]Patent: US2014/179917,2014,A1 .Location in patent: Paragraph 0474; 0475
[6]Patent: WO2014/94527,2014,A1 .Location in patent: Page/Page column 32-33
[7]Patent: US2014/178415,2014,A1 .Location in patent: Paragraph 0446; 0447

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[ 55750-53-3 ]
[ 1375089-56-7 ]

Yield	Reaction Conditions	Operation in experiment
62.6%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Example 5 Condensation of N-Me-L-Ala-MDC with MA-ACP (L-3AA-MDC) [0183] [0184] Into above prepared N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0 C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250×20 mm, S 10 mum) gave the desired L-3AA-MDC (N2?-deacetyl-N-2?(3-mercapto-1-oxopropyl)-maytansine): white solid (0.280 g, 62.6% overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min.
62.6%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Example 5 Condensation of N-Me-L-Ala-MDC with MA-ACP (L-3AA-MDC) [0183] [0184] Into above prepared N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0 C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250×20 mm, S 10 mum) gave the desired L-3AA-MDC (N2?-deacetyl-N-2?(3-mercapto-1-oxopropyl)-maytansine): white solid (0.280 g, 62.6% overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min.
0.28 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Into above prepared N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0 C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EA, washed with brine, dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column, 250×20 mm, S 10 mum) gave the desired L-3AA-MDC: white solid (0.280 g, 62.6% overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min.

0.28 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Into above prepared N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0 C was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, dried over Na2S04. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2 MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250x20 mm, S 10 muiotaeta) gave the desired L-3AA-MDC: white solid (0.280 g, 62.6% overall yield over two steps), 1H NMR (400 MHz, CDC13): 50.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s),1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J = 12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J= 9.6 Hz), 3.10 (1H, d, J = 8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J = 6.8Hz), 3.48 (2H, d, J= 6.8 Hz), 3.62 (1H, d, J = 12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J = 11.2 Hz), 4.76 (lH,d, J = 11.6 Hz), 5.36 (1H, q, J = 6.8 Hz), 5.65 ( 1H, dd, J = 15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J= 15.2 , 1 1.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J= 11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc: 865.3, found: 865.3. Rt: 6.59 min.
0.28 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 0 - 20℃;	Into above prepared N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g, 2.123 mmol) in DMF (25 mL) under 0 C. was added EDC (0.407 g, 2.123 mmol). The mixture was stirred at r.t. overnight, quenched with water, extracted with EtOAc, washed with brine, dried over Na2SO4. The solvent was removed under reduced pressure. Chromatography (silica gel: CH2Cl2/MeOH 30:1) gave the crude product. Further purification by preparative HPLC on a YMC C-18 column (250×20 mm, S 10 mum) gave the desired L-3AA-MDC: white solid (0.280 g, 62.6% overall yield over two steps), 1H NMR (400 MHz, CDCl3): delta0.79 (3H, s), 1.17-1.32 (3H, m), 1.27 (3H, s), 1.29 (3H, s), 1.40-1.76 (7H, m), 2.12-2.23 (2H, m), 2.31-2.45 (1H, m), 2.59 (1H, t, J=12.8 Hz), 2.82 (3H, s), 3.01 (1H, d, J=9.6 Hz), 3.10 (1H, d, J=8.8 Hz), 3.17 (3H, s), 3.34 (3H, s), 3.42 (2H, t, J=6.8 Hz), 3.48 (2H, d, J=6.8 Hz), 3.62 (1H, d, J=12.8 Hz), 3.97 (3H, s), 4.27 (1H, t, J=11.2 Hz), 4.76 (1H, d, J=11.6 Hz), 5.36 (1H, q, J=6.8 Hz), 5.65 (1H, dd, J=15.2, 9.2 Hz), 6.25 (1H, s), 6.41 (1H, dd, J=15.2, 11.2 Hz), 6.64 (1H, s), 6.65 (2H, s), 6.72 (1H, d, J=11.2 Hz), 6.82 (1H, s). LC-MS (M+Na+) calc.: 865.3. found: 865.3. Rt: 6.59 min.
0.28 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃;	Crude N-Me-L-Ala-MDC (0.5307 mmol) and MA-ACP (0.448 g) prepared in the previous step 2.123 mmol) was dissolved in DMF (25 mL), cooled in an ice-water bath and EDC (0.407 g, 2.123 mmol) was added. Reaction mixture at room temperature The mixture was stirred overnight, quenched with water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation. Column chromatography (silica gel, CH2C12/Me0H 30:1) gave the expected product L-3AA-MDC: white solid (0.280 g, two steps total Yield 62.6%)

Reference： [1]Patent: US2014/178413,2014,A1 .Location in patent: Paragraph 152; 153
[2]Patent: US2014/178412,2014,A1 .Location in patent: Paragraph 183; 184
[3]Journal of Medicinal Chemistry,2014,vol. 57,p. 7890 - 7899
[4]Patent: US2014/179917,2014,A1 .Location in patent: Paragraph 0476; 0477
[5]Patent: WO2014/94527,2014,A1 .Location in patent: Page/Page column 33-34
[6]Patent: US2014/178415,2014,A1 .Location in patent: Paragraph 0449
[7]Patent: CN107899020,2018,A .Location in patent: Paragraph 0197-0200

21
[ 6066-82-6 ]
[ 55750-53-3 ]
[ 55750-63-5 ]

Yield	Reaction Conditions	Operation in experiment
95%	With dicyclohexyl-carbodiimide; In acetonitrile; at 0 - 20℃;Inert atmosphere;	Under nitrogen protection, 4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile.Another 4.5 g (22 mmol) of DCC was dissolved in 25 ml of acetonitrile,Keeping the internal temperature around 0C,It was slowly dropped into the reaction solution.The reaction solution was reacted at 0C for 2 hours and then reacted overnight at room temperature.filter,The filter cake was washed with acetonitrile 10ml×3.The filtrate was concentrated to dryness under reduced pressure.The resulting oil was dried under reduced pressure at room temperature for 6 h to give 6.4 g of light brown solid.Yield 95%.
95%	With dicyclohexyl-carbodiimide; In acetonitrile; at 0 - 20℃;Inert atmosphere;	4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile under nitrogen. Another 4.5g (22mmol) DCC was dissolved in 25ml acetonitrile.The internal temperature was kept at about 0 C, and it was slowly dropped into the reaction liquid. The reaction solution was reacted at 0 C for 2 hours and then allowed to react at room temperature overnight. filter,The filter cake was washed with acetonitrile 10 ml × 3The filtrate was concentrated to dryness under reduced pressure. The obtained oil was dried under reduced pressure at room temperature for 6 h to give 6.4 g of pale brown solid. The yield was 95%.
91.9%	With dicyclohexyl-carbodiimide; In dichloromethane; at 20℃; for 4h;	The method for preparing biotin maleimide according to the present example includes the following steps: <strong>[55750-53-3]6-maleimidocaproic acid</strong> compound 1 (1.28 g, 6 mmol, 1 eq), and N-hydroxysuccinimide (0.7 g, 6 mmol, 1 eq) were sequentially added to a 50 ml single-necked flask and 10 ml of dichloromethane, dissolved and clarified, and added dicyclohexylcarbodiimide DCC (1.25 g, 6 mmol, 1 eq) at room temperature, and stirred at room temperature for 4 hours.A large amount of dicyclohexylurea DCU was produced, and TLC monitored the reaction of the starting material completely.After cooling to room temperature, filtration, the filtrate was added with silica gel, and the column was chromatographed, and the product was eluted with PE: EtOAc = 1:1.1.7 g of an oily liquid was obtained as 6-maleimidohexanoic acid N-hydroxysuccinimide ester compound 2, MS+1: 309.10, yield 91.9percent.

70%	With dicyclohexyl-carbodiimide; In dichloromethane; at 20℃; for 3h;	Dicyclohexyl-carbodiimide (2.68 g, 13.02 mmol) and N-hydroxysuccinimide (1 .36 g, 1 1 .84 mmol) are added at room temperature to a stirrer solution of compound [4] (2.5 g, 1 1 .84 mmol) in anhydrous dichloromethane (15 mL) and the mixture stirred at room temperature for 3 hours. The white solid is filtered with dichloromethane to remove the dicyclohexylurea, the organic phase is washed with HCI 0.1 N and water, then dried over anhydrous sodium sulfate and the solvent removed by rotatory evaporation. The resulting residue is subjected to flash column chromatography with a medium pressure system Sepacore Buchi (silica gel; gradient A: petroleum ether/B: ethyl acetate; B% 0-80 in 15 minutes) to give the activated acid [5] as a white solid, 2.4 g (70%). MS: m/z 309 [M+H]+.1H NMR (400 MHz, MeOD) delta 6.78 (s, 2H), 3.49 - 3.46 (m, 2H), 2.82 (s, 4H), 2.62 - 2.59 (m, 2H), 1 .78 - 1 .65 (m, 2H), 1 .64 - 1 .50 (m, 2H), 1 .46 - 1 .26 (m, 2H).13C NMR (100 MHz, MeOD) delta 169.8, 169.0, 167.3, 132.5, 35.4, 28.6, 26.1 , 23.8, 23.6, 22.3.
48%	With dicyclohexyl-carbodiimide; In ethyl acetate; at 0 - 20℃; for 20h;	To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 C, was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed to warm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48%) as a clear oil that solidified slowly to a waxy white solid. NMR (400 MHz, Chloroform-i/) delta 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J= 7.4 Hz, 2H), 1.73 - 1.57 (m, 2H), 1.50 - 1.35 (m, 2H). m/z calcd. for C14H16N206 = 308.10. Found [M+H]+ = 309.13. Rf = 0.28 (50% EtOAc/Hex).
48%	With dicyclohexyl-carbodiimide; In ethyl acetate; at 0 - 20℃; for 20h;	To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 C,was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed towarm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48%) as a clear oil that solidified slowly to a waxy white solid. ?H NMR (400 MHz, Chloroform-d) oe 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J = 7.4 Hz, 2H), 1.73 - 1.57 (m,2H), 1.50 1.35 (m, 2H). m/z calcd. for C,4H,6N206 = 308.10. Found [M+H] = 309.13. Rf= 0.28 (50% EtOAc/Hex)
	With dicyclohexyl-carbodiimide; In acetonitrile; at 0℃; for 2h;Inert atmosphere;	4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile under nitrogen.Another 4.5g (22mmol) DCC was dissolved in 25ml acetonitrile.The internal temperature was kept at about 0 C, and it was slowly dropped into the reaction liquid.The reaction solution was reacted at 0 C for 2 hours and then allowed to react at room temperature overnight. filter,The filter cake was washed with acetonitrile (10 ml × 3) and the filtrate was concentrated to dryness.The obtained oil was dried under reduced pressure at room temperature for 6 h.Obtained 6.4 g of light brown solid.The yield was 95%.

Reference： [1]Patent: CN107789630,2018,A .Location in patent: Paragraph 0064; 0065; 0066
[2]Patent: CN108714220,2018,A .Location in patent: Paragraph 0011-0013
[3]Patent: CN109485655,2019,A .Location in patent: Paragraph 0025; 0040-0042; 0046; 0048
[4]Patent: WO2018/178060,2018,A1 .Location in patent: Page/Page column 58; 60; 61
[5]International Journal of Molecular Sciences,2017,vol. 18
[6]Patent: WO2015/95953,2015,A1 .Location in patent: Page/Page column 118
[7]Patent: WO2016/41082,2016,A1 .Location in patent: Page/Page column 97; 98
[8]Organic and Biomolecular Chemistry,2014,vol. 12,p. 6624 - 6633
[9]Patent: CN108743968,2018,A .Location in patent: Paragraph 0007; 0008; 0009

22
[ 55750-53-3 ]
[ 76-05-1 ]
[ 151038-94-7 ]

Reference： [1]Journal of Controlled Release,2014,vol. 192,p. 29 - 39

23
[ 55750-53-3 ]
[ 159857-80-4 ]

Reference： [1]Patent: WO2014/191578,2014,A1
[2]Patent: WO2017/66668,2017,A1
[3]International Journal of Molecular Sciences,2017,vol. 18
[4]Patent: CN107789630,2018,A
[5]Patent: CN108743968,2018,A
[6]Tetrahedron Letters,2018,vol. 59,p. 3594 - 3599
[7]Patent: WO2020/14541,2020,A2
[8]Patent: WO2020/84115,2020,A1

24
[ 55750-53-3 ]
[ 1343476-44-7 ]
[ 1870916-87-2 ]

Yield	Reaction Conditions	Operation in experiment
52%	With N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate In N,N-dimethyl-formamide at 20℃; for 2h;
	With 4-methyl-morpholine; N-[(1H-1,2,3-benzotriazol-1-yloxy)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate In N,N-dimethyl-formamide

Reference： [1]Audisio, Davide; Babin, Victor; Barbe, Peggy; Beau, Fabrice; Correia, Isabelle; Devel, Laurent; Dubois, Steven; Feuillastre, Sophie; Ge, Xin; Keck, Mathilde; Lee, Ki Baek; Lequin, Olivier; Malgorn, Carole; Palazzolo, Alberto; Pieters, Gregory; Sallustrau, Antoine; Thai, Robert; Cahuzac, Héloïse; Garcia-Argote, Sébastien; Nozach, Hervé [Journal of Medicinal Chemistry, 2022]
[2]Current Patent Assignee: MAGENTA THERAPEUTICS INC - US2014/363454, 2014, A1 Location in patent: Paragraph 0341

25
[ 55750-53-3 ]
[ 21075-83-2 ]
C₁₆H₂₅N₃O₅ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
80%	With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃;	100 mg (473 mumol) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid were dissolved in 71 mul of DMF and then admixed with 139 mg (947 mumol) of tert-butyl 1-methylhydrazinecarboxylate, 182 mg (947 mumol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 145 mg (947 mumol) of 1-hydroxy-1H-benzotriazole hydrate. The mixture was stirred at RT overnight and then concentrated under reduced pressure. The remaining residue was purified by means of preparative HPLC. After lyophilization from dioxane/water, 129 mg (80percent of theory) of the protected intermediate were obtained as a colourless foam. [1473] Subsequently, the 129 mg (380 mumol) were deblocked with 2 ml of trifluoroacetic acid in 8 ml of dichloromethane. After stirring at RT for 1 h, the reaction mixture was concentrated under reduced pressure. The residue was lyophilized from acetonitrile/water, which left 125 mg (83percent of theory) of the title compound as a colourless foam. [1474] LC-MS (Method 1): Rt=0.38 min; MS (ESIpos): m/z=240 (M+H)+
80%	With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃;	[3848] 100 mg (473 f.tmol) of 6-(2,5-dioxo-2,5-dihydrolH-pyrrol-1-yl)hexanoic acid were dissolved in 71 fll ofDMFand then admixed with 139 mg (947 f.tmol) oftert-butyl-1-methylhydrazinecarboxylate, 182 mg (947 f.tmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorideand 145 mg (947 flillOI) of 1-hydroxy-lH-benzotriazolehydrate. The mixture was stirred at RT overnight and thenconcentrated in vacuo. The remaining residue was purified bymeans of preparative HPLC. After lyophilization from dioxane/water, 129 mg (80percent of theory) of the protected intermediatewere obtained as a colourless foam.

Reference： [1]Patent: US2015/23989,2015,A1 .Location in patent: Paragraph 1472-1474
[2]Patent: US2015/246136,2015,A1 .Location in patent: Paragraph 3847-3848

26
[ 55750-53-3 ]
[ 3017-32-1 ]
C₂₂H₃₅N₃O₇ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
93%	Stage #1: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane for 1h; Cooling with ice; Inert atmosphere; Stage #2: (S)-methyl 2-amino-6-(tert-butoxycarbonylamino)hexanoate With N-ethyl-N,N-diisopropylamine In dichloromethane at 20℃; for 12h; Inert atmosphere;	1 Synthesis of a Maleimide-Hydrocarbon with Blocked Lysine Intermediate (2) To an ice cooled solution of 6-maleimidocaproic acid (1) in CH2Cl2 (5 mL) under nitrogen atmosphere, EDCI (544.5 mg, 2.8407 mmol) and HOBT (383.7 mg, 2.8407 mmol) was added successively and stirred for 1 h. To this activated acid solution, protected lysine (630 mg, 2.1305 mmol) was added. The reaction mixture was basified with DIPEA and stirred for 12 h at room temperature, and analysed by TLC. After completion, the reaction mixture was quenched with H2O (10 mL) and twice with 0.1N HCl (25 mL), the intermediate compound 2 was extracted with twice with CHCl3 (15 mL each). The combined organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure and purified by silica gel chromatography to obtain coupling product 2 with 93% yield (602 mg). [0268] 1H NMR (500 MHz, CDCl3) δ: 6.71 (s, 2H), 6.07 (d, J=7.1 Hz, 1H), 4.61 (dt, J=12.8, 6.5 Hz, 1H), 3.76 (s, 3H), 3.53 (dd, J=14.1, 6.9 Hz, 2H), 3.12 (s, 2H), 2.33-2.15 (m, 2H), 1.92-1.80 (m, 2H), 1.78-1.57 (m, 4H), 1.56-1.48 (m, 2H), 1.48-1.41 (m, 11H), 1.41-1.24 (m, 2H). ESIMS m/z=476 [M+Na]+ for C22H35N3O7Na.

