Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates

Article abstract of DOI:10.1021/jm040137q

Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs (MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at the 5-position on the B-ring, since model studies showed that attachment of an electron-withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule. Because of the hydrophobic nature of the MGBs, several measures were required to overcome their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer. The resulting ADCs were completely aggregated. Removal of the self-immolative spacer, introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyl-eneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated, even with as many as eight drugs per mAb. These results were extended to include the hydroxy aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays established that the mAb-MGB drug conjugates were highly cytotoxic and effected immunologically specific cell kill at subsaturating doses. The results provide a general strategy for MGB prodrug design and illustrate the importance of linker hydrophilicity in making nonaggregated, active mAb-MGB conjugates.

Full text of DOI:10.1021/jm040137q

1344
J. Med. Chem. 2005, 48, 1344-1358
Design, Synthesis, and in Vitro Evaluation of Dipeptide-Based Antibody Minor
Groove Binder Conjugates
Scott C. Jeffrey,*,† Michael Y. Torgov,† Jamie B. Andreyka,† Laura Boddington,† Charles G. Cerveny,†
William A. Denny,‡ Kristine A. Gordon,† Darin Gustin,† Jennifer Haugen,† Toni Kline,† Minh T. Nguyen,† and
Peter D. Senter†
Seattle Genetics, 21823 30th Drive SE, Bothell, Washington 98021, and Auckland Cancer Society Research Centre,
University of Auckland School of Medicine, Private Bag 92019, Auckland, New Zealand
Received July 20, 2004
Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs
(MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release
drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at
the 5-position on the B-ring, since model studies showed that attachment of an electron-
withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule.
Because of the hydrophobic nature of the MGBs, several measures were required to overcome
their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series
of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide
sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer.
The resulting ADCs were completely aggregated. Removal of the self-immolative spacer,
introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyl-
eneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated,
even with as many as eight drugs per mAb. These results were extended to include the hydroxy
aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl
ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the
hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays
established that the mAb-MGB drug conjugates were highly cytotoxic and effected im-
munologically specific cell kill at subsaturating doses. The results provide a general strategy
for MGB prodrug design and illustrate the importance of linker hydrophilicity in making
nonaggregated, active mAb-MGB conjugates.
Introduction
other ADCs are in various stages of preclinical or clinical
development. These include conjugates of doxorubicin,5,6
a maytansine derivative,7 potent taxol derivatives,8
conjugates of CC-1065 and duocarmycin minor groove
binders,9-11 and the auristatin antimitotic drugs.12,13
Collectively, these studies have led to the identification
of such issues as drug potency, linker stability, and
efficient release of active drug at the tumor site that
affect ADC efficacy, potency, and toxicity. We have
recently reported the effects of ADCs consisting of the
antimitotic drug monomethylauristatin E (MMAE),
which incorporated an enzymatically labile dipeptide
linker connected to MMAE through a self-immolative
para-aminobenzyl carbamate (PABC) spacer.12,13 Upon
treatment with a lysosomal enzyme such as cathepsin
B, the bond between the peptide and the PABC group
was hydrolyzed, leading to release of MMAE. The
conjugates were potent, selective, and highly active in
carcinoma and hematologic xenograft models. Most
importantly, cures and regressions of established tu-
mors were obtained without toxicity, in some cases as
The effectiveness of drugs for cancer chemotherapy
generally relies on differences in growth rates, biochemi-
cal pathways, and physiological characteristics between
cancer and normal tissues. Consequently, most standard
chemotherapeutics are relatively nonspecific and exhibit
dose-limiting toxicities that contribute to suboptimal
therapeutic effects. Considerable effort has been di-
rected toward the use of monoclonal antibodies (mAbs)
for targeted drug delivery due to their high selectivities
for tumor-associated antigens, favorable pharmaco-
kinetics, and relatively low intrinsic toxicities. The
mAb-drug conjugates (ADCs) are formed by covalently
linking anticancer drugs to mAbs, usually through a
conditionally stable linker system. Upon binding to cell
surface antigens, mAbs used for most ADCs are actively
transported to lysosomes or other intracellular compart-
ments, where enzymes, low pH, or reducing agents
facilitate drug release.1,2
The only clinically approved ADC is Mylotarg, which
is used in the treatment of acute myeloid leukemia.3,4
This drug is comprised of an anti-CD33 mAb linked to
the highly potent DNA minor groove binder calecheami-
cin through an acid-labile hydrazone linker. Several
1
low as /60 the maximum tolerated dose (MTD).
We wished to extend these findings to include drugs
with complementary mechanisms of activity. The cy-
clopropylindole DNA minor groove binder (MGB) alky-
lating agents attracted our interest due to their high
potencies, mechanisms of activity, circumvention of
common MDR mechanisms, and compatibility with the
linker strategy developed for MMAE. Representative
* To whom correspondence should be addressed. Phone: 425-527-
4738. Fax: 425-527-4109. E-mail: sjeffrey@seagen.com.
† Seattle Genetics.
‡ University of Auckland School of Medicine.
10.1021/jm040137q CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/05/2005

Antibody Minor Groove Binder Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1345
Scheme 1
MGBs include such natural products as CC-1065,14 the
duocarmycins,15 and synthetic analogues such as the
racemic seco chloride amino-CBI compound (1)16,17 and
racemic seco chloride hydroxy aza-CBI compound (2).18
It has previously been shown that ADCs of CC-1065
analogues displayed both in vitro and in vivo activity.9-11
This was in spite of the fact that the reactivity of the
alkylating portion of the drug was not attenuated,
making the drug susceptible to hydrolysis, alkylation,
and inactivation while still attached to the mAb. In
addition, the linker between the drug and the mAb
involved a sterically hindered disulfide. Linkers of this
class are known to have limited stability in serum.19 The
approach we describe here employs a linking strategy
that inactivates the MGB while it is conjugated to the
mAb and uses a peptide linker with high serum stabil-
ity. Since this class of drugs is quite hydrophobic,
particular attention was focused on developing hydro-
philic peptide-linker derivatives that allowed conjugates
to be produced without causing mAb aggregation. The
resulting monomeric ADCs exhibited potent and specific
in vitro cell kill. The approaches described should be
broadly applicable to the general class of seco halide CC-
1065-like MGBs.
4 as well as higher order species, which were assigned
as dimers and trimers according to LC-MS analysis.
The hydroxy aza-CBI 2 was also susceptible to hydroly-
sis (5) and, in addition, formed the cyclopropyl adduct
6, through the loss of HCl. Presumably, the hydroxy
compound 5 is formed via hydrolysis of the cyclopropyl
intermediate 6. Similarly, compound 4 is likely formed
through its corresponding cyclopropyl adduct.
These data prompted us to devise methods of linking
the drugs to mAbs in a manner that would significantly
attenuate unwanted drug reactivity, since mAb-drug
conjugates may circulate in blood for several days before
binding to tumor cells.1,2 Functional groups that inhibit
cyclopropyl group formation are known to significantly
reduce MGB reactivity and, as a consequence, drug
potency.20,21 To test this with the MGB classes described
here, we prepared the tert-butyl carbamate 7 from 1 and
analyzed this compound, as well as the benzyl ether 8,
to determine their stabilities in aqueous buffers at pH
7. Both of these compounds were highly stable, giving
<5% hydrolysis in 24 h. Thus, significant attenuation
of MGB chemical reactivity is achieved by alkylation
or acylation of the activating heteroatom at the drugs’
5-position.
On the basis of the stability data, three different
strategies for linking MGBs to mAbs, as depicted in
Figure 1, were explored. Approach A incorporates a
para-aminobenzyl carbamate (PABC) self-immolative
spacer between the enzymatically reactive peptide
linker and the drug. This approach has previously been
used for the release of doxorubicin,22,23 MMAE,12,13 and
Results
Design of Drug-Linker Constructs. Compounds
2 and 3 proved to be unstable in aqueous solutions
when incubated at 37 °C (Scheme 1). The amino-CBI 3
underwent significant decomposition at both pH 5 and
7 (>25% in 24 h), forming the hydroxymethyl compound

1346 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Jeffrey et al.
Figure 1. General approaches for the attachment of MGBs to mAbs. A para-aminobenzyl carbamate (PABC)-containing self-
immolated spacer (A) was used for the release of the amino-CBI 1 through proteolytic cleavage followed by 1,6-elimination. Direct
attachment of a dipeptide (B) was anticipated to release 1 directly. The para-aminobenzyl ether (PABE) self-immolative spacer
was employed (C) for the release of the hydroxy aza-CBI 2.
camptothecin24 from ADCs upon treatment with lyso-
somal preparations or purified lysosomal enzymes. The
PABC spacer was incorporated into these ADCs to
minimize the effects of drug steric interactions on
peptide bond hydrolysis. In approach B, the drug is
linked directly to the peptide without the use of a self-
immolative spacer. It was reasoned that the aniline
amide would provide high drug stability while attached
to the mAb and that the drug could be proteolytically
cleaved, since the aminonaphthalene leaving group in
the amino-CBI class is structurally similar to the PABC
spacer aniline in approach A. In approach C, a para-ami-
nobenzyl ether (PABE) group is used as a self-immola-
tive spacer between the drug and the peptide. This
approach extends previous work from our laboratory
showing that para-amidobenzyl ethers of phenolic drugs
were fragmented upon amide bond hydrolysissleading
to the release of the free phenolic drug.25 This approach
should be broadly applicable to seco halide CC-1065-
like MGB drugs, since the vast majority have a phenolic
hydroxyl group as the activating heteroatom.26
Approach A: PABC Constructs Using Amino-
CBI. The synthesis of amino-CBI 1 was carried out by
modification of a previously described route.27 The final
step in the synthesis of 1 involves removal of the allyl
protecting group from the aniline nitrogen of 9, a
reaction that is reported to occur in low yields (42%)
using Grubbs’ catalyst. When a palladium catalyst for
allyl group removal was used,28 it was possible to
improve the yield of 1 to 83%. Incorporation of the
PABC-linker moiety was accomplished as shown in
Scheme 2. Briefly, the amino-CBI compound 1 was
reacted with excess phosgene to form the corresponding
isocyanate in situ, and 10 was added providing the
carbamate 11. Removal of the Fmoc group gave 12,
which was coupled directly to the N-hydroxysuccinimide
ester of maleimidocaproic acid (MC-OSu, 13) to afford
the final product 14.
Approach B: Directly Attached Constructs Us-
ing Amino-CBI. Dipeptide drug-linkers lacking the
para-aminobenzyl carbamate were prepared according
to the synthetic route shown in Scheme 3. Coupling of
the Fmoc-protected amino acid residues 15 and 16 to 1
was accomplished using a HATU/HOAt coupling sys-
tem29,30 to provide 17a and 17b. The Fmoc group was
cleaved, yielding the free amines 18a and 18b. A second
peptide-coupling reaction, using Fmoc-Val-OSu (19),
afforded 20a and 20b, and amine group deprotection
gave the dipeptides 21a and 21b. Compounds 21a and
21b were reacted with MC-OSu (13) to provide 22a and
22b, respectively. Deprotection of 22a in cold TFA
afforded the desired product 23.
A compound with potentially greater water solubility
was formed from 21a using the tetraethyleneglycol
(TEG) acid 24 as a more hydrophilic spacer than
maleimidocaproyl. This yielded 25, which was subse-
quently deprotected to provide 26. Treatment of this
compound with bromoacetyl bromide gave 27, and this
was followed by removal of the Boc group which afforded
28.
Ultimately it was discovered that the bromoacetamide
functional group used in 28 was less suitable than a

Antibody Minor Groove Binder Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1347
Scheme 2
Scheme 3
maleimide for antibody thiol alkylation. This was due
to its instability at pH 9, the pH required for mAb
alkylation. The maleimide-containing drug-linkers could
be reacted with reduced antibodies under essentially
neutral conditions and were stable. This resulted in
more efficient loading of the mAb with drug-linker.
Approach C: PABE Construct Using Hydroxy
Aza-CBI. A third approach for preparing a MGB

1348 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Jeffrey et al.
Scheme 4
derivative for mAb attachment involved O-alkylation
of molecules in the hydroxy aza-CBI family to form
ethers. The key step in the synthesis of 29 involved
formation of an ether bond through O-alkylation of
aza-CBI core 30 with the bromide 31. The synthesis
of bromide 31 began with coupling of the protected
lysine acid 32 to para-aminobenzyl alcohol (33) using
EEDQ as a coupling reagent (Scheme 4).31 Next, the
alcohol 34 was converted to the TBS ether 35.32 This
was followed by alkylation of the anilide nitrogen of 35
with SEMCl and sodium hydride33 to afford the fully
protected adduct 36. Removal of the TBS protecting
group with TBAF afforded the benzyl alcohol 37, which
was converted to the desired benzyl bromide 31 using
carbon tetrabromide and triphenylphosphine. The SEM
protecting group served to stabilize 31 against base-
promoted 1,6-elimination of bromide.
Coupling of 31 to 30 (prepared through hydrogenation
of 3818) proceeded smoothly using potassium carbonate
as base, to give the key intermediate 39 in good yield
(Scheme 5). Both the SEM and Boc protecting groups
were removed through treatment of 39 with ice-cold
trifluoroacetic acid, resulting in the formation of the
hydroxymethyl adduct 40, as determined by LC-MS
analysis. Workup with aqueous ammonia resulted in the
formation of the free anilide 41. The primary amino
group proved to be more nucleophilic than the secondary
aniline, allowing for selective coupling with Fmoc-Val-
OSu (19), which afforded 42. Acylation of the secondary
aniline of 42 with 43 led to the trimethoxyindole 44.
Transesterification in acidic methanol afforded the
alcohol 45, which was converted to the corresponding
chloride 46 through treatment with carbon tetrachloride
and triphenylphosphine.
ide 52. Finally, deprotection of the lysine ꢀ-amine
with cold TFA led to the formation of the target
compound 29.
The purity of drug-linkers 14, 22b, 23, 28, and 29
was assessed by analytical HPLC analysis using both
neutral and acidic mobile phases and a diode array
detector. Each compound was obtained in high purity
as was determined by analysis of each compound’s
chromatogram at multiple wavelengths.
Preparation of Immunoconjugates. The mAbs
used in these investigations were cAC10, a chimeric
IgG1 against the CD30 antigen,34 1F6, a murine IgG1
against the CD70 antigen,35,36 and cBR96, a chimeric
IgG1 against the Ley carbohydrate antigen.5 Conjugates
were formed as previously described12,13 by reducing
interchain disulfides under mild conditions with dithio-
threitol (DTT), adding the drugs 14, 22b, 23, 28, and
29, and purifying the resulting conjugate away from free
drug either by size-exclusion or by hydroxyapatite
chromatography. The extent of mAb reduction, and
subsequent drug incorporation, was a function of how
much DTT was used to reduce the interchain disulfides.
For the cAC10 and cBR96 mAbs with four interchain
disulfides, reduction methods allowed for an average of
one to four disulfide bonds to be broken. Under nonde-
naturing conditions, the mAb chains remain tightly
associated, even in the absence of intact disulfides
(unpublished results).
The cAC10 conjugate derived from compound 14 was
highly aggregated, even with low levels of drug substi-
tution (Table 1). With two drugs per mAb, 48% of the
conjugate was aggregated, and this level increased with
increasing drug/mAb ratios. As with cBR96-doxorubi-
cin conjugates,22 the soluble aggregates were noncova-
lent according to SDS-PAGE (data not shown) and
were most likely due to hydrophobic interactions be-
tween the drugs.22 Attempts to circumvent aggregation
by altering reaction conditions or using different buffers,
cosolvents, and detergents were without success.
Incremental improvements in the aggregation state
of the cAC10 mAb conjugates were obtained using drugs
that were less hydrophobic than 14. Conjugates formed
from 22b, a drug lacking the hydrophobic PABC spacer,
also had a pronounced tendency to aggregate but to a
slightly lesser degree than cAC10-14. Aggregation was
reduced even further in cAC10-23 conjugates, in which
the citrulline residue in 22b was replaced with a lysine.
A lysine residue ꢀ-amine protecting group exchange
was needed next. This would eliminate the need for
purification of the final deprotected molecule. Removal
of the Alloc group of 46 with a palladium catalyst gave
47,28 and the newly liberated ꢀ-amine was capped using
Boc-anhydride to afford 48. Both steps were high-
yielding.
Standard peptide coupling chemistry was all that was
required to complete the synthesis of 29. The removal
of the Fmoc group of 48 gave 49, and this was coupled
to the tetraethylene glycol (TEG) acid 24 to afford 50
in high yield. The Fmoc group was removed to give 51,
and a reaction with MC-OSu (13) afforded the maleim-

Antibody Minor Groove Binder Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1349
Scheme 5
The aggregate content of cAC10-23 with four drugs per
mAb was only 22% compared to 44% with the corre-
sponding citrulline conjugate cAC10-22b.
by the change in 320 nm versus 280 nm absorbances
(drug chromophore absorbance versus mAb absorbance)
from the conjugates’ SEC HPLC trace. After 6 h,
approximately 30% of the drug was released from the
cAC10-29 conjugate, whereas no drug release was
observed from cAC10-28.
A significant improvement was realized with 28, a
drug that contains a hydrophilic TEG spacer between
the thiol-reactive functionality and the Val-Lys dipep-
tide. The cAC10-28 conjugates were formed with as
many as eight drugs per mAb, representing maximal
substitution of available cysteines, with only 5% ag-
gregated protein. Similar results were obtained with
conjugates of 29 which contained the hydrophobic PABE
spacer. This further underscores the importance of the
hydrophilic TEG linker in the formation of monomeric
conjugates with high levels of drug loading.
The tendency for a drug-linker to cause mAb ag-
gregation was largely independent of the mAb. For
example, conjugation of 14 to cBR96 gave essentially
identical results to that described for cAC10. Similar
to the cAC10 results, conjugation of compounds 28 and
29 to cBR96 gave little mAb aggregation.
The results with cAC10-29 are consistent with
cathepsin B cleavage experiments involving a cysteine-
quenched derivative of 29, in which efficient cleavage
was observed (data not shown). In addition, observations
previously published from this laboratory showed dipep-
tide-containing para-aminobenzyl ethers (PABE) of
phenolic drugs were readily cleaved under the action
of cathepsin B.25 The failure of cAC10-28 to cleave with
cathepsin B is likely due to steric hindrance from the
amino-CBI core. This result is in line with studies using
a cysteine-quenched, close structural analogue of 28
(contained a phenylalanine-lysine dipeptide as opposed
to the valine-lysine sequence in 28) which was not a
substrate for cathepsin B (data not shown). These
results were extrapolated to other mAb conjugates of
28 and 29.
Human Cathepsin B Mediated Cleavage. Expo-
sure of conjugates cAC10-28 and cAC10-29 to hu-
man cathepsin B resulted in the observed loss of drug
from only the cAC10-29 conjugate (Figure 2). In both
cases, drug loss from the conjugate was monitored
In Vitro Cytotoxicity. Compounds 1 and 2 were
highly cytotoxic on a panel of cancer cell lines (Table
2). The IC50 values are consistent with previous reports

1350 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Jeffrey et al.
Table 1. Structures of mAb-MGB ADCs and the Effects of Drug Loading on Aggregation
a The drug was attached to mAb sulfhydryl groups through the formation of a maleimido-thioether bond. b Aggregates consisted of
mAb dimers and higher species as determined by size-exclusion HPLC.
for these molecules16,18 and are 50- to 200-fold more
potent than mitomycin C, a clinically approved minor
groove binding agent.
compared to the corresponding free drugs. Neither
cAC10 nor m1F6 displays significant cytotoxic proper-
ties in their unconjugated form (data not shown).
Additional studies were undertaken in the human
small cell lung carcinoma cell line NCI-H69 and a drug
resistant variant of this cell line, NCI-H69/LX4 that was
selected after prolonged exposure to doxorubicin.37
FACS analysis indicated that the variant cell line
overexpressed P-glycoprotein (P-gp), accounting for its
24-fold resistance to doxorubicin compared to the pa-
rental cell line (Table 3). As shown, P-gp did not impart
significant resistance to the alkylating agent cisplatin
or to 1. Consistent with this, drug resistant NCI-H69/
LX4 cells were sensitive to BR96-28 and in fact were
slightly more sensitive to the conjugate than the pa-
rental cell line. While this may reflect the 2.9-fold
difference in Lewis-Y expression, the results indicate
The activities of mAb-28 and mAb-29 conjugates
against two different cell lines are shown in Figure 3.
The cAC10 conjugates (anti-CD30) and 1F6 conjugates
(anti-CD70) were specifically cytotoxic against CD30-
positive Karpas human anaplastic large cell lymphoma
cells (Figure 3A) and Caki-1, a CD70-positive human
renal cell carcinoma line (Figure 3B), respectively. The
conjugates’ cytotoxic effects were immunologically spe-
cific, since nonbinding control conjugates were signifi-
cantly less cytotoxic than corresponding conjugates that
bound to cell-surface antigens. On the basis of drug
concentration, the binding conjugates and free drugs
were nearly equipotent, indicating that the efficiency
of drug delivery by the conjugates was not reduced

Antibody Minor Groove Binder Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1351
Figure 2. Hydrolysis of cAC10-28 and cAC10-29 by human
cathepsin B. Conjugates were added to activated enzyme
followed by incubation at 37 °C (pH 5). Drug release from the
conjugates was monitored by SEC HPLC (320 nm) relative to
mAb absorbance (280 nm).
Table 2. Cytotoxic Activities of Amino-CBI (1) and Hydroxy
Aza-CBI (2) on Human Karpas 299 Anaplastic Large Cell
Lymphoma and Human Caki-1 Renal Cell Carcinoma Cell
Linesa
IC50 (nM)
Karpas 299 (CD30+)
4 h 96 h
Caki-1 (CD70+)
4 h 96 h
free drug
1
2
0.34 (1) 1.17 ( 0.24 (9) 0.59 (1) 0.68 (1)
0.28 (1) 0.18 (1)
0.55 (1) 0.45 (1)
Figure 3. Cytotoxic activities of ADCs on cancer cell lines.
(A) Karpas 299 cells (CD30+) were treated with the anti CD30
ADCs, cAC10-28, and cAC10-29, for 4 and 96 h, and the
cytotoxic effects were compared to cells treated with the
nonbinding control conjugates cBR96-28 and cBR96-29. (B)
Cytotoxic activities of murine 1F6-28 and 1F6-29 conjugates
on Caki-1 cells (CD70+) were treated for 4 h with the anti-
CD70 ADCs, 1F6-28, and 1F6-29, and the activities were
compared to nonbinding control conjugates cAC10-28 and
cAC10-29. Results are shown as mean values ( SD.
mitomycin C 525 (1) 133 (12 (39)
251 (1) 40 ( 6.2 (14)
a Values are the mean ( SEM (number of experiments).
that targeted MGBs might not be susceptible to at least
one common form of multidrug resistance.
Discussion
Several recent studies have provided convincing
evidence that drug potency can strongly influence ADC
activity, particularly in the treatment of solid tumors.
This is exemplified by comparing mAb-doxorubicin
conjugates5 with corresponding mAb-auristatin conju-
gates.12,13 It was shown that the highly potent auristatin
ADCs effected cures and regressions of established
disulfide linker bridging the drug to the mAb, and the
drug was not inactivated while attached to the mAb.
In developing newer generation mAb-MGB conjugates,
we felt that it was critical to incorporate linker tech-
nologies that were considerably more stable than hin-
dered disulfides and to attach the drug in a manner
that suppressed reactivity while attached to the mAb
carrier.
We have demonstrated that two classes of MGBs,
amino-CBIs as represented by 1 and hydroxyl aza-CBI
as represented by 2, were labile in the free forms but
were considerably more stable when the activating
heteroatoms were modifiedseither acylated in the case
of the 1 or alkylated in the case of 2 (Scheme 1).
Consequently, peptide-containing linkers were attached
to the activating heteroatoms, leading to ADCs with or
without self-immolative spacers. The mode of attach-
ment of the self-immolative spacer to the drugs was
either through a carbamate bond to the amino-CBI
tumors at doses that were not only well-tolerated but
were
1
/
of that needed to achieve similar levels of
100
efficacy with the doxorubicin conjugates. These results
prompted us to investigate other highly potent drug
classes, specifically the MGBs 1 and 2 described in this
paper.
MGBs are of great interest for targeted drug delivery.
In general, these drugs are highly potent, amenable to
a variety of chemical manipulations, and are active
against many cancer cell lines. Previously, Chari and
co-workers9 described mAb-MGB conjugates that were
efficacious against metastatic human B-cell lymphoma
(Namalwa) at doses just below the MTD. This was in
spite of the fact that the ADCs contained a labile
Table 3. Cytotoxic Activities of Amino-CBI 1 and CBR96-28 on MDR+ Cellsa
MFIb
IC50 (n ) 3) (nM)
cisplatin
cell line
LeY
P-gp
Doxc
1
cBR96-28f
NCI-H69d
224
640
2.9
14
78
5.6
11.3 ( 1.6
1800 ( 400
74
683 ( 185
2820 ( 637
4.1
0.87 (0.61
2.87 ( 0.64
3.3
13.4 ( 5
20.7 ( 6
1.54
NCI-H69/LX4e
fold difference
a Values are the mean ( SEM. b Mean fluorescence intensity (MFI) was obtained from at least four measurements after a background
fluorescence was subtracted. LeY and P-gp expression were determined using cBR96 and MRK-16 mAbs, respectively. c Doxorubicin.
d Human small cell lung carcinoma cell line. e Derived from parental NCI-H69 cells upon prolonged drug exposure.37 f Concentration
based on drug payload (8 drugs per mAb).

1352 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Jeffrey et al.
HCO2H; 10-95% over 8 min) in A (0.05% aqueous TFA) or
neutral linear gradients (designated gradient N) of mobile
phase B (CH3CN; 10-90% over 10 min, then hold at 90% for
5 min) in A (5.0 mM NH4H2PO4) at a flow rate of 1.0 mL/min.
For preparative HPLC, the stationary phase used was a
Phenomonex Synergi 10 mm × 250 mm, 4 µm, 80 Å semi-
preparative column, using the acidic gradient A at a flow rate
of 4.6 mL/min. All peaks were monitored at 340 and 215 nm.
Radial chromatography was performed on a Chromatotron
instrument (Harrison Research, Palo Alto, CA); preparative
thin-layer chromatography was performed on Whatman 20 cm
× 20 cm, 500 µm, 60 Å silica gel plates, and all other
preparative normal phase purifications were done by standard
flash silica gel chromatography using Whatman Science 60 Å
230-400 mesh silica gel as adsorbent.
{1-Chloromethyl-3-[3-(4-methoxy-phenyl)acryloyl]-2,3-
dihydro-1H-benzo[e]indol-5-yl}-carbamic Acid 4-{2-[2-
(9H-Fluoren-9-ylmethoxycarbonylamino)-3-methyl-bu-
tyrylamino]-5-ureido-pentanoylamino}-benzyl Ester (11).
A solution of 1 (331 mg, 0.842 mmol) in dichloromethane (5
mL) was treated with a solution of phosgene in toluene (4 mL,
20 wt %, Fluka). The mixture was stirred in a sealed vessel
for 17 h, then concentrated and dried under high vacuum for
at least 30 min. A solution of 10 (495 mg, 0.822 mmol) in DMF
(5 mL) was stirred with 4 Å molecular sieves (2 g) for 2 h,
then combined with the residue obtained above. The resulting
solution was heated to 45 °C for at least 2 h, then allowed to
cool, filtered, and concentrated in vacuo. The residue so
obtained was purified by SiO2 PTLC (20 cm × 20 cm plate, 1
mm thickness, partially eluted with 20% MeOH/CH2Cl2, then
fully eluted with 5% MeOH/CH2Cl2 and 10% MeOH/CH2Cl2)
to give 11 (211 mg, 25%) as an amorphous solid. 1H NMR
(DMF-d7) δ 0.96-1.00 (m, 6H), 1.55 (m, 2H), 1.73 (m, 1H), 1.88
(m, 1H), 2.18 (m, 1H), 3.06 (m, 1H), 3.26 (m, 1H), 3.88 (s, 3H),
3.99 (dd, J ) 11.5, 8.2 Hz, 1H), 4.12 (dd, J ) 11.5, 3.5 Hz,
1H), 4.16 (dd, J ) 8.6, 6.5 Hz, 1H), 4.33-4.25 (m, 2H), 4.64
(bs, 4H), 5.20 (s, 2H), 5.67 (bs, 2H), 6.27 (bs, 1H), 7.05 (d, J )
8.8 Hz, 2H), 7.24 (d, J ) 15.3 Hz, 1H), 7.47-7.32 (m, 10H),
7.58 (t, J ) 7.0 Hz, 2H), 7.85-7.72 (m, 12 H), 7.92 (d, J ) 7.6
Hz, 2H), 7.98 (m (obsd), 2H), 8.24 (d, J ) 7.4 Hz, 2H), 8.99
(bs, 1H), 9.72 (bs, 1H), 10.21 (s, 1H); LC-MS m/z (ES+), 1042.3
(M + Na)+.
{1-Chloromethyl-3-[3-(4-methoxy-phenyl)-acryl-
oyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}-carbamic Acid
4-(2-{2-[6-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)hexanoylami-
no]-3-methyl-butyrylamino}-5-ureido-pentanoylamino)-
benzyl Ester (14). Carbamate 11 (195 mg, 0.191 mmol) was
dissolved in DMF (7.5 mL) and treated with a solution of 20%
piperidine in DMF (4 mL). The solution was stirred for at least
2 min, then partitioned between EtOAc (200 mL) and satu-
rated ammonium bicarbonate (50 mL). The EtOAc layer was
washed with water (3 × 75 mL) and brine (75 mL), dried over
Na2SO4, and concentrated. Purification of the crude product
by PTLC (SiO2, 20 cm × 20 cm plate, 1 mm thickness, eluted
with 10% MeOH/CH2Cl2) gave amine 12 (51 mg, 33%). The
purified amine (51 mg, 0.064 mmol) was dissolved in DMF (1
mL), and the solution was treated with MCOSu (13) (34 mg,
0.11 mmol). The mixture was stirred for 17 h, then concen-
trated to dryness. Purification of the residue by PTLC (SiO2,
20 cm × 20 cm plate, 1 mm thickness, eluted with 10% MeOH/
CH2Cl2) gave 14 (58 mg, 91%). 1H NMR (DMF-d7) δ 0.95-0.98
(m, 6H), 1.26 (m, 1H), 1.49-1.61 (m, 9H), 1.72 (m, 1H), 1.88
(m, 1H), 2.14 (m, 2H), 2.23-2.33 (m, 3H), 3.05 (m, 1H), 3.20
(m, 2H), 3.42 (t, J ) 7.0 Hz, 1H), 3.87 (s, 3H), 3.99 (dd, J )
11.1, 8.0 Hz, 1H), 4.11 (dd, J ) 11.1, 3.1 Hz, 1H), 4.34 (dd, J
) 8.4, 6.3 Hz, 1H), 4.57-4.64 (m, 3H), 5.19 (s, 2H), 5.67 (s,
2H), 6.42 (bs, 1H), 7.00 (s, 2H), 7.05 (d, J ) 8.8 Hz, 1H), 7.24
(d, J ) 15.2 Hz, 1H), 7.43-7.46 (m, 3H), 7.57 (t, J ) 7.22 Hz,
1H), 7.76 (d, J ) 15.2 Hz, 1H), 7.83 (m, 2H), 7.98 (m (obs),
1H), 8.24 (d, J ) 8.2 Hz, 1H), 8.31 (d, J ) 7.6 Hz, 1H), 8.99 (s,
1H), 9.75 (s, 1H), 10.26 (s, 1H); UV λmax 316, 241 nm; anal.
HPLC (gradient A) tR ) 6.65 min, 93% AUC340, (gradient N)
tR ) 9.72 min, 95% AUC340; LC-MS m/z (ES+) found, 991.1
molecules or through an ether bond to molecules in the
hydroxy aza-CBI class.
Because of the hydrophobicity of the MGB class of
molecules, several modifications from 14 were required
to form conjugates that were not aggregated. Incorpora-
tion of a hydrophilic peptide linker, together with a TEG
chain between the peptide and the mAb, allowed us to
attach as many as eight amino-CBI- or hydroxy aza-
CBI-based molecules to cAC10, leading to cAC10-28
and cAC10-29, respectively. The conjugates were al-
most exclusively monomeric and were both highly
potent and immunologically specific. The finding that
both conjugates were active in cells, while only
cAC10-29 was a cathepsin B substrate, strongly argues
for the involvement of enzymes other than cathepsin
B in drug release. The activities of cAC10-28 and
cAC10-29 compared favorably to those of the
cAC10-Val-Cit-PABC-MMAE conjugates, which are
highly active in several in vivo tumor models.12 The
m1F6-28 and m1F6-29 ADCs were also found to be
active against Caki-1, a renal cell carcinoma line,
indicating that this approach may be applicable to a
wide array of tumor types. We note that the work
described here comprises a general method for attaching
seco halide MGBs in the CC-1065 family to mAbs, since
it applies to both hydroxyl- and amino-containing MGB
analogues. Furthermore, this is the first application of
the PABE technology for ADC formation.25
The potential of mAb-MGB conjugates is further
underscored by the demonstration that cBR96-28 is
active against a cell line that has high levels of P-gp
(Table 3). These results are in stark contrast to Mylo-
targ, which contains the MGB calicheamicin and is
known to be highly susceptible to the MDR pheno-
type.3,4,38 This is an important advantage in using drugs
in the CC-1065 family, since clinical studies have
correlated MDR with resistance to Mylotarg. Taken
together, the in vitro results provide a strong basis for
ongoing in vivo studies designed to explore the thera-
peutic potential of mAb-28 and mAb-29 conjugates
described here.
Experimental Section
Unless otherwise indicated, all anhydrous solvents were
commercially obtained and stored in Sure-seal bottles under
nitrogen. All other reagents and solvents were purchased as
the highest grade available and used without further purifica-
tion. NMR spectra were recorded on Varian Mercury 400 MHz
instrument. Chemical shifts (δ) are reported in parts per
million (ppm) referenced to tetramethylsilane at 0.00, and
coupling constants (J) are reported in hertz. Low-resolution
mass spectral data were acquired on a Micromass ZMD mass
spectrometer interfaced with an HP Agilent 1100 high-
performance liquid chromatography instrument for LC-MS.
Products were eluted on a Phenomonex Synergi 2.0 mm × 150
mm, 4 µm, 80 Å MAX RP column using a linear gradient of
mobile phase B (CH3CN with 0.05% HCO2H) in A (0.05%
aqueous HCO2H) at 0.4 mL/min. Unless otherwise specified,
the reported retention times (tR) are those from LC-MS. High-
resolution (exact mass) data were obtained at the University
of Washington Medicinal Chemistry Mass Spectrometry Cen-
ter on a Bruker APEXIII 47e [FT(ICR)]MS mass spectrometer.
Analytical HPLC was conducted on a Waters 2695 instrument
using a Waters 2996 PDA and Millenium32 software. For
analytical HPLC, the stationary phase used was a Phenom-
onex Synergi 4.6 mm × 150 mm, 4 µm, 80 Å MAX RP column.
Products were eluted on either acidic linear gradients (desig-
nated gradient A) of mobile phase B (CH3CN with 0.05%

Antibody Minor Groove Binder Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1353
(M + H)+; HRMS m/z for C52H59ClN8O10Na+ (M + Na)+ calcd,
The reaction mixture was directly aspirated onto a 1-mm
radial Chromatotron plate and was eluted with a 2-5%
methanol/dichloromethane solvent gradient. The major 254-
nm UV-active band was collected and concentrated under
reduced pressure to give 30 mg (63%) of 25 as an amorphous
yellow solid. Material was not characterized but was used
directly in the preparation of 26.
Amine 26. See preparation of 18a for a general Fmoc-
deprotection procedure and method of purification. Deprotec-
tion of 25 (30 mg, 25.3 µmol) gave 15 mg (62%) of 26 as an
amorphous solid: LC-MS m/z (ES+) 967.6 (M + H)+.
Bromoacetamide 27. To a 0 °C solution of 26 (15.4 mg,
16 µmol) in dichloromethane (4 mL) was added DIPEA (8.3
µL, 48 µmol). This was followed by the addition of 1 mL of a
bromoacetyl bromide/dichloromethane solution, prepared by
dissolving bromoacetyl bromide (32 µL) into dichloromethane
(10 mL). The reaction was stirred for 5 min before being
aspirated directly onto a 1-mm radial Chromatotron plate. The
product was eluted using a 5-7% methanol/dichloromethane
solvent gradient, and the major 254-nm band was collected
and concentrated. This gave 14.5 mg (83%) of 27 as a yellow
solid: LC-MS m/z (ES+) 1087.5 (M + H)+.
1013.3940; found, 1013.3940.
(5-Amino-5-{1-chloromethyl-3-[3-(4-methoxy-phenyl)-
acryloyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamoyl}-
pentyl)carbamic Acid tert-Butyl Ester (17a). To an ice-
cold solution of 1 (105 mg, 0.27 mmol) in 2 mL of dichlorometh-
ane was added, sequentially, 1-hydroxy-7-azabenzotriazole
ꢀ
(HOAt, 44 mg, 0.32 mmol), NRFmocN Boc-lysine (15, 150 mg,
0.32 mmol), DIPEA (0.11 mL, 0.64 mmol), and O-(7-azaben-
zotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophos-
phate (HATU) (122 mg, 0.32 mmol). The reaction was allowed
to equilibrate to an ambient temperature and was stirred for
24 h. The mixture was diluted with dichloromethane, and the
organic phase was washed successively with 0.1 mM citric acid,
NaHCO3, and brine. The organic phase was dried and con-
centrated in vacuo to a residual oil that was purified on flash
silica (97:3 CHCl3-MeOH) to give 17a as a yellow solid (220
mg, 97%): HPLC tR ) 13.50 min; 1H NMR (CDCl3) δ 8.85 (br,
1H), 7.50 (br, 1H), 7.00-7.20 (m, 18 H), 6.90 (d, 1H), 4.90 (br,
1H), 4.00-4.60 (m, 9H), 3.83 (s, 3H), 3.15-3.20 (m, 2H), 1.45-
2.20 (m, 4H), 1.40 (s, 9H), 10.10-1.150 (m, 2H); LC-MS m/z
(ES+) 843.3 (M + H)+; tR ) 5.28 min. Anal. (C49H51ClN4O7) C,
H, N.
Amine-TFA Salt 28. To a flask containing 27 (14.5 mg, 13.3
µmol) and cooled in an ice-water bath was added TFA (4 mL,
precooled in an ice-water bath). The reaction mixture was
stirred for 15 min and was concentrated under reduced
pressure. The resulting residue was redissolved in dichlo-
romethane (5 mL) and concentrated 2 additional times under
reduced pressure to give a solid residue 16.4 mg of 28 as a
TFA salt complex: 1H NMR (DMF-d7) δ 0.99 (m, 6H), 1.58-
1.75 (m, 2H), 1.80-1.95 (m, 3H), 2.05-2.21 (m, 2H), 2.50-
2.64 (m, 2H), 3.13 (m, 2H), 3.35 (q, J ) 5.7 Hz, 2H), 3.51 (t, J
) 5.9 Hz, 2H), 3.56 (s, 12H), 3.69 (t, J ) 6.5 Hz, 2H), 3.88 (s,
3H), 3.96 (s, 2H), 4.01 (m, 1H), 4.12 (dd, J ) 3.1, 11.3 Hz, 1H),
4.35 (dd, J ) 1.8, 8.0 Hz, 1H), 4.41 (bs, 1H), 4.66 (bs, 2H),
4.78 (bs, 1H), 7.05 (d, J ) 8.8 Hz, 2H), 7.24 (d, J ) 15.1 Hz,
1H), 7.45 (t, J ) 6.9 Hz, 1H), 7.59 (t, J ) 7.2 Hz, 1H), 7.75 (d,
J ) 15.3 Hz, 2H), 7.83 (d, J ) 8.7 Hz, 1H), 8.06 (dd, J ) 2.7,
7.8 Hz, 2H), 8.16 (m, 1H), 8.26 (m, 3H), 8.36 (m, 2H), 8.91
(bs, 1H), 10.01 (bs, 1H); UV λmax 317 nm; anal. HPLC (gra-
dient A) tR ) 4.55 min, 93% AUC340, (gradient N) tR ) 10.67
min, 96% AUC340; LC-MS m/z (ES+) 987.4 (M + H); HRMS
(5-Amino-5-{1-chloromethyl-3-[3-(4-methoxy-phenyl)-
acryloyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamoyl}-
pentyl)carbamic Acid tert-Butyl Ester (18a). Intermediate
17a was dissolved in 1 mL of DMF and treated at an ambient
temperature with 20% v/v piperidine in DMF (20 mL) for 2.5
h. The reaction mixture was poured into a 200-mL 4:1 brine/
ethyl acetate solution, and the organic phase was separated,
washed an additional 3 times with brine, dried, and concen-
trated in vacuo to a residual oil that was purified on flash silica
(97:3 CHCl3-MeOH) to give 18a (108 mg, 67%): HPLC tR
)
1
6.36 min; H NMR (CDCl3) δ 9.00 (br, 1H), 7.40-7.80 (m, 8
H), 6.90-7.00 (m, 3H), 6.80 (br, 1H), 4.60-4.70 (m, 1H), 4.50-
4.60 (m, 1H), 4.35-4.45 (m, 1H), 3.80-4.00 (m, 1H), 3.83 (s,
3H), 3.60-3.70 (m, 1H), 3.40-3.50 (m, 1H), 3.15-3.20 (m, 2H),
1.70-2.20 (m, 6H), 1.50-1.60 (m, 2H), 1.40 (s, 9H); LC-MS
m/z (ES+) 621.2 (M + H)+, 521.4 (M - Boc + H)+; tR ) 3.96
min. Anal. (C34H41ClN4O5) C, H, N.
{5-{1-Chloromethyl-3-[3-(4-methoxy-phenyl)acryl-
oyl]-2,3-dihydro-1H-benzo[e]indol-5-ylcarbamoyl}-5-[2-
(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-bu-
tyrylamino]-pentyl}-carbamic Acid tert-Butyl Ester (20a).
To a solution of 18a (41 mg, 66 µmol) in DMF (2 mL) was
added Fmoc-Val-OSu (19) (87 mg, 0.2 mmol), and the reaction
mixture was stirred for 16 h. The reaction mixture was
concentrated under reduced pressure and was purified via
radial chromatography on a 1 mm plate, eluting with a
25-50% ethyl acetate/hexanes gradient. The major UV-active
254-nm band was collected and concentrated under reduced
pressure to give 54 mg (87%) of 20a as a yellow solid: 1H NMR
(DMSO-d6) δ 0.88 (d, J ) 6.5 Hz, 3H), 0.91 (d, J ) 6.3 Hz,
3H), 1.26 (m, 1H), 1.36 (s, 9H), 1.38-1.52 (m, 3H), 1.78 (m,
1H), 1.90 (m, 1H), 2.94 (m, 2H), 3.83 (s, 3H), 3.88-4.07 (m,
4H), 4.20-4.40 (m, 5H), 4.51 (m, 3H), 4.624 (m, 1H), 6.53 (bs,
1H), 7.01 (d, J ) 8.8 Hz, 2H), 7.06 (d, J ) 15.5 Hz, 1H), 7.23
(bs, 1H), 7.30 (t, J ) 7.6 Hz, 2H), 7.40 (dd, J ) 8.2, 15.3 Hz,
2H), 7.55 (t, J ) 7.2 Hz, 1H), 7.64 (d, J ) 15.3 Hz, 1H),
7.67 (m, 1H), 7.74 (d, J ) 8.4 Hz, 2H), 7.86 (d, J ) 7.2
Hz, 2H), 7.92 (8.4 Hz, 1H), 8.01 (d, J ) 8.4 Hz, 2H), 8.61 (bs,
1H), 9.88 (bs, 1H); LC-MS m/z (ES+) 942 (M + H)+. Anal.
(C54H60ClN5O8) C, H, N.
(5-(2-Amino-3-methyl-butyrylamino)-5-{1-chloromethyl-
3-[3-(4-methoxy-phenyl)acryloyl]-2,3-dihydro-1H-benzo-
[e]indol-5-ylcarbamoyl}-pentyl)carbamic Acid tert-Butyl
Ester (21a). See the preparation of 18a for general Fmoc-
deprotection procedure. Deprotection of 20a (54 mg, 57 µmol)
gave 33 mg (80%) of 21a: LC-MS m/z (ES+) 720 (M + H)+;
Anal. (C39H50ClN5O6) C, H, N.
Fmoc-TEG 25. To a mixture of 21a (29 mg, 40 µmol) and
Fmoc-TEG-OH (24) (25.3 mg, 52 µmol) in dichloromethane (2
mL) was added DIPEA (21 µL, 0.12 mmol) followed by HATU
(23.3 mg, 61 µmol). The reaction mixture was stirred for 1 h.
m/z (M + H)+ for C47H65BrClN6O10 calcd, 987.3634; found,
+
987.3597.
Maleimide 22a. To a DMF (0.2 mL) solution of 21a (10
mg, 14 µmol) was added MCOSu (13) (10 mg, 42 µmol), and
the mixture was stirred for 16 h at an ambient temperature.
The reaction mixture was concentrated under reduced pres-
sure, and the resulting residue was chromatographed on a
1-mm radial Chromatotron plate, eluting with a 1-5% metha-
nol/dichloromethane gradient. A single 254-nm UV-active band
was collected and concentrated to give 22a, 6.4 mg (50%) as
an amorphous solid. MS m/z (ES+) 913 (M + H)+.
6-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)hexanoic Acid [1-(1-
{1-Chloromethyl-3-[3-(4-methoxy-phenyl)acryloyl]-2,3-
dihydro-1H-benzo[e]indol-5-ylcarbamoyl}-4-ureido-bu-
tylcarbamoyl)-2-methyl-propyl]-amide (22b). This com-
pound was prepared in a manner very similar to 22a and
starting from 1: UV λmax 317 nm; anal. HPLC (gradient A) tR
) 6.28 min, 95% AUC340, (gradient N) tR ) 9.05 min, 95%
AUC340; HRMS m/z for C44H52ClN7O8Na (M + Na)+ calcd,
864.3464; found, 864.3444.
Amine-TFA Salt 23. See procedure for synthesis of 28 for
Boc-removal. The conversion of 22a (6.4 mg, 7 µmol) gave 12.5
mg of 23 as an amorphous TFA salt complex: 1H NMR
(DMSO-d6) δ 0.84 (m, 6H), 1.12-1.28 (m, 3H), 1.33-1.65 (m,
8H), 1.64-1.8 (m, 1H), 1.82-2.03 (m, 2H), 2.05-2.22 (m, 2H),
2.80 (m, 2H), 3.34 (m, 2H), 3.80 (s, 3H), 3.91 (t, J ) 10.6 Hz,
1H), 4.01 (d, J ) 8.6 Hz, 1H), 4.19 (m, 1H), 4.36 (bs, 1H), 4.44-
4.59 (m, 3H), 6.99 (m, 4H), 7.08 (d, J ) 15.5 Hz, 1H), 7.39 (t,
J ) 6.5 Hz, 1H), 7.54 (t, J ) 7.4 Hz, 1H), 7.51 (d, J ) 15.1 Hz,
1H), 7.67 (bs, 2H), 7.75 (d, J ) 8.8 Hz, 2H), 7.84-7.96 (m,
3H), 8.24 (m, 1H), 8.61 (m, 1H), 10.0 (bs, 1H); UV λmax 317
nm; anal. HPLC (gradient A) tR ) 4.68 min, 96% AUC340
,

1354 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
(gradient N) tR ) 12.10 min, 95% AUC340; MS m/z (ES+) 813.4
Jeffrey et al.
eluting with 25% ethyl acetate/hexanes. The product (Rf ) 0.7
TLC using 50% ethyl acetate/hexanes) eluted as the first band,
followed by residual starting material. (Note: the 254-nm UV
absorbance of product is greatly diminished relative to starting
material.) The product-containing fractions were combined and
concentrated under reduced pressure to give 538 mg (78%) of
36 as a clear oil: 1H NMR (CDCl3) δ 0.00 (s, 9H), 0.12 (d, J )
1.4 Hz, 6H), 0.95-0.97 (m, 11H), 1.08-1.26 (m, 2H), 1.35-
1.53 (m, 11H), 2.99 (m, 1H), 3.63 (dd, J ) 8.6, 8.6 Hz, 2H),
4.33 (m, 1H), 4.53 (d, J ) 5.5 Hz, 2H), 4.76 (s, 2H), 4.96 (d, J
) 9.8 Hz, 1H), 5.14 (d, J ) 10 Hz, 1H), 5.20 (m, 2H), 5.28 (dd,
(M + H); HRMS m/z (M + H)+ for C44H54ClN6O7 calcd,
+
813.3743; found, 813.3715.
1-(5-Amino-1-chloromethyl-1,2-dihydro-benzo[e]indol-
3-yl)-3-(4-methoxy-phenyl)propenone (1). To a solution of
9 (691 mg, 1.6 mmol) in dichloromethane (20 mL) were added
PhSO2Na (524 mg, 3.2 mmol) and camphorsulfonic acid (742
mg, 3.2 mmol). The (Ph3P)4Pd (92 mg, 0.08 mmol) was added,
and the reaction mixture was stirred under an inert atmo-
sphere and at ambient temperatures. After 15 min, the mix-
ture was directly aspirated onto a 4-mm Chromatotron plate,
and the product was eluted as the major yellow band with 25%
ethyl acetate/hexanes. The combined fractions were concen-
trated under reduced pressure to give 547 mg of 1 (87%). This
material was identical in all respect to an authentic sample
of 1.17
{1-Chloromethyl-3-[3-(4-methoxy-phenyl)acryloyl]-2,3-
dihydro-1H-benzo[e]indol-5-yl}-carbamic Acid tert-Butyl
Ester (7). To a mixture of 1 (100 mg, 0.51 mmol) in THF (2.5
mL) was added Boc2O (330 mg, 1.53 mmol), and the mixture
was heated at reflux for 7 h. The reaction mixture was
concentrated under reduced pressure, and the resulting resi-
due was dissolved in ethyl acetate (5 mL) and precipitated by
the addition of hexanes (30 mL). The resulting light yellow
solid was isolated by filtration to give 109 mg (87%) of 7:
LC-MS m/z (ES+) 493 (M + H)+.
[5-tert-Butoxycarbonylamino-5-(4-hydroxymethyl-
phenylcarbamoyl)pentyl]-carbamic Acid Allyl Ester (34).
To a solution of Boc-Lys (Alloc)-OH (32) (100 mg, 0.3 mmol)
and para-aminobenzyl alcohol (33) (37 mg, 0.3 mmol) in
chloroform (5 mL) was added EEDQ (88 mg, 0.36 mmol). The
mixture was stirred at an ambient temperature for 2 h and
was poured into a 0.1 N HCl solution (50 mL), and the
resulting mixture was extracted with ethyl acetate (3 × 25
mL). The combined extracts were washed with water and brine
and were dried over magnesium sulfate, before being filtered
and concentrated under reduced pressure. The resulting resi-
due was purified via radial chromatography on a 2-mm plate
eluting with a 25% ethyl acetate/hexanes to 100% ethyl acetate
gradient. The major 254-nm UV-active band was collected and
concentrated under reduced pressure to give 100 mg (77%) of
34 as a viscous oil: 1H NMR (DMSO-d6) δ 1.22-1.53 (m, 13H),
1.55-1.71 (m, 2H), 2.98 (q, J ) 6.3 Hz, 2H), 4.04 (m, 1H), 4.45
(m, 4H), 4.92 (t, J ) 5.5 Hz, 1H), 5.15 (d, J ) 10.4 Hz, 1H),
5.26 (dd, J ) 1.8, 7.2 Hz, 1H), 5.90 (m, 1H), 6.70 (bs, 1H), 6.98
(bs, 1H), 7.24 (d, J ) 8.0 Hz, 2H), 7.52 (d, J ) 8.4 Hz, 2H),
9.68 (s, 1H); LC-MS m/z (ES+) 458 (M + Na)+.
J ) 1.5, 17.2 Hz, 1H), 5.91 (m, 1H), 7.26 (d, J ) 8.4 Hz, 1H),
+
7.40 (d, J ) 8.4 Hz, 1H); LC-MS m/z (ES+) 702 (M + Na)
.
{5-tert-Butoxycarbonylamino-5-[(4-hydroxymethyl-
phenyl)-(2-trimethylsilanyl-ethoxymethyl)carbamoyl]-
pentyl}-carbamic Acid Allyl Ester (37). To a solution of
36 (530 mg, 0.78 mmol) in THF (20 mL) was added TBAF (1
mL of a 1.0 M solution, 1 mmol). After 15 min, the reaction
mixture was concentrated under reduced pressure and redis-
solved in dichloromethane (3 mL). This mixture was directly
aspirated onto a 4-mm radial Chromatotron plate and eluted
with 50% ethyl acetate/hexanes to 100% ethyl acetate solvent
gradient. A single 254-nm UV-active band (Rf ) 0.2, 50% ethyl
acetate/hexanes) was collected and concentrated under reduced
pressure to give 436 mg (95%) of 37 as a clear oil: 1H NMR
(CDCl3) δ 0.00 (s, 9H), 0.96 (m, 2H), 0.99-1.28 (m, 4H), 1.33-
1.54 (m, 11H), 2.85 (m, 2H), 3.63 (dd, J ) 8.0, 8.0 Hz, 2H),
4.34 (m, 1H), 4.54 (d, J ) 5.2 Hz, 2H), 4.71 (s, 2H), 4.82 (bs,
1H), 4.98 (d, J ) 10.0 Hz, 2H), 5.08-5.25 (m, 2H), 5.27 (dd, J
) 1.6, 15.7 Hz, 1H), 5.94 (m, 1H), 7.31 (d, J ) 8.4 Hz, 2H),
+
7.47 (d, J ) 8.4 Hz, 2H); LC-MS m/z (ES+) 588 (M + Na)
.
{5-[(4-Bromomethyl-phenyl)-(2-trimethylsilanyl-
ethoxymethyl)carbamoyl]-5-tertbutoxycarbonylamino-
pentyl}-carbamic Acid Allyl Ester (31). To a solution of
37 (450 mg, 0.8 mmol) in dichloromethane (25 mL) was added
triphenylphosphine (631 mg, 2.4 mmol) followed by carbon
tetrabromide (794 mg, 2.4 mmol). The reaction mixture was
stirred for 1 h and was directly aspirated onto a 4-mm radial
Chromatotron plate and eluted with 50% ethyl acetate/
hexanes. Product-containing fractions were concentrated under
reduced pressure to give 450 mg (68%) of 31 as a clear oil: 1H
NMR (CDCl3) δ (ppm) 0.00 (s, 9H), 0.94 (m, 2H), 1.05-1.28
(m, 4H), 1.35-1.54 (m, 11H), 2.99 (m, 2H), 3.63 (dd, J ) 8.0,
8.0 Hz, 2H), 4.30 (m, 1H), 4.49 (s, 2H), 4.53 (d, J ) 5.2 Hz,
2H), 4.73 (bs, 1H), 4.95 (d, J ) 10.0 Hz, 2H), 5.14-5.21 (m,
2H), 5.27 (ddd, J ) 1.6, 1.6, 17.2 Hz, 1H), 5.91 (m, 1H), 7.30
(d, J ) 7.6 Hz, 2H), 7.48 (d, J ) 8.4 Hz, 2H); LC-MS m/z
+
(ES+) 650 (M + Na)
.
{5-Allyloxycarbonylamino-1-[4-(tert-butyl-dimethyl-si-
lanyloxymethyl)phenylcarbamoyl]-pentyl}-carbamic Acid
tert-Butyl Ester (35). To a DMF (3 mL) solution of 34 (500
mg, 1.15 mmol) was added DIPEA (1 mL, 5.76 mmol) followed
by TBSCl (517 mg, 3.45 mmol). The reaction mixture was
stirred for 10 min, before being concentrated under reduced
pressure. The residue was dissolved in dichloromethane/
hexanes (1:1, 20 mL) and was directly aspirated onto a 4-mm
radial Chromatotron plate and eluted with a 10-50% ethyl
acetate/hexanes solvent gradient. The major 254-nm UV-active
band was collected and concentrated to give 547 mg (87%) of
35 as a clear oil: 1H NMR (CDCl3) δ 0.05 (s, 6H), 0.89 (s, 9H),
1.42 (s, 9H), 1.48-1.75 (m, 6H), 1.92 (m, 1H), 3.17 (q, J ) 6.3
Hz, 2H), 4.13 (bs, 1H), 4.53 (d, J ) 5.3 Hz, 2H), 4.66 (s, 2H),
4.81 (m, 1H), 5.17 (m, 2H), 5.26 (dq, J ) 1.6, 15.7 Hz, 1H),
5.87 (m, 1H), 7.28 (d, J ) 8.4 Hz, 2H), 7.45 (d, J ) 8.4 Hz,
2H), 8.26 (bs, 1H); LC-MS m/z (ES+) 572 (M + Na)+.
{5-tert-Butoxycarbonylamino-5-[[4-(tert-butyl-dimeth-
yl-silanyloxymethyl)phenyl]-(2-trimethylsilanyl-eth-
oxymethyl)carbamoyl]-pentyl}-carbamic Acid Allyl Es-
ter (36). To a solution of 35 (559 mg, 1.0 mmol) in THF (10
mL) was added NaH (40 mg of a 60% dispersion in mineral
oil, 1.0 mmol). After 10 min, neat SEMCl (185 µL, 1.12 mmol)
was added, and the reaction mixture was stirred for 30 min.
The crude reaction mixture was concentrated under reduced
pressure, and the resulting oil was dissolved in hexanes and
directly aspirated onto a 4-mm radial Chromatotron plate,
1-Acetoxymethyl-5-hydroxy-1,2-dihydro-pyrrolo[3,2-f]-
quinoline-3-carboxylic-acid tert-Butyl Ester (30). To a 0
°C solution of 3818 (350 mg, 0.78 mmol) in THF (20 mL) and
saturated aqueous ammonium bicarbonate (4 mL) was added
10% Pd/carbon (350 mg). The reaction vessel was equipped
with a balloon containing hydrogen gas, and the reaction
mixture was stirred vigorously for 1.5 h. The reaction mixture
was filtered through a plug of Celite, and the plug was washed
with ethyl acetate (20 mL) and ethanol (20 mL). The reaction
mixture was concentrated to approximately 20 mL, and the
material was filtered through a Millipore filter (0.2 µm, syringe
filter) to remove residual catalyst and carbon. Concentration
gave 271 mg (97%) of 30, crude yield. The material was used
without purification: 1H NMR (CDCl3) δ (ppm) 1.61 (s, 9H),
2.09 (s, 3H), 3.89 (m, 2H), 4.09 (m, 2H), 4.46 (m, 1H), 7.41
(dd, J ) 3.9, 4.5 Hz), 8.16 (d, J ) 8.8 Hz, 1H), 8.61 (s, 1H);
LC-MS m/z (ES+) 359 (M + H)+.
1-Acetoxymethyl-5-{4-[(6-allyloxycarbonylamino-2-tert-
butoxycarbonylamino-hexanoyl)-(2-trimethylsilanyl-
ethoxymethyl)amino]-benzyloxy}-1,2-dihydro-pyrrolo-
[3,2-f]quinoline-3-carboxylic Acid tert-Butyl Ester (39).
To a mixture of 31 (171 mg, 0.273 mmol) and phenol 30 (117
mg, 0.327 mmol) in DMF (2 mL) was added potassium
carbonate (42 mg, 0.304 mmol), and the reaction mixture was
stirred for 16 h at an ambient temperature. The mixture was
concentrated under reduced pressure, and the resulting resi-

