Studies toward the duocarmycin prodrugs for the antibody prodrug therapy approach

Article abstract of DOI:10.1016/j.tetlet.2009.03.205

A tricyclic precursor for the synthesis of the prodrugs of pro-1,2,9,9a-tetrahydrocyclopropa[c]benz-[e]indole-4-one tetramethoxyindolecarboxamide (CBI-TMI) was prepared using the ring-closing metathesis approach. The tricyclic intermediate was converted to an advanced precursor of a CBI-TMI prodrug equipped with a linker presumably suitable for activation using the aldolase catalytic antibody 38C2. An attempted 38C2-catalyzed two-step activation of the hydroxy-pro-CBI intermediate involving retro-aldol and the β-elimination reactions was also examined.

Full text of DOI:10.1016/j.tetlet.2009.03.205

Tetrahedron Letters 50 (2009) 2932–2935
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Studies toward the duocarmycin prodrugs for the antibody prodrug
therapy approach
*
Lian-Sheng Li, Subhash C. Sinha
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A tricyclic precursor for the synthesis of the prodrugs of pro-1,2,9,9a-tetrahydrocyclopropa[c]benz-
[e]indole-4-one tetramethoxyindolecarboxamide (CBI-TMI) was prepared using the ring-closing metath-
esis approach. The tricyclic intermediate was converted to an advanced precursor of a CBI-TMI prodrug
equipped with a linker presumably suitable for activation using the aldolase catalytic antibody 38C2.
An attempted 38C2-catalyzed two-step activation of the hydroxy-pro-CBI intermediate involving retro-
aldol and the b-elimination reactions was also examined.
Received 5 February 2009
Revised 24 February 2009
Accepted 30 March 2009
Available online 5 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Selective delivery of cytotoxins to tumor and cancer-associated
cells without affecting the normal cells remains one of the impor-
tant unmet goals.1 We are developing several alternative ap-
proaches, including the chemical programing2 of the catalytic
aldolase antibody (Ab) 38C23 using inhibitors of the cancer-asso-
ciated antigens/receptors, and use of the chemically programmed
Ab 38C2 (cpAb or cp38C2)4 and inhibitors to selectively deliver a
cytotoxin to tumor site. In one approach, we are using chemically
labