Design, synthesis, and biophysical and biological evaluation of a series of pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugates

5442

J. Med. Chem. 2006, 49, 5442-5461

Design, Synthesis, and Biophysical and Biological Evaluation of a Series of

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Geoff Wells,^†Christopher R. H. Martin,^‡,§Philip W. Howard,^†,‡Zara A. Sands,^|Charles A. Laughton,^|Arnaud Tiberghien,^†,‡

,‡

Chi Kit Woo,^‡Luke A. Masterson,^†,‡Marissa J. Stephenson,^‡John A. Hartley, Terence C. Jenkins,^#,∇Steven D. Shnyder,^X

Paul M. Loadman,^XMichael J. Waring,^§and David E. Thurston*^,†,‡

Cancer Research UK Gene Targeted Drug Design Research Group, The School of Pharmacy, UniVersity of London, 29/39 Brunswick Square,

London WC1N 1AX, U.K., Spirogen Ltd, 2 Royal College Street, London NW1 0NH, U.K., Department of Pharmacology, UniVersity of

Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK, School of Pharmacy, UniVersity of Nottingham, UniVersity Park, Nottingham NG7

2RD, U.K., Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, Royal Free and UniVersity College

Medical School, UCL, 91 Riding House Street, London W1W 7BS, U.K., School of Pharmacy and Pharmaceutical Sciences, UniVersity of

Manchester, Oxford Road, Manchester M13 9PL, U.K., and Cancer Research UK Cancer Research Unit, Institute of Cancer Therapeutics,

UniVersity of Bradford, All Saints Road, Bradford, West Yorkshire BD7 3AY, U.K.

ReceiVed NoVember 29, 2005

A novel series of methyl ester-terminated C8-linked pyrrolobenzodiazepine (PBD)-poly(N-methylpyrrole)

conjugates (50a-f) has been synthesized and their DNA interaction evaluated by thermal denaturation,

DNA footprinting, and in vitro transcription stop assays. The synergistic effect of attaching a PBD unit to

a polypyrrole fragment is illustrated by the large increase in DNA binding affinity (up to 50-fold) compared

to the individual PBD and pyrrole components. 50a-f were found to bind mainly to identical DNA sequences

but with apparent binding site widths increasing with molecular length and the majority of sites conforming

to the consensus motif 5’-XGXW_z(z ) 3 ( 1; W ) A or T; X ) any base but preferably a purine). They

also provided robust sequence-selective blockade of transcription at sites corresponding approximately to

their DNA footprints. 50a-f were shown to have good cellular/nuclear penetration properties, and a degree

of correlation between cytotoxicity and DNA-binding affinity was observed.

Introduction

Compounds that bind in the minor groove of DNA have found

use in the experimental treatment of cancer and certain infectious

diseases. Furthermore, agents which target and disrupt distinct

DNA sequences have the potential to offer selective therapies

by modulating the activity of specific transcription factors or

genes. For this reason, a number of sequence-selective DNA-

binding agents have been evaluated with a range of affinities

and recognition fidelities. Of particular note are the hairpin

polyamides¹that combine high affinity DNA binding with good

sequence discrimination, although variable nuclear penetration

characteristics in cells have to date slowed their development

as therapeutic agents.^2,3The addition of a covalent binding motif

to such molecules offers the possibility of targeting the coding

regions of genes where the molecules may otherwise be

dislodged by the advancing replication fork.⁴Such a covalent

moiety may also have innate DNA sequence-recognition proper-

ties. In this respect, the pyrrolo[2,1-c][1,4]benzodiazepines

(PBDs^a)⁵(e.g., 1-3 in Figure 1) are of interest as they bind

to guanine residues in the minor groove with a preference for

Pu-G-Pu sequences.

Figure 1. Structures of the PBD monomers (1, 2, 3, 5, and 6), the

PBD dimer 4, and distamycin and netropsin (7 and 8).

ation of conjugates containing a PBD moiety linked to various

combinations of pyrroles and imidazoles. Lown has reported⁹

the synthesis of PBD conjugates (termed PBD-lexitropsin

conjugates) 9a-c (Figure 2) with a four-carbon linker between

the PBD C8-position and first heterocycle, and with a 3-(di-

methylamino)propyl terminus. Although stated to be lexitropsin

derivatives with the n ) 2 and 3 analogues mimicking the linked

N-methylpyrrole rings of 8 (netropsin) and 7 (distamycin),

respectively, the 3-(dimethylamino)propyl tail differs from the

amidinoalkyl tails of the natural products. Conjugates 9a-c were

There has been significant recent research activity in the PBD

area,^6-8including previous reports of the synthesis and evalu-

* Corresponding author. Phone: +44 0207 753 5931 (Direct) or +44

0207 753 5932 (Secretary) Fax: +44 0207-753-5935. E-Mail:

david.thurston@pharmacy.ac.uk.

^†University of London.

^‡Spirogen Ltd.

^§University of Cambridge.

^aAbbreviations: CT-DNA, calf thymus DNA; EMSA, electrophoretic

mobility shift assay; ER, estrogen receptor; GI₅₀, concentration causing 50%

growth inhibition; LC₅₀, concentration causing 50% cell kill; LTR, long

terminal repeat; MD, molecular dynamics; PBD, pyrrolo[2,1-c][1,4]-

benzodiazepine; TGI, concentration causing 100% growth inhibition; T_m,

DNA helix to coil transition (melting) temperature; T-stop assay, transcrip-

tion stop assay.

^|University of Nottingham.

Royal Free and University College Medical School.

^#University of Manchester.

^XUniversity of Bradford.

^∇Present address: Morvus Technology Limited, Building 115, Porton

Down Science Park, Salisbury, Wiltshire SP4 0JQ, U.K.

Published on Web 08/05/2006

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5443

reportedly prepared in overall 28-30% yields as a mixture of

their N10-C11 imine and carbinolamine methyl ether forms

which were highly polar and soluble only in a CHCl₃-MeOH

mixture but not sufficiently soluble in either solvent alone to

allow separation of the pure forms. Compounds 9b and 9c had

modest cytotoxicity in the NCI panel with average LC₅₀values

ranging from 7.5 to 86.5 µM for 9b (n ) 2) to 0.9-93 µM for

9c (n ) 3).¹⁰Lown has also reported^11,12the synthesis of three

equivalent imidazole analogues (10a-c; n ) 1-3) and two

mixed pyrrole-imidazole analogues (11a,b; n ) 1-2). These

compounds, obtained in overall 35-40% yields, existed as 1:1

mixtures of imines and carbinolamine methyl ethers. Com-

pounds 10a-c (n ) 1-3) were not significantly active in the

NCI 60 cell-line panel, with mean GI₅₀, TGI, and LC₅₀values

in the range of 16.2-95.5 µM, but conjugates 10b and 10c gave

IC₅₀values of 14.4 and 4.4 µM, respectively, in K562 cells (48

h exposure). Similarly, 11a,b showed no significant cytotoxicity

with mean NCI GI₅₀, TGI, and LC₅₀values in the 20.9-95.5

µM range (5.2 µM in K562).

Baraldi and co-workers have reported¹³the synthesis of PBD-

distamycin conjugates (12c, n ) 3) and related analogues 12a,b

(n ) 1 or 2) and 12d (n ) 4) which differ from the conjugates

synthesized by Lown in having a three-carbon rather than a four-

carbon linker, and in possessing the amidine terminus found in

distamycin and netropsin rather than a 3-(dimethylamino)propyl

residue. Although NCI data are not reported for these conjugates,

12c is more cytotoxic (IC₅₀) 0.2 µM in K562 cells) than the

Lown conjugates and slightly more potent than either the PBD

fragment alone in the form of an ester (5, 1.0 µM) or distamycin

A (>30 µM). Interestingly, 12d was the most active conjugate

reported with an IC₅₀of 0.04 µM in K562 cells (>100 µM and

6.0 µM for 12a and 12b, respectively). In the HL3T1 cell line

(72 h incubation), 12a-d had IC₅₀values of 50.0, 12.0, 2.5,

and 1.0 µM, respectively. However, cytotoxicity evaluations in

K562 cells (>100 µM [n ) 1] to 0.04 µM [n ) 4]) and Jurkat

cells (80 µM [for n ) 1] to 0.07 µM [for n ) 4]) suggested

that increasing the length of the polypyrrole backbone leads to

enhanced in vitro activity. Only the n ) 3 and n ) 4 compounds

were more potent than either the PBD fragment alone (5) or

the relevant PBD-free polypyrrole.

unlike distamycin A which inhibited PCR in only the A/T-rich

sequence (or control PBD 5 which failed to inhibit PCR in either

sequence), 12c (n ) 3) was equipotent in inhibiting PCR in

both the G/C-rich and A/T-rich DNAs at a level of potency

6-fold higher than distamycin in the A/T-rich sequence. This

demonstrated that the hybrid was much more active than either

of its constituent parts in terms of DNA binding affinity. It also

suggested that the sequence-selectivity of the hybrid was

different from distamycin in binding to mixed rather than only

A/T-rich sequences. For the extended set 12a-d, Baraldi also

investigated¹⁵sequence selectivity and stability of the DNA-

drug complexes, and performed DNase I footprinting and

arrested PCR assays on fragments of the human c-myc oncogene

and human immunodeficiency virus type 1 long terminal repeat

(HIV-1 LTR) (both G/C-rich), and the ER gene (A/T-rich)

sequence. It was found that the ability of 12a-d to arrest PCR

of the c-myc (IC₅₀) 2-6 µM) and HIV (IC₅₀) 0.8-2.0 µM)

gene targets was greater than for distamycin A (25 µM and 50

µM for c-myc and HIV, respectively), suggesting that the

presence of the PBD might favor a shift towards G/C recogni-

tion. Interestingly, for the ER gene, 12a,b were similar in activity

(IC₅₀) 3.0 µM and 2.0 µM, respectively) to distamycin (IC₅₀

) 5µM), whereas 12c-d were marginally more active (0.8 µM

and 0.2 µM, respectively) suggesting greater A/T-selectivity.

Analysis of arrest sites for ER PCR suggested that 12a arrests

at 5′-AGTTTAAA-3′, whereas 12b-d cause arrest at the same

site but also at 5′-CATATATGTGTG-3′. Comparative foot-

printing experiments for 12a-d suggested that changes in the

number of pyrrole rings did not produce significant changes in

sequence recognition; however, it was noted that the footprints

generated by 12d were larger than that generated by distamycin.

Using a PCR-based dialysis experiment, it was demonstrated

that hybrids 12a-d exhibit different DNA-binding character-

istics compared to either 7 (distamycin A) or the PBD (5). In

addition, a direct relationship was found between the number

of pyrrole rings present in the hybrids and the stability of the

DNA-ligand complexes. Confirming the previous studies of

Baraldi,¹⁵Gambari and co-workers reported¹⁶the effects of

12a-d on the interaction of purified NF-κB and [³²P]-labeled

oligomers that mimic the NF-κB HIV-1 LTR binding sites using

both gel retardation (EMSA) and filter binding assays. The

results showed that conjugates 12a-d were effective in inhibit-

ing NF-κB p52/ NF-κB DNA interactions by the EMSA assay

although only 12b-d were active according to the filter assay.

Similarly, conjugates 12c-d (but not 12a) were shown to

efficiently inhibit HIV-1 LTR-driven transcription in vitro

whereas the PBD fragment 5 alone could not. Baraldi and

co-workers¹⁷also reported that 12c inhibits binding of the

transcription factor Sp1, a protein important for the control

of transcription of cellular and viral genes, to its cognate DNA

sequence. Nuclear proteins were isolated from K562 cells and

immobilized on a filter after electrophoresis. The ability of

12c to inhibit the binding of [³²P]-labeled Sp1 oligomer to

the filter was then studied. Although the PBD fragment (5) or

distamycin A failed to inhibit binding at concentrations of

up to 50 µM, conjugate 12c completely blocked the Sp1/DNA

binding interaction at 10 µM, a result which was confirmed

by gel shift experiments. Gambari and co-workers also re-

ported¹⁸IC₅₀values for 12a-d in HL3T1 cells (72 h),

confirming earlier observed trends of greater cytotoxicity with

increasing numbers of attached pyrrole units. Interestingly, they

also found that 12a-d bind to TAR-RNA (particularly the

structured TAR-RNA of HIV-1) and can inhibit TAR/protein

interaction.

Finally, in an effort to enhance water solubility, Lown and

co-workers more recently reported¹⁴a set of sixteen PBD-

heterocycle conjugates (13a-h) containing various numbers of

pyrrole and imidazole units but with ring N-glycosylated

heterocycles. In eight of these molecules the hydroxyl moieties

of the glycosyl units were fully acetylated (R ) Ac), and in the

other eight all acetyl groups were absent (R ) H). The acetylated

and deacetylated analogues are directly comparable in structure

to 9a-c and 10a-c but contain either one or two glycosyl units

per molecule. Data from the NCI panel indicate that all

molecules in the series 13a-h, whether glycosylated or not,

are significantly less cytotoxic than 9b,c (n ) 2 or 3). Also,

although as a group, 13a-h (R ) H) are designed to be more

water-soluble than their acetylated counterparts 13a-h (R )

Ac), the average IC₅₀values show only a marginal improvement

due to just one compound 13h (R ) H). This conjugate shows

a marked improvement compared to its acetylated equivalent

(i.e., mean LC₅₀) 0.59 versus 93.3 µM for 13h [R ) H] and

13h [R ) Ac], respectively), although both compounds had IC₅₀

values of >100 µM in K562 cells.

Limited DNA-binding data have been reported for the

conjugates described above. For example, using a polymerase

chain reaction (PCR) assay against the Ha-ras oncogene

(G/C-rich) or estrogen receptor (ER) (A/T-rich) sequences,

5444 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

Figure 2. Structures of PBD-polypyrrole conjugates previously reported by Baraldi and Lown.

To further explore this class of molecules, we set out to extend

the number of pyrroles attached to a PBD via the C8-position

to six heterocyclic units and, as part of this process, to use

molecular modeling to optimize the linkage between the PBD

and polyheterocycle chain. Furthermore, we sought to explore

a different tail (methyl ester) for the conjugates in contrast to

the earlier reported 3-(dimethylamino)propyl or amidine termini.

This has allowed us to show that the four-carbon link between

the PBD and heterocycle tail is preferred as the conjugates are

maintained in an isohelical structure more suitable for accom-

modation in the minor groove of DNA. We have used thermal

denaturation, DNA footprinting, and in vitro transcription

inhibition methodologies to evaluate both DNA-reactivity and

sequence-selectivity and have also determined nuclear/cellular

penetration and cytotoxicity in a number of human tumor cell

lines. The results have revealed a surprisingly large synergistic

effect upon joining the PBD and methyl ester-terminated

polyheterocycle units through a four-carbon linker in terms of

both the rate and extent of DNA binding and their in vitro

cytotoxic potency.

appropriately for interaction, while other favorable and poten-

tially sequence-reading interactions were made by the remainder

of the molecule. However, extensive manual docking investiga-

tions made it clear that the limited flexibility of the three-carbon

linker between the PBD and first heterocycle made it difficult

to obtain a conformation of the conjugate that fully satisfied

these requirements. In particular, conformations in which the

three N-methylpyrrole rings fitted neatly and isohelically within

the minor groove always resulted in the PBD unit rotating

partially out of the groove, so that the six-membered A-ring of

the PBD made poor contacts with the groove base. Conversely,

if optimal interaction of the PBD moiety within the minor

groove was enforced, the first N-methypyrrole ring ‘bulged’ out

of the groove. One of the best manually docked conformations

was subjected to a molecular dynamics (MD)-based relaxation

and conformational sampling process in order to see if this could

be improved. For this we used a Generalized Born (GB)

solvation model and restrained the DNA close to a canonical

B-form helix so that the ‘fitness’ of the ligand for the DNA

was the key factor, rather than any plasticity in the DNA itself.

Use of the GB model allowed a reasonable sampling of

conformational space by the ligand within a limited simulation

time frame, due to the absence of solvent damping effects. The

PBD portion of the ligand remained suitably poised for

interaction between the imine center and the guanine N2, but

the overall orientation of 38 in the minor groove was still poor

(Figure 3A).

Results and Discussion

Molecular Modeling. Before embarking on the synthesis

program, initial molecular modeling studies were carried out

to determine the optimum length for the linker joining the C8-

position of the PBD to the amino group of the first heterocycle.

The choices were a three-carbon link as used by Baraldi and

co-workers (e.g. 12, n ) 1-4 in Figure 2) and as featured in

38, or a four-carbon link as used by Lown and colleagues (e.g.,

9-11 and 13a-h) and as adopted for 50a-f. Initial studies

focused on the interaction of 38 within the DNA minor groove.

Since covalent binding (“fixation”) of the PBD unit to guanine

is essentially irreversible at physiological pH, sequence-selective

positioning of the ligand must occur prior to this event.

Therefore, we sought to produce a model for the complex in

which the imine functionality of the PBD was positioned

This led us to investigate the effect of adding an additional

methylene unit to the linker between the PBD and the poly-

pyrrole sections of the ligand (i.e., 50c). Manual docking of

50c into the DNA minor groove was performed as before, and

it was immediately evident that a much more isohelical ligand

conformation was readily achievable. The docked conformation

was subjected to the same MD analysis as before. Again,

through dynamics the ligand explored a reasonable region of

conformational space, but remained suitably placed for the

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5445

Figure 3. Time-averaged structures of 38 (A) and 50c (B) bound to the 17-mer DNA duplex 5′-TTTTGCAAAAAGCTTCA-3′ (guanine adjacent

to the PBD C11-position is underlined). In each case, sections of the molecular surface of the DNA less than 5 Å from the ligand are colored red

to emphasize the superior fit of 50c (B) in the minor groove compared to 38 (A).

Scheme 1^a

^a(a) EDCI, DMAP, CH₂Cl₂, 18 h, 48-94%; (b) NaOH, H₂O, MeOH, 74-100%; (c) 4M HCl in dioxane, 30 min; (d) EDCI, DMAP, HCO₂H, CH₂Cl₂,

18 h, 60%; (e) EDCI, DMAP, 4-(dimethylamino)butyric acid, CH₂Cl₂, 96 h, 81%. Compounds 18 and 21 were formed in situ and used directly in the next

steps.^22-24

Scheme 2^a

alkylation reaction and with a highly isohelical geometry (Figure

3B). The apparent superiority of 50c was confirmed by the

calculation of relative binding affinities, using the MM-GBSA

method.¹⁹Binding energies were estimated to be -14 ( 3 kcal

mol^-1and -26 ( 6 kcal mol^-1for 38 and 50c, respectively.

This method does not represent a ‘state-of-the-art’ attempt

to predict the structural and energetic features of the ligand-

DNA recognition process, as the simulations were short, the

DNA was restrained, and an approximate solvation model was

used. Nevertheless, these calculations do provide a good

qualitative indication that a four-carbon linker should be superior

in terms of binding behavior. On this basis, the synthesis of the

50a-f series was planned in conjunction with the appropriate

controls 6, 22, 23, and 56 for comparison.

Synthesis. The pyrrole amino ester (14) and Boc-protected

^a(a) EDCI, DMAP, CH₂Cl₂, 48 h, 54-90%; (b) 4M HCl in dioxane, 30

pyrrole amino acid (15) prepared as described previously²⁰were

min. Compounds 25, 27, and 29 were formed in situ and used directly in

coupled using a solution-phase method (EDCI and DMAP) so

that the reaction could be easily monitored, with workup

involving simple solvent extraction to furnish the product in

sufficient yield and purity to use in subsequent steps²¹(Scheme

1). The resulting N-Boc dipyrrole (16) was deprotected to give

18^22-24which was followed by a second coupling reaction with

15 to give the Boc-protected trimer pyrrole (19). Saponification

or Boc-deprotection of esters 16 and 19 provided the corre-

sponding dimer and trimer acids (17 and 20) or the amino esters

(18 or 21), respectively, that were used to make the higher

homologues in the series. Reaction of the amino (18) and acid

the next steps.^22-24

(17) dimers afforded the tetrapyrrole fragment 24 which could

be Boc-deprotected to give 25^22-24(Scheme 2). Similarly, the

Boc-deprotected trimer 21^22-24could be reacted with either the

acid dimer (17) or trimer (20) to give the Boc-protected

pentapyrrole (26) or hexapyrrole (28) molecules. These could

be Boc-deprotected to give 27 or 29,^22-24respectively. It should

be noted that 18, 21, 25, 27, and 29 were prepared in situ and

used directly in the next steps. The control molecules 23 and

5446 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

Scheme 3^a

^a(a) PhCH₂OH, cat. 4-TsOH, toluene, 3.5 h, 56%; (b) (i) (COCl)₂, DMF, CH₂Cl₂, 18 h; (ii) (2S)-(+)-pyrrolidinemethanol, Et₃N, -30 °C to RT, then

overnight, 100%; (c) SnCl₂‚2H₂O, MeOH, reflux, 6 h; (d) (Boc)₂O, THF, reflux, 18 h; (e) pyridinium dichromate, CH₂Cl_2,4 Å sieves, 4 h, 27% (three steps);

(f) 10% Pd/C, H₂(16 psi), EtOH, 100%.

Scheme 4^a

in MeOH led to racemization at the C11a position, a phenom-

enon first noted by Tercel and co-workers.²⁹This is thought to

arise from ring opening, enolization, and subsequent ring closure

under the basic conditions of the reaction which results in loss

of stereochemistry at C11a. To prevent this, and also to

overcome the problem of competing ester formation during

coupling reactions, a number of protecting group strategies were

investigated for the C11-OH group. While protection with

silicon-based protecting groups (i.e. TBDMS or TIPS) or acetate

could be achieved, these groups were not stable to ester

saponification and also led to racemization. Methylation at C11-

OH could be achieved using MeI and Ag₂O; however, the

AgOH byproduct induced partial (i.e., ∼10%) loss of C11a-

stereochemistry as observed by chiral HPLC. Also, the methyl

group was resistant to the ester hydrolysis step, and no further

racemization was seen during this process. However, the most

effective protecting group regarding the preservation of C11a

stereochemistry was found to be THP, which was introduced

easily using DHP and a catalytic amount of 4-toluenesulfonic

acid to give 47 in quantitative yield. Although this product was

a mixture of diastereomers due to the mixed stereochemistry at

the pyran C2-position of the THP protecting group, ester

hydrolysis gave the desired acid (48) in chirally pure form at

C11a. This was confirmed by re-esterification followed by THP

removal to give material that was equivalent by chiral HPLC

to compound 46. Treatment of the methyl ester 47 with Pd-

(Ph₃P)₄and pyrrolidine removed the Alloc and C11-O-THP

protecting groups to afford the PBD 6 in 98% yield.

^a(a) EDCI, DMAP, CH₂Cl₂, 18 h, 65%; (b) 95% aq. TFA, -10°C, 3 h,

96%.

22, chosen to represent the pyrrole portions of the conjugates

with and without a four-carbon linker, were prepared by reacting

the tripyrrole amine (21) with either formic acid or 4-(dimethyl-

amino)butyric acid, respectively²¹(Scheme 1).

The Boc-PBD C8-propanoic acid (36) was synthesized using

a modified Fukuyama approach (Scheme 3).²⁵The side-chain

carboxylic acid of 30²⁶was selectively esterified with PhCH₂-

OH under acidic conditions to give 31, and this was coupled to

(S)-pyrrolidine methanol at -30 °C to furnish the nitro alcohol

32. Reduction of the nitro group was followed by Boc protection

of the resulting amine (33) to give 34. Treatment of the primary

alcohol with pyridinium dichromate effected oxidation and

spontaneous ring closure to provide the N10-Boc-protected PBD

35 in 27% yield over three steps. Removal of the benzyl group

from the C8 side-chain by catalytic hydrogenation gave the C8-

propanoic acid PBD (36)²⁷in quantitative yield. The PBD-

tripyrrole conjugate with the three-carbon linker (38) was

prepared by coupling the tripyrrole amino ester fragment (21),

prepared in situ from 19, to the PBD capping unit (36) (Scheme

4). The resulting conjugate intermediate (37) was Boc-depro-

tected to give the imine-containing PBD conjugate 38.

The set of conjugates 50a-f with each containing the four-

carbon linker required the synthesis of a different PBD capping

unit (48) as shown in Scheme 5. Vanillin (39) was initially

coupled to methyl 4-bromobutyrate to give the ester aldehyde

40.²⁸Subsequent nitration (41²⁸), oxidation (42), and coupling

to (S)-pyrrolidine methanol gave the key nitro alcohol 43, which

was subsequently reduced (44) and N-Alloc-protected (45).

Oxidative cyclization under Swern conditions gave the cyclized

N10-Alloc-protected PBD ring system (46²⁹). At this stage,

hydrolysis of the methyl ester was required to provide the free

acid capping unit. However, saponification with aqueous NaOH

The C11-O-THP PBD acid capping unit 48 was coupled to

the six individual amino acid pyrrole oligomers 14, 18, 21, 25,

27, and 29 to generate conjugates 49a-f (Scheme 6). Treatment

with Pd(Ph₃P)₄and pyrrolidine resulted in concerted removal

of the N10-Alloc and C11-O-THP protecting groups to give

the pure target PBD imine conjugates 50a-f in 50-95% yield

after purification by chromatography.

The non-imine-containing PBD-tripyrrole dilactam conjugate

56 was synthesized by a similar route (Scheme 7). The

benzylated nitro vanillic acid 51 was coupled to L-proline methyl

ester to give 52, which cyclized to dilactam 53 in 88% yield

after hydrogenation over 10% Pd/C in EtOH. The C8-butanoic

acid linker was installed by reaction with benzyl 4-bromobu-

tyrate/K₂CO₃to give 54, which was subsequently debenzylated

by hydrogenation to give the free acid (55). Reaction of this

with the amino ester tripyrrole fragment 21 afforded the

tripyrrole PBD dilactam 56.

Thermal Denaturation Studies. The relative reactivity and

affinity of the hybrid molecules toward double-stranded DNA

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5447

Scheme 5^a

^a(a) Br(CH₂)₃CO₂CH₃, K₂CO₃, DMF, 16 h, 85%; (b) 70% HNO₃, Ac₂O, 2.5 h; (c) KMnO_4,acetone, H₂O, 1 h, 50% (two steps); (d) (i) (COCl)₂, DMF,

CH₂Cl₂, 18 h; (ii) (2S)-(+)-pyrrolidinemethanol, Et₃N, -30 °C to RT, then overnight, 100%; (e) 10% Pd/C, H₂(45 psi)_,EtOH; (f) Alloc-Cl, CH₂Cl₂, 18 h,

used without further purification; (g) DMSO, (COCl)₂, Et₃N, -40 °C, 62% (three steps); (h) DHP, cat. 4-TsOH, EtOH, 2 h, 100%; (i) NaOH, H₂O, 15 min,

98%; (j) Pd(PPh₃)₄, pyrrolidine, CH₂Cl₂, 1 h, 98%.

Scheme 6^a

markedly increased ∆T_mvalues observed upon attaching

between one to six pyrrole units (i.e., 50a f 50f) suggests that

these heterocycles are also interacting in the minor groove and

further enhancing DNA stabilization. Also the progressive

increases in ∆T_mfor each conjugate upon incubation clearly

demonstrates that the pyrrole units do not interfere with the

covalent DNA-binding ability of the PBD unit.

The second type of control molecules were the tripyrrole

analogues 23 and 22, comprising the tripyrrole unit of 38 and

50c but with either 4-(dimethylamino)butyryl or formyl residues

added to the N-terminus. These compounds, designed to gauge

the DNA-binding efficiency of the polypyrrole fragment alone

with or without a linker, increased the melting temperature of

DNA by 5.5 and 4.3 °C, respectively (with no incubation). No

significant increases in ∆T_mwere seen after incubation as would

be expected for a reversible, noncovalent mode of binding. The

third type of control molecule, 56, is a modified version of 50c

(i.e., a PBD conjugate with three pyrrole units) where the N10-

C11 imine moiety is replaced with an inert amide (i.e., a PBD

dilactam³⁹) that cannot interact with DNA in a covalent manner.

The time-independent behavior of ∆T_mfor this dilactam upon

incubation indicates that it binds reversibly without a slow

secondary covalent component to its binding profile, as would

be expected for an inactivated PBD. We have previously

reported analogous behavior (i.e., ∆T_m) 2.9-3.3 °C) for simple

PBD dilactams that contain C-ring functionalization.³⁹The

ligand-induced ∆T_mshift for 56 is significantly smaller than

that seen for the tripyrrole control molecules 22 and 23,

suggesting that the “neutralized” PBD unit presented by the

dilactam may actually hinder accommodation of the oligopyrrole

tail in the minor groove.

^a(a) EDCI, DMAP, CH₂Cl₂, 24 h, 23-88%; (b) Pd[PPh₃]₄, pyrrolidine,

CH₂Cl₂, 2 h, 50-95%.

was assessed from their induced effects upon the melting

behavior (T_m) of calf thymus DNA. The thermal denaturation

data shown in Table 1 provide unequivocal evidence that the

PBD and polyheterocycle fragments contribute to overall effect

in a synergistic manner. First, it is important to note the greater

∆T_mshifts for 50c compared to 38, thus confirming our

modeling predictions for superiority with the four- rather than

the three-carbon linker. The ∆T_mvalues for 50c are >2-fold

greater than those for 38 (following 0, 4, or 18 h incubation at

37 °C), indicating that the longer homologue has a greater

stabilizing effect on the DNA duplex. Furthermore, the increased

∆T_mshifts seen for 50a-f and 38 upon longer incubation are

consistent with a time-dependent covalent mode of DNA

interaction (i.e. “fixation” or bonding), following the faster initial

component of reversible binding. This behavior is expected for

any molecule containing a PBD unit.^26,30-38

The ∆T_mvalues for the series 50a-f reveal an increase in

binding-induced DNA stabilization upon lengthening from one

to three pyrrole units (i.e., 50a f 50c), followed by a subsequent

decline to lower but still highly significant values as the side-

chain is further extended. Interestingly, the control molecule 6,

which is a PBD with a similarly saturated and unsubstituted

C-ring and a four-carbon linker but without pyrrole units, gives

modest ∆T_mvalues of only 0.2, 0.3, and 0.4 °C (after 0, 4, and

18 h). The progressive kinetic increase in ∆T_mupon incubation

indicates that this molecule interacts covalently with DNA,

although conferring a much reduced level of overall stabilization.

As expected, the ∆T_mshifts induced by 6 are similar to those

obtained for the naturally occurring PBD monomer 1 (DC-81,

Figure 1). The slightly higher effect may reflect an enhanced

stability for the DNA adduct due to accommodation of the ester-

terminated C8-linker in the host minor groove. However, the

Table 1 illustrates the pronounced synergistic effect on ∆T_m

of linking pyrrole units to the PBD moiety through the C8-

position of the A-ring. For example, subunits 6, 22, and 23 give

∆T_mvalues (after 0-18 h incubation) of 0.2-0.4, 4.2-4.3, and

5.5-5.6 °C, respectively, whereas induced ∆T_mshifts of 17.3,

19.6, and 21.2 °C (at 0, 4, and 18 h) are obtained when two of

these components are combined to give 50c. This remarkable

synergy was unexpected and is most likely due to either (i) an

increased length of spanned DNA and enhanced covalent

binding affinity, or (ii) the formation of “bis” adducts consisting

of two molecules binding in antiparallel mode at the site. To

put the ligand-induced ∆T_mshifts for the highest-affinity

compound 50c in context, the PBD dimer 4 (SJG-136, Figure

1), a sequence-selective covalent interstrand DNA cross-linking

agent, was recently reported³³to induce the greatest known

effect upon T_mto date. Interestingly, 50c stabilizes DNA toward

5448 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

Scheme 7^a

^a(a) (i) (COCl)₂, DMF, CH₂Cl₂; (ii) L-proline methyl ester hydrochloride, Et₃N, CH₂Cl₂, -30 °C, 97% (over two steps); (b) 10% Pd/C, H₂, EtOH, 88%;

(c) K₂CO₃, 4-bromobutanoic acid benzyl ester, DMF, overnight, 62%; (d) 10% Pd/C, H₂, EtOH, 100%; (e) EDCI, DMAP, CH₂Cl₂, 24 h, 6% (as a racemic

mixture after preparative HPLC).

thermal denaturation even more effectively than 4 but without

any requirement to form covalent interstrand DNA cross-links.

DNA Footprinting. The sequence selectivity of the six

PBD-pyrrole conjugates (50a-f) and the control molecules (6,

22, 23 and 56) was evaluated by standard DNase I footprinting

methods using the MS2 fragment which was designed by Fox

and co-workers to contain every possible combination of

tetranucleotides.⁴⁰Conjugates 50a-f were found to bind to the

MS2 fragment at several locations. However, despite differences

in binding affinity between each compound in the set, the

footprinting patterns were surprisingly similar. A DNase I

footprinting gel for 50c, the conjugate effecting the largest ∆T_m

shifts, on the MS2F (forward labeled) DNA fragment is shown

in Figure 4. Footprinting gels for the other conjugates 50a,b

and 50d-f for the MS2F fragment are shown in Supporting

Information (S1, A-D). An example of a footprint on a MS2R

(reversed labeled) fragment is shown (for 50e) in S1-C.

The vast majority of footprint sites are common features in

the binding profiles of all six conjugates, with only a small

number of sites being footprinted by a subset of the family.

Even more unexpectedly, no site is footprinted by only one

molecule (in fact, the fewest number of conjugates that bind at

any single site is four). The differential cleavage plot (Figure

5) provides footprinting profiles (color-coded for each conjugate)

which illustrate a striking degree of overlap. Although there is

no conspicuous change in footprinting patterns as the number

of pyrrole units in each conjugate increases, there are changes

in two other features, namely, the apparent binding affinity and

the width of the footprinted site. The binding affinity of each

molecule at a particular site was estimated (using the individual

DNase I footprint images) as the concentration of conjugate

providing 50% inhibition of DNase I-mediated cleavage at that

site (i.e., DNase IC₅₀). To simplify comparison between

molecules, one significant footprint site (5’-⁶²CAATACACA⁷⁰-

3’/3′-GTTATGTGT-5′) was selected for comparison (Band “C”

in Figures 4 and 5). Using this approach, 50d (four pyrroles)

appears to be the strongest binder with a DNase IC₅₀of around

30 nM. Conjugates 50c (three pyrroles) and 50e (five pyrroles)

follow closely with affinities in the region of 30-100 nM.

Conjugates 50b (2 pyrroles) and 50f (6 pyrroles) are poorer

binders but still exhibit nanomolar affinities in the region of

100-300 nM and 300 nM, respectively. Finally, 50a (one

pyrrole) is a weak footprinting molecule with a DNase IC₅₀

value of approximately 10 µM. In terms of a possible relation-

ship between binding site width and length of the PBD

conjugates, inspection of sites B and C in Figure 5 suggests

that, compared to the shortest conjugate 50a (red line), the longer

conjugates appear to have larger binding site widths, although

there is not a perfect correlation.

The binding characteristics of the 50a-f series at all thirteen

sites within the MS2 DNA fragment are provided in detail in

Figures S2A and S2B (Supporting Information). The same site

(“C”; 5’-⁶²CAATACACA⁷⁰-3’) and its close neighbor 5’-⁵⁰-

ATCCATATGCG⁶⁰-3’ (“B”) were also chosen and analyzed

in order to assess the effect of increasing the size of the

molecules on the length of the bound sequence. It appears that,

as additional pyrroles are added to the PBD, there is a

consequent rise in the number of base pairs within the associated

binding site. This positive correlation is suggestive of larger

tracts of DNA becoming bound by molecules of increasing

length, although it is not yet known whether the effect observed

at any given site results from the binding of single molecules

or more.

To assess the relative contributions of both the PBD subunit

and the polypyrrole fragment in the 50a-f series, the two control

molecules 6 and 23 were examined and compared to 50c (Figure

4). Interestingly, 6 does not produce any inhibition of DNase

I-mediated cleavage at concentrations of up to 100 µM (cf. 50c

binding at 30-100 nM). In contrast, polypyrrole 23 produces a

vivid footprinting pattern that shows significant overlap with

that of 50c apart from a few deviations (Figure 4, and detailed

analysis of binding sites in S3, Supporting Information). These

differences include the areas around positions 70-75 (5’-

ATGGCC-3’), 90-95 (5’-CTAGTC-3’), 178-185 (5’-GGTCT-

GTT-3’), and 197-202 (5’-GCCTCG-3’). One site (5’-¹⁸⁵

-

TTTGTC¹⁹⁰-3’) is bound strongly by 23 alone. It is also

interesting to note that the DNase IC₅₀value of 23 is 100-fold

higher (∼10 µM) than that of 50c (∼100 nM) at sites to which

both molecules bind, presumably reflecting the covalent interac-

tion of the PBD unit.

Given the failure of 6 to yield a useful footprint, anthramycin

itself (3 in Figure 1) was evaluated under similar conditions

(S4, Supporting Information). This molecule produced several

impressive footprints as previously documented in the literature,⁵

although few coincided with those of 50a-f (or 23). Nearly all

of the footprints produced by anthramycin correspond to the

well-established 5′-PuGPu-3′ binding motif.⁵The most probable

footprint sites included 5′-⁵¹TCC⁵³-3′ (denoted B′; 5′-⁵³GGA⁵¹-

3′ on the complementary labeled strand), 5′-¹²³TCT¹²⁵-3′ and

5′-¹²⁸CCT¹³⁰-3′ (K′ and L′; 5′-¹²⁵AGA¹²³-3′ and 5′-¹³⁰AGG¹²⁸

3′ on the lower strand) and 5′-²¹⁴TCT²¹⁶-3′ which is 5′-²¹⁶

-

AGA²¹⁴-3′ (U′) on the lower strand. Some footprints are visible

at less favored 5’-PyGPu-3′ sequences, e.g., 5′-⁷¹TGG⁷³-3′ (C′)

and 5′-⁸⁶TGC⁸⁸-3′ (E′). For the full list of 23 footprints see S4

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5449

Table 1. Cytotoxicity and Thermal Denaturation Data for the PBD-Pyrrole Conjugates 38 and 50a-f, the Control Molecules 6, 22, 23, and 56, and the

PDB Monomer 1 and Dimer 4

^aFor CT-DNA alone, ∆T_m) 67.44 ( 0.07 °C (mean from >80 experiments). All ∆T_mvalues are ( 0.1-0.2 °C. ^bFor a fixed 1:10 molar ratio of

[ligand]/[DNA], DNA concentration ) 50 µM (bp) (i.e., 100 µM in DNAp) and ligand concentration ) 5 µM, in aqueous sodium phosphate buffer [10 mM

sodium phosphate + 1 mM EDTA, pH 7.00 ( 0.01]. ^cValue of 21.5 °C determined after incubation for 72 h. ^dValue of 19.2 °C determined after incubation

for 72 h. ^eValues of 0.3, 0.5, 0.7, and 0.8 °C were determined for 1 at a higher 1:5 molar ratio after incubation for 0, 4, 18, and 72 h, respectively. ^fValues

of 10.2, 13.1, 15.1, and 15.5 °C were determined for DSB-120 at a higher 2:5 [ligand]/[DNA(bp)] molar ratio after incubation for 0, 4, 18, and 72 h,

respectively.^30,31,52^gValues of 21.3/25.7, 25.1/31.9, 28.3/33.6, and 29.4/34.4 °C were determined for 4 at higher 1:5/2:5 molar ratios after incubation for

0, 4, 18, and 72 h, respectively.³³NA ) Not available.

5450 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

Figure 4. DNase I footprinting gels showing the binding profiles of 6 (left), 50c (center), and 23 (right) on forward-labeled MS2 DNA. Differential

Cleavage Plots for both this fragment (between positions 30-130) and the reverse fragment (between positions 130-230; not indicated) are shown

in Figure 5. “G + A” represents a standard Maxim-Gilbert purine marker lane, and ‘Uncut’ represents DNA not treated with DNase I. The lettering

system and vertical bars refer to the footprinted regions on the forward fragment as identified in the legend to Figure 5.

(Supporting Information). However, not all 5′-PuGPu-3′ se-

quences were footprinted, suggesting that local sequence and

structure are crucial factors in determining the overall sequence-

selectivity of these agents. The comparison of control molecule

6 to anthramycin in these footprinting experiments indicates the

importance of C2-C3 unsaturation and C2-substitution in

enhancing the DNA-binding reactivity.^41,42

Conjugate 38 (the predecessor to 50c and containing only

three carbon atoms in the linkage between the PBD and

polypyrrole tail) was also assessed for DNA binding by DNase

I footprinting (S5, Supporting Information). The results confirm

the predictions from molecular modeling and the indication from

T_mstudies that 38 should have a poorer fit in the DNA minor

groove compared to 50c and thus a lower reactivity toward

DNA. The gel provided in S5 shows that for 38 footprints first

appear at ∼3 µM which is 30-fold higher than the concentration

at which footprints first appear for 50c (∼0.1µM; see Figure

4). Furthermore, differential cleavage analysis (Supporting

Information, S6) shows, as expected, that the actual pattern of

footprints produced by 38 is similar to 50c, although there are

a few differences such as K and L (footprints that are weaker

or lacking for 38). Also, comparing the images (Figures 5 and

S6) there are distinct differences at positions M and G at which

compound 38 does not footprint.

Taking all of these data into account (e.g., see legend to

Figure 5), the majority of these sites conform to a consensus

motif (5’-XGXW_z, z ) 3 ( 1; W ) A or T; X ) any base but

preferably a purine) that correlates well with the already

established individual sequence-selective preferences of both

the PBD and polypyrrole components. Eleven of the thirteen

sites conform to this consensus motif as follows (reactive GC

base pair underlined): 5′-²⁰TGATTACGA²⁸-3′, 5′-⁵⁰ATC-

CATATGCG⁶⁰-3′, 5′-⁶²CAATACACA⁷⁰-3′, 5′-⁷⁷ATTTCC-

A⁸³-3′, 5′-¹⁰⁹GGTTAA¹¹⁴-3′, 5′-¹²⁴CTATC¹²⁸-3′, 5′-¹³⁶AG-

CAATTAGGG¹⁴⁶-3′, 5′-¹⁵⁶TTATGTAAAGTACG¹⁶⁹-3′, 5′-¹⁸²TG-

TTTTGTCAT¹⁹²-3′, 5′-²⁰¹CGAATGCGGAT²¹¹-3′ and 5′-²³⁴ATG-

CAAGCTT²⁴³-3′), while the other two do not (5′-⁹⁰CTAGT-

CG⁹⁶-3′ and 5′-²¹⁶TAGAGT²²¹-3′).

In Vitro Transcription Assay. Conjugate ligands 50a-f

were subjected to an in vitro transcription assay⁴⁰to establish

whether any could inhibit transcription. As with the DNase I

footprinting results, each member produced similar T-stop

patterns. Results for 50c and 50a,b,d-f are shown in Figure 6

and S7 of Supporting Information, respectively. It should be

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5451

Figure 5. Differential Cleavage Plots showing the DNase I footprinting profiles of 50a-f for the forward (A-G) and reverse (H-M) strands.

Approximate positions of footprints are annotated with horizontal black bars: A: 5′-²⁰TGATTACGA²⁸-3′; B: 5′-⁵⁰ATCCATATGCG⁶⁰-3′; C: 5′-

⁶²CAATACACA⁷⁰-3′; D: 5′-⁷⁷ATTTCCA⁸³-3′; E: 5′-⁹⁰CTAGTCG⁹⁶-3′; F: 5′-¹⁰⁹GGTTAA¹¹⁴-3′; G: 5′-¹²⁴CTATC¹²⁸-3′; H: 5′-¹³⁶AGCAATTAGGG¹⁴⁶

-

3′; I: 5′-¹⁵⁶TTATGTAAAGTACG¹⁶⁹-3′; J: 5′-¹⁸²TGTTTTGTCAT¹⁹²-3′: K: 5′-²⁰¹CGAATGCGGAT²¹¹-3′; L: 5′-²¹⁶TAGAGT²²¹-3′; M: 5′-

²³⁴ATGCAAGCTT²⁴³-3′. Note that the concentrations of drugs used are different in the top and bottom panels. However, in every experiment the

concentration was above the approximate IC₅₀value for each compound.

noted that the intensities of the T-stops are proportional to their

radiolabeled cytidine content. All seven observed T-stops

localize within a few bases of prominent footprints produced

by the same compounds, and the correlation is highlighted in

Figure 7 where the T-Stops are depicted as asterisks. Those

with transcript lengths in nucleotides (sequence position in

brackets) of 55 (51), 64 (60), 95 (91), 111 (107) and 142 (138)

nucleotides are found on the 5’-side of the likely binding sites.

The two remaining T-stops, 87 (8) and 132 (128), are located

only one or two base pairs to the 3′-side of the nearest footprint.

In general, all compounds provide T-stops within the same

concentration range, producing 50% inhibition of full-length

transcript synthesis at around 5 µM (for comparison, SJG-136

requires a dose of 1-2 µM⁴⁰). However, the use of this particular

assay in determining, or even estimating, affinity constants has

not been validated and therefore only sequence data can be

analyzed. Although 6 did not produce any footprints at

concentrations up to 100 µM, its efficacy in the in Vitro

transcription assay was assessed as it is known that this assay

is more sensitive than footprinting.⁴⁰Figure 6 shows that control

PBD 6 is able to inhibit RNA production, although at concen-

trations approximately 10-fold higher than 50c (i.e. first T-stops

appearing at 1.5µM vs 0.15µM, respectively). By comparison,

3 (anthramycin) provides a robust blockade to viral RNA

polymerase with several intense T-stops appearing at concentra-

tions as low as 0.005 µM (S8, Supporting Information). In

addition to the T-stops observed in S8, there is evidence of much

weaker sites at 95, 100, 111, and 122 nucleotides. The most

intense T-stops (lengths 55, 87, 128, 132, 205, 220, and 231

corresponding to positions 51, 83, 124, 128, 201, 216, and 227,

respectively) are all located either 5’- to or within the closest

footprinted site (55 - B, 87 - D, 128 - K, 132 - L, 205 - R,

220 - U, 231 - W; lettered according to Figure 4). Interestingly,

these are all 5′-PuGPu-3′ sites with no bias toward the upper

or lower strand. The less-favored 5′-PyGPu-3′ sites do not

immediately appear to be associated with any T-stops.

It is interesting that 6 does not prevent full-length transcript

production to the same extent as either the 50a-f conjugates

or 3. For example, at 50 µM of 6 there is very little change in

the amount of 266-nt product, whereas complete inhibition

occurs at ∼5 µM anthramycin (Figure S8) and <15 µM 50c

(Figure 6). Also, 6 produces T-stops at identical sites to

anthramycin, but many others in addition. These extra T-stops

include a mixture of 5′-PuGPu-3’ sites (5′-¹⁰⁸AGG¹¹⁰-3′ and

5′-¹⁹²TCT¹⁹⁴-3′ [5′-AGA-3′ on the complementary strand]), two

5′-PuGPy-3′ (5′-⁷²GGC⁷⁴-3′ and 5′-⁹²AGT⁹⁴-3′) and what are

most likely to be 5′-PyGPy-3′ sites (5′-⁶⁶ACA⁶⁸-3′ [5′-TGT-3′

on the lower strand], 5′-⁸⁶TGC⁸⁸-3′ and 5′⁹⁵CGT⁹⁷-3′). The

finding that 50c is able to block transcription at a 10-fold lower

concentration than the PBD unit (6) alone (i.e. significant stops

first appear at 0.5 µM and 5 µM for 50c and 6, respectively),

5452 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

Figure 6. Inhibition of viral T7 RNA polymerase-catalyzed transcription by 6 (left), 50c (center), and 23 (right). The nearest likely binding sites

corresponding to robust T-stops are indicated to the left of each gel. The full-length fragment contains 266 nt, and the ‘marker’ lanes show the

length (in nucleotides) of the enzymatically created markers which are identical for all three gels. The presence of a 60 nucleotide marker and the

absence of a 56 nucleotide marker (as present in Figures S7A-E in Supporting Information) is due to the use of an alternative enzyme (see

Experimental Section of main text). Note that 23 fails to block transcription as it binds noncovalently to DNA.

Figure 7. Differential cleavage analysis of 50c (1 µM) on the central base portion (positions 40-150) of the MS2 fragment. Vertical bars represent

the extent of cleavage at each nucleotide compared to control. Positive and negative deflections indicate enhanced and reduced DNase I cleavage,

respectively. Negative deflections are indicative of regions protected by the bound drug. The positions of the T-stops derived from Figure 6 are

indicated by asterisks.

suggests that the polypyrrole part of the conjugate 50c is driving

the reaction with DNA.

one exception; the lack of a T-stop corresponding to the 132-nt

transcript which corresponds well with the lack of footprinting

around this site by 38. The range of concentrations over which

38 exerts its effect is similar to that of 50c, however, the use of

this assay to compare effective concentration ranges has not

been validated.

In Vitro Cytotoxicity. Conjugates 50a-f were all signifi-

cantly cytotoxic across the cell lines studied and, although there

is not a perfect correlation with the thermal denaturation data,

those compounds with the highest ∆T_mvalues (50c-e) have

the lowest average TGI and LC₅₀values across the 60 cells

As anticipated, given its noncovalent interaction with DNA,

23 is unable to produce sequence-selective T-stops at concentra-

tions of up to 500 µM. However, production of the full-length

transcript does reduce as the concentration of 23 increases from

0.5 µM to 500 µM which is thought to be due to concentration-

dependent binding of 23 to the T7 promoter sequence which

gradually inhibits the RNA polymerase from binding. In

accordance with the DNase I footprinting data, 38 produces

T-stops at identical positions to 50a-f (data not shown) with

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5453

are shown in Figure 8. Although high concentrations of

conjugates were used in these preliminary experiments, this did

not appear to be detrimental to the cells which, over a 24 h

period, remained attached while maintaining a similar morphol-

ogy to drug free controls.

Conclusions

The systematic studies of the PBD-pyrrole conjugates 50a-f

(together with the appropriate control molecules) using molec-

ular modeling, thermal denaturation, DNA footprinting, in vitro

transcription stop assays, confocal microscopy, and cytotoxicity

evaluations show that a four-carbon linkage is markedly superior

to a three-carbon one. This separation between the C8-position

of the PBD and the N-terminal of the first heterocycle was first

suggested by Lown to be preferable to the three-carbon link

used by Baraldi in terms of maximizing DNA-binding efficacy.

The superiority of the four-carbon link in improving the

isohelical fit of the molecules in the minor groove can now be

explained by molecular modeling. Furthermore, based on the

higher cytotoxicity values for members of the 50a-f series

compared to the Lown and Baraldi conjugates it appears that,

although high cytotoxicity is achievable with 3-(dimethylamino)-

propyl or amidino termini, a methyl ester terminus is preferable.

This may be due to greater cellular and/or nuclear penetration

by the ester derivatives. Further advantages of the methyl ester

compounds are that they can be isolated in the N10-C11 imine

form and do not appear to suffer from the insolubility problem

experienced with the 3-(dimethylamino)propyl-terminated con-

jugates, an issue that prompted the development of glycosylated

analogues (i.e., 13a-h in Figure 2) which had improved water

solubility but poorer IC₅₀values (in the 0.6 to 100 µM range)

along with inherent synthetic challenges. The fact that 38 and

50a-f can be isolated as N10-C11 imines appears to be due

solely to the ester moiety, as earlier conjugates differ mainly in

this aspect. The Lown conjugates all have a 3-(dimethylamino)-

propyl terminus which presumably raises polarity when proto-

nated, thus inhibiting cellular penetration and reducing cytotoxic

potency.

Figure 8. Nuclear uptake of conjugates following incubation with

MCF-7 cells for 24 h at 100 µM. A: Drug-free control, B: 50a, C:

50c, D: 50f.

lines of the NCI 60 panel. It is significant that, despite its high

molecular weight (i.e., 1079), 50f is still highly cytotoxic. The

activity appears to fall off slightly for 50b although it is

particularly potent in K562 (7.1 nM). 50a is the least potent

member of the series, although it still significantly active across

all cell lines. Conjugate 38 with the three-carbon linker is less

cytotoxic than any of the 50a-f series which was anticipated

from the modeling and DNA thermal denaturation data.

As anticipated from the thermal denaturation, footprinting and

in vitro transcription data, the control polypyrroles 22 and 23

were relatively noncytotoxic with IC₅₀values of >10 µM.

Similarly, the PBD control molecule 6 was significantly less

cytotoxic (e.g., IC₅₀) 0.526 µM in K562 cells) compared to

50a-f, as was the control PBD dilactam 56 (IC₅₀>10 µM in

K562). Taken together, these results further illustrate the synergy

of joining a PBD unit to the polypyrrole fragments whereby

the combination provides dramatically enhanced cytotoxicity

and DNA-binding affinity compared to the component units

alone. Another interesting finding is that 50a-f are significantly

more cytotoxic than the conjugates reported by Baraldi and

Lown, with the possible exception of 12d (n ) 4) which has a

reported IC₅₀of 0.04 µM in K562 cells.¹⁰One possible

conclusion from these studies is that the positively charged

3-(dimethylamino)propyl and amidino termini of the earlier

conjugates reduce their cellular penetration efficiency compared

to the neutral esters reported here.

The results of this study also highlight the remarkable synergy

of joining a PBD unit to a polypyrrole fragment in terms of

DNA reactivity, helix stabilization (T_mvalues), and cytotoxicity.

The DNA footprinting and in vitro transcription studies dem-

onstrate that conjugates 50a-f take on the properties of both

component fragments in terms of targeting a run of A/T base

pairs (through the polypyrrole fragment) adjacent to a Pu-G-

Pu site which becomes alkylated by the PBD. Unexpectedly,

all six members (50a-f) were found to bind, for the most part,

to similar sequences within the MS2 fragment. The majority of

these sites conformed to a consensus motif (5’-XGXW_z, z ) 3

( 1; W ) A or T; X ) any base but preferably a purine) that

correlated well with the individual established sequence-selective

preferences of the PBD and polypyrrole components. The data

indicate that a molecule containing three or four N-methylpyrrole

rings is optimal for DNA stabilization and that a four-carbon

rather than a three-carbon linker between the PBD and N-

terminus of the first polypyrrole is preferable. There also appears

to be a correlation between the overall length of the binding

site and the molecular length of the hybrid conjugate.

Cellular and Nuclear Penetration. The preliminary data

reported here indicate that, at the highest drug concentrations

used and an exposure time of 24 h, it is clear that all compounds

are taken up into MCF-7 cells. Cellular uptake was evident for

all conjugates at a concentration of 100 µM, with particularly

strong nuclear fluorescence observed for 50a-c. The fluores-

cence appeared more diffuse throughout the cell for the longer

conjugates 50d-f, suggesting poorer nuclear penetration.

Furthermore, the length of the molecules appeared to have an

effect on their rate of uptake, with 50a being taken up very

rapidly (<1 h) but longer members (50b and 50c) detectable

only after 3 h. Representative images from these experiments

The in vitro transcription studies show that all six conjugates

50a-f efficiently inhibit transcription at sites that mostly

correspond to the DNA footprints, and that the PBD subunit is

critical for realizing this. The addition of a polypyrrole fragment

to a PBD subunit significantly reduces the number of adducts

5454 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

eter or an AEI MS-902 spectrometer using the electrospray

ionization technique. Flash column chromatography refers to

medium-pressure silica gel (C60, 40-60 µm) preparative column

chromatography. Procedures for the synthesis of intermediates 16,

17, 19, 20, 24, 26, 28, 31, 32, 40-45, 52-54 are provided in

Supporting Information (S9). ¹³C NMR and IR data for compounds

6, 22, 35, 38, 46-48, and 50a-f are provided in Supporting

Information (S10). Compounds 18, 21, 25, 27, and 29 were formed

in situ and used directly in the next step.^22-24New compounds were

judged to be pure for testing by analysis of their NMR, HRMS,

and LCMS/HPLC data (summarized in Supporting Information,

S11).

formed on the MS2 fragment, thus indicating an increase in

sequence-selectivity compared to a simple PBD monomer.

However, the fact that 50c is able to block transcription at a

10-fold lower concentration than the PBD unit (6) alone suggests

that the polypyrrole part of the conjugate 50c is driving the

reaction with DNA.

Overall, the PBD-polypyrrole conjugates 50a-f show

characteristics highly desirable for gene-targeting purposes,

including the ability to be isolated in the N10-C11 imine form,

adequate water solubility and cellular/nuclear penetration char-

acteristics, sequence-selective DNA binding with surprisingly

high affinity, and the ability to robustly block RNA transcription

at well-defined sequences. Further work is underway to establish

the structure of the DNA adducts being formed, and the possible

consequences of hydrolysis of the methyl ester termini in vivo.

Versions of 50a-f containing alternative non-pyrrole hetero-

cyclic units and extended analogues of greater length are also

being evaluated. Results from these studies will be reported in

due course, together with in vivo data for selected compounds.

Methyl 4-({4-[(4-Formamino-1-methyl-1H-pyrrole-2-carbo-

nyl)amino]-1-methyl-1H-pyrrole-2-carbonyl}amino)-1-methyl-

1H-pyrrole-2-carboxylate (22). The Boc pyrrole trimer 19 (0.1

g, 0.20 mmol) in a dry round-bottomed flask was treated with 4M

HCl in dioxane (2 mL). The reaction mixture was stirred for 30

min during which time a precipitate formed. The solvent was then

removed and the residue dried in vacuo. The residue was dissolved

in anhydrous CH₂Cl₂(2 mL) then the formic acid (0.014 g, 0.41

mmol, 2.0 equiv) was added followed by EDCI (0.153 g, 0.80

mmol, 4.0 equiv) (effervescence observed) and DMAP (0.122 g,

1.00 mmol, 5.0 equiv). The reaction mixture was stirred for 18 h

then diluted with EtOAc (20 mL) and washed with 1M HCl solution

(2 × 15 mL) then saturated NaHCO₃solution (3 × 15 mL). The

organic layer was dried (MgSO₄) and concentrated in vacuo to give

an off white solid, 22 (0.051 g, 60%). ¹H NMR (d₆-acetone) δ 9.23

(1H, s, NCH₃), 9.20 (1H, s, NH), 9.04 (1H, s, NH), 8.20 (1H, d, J

) 1.6 Hz, CHO), 7.49 (1H, d, J ) 2.0 Hz, Py-H), 7.21 (1H, d, J

) 1.9 Hz, Py-H), 7.18 (1H, d, J ) 1.9 Hz, Py-H), 6.97 (1H, d, J

) 1.9 Hz, Py-H), 6.92 (1H, d, J ) 2.0 Hz, Py-H), 6.82 (1H, d, J

) 1.9 Hz, Py-H), 3.94 (3H, s, O/NCH₃), 3.91 (3H, s, O/NCH₃),

3.76 (3H, s, OCH₃); MS (ES⁺) m/z (relative intensity) 427 ([M +

H]^+•, 100%); Acc. Mass C₂₀H₂₂N₆O₅calc. 427.1725 found 427.1713.

Methyl 4-[(4-{[4-(4-Dimethylaminobutyrylamino)-1-methyl-

1H-pyrrole-2-carbonyl]amino}-1-methyl-1H-pyrrole-2-carbonyl)-

amino]-1-methyl-1H-pyrrole-2-carboxylate (23).²¹The Boc pyr-

role trimer 19 (0.1 g, 0.20 mmol) in a dry round-bottomed flask

was treated with 4M HCl in dioxane (2 mL). The reaction mixture

was stirred for 30 min during which time a precipitate formed. The

solvent was then removed and the residue dried in vacuo. The

residue was dissolved in anhydrous DMF (1 mL) and N,N-

dimethylaminobutyric acid (0.026 g, 0.20 mmol, 1.0 equiv) was

added followed by EDCI (0.096 g, 0.50 mmol, 2.5 equiv) then

DMAP (0.048 g, 0.40 mmol, 2.0 equiv). The reaction mixture was

stirred for 96 h then the solvent was removed in vacuo. The residue

was diluted with water (10 mL) and extracted with EtOAc (4 × 5

mL). The combined organic layers were dried (MgSO₄) and

concentrated in vacuo to give a solid which was suspended in Et₂O

(10 mL). Collection of the precipitate and vacuum-drying gave 23

Experimental Section

Molecular Modeling. Initial models for 38 and 50c were built

using InsightII (Biosym Inc., San Diego, CA). For parametrization

purposes the molecules were divided into fragments: the terminal

methoxy group, the pyrrole unit, and the PBD (including the linker).

Each fragment was capped with methylamino and/or acetyl groups

as appropriate and geometry-optimized at the HF/6-31G* level using

Gaussian98.⁴³All further molecular modeling was performed using

programs from the Amber7 suite⁴⁴running on Silicon Graphics

workstations. Partial charges for the fragments were calculated using

the RESP procedure.⁴⁵Missing force field parameters were

estimated from the optimized geometry or by analogy to similar

parameters in the parm99 database.⁴⁶Molecular mechanics energy

minimizations on the capped fragments were used to check that

the ab initio geometries were maintained. The molecular model

for the 17-mer DNA target sequence 5′-TTTTGCAAAAAGCT-

TCA-3′, found to be a favored site for binding in early footprinting

studies (results not shown) was built in standard B-form geometry

using the Amber program Nucgen (covalently modified guanine

underlined). Alternative DNA sequences were not investigated

because, although DNA helical parameters are sequence dependent,

the range of variation is small compared to what would be required

to qualitatively alter the conclusion regarding the relative isohelicity

of the two ligands. Thus a quantitative analysis of sequence-

dependent effects was not carried out.

Initial docking of 38 and 50c to the minor groove of the target

DNA was performed manually. The PBD unit was oriented

appropriately (i.e., A-ring toward the 3′-end of the covalently bound

strand⁵and the pyrrole units residing within the A₅tract) for attack

by the exocyclic C2-amino group of guanine-5 (underlined) on the

N10-C11 imine function of the PBD. The torsion angles in the

linker and between the pyrrole units were adjusted to obtain, as far

as possible, an isohelical conformation with the DNA. Energy

minimizations and molecular dynamics simulations on the complex

were performed using the Generalized Born solvation model.⁴⁷The

protocol consisted of: initial energy minimization of the ligand

alone; 10 ps MD on the complex gradually warming to 300 K with

position restraints of 10 kcal‚mol^-1‚Å^-2on the DNA; then three

more 10 ps MD simulations at 300 K with restraints on the DNA

reduced to 5, then 2.5, then 1 kcal‚mol^-1‚Å^-2. The final ‘production’

MD run was for 100 ps, maintaining the 1 kcal‚mol^-1‚Å^-2restraint

on DNA atoms. Energetic analysis of the MD run was performed

using the MM-GBSA module¹⁹of Amber.⁴⁸

1

as a solid (0.082 g, 81%). H NMR (d₆-DMSO) δ 9.91 (1H, s,

NH), 9.89 (1H, s NH), 9.81 (1H, s, NH), 7.47 (1H, d, J ) 1.9 Hz,

Py-H), 7.23 (1H, d, J ) 1.8 Hz, Py-H), 7.16 (1H, d, J ) 1.8 Hz,

Py-H), 7.06 (1H, d, J ) 1.8 Hz, Py-H), 6.91 (1H, d, J ) 1.9 Hz,

Py-H), 6.87 (1H, d, J ) 1.9 Hz, Py-H), 3.85 (3H, s, NCH₃), 3.84

(3H, s, NCH₃), 3.83 (3H, s, NCH₃), 3.75 (OCH₃), 2.33-2.24 (4H,

2 × t, J ) 7.3 Hz, side-chain H-2,4), 2.20 (6H, s, N[CH₃]₂), 1.72

(2H, p, J ) 7.3 Hz, side-chain H-3).

3-[5-Amino-4-((2S)-2-hydroxymethyl-pyrrolidine-1-carbonyl)-

2-methoxyphenoxy]propionic Acid Benzyl Ester (33). Nitro

compound 32 (59 g, 0.128 mol) was dissolved in MeOH (650 mL).

Tin(II) chloride dihydrate (130 g, 0.577 mol, 4.5 equiv) was added,

and the mixture was heated under reflux for 6 h. The solvent was

removed in vacuo, and the residue was dissolved in EtOAc (1 L)

in a 5 L flask, and saturated NaHCO₃solution (2 L) was added

carefully. The mixture was stirred for 2 h, and the resulting emulsion

(pH 8) was left to settle overnight then filtered through Celite. The

Celite pad was washed twice with EtOAc and the aqueous layer

extracted with further EtOAc. The organic layers were combined,

dried (MgSO₄), and concentrated in vacuo to give crude 33 as a

black tar (50 g). The product was used in the next step with no

Chemistry. General. Melting points (mp) were obtained using

a Gallenkamp melting point apparatus and are uncorrected. IR

spectra were measured using a Mattson 2020 Galaxy Series FT-IR

spectrometer. ¹H and ¹³C NMR spectra were acquired using a

Bru¨ker Avance 400 spectrometer at 400 and 100 MHz, respectively.

Mass spectra were recorded with a Micromass Platform Spectrom-

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5455

further purification. ¹H NMR (CDCl₃) δ 7.40-7.32 (5H, m, benzyl),

6.74 (1H, s, phenyl H-3), 6.28 (1H, s, phenyl H-6), 5.19 (2H, s,

benzyl CH₂), 4.33-4.23 (3H, m, side-chain H-1, pyrrolidine H-2),

3.73 (3H, s, OCH₃), 3.77-3.50 (4H, m, pyrrolidine H-5, CH₂-

OH), 2.91 (2H, t, J ) 6.2 Hz, side-chain H-2), 2.20-2.10 (1H, m,

pyrrolidine H-3), 1.88-1.60 (3H, m, pyrrolidine H-3,4).

residue was dissolved in anhydrous DMF (2.5 mL) then the Boc-

PBD acid 36 (0.139 g, 0.32 mmol, 1 equiv) was added followed

by EDCI (0.122 g, 0.64 mmol, 2.0 equiv) and DMAP (0.096 g,

0.80 mmol, 2.5 equiv). The reaction mixture was stirred for 18 h

then diluted with EtOAc (40 mL) and washed with 10% citric acid

solution (3 × 50 mL) then saturated NaHCO₃solution (3 × 50

mL). The organic layer was dried (MgSO₄) and concentrated in

3-[5-tert-Butoxycarbonylamino-4-((2S)-2-hydroxymethylpyr-

rolidine-1-carbonyl)-2-methoxy-phenoxy]propionic Acid Benzyl

Ester (34). Crude amine 33 (39 g, 52.3 mmol) was dissolved in

THF (650 mL), and di-tert-butyl dicarbonate (22.4 g, 0.103 mol)

was added in one portion. The mixture was heated under reflux

for 18 h, after which TLC (EtOAc) showed completion of the

reaction. The volatiles were removed in vacuo, and the residue was

dissolved in EtOAc (500 mL) and washed with water (2 × 200

mL) and brine (200 mL). After drying (MgSO₄) and concentration

in vacuo, crude 34 was obtained as a brown foam (53.2 g) slightly

contaminated with tert-butyl alcohol and solvent. This material was

used in the next step without further purification. ¹H NMR (CDCl₃)

δ 8.41 (1H, br s, NH), 7.82 (1H, s, phenyl H-6), 7.38-7.30 (5H,

m, benzyl), 6.81 (1H, s, phenyl H-3), 5.18 (2H, s, benzyl CH₂),

4.44-4.34 (3H, m, pyrrolidine H-2, side-chain H-1), 3.76 (3H, s,

OCH₃), 3.77-3.47 (4H, m, pyrrolidine H-5, CH₂-OH), 2.93 (2H,

t, J ) 6.2 Hz, side-chain H-2), 2.10-2.00 (1H, m, pyrrolidine H-3),

1.88-1.60 (3H, m, pyrrolidine H-3,4), 1.50 (9H, s, C[CH₃]₃).

(11S,11aS)-8-(2-Benzyloxycarbonylethoxy)-11-hydroxy-7-meth-

oxy-5-oxo-2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-10-carboxylic Acid tert-Butyl Ester (35). The crude

Boc amine 34 (50 g, 0.094 mol) was dissolved in anhydrous CH₂-

Cl₂(500 mL) at room temperature under N₂. 4 Å molecular sieves

(47 g) and pyridinium dichromate (43 g, 0.114 mol, 1.2 equiv) were

added, and the mixture was stirred for 4 h. Once the initial exotherm

had subsided, the mixture was sampled by TLC. Upon completion

of the reaction, EtOAc (1 L) was added with stirring. The black

mixture was filtered through Celite, and the residue was mixed with

warm EtOAc (3 × 200 mL) and refiltered. Evaporation of the

combined organic phase gave a black foam which was subjected

to column chromatography (EtOAc-petroleum ether, 70/30 v/v)

to give 35 as a pink-colored foam (16 g). Further fractions gave 2

g of additional product (27% yield, 3 steps). [R]²⁵_D) +335° (c )

0.212, CHCl₃); ¹H NMR (CDCl₃) δ 7.37-7.29 (5H, m, benzyl Ph-

H), 7.22 (1H, s, H-6), 6.66 (1H, s, H-9), 5.55 (1H, d, J ) 6.4 Hz,

H-11) 5.18 (2H, s, benzyl CH₂), 4.40-4.23 (2H, m, side-chain H-1),

3.88 (3H, s, OCH₃), 3.76-3.44 (4H, m, H-3,11a, OH), 2.93 (2H,

t, J ) 6.7 Hz, side-chain H-2), 2.14-1.98 (4H, m, H-1,2), 1.36

(9H, s, C[CH₃]₃); MS (ES⁺) m/z (relative intensity) 527 ([M +

H]^+•, 30%), 471 (50%), 409 (100%).

1

vacuo to give a foamy solid, 37 (0.170 g, 65%). H NMR (d₆-

DMSO) δ 10.07 (1H, s, NH), 9.96 (2H, s, NH), 7.49 (1H, d, J )

1.8 Hz, Py-H), 7.26 (1H, d, J ) 1.6 Hz, Py-H), 7.21 (1H, d, J )

1.6 Hz, Py-H), 7.07 (1H, d, J ) 1.6 Hz, Py-H), 7.06 (1H, s, H-9),

6.93 (1H, s, Py-H), 6.92 (1H, d, J ) 1.8 Hz, Py-H), 6.76 (1H, s,

H-6), 6.46 (1H, bs, OH), 5.47-5.41 (1H, m, H-11), 4.30-4.16 (2H,

m, side-chain H-1), 3.85 (6H, s, O/NCH₃), 3.78 (3H, s, O/NCH₃),

3.76 (3H, s, O/NCH₃), 3.74 (3H, s, O/NCH₃), 3.49-3.36 (1H, m,

H-11a), 3.28-3.18 (2H, m, H-3), 2.80-2.77 (2H, m, side-chain

H-2), 2.03-1.90 (4H, m, H-1,2), 1.19 (9H, s, C[CH₃]₃).

(11aS) Methyl 4-{[4-({4-[4-(7-Methoxy-5-oxo-2,3,5,11a-tetra-

hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)propio-

nylamino]-1-methyl-1H-pyrrole-2-carbonyl]amino)-1-methyl-1H-pyr-

role-2-carbonyl]amino}-1-methyl-1H-pyrrole-2-carboxylate (38).

The Boc-PBD conjugate 37 (0.053 g, 0.12 mmol) dissolved in a

solution of 95% aq. TFA (2.5 mL) cooled to -10 °C. The reaction

mixture was stirred at this temperature for 3 h then poured into a

flask containing ice (∼20 g). The mixture was adjusted to pH∼8

by careful addition of saturated NaHCO₃solution. The aqueous

phase was extracted with chloroform (3 × 20 mL). The combined

organic layers were dried (MgSO₄) and concentrated in vacuo to

give a pale yellow foam, 38 (0.044 g, 96%). [R]^26.3_D) +89° (c )

0.027, CHCl₃); ¹H NMR d₆-DMSO (d₆-DMSO) δ 9.99 (1H, s, NH),

9.91 (1H, s, NH), 9.90 (1H, s, NH), 7.79 (1H, d, J ) 4.4 Hz, H-11),

7.46 (1H, d, J ) 1.8 Hz, Py-H), 7.33 (1H, s, H-9), 7.23 (1H, d, J

) 1.8 Hz, Py-H), 7.20 (1H, d, J ) 1.7 Hz, Py-H), 7.18 (1H, d, J

) 1.7 Hz, Py-H), 7.05 (1H, d, J ) 1.8 Hz, Py-H), 6.90 (1H, d, J

) 1.7 Hz, Py-H), 6.88 (1H, s, H-6), 4.17-4.00 (2H, m, side-chain

H-1), 3.84 (3H, s, O/NCH₃), 3.84 (6H, s, O/NCH₃), 3.79 (3H, s,

O/NCH₃), 3.73 (3H, s, O/NCH₃), 3.69-3.57 (2H, m, H-3,11a),

3.45-3.35 (1H, m, H-3), 2.79-2.73 (2H, m, side-chain H-2), 2.29-

2.21 (2H, m, H-1), 2.01-1.92 (2H, m, H-2); MS (ES⁺) m/z (relative

intensity) 699 ([M + H]^+•, 100%); Acc. Mass C₃₅H₃₈N₈O₈calc.

699.2885 found 699.2872.

(11S,11aS) 11-Hydroxy-7-methoxy-8-(3-methoxycarbonylpro-

poxy)-5-oxo-2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-c][1,4]-

benzodiazepine-10-carboxylic Acid Allyl Ester (46).²⁹Oxalyl

chloride (17.87 g, 12.28 mL, 1.8 equiv) in anhydrous CH₂Cl₂(200

mL) was cooled to -40 °C, and a solution of anhydrous DMSO

(16.23 g, 16.07 mL, 3.6 equiv) in anhydrous CH₂Cl₂(200 mL)

was added dropwise over 2 h, maintaining the temperature below

-37 °C. A white suspension formed then redissolved. The crude

Alloc-protected amine 45 (26 g, 57.7 mmol) in anhydrous CH₂Cl₂

(450 mL) was added dropwise over 3 h maintaining the temperature

below -37 °C. The mixture was stirred at -40 °C for a further 1

h. A solution of DIPEA (32.1 g, 43.2 mL, 4.3 equiv) in anhydrous

CH₂Cl₂(100 mL) was added dropwise over 1 h, and the reaction

was allowed to warm to room temperature. The mixture was

extracted with a concentrated aqueous solution of citric acid. (pH

2-3 after extraction). The organic phase was washed with water

(2 × 400 mL) and brine (300 mL), then dried (MgSO₄). Solvent

removal in vacuo gave a paste; column chromatography (EtOAc-

petroleum ether, 70/30 v/v) gave 46 (17.0 g, 62%); [R]²⁶_D) +122°

(c ) 0.2, CHCl₃); ¹H NMR (CDCl₃) δ 7.23 (1H, s, H-9), 6.69 (1H,

s, H-6), 5.72-5.88 (1H, m, allyl H-2), 5.57-5.68 (1H, m, H-11),

5.15 (2H, d, J ) 12.9 Hz, allyl H-3), 4.69-4.43 (2H, m, allyl H-1),

4.00-4.13 (2H, m, side-chain H-1), 3.84-3.95 (4H, m, OCH₃, OH),

3.62-3.72 (4H, m, OCH₃, H-3), 3.58-3.45 (2H, m, H-3,11a), 2.53

(2H, t, J ) 7.2 Hz, side-chain H-3), 2.18-1.94 (6H, m, side-chain

H-2, H-1,2); MS (ES⁺) m/z 449 (M + H, 100%).

(11S,11aS)-8-(2-Carboxyethoxy)-11-hydroxy-7-methoxy-5-

oxo-2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-c][1,4]benzodiaze-

pine-10-carboxylic Acid tert-Butyl Ester (36).²⁷Benzyl ester 35

(9 g, 17.1 mmol) was dissolved in EtOH (120 mL), and a slurry of

10% Pd/C (0.5 g) in EtOH was added. The mixture was hydroge-

nated in a Parr apparatus at 16 psi until the H₂uptake ceased (2 h).

The reaction mixture was filtered through Celite and the residue

washed with EtOH. The solvent was removed in vacuo to give 36

as a white foam (quantitative yield). [R]^24.9_D) +104° (c ) 0.920,

1

CHCl₃) H NMR (d₆-DMSO) δ 7.24 (1H, s, H-6), 6.68 (1H, s,

H-9), 5.60 (1H, d, ;) 10.0 Hz, H-11), 4.31 (2H, t, J ) 7.5 Hz,

side-chain H-1), 3.90 (3H, s, OCH₃), 3.76-3.44 (3H, m, H-3,11a),

2.92 (2H, t, J ) 6.2 Hz, side-chain H-2), 2.14-1.98 (4H, m, H-1,2),

1

1.38 (9H, s, C[CH₃]₃). Based on the H NMR and the method

employed, the product was found to be identical to the literature

description.²⁷

(11S,11aS)-11-Hydroxy-7-methoxy-8-(3-{5-[5-(5-methoxycar-

bamoyl)-1-methyl-1H-pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyr-

rol-3-ylcarbamoyl]-1-methyl-1H-pyrrol-3-ylcarbamoyl}-ethoxy)-

5-oxo-2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-c][1,4]benzo-

diazepine-10-carboxylate tert-Butyl Ester (37). The Boc pyrrole

trimer 19 (0.16 g, 0.32 mmol) in a dry round-bottomed flask was

treated with 4M HCl in dioxane (2.5 mL). The reaction mixture

was stirred for 30 min during which time a precipitate formed. The

solvent was then removed and the residue dried in vacuo. The

(11S,11aS)-7-Methoxy-8-(3-methoxycarbonylpropoxy)-5-oxo-

11-(tetrahydropyran-2-yloxy)-2,3,11,11a-tetrahydro-1H,5H-pyr-

rolo[2,1-c][1,4]benzodiazepine-10-carboxylic Acid Allyl Ester

(47). A solution of dihydropyran (4.22 mL, 46.2 mmol) and

5456 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

4-toluenesulfonic acid (catalytic quantity, 0.020 g) in EtOAc (30

mL) was stirred for 10 min. Ester 46 (2.0 g, 4.62 mmol) was then

added in one portion, and the mixture was stirred for 2 h. The

solution was diluted with EtOAc (70 mL) and washed with saturated

aqueous NaHCO₃(50 mL), followed by brine (50 mL). The organic

layer was dried (MgSO₄), and the volatiles were removed under

vacuum. The oily residue of 47 (2.38 g, 100%) was pure by TLC

(EtOAc) and was used directly in the next step. ¹H NMR (CDCl₃)

mixture (4:5) of diastereoisomers δ 7.24-7.21 (1H, s × 2, H-9),

6.88-6.60 (1H, s × 2, H-6), 5.89-5.73 (2H, m allyl H-2, H-11),

5.15-5.04 (2H, m, allyl H-3), 4.96-4.81 (1H, m, pyran H-2),

4.68-4.35 (2H, m, allyl H-1), 4.12-3.98 (2H, m, side-chain H-1),

3.98-3.83 (4H, m, OCH_3,H11a), 3.74-3.63 (4H, m, OCH_3,H-3),

3.60-3.40 (3H, m, pyran H-6, H-3), 2.56-2.50 (2H, m, side-chain

H-3), 2.23-1.68 (6H, m side-chain H-2, H-1,2), 1.66-1.48 (6H,

m, pyran H-3,4,5); MS (ES⁺) m/z (relative intensity) 533 ([M +

H]^+., 100%).

mixture of diastereomers δ 9.09 (1H, s, NH), 7.39 (1H, d, J ) 2.0

Hz, Py-H), 7.14 (1H, s, H-6), 7.12 (1H, s, H-6), 6.96 (1H, s, H-9),

6.76 (1H, d, J ) 2.0 Hz, Py-H), 5.86-5.75 (2H, m, H-11, allyl

H-2), 5.13-4.84 (3H, m, pyran H-2, allyl H-3), 4.61-4.21 (2H,

m, allyl H-1), 4.06-3.88 (3H, m, side-chain H-1, pyran H-6), 3.87

(3H, s, O/NCH₃), 3.87 (3H, s, O/NCH₃), 3.86 (3H, s, O/NCH₃),

3.74 (3H, s, OCH₃), 3.53-3.44 (3H, m, H-11a, H-3), 2.55-2.45

(2H, m, side-chain H-3), 2.13-1.88 (6H, m, H-1,2, side-chain H-2),

1.70-1.39 (6H, m, pyran H-3,4,5).

(11S,11aS)-7-Methoxy-8-{3-[5-(5-methoxycarbonyl-1-methyl-

1H-pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-ylcarbamoyl]-

propoxy}-5-oxo-11-(tetrahydropyran-2-yloxy)-2,3,11,11a-tet-

rahydro-1H,5H-pyrrolo[2,1-c][1,4]benzodiazepine-10-

carboxylic Acid Allyl Ester (49b). The Boc-pyrrole dimer 16

(0.109 g, 0.29 mmol) was treated with 4M HCl in dioxane (2 mL).

The reaction mixture was stirred at room temperature for 30 min

during which time a precipitate formed. The solvent was removed

and the residue dried in vacuo. The residue was dissolved in

anhydrous CH₂Cl₂(2 mL), and Alloc-THP-PBD acid 48 (0.150 g,

0.29 mmol, 1 equiv) was added followed by EDCI (0.111 g, 0.58

mmol, 2 equiv) and DMAP (0.088 g, 0.72 mmol, 2.5 equiv). After

stirring for 24 h the solvent was removed in vacuo and the residue

diluted with EtOAc (25 mL) and washed with 1M HCl solution (3

× 10 mL) then saturated NaHCO₃solution (3 × 10 mL). The

organic fraction was dried (MgSO₄) and concentrated in vacuo, to

give a solid, (0.232 g) which was purified by column chromatog-

raphy (eluted with CHCl₃97%, MeOH 3%) to give a foam, 49b

(0.115 g, 51%). ¹H NMR (d₆-acetone) mixture of diastereomers δ

9.20 (2H, s, NH), 7.33 (1H, d, J ) 1.8 Hz, Py-H), 7.14 (1H, s,

H-6), 7.13 (1H, s, H-6), 6.94 (1H, s, H-9), 6.92-6.90 (1H, m, Py-

H), 6.90-6.89 (1H, m, Py-H), 6.81-6.80 (1H, m, Py-H), 5.86-

5.75 (2H, m, H-11, allyl H-2), 5.04-4.85 (3H, s, pyran H-2, allyl

H-3), 4.64-4.26 (2H, m, allyl H-1), 4.07-3.87 (4H, s, side-chain

H-1, pyran H-6), 3.86 (3H, s, O/NCH₃), 3.86 (3H, s, O/NCH₃),

3.85 (3H, s, O/NCH₃), 3.77 (1H, s, OCH₃), 3.59-3.46 (3H, m,

H-11a, H-3), 2.56-2.45 (2H, m, side-chain H-3), 2.15-1.92 (6H,

m, H-1,2, side-chain H-2), 1.71-1.40 (6H, m, pyran H-3,4,5).

(11S,11aS)-7-Methoxy-8-(3-{5-[5-(5-methoxycarbonyl-1-methyl-

1H-pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-ylcarbamoyl]-

1-methyl-1H-pyrrol-3-ylcarbamoyl}propoxy)-5-oxo-11-(tetrahy-

dropyran-2-yloxy)-2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-

c][1,4]benzodiazepine-10-carboxylic Acid Allyl Ester (49c). A

solution of Boc-pyrrole trimer 19 (0.144 g, 0.29 mmol) was treated

with 4M HCl in dioxane (2 mL). The reaction mixture was stirred

at room temperature for 30 min during which time a precipitate

formed. The solvent was removed and the residue dried in vacuo.

The residue was dissolved in anhydrous CH₂Cl₂(2 mL), and Alloc-

THP-PBD acid 48 (0.150 g, 0.29 mmol, 1 equiv) was added

followed by EDCI (0.111 g, 0.58 mmol, 2 equiv) and DMAP (0.088

g, 0.72 mmol, 2.5 equiv). After stirring for 24 h the solvent was

removed in vacuo and the residue diluted with EtOAc (25 mL)

and washed with 1M HCl solution (3 × 10 mL) then saturated

NaHCO₃solution (3 × 10 mL). The organic fraction was dried

(MgSO₄) and concentrated in vacuo, to give an off white foam,

49c (0.153 g, 59%). ¹H NMR (d₆-acetone) mixture of diastereomers

δ 9.28 (1H, s, NH), 9.19 (1H, s, NH), 9.02 (1H, s, NH), 7.50 (1H,

d, J ) 1.7 Hz, Py-H), 7.23 (1H, d, J ) 1.7 Hz, Py-H), 7.16 (1H,

d, J ) 1.7 Hz, Py-H), 7.15 (1H, s, H-6), 7.13 (1H, s, H-6), 6.99

(1H, d, J ) 1.7 Hz, Py-H), 6.92 (1H, d, J ) 1.9 Hz, Py-H), 6.91

(1H, s, H-9), 6.81 (1H, s, Py-H), 5.89-5.76 (2H, m, H-11, allyl

H-2), 5.20-4.84 (3H, m, pyran H-2, allyl H-3), 4.61-4.25 (2H,

m, allyl H-1), 4.11-3.95 (3H, m, side-chain H-1, pyran H-6), 3.94

(3H, s, O/NCH₃), 3.93 (3H, s, O/NCH₃), 3.91 (3H, s, O/NCH₃),

3.87 (3H, s, O/NCH₃), 3.76 (3H, s, OCH₃), 3.57-3.45 (3H, m,

H-3, H-11a), 2.54-2.45 (2H, m, side-chain H-3), 2.12-1.88 (6H,

m, H-1,2, side-chain H-2), 1.69-1.39 (6H, m, pyran H-3,4,5).

(11S,11aS)-7-Methoxy-8-[3-(5-{5-[5-(5-methoxycarbonyl-1-

methyl-1H-pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-ylcar-

bamoyl]-1-methyl-1H-pyrrol-3-ylcarbamoyl}-1-methyl-1H-pyrrol-

3-ylcarbamoyl)propoxy]-5-oxo-11-(tetrahydropyran-2-yloxy)-

2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-c][1,4]benzodiazepine-

(11S,11aS)-8-(3-Carboxypropoxy)-7-methoxy-5-oxo-11-(tet-

rahydropyran-2-yloxy)-2,3,11,11a-tetrahydro-1H,5H-pyrrolo-

[2,1-c][1,4]benzodiazepine-10-carboxylic Acid Allyl Ester (48).

Methyl ester 47 (2.2 g, 4.26 mmol) was dissolved in MeOH (30

mL). A solution of sodium hydroxide (0.34 g, 8.5 mmol) in water

(7 mL) was then added and the mixture was stirred at 70 °C for 15

min. The volatiles were then removed in vacuo, and water (20 mL)

was added. The aqueous solution was allowed to return to room

temperature, and the pH was adjusted to <4 by addition of a 5%

aqueous citric acid solution. The precipitate was extracted with

EtOAc (100 mL). The organic layer was washed with brine (30

mL) and dried (MgSO₄). The solvent was removed under vacuum,

then Et₂O (50 mL) was added to the residue and removed in vacuo.

1

Vacuum drying gave 48 (2.10 g, 98%) as a white foam. H NMR

(d₆-DMSO) mixture (4:5) of diastereoisomers δ 7.10 (1H, s × 2,

H-9), 6.90-6.84 (1H, s × 2, H-6), 5.84-5.68 (2H, m, allyl H-2,

H-11), 5.45-4.91 (3H, m, allyl H-3, pyran H-2), 4.72-4.30 (2H,

m, allyl H-1), 4.09-3.93 (2H, m, side-chain H-1), 3.91-3.75 (4H,

m, OCH₃), 3.60-3.44 (2H, m, H-3), 3.44-3.22 (2H, m, pyran H-6),

2.46-2.33 (2H, m, side-chain H-3), 2.20-1.76 (6H, m, side-chain

H-2, H-1,2), 1.76-1.31 (6H, m, pyran H-3,4,5); MS (ES⁺) m/z

(relative intensity) 519 ([M + H]^+•, 100%). This material was shown

to be optically pure at C11a by re-esterification (EDCI, HOBt, then

MeOH), THP removal (AcOH/THF/H₂O), and analysis by chiral

HPLC (data not shown).

(11aS) Methyl 4-(7-Methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-

pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanoate (6). A mix-

ture of 47 (0.086 g, 162 µmol), palladium tetrakis[triphenylphos-

phine] (0.002 g, 1.7 µmol), and pyrrolidine (14.7 µL, 176 µmol)

in anhydrous CH₂Cl₂(10 mL) was stirred at room temperature for

1 h. The solvent was removed in vacuo and the residue purified by

flash chromatography (eluted with EtOAc). The pure fractions were

combined to provide 6, (0.055 g, 98%). [R]²⁶_D) +482° (c ) 0.08,

CHCl₃); ¹H NMR (CDCl₃) δ 7.66 (1H, d, J ) 4.4 Hz, 1H, H-11),

7.51 (1H, s, H-9), 6.80 (1H, s, H-6), 4.21-4.02 (2H, m, side-chain

H-1), 3.93 (1H, s, OCH₃), 3.86-3.76 (1H, m, H-3), 3.76-3.64 (4H,

m, ester OCH₃, H11a), 3.63-3.51 (1H, m, H-3), 2.54 (2H, t, J )

7.3 Hz, side-chain H-3), 2.39-2.34 (2H, m, H-1), 2.24-2.13 (2H,

m, side-chain H-2), 2.12-1.95 (2H, m, H-2); MS (ES⁺) m/z (relative

intensity) 347 ([M + H]^+•, 100%); Acc. Mass C₁₈H₂₂N₂O₅calc.

347.1602 found 347.1612.

(11S,11aS)-7-Methoxy-8-[3-(5-methoxycarbonyl-1-methyl-

1H-pyrrol-3-ylcarbamoyl)propoxy]-5-oxo-11-(tetrahydropyran-

2-yloxy)-2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-c][1,4]-

benzodiazepine-10-carboxylic Acid Allyl Ester (49a). A solution

of pyrrole methyl ester 14 (0.055 g, 0.29 mmol) and Alloc-THP-

PBD acid 48 (0.150 g, 0.29 mmol, 1 equiv) dissolved in anhydrous

CH₂Cl₂(2 mL) was treated with EDCI (0.111 g, 0.58 mmol, 2

equiv) and DMAP (0.088 g, 0.72 mmol, 2.5 equiv). After stirring

for 24 h the solvent was removed in vacuo and the residue was

diluted with EtOAc (25 mL) and washed with 1M HCl solution (3

× 10 mL) then saturated NaHCO₃solution (3 × 10 mL). The

organic fraction was dried (MgSO₄) and concentrated in vacuo, to

give an off white foam, 49a (0.167 g, 88%). ¹H NMR (d₆-acetone)

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5457

10-carboxylic Acid Allyl Ester (49d). A solution of Boc-pyrrole

tetramer 24 (0.180 g, 0.29 mmol) was treated with 4M HCl in

dioxane (2 mL). The reaction mixture was stirred at room

temperature for 30 min during which time a precipitate formed.

The solvent was removed and the residue dried in vacuo. The

residue was dissolved in anhydrous CH₂Cl₂(2 mL), and Alloc-

THP-PBD acid 48 (0.150 g, 0.29 mmol, 1 equiv) was added

followed by EDCI (0.111 g, 0.58 mmol, 2 equiv) and DMAP (0.088

g, 0.72 mmol, 2.5 equiv). After stirring for 24 h the solvent was

removed in vacuo and the residue diluted with EtOAc (25 mL)

and washed with 1M HCl solution (3 × 10 mL) then saturated

NaHCO₃solution (3 × 10 mL). The organic fraction was dried

(MgSO₄) and concentrated in vacuo, to give an off white foam,

49d (0.068 g, 23%). ¹H NMR (d₆-acetone) mixture of diastereomers

δ 9.28 (1H, s, NH), 9.25 (1H, s, NH), 9.18 (1H, s, NH), 9.03 (1H,

s, NH), 7.50 (1H, d, J ) 1.9 Hz, Py-H), 7.23 (2H, d, J ) 1.4 Hz,

Py-H), 7.15 (1H, s, H-6), 7.14 (1H, s, H-6), 6.99 (1H, J ) 2.0 Hz,

Py-H), 6.96 (1H, s, H-9), 6.93 (1H, d, J ) 1.9 Hz, Py-H), 6.90

(1H, s, Py-H), 6.83 (1H, s, Py-H), 6.81 (1H, s, Py-H), 5.87-5.77

(2H, m, H-11, allyl H-2), 5.14-4.85 (3H, m, pyran H-2, allyl H-3),

4.62-4.22 (2H, m, allyl H-1), 4.09-3.95 (3H, m, side-chain H-1,

pyran H-6), 3.94-3.87 (15H, 5 × s, O/NCH₃), 3.74 (3H, s, OCH₃),

3.57-3.44 (3H, m, H-3,11a), 2.49 (2H, d, J ) 7.0 Hz, side-chain

H-3), 2.13-1.89 (6H, m, H-1,2, side-chain H-2), 1.64-1.39 (6H,

m, pyran H-3,4,5).

(11S,11aS)-7-Methoxy-8-{3-[5-(5-{5-[5-(5-methoxycarbonyl-

1-methyl-1H-pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-yl-

carbamoyl]-1-methyl-1H-pyrrol-3-ylcarbamoyl}-1-methyl-1H-

pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-ylcarbamoyl]-

propoxy}-5-oxo-11-(tetrahydropyran-2-yloxy)-2,3,11,11a-tetrahydro-

1H,5H-pyrrolo[2,1-c][1,4]benzodiazepine-10-carboxylic Acid Al-

lyl Ester (49e). A solution of Boc-pyrrole pentamer 26 (0.150 g,

0.20 mmol) was treated with 4M HCl in dioxane (2 mL). The

reaction mixture was stirred at room temperature for 30 min during

which time a precipitate formed. The solvent was removed and

the residue dried in vacuo. The residue was dissolved in anhydrous

CH₂Cl₂(2 mL) and Alloc-THP-PBD acid 48 (0.150 g, 0.2 mmol,

1 equiv) was added followed by EDCI (0.111 g, 0.40 mmol, 2

equiv) and DMAP (0.088 g, 0.50 mmol, 2.5 equiv). After stirring

for 24 h the solvent was removed in vacuo and the residue diluted

with EtOAc (25 mL) and washed with 1M HCl solution (3 × 10

mL) then saturated NaHCO₃solution (3 × 10 mL). The organic

fraction was dried (MgSO₄) and concentrated in vacuo, to give an

off white foam, 49e (0.164 g, 71%). ¹H NMR (d₆-acetone) mixture

of diastereomers δ 9.26-9.20 (5H, 5 × s, NH), 7.50 (1H, d, J )

1.6 Hz, Py-H), 7.23 (3H, d, J ) 1.7 Hz, Py-H), 7.15 (1H, s, H-6),

6.98-6.97 (2H, m, Py-H), 6.93 (2H, d, J ) 1.8 Hz, Py-H), 6.90

(1H, s, H-9), 6.84 (1H, d, J ) 2.0 Hz, Py-H), 6.80 (1H, d, J ) 2.0

Hz, Py-H), 5.89-5.77 (2H, m, H-11, allyl H-2), 5.14-4.86 (3H,

m, pyran H-2, allyl H-3), 4.60-4.21 (2H, m, allyl H-1), 4.10-

3.95 (3H, m, side-chain H-1, pyran H-6), 3.94-3.87 (18H, 6 × s,

O/NCH₃), 3.76 (3H, s, OCH₃), 3.54-3.43 (3H, m, H-3,11a), 2.54-

2.45 (2H, m, side-chain H-3), 2.13-1.89 (6H, m, H-1,2, side-chain

H-2), 1.68-1.38 (6H, m, pyran H-3,4,5).

NaHCO₃solution (3 × 10 mL). The organic fraction was dried

(MgSO₄) and concentrated in vacuo, to give an off white foam,

1

49f (0.174 g, 77%). H NMR (d₆-acetone, 500 MHz) mixture of

diastereomers δ 9.28-9.16 (6H, 6 × s, NH), 7.50 (1H, d, J ) 1.8

Hz, Py-H), 7.24 (3H, d, J ) 1.5 Hz, Py-H), 7.16 (1H, s, H-6), 7.14

(2H, s, H-6, Py-H), 6.99 (1H, d, J ) 1.7 Hz, Py-H), 6.96 (1H, s,

H-9), 6.93 (4H, d, J ) 1.9 Hz, Py-H), 6.83 (1H, d, J ) 2.3 Hz,

Py-H), 6.79 (1H, s, Py-H), 5.89-5.77 (2H, m, H-11, allyl H-2),

5.14-4.83 (3H, m, pyran H-2, allyl H-3), 4.62-4.22 (2H, m, allyl

H-3), 4.12-3.95 (3H, m, side-chain H-1, pyran H-6), 3.94-3.81

(21H, 7 × s, O/NCH₃), 3.75 (3H, s, OCH₃), 3.54-3.46 (3H, m,

H-3,11a), 2.54-2.46 (2H, m, side-chain H-3), 2.12-1.88 (6H, m,

H-1,2, side-chain H-2), 1.68-1.38 (6H, m, pyran H-3,4,5).

(11aS) Methyl 4-[4-(7-Methoxy-5-oxo-2,3,5,11a-tetrahydro-

5H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butyrylamino]-1-

methyl-1H-pyrrole-2-carboxylate (50a). A solution of Alloc-THP-

PBD conjugate 49a (0.157 g, 0.24 mmol) dissolved in anhydrous

CH₂Cl₂(2 mL) under a nitrogen atmosphere was treated with

pyrrolidine (22 µL, 0.26 mmol, 1.1 equiv) and then palladium

tetrakis[triphenylphosphine] (0.014 g, 0.012 mmol, 0.05 equiv). The

reaction mixture was stirred at room temperature for 2 h and the

product purified directly by column chromatography (eluted with

CHCl₃96%, MeOH 4%) to give the product as a glassy solid, 50a

1

(0.093 g, 83%). [R]^27.2) +351° (c ) 0.08, CHCl₃); H NMR

D

(d₆-DMSO) δ 9.94 (1H, s, NH), 7.83 (1H, d, J ) 4.4 Hz, H-11),

7.39 (1H, d, J ) 2.0 Hz, Py-H), 7.39 (1H, s, H-6), 6.88 (1H, s,

H-9), 6.76 (1H, d, J ) 2.0 Hz, Py-H), 4.17-4.08 (2H, m, H-1

side-chain H-1), 3.87 (3H, s, O/NCH₃), 3.86 (3H, s, O/NCH₃), 3.77

(3H, s, OCH₃), 3.76-3.65 (2H, m, H-3,11a), 3.49-3.39 (1H, m,

H-3), 2.49-2.45 (2H, m, side-chain H-3), 2.34-2.29 (2H, m, H-1),

2.13-2.05 (2H, m, side-chain H-2), 2.03-1.97 (2H, m, H-2); MS

(ES⁺) m/z (relative intensity) 469 ([M + H]^+•, 100%) Acc. Mass

C₂₄H₂₈N₄O₆calc. 469.2082 found 469.2085.

(11aS) Methyl 4-({4-[4-(7-Methoxy-5-oxo-2,3,5,11a-tetrahy-

dro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butyrylamino]-

1-methyl-1H-pyrrole-2-carbonyl}amino)-1-methyl-1H-pyrrole-

2-carbxylate (50b). A solution of Alloc-THP-PBD conjugate 49b

(0.093 g, 0.12 mmol) dissolved in anhydrous CH₂Cl₂(2 mL) under

a nitrogen atmosphere was treated with pyrrolidine (11 µL, 0.13

mmol, 1.1 equiv) and then palladium tetrakis[triphenylphosphine]

(0.007 g, 0.006 mmol, 0.05 equiv). The reaction mixture was stirred

at room temperature for 2 h and the product purified directly by

column chromatography (eluted with CHCl₃96%, MeOH 4%) to

give the product as a glassy solid, 50b (0.067 g, 95%). [R]^27.1

)

D

1

+348° (c ) 0.022, CHCl₃); H NMR (d₆-DMSO) δ 9.88 (1H, s,

NH), 7.78 (1H, d, J ) 4.3 Hz, H-11), 7.45 (1H, d, J ) 1.7 Hz,

Py-H), 7.34 (1H, s, H-6), 7.16 (1H, d, J ) 1.6 Hz, Py-H), 6.90

(1H, d, J ) 1.9 Hz, Py-H), 6.88 (1H, d, J ) 1.8 Hz, Py-H), 6.83

(1H, s, H-9), 4.10-3.97 (2H, m, side-chain H-1), 3.84 (6H, s,

O/NCH₃), 3.83 (3H, s, O/NCH₃), 3.74 (3H, s, OCH₃), 3.68-3.60

(2H, m, H-3,11a), 3.44-3.35 (1H, m, H-3), 2.45-2.42 (1H, m,

side-chain H-3), 2.27-2.20 (2H, m, H-1), 2.09-1.93 (4H, m, H-2,

side-chain H-2); MS (ES⁺) m/z (relative intensity) 591 ([M + H]^+•,

100%); Acc. Mass C₃₀H₃₄N₆O₇calc. 591.2562 found 591.2535.

(11aS) Methyl 4-{[4-({4-[4-(7-Methoxy-5-oxo-2,3,5,11a-tet-

rahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butyryl-

amino]-1-methyl-1H-pyrrole-2-carbonyl]amino)-1-methyl-1H-

pyrrole-2-carbonyl]amino}-1-methyl-1H-pyrrole-2-carboxy-

late (50c). A solution of Alloc-THP-PBD conjugate 49c (0.140 g,

0.16 mmol) dissolved in anhydrous CH₂Cl₂(2 mL) under a nitrogen

atmosphere was treated with pyrrolidine (15 µL, 0.17 mmol, 1.1

equiv) and then palladium tetrakis[triphenylphosphine] (0.009 g,

0.008 mmol, 0.05 equiv). The reaction mixture was stirred at room

temperature for 2 h and the product purified directly by column

chromatography (eluted with CHCl₃96%, MeOH 4%) to give the

product as a glassy solid, 50c (0.076 g, 68%). [R]^27.1_D) +185° (c

(11S,11aS)-7-Methoxy-8-(3-{5-[5-(5-{5-[5-(5-methoxycarbo-

nyl-1-methyl-1H-pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-

ylcarbamoyl]-1-methyl-1H-pyrrol-3-ylcarbamoyl}-1-methyl-

1H-pyrrol-3-ylcarbamoyl)-1-methyl-1H-pyrrol-3-ylcarbamoyl]-

1-methyl-1H-pyrrol-3-ylcarbamoyl}propoxy)-5-oxo-11-(tetra-

hydropyran-2-yloxy)-2,3,11,11a-tetrahydro-1H,5H-pyrrolo[2,1-c]-

[1,4]benzodiazepine-10-carboxylic Acid Allyl Ester (49f). A

solution of Boc-pyrrole hexamer 28 (0.155 g, 0.18 mmol) was

treated with 4M HCl in dioxane (2 mL). The reaction mixture was

stirred at room temperature for 30 min during which time a

precipitate formed. The solvent was removed and the residue dried

in vacuo. The residue was dissolved in anhydrous CH₂Cl₂(2 mL),

and Alloc-THP-PBD acid 48 (0.093 g, 0.18 mmol, 1 equiv) was

added followed by EDCI (0.068 g, 0.36 mmol, 2 equiv) and DMAP

(0.054 g, 0.45 mmol, 2.5 equiv). After stirring for 24 h the solvent

was removed in vacuo and the residue diluted with EtOAc (25 mL)

and washed with 1M HCl solution (3 × 10 mL) then saturated

1

) 0.077, CHCl₃); H NMR (d₆-DMSO) δ 9.92 (1H, s, NH), 9.90

(1H, s, NH), 9.88 (1H, s, NH), 7.78 (1H, d, J ) 4.4 Hz, H-11),

7.47 (1H, d, J ) 1.9 Hz, Py-H), 7.34 (1H, s, H-6), 7.24 (1H, d, J

) 1.7 Hz, Py-H), 7.17 (1H, d, J ) 1.7 Hz, Py-H), 7.06 (1H, d, J

) 1.8 Hz, Py-H), 6.91 (1H, d, J ) 1.9 Hz, Py-H), 6.89 (1H, d, J

5458 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

Wells et al.

give the product as a glassy solid, 50f (0.084 g, 57%). [R]^27.1

) 1.8 Hz, Py-H), 6.83 (1H, s, H-9), 4.14-4.05 (2H, m, side-chain

H-1), 3.85 (3H, s, O/NCH₃), 3.84 (3H, s, O/NCH₃), 3.84 (3H, s,

O/NCH₃), 3.83 (3H, s, O/NCH₃), 3.74 (3H, s, OCH₃), 3.67-3.61

(1H, m, H-3,11a), 3.49-3.32 (1H, m, H-3), 2.47-2.44 (2H, m,

side-chain H-3), 2.30-2.23 (2H, m, H-1), 2.05-1.95 (4H, m, H-2,

side-chain H-2); MS (ES⁺) m/z (relative intensity) 713 ([M + H]^+•,

100%) Acc. Mass C₃₆H₄₀N₈O₈calc. 713.3042 found 713.3022.

(11aS) Methyl 4-[(4-{[4-({4-[4-(7-Methoxy-5-oxo-2,3,5,11a-

tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)bu-

tyrylamino]-1-methyl-1H-pyrrole-2-carbonyl]amino)-1-methyl-

1H-pyrrole-2-carbonyl]amino}-1-methyl-1H-pyrrole-2-carbonyl)-

amino]-1-methyl-1H-pyrrole-2-carboxylate (50d). A solution of

Alloc-THP-PBD conjugate 49d (0.065 g, 0.06 mmol) dissolved in

anhydrous CH₂Cl₂(2 mL) under a nitrogen atmosphere was treated

with pyrrolidine (5 µL, 0.07 mmol, 1.1 equiv) and then palladium

tetrakis[triphenylphosphine] (0.004 g, 0.003 mmol, 0.05 equiv). The

reaction mixture was stirred at room temperature for 2 h and the

product purified directly by column chromatography (eluted with

)

D

1

+107° (c ) 0.084, CHCl₃); H NMR (d₆-DMSO) δ 9.96 (2H, s,

NH), 9.95 (1H, s, NH), 9.94 (1H, s, NH), 9.91 (1H, s, NH), 9.89

(1H, s, NH), 7.78 (1H, d, J ) 4.4 Hz, H-11), 7.35 (1H, s, H-6),

7.27-7.25 (4H, m, Py-H), 7.17 (1H, d, J ) 1.6 Hz, Py-H), 7.09

(2H, d, J ) 1.5 Hz, Py-H), 7.08 (2H, d, J ) 1.7 Hz, Py-H), 6.92

(1H, d, J ) 1.9 Hz, Py-H), 6.91 (1H, d, J ) 1.8 Hz, Py-H), 6.84

(1H, s, H-9), 4.14-4.05 (2H, m, side-chain H-1), 3.87-3.83 (21H,

7 × s, O/NCH₃), 3.75 (3H, s, OCH₃), 3.68-3.61 (2H, m, H-3,-

11a), 3.48-3.32 (1H, m, H-3), 2.46-2.43 (2H, m, side-chain H-3),

2.29-2.23 (2H, m, H-1), 2.06-1.94 (4H, m, H-2, side-chain H-2);

MS (ES⁺) m/z (relative intensity) 1079 ([M + H]^+•, 50%); Acc.

mass C₅₄H₅₈N₁₄O₁₁calc. 1079.4482 found 1079.4542.

(11aS)-4-(7-Methoxy-5,11-dioxo-2,3,4,10,11a-hexahydro-1H-

pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)butanoic acid (55).

The benzyl ester 54 (3.58 g, 8.17 mmol) was dissolved in EtOH

(40 mL), and a suspension of 10% palladium on charcoal (0.358

g) in EtOAc (5 mL) was added. The reaction mixture was agitated

under a hydrogen atmosphere (50 psi) for 2.5 h then filtered through

a Celite pad. The Celite was washed with EtOAc then the combined

filtrates were concentrated in vacuo, then dried in vacuo to give

the product as a white foam 55 (2.44 g, 100%). ¹H NMR

(d₆-DMSO) δ 11.96 (1H, s, COOH), 10.21 (1H, s, NH), 7.23 (1H,

s, H-6), 6.68 (1H, s, H-9), 4.06 (2H, d, J ) 7.3 Hz, side-chain

H-1), 4.03-3.94 (2H, m, H-11a), 3.78 (3H, s, OCH₃), 3.58-3.52

(1H, m, H-3), 3.48-3.42 (1H, m, H-3), 2.49-2.44 (1H, m, H-1),

2.39 (2H, t, J ) 7.2 Hz, side-chain H-3), 1.96 (2H, p, J ) 7.2 Hz,

side-chain H-2), 1.94-1.76 (3H, m, H-1,2).

CHCl₃96%, MeOH 4%) to give the product as a glassy solid, 50d

1

(0.029 g, 55%). [R]^26.5) +129° (c ) 0.031, CHCl₃); H NMR

D

(d₆-DMSO) δ 9.94 (1H, s, NH), 9.93 (1H, s, NH), 9.90 (1H, s,

NH), 9.88 (1H, s, NH), 7.78 (1H, d, J ) 4.4 Hz, H-11), 7.48 (1H,

d, J ) 1.3 Hz, Py-H), 7.35 (1H, s, H-6), 7.25 (2H, s, Py-H), 7.17

(1H, d, J ) 0.8 Hz, Py-H), 7.08 (1H, d, J ) 1.1 Hz, Py-H), 7.06

(1H, d, J ) 0.9 Hz, Py-H), 6.92 (1H, d, J ) 1.2 Hz, Py-H), 6.90

(1H, s, Py-H), 6.83 (1H, s, H-9), 4.14-4.05 (2H, m, side-chain

H-1), 3.86-3.83 (15H, 5 × s, O/NCH₃), 3.75 (3H, s, OCH₃), 3.68-

3.61 (2H, m, H-3,11a), 3.46-3.30 (1H, m, H-3), 2.47-2.43 (2H,

m, side-chain H-3), 2.26-2.18 (2H, m, H-1), 2.05-1.94 (4H, m,

H-2, side-chain H-2); MS (ES⁺) m/z (relative intensity) 835 ([M +

H]^+•, 100%); Acc. Mass C₄₂H₄₆N₁₀O₉calc. 835.3522 found

835.3497.

4-{[4-({4-[4-((11aS)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-

pyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione)-8-yloxy)butyryl-

amino]-1-methyl-1H-pyrrole-2-carbonyl}amino)-1-methyl-1H-

pyrrole-2-carbonyl]-amino}-1-methyl-1H-pyrrole-2-carboxylic

Acid Methyl Ester (56). A solution of Boc pyrrole trimer 19 (0.200

g, 0.40 mmol) was treated with 4M HCl in dioxane (2 mL). The

reaction mixture was stirred at room temperature for 30 min during

which time a precipitate formed. The solvent was removed and

the residue dried in vacuo. The residue was dissolved in anhydrous

CH₂Cl₂, and 55 (0.140 g, 0.4 mmol, 1 equiv) was added followed

by EDCI (0.115 g, 0.6 mmol, 1.5 equiv) and DMAP (0.059 g, 0.48

mmol, 1.2 equiv). The reaction mixture was stirred for 24 h then

the solvent was removed in vacuo and the residue diluted with

EtOAc (25 mL) and washed with 1M HCl solution (3 × 10 mL)

then saturated NaHCO₃solution (3 × 10 mL). The organic fraction

was dried (MgSO₄) and concentrated in vacuo, to give a solid, which

was purified by column chromatography (eluted with EtOAc-

hexane 30:70-75:25) and by preparative LC-MS (Phenomenex

Luna-Combi-HTS 5 × 100 mm × 21.2 mm column, flow rate 32

mL/min and a gradient solvent system going from 90:10 solvent

A:B at time 0-1.35 min to 10:90 A:B at 5.85 min after sample

injection then maintained at 10:90 until 6.85 min. Solvent A -

0.1% formic acid in water, solvent B - 0.1% formic acid in

acetonitrile) and lyophilized to afford a white solid 56, (0.016 g,

(11aS) Methyl 4-({4-[(4-{[4-({4-[4-(7-Methoxy-5-oxo-2,3,5,-

11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yloxy)-

butyrylamino]-1-methyl-1H-pyrrole-2-carbonyl]amino)-1-methyl-

1H-pyrrole-2-carbonyl]amino}-1-methyl-1H-pyrrole-2-carbo-

nyl)amino]-1-methyl-1H-pyrrole-2-carbonyl}amino)-1-methyl-1H-pyr-

role-2-carboxylate (50e). A solution of Alloc-THP-PBD conjugate

49e (0.164 g, 0.14 mmol) dissolved in anhydrous CH₂Cl₂(2 mL)

under a nitrogen atmosphere was treated with pyrrolidine (13 µL,

0.16 mmol, 1.1 equiv) and then palladium tetrakis[triphenylphos-

phine] (0.008 g, 0.007 mmol, 0.05 equiv). The reaction mixture

was stirred at room temperature for 2 h and the product purified

directly by column chromatography (eluted with CHCl₃96%,

MeOH 4%) to give the product as a glassy solid, 50e (0.068 g,

50%). [R]^26.7_D) +90° (c ) 0.068, CHCl₃); ¹H NMR (d₆-DMSO)

δ 9.95 (1H, s, NH), 9.95 (1H, s, NH), 9.94 (1H, s, NH), 9.91 (1H,

s, NH), 9.89 (1H, s, NH), 7.78 (1H, d, J ) 4.4 Hz, H-11), 7.48

(1H, d, J ) 1.8 Hz, Py-H), 7.35 (1H, s, H-6), 7.25 (3H, s, Py-H),

7.17 (1H, d, J ) 1.6 Hz, Py-H), 7.09 (1H, d, J ) 2.1 Hz, Py-H),

7.08 (1H, s, Py-H), 7.07 (1H, d, J ) 1.6 Hz, Py-H), 6.92 (1H, d,

J ) 1.9 Hz, Py-H), 6.91 (1H, d, J ) 1.8 Hz, Py-H), 6.83 (1H, s,

H-9), 4.14-4.05 (2H, m, side-chain H-1), 3.87-3.83 (18H, 6 × s,

O/NCH₃), 3.75 (3H, s, OCH₃), 3.68-3.60 (2H, m, H-3,11a), 3.47-

3.31 (1H, m, H-3), 2.47-2.44 (2H, m, side-chain H-3), 2.30-2.23

(2H, m, H-1), 2.06-1.94 (4H, m, H-2, side-chain H-2); MS (ES⁺)

m/z (relative intensity) 957 ([M + H]^+•, 90%); Acc. Mass

C₄₈H₅₂N₁₂O₁₀calc. 957.4002 found 957.4010.