Antibody Minor Groove Binder Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1355
due was suspended in dichloromethane (3 mL) and was
directly aspirated onto a 2-mm radial Chromatotron plate. The
product was eluted with ammonia-saturated dichloromethane,
and the fractions were combined and concentrated. The
partially pure product was purified a second time on a 1-mm
radial Chromatotron plate eluting with a 5% methanol/
dichloromethane mixture. The product-containing fractions
were combined and concentrated under reduced pressure to
give 179 mg (68%) of 39 as a white solid: 1H NMR (DMSO-d6)
δ (ppm) 0.01 (s, 6H), 0.88 (dd, J ) 1.3, 9.1 Hz, 2H), 0.092-
1.29 (m, 4H), 1.38 (s, 9H), 1.44 (m, 2H), 1.56 (s, 9H), 1.96 (s,
3H), 2.84 (m, 2H), 3.59 (hex, 8.0 Hz, 2H), 3.94-4.17 (m, 5H),
4.36 (dd, J ) 4, 10.4 Hz, 1H), 4.43 (d, J ) 5.6 Hz, 2H), 4.95 (d,
J ) 10.4 Hz, 1H), 5.06 (m, 1H), 5.12 (m, 1H), 5.22 (dd, J )
1.4, 17.1 Hz, 1H), 5.34 (s, 2H), 5.87 (m, 1H), 6.71 (m, 1H), 6.84
(m, 1H), 7.40 (d, J ) 5.4 Hz, 2H), 7.50-7.58 (m, 3H), 7.61-
7.66 (m 3H), 7.87 (bs, 1H), 8.27 (dd, J ) 1.6, 8.5 Hz, 1H), 8.75
(t, J ) 1.6 Hz, 1H); LC-MS m/z (ES+) 906 (M + H)+. Anal.
(C47H67N5O11Si) C, H, N.
Acetic Acid 5-[4-(6-Allyloxycarbonylamino-2-amino-
hexanoylamino)benzyloxy]-2,3-dihydro-1H-pyrrolo[3,2-
f]quinolin-1-ylmethyl Ester (41). To TFA (20 mL) at 0 °C
was added neat 39 (176 mg, 0.194 mmol) followed by stirring
for 1 h. The mixture was poured into an ice-water bath, and
the pH was adjusted to 9-10 with aqueous ammonium
hydroxide. The solution was extracted with ethyl acetate (3 ×
50 mL). The combined extracts were washed with water and
brine and were dried over magnesium sulfate. Filtration and
concentration under reduced pressure afforded 116 mg of crude
41 that was carried on without purification: LC-MS m/z (ES+)
776 (M + H)+.
Acetic Acid 5-(4-{6-Allyloxycarbonylamino-2-[2-(9H-
fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl-
amino]-hexanoylamino}-benzyloxy)-2,3-dihydro-1H-pyr-
rolo[3,2-f]quinolin-1-ylmethyl Ester (42). To a solution of
41 (116 mg, 0.20 mmol) in DMF (3 mL) was added Fmoc-Val-
OSu (19, 263 mg, 0.61 mmol), and the mixture was stirred at
an ambient temperature for 16 h. The reaction mixture was
concentrated under reduced pressure, and the resulting resi-
due was dissolved in dichloromethane (5 mL) and directly
aspirated onto a 2-mm radial Chromatotron plate. The product
was eluted using a 1-5% methanol/dichloromethane gradient
and the product-containing fractions were combined and
concentrated to give 101 mg (58% for two steps) of 42. The
material was carried forward without characterization.
Acetic Acid 5-(4-{6-Allyloxycarbonylamino-2-[2-(9H-
fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyryl-
amino]-hexanoylamino}-benzyloxy)-3-(5,6,7-trimethoxy-
1H-indole-2-carbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quin-
olin-1-ylmethyl Ester (44). To a mixture of 42 (101 mg, 0.113
mmol) and trimethoxy indole carboxylic acid 43 (43 mg, 0.169
mmol) in DMA (2 mL) was added EDCI-HCl (52 mg, 0.27
mmol), and the reaction mixture was stirred at an ambient
temperature for 3 h. An additional portion of the trimethoxy
indole carboxylic acid 43 (33.5 mg, 0.113 mmol) was added,
and the reaction mixture was stirred for an additional 1.5 h.
An additional portion of EDCI-HCl (22 mg, 0.113 mmol) was
added, and the reaction mixture was stirred at an ambient
temperature for 16 h. The reaction mixture was poured into
saturated aqueous sodium bicarbonate (50 mL), and the
mixture was extracted with ethyl acetate (3 × 100 mL). The
combined extracts were washed with water and brine and were
dried over magnesium sulfate. Filtration and concentration
resulted in a residue that was dissolved in dichloromethane
(5 mL, with several drops of methanol) and aspirated directly
onto a 2-mm radial Chromatotron plate. The product was
eluted with a 1-5% methanol/dichloromethane gradient. The
product-containing fractions were combined and concentrated
to give 52 mg (41%) of 44 as an off-white solid: 1H NMR
(DMSO-d6) δ (ppm) 0.86 (m, 6H), 1.23-1.46 (m, 4H), 1.55-
1.77 (m, 2H), 1.91 (s, 3H), 2.00 (m, 1H), 2.95 (q, J ) 6.1 Hz,
2H), 3.80 (s, 3H), 3.82 (s, 3H), 3.84-4.92 (m, 4H), 4.06-4.18
(m, 3H), 4.20-4.38 (m, 4H), 4.38-4.45 (m, 3H), 4.65 (m, 1H),
5.11 (d, J ) 6.8 Hz, 1H), 5.19-5.23 (m, 3H), 5.86 (m, 1H), 6.92
(bs, 1H), 6.95 (s, 1H), 7.02 (s, 1H), 7.19 (bs, 1H), 7.29 (t, J )
7.2 Hz, 2H), 7.35-7.43 (m, 4H), 7.52-7.63 (m, 3H), 7.63-7.75
(m, 2H), 7.84-7.91 (m, 3H), 8.03 (bs, 1H), 8.33 (dd, J ) 1.6,
8.4 Hz, 1H), 8.77 (dd, J ) 1.3, 4.1 Hz, 1H), 9.89 (bs, 1H), 11.28
(bs, 1H); LC-MS m/z (ES+) 1130 (M + H)+. Anal. (C63H67N7O13)
C, H, N.
(5-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-meth-
yl-butyrylamino]-5-{4-[1-hydroxymethyl-3-(5,6,7-tri-
methoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-pyrrolo-
[3,2-f]quinolin-5-yloxymethyl]-phenylcarbamoyl}-
pentyl)carbamic Acid Allyl Ester (45). Methanol (10 mL)
was treated with acetyl chloride (30 µL), and the resulting
mixture was added to 44 (47 mg, 41.6 µmol). The mixture was
stirred for 16 h at an ambient temperature and was then
heated at 50 °C for 6 h. The reaction mixture was neutralized
with DIPEA until the pH was 7. The mixture was concentrated
under reduced pressure, and the resulting residue was dis-
solved in dichloromethane (3 mL) and directly aspirated onto
a 1-mm radial Chromatotron plate. The product was eluted
with a 0-5% methanol/dichloromethane gradient, and the
product-containing fractions were combined and concentrated
to give 32 mg (71%) of 45 as a white solid: 1H NMR (DMSO-
d6) δ 0.86 (m, 6H), 1.24-1.47 (m, 4H), 1.58-1.75 (m, 2H), 2.00
(spt, J ) 6.7 Hz, 1H), 2.95 (q, J ) 6.3 Hz, 2H), 3.48 (m, 1H),
3.70 (m, 1H), 3.78 (s, 3H), 3.81 (s, 3H), 3.85-3.95 (m, 5H),
4.09-4.37 (m, 3H), 4.41 (d, J ) 5.2 Hz, 2H), 4.49 (dd, J ) 2.5,
11 Hz, 1H), 4.60 (t, J ) 9.2 Hz, 1H), 4.84 (dd, J ) 5.3, 5.3 Hz,
1H), 5.11 (dd, J ) 1.6, 10.6 Hz, 1H), 5.19-5.24 (m, 3H), 5.86
(m, 1H), 6.91 (bs, 1H), 6.96 (s, 1H), 7.04 (d, J ) 2.0 Hz, 1H),
7.19 (s, 1H), 7.29 (t, J ) 7.4 Hz, 2H), 7.39 (m, 2H), 7.43 (d, J
) 9.3 Hz, 2H), 7.51 (dd, J ) 4.1, 8.4 Hz, 1H), 7.59 (d, J ) 8.6
Hz, 2H), 7.67-7.72 (m, 2H), 7.83-7.88 (m, 3H), 8.15 (bs, 1H),
8.30 (dd, J ) 1.8, 6.4 Hz, 1H), 8.75 (dd, J ) 1.9, 4.1 Hz, 1H),
9.88 (bs, 1H), 11.22 (bs, 1H); LC-MS m/z (ES+) 1088 (M +
H)+.
{5-{4-[1-Chloromethyl-3-(5,6,7-trimethoxy-1H-indole-
2-carbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-
yloxymethyl]-phenylcarbamoyl}-5-[2-(9H-fluoren-9-yl-
methoxycarbonylamino)-3-methyl-butyrylamino]-pentyl}-
carbamic Acid Allyl Ester (46). To a mixture of 45 (32 mg,
29 µmol) in dichloromethane (1.5 mL) was added triph-
enylphospine (23 mg, 87 µmol) and carbon tetrachloride (25
µL, 260 µmol). The reaction mixture was heated at reflux for
30 min. An additional quantity of both triphenylphospine (23
mg, 87 µmol) and carbon tetrachloride (25 µL, 260 µmol) was
added, and the reaction mixture was heated for an additional
1 h. The reaction mixture was directly aspirated onto a 1-mm
radial Chromatotron plate, and the product was eluted with
a 0-3% methanol/dichloromethane gradient to give 27.4 mg
(86%) of 46 as a white solid: 1H NMR (DMSO-d6) δ 0.86 (t, J
) 7.2 Hz, 6H), 1.25-1.43 (m, 4H), 1.57-1.72 (m, 2H), 2.01 (m,
1H), 2.95 (q, J ) 7.2 Hz, 2H), 3.80 (s, 3H), 3.82 (s, 3H), 3.88-
3.94 (m, 4H), 4.02 (dd, J ) 3.5, 10.8 Hz, 1H), 4.21-4.32 (m,
5H), 4.41 (d, J ) 5.3 Hz, 2H), 4.50 (d, J ) 10.5 Hz, 1H), 4.73
(t, J ) 11.0 Hz, 1H), 5.09 (d, J ) 10.6 Hz, 1H), 5.19-5.23 (m,
3H), 5.86 (m, 1H), 6.91 (bs, 1H), 6.96 (s, 1H), 7.04 (s, 1H), 7.18
(b s, 1H), 7.29 (t, J ) 7.6 Hz, 2H), 7.34 (t, J ) 7.0 Hz, 2H),
7.42 (d, J ) 8.2 Hz, 2H), 7.53 (dd, J ) 4.3, 8.6 Hz, 1H), 7.59
(d, J ) 8.6 Hz, 2H), 7.68 (d, J ) 7.8 Hz, 2H), 7.80-7.88 (m,
3H), 8.09 (bs, 1H), 8.35 (d, J ) 8.6 H, 1H), 8.77 (t, J ) 1.6 Hz,
1H), 9.98 (bs, 1H), 11.26 (bs, 1H); LC-MS m/z (ES+) 1106 (M
+ H)+.
[1-(5-Amino-1-{4-[1-chloromethyl-3-(5,6,7-trimethoxy-
1H-indole-2-carbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quin-
olin-5-yloxymethyl]-phenylcarbamoyl}-pentylcarbamoyl)-
2-methyl-propyl]-carbamic Acid 9H-fluoren-9-ylmethyl
Ester (47). To a solution of 46 (8.6 mg, 7.8 µmol) in dichlo-
romethane (300 µL) was added camphor sulfonic acid (4 mg,
16 µmol) and NaSO2Ph (3 mg, 16 µmol). Tetrakistriphen-
ylphospine palladium (1 mg, 0.78 µmol) was added. After 10
min, the reaction mixture was directly aspirated onto a 1-mm
radial Chromatotron plate and eluted with a 1-5% methanol/
ammonia-saturated dichloromethane gradient to give a single