Reference： [1]Current Patent Assignee: INVICTUS ONCOLOGY PVT LTD - US2015/45428, 2015, A1 Location in patent: Paragraph 0266-0267

27
[ 55750-53-3 ]
[ 474645-27-7 ]
[ 863971-24-8 ]

Yield	Reaction Conditions	Operation in experiment
36%	With diethyl cyanophosphonate; N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃; for 12h;Inert atmosphere;	To a suspension of MMAE (0.05 g, 0.0694 mmol) in freshly distilled dry DCM (2 ml), 6-maleimidocaproic acid (0.0221 g, 0.104 mmol) was added followed by diethylcyanophosphonate (21 muIota, 0.139 mmol) and DIPEA (37 muIota, 0.208 mmol). On addition of DIPEA, the reaction mixture became clear and was stirred at room temperature under nitrogen for 12 h, TLC [silica gel: 5percent MeOH/DCM, Rf 0.31]. The reaction mixture was diluted with DCM (30 ml) and washed with 10percent citric acid (2 X 20 ml), water (20 ml), brine (20 ml) and concentrated to dryness. The crude was purified by flash column chromatography [silica gel: 5percent MeOH/DCM] to give 6- Maleimidocaproyl-MMAE 37 as a white solid 0.023 g (36percent). MS (m/z) found 911.58 (M+1) calculated for C49H79N6O10

Reference： [1]Patent: WO2016/46574,2016,A1 .Location in patent: Page/Page column 75

28
[ 55750-53-3 ]
[ 5581-32-8 ]
C₄₁H₅₀N₂O₁₂ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
79%	With 2-methylbenzene-1,4-diol In toluene at 60℃;	Preparation of Inventive Bismaleimide resin 2 In a 1L 4 necked flask equipped with a mechanical stirrer were taken bisphenol A diglycidyl ether (122g, 358mmol), 6-maleimidocaproic acid (159g, 752mmol) in toluene (200mL). The mixture was stirred at 60°C until it became homogenous. Hycat 2000S (2.81g, 1 wt%) was added and the mixture stirred at the same temperature overnight. 500mL of ethyl acetate was added and the mixture decanted to a separatory funnel and washed twice with saturated aq. NaHC03 solution (200mlx2) and several times with deionized water. After drying the organic layer over anhydrous Na2S04, the organic layer was passed through silica gel and the solvent evaporated to give Bisphenol A based bismaleimide resin 2 (220g, 79%).

Reference： [1]Current Patent Assignee: SUZHOU RAINBOW MATERIALS CO LTD - WO2017/11207, 2017, A1 Location in patent: Paragraph 0041

29
[ 55750-53-3 ]
[ 81186-33-6 ]

Yield	Reaction Conditions	Operation in experiment
	Stage #1: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid With t-butoxycarbonylhydrazine; benzotriazol-1-ol; dicyclohexyl-carbodiimide In N,N-dimethyl acetamide Stage #2: With hydrogenchloride In diethyl ether; water; ethyl acetate
	Multi-step reaction with 2 steps 1: dicyclohexyl-carbodiimide / dichloromethane / 4 h / 20 °C 2: hydrazine hydrate / tetrahydrofuran / 4 h / 20 °C
	Multi-step reaction with 3 steps 1: thionyl chloride; N,N-dimethyl-formamide / ethyl acetate / 3 h / Reflux 2: triethylamine / dichloromethane / 4 h / 10 - 15 °C 3: hydrogenchloride / tetrahydrofuran / 4 h / Reflux

Reference： [1]Current Patent Assignee: GEORGETOWN UNIVERSITY - WO2017/95924, 2017, A1 Location in patent: Sheet 5/7
[2]Current Patent Assignee: HEBEI BAILING WEICHAO FINE MAT - CN109485655, 2019, A
[3]Current Patent Assignee: SUZHOU HIGHFINE BIOTECH - CN109535058, 2019, A

30
[ 55750-53-3 ]
C₂₃H₃₇N₃O₈ [ No CAS ]
C₃₃H₄₈N₄O₁₁ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
66%		To a solution of 6-maleimidohexanoic acid (1.73 g, 8.19 mmol) in THF (70 mL), PyBOP (4.26 g, 8.19 mmol) and DIEA (3.89 mL, 22.33 mmol) were added. Then, the reaction mixture was stirred for 2 h at rt. Afterwards, 29a (3.60 g, 7.44 mmol) was dissolved in THF (10 mL) and added to the reaction mixture. The reaction was stirred at rt overnight. Then,methyl-tert-butylether (300 mL) was added. The organic phase was washed 2 x with 0.1 M HCl solution (200 mL). The combined aqueous phases were extracted 2 x with methyl-tertbutylether (200 mL). The combined organic phases were washed with brine (150 mL), dried over Na2SO4 and filtrated. The solvent was evaporated in vacuo. 29b was purified using flash chromatography.Yield:3.34 g (4.94 mmol, 66percent)m/z 677.34 = [M+H]+, (calculated monoisotopic mass =676.33)
66%		To a solution of 6-maleimidohexanoic acid (1.73 g, 8.19 mmol) in THF (70 mL), PyBOP (4.26 g, 8.19 mmol) and DIEA (3.89 mL, 22.33 mmol) were added. Then, the reaction mixture was stirred for 2 h at rt. Afterwards, 29a (3.60 g, 7.44 mmol) was dissolved in THF (10 mL) and added to the reaction mixture. The reaction was stirred at rt overnight. Then, methyl-tert-butylether (300 mL) was added. The organic phase was washed 2 x with 0.1 M HC1 solution (200 mL). The combined aqueous phases were extracted 2 x with methyl-tert- butylether (200 mL). The combined organic phases were washed with brine (150 mL), dried over Na2S04 and filtrated. The solvent was evaporated in vacuo. 29b was purified using flash chromatography. Yield: 3.34 g (4.94 mmol, 66percent). MS:mlz 677.34 = [M+H], (calculated monoisotopic mass =676.33).
3.34 g		To a solution of 6-maleimidohexanoic acid (1.73 g, 8.19 mmol) in THF (70 mL), PyBOP (4.26 g, 8.19 mmol) and DIEA (3.89 mL, 22.33 mmol) were added. Then, the reaction mixture was stirred for 2 h at rt. Afterwards, 29a (3.60 g, 7.44 mmol) was dissolved in THF (10 mL) and added to the reaction mixture. The reaction was stirred at rt overnight. Then, methyl-tert-butylether (300 mL) was added. The organic phase was washed 2 x with 0.1 M HCl solution (200 mL). The combined aqueous phases were extracted 2 x with methyl-tert- butylether (200 mL). The combined organic phases were washed with brine (150 mL), dried over Na2SO4 and filtrated. The solvent was evaporated in vacuo.29b was purified using flash chromatography. Yield: 3.34 g (4.94 mmol, 66percent) MS: m/z 677.34 = [M+H]+ , (calculated monoisotopic mass =676.33).

Reference： [1]Patent: WO2017/118707,2017,A1 .Location in patent: Page/Page column 213-214
[2]Patent: WO2017/118700,2017,A1 .Location in patent: Page/Page column 213-215
[3]Patent: WO2017/118703,2017,A1 .Location in patent: Page/Page column 217-218

31
[ 55750-53-3 ]
[ 1870916-87-2 ]

Yield	Reaction Conditions	Operation in experiment
	Multi-step reaction with 2 steps 1: trifluoroacetic anhydride; 2,4,6-trimethyl-pyridine / tetrahydrofuran / 1.5 h / 0 - 20 °C 2: N,N-dimethyl-formamide / 6 h / 20 °C
	Multi-step reaction with 2 steps 1: 2,4,6-trimethyl-pyridine; trifluoroacetic anhydride / tetrahydrofuran / 1 h / 20 °C / Cooling 2: N,N-dimethyl-formamide / 20 °C

Reference： [1]Wang, Yanming; Fan, Shiyong; Zhong, Wu; Zhou, Xinbo; Li, Song [International Journal of Molecular Sciences, 2017, vol. 18, # 9]
[2]Xiao, Dian; Luo, Longlong; Li, Jiaguo; Wang, Zhihong; Liu, Lianqi; Xie, Fei; Feng, Jiannan; Zhou, Xinbo [Bioorganic Chemistry, 2021, vol. 116]

32
[ 55750-53-3 ]
[ 1639939-40-4 ]

Yield	Reaction Conditions	Operation in experiment
	Multi-step reaction with 3 steps 1: trifluoroacetic anhydride; 2,4,6-trimethyl-pyridine / tetrahydrofuran / 1.5 h / 0 - 20 °C 2: N,N-dimethyl-formamide / 6 h / 20 °C 3: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 5 h / 20 °C
	Multi-step reaction with 3 steps 1: 2,4,6-trimethyl-pyridine; trifluoroacetic anhydride / tetrahydrofuran / 1 h / 20 °C / Cooling 2: N,N-dimethyl-formamide / 20 °C 3: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 5 h / 20 °C

33
[ 55750-53-3 ]
[ 86060-81-3 ]
C₁₆H₂₃N₃O₆S [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
		General procedure: Resin loading: In a 10 mL solid phase reaction vessel (plastic syringe with PET frit) was added 0.15 g of 2-Cl-Tritylchloride resin (0.225 mmol) followed by a solution of Fmoc- amino acid or Fmoc-amino-PEG24-COOH (0.225 mmol, 1.0 equiv) and N,N- diisopropylethylamine (DIPEA) (0.338 mmol, 1.5 equiv) in 3 mL of dry CH2C12. The vessel was shaken for 5 min, then more DIPEA (0.225 mmol, 1.0 equiv) was added, and the vessel was shaken for additional 30 min. Unreacted resin was quenched by adding MeOH (1.0 mL) for 5 min. Resin was then washed with DMF (5 x 5 mL), CH2C12 (5 x 5 mL), diethyl ether (5 x 5 mL) and dried in vacuo.Fmoc removal procedure: Resin containing Fmoc-protected peptide was treated with 20 percent piperidine in DMF (5 x 3 mL) for 30 min total. The resin was then washed with DMF (5 x 5 mL) prior to further manipulation.Standard coupling procedure: To the resin deprotected N-terminus amino acid (1 equiv), a solution containing Fmoc-amino acid or maleimido-acid (3 equiv), HATU (3 equiv), and DIPEA (6 equiv) in DMF (5 mL) was added. The reaction vessel was shaken for 1 hr. The resin was then washed with DMF (5 x 5 mL). Fmoc-Cys amino acids (1 equiv) were coupled by adding a solution containing Fmoc-Cys (4 equiv), hydroxybenzotriazole (HOBT) (4 equiv), and Nu,Nu'-diisopropylcarbodiimide (DIC) (4 equiv).Cleavage from resin: To dried resin was added 2 mL of a solution of 10 percenttrifluoroacetic acid(TFA) in CH2C12 that also contained 2.5 percent H20 and 2.5 percent triisopropyl silane in a 5 mL plastic syringe. After 1 min, the reaction mixture was transferred to a 20 mL borosilicate glass scintillation vial. This procedure was repeated three times. The cleavage solution was dried under a stream of N2, washed 3x with 0.5 mL diethyl ether, then dried in vacuo. The crude products were either used without subsequent purification or purified by reverse-phase FfPLC using the procedure described above. Expected exact mass: 385.13; observed m/z: 384.3 (M+H)+, LC-MS tR= 0.66 min. The crude product was judged to be > 95percent pure and was used for antibody conjugation with no subsequent purification.

Reference： [1]Patent: WO2018/112253,2018,A1 .Location in patent: Paragraph 0252-0257

34
[ 5026-74-4 ]
[ 55750-53-3 ]
C₂₅H₃₂N₂O₈ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With 2-methylbenzene-1,4-diol In ethyl acetate at 65℃; for 4h;	1 Example 1 To a 500mL 3 necked flask equipped with a magnetic stir bar were added N,Ndiglycidyl-4-glycidyloxyaniline (24.18g. 87mmol), 6-maleimidocaproic acid (1 8.42g, 87mmol), Hycat 2000S (340mg, O.8wt%) and methyihydroquinone (42mg, l000ppm) in ethyl acetate (42mL). The mixture was stirred at 65°C for 4h. After cooling, 300mL of ethyl acetate was added and the organic layer washed once with aq. NaHCO3 solution, once with 5% aq. NaOH solution and 3 times with deionized water. After drying over anhydrous Na2SO4, the solution was stirred with 5g (10 wt %) silica and 5g of sillitin for lh. The solution was filtered off and washed with 1 O0mL of ethyl acetate. 70mg of methylhydroquinone was added (total 2000ppm) and the solvent evaporated to yield a resin as a liquid (35g, 83%). ‘H NMR indicated the presence of a mixture of the N-glycidyl and 0-glycidyl adducts of maleimidocaproic acid.

Reference： [1]Current Patent Assignee: HENKEL AG & CO. KGAA - WO2018/213668, 2018, A1 Location in patent: Paragraph 0037

35
[ 6258-63-5 ]
[ 55750-53-3 ]
C₁₅H₁₉NO₄S₂ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
24.8%	In methanol at 60℃; for 72h;	3 For the cycloaddition between 6-maleimidohexanoic acid and 2-furanmethanethiol, 4.18 g of the dienophile, the maleimide, was combined with 1 mL of the diene reagent, in a 1 : 1 (v/v) dichloromethane/methanol ("DCM:MeOH") solvent mixture. The reaction was allowed to proceed for 7 days under agitating conditions at room temperature in a sealed container. For the Diels-Alder reactions between the 2-thienylmethanethiol (0.5 mL) and the 6-maleimidohexanoic acid (2.11 g); and the pyrrole-2-carboxylic acid (0.555 g) and 6-maleimidohexanoic acid (2.11 g), the reagents were again mixed together in MeOH-only solvent. Both the reactions for the pyrrole and thiophene were carried out in an oil bath at 60°C for 3 days under controlled ventilation. The bicyclic products between the different dienes and dienophile were purified by HPLC and characterized via MALDI-MS, and 13C NMR.

Reference： [1]Current Patent Assignee: COMMONWEALTH SYSTEM OF HIGHER EDUCATION - WO2019/14549, 2019, A1 Location in patent: Page/Page column 21; 22; 23

36
[ 55750-53-3 ]
[ 50-65-7 ]
(4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
82%	With triethylamine; dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 0.25h;Inert atmosphere;	Specifically, NIC (1.032 g, 3.16 mmol) and dry DMF (5 mL) were added to a dry vial. Next, Et3N (0.4 mL, 2.84 mmol) was added to the suspension, and the mixture was sonicated to produce a red-colored homogeneous solution. DCC (1.95 g, 9.47 mmol), dry DMF (5 mL) and 6-Maleimidohexanoic acid (1.99 g, 9.47 mmol) were added to a dry round-bottomed flask equipped with a magnetic stir bar under an Argon atmosphere. The red DMF suspension of NIC was added dropwise over 2 min to this solution at room temperature, and the vial was rinsed with twice with 1 mL dry DMF that was added to the flask. After 5% hours, an additional 0.2 mL of Et3N was added, the reaction mixture was stirred for an additional 15 min, and then filtered to remove a white precipitate that formed during the course of the reaction. The filtrate was poured into 200 mL of 0.1 M NaH2PO4 solution at pH 4-5, and the mixture was extracted twice by 75 mL ethyl acetate. The ethyl acetate solutions were combined and washed three times with PBS (pH 4-5), three times with water, twice with 3% sodium bicarbonate (freshly prepared), once with saturated sodium chloride solution, then dried over sodium sulfate and filtered. To the filtrate was added 5 mL of silica gel, and ca. 25 mL heptane, and the mixture was concentrated to dryness on a rotary evaporator. The solids were loaded onto a 120 mL silica gel column packed in 1% ethyl acetate/chloroform, and eluted with a gradient of 1-4% ethyl acetate/chloroform. The fractions containing the desired material (Rf=0.2 in 4% EtOAc/CHCl3) were combined and concentrated to give 1.34 g (82%) of the 6-Maleimidohexanoic ester of NIC (4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) delta=10.44 (br. s, 1H), 8.36 (d, J=2.54 Hz, 1H), 8.23 (dd, J=2.60, 9.20 Hz, 1H), 8.05 (d, J=9.08 Hz, 1H), 7.79 (d, J=2.54 Hz, 1H), 7.66 (dd, J=2.54, 8.7 Hz, 1H), 7.30 (d, J=8.7 Hz, 1H), 6.96 (br. s, 2H), 3.28 (t, J=7.2 Hz, partial overlap with H2O peak), 2.50 (t, J=7.3 Hz partial overlap with DMSO peak), 1.47-1.63 (m, 2H), 1.30-1.45 (m, 2H), 1.12-1.26 (m, 2H). MS (ESI) m/z=518 (M-1). FTIR (thin film, cm-1) u=3369 (br, med), 1770 (med), 1702 (st).

Reference： [1]Patent: US2019/83638,2019,A1 .Location in patent: Paragraph 0253-0254

37
[ 55750-53-3 ]
[ 68858-20-8 ]
(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid [ No CAS ]
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-L-ornithine [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
73%	Stage #1: (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid With N-ethyl-N,N-diisopropylamine In dichloromethane; N,N-dimethyl-formamide for 2h; Stage #2: With piperidine In N,N-dimethyl-formamide for 0.0833333h; Stage #3: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid; Fmoc-Val-OH Further stages;
71%	Stage #1: (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid With N-ethyl-N,N-diisopropylamine In dichloromethane for 1.16667h; Stage #2: With piperidine In N,N-dimethyl-formamide for 0.35h; Stage #3: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid; Fmoc-Val-OH Further stages;	a (a) Preparation of LIN 1-1 : MC-Val-Cit-OH LIN 1-1 Cl-T rtCI-resin (20 g, 1 .49 mmol/g) (Iris Biotech, Ref.: BR-1065, 2-Chlorotrityl chloride resin (200-400 mesh, 1% DVB, 1.0-1.6 mmol/g), CAS 42074-68-0) was placed in a filter plate. 100 mL of DCM was added to the resin and the mixture was stirred for 1 h. The solvent was eliminated by filtration under vacuum. A solution of Fmoc-Cit-OH (11.83 g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was added and the mixture was stirred for 10 min. After that DIPEA (34.82 mmol, 199.98 mmol) was added and the mixture was stirred for 1 h. The reaction was terminated by addition of MeOH (30 mL) after stirring for 15 minutes. The Fmoc-Cit-O-TrtCI-resin produced as a result was subjected to the following washing/treatments: DCM (5 x 50 mL x 0.5 min), DMF (5 x 50 mL x 0.5 min), piperidine:DMF (1 :4, 1 x 1 min, 2 x 10 min), DMF (5 x 50 mL x 0.5 min), DCM (5 x 50 mL x 0.5 min). The final piperidine wash gave NH2-Cit-0-TrtCI-resin. The loading was calculated: 1.15 mmol/g. (1562) The NH2-Cit-0-TrtCI-resin produced above was washed with DMF (5 x 50 mL x 0.5 min) and a solution of Fmoc-Val-OH (31.22 g, 91.98 mmol), HOBt (11.23 g, 91.98 mmol) in DMF (100 mL) was added to the NH2-Cit-0-TrtCI-resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min). The Fmoc-Val-Cit-O-TrtCI-resin thus produced was treated with piperidine:DMF (1 :4, 1 x 1 min, 2 x 10 min) and washed with DMF (5 x 50 mL x 0.5 min). The final piperidine wash gave NH2-Val-Cit-0-TrtCI-resin. A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt (6.21 g, 45.92 mmol) in DMF (100 mL) was added to the Nh -Val-Cit-O-TrtCI-resin produced above, stirred and DIPCDI (7.12 mL, 45.92 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min) and DCM (5 x 50 mL x 0.5 min). (1563) The peptide was cleaved from the resin by treatments with TFA:DCM (1 :99, 5 x 100 mL). The resin was washed with DCM (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure and the solid obtained was triturated with Et20 and filtrated to obtain LIN 1-1 (7.60 g, 71%) as a white solid. (1564) 1H NMR (500 MHz, DMSO-ofe): d 12.47 (s, 1H), 8.13 (d, J = 7.3 Hz, 1H), 7.74 (d, J= 9.0 Hz, 1 H), 6.99 (s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd, J= 9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J = 7.0 Hz, 2H), 3.00-2.88 (m, 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1 H), 1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.50-1.31 (m, 6H), 1.21-1.11 (m, 2H), 0.84 (d, J= 6.8 Hz, 3H), 0.80 (d, J= 6.8 Hz, 3H). (1565) ESI-MS m/z : Calcd. for C21H33N5O7: 467.2. Found: 468.3 (M+H)+.
71%	Stage #1: (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid With N-ethyl-N,N-diisopropylamine In dichloromethane for 1.16667h; Stage #2: With piperidine In N,N-dimethyl-formamide Stage #3: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid; Fmoc-Val-OH Further stages;	Cl-TrtCl-resin (20 g, 1.49 mmol/g) (Iris Biotech, Ref.: BR-1065, 2-Chlorotrityl chloride resin (200-400 mesh, 1% DVB, 1.0-1.6 mmol/g), CAS 42074-68-0) was placed in a filter plate. 100 mL of DCM was added to the resin and the mixture was stirred for 1 h. The solvent was eliminated by filtration under vacuum. A solution of Fmoc-Cit-OH (11.83 g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was added and the mixture was stirred for 10 min. After that DIPEA (34.82 mmol, 199.98 mmol) was added and the mixture was stirred for 1 h. The reaction was terminated by addition of MeOH (30 mL) after stirring for 15 minutes. The Fmoc-Cit-O-TrtCl-resin produced as a result was subjected to the following washing/treatments: DCM (5 x 50 mL x 0.5 min), DMF (5 x 50 mL x 0.5 min), piperidine:DMF (1:4, 1 x 1 min, 2 x 10 min), DMF (5 x 50 mL x 0.5 min), DCM (5 x 50 mL x 0.5 min). The final piperidine wash gave NH2-Cit-O-TrtCl-resin. The loading was calculated: 1.15 mmol/g. The NH2-Cit-O-TrtCl-resin produced above was washed with DMF (5 x 50 mL x 0.5 min) and a solution of Fmoc-Val-OH (31.22 g, 91.98 mmol), HOBt (11.23 g, 91.98 mmol) in DMF (100 mL) was added to the NH2-Cit-O-TrtCl-resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min). The Fmoc-Val-Cit-O-TrtCl-resin thus produced was treated with piperidine:DMF (1:4, 1 x 1 min, 2 x 10 min) and washed with DMF (5 x 50 mL x 0.5 min). The final piperidine wash gave NH2-Val-Cit-O-TrtCl-resin. A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt (6.21 g, 45.92 mmol) in DMF (100 mL) was added to the NH2-Val-Cit-O-TrtCl-resin produced above, stirred and DIPCDI (7.12 mL, 45.92 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min) and DCM (5 x 50 mL x 0.5 min). The peptide was cleaved from the resin by treatments with TFA:DCM (1:99, 5 x 100 mL). The resin was washed with DCM (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure and the solid obtained was triturated with Et2O and filtrated to obtain LIN 1-1 (7.60 g, 71%) as a white solid. 1H NMR (500 MHz, DMSO-d6): d 12.47 (s, 1H), 8.13 (d, J = 7.3 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 6.99 (s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd, J = 9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J = 7.0 Hz, 2H), 3.00-2.88 (m, 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.50-1.31 (m, 6H), 1.21-1.11 (m, 2H), 0.84 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H). ESI-MS m/z: 468.3 (M+H)+.