(11aS) Methyl 4-{[4-({4-[(4-{[4-({4-[4-(7-Methoxy-5-oxo-

2,3,5,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-8-

yloxy)butyrylamino]-1-methyl-1H-pyrrole-2-carbonyl]amino)-

1-methyl-1H-pyrrole-2-carbonyl]amino}-1-methyl-1H-pyrrole-

2-carbonyl)amino]-1-methyl-1H-pyrrole-2-carbonyl}amino)-1-

methyl-1H-pyrrole-2-carbonyl]amino}-1-methyl-1H-pyrrole-2-

carboxylate (50f). A solution of Alloc-THP-PBD conjugate 49f

(0.174 g, 0.14 mmol) dissolved in anhydrous CH₂Cl₂(2 mL) under

a nitrogen atmosphere was treated with pyrrolidine (13 µL, 0.15

mmol, 1.1 equiv) and then palladium tetrakis[triphenylphosphine]

(0.008 g, 0.007 mmol, 0.05 equiv). The reaction mixture was stirred

at room temperature for 2 h and the product purified directly by

column chromatography (eluted with CHCl₃96%, MeOH 4%) to

1

6%) as a racemic mixture. H NMR (d₆-DMSO) δ 10.20 (1H, s,

NH), 9.92 (1H, s, NH), 9.90 (1H, s, NH), 9.88 (1H, s, NH), 7.46

(1H, d, J ) 2.0 Hz, Py-H), 7.25 (1H, s, H-6), 7.23 (1H, d, J ) 1.6

Hz, Py-H), 7.17 (1H, d, J ) 2.0 Hz, Py-H), 7.05 (1H, d, J ) 1.6

Hz, Py-H), 6.90 (1H, d, J ) 2.0 Hz, Py-H), 6.87 (1H, d, J ) 1.6

Hz, Py-H), 6.69 (1H, s, H-9), 4.06-4.03 (2H, m, side-chain H-1),

4.01-3.97 (2H, m, side-chain H-3), 3.84 (3H, s, NCH₃), 3.84 (3H,

s, NCH₃), 3.83 (3H, s, NCH₃), 3.79 (3H, s, OCH₃), 3.74 (3H, s,

OCH₃), 3.71-3.64 (1H, m, H-11a), 3.57-3.51 (2H, m, H-3), 3.47-

3.41 (2H, m, H-4), 2.09-2.02 (2H, m, side-chain H-2), 1.94-1.87

(2H, m, H-2); Acc. Mass C₃₆H₄₀N₈O₉calc. 729.2991 found

729.2988.

Thermal Denaturation Studies. The PBD agents were subjected

to DNA thermal melting (denaturation) studies^33,39,49using duplex-

form calf thymus DNA (CT-DNA, type-I, highly polymerized

sodium salt; 42% G+C [Sigma]) at a fixed 100 µM (DNAp

equivalent to 50 µM in bp) concentration, determined using an

extinction coefficient of 6600 (M phosphate)^-1cm^-1at 260 nm.⁵⁰

Solutions were prepared in pH 7.00 ( 0.01 aqueous buffer

Pyrrolobenzodiazepine-Poly(N-methylpyrrole) Conjugates

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5459

containing 10 mM NaH₂PO₄/Na₂HPO₄and 1 mM EDTA. Working

solutions containing CT-DNA and the test compound (5 µM) were

incubated at 37.0 ( 0.1 °C at 0, 4, or 18 h intervals using an external

water bath. Samples were monitored at 260 nm using a Varian-

Cary 4000 spectrophotometer fitted with a Peltier heating accessory.

Heating was applied at a rate of 1 °C/min in the 45-98 °C

temperature range, following equilibration of samples at 45 °C for

15 min, with optical and temperature data sampling at 100 ms

intervals. A separate experiment was carried out using buffer alone,

and this baseline was subtracted from each DNA melting curve

before data treatment. Optical data were imported into the Origin

5 program (MicroCal Inc., Northampton, MA) for analysis. DNA

helixfcoil transition temperatures (T_m) were determined at the

midpoint of the normalized melting profiles using a published

analytical procedure.³⁹Results are given as the mean ( standard

deviation from at least three determinations. Ligand-induced

presence of drug and f_cis the fractional cleavage of the same bond

in the control. Maxam-Gilbert G + A marker lanes were prepared

in the usual fashion.

In Vitro Transcription. An extended 282-bp DNA fragment,

containing the 262-bp sequence used in the footprinting assays and

a suitably positioned 5′ T7 promoter, was amplified from the MS2

plasmid by PCR with MS2-T7 forward primer (5′-TAATAC-

GACTCACTATAGGGCAGGAAACAGCTATGAC-3′) and re-

verse primer (see DNA Footprinting section above). The 282-mer

product was purified by phenol-CHCl₃extraction and EtOH

precipitation before being resuspended in nuclease-free water to a

fixed concentration (1 µg/µL).

The 282-mer (1 µL) was incubated overnight with either 1.25x

drug solution (4 µL) or water as a control. Transcription mix [5

µL total containing 5x transcription buffer (2 µL), RNasin RNase

inhibitor (0.5 µL of 40 U/µL), T7 RNA polymerase (0.25 µL of

20 U/µL), DTT (0.25 µL, 100 mM; all Promega), NTPs (0.5 µL of

25 mM A, C, G and T, Amersham Pharmacia), (R-³²P)-CTP (0.25

µL of 10 µCi/µL, 3000 Ci/mmol, Perkin-Elmer Life Sciences) and

nuclease-free water (1.25 µL)] was added, and synthesis of RNA

transcripts was allowed to proceed at 37 °C for 90 min. Following

the addition of loading dye (10 µL), samples were heated to 94 °C

for 4 min, briefly cooled on ice, and then loaded on to a preheated

8% denaturing polyacrylamide gel. Electrophoresis was performed

for 80 min in 0.5x TBE buffer at 60 W (2000-2250 V). The gel

was then blotted onto Whatman 3MM paper and dried under

vacuum at 80 °C for 45 min. Exposure to phosphor storage screens

and data collection were carried out as for the footprinting gels.

Marker lanes were created by restriction enzyme digestion of

the 282-mer. Ten different restriction enzymes, each with a single

cutting site on the 282-mer, were employed to cleave the template

282-mer DNA in separate reactions according to the manufacturer’s

guidelines. Cut DNA was pooled and purified by phenol-CHCl₃

extraction and EtOH precipitation. The DNA was resuspended in

nuclease-free water to give a fixed concentration (1 µg/µL). Marker

DNA (1 µL) was used as a template for transcription as described

above, yielding RNA transcripts of known length depending on

the enzyme cutting site. Therefore, the marker lengths differ for

particular experiments depending upon the enzymes chosen.

The lengths of attenuated transcripts produced in the presence

of drug were calculated by a graphical method. The electrophoresed

distance was accurately measured for each marker band, and a plot

of the length of the transcript against this distance was curve-fitted.

The electrophoresed distance of each attenuated transcript was then

used to determine its approximate length and hence the position of

the transcription stop site on the DNA template.

alterations in DNA melting behavior (∆T_m) are given by ∆T_m

)

T_m(DNA + ligand) - T_m(DNA), where the T_mvalue determined

for free CT-DNA is 67.82 ( 0.06 °C (averaged from >100 runs).

All assay compounds were dissolved in HPLC-grade DMSO to give

working solutions containing e0.2% v/v DMSO; T_mresults were

corrected for the effects of DMSO cosolvent by using a linear

correction term. Other [DNAp]/[ligand] molar ratios (i.e., 20:1, 5:1

and 2:1) were examined to ensure that the fixed 10:1 ratio used in

this assay did not result in saturation of the host CT-DNA duplex.

DNA Footprinting. Forward (5′-CAGGAAACAGCTATGAC-

3′) or reverse (5′-GTAAAACGACGGCCAGT-3′) primer was 5′-

end labeled with ³²P by T4 polynucleotide kinase. Labeling mixture

contained primer (5 pmol; Sigma-Genosys), 10x kinase buffer (1

µL; Promega), (γ-³²P)-ATP (2 µL of 10 µCi/µL, 6000 Ci/mmol;

Perkin-Elmer Life Sciences) and T4 polynucleotide kinase (10 U;

Promega) in a 10 µL final volume. This mixture was incubated at

37 °C for 30 min followed by inactivation of the kinase at 70 °C

for 10 min. A 262-bp DNA fragment (MS2F or MS2R) was

amplified from the MS2 plasmid using PCR with either labeled

forward or reverse primer and the unlabeled primer as required.

Reactions were comprised of labeled primer mix (10 µL), 10x PCR

buffer (5 µL; Sigma), dNTPs (5 µL; 2.5 mM each of A, C, G and

T, Amersham Pharmacia), unlabeled primer as required (5 pmol),

MS2 plasmid template DNA (10 ng) and Taq DNA polymerase (5

U; Sigma) in a 50 µL final volume. Radiolabeled 262-mer was

purified by nondenaturing polyacrylamide gel electrophoresis,

elution, filtration, and EtOH precipitation, and was then resuspended

in a solution of Tris-HCl (10 mM) pH 7.4/NaCl (10 mM) to yield

an activity of 200 counts per second per 5 µL when held up, in a

pipet tip, to a capped Morgan series 900 mini-monitor.

For DNase I footprinting, radiolabeled 262-mer (2 µL) was

incubated overnight (16-18 h) with either drug solution (388 µL)

or an aqueous buffer [HEPES (20 mM) pH 7.9, NaCl (50 mM),

MgCl₂(1 mM), DTT (5 mM), 10% glycerol] for control reactions.

Enzymatic cleavage was initiated by addition of DNase I solution

(10 µL of 0.05 U/mL; Sigma) in aqueous NaCl (200 mM), MgCl₂

(20 mM) and MnCl₂(20 mM), and halted with stop solution [40

µL of NaCl (2.25 M), EDTA (150 mM) pH 8.0, 0.57 µg/µL

glycogen (0.57 µg/µL; Roche), poly(dI-dC)‚poly(dI-dC) DNA (19.3

ng/µL; Amersham Pharmacia)]. From this mixture, digested DNA

fragments were prepared for electrophoresis following the protocol

of Trauger & Dervan⁵¹and were loaded on to a preheated 8%

denaturing polyacrylamide gel. Electrophoresis in 1x TBE buffer

was allowed to proceed for 100 min at 70 W (1600-2000 V). The

gel was then fixed in acetic acid (10% v/v) and CH₃OH (10% v/v)

for 15 min followed by blotting onto Whatman 3MM paper and

vacuum-drying at 80 °C for 45 min.

Dried gels were stored at room temperature in phosphor storage

screens (Molecular Dynamics) for a minimum period of 12 h. Data

were collected from exposed screens using a Molecular Dynamics

425E PhosphorImager and transferred to ImageQuant v1.2 software

(Molecular Dynamics) for analysis. Data were expressed as the

differential cleavage between control and sample lanes. Peaks were

integrated and differential cleavage calculated as ln(f_d) - ln(f_c),

where f_dis the fractional cleavage at any particular bond in the

Determination of In Vitro Cytotoxicity. K562 human chronic

myeloid leukemia cells were maintained in RPMI 1640 medium

supplemented with 10% fetal calf serum and glutamine (2 mM) at

37 °C in a humidified atmosphere containing 5% CO₂. Cells were

plated at 1 × 10⁴cells per well in media (190 µL) in 96-well plates.

Compounds were serially diluted in 0.6% DMSO and added to the

cell plates in triplicate (10 µL per well). Control wells containing

vehicle control of 0.6% DMSO were included, and outer columns

were used as blanks. Plates were kept in the dark at 37 °C in a

humidified atmosphere containing 5% CO₂. Following a 4-day

incubation (to allow control cells to increase in number by

approximately 10-fold), the MTT assay was used to determine cell

viability. This assay is based on the ability of viable cells to reduce

a yellow soluble tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich), to an

insoluble purple formazan precipitate. MTT solution (25 µL of

5 mg/mL in phosphate-buffered saline) was added to each well

and the plates further incubated for 4 h. The bulk of the medium

was then pipetted from the cell pellet. DMSO (200 µL) was added

to each well in order to solubilize the purple formazan dye. The

optical density was then read at 540 nm on a Fusion plate reader

(Perkin-Elmer). A dose-response curve was constructed using

GraphPad Prism 4.01 (GraphPad Software Inc.) from n ) 2 data

(from two individual experiments). An IC₅₀value was read as the

5460 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18

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dose required to reduce the final optical density to 50% of the

control value.

Cellular and Nuclear Penetration into MCF-7 Human Mam-

mary Cells. Cellular uptake and nuclear incorporation of drug into

MCF-7 human mammary adenocarcinoma cells (ATCC Collection,

LGC Promochem) was visualized using confocal microscopy.

Solutions of conjugates 50a-f were prepared in DMSO at 20 mM

and diluted in Hank’s Balanced Salt Solution (Sigma) to the

appropriate concentration. Freshly harvested MCF7 cells at 5 ×

10⁴cells/mL were placed in 200 µL of complete RPMI1640

(containing 10% FCS) contained in the wells of eight-well

chambered cover-glasses. The cells were incubated overnight to

adhere at 37 °C. Cellular preparations were then spiked with the

conjugates to give final concentrations of 1, 10, and 100 µM,

ensuring that final DMSO concentrations were <1% v/v. At 1, 3,

5, and 24 h after addition of the conjugates, the cells were examined

using a Zeiss LSM510 inverted microscope with UV laser excita-

tion. Samples were viewed under oil immersion with a 60×

objective lens. Representative images for conjugates 50a, 50c, and

50f are shown in Figure 8.

Acknowledgment. This work was supported by Cancer

Research UK (C180/A1060, SP1938/0402, SP1938/0201 and

SP1938/0301 to D.E.T., J.A.H., and T.C.J., C459/A2579 to

S.D.S. and P.M.L.) and by Spirogen Ltd. Z.A.S. was supported

by studentship 00A1E06372 from the BBSRC. Dr. Robert

Schultz of the National Cancer Institute (NCI) is thanked for

providing the 60-cell line data shown in Table 1. Dr. Fatima

A. Delmani is acknowledged for providing preliminary cyto-

toxicity data not appearing in this paper, as is Dr. Tom Ellis

for his contribution to the development of the gel-based assays.

The EPSRC is thanked for funding an NMR upgrade (GR/

R47646/01) in the School of Pharmacy.

Supporting Information Available: S1-A: Footprinting Gels

for 50a and 50b (Forward-labeled MS2F DNA fragment); S1-B:

Footprinting Gel for 50d (Forward-labeled MS2F DNA fragment);

S1-C: Footprinting Gels for 50d (Forward- and Reverse-labeled

MS2 DNA fragments); S1-D: Footprinting Gel for 50f (Forward-

labeled MS2F DNA Fragment); S2-A: Analysis of footprints from

the MS2F DNA (Forward-labeled DNA fragment); S2-B: Analysis

of footprints from the MS2R DNA (Reverse-labeled DNA frag-

ment); S3: Differential cleavage plots showing the relative positions

of footprints produced by 50c, 23, and 3 (anthramycin methyl ether);

S4: DNase I footprinting gels of 3 binding to both MS2F (Forward-

labeled) and MS2R (Reverse-labeled) DNA fragments; S5: DNase

I footprinting gels of compound 38 binding to both MS2F (Forward-

labeled) and MS2R (Reverse-labeled) DNA fragments; S6: Com-

parative differential cleavage plots for compounds 50c and 38; S7-

A: In vitro transcription gel showing the T-stops produced by 50a

on the MS2 DNA fragment; S7-B: In vitro transcription gel showing

the T-stops produced by 50b on the MS2 DNA fragment; S7-C:

In vitro transcription gel showing the T-stops produced by 50d on

the MS2 DNA fragment; S7-D: In vitro transcription gel showing

the T-stops produced by 50e on the MS2 DNA fragment; S7-E: In

vitro transcription gel showing the T-stops produced by 50f on the

MS2 DNA fragment; S8: In vitro transcription gel showing the

T-stops produced by 3 on the MS2 DNA fragment; S9: Experi-

mental details (and references) for synthesis of compounds 16, 17,

19, 20, 24, 26, 28, 31, 32, 40-45, and 52-54; S10: ¹³C NMR and

IR data for compounds 6, 22, 35, 38, 46-48, 50a-f; S11: HPLC

data for key compounds 6, 22, 38, 50a-f, and 56. This material is

available free of charge via the Internet at http://pubs.acs.org.

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JM051199Z

Article Doi

Design, synthesis, and biophysical and biological evaluation of a series of pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugates

DOI: 10.1021/jm051199z

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Article abstract of DOI:10.1021/jm051199z

Full text of DOI:10.1021/jm051199z

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