1356 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Jeffrey et al.
254-nm UV-active band, which was collected and concentrated
to give 6.5 mg (82%) of 47 as a solid: LC-MS m/z (ES+) 1022
(M + H)+.
7.2 Hz, 1H), 8.09 (bs, 1H), 8.35 (dd, J ) 1.6, 8.5 Hz), 8.77 (d,
J ) 2.5 Hz, 1H), 9.78 (bs, 1H), 11.41 (bs, 1H); LC-MS m/z
(ES+) 1147 (M + H)+.
Maleimide 52. To a solution of 51 (4.9 mg, 4.9 µmol) in
DMF (0.2 mL) was added maleimidocaproyl NHS ester 13 (4.2
mg, 12.8 µmol). The mixture was stirred for 16 h at an ambient
temperature, and the reaction mixture was concentrated. The
resulting residue was purified via radial chromatography on
a 1-mm plate. The product was eluted with a 1-5% methanol/
dichloromethane gradient, and a single UV-active band at 254
nm was collected to give 5.2 mg (79%) of 52 as a solid. This
material was carried forward without characterization.
Amine-TFA Salt 29. See Boc-removal procedure for the
synthesis of 28. Compound 52 (8.5 mg, 6.3 µmol) was depro-
tected to provide 10.4 mg of 29 as a solid TFA salt complex:
1H NMR (DMSO-d6) δ 0.83 (d, J ) 6.6 Hz, 3H), 0.86 (d, J )
7.0 Hz, 3H), 0.92-1.00 (m, 4H), 1.12-1.20 (m, 3H), 1.28-1.79
(m, 10H), 1.96 (q, J ) 7.7 Hz, 1H), 2.03 (t, J ) 7.4 Hz, 1H),
2.32-2.40 (m, 1H), 2.74-2.81 (m, 2H), 3.16 (q, J ) 5.9 Hz,
2H), 3.33-3.39 (m, 5H), 3.49 (m, 12H), 3.59 (m, 2H), 3.80 (s,
3H), 3.82 (m, 3H), 3.86-3.95 (m, 4H), 4.04 (m, 1H), 4.20 (dd,
J ) 8.0, 15.3 Hz, 1H), 4.30 (m, 1H), 4.37 (m, 1H), 5.50 (m,
1H), 4.78 (m, 1H), 5.23 (s, 2H), 6.98-7.0 (m, 2H), 7.10 (d, J )
2.2 Hz, 1H), 7.37-7.50 (m, 2H), 7.56-7.67 (m, 4H), 7.80 (t, J
) 15.3 Hz, 1H), 7.91 (d, J ) 8.6 Hz, 1H), 8.15 (d, J ) 8.0 Hz,
1H), 8.45 (m, 1H), 8.78 (dd, J ) 1.6, 4.1 Hz, 1H), 10.02 (s, 1H),
11.51 (s, 1H); UV λmax 312, 250 nm; anal. HPLC (gradient A)
tR ) 4.33 min, 91% AUC340, (gradient N) tR ) 10.29 min, 93%
AUC340; LC-MS m/z (ES+) 1240 (M + H)+; HRMS m/z for
C63H83ClN9O15+ (M + H)+ calcd, 1240.5697; found, 1240.5679.
Conjugate Preparation. The mAbs (>5 mg/mL) in PBS
containing 50 mM sodium borate, pH 8.0, were treated with
dithiothreitol (10 mM final) at 37 °C for 30 min. After gel
filtration (G-25, PBS containing 1mM DTPA), thiol determi-
nation using 5,5′-dithiobis(2-nitrobenzoic acid) indicated that
there were approximately eight thiols per mAb. To the reduced
mAb at 4 °C was added the maleimide or bromoacetamide (0.5
M sodium borate buffer pH 9 was required to promote mAb
alkylation with bromoacetamide) drug derivatives (1.2 equiv/
SH group) in cold DMSO (20% v/v). After 1 h, the reactions
were quenched with excess cysteine; the conjugates were
concentrated by centrifugal ultrafiltration, gel filtered (G-25,
PBS), and sterile filtered. Protein concentration and drug
loading were determined by spectral analysis at 280 and 320
nm, respectively. Size-exclusion HPLC was used to determine
percent monomer of each conjugate prepared, and RP-HPLC
established that there was less than 0.5% unconjugated
cysteine-quenched drug.
Cathepsin B Drug Release Assay. Human liver cathepsin
B (Calbiochem no. 219364, 5U) as provided (49.1 µL) was
constituted into a glycerol (46 µL), 0.5 M sodium acetate (4
µL), and 0.5 M EDTA (0.2 µL) to a final enzyme concentration
of 0.275 mg/mL. The specific activity was determined to be 75
(µg/min)/mg.39 The enzyme solution (6 µL) was diluted into
water (168 µL), buffered with 0.5 M sodium acetate pH 5 (60
µL), and treated with 0.1 M DTT (12 µL). The solution was
incubated at 37 °C for 15 min. The conjugates cAC10-28 and
cAC10-29 (56 µL of a 1-mg/mL solution) were added, and the
mixtures were incubated at 37 °C. Aliquots (10 µL) were taken
at 30 min, 1 h, 2.5 h, and 6 h and analyzed by SEC HPLC.
Drug release from the conjugates was determined by measur-
ing the AUC for drug chromophore (320 nm) relative to protein
absorbance (280 nm) associated with the eluting conjugate.
{5-{4-[1-Chloromethyl-3-(5,6,7-trimethoxy-1H-indole-
2-carbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-
yloxymethyl]-phenylcarbamoyl}-5-[2-(9H-fluoren-9-yl-
methoxycarbonylamino)-3-methyl-butyrylamino]-pentyl}-
carbamic Acid tert-Butyl Ester (48). To a THF (0.5 mL)
solution of 47 (6.5 mg, 6.4 µmol) was added Boc anhydride (50
µL). The reaction mixture was stirred for 10 min and was
directly aspirated onto a 1-mm radial Chromatotron plate, and
the product was eluted with a 0-3% methanol/dichloromethane
gradient. A single band was collected, and the fractions were
concentrated to give 7.0 mg (98%) of 48 as an amorphous white
solid: 1H NMR (DMSO-d6) δ 0.85 (d, J ) 7.0 Hz 3H), 0.88 (d,
J ) 8.4 Hz, 3H), 1.25-1.44 (m, 13H), 1.56-1.75 (m, 2H), 1.99
(m, 1H), 2.88 (q, J ) 5.7 Hz, 2H), 2.93 (bs, 1H), 3.80 (s, 3H),
3.83 (s, 3H), 3.88-3.94 (m, 5H), 4.04 (dd, J ) 3.7, 11.0 Hz,
1H), 4.20-4.33 (m, 4H), 4.42 (m, 1H), 4.50 (d, J ) 11.2 Hz,
1H), 4.77 (t, J ) 9.0 Hz, 1H), 5.21 (s, 2H), 6.62 (bs, 1H), 6.96
(s, 1H), 7.07 (d, J ) 2.0 Hz, 1H), 7.31 (t, J ) 7.0 Hz, 2H), 7.38-
7.45 (m, 3H), 7.56 (dd, J ) 4.1, 8.4 Hz, 2H), 7.61 (d, J ) 8.6
Hz, 2H), 7.72 (t, J ) 8.0 Hz, 1H), 7.86 (d, J ) 7.6 Hz, 1H),
7.97 (d, J ) 7.2 Hz, 1H), 8.13 (bs, 1H), 8.39 (dd, J ) 1.9, 8.7
Hz, 1H), 8.78 (dd, J ) 1.6, 4.1 Hz, 1H), 9.98 (bs, 1H), 11.41
(bs, 1H); LC-MS m/z (ES+) 1122 (M + H)+.
(5-(2-Amino-3-methyl-butyrylamino)-5-{4-[1-chloro-
methyl-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-di-
hydro-1H-pyrrolo[3,2-f]quinolin-5-yloxymethyl]-phenyl-
carbamoyl}-pentyl)carbamic acid tert-Butyl Ester (49).
To a 0 °C solution of 48 (17 mg, 15 µmol) in dichloromethane
(4 mL) was added piperidine (1 mL). The reaction mixture was
stirred for 5 min, before being concentrated under reduced
pressure. The resulting residue was dissolved in dichlo-
romethane (1 mL) and aspirated directly onto a 1-mm radial
Chromatotron plate. The product was eluted with a 1-5%
methanol/ammonia-saturated dichloromethane gradient. The
second major 254-nm UV-active band observed at 254 nm was
collected (first band dibenzofulvene) to give 13.1 mg (88%) of
49 as a solid: 1H NMR (DMSO-d6) δ 0.78 (d, J ) 6.5 Hz, 3H),
0.89 (d, J ) 6.8 Hz, 3H), 1.24-1.42 (m, 13H), 1.57-1.75 (m,
2H), 1.95 (m, 1H), 2.89 (q, J ) 6.4 Hz, 2H), 2.97 (bs, 2H), 3.02
(m, 1H), 3.80 (s, 3H), 3.83 (s, 3H), 3.90 (dd, J ) 7.0, 11.2 Hz,
1H), 3.94 (s, 3H), 4.04 (dd, J ) 3.5, 11.2 Hz, 1H), 4.29 (m, 1H),
4.45 (m, 1H), 4.76 (t, J ) 10 Hz, 1H), 5.21 (s, 2H), 6.64 (bs,
1H), 6.98 (s, 1H), 7.07 (d, J ) 1.6, 1H), 7.45 (d, J ) 8.4 Hz,
2H), 7.56 (dd, J ) 4.9, 8.4 Hz, 1H), 7.62 (d, J ) 8.4 Hz, 2H),
8.03 (bs, 1H), 8.12 (bs, 1H), 8.38 (d, J ) 7.2 Hz, 1H), 8.77 (t,
J ) 1.4 Hz, 1H), 10.04 (bs, 1H), 11.42 (bs, 1H); LC-MS m/z
(ES+) 900 (M + H)+.
Tetraethylene Glycol 50. To a solution of 49 (13 mg, 14.4
µmol) and the TEG acid 24 (8.3 mg, 17 µmol) in chloroform
(0.5 mL) was added EEDQ (4.2 mg, 17 µmol), and the reaction
mixture was stirred at an ambient temperature for 16 h. The
mixture was directly aspirated onto a 1-mm radial Chroma-
totron plate, and the product was eluted with a 1-5%
methanol/dichloromethane gradient. A single UV-active band
at 254 nm was collected and concentrated to give 17.8 mg
(90%) of 50 as an amorphous solid: LC-MS m/z (ES+) 1369
(M + H)+.
Amine 51. See procedure for the synthesis of 49 for a
general procedure for Fmoc group deprotection. Compound 50
(17.8 mg, 13 µmol) was deprotected to give 13.1 mg (88%) of
51 as an amorphous solid: 1H NMR (DMSO-d6) δ 0.84 (d, J )
6.8 Hz, 3H), 0.86 (d, J ) 6.8 Hz, 3H), 1.23-1.42 (m, 13H), 1.62
(m, 1H), 1.73 (m, 1H), 2.00 (sept, J ) 6.5 Hz, 1H), 2.40 (m,
1H), 2.63 (t, J ) 5.1 Hz, 1H), 2.89 (q, J ) 5.9 Hz, 2H), 3.34 (t,
J ) 3.3 Hz, 2H), 3.47-3.51 (m, 16H), 3.62 (m, 2H), 3.80 (s,
3H), 3.82 (s, 3H), 3.88 (dd, J ) 3.5, 11.0 Hz, 1H), 3.94 (s, 3H),
4.02 (dd, J ) 3.3, 10.8 Hz, 1H), 4.19 (t, J ) 8.6 Hz, 1H), 4.26
(m, 1H), 4.35 (m, 1H), 4.49 (d, J ) 11 Hz, 1H), 4.73 (t, J )
11.1 Hz, 1H), 6.97 (s, 2H), 6.51 (bs, 1H), 6.97 (s, 1H), 7.04 (s,
1H), 7.43 (d, J ) 8.2 Hz, 2H), 7.53 (dd, J ) 4.1, 8.4 Hz, 1H),
7.60 (d, J ) 8.6 Hz, 2H), 7.70 (d, J ) 7.2 Hz, 1H), 7.83 (d, J )
Stability Assay. Both compounds 2 and 3 were dissolved
in DMSO (1 mM solutions), and 100 µL of each was added to
both pH 5 and pH 7 ammonium phosphate buffers (900 µL).
The mixtures were vortexed, and all four samples were
incubated at 37 °C and monitored by HPLC-MS at various
intervals up to and beyond 24 h. Remaining starting materials,
and new product formation, were calculated based on percent-
age of the AUC.
Cell Culture. The CD30+ cell line Karpas 299 was pur-
chased from the DSMZ (Braunschwieg, Germany) and grown
in RPMI 1640 + 10% FBS (both Invitrogen, Carlsbad, CA).

Antibody Minor Groove Binder Conjugates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1357
(8) Ojima, I.; Geng, X.; Wu, X.; Qu, C.; Borella, C. P.; Xie, H.;
Wilhelm, S. D.; Leece, B. A.; Bartle, L. M.; Goldmacher, V. S.;
Chari, R. V. Tumor-specific novel taxoid-monoclonal antibody
conjugates. J. Med. Chem. 2002, 45, 5620-5623.
The cell line pair NCI-H69 and its P-glycoprotein-expressing
subline NCI-H69/LX4 were purchased from the ECACC (Sal-
isbury, England) and grown in RPMI 1640 supplemented with
2 mM L-glutamine and 10% FBS. NCI-H69/LX4 growth
medium also contained 0.4 µg/mL doxorubicin as directed by
the manufacturer. Bulk cultures of cells were routinely pas-
saged 2 times per week, and all assays were initiated while
cells were in log phase.
(9) Chari, R. V.; Jackel, K. A.; Bourret, L. A.; Derr, S. M.; Tadayoni,
B. M.; Mattocks, K. M.; Shah, S. A.; Liu, C.; Blattler, W. A.;
Goldmacher, V. S. Enhancement of the selectivity and antitumor
efficacy of
a CC-1065 analogue through immunoconjugate
formation. Cancer Res. 1995, 55, 4079-4084.
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selectivity of adriamycin-monoclonal antibody conjugate via a
poly(ethylene glycol)-based cleavable linker. J. Controlled Re-
lease 2002, 79, 229-242.
In Vitro Growth Inhibition. For assays to assess the
activity of cAC10 mAb-ADCs, cells were collected and plated
in 96-well black-sided plates at a density of 10 000 cells/well
in 150 µL. Serial dilutions of mAb-ADC were made in medium
at a 4× working concentration, and 50 µL of the dilution was
transferred from the dilution plate to the cultures. After
addition of ADC, cultures were incubated to 96 h at 37 °C.
Four hours before the end of the assay, 50 µL of 250 µM
resazurin (Sigma, St. Louis, MO) in medium was added to each
well (final concentration, 50 µM), and the plates were returned
to the incubator. At the completion of the assay, plates were
read on a Fusion HT microplate reader (Packard, Meriden,
CT) using an excitation wavelength of 525 nm and an emission
wavelength of 590 nm. For experiments to determine if the
drug was a substrate for P-glycoprotein, the NCI-H69 cell line
and the NCI-H69/LX4 subline were used.37 Cells were collected
and plated at 5000 (NCI-H69) or 8000 (NCI-H69/LX4) cells/
well in 150 µL of medium. After incubating 24 h to allow
surface protein reconstitution, serial dilutions of cBR96 mAb-
ADC were added as above, and the cultures were incubated
for 6 days at 37 °C. Cultures were then labeled with resazurin
as described above. Data from all assays were reduced using
GraphPad Prism version 4 for Windows (GraphPad Software,
San Diego, CA). The IC50 concentrations were determined from
four-parameter curve fits and are defined as the concentration
that inhibits cellular growth by 50% compared with untreated
cultures.
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Acknowledgment. The authors would like to thank
Brian Toki for many useful discussions pertaining to
para-aminobenzyl ether cleavage chemistry, Damon
Meyer for his assistance with drug-linker conjugation,
Che-Leung Law for his support of m1F6-MGB conjugate
exploration, and Alan Wahl for biochemistry support
and guidance.
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(19) Xie, H.; Audette, C.; Hoffee, M.; Lambert, J. M.; Blattler, W. A.
Pharmacokinetics and biodistribution of the antitumor immu-
noconjugate, cantuzumab mertansine (huC242-DM1), and its
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Supporting Information Available: Elemental analysis
results for compounds 17a, 18a, 20a, 21a, 39, and 44. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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mate prodrugs for use with nitroreductase-mediated gene-
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(22) King, H. D.; Dubowchik, G. M.; Mastalerz, H.; Willner, D.;
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(23) Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner, D.;
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