71%	Stage #1: (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid With N-ethyl-N,N-diisopropylamine In dichloromethane for 2.16667h; Stage #2: With piperidine In dichloromethane; N,N-dimethyl-formamide Stage #3: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid; Fmoc-Val-OH Further stages;	2.a (a) Preparation of LIN 1-1: MC-Val-Cit-OH LIN 1-1 Cl-TrtCl-resin (20 g, 1.49 mmol/g) (Iris Biotech, Ref.: BR-1065, 2-Chlorotrityl chloride resin (200-400 mesh, 1% DVB, 1.0-1.6 mmol/g), CAS 42074-68-0) was placed in a filter plate. 100 ml, of DCM was added to the resin and the mixture was stirred for 1 h. The solvent was eliminated by filtration under vacuum. A solution of Fmoc-Cit-OH (11.83 g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was added and the mixture was stirred for 10 min. After that DIPEA (34.82 mmol, 199.98 mmol) was added and the mixture was stirred for 1 h. The reaction was terminated by addition of MeOH (30 mL) after stirring for 15 minutes. The Fmoc-Cit-O-TrtCl-resin produced as a result was subjected to the following washing/treatments: DCM (5 x 50 mL x 0.5 min), DMF (5 x 50 mL x 0.5 min), piperidine:DMF (1:4, l x l min, 2 x 10 min), DMF (5 x 50 mL x 0.5 min), DCM (5 x 50 mL x 0.5 min). The final piperidine wash gave NFb-Cit-O-TrtCl-resin. The loading was calculated: 1.15 mmol/g.The NFF-Cit-O-TrtCl-resin produced above was washed with DMF (5 x 50 mL x 0.5 min) and a solution of Fmoc-Val-OH (31.22 g, 91.98 mmol), HOBt (11.23 g, 91.98 mmol) in DMF (100 mL) was added to the NFL-Cit-O-TrtCl-resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min). The Fmoc-Val-Cit-O-TrtCl -resin thus produced was treated with piperidine: DMF (1:4, l x l min, 2 x 10 min) and washed with DMF (5 x 50 mL x 0.5 min). The final piperidine wash gave NFL-Val-Cit-O-TrtCl-resin.A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt (6.21 g, 45.92 mmol) in DMF (100 mL) was added to the NH2-Val-Cit-O-TrtCl-resin produced above, stirred and DIPCDI (7.12 mL, 45.92 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min) and DCM (5 x 50 mL x 0.5 min).The peptide was cleaved from the resin by treatments with TFA:DCM (1:99, 5 x 100 mL). The resin was washed with DCM (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure and the solid obtained was triturated with Et20 and filtrated to obtain LIN 1-1 (7.60 g, 71%) as a white solid. NMR (500 MHz, DMSO-d6): d 12.47 (s, 1H), 8.13 (d, /= 7.3 Hz, 1H), 7.74 (d, /= 9.0 Hz, 1H), 6.99 (s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd, J= 9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J = 7.0 Hz, 2H), 3.00-2.88 (m, 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.50-1.31 (m, 6H), 1.21-1.11 (m, 2H), 0.84 (d, J= 6.8 Hz, 3H), 0.80 (d, J= 6.8 Hz, 3H).ESI-MSm/z: Calcd. for C21H33N5O7: 467.2. Found: 468.3 (M+H)+.
71%	Stage #1: (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5-ureidopentanoic acid With N-ethyl-N,N-diisopropylamine In dichloromethane for 2.16667h; Stage #2: With piperidine In dichloromethane; N,N-dimethyl-formamide Stage #3: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid; Fmoc-Val-OH Further stages;	2.a (a) Preparation of LIN 1-1: MC-Val-Cit-OH LIN 1-1 Cl-TrtCl-resin (20 g, 1.49 mmol/g) (Iris Biotech, Ref.: BR-1065, 2-Chlorotrityl chloride resin (200-400 mesh, 1% DVB, 1.0-1.6 mmol/g), CAS 42074-68-0) was placed in a filter plate. 100 ml, of DCM was added to the resin and the mixture was stirred for 1 h. The solvent was eliminated by filtration under vacuum. A solution of Fmoc-Cit-OH (11.83 g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was added and the mixture was stirred for 10 min. After that DIPEA (34.82 mmol, 199.98 mmol) was added and the mixture was stirred for 1 h. The reaction was terminated by addition of MeOH (30 mL) after stirring for 15 minutes. The Fmoc-Cit-O-TrtCl-resin produced as a result was subjected to the following washing/treatments: DCM (5 x 50 mL x 0.5 min), DMF (5 x 50 mL x 0.5 min), piperidine:DMF (1:4, l x l min, 2 x 10 min), DMF (5 x 50 mL x 0.5 min), DCM (5 x 50 mL x 0.5 min). The final piperidine wash gave NFb-Cit-O-TrtCl-resin. The loading was calculated: 1.15 mmol/g.The NFF-Cit-O-TrtCl-resin produced above was washed with DMF (5 x 50 mL x 0.5 min) and a solution of Fmoc-Val-OH (31.22 g, 91.98 mmol), HOBt (11.23 g, 91.98 mmol) in DMF (100 mL) was added to the NFL-Cit-O-TrtCl-resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min). The Fmoc-Val-Cit-O-TrtCl -resin thus produced was treated with piperidine: DMF (1:4, l x l min, 2 x 10 min) and washed with DMF (5 x 50 mL x 0.5 min). The final piperidine wash gave NFL-Val-Cit-O-TrtCl-resin.A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt (6.21 g, 45.92 mmol) in DMF (100 mL) was added to the NH2-Val-Cit-O-TrtCl-resin produced above, stirred and DIPCDI (7.12 mL, 45.92 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5 x 50 mL x 0.5 min) and DCM (5 x 50 mL x 0.5 min).The peptide was cleaved from the resin by treatments with TFA:DCM (1:99, 5 x 100 mL). The resin was washed with DCM (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure and the solid obtained was triturated with Et20 and filtrated to obtain LIN 1-1 (7.60 g, 71%) as a white solid. NMR (500 MHz, DMSO-d6): d 12.47 (s, 1H), 8.13 (d, /= 7.3 Hz, 1H), 7.74 (d, /= 9.0 Hz, 1H), 6.99 (s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd, J= 9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J = 7.0 Hz, 2H), 3.00-2.88 (m, 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.50-1.31 (m, 6H), 1.21-1.11 (m, 2H), 0.84 (d, J= 6.8 Hz, 3H), 0.80 (d, J= 6.8 Hz, 3H).ESI-MSm/z: Calcd. for C21H33N5O7: 467.2. Found: 468.3 (M+H)+.

Reference： [1]Wu, Chunlei; Cheng, Zhehong; Lu, Danyi; Liu, Ke; Cheng, Yulian; Wang, Pengxin; Zhou, Yimin; Li, Meiqing; Shao, Ximing; Li, Hongchang; Su, Wu; Fang, Lijing [Journal of Medicinal Chemistry, 2021, vol. 64, # 2, p. 991 - 1000]
[2]Current Patent Assignee: PHARMA MAR SA - WO2020/84115, 2020, A1 Location in patent: Page/Page column 227; 228
[3]Current Patent Assignee: PHARMA MAR SA - WO2021/43951, 2021, A1 Location in patent: Page/Page column 117-118
[4]Current Patent Assignee: PHARMA MAR SA - WO2021/214126, 2021, A1 Location in patent: Page/Page column 122-123
[5]Current Patent Assignee: PHARMA MAR SA - WO2021/214126, 2021, A1 Location in patent: Page/Page column 122-123

38
[ 55750-53-3 ]
[ 144875-48-9 ]
[ 2832198-91-9 ]

Yield	Reaction Conditions	Operation in experiment
27.6%	Stage #1: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid With 2,6-dimethylpyridine; benzotriazol-1-ol In N,N-dimethyl acetamide at 20℃; for 0.25h; Stage #2: resquimod With 2,6-dimethylpyridine In N,N-dimethyl acetamide at 20℃; for 72h;	6 LP5: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(ethoxymethyl)-1-(2-hydroxy- 2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)hexanamide [mc-resiquimod] To a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (13.0 mg, 61.5 µmol) in DMA (400 µL) was added 2,6-dimethylpyridine (21.4 µL, 185 µmol) and HOBt (13.2 mg, 86.2 µmol). This mixture was stirred for 15 min at rt and treated with a solution of resiquimod (21.3 mg, 67.7 µmol) and 2,6-dimethylpyridine (21.4 µL, 185 µmol) in DMA (300 µL). After 72h at rt, the crude mixture was purified on prep-HPLC, producing 8.6 mg (27.6%) of E130 as a white solid, LC-MS (Protocol A): m/z 764.5 [M+1]+; Retention time = 2.80 min. 1H NMR (400 MHz, DMSO) δH/ppm 8.74 (d, J = 8.3 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.01 (s, 2H), 4.85 - 4.79 (m, 1H), 3.58 (dd, J = 14.0, 7.0 Hz, 6H), 3.42 (t, J = 7.0 Hz, 3H), 2.80 (t, J = 7.3 Hz, 2H), 1.75 - 1.66 (m, 2H), 1.56 (dt, J = 14.8, 7.3 Hz, 3H), 1.39 - 1.29 (m, 3H), 1.21 (s, 1H), 1.16 (t, J = 7.0 Hz, 5H).
27.6 %	Stage #1: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid With 2,6-dimethylpyridine; benzotriazol-1-ol In N,N-dimethyl acetamide at 20℃; Stage #2: resquimod With 2,6-dimethylpyridine In N,N-dimethyl acetamide at 20℃;	6 LP5: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(ethoxymethyl)-1-(2-hydroxy- 2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)hexanamide [mc-resiquimod] To a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (13.0 mg, 61.5 µmol) in DMA (400 µL) was added 2,6-dimethylpyridine (21.4 µL, 185 µmol) and HOBt (13.2 mg, 86.2 µmol). This mixture was stirred for 15 min at rt and treated with a solution of resiquimod (21.3 mg, 67.7 µmol) and 2,6-dimethylpyridine (21.4 µL, 185 µmol) in DMA (300 µL). After 72h at rt, the crude mixture was purified on prep-HPLC, producing 8.6 mg (27.6%) of E130 as a white solid, LC-MS (Protocol A): m/z 764.5 [M+1]+; Retention time = 2.80 min. 1H NMR (400 MHz, DMSO) δH/ppm 8.74 (d, J = 8.3 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.01 (s, 2H), 4.85 - 4.79 (m, 1H), 3.58 (dd, J = 14.0, 7.0 Hz, 6H), 3.42 (t, J = 7.0 Hz, 3H), 2.80 (t, J = 7.3 Hz, 2H), 1.75 - 1.66 (m, 2H), 1.56 (dt, J = 14.8, 7.3 Hz, 3H), 1.39 - 1.29 (m, 3H), 1.21 (s, 1H), 1.16 (t, J = 7.0 Hz, 5H).
27.6 %	Stage #1: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid With 2,6-dimethylpyridine; benzotriazol-1-ol In N,N-dimethyl acetamide at 20℃; Stage #2: resquimod With 2,6-dimethylpyridine In N,N-dimethyl acetamide at 20℃;	6 LP5: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(ethoxymethyl)-1-(2-hydroxy- 2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)hexanamide [mc-resiquimod] To a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (13.0 mg, 61.5 µmol) in DMA (400 µL) was added 2,6-dimethylpyridine (21.4 µL, 185 µmol) and HOBt (13.2 mg, 86.2 µmol). This mixture was stirred for 15 min at rt and treated with a solution of resiquimod (21.3 mg, 67.7 µmol) and 2,6-dimethylpyridine (21.4 µL, 185 µmol) in DMA (300 µL). After 72h at rt, the crude mixture was purified on prep-HPLC, producing 8.6 mg (27.6%) of E130 as a white solid, LC-MS (Protocol A): m/z 764.5 [M+1]+; Retention time = 2.80 min. 1H NMR (400 MHz, DMSO) δH/ppm 8.74 (d, J = 8.3 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.01 (s, 2H), 4.85 - 4.79 (m, 1H), 3.58 (dd, J = 14.0, 7.0 Hz, 6H), 3.42 (t, J = 7.0 Hz, 3H), 2.80 (t, J = 7.3 Hz, 2H), 1.75 - 1.66 (m, 2H), 1.56 (dt, J = 14.8, 7.3 Hz, 3H), 1.39 - 1.29 (m, 3H), 1.21 (s, 1H), 1.16 (t, J = 7.0 Hz, 5H).

27.6 %

Stage #1: 2,5-Dihydro-2,5-dioxo-1H-pyrrole-1-hexanoic acid With 2,6-dimethylpyridine; benzotriazol-1-ol In N,N-dimethyl acetamide at 20℃; Stage #2: resquimod With 2,6-dimethylpyridine In N,N-dimethyl acetamide at 20℃;

6 LP5: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(ethoxymethyl)-1-(2-hydroxy- 2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl)hexanamide [mc-resiquimod] To a solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (13.0 mg, 61.5 µmol) in DMA (400 µL) was added 2,6-dimethylpyridine (21.4 µL, 185 µmol) and HOBt (13.2 mg, 86.2 µmol). This mixture was stirred for 15 min at rt and treated with a solution of resiquimod (21.3 mg, 67.7 µmol) and 2,6-dimethylpyridine (21.4 µL, 185 µmol) in DMA (300 µL). After 72h at rt, the crude mixture was purified on prep-HPLC, producing 8.6 mg (27.6%) of E130 as a white solid, LC-MS (Protocol A): m/z 764.5 [M+1]+; Retention time = 2.80 min. 1H NMR (400 MHz, DMSO) δH/ppm 8.74 (d, J = 8.3 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.01 (s, 2H), 4.85 - 4.79 (m, 1H), 3.58 (dd, J = 14.0, 7.0 Hz, 6H), 3.42 (t, J = 7.0 Hz, 3H), 2.80 (t, J = 7.3 Hz, 2H), 1.75 - 1.66 (m, 2H), 1.56 (dt, J = 14.8, 7.3 Hz, 3H), 1.39 - 1.29 (m, 3H), 1.21 (s, 1H), 1.16 (t, J = 7.0 Hz, 5H).

Reference： [1]Current Patent Assignee: STATE UNIVERSITY OF NEW YORK - WO2022/187809, 2022, A1 Location in patent: Paragraph 0222; 0227-0228
[2]Current Patent Assignee: STATE UNIVERSITY OF NEW YORK - WO2022/187809, 2022, A1 Location in patent: Paragraph 0222; 0227-0228
[3]Current Patent Assignee: STATE UNIVERSITY OF NEW YORK - WO2022/187809, 2022, A1 Location in patent: Paragraph 0222; 0227-0228
[4]Current Patent Assignee: STATE UNIVERSITY OF NEW YORK - WO2022/187809, 2022, A1 Location in patent: Paragraph 0222; 0227-0228

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[ 55750-53-3 ]

A100170[ 151038-94-7 ]

