Synthesis and intermediates of pyrrolobenzodiazepine derivatives for conjugation

Abstract

A method of synthesing a compound of formula (I) from a compound of formula (III). ##STR00001##

Claims

1. A method of synthesizing a compound of formula III:, ##STR00068## from a compound of formula VI: ##STR00069## which proceeds via a compound of formula VIIa or VI lb: ##STR00070## wherein: R.sup.2 is selected from a group of: (a) formula IIa: ##STR00071## where A is a phenyl or C.sub.5-7 heteroaryl group, and either: (i) Q.sup.1 is a single bond, and Q.sup.2 is selected from a single bond and —Z—(CH.sub.2).sub.n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3, or (ii) Q.sup.1 is —CH═CH—, and Q.sup.2 is a single bond; (b) formula IIb: ##STR00072## R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and unsubstituted C.sub.1-2 alkyl; (c) formula IIc: ##STR00073## L.sup.2 is selected from a single bond and a group of: (a) ##STR00074## wherein n is 0 to 3; (b) ##STR00075##  wherein n is as defined above; (c) ##STR00076##  wherein n is as defined above; and (d) ##STR00077##  wherein n is as defined above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or CR; L.sup.1 is: ##STR00078##  where X is such that L.sup.1 is an amino-acid residue, a dipeptide residue or a tripeptide residue; Prot is selected from Fmoc (fluorenylmethyloxycarbonyl), Teoc (2-(trimethylsilyl)ethoxycarbonyl) and Boc (t-butoxycarbonyl); R.sup.12 is selected from: (a) C.sub.5-10 heteroaryl, phenyl, or naphthyl group, optionally substituted by one or more substituents selected from the group consisting of: halo, nitro, cyano, C.sub.1-7 alkoxy, C.sub.3-20 heterocyclyloxy, phenoxy, naphthyloxy, C.sub.5-20 heteroaryloxy, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; (b) C.sub.1-5 saturated aliphatic alkyl; (c) C.sub.3-6 saturated cycloalkyl; (d) ##STR00079##  wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5; (e) ##STR00080##  wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (f) ##STR00081##  where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; R.sup.6, R.sup.6 ′, R.sup.9 and R.sup.9′ are H; R.sup.7 is C.sub.1-7, alkoxy; R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms selected from O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings selected from benzene and pyridine; Y and Y′ are selected from O, S, and NH; SEM is 2-((trimethylsilyl)ethoxy)methyl; OTf is CF.sub.3SO.sub.3; C.sub.3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms selected from O, S, and N; C.sub.3-7 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms selected from O, S, and N; C.sub.5-20 heteroaryl is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of a heteroaromatic compound, which moiety has from 5 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms selected from O, S, and N; C.sub.5-10 heteroaryl is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of a heteroaromatic compound, which moiety has from 5 to 10 ring atoms, of which from 1 to 4 are ring heteroatoms selected from O, S, and N; C.sub.5-7 heteroaryl is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of a heteroaromatic compound, which moiety has from 5 to 20 ring atoms, of which from 1 to 4 are ring heteroatoms selected from O, S, and N; and R.sup.7′ is selected from the same groups as R.sup.7; wherein the R.sup.2 group is introduced by coupling a compound of formula XIa, XIb or XIc: ##STR00082## (a) ##STR00083## (b) ##STR00084## (c) wherein the R.sup.12 group is introduced by coupling a derivative comprising R.sup.12 which, when R.sup.12 is ##STR00085##  is ##STR00086##  or otherwise the derivative is R.sup.B′ —R.sup.12, where R.sup.B represents boronic acid or a boronate and R.sup.B′ represents boronic acid or a boronate.

2. The method according to claim 1, wherein Y and Y′ are O and R″ is a C.sub.3-7 alkylene group with no substituents.

3. The method according to claim 2, wherein R″ is a C.sub.3, C.sub.5 or C.sub.7 alkylene group.

4. The method according to claim 1, wherein R.sup.7 is methoxy.

5. The method according to claim 1, wherein R.sup.12 is selected from: (a) phenyl; (b) methyl, ethyl or propyl; (c) ##STR00087## (d) ##STR00088##  wherein one of R.sup.25a and R.sup.25b is H and the other is phenyl; and (e) ##STR00089##  wherein R.sup.24 is selected from H and methyl.

6. The method according to claims 1, wherein R.sup.2 is a group of formula IIa, and Q.sup.1 is a single bond.

7. The method according to claim 6, wherein Q.sup.2 is a single bond.

8. The method according to claim 6, wherein Q.sup.2 is a —Z—(CH.sub.2).sub.n—, Z is selected from 0 and S, and n is selected from 1 or 2.

9. The method according to claim 1, wherein R.sup.2 is selected from: (a) a group of formula IIa, and Q.sup.1 is —CH═CH—; (b) a group of formula IIb, and R.sup.C1, R.sup.C2 and R.sup.C3 are all H; (c) a group of formula IIb, and R.sup.C1, R.sup.C2 and R.sup.C3 are all methyl; and (d) a group of formula IIb, and R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and methyl.

10. The method according to claim 1, wherein L.sup.2 is a single bond.

11. The method according to claim 1, wherein L.sup.2 is selected from: (a) ##STR00090##  and n is 0 or 1; (b) ##STR00091##  and n is 0 or 1; (c) ##STR00092##  and n is 0 or 1; (d) ##STR00093##  and n is 0 or 1.

12. The method according to any claim 1, wherein L.sup.1 comprises a dipeptide residue.

13. The method according to claim 12, wherein L.sup.1 is selected from: .sup.Prot-Phe-Lys-.sup.L2, .sup.Prot-Val-Ala-.sup.L2, .sup.Prot-Val-LyS-.sup.L2, .sup.Prot-Ala-Lys-.sup.L2, .sup.Prot-Val-Cit-.sup.L2, .sup.Prot-Phe-Cit-.sup.L2, .sup.Prot-Leu-Cit-.sup.L2, .sup.Prot-Ile-Cit-.sup.L2, .sup.Prot-Phe-Arg-.sup.L2, and .sup.Prot-Trp-Cit-.sup.L2.

14. The method according to claim 13, wherein L.sup.1 is selected from: .sup.Prot-Phe-Lys-.sup.L2, .sup.Prot-Val-Ala- .sup.L2, .sup.Prot-Val-Lys- .sup.L2, .sup.Prot-Ala-Lys- .sup.L2, and .sup.Prot-Val-Cit- .sup.L2.

15. The method according to claim 14, wherein L.sup.1 is selected from .sup.Prot-Phe-Lys-.sup.L2, .sup.Prot-Val-Cit-.sup.L2 or .sup.Prot-Val-Ala-.sup.L2.

16. The method according to claim 1, wherein Prot is Fmoc or Teoc.

17. The method according to claim 1, wherein R.sup.B is selected from: (a) —B(OH).sub.2; (b) —B(OR).sub.2, where R is selected from C.sub.1-4 alkyl; (c) —B(—O—R′—O—), where R′ is an C.sub.2-10 alkylene group having between 2 and 4 carbon atoms in the chain between the oxygen atoms; (d) B.sup.−F.sub.3K.sup.+; and (e) ##STR00094##

Description

EXAMPLES

(1) General Experimental Methods

(2) Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100 mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. .sup.1H and .sup.13C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (° C.), 100; Desolvation Temperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive W-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F.sub.254), and flash chromatography utilised silica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt (NovaBiochem) and solid-supported reagents (Argonaut), all other chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied without further purification. Anhydrous solvents were prepared by distillation under a dry nitrogen atmosphere in the presence of an appropriate drying agent, and were stored over 4 Å molecular sieves or sodium wire. Petroleum ether refers to the fraction boiling at 40-60° C.

(3) General LC/MS Conditions:

(4) Method 1 (default method, used unless stated otherwise)

(5) The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B held over 1.0 min, then increase from 5% B to 95% B over a 3 min period. The composition was held for 0.1 min at 95% B, then returned to 5% B in 0.03 minutes and hold there for 0.87 min. Total gradient run time equals 5 minutes.

(6) Method 2

(7) The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B held over 1.0 minute, then increase from 5% B to 95% B over a 2.5 minute period. The composition was held for 0.5 minutes at 95% B, then returned to 5% B in 0.1 minutes and hold there for 0.9 min. Total gradient run time equals 5 minutes.

(8) For Both Methods

(9) Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex Onyx Monolithic C18 50×4.60 mm.

(10) Method 3

(11) LC/MS (Shimazu LCMS-2020) using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

(12) Gradient: initial composition 5% B held over 0.25 min, then increase from 5% B to 100% B over a 2 min period. The composition was held for 0.50 min at 100% B, then returned to 5% B in 0.05 minutes and hold there for 0.05 min. Total gradient run time equals 3 min. Flow rate 0.8 mL/min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50° C. Column: Waters Acquity UPLC BEH Shield RP18 1.7 μm 2.1×50 mm.

(13) Preparative HPLC:

(14) Method 1 (Default, Unless Otherwise Specified)

(15) The reverse phase flash purification conditions were as follows: The Flash purification system (Varian 971-Fp) was run using a mobile phase of water (A) and acetonitrile (B). Gradient: initial composition 5% B over 20 C.V. (Column Volume) then 5% B to 70% B within 60 C.V. The composition was held for 15 C.V. at 95% B, and then returned to 5% B in 5 C.V. and held at 5% B for 10 C.V. Total gradient run time equals 120 C.V. Flow rate 6.0 mL/min. Wavelength detection range: 254 nm. Column: Agilent AX1372-1 SF10-5.5 gC8.

(16) Method 2

(17) HPLC (Shimadzu UFLC) was run using a mobile phase of water (0.1% formic acid) A and acetonitrile (0.1% formic acid) B. Wavelength detection range: 254 nm. Column: Phenomenex Gemini 5μ C18 150×21-20 mm. Gradient:

(18) TABLE-US-00002 B t = 0 13% t = 15.00 95% t = 17.00 95% t = 17.10 13% t = 20.00 13%

(19) Total gradient run time is 20 min; flow rate 20.00 mL/min.

Example 1

(20) ##STR00062## ##STR00063##
(a) (R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido) propanoic acid (2)

(21) HO-Ala-Val-H 1 (350 mg, 1.86 mmol) and Na.sub.2CO.sub.3 (493 mg, 4.65 mmol) were dissolved in distilled H.sub.2O (15 mL) and the mixture was cooled to 0° C. before dioxane (15 mL) was added (partial precipitation of the amino acid salt occurred). A solution of Fmoc-CI (504 mg, 1.95 mmol) in dioxane (15 mL) was added dropwise with vigorous stirring over 10 minutes. The resulting mixture was stirred at 0° C. for 2 hours before the ice bath was removed and stirring was maintained for 16 hours. The solvent was removed by rotary evaporation under reduced pressure and the residue dissolved in water (150 mL). The pH was adjusted from 9 to 2 with 1N HCl and the aqueous layer was subsequently extracted with EtOAc (3×100 mL). The combined organics were washed with brine (100 mL), dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure to afford pure HO-Ala-Val-Fmoc 2 (746 mg, 97% yield). LC/MS 2.85 min (ES+) m/z (relative intensity) 410.60; .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.79 (d, J=7.77 Hz, 2H), 7.60(d, J=7.77 Hz, 2H), 7.43(d, J=7.5 Hz, 2H), 7.34 (d, J=7.5 Hz, 2H), 6.30 (bs, 1H), 5.30 (bs, 1H), 4.71-7.56 (m, 1H), 4.54-4.36 (m, 2H), 4.08-3.91 (m, 1H), 2.21-2.07 (m, 1H), 1.50 (d, J=7.1 Hz, 3H), 1.06-0.90 (m, 6H).

(22) (b) (9H-fluoren-9-yl)methyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)propan-2-yl)amino)butan-2-yl)carbamate (3)

(23) 4-Aminophenylboronic acid pinacol ester was added (146.9 mg, 0.67 mmol) was added to a solution of HO-Ala-Val-Fmoc 2 (330 mg, 0.8 mmol), DCC (166 mg, 0.8 mmol) and DMAP (5 mg, cat.) in dry DCM (8 mL) previously stirred for 30 minutes at room temperature in a flask flushed with argon. The reaction mixture was then allowed to stir at room temperature overnight. The reaction was followed by LCMS and TLC. The reaction mixture was diluted with CH.sub.2Cl.sub.2 and the organics were washed with H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dryloaded on a silicagel chromatography column (Hexane/EtOAc, 6:4) and pure product 3 was isolated as a white solid in 88% yield (360 mg).

(24) Alternative Synthesis of 3

(25) 4-Aminophenylboronic acid pinacol ester was added (444 mg, 2.02 mmol) was added to a solution of HO-Ala-Val-Fmoc 2 (1 g, 2.43 mmol) and EEDQ (600 mg, 2.43 mmol) in dry DCM (20 mL) at room temperature in a flask flushed with argon. The reaction mixture was then allowed to stir at room temperature for 3.5 hours (or until complete). The reaction was followed by LCMS and TLC. The reaction mixture was diluted with CH.sub.2Cl.sub.2 and the organics were washed with H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dryloaded on a silica gel chromatography column (Hexane/EtOAc, 6:4) and pure product 3 was isolated as a white solid in 58% yield (876 mg).

(26) (c) 8-(3-((2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl trifluoromethanesulfonate (5)

(27) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione] 4 (2.03 g, 1.81 mmol), boronic pinacol ester (1 g, 1.63 mmol) and Na.sub.2CO.sub.3 (881 mg, 8.31 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (40 mL). The reaction flask was purged and filled with argon three times before tetrakis(triphenylphosphine)palladium(0) (41 mg, 0.035 mmol) was added and the reaction mixture heated to 30° C. overnight. The solvents were removed under reduce pressure and the residue was taken up in H.sub.2O (100 mL) and extracted with EtOAc (3×100 mL). The combined organics were washed with brine (100 mL), dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel chromatography column (Hexane/EtOAc, 8:2 to 25:75) to afford pure 5 in 33% yield (885 mg). LC/MS 3.85 min (ES+) m/z (relative intensity) 1452.90; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.78-7.16 (m, 17H), 7.13 (s, 1H), 6.51-6.24 (m, 1H), 5.51 (dd, J=10.0, 5.1 Hz, 2H), 5.36-5.11 (m, 1H), 4.74 (dd, J=10.1, 4.4 Hz, 2H), 4.70-4.53 (m, 2H), 4.47 (d, J=6.4 Hz, 1H), 4.37 (d, J=7.2 Hz, 1H), 4.27 (m, 4H), 4.20-4.14 (m, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.77 (ddd, J=16.7, 9.0, 6.4 Hz, 3H), 3.71-3.61 (m, 2H), 3.24-2.91 (m, 3H), 2.55-2.33 (m, 2H), 2.22-2.07 (m, 1H), 1.52-1.37 (m, 3H), 1.04-0.86 (m, 10H), 0.00 (s, 18H).

(28) (d) (9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (6)

(29) Triphenylarsine (42 mg, 0.137 mmol) was added to a mixture of PBD-triflate 5 (250 mg, 0.172 mmol), cyclopropylboronic acid (73.9 mg, 0.86 mmol), silver oxide (159 mg, 0.688 mmol) and potassium phosphate tribasic (438 mg, 2.06 mmol) in dry dioxane (10 mL) under an argon atmosphere. The reaction was flushed with argon 3 times and bis(benzonitrile)palladium(II) chloride (13.2 mg, 0.034 mmol) was added. The reaction was flushed with Argon 3 more times before being warmed to 75° C. and stirred for 10 minutes. The reaction mixture was filtered through a pad of celite which was subsequently rinsed with ethyl acetate. The solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 1% methanol/chloroform). Pure fractions were collected and combined, and excess eluent was removed by rotary evaporation under reduced pressure to afford the desired product 6 (132 mg, 50% yield). LC/MS 3.83 min (ES+) m/z (relative intensity) 1345.91; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.88-7.14 (m, 17H), 6.69 (s, 1H), 6.45-6.25 (m, 1H), 5.57-5.41 (m, 2H), 5.34-5.14 (m, 1H), 4.78-4.67 (m, 2H), 4.62-4.55 (m, 1H), 4.50-4.45 (m, 2H), 4.51-4.44 (m, 1H), 4.31-4.21 (m, 4H), 4.16 (m, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.82-3.71 (m, 2H), 3.66 (m, 3H), 3.40-3.28 (m, 1H), 3.07 (m, 1H), 2.70-2.57 (m, 1H), 2.47-2.36 (m, 2H), 2.15 (m, 1H), 1.51-1.40 (m, 3H), 1.03-0.87 (m, 11H), 0.77-0.71 (m, 2H), 0.60-0.54 (m, 2H), 0.00 (t, J=3.0 Hz, 18H).

(30) (e) (9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1 H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (7)

(31) A solution of Super-Hydride® (0.5 mL, 1M in THF) was added dropwise to a solution of SEM dilactam 6 (265 mg g, 0.19 mmol) in THF (10 mL) at −78° C. under an argon atmosphere. The addition was completed over 5 minutes in order to maintain the internal temperature of the reaction mixture constant. After 20 minutes, an aliquot was quenched with water for LC/MS analysis, which revealed that the reaction was complete. Water (20 mL) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with EtOAc (3×30 mL) and the combined organics were washed with brine (50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in MeOH (12 mL), CH.sub.2Cl.sub.2 (6 mL), water (2 mL) and enough silica gel to form a thick stirring suspension. After 5 days, the suspension was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (200 mL) until the elution of the product was complete. The organic layer was washed with brine (2×70 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCl.sub.3 to 96% CHCl.sub.3/4% MeOH) afforded the product 7 as a yellow solid (162 mg, 78%). LC/MS 3.02 min (ES+) m/z (relative intensity) 1052.37.

(32) (f) (2S)-2-amino-N-((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (8)

(33) Excess piperidine was added (0.2 mL, 2 mmol) to a solution of SEM-dilactam 7 (76 mg, 0.073 mmol) in DMF (1 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH.sub.2Cl.sub.2 (75 mL) and the organic phase was washed with H.sub.2O (3×75 mL) until complete piperidine removal. The organic phase was dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude product 8 which was used as such in the next step. LC/MS 2.32 min (ES+) m/z (relative intensity) 830.00.

(34) (g) N-((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (9)

(35) EDCI hydrochloride (14 mg, 0.0732 mmol) was added to a suspension of Maleimide-PEG.sub.8-acid (43.4 mg, 0.0732 mmol) in dry CH.sub.2Cl.sub.2 (5 mL) under argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 8 (60.7 mg, 0.0732 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH.sub.2Cl.sub.2 and the organic phase was washed with H.sub.2O and brine before being dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCl.sub.3 up to 9:1 CHCl.sub.3/MeOH) followed by reverse phase chromatography to remove unreacted maleimide-PEG.sub.8-acid. The product 9 was isolated in 17.6% (21.8 mg). LC/MS 2.57 min (ES+) m/z (relative intensity) 1405.30; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91 (t, J=3.5 Hz, 1H), 7.80 (d, J=4.0 Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H), 7.39-7.31 (m, 2H), 6.87 (d, J=10.5 Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J=10.2, 5.2 Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J=16.1, 4.1 Hz, 1H), 2.62-2.49 (m, 4H), 2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J=9.2, 5.3 Hz, 2H), 0.57 (dd, J=9.2, 5.3 Hz, 2H), NH were not observed.

Example 2

(36) ##STR00064## ##STR00065##
(a) (S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl trifluoromethanesulfonate (10)

(37) Pd(PPh.sub.3).sub.4 (20.6 mg, 0.018 mmol) was added to a stirred mixture of the bis-enol triflate 4 (500 mg, 0.44 mmol), N-methyl piperazine boronic ester (100 mg, 0.4 mmol), Na.sub.2CO.sub.3 (218 mg, 2.05 mmol), MeOH (2.5 mL), toluene (5 mL) and water (2.5 mL). The reaction mixture was allowed to stir at 30° C. under a nitrogen atmosphere for 24 hours after which time all the boronic ester has consumed. The reaction mixture was then evaporated to dryness before the residue was taken up in EtOAc (100 mL) and washed with H.sub.2O (2×50 mL), brine (50 mL), dried (MgSO.sub.4), filtered and evaporated under reduced pressure to provide the crude product. Purification by flash chromatography (gradient elution: 80:20 v/v Hexane/EtOAc to 60:40 v/v Hexane/EtOAc) afforded product 10 as a yellowish foam (122.6 mg, 25%). LC/MS 3.15 min (ES+) m/z (relative intensity) 1144 ([M+H].sup.+; 20%).

(38) (b) (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (11)

(39) PBD-triflate 10 (359 mg, 0.314 mmol), boronic pinacol ester 3 (250 mg, 0.408 mmol) and triethylamine (0.35 mL, 2.51 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (3 mL). The microwave vessel was purged and filled with argon three times before tetrakis(triphenylphosphine)palladium(0) (21.7 mg, 0.018 mmol) was added and the reaction mixture placed in the microwave at 80° C. for 10 minutes. Subsequently, CH.sub.2Cl.sub.2 (100 mL) was added and the organics were washed with water (2×50 mL) and brine (50 mL) before being dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel chromatography column (CHCl.sub.3/MeOH, 100% to 9:1) to afford pure 11 (200 mg, 43% yield). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478 ([M+H].sup.+; 100%).

(40) (c) (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (12)

(41) A solution of Super-Hydride® (0.34 mL, 1M in THF) was added dropwise to a solution of SEM-dilactam 11 (200 mg, 0.135 mmol) in THF (5 mL) at −78° C. under an argon atmosphere. The addition was completed over 5 minutes in order to maintain the internal temperature of the reaction mixture constant. After 20 minutes, an aliquot was quenched with water for LC/MS analysis, which revealed that the reaction was complete. Water (20 mL) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with EtOAc (3×30 mL) and the combined organics were washed with brine (50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in MeOH (6 mL), CH.sub.2Cl.sub.2 (3 mL), water (1 mL) and enough silica gel to form a thick stirring suspension. After 5 days, the suspension was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (100 mL) until the elution of the product was complete. The organic layer was washed with brine (2×50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCl.sub.3 to 96% CHCl.sub.3/4% MeOH) afforded the product 12 as a yellow solid (100 mg, 63%). LC/MS 2.67 min (ES+) m/z (relative intensity) 1186 ([M+H].sup.+; 5%).

(42) (d) (S)-2-amino-N—((S)-1-((4-((R)-7-methoxy-8-(3-(((R)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (13)

(43) Excess piperidine was added (0.1 mL, 1 mmol) to a solution of PBD 12 (36.4 mg, 0.03 mmol) in DMF (0.9 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL) and the organic phase was washed with H.sub.2O (3×50 mL) until complete piperidine removal. The organic phase was dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude product 13 which was used as such in the next step. LC/MS 2.20 min (ES+) m/z (relative intensity) 964 ([M+H].sup.+; 5%).

(44) (e) 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (14)

(45) EDCI hydrochloride (4.7 mg, 0.03 mmol) was added to a suspension of 6-maleimidohexanoic acid (6.5 mg, 0.03 mmol) in dry CH.sub.2Cl.sub.2 (3 mL) under argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 13 (34 mg, crude) was added. Stirring was maintained until the reaction was complete (6 hours). The reaction was diluted with CH.sub.2Cl.sub.2 and the organic phase was washed with H.sub.2O and brine before being dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCl.sub.3 up to 9:1 CHCl.sub.3/MeOH) followed by reverse phase chromatography to remove unreacted maleimide-PEG.sub.8-acid. The product 14 was isolated in 41% over two steps (14.6 mg). LC/MS 2.40 min (ES+) m/z (relative intensity) 1157 ([M+H].sup.+5%)

Example 3

Alternative Synthesis of Compound 11

(46) ##STR00066##

(47) PBD-triflate 5 (469 mg, 0.323 mmol), boronic pinacol ester (146.5 mg, 0.484 mmol) and Na.sub.2CO.sub.3 (157 mg, 1.48 mmol) were dissolved in a mixture of toluene/MeOH/H.sub.2O, 2:1:1 (10 mL). The reaction flask was purged with argon three times before tetrakis(triphenylphosphine)palladium(0) (7.41 mg, 0.0064 mmol) was added and the reaction mixture heated to 30° C. overnight. The solvents were removed under reduced pressure and the residue was taken up in H.sub.2O (50 mL) and extracted with EtOAc (3×50 mL). The combined organics were washed with brine (100 mL), dried with MgSO.sub.4, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography (CHCl.sub.3 100% to CHCl.sub.3/MeOH 95%:5%) to afford pure 11 in 33% yield (885 mg). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478 ([M+H].sup.+; 100%).

Example 4

(48) ##STR00067##
(a) 9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (15)

(49) The triflate 5 (0.5 g, 0.35 mmol, 1 equiv.), 3,4-(methylenedioxy)phenyl boronic acid (75 mg, 0.45 mmol, 1.3 equiv.) and Na.sub.2CO.sub.3 (0.17 g, 1.6 mmol, 4.5 equiv.) were dissolved in toluene (11 mL), EtOH (5.5 mL) and water (5.5 mL) under an Ar atmosphere. The flask was evacuated and flushed with Ar three times. Pd(PPh.sub.3).sub.4 (24 mg, 0.02 mmol, 0.06 equiv.) was added and again the flask was evacuated and flushed with Ar three times. This was heated to 30° C. and left stirring overnight. Analysis by LC/MS showed complete loss of starting material. The solvent was removed in vacuo and the residue dissolved in water (60 mL) before washing with ethyl acetate (60 mL×3). The combined organic layers were washed with brine (50 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by column chromatography (50:50 to 25:75 v/v hexane/ethyl acetate) afforded the product 15 as a yellow solid (310 mg, 64%). LC/MS (method 3)(1.44 min (ES.sup.−) m/z (relative intensity) 1423.35 ([M−H].sup.−; 79).

(50) (b) (9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (16)

(51) SEM dilactam 15 (0.31 g, 0.22 mmol, 1 equiv.) was dissolved in THF (10 mL) and cooled to −78° C. under an Ar atmosphere. Super-Hydride® (0.5 mL, 1 M in THF, 2.5 equiv.) was added drop wise over 5 minutes while monitoring the temperature. After 30 minutes a small sample was taken and worked-up for LC/MS analysis. Water (50 mL) was added, the cold bath was removed and the solution washed with ethyl acetate (50 mL). The organic layer was extracted and washed with brine (60 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. The crude product was dissolved in EtOH (13.2 mL), CH.sub.2Cl.sub.2 (6.6 mL) and water (2.2 mL) and enough silica gel was added until it was a thick suspension. After 5 days stirring, it was filtered through a sintered funnel and washed with CH.sub.2Cl.sub.2/MeOH (9:1) (100 mL) until product ceased to be eluted. The organic layer was washed with brine (2×50 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. Purification by silica gel column chromatography (CHCl.sub.3 with 1% to 4% MeOH gradient) afforded the pure product 16 as a yellow solid (185 mg, 75%). LC/MS (method 3) (1.70 min (ES.sup.+) m/z (relative intensity) 1132.85 ([M+H].sup.+; 60).

(52) (c) (S)-2-Amino-N—((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (17)

(53) The imine 16 (82 mg, 0.07 mmol, 1 equiv.) was dissolved in DMF (1 mL) before piperidine (0.2 mL, 2 mmol, excess) was added slowly. This solution was left to stir at room temperature for 20 minutes until LC/MS analysis showed complete consumption of starting material. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL), washed with water (50 mL×4), dried with MgSO.sub.4, filtered and the solvent removed in vacuo. The product 17 was used without further purification in the next step. LC/MS (method 3) (1.15 min (ES.sup.+) m/z (relative intensity) 910.60 ([M+H].sup.+; 58).

(54) (D) N—((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(Benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (18)

(55) The imine 17 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHCl.sub.3 (6 mL) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG.sub.8-acid (53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14 mmol, 1.5 equiv.). This was left to stir vigorously at room temperature under Ar for 4 days until LC/MS analysis showed majority product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHCl3 with 1% to 10% MeOH gradient) yielding 18 (81 mg). The material was purified further by preparative HPLC (method 2) to give 18 as a yellow solid (26.3 mg, 18%). Fast Formic run: LC/MS (method 3)(1.39 min (ES+) m/z (relative intensity) 1485.00 ([M+H]+., 64).

(56) Abbreviations

(57) Ac acetyl Acm acetamidomethyl Alloc allyloxycarbonyl Boc di-tert-butyl dicarbonate t-Bu tert-butyl Bzl benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is methylbenzene Cbz or Z benzyloxy-carbonyl, where Z—CI and Z—Br are chloro- and bromobenzyloxy carbonyl respectively DMF N,N-dimethylformamide Dnp dinitrophenyl DTT dithiothreitol Fmoc 9H-fluoren-9-ylmethoxycarbonyl imp N-10 imine protecting group: 3-(2-methoxyethoxyl)propanoate-Val-Ala-PAB MC-OSu maleimidocaproyl-O—N-succinimide Moc methoxycarbonyl MP maleimidopropanamide Mtr 4-methoxy-2,3,6-trimethtylbenzenesulfonyl PAB para-aminobenzyloxycarbonyl PEG ethyleneoxy PNZ p-nitrobenzyl carbamate Psec 2-(phenylsulfonyl)ethoxycarbonyl TBDMS tert-butyldimethylsilyl TBDPS tert-butyldiphenylsilyl Teoc 2-(trimethylsilyl)ethoxycarbonyl Tos tosyl Troc 2,2,2-trichlorethoxycarbonyl chloride Trt trityl Xan xanthyl