6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide 2,2,2-trifluoroacetate

Reason: Free-Salt

Purity	Size	Price	VIP Price	USA Stock *0-1 Day	Global Stock *5-7 Days	Quantity
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Precautionary Statements-General
Code	Phrase
P101	If medical advice is needed,have product container or label at hand.
P102	Keep out of reach of children.
P103	Read label before use
Prevention
Code	Phrase
P201	Obtain special instructions before use.
P202	Do not handle until all safety precautions have been read and understood.
P210	Keep away from heat/sparks/open flames/hot surfaces. - No smoking.
P211	Do not spray on an open flame or other ignition source.
P220	Keep/Store away from clothing/combustible materials.
P221	Take any precaution to avoid mixing with combustibles
P222	Do not allow contact with air.
P223	Keep away from any possible contact with water, because of violent reaction and possible flash fire.
P230	Keep wetted
P231	Handle under inert gas.
P232	Protect from moisture.
P233	Keep container tightly closed.
P234	Keep only in original container.
P235	Keep cool
P240	Ground/bond container and receiving equipment.
P241	Use explosion-proof electrical/ventilating/lighting/equipment.
P242	Use only non-sparking tools.
P243	Take precautionary measures against static discharge.
P244	Keep reduction valves free from grease and oil.
P250	Do not subject to grinding/shock/friction.
P251	Pressurized container: Do not pierce or burn, even after use.
P260	Do not breathe dust/fume/gas/mist/vapours/spray.
P261	Avoid breathing dust/fume/gas/mist/vapours/spray.
P262	Do not get in eyes, on skin, or on clothing.
P263	Avoid contact during pregnancy/while nursing.
P264	Wash hands thoroughly after handling.
P265	Wash skin thouroughly after handling.
P270	Do not eat, drink or smoke when using this product.
P271	Use only outdoors or in a well-ventilated area.
P272	Contaminated work clothing should not be allowed out of the workplace.
P273	Avoid release to the environment.
P280	Wear protective gloves/protective clothing/eye protection/face protection.
P281	Use personal protective equipment as required.
P282	Wear cold insulating gloves/face shield/eye protection.
P283	Wear fire/flame resistant/retardant clothing.
P284	Wear respiratory protection.
P285	In case of inadequate ventilation wear respiratory protection.
P231 + P232	Handle under inert gas. Protect from moisture.
P235 + P410	Keep cool. Protect from sunlight.
Response
Code	Phrase
P301	IF SWALLOWED:
P304	IF INHALED:
P305	IF IN EYES:
P306	IF ON CLOTHING:
P307	IF exposed:
P308	IF exposed or concerned:
P309	IF exposed or if you feel unwell:
P310	Immediately call a POISON CENTER or doctor/physician.
P311	Call a POISON CENTER or doctor/physician.
P312	Call a POISON CENTER or doctor/physician if you feel unwell.
P313	Get medical advice/attention.
P314	Get medical advice/attention if you feel unwell.
P315	Get immediate medical advice/attention.
P320
P302 + P352	IF ON SKIN: wash with plenty of soap and water.
P321
P322
P330	Rinse mouth.
P331	Do NOT induce vomiting.
P332	IF SKIN irritation occurs:
P333	If skin irritation or rash occurs:
P334	Immerse in cool water/wrap n wet bandages.
P335	Brush off loose particles from skin.
P336	Thaw frosted parts with lukewarm water. Do not rub affected area.
P337	If eye irritation persists:
P338	Remove contact lenses, if present and easy to do. Continue rinsing.
P340	Remove victim to fresh air and keep at rest in a position comfortable for breathing.
P341	If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing.
P342	If experiencing respiratory symptoms:
P350	Gently wash with plenty of soap and water.
P351	Rinse cautiously with water for several minutes.
P352	Wash with plenty of soap and water.
P353	Rinse skin with water/shower.
P360	Rinse immediately contaminated clothing and skin with plenty of water before removing clothes.
P361	Remove/Take off immediately all contaminated clothing.
P362	Take off contaminated clothing and wash before reuse.
P363	Wash contaminated clothing before reuse.
P370	In case of fire:
P371	In case of major fire and large quantities:
P372	Explosion risk in case of fire.
P373	DO NOT fight fire when fire reaches explosives.
P374	Fight fire with normal precautions from a reasonable distance.
P376	Stop leak if safe to do so. Oxidising gases (section 2.4) 1
P377	Leaking gas fire: Do not extinguish, unless leak can be stopped safely.
P378
P380	Evacuate area.
P381	Eliminate all ignition sources if safe to do so.
P390	Absorb spillage to prevent material damage.
P391	Collect spillage. Hazardous to the aquatic environment
P301 + P310	IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician.
P301 + P312	IF SWALLOWED: call a POISON CENTER or doctor/physician IF you feel unwell.
P301 + P330 + P331	IF SWALLOWED: Rinse mouth. Do NOT induce vomiting.
P302 + P334	IF ON SKIN: Immerse in cool water/wrap in wet bandages.
P302 + P350	IF ON SKIN: Gently wash with plenty of soap and water.
P303 + P361 + P353	IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower.
P304 + P312	IF INHALED: Call a POISON CENTER or doctor/physician if you feel unwell.
P304 + P340	IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for breathing.
P304 + P341	IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing.
P305 + P351 + P338	IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
P306 + P360	IF ON CLOTHING: Rinse Immediately contaminated CLOTHING and SKIN with plenty of water before removing clothes.
P307 + P311	IF exposed: call a POISON CENTER or doctor/physician.
P308 + P313	IF exposed or concerned: Get medical advice/attention.
P309 + P311	IF exposed or if you feel unwell: call a POISON CENTER or doctor/physician.
P332 + P313	IF SKIN irritation occurs: Get medical advice/attention.
P333 + P313	IF SKIN irritation or rash occurs: Get medical advice/attention.
P335 + P334	Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages.
P337 + P313	IF eye irritation persists: Get medical advice/attention.
P342 + P311	IF experiencing respiratory symptoms: call a POISON CENTER or doctor/physician.
P370 + P376	In case of fire: Stop leak if safe to Do so.
P370 + P378	In case of fire:
P370 + P380	In case of fire: Evacuate area.
P370 + P380 + P375	In case of fire: Evacuate area. Fight fire remotely due to the risk of explosion.
P371 + P380 + P375	In case of major fire and large quantities: Evacuate area. Fight fire remotely due to the risk of explosion.
Storage
Code	Phrase
P401
P402	Store in a dry place.
P403	Store in a well-ventilated place.
P404	Store in a closed container.
P405	Store locked up.
P406	Store in corrosive resistant/ container with a resistant inner liner.
P407	Maintain air gap between stacks/pallets.
P410	Protect from sunlight.
P411
P412	Do not expose to temperatures exceeding 50 oC/ 122 oF.
P413
P420	Store away from other materials.
P422
P402 + P404	Store in a dry place. Store in a closed container.
P403 + P233	Store in a well-ventilated place. Keep container tightly closed.
P403 + P235	Store in a well-ventilated place. Keep cool.
P410 + P403	Protect from sunlight. Store in a well-ventilated place.
P410 + P412	Protect from sunlight. Do not expose to temperatures exceeding 50 oC/122oF.
P411 + P235	Keep cool.
Disposal
Code	Phrase
P501	Dispose of contents/container to ...
P502	Refer to manufacturer/supplier for information on recovery/recycling

Physical hazards
Code	Phrase
H200	Unstable explosive
H201	Explosive; mass explosion hazard
H202	Explosive; severe projection hazard
H203	Explosive; fire, blast or projection hazard
H204	Fire or projection hazard
H205	May mass explode in fire
H220	Extremely flammable gas
H221	Flammable gas
H222	Extremely flammable aerosol
H223	Flammable aerosol
H224	Extremely flammable liquid and vapour
H225	Highly flammable liquid and vapour
H226	Flammable liquid and vapour
H227	Combustible liquid
H228	Flammable solid
H229	Pressurized container: may burst if heated
H230	May react explosively even in the absence of air
H231	May react explosively even in the absence of air at elevated pressure and/or temperature
H240	Heating may cause an explosion
H241	Heating may cause a fire or explosion
H242	Heating may cause a fire
H250	Catches fire spontaneously if exposed to air
H251	Self-heating; may catch fire
H252	Self-heating in large quantities; may catch fire
H260	In contact with water releases flammable gases which may ignite spontaneously
H261	In contact with water releases flammable gas
H270	May cause or intensify fire; oxidizer
H271	May cause fire or explosion; strong oxidizer
H272	May intensify fire; oxidizer
H280	Contains gas under pressure; may explode if heated
H281	Contains refrigerated gas; may cause cryogenic burns or injury
H290	May be corrosive to metals
Health hazards
Code	Phrase
H300	Fatal if swallowed
H301	Toxic if swallowed
H302	Harmful if swallowed
H303	May be harmful if swallowed
H304	May be fatal if swallowed and enters airways
H305	May be harmful if swallowed and enters airways
H310	Fatal in contact with skin
H311	Toxic in contact with skin
H312	Harmful in contact with skin
H313	May be harmful in contact with skin
H314	Causes severe skin burns and eye damage
H315	Causes skin irritation
H316	Causes mild skin irritation
H317	May cause an allergic skin reaction
H318	Causes serious eye damage
H319	Causes serious eye irritation
H320	Causes eye irritation
H330	Fatal if inhaled
H331	Toxic if inhaled
H332	Harmful if inhaled
H333	May be harmful if inhaled
H334	May cause allergy or asthma symptoms or breathing difficulties if inhaled
H335	May cause respiratory irritation
H336	May cause drowsiness or dizziness
H340	May cause genetic defects
H341	Suspected of causing genetic defects
H350	May cause cancer
H351	Suspected of causing cancer
H360	May damage fertility or the unborn child
H361	Suspected of damaging fertility or the unborn child
H361d	Suspected of damaging the unborn child
H362	May cause harm to breast-fed children
H370	Causes damage to organs
H371	May cause damage to organs
H372	Causes damage to organs through prolonged or repeated exposure
H373	May cause damage to organs through prolonged or repeated exposure
Environmental hazards
Code	Phrase
H400	Very toxic to aquatic life
H401	Toxic to aquatic life
H402	Harmful to aquatic life
H410	Very toxic to aquatic life with long-lasting effects
H411	Toxic to aquatic life with long-lasting effects
H412	Harmful to aquatic life with long-lasting effects
H413	May cause long-lasting harmful effects to aquatic life
H420	Harms public health and the environment by destroying ozone in the upper atmosphere

[ CAS No. 55750-53-3 ] {[proInfo.proName]}

Quality Control of [ 55750-53-3 ]

Related Doc. of [ 55750-53-3 ]

Product Details of [ 55750-53-3 ]

Calculated chemistry of [ 55750-53-3 ]

Physicochemical Properties

Pharmacokinetics

Lipophilicity

Druglikeness

Water Solubility

Medicinal Chemistry

Safety of [ 55750-53-3 ]

Application In Synthesis of [ 55750-53-3 ]

[ 55750-53-3 ] Synthesis Path-Upstream 1~14

[ 55750-53-3 ] Synthesis Path-Downstream 1~38

Similar Product of [ 55750-53-3 ]

Precautionary Statements-General

Prevention

Response

Storage

Disposal

Physical hazards

Health hazards

Environmental hazards

Inquiry

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[ 55750-53-3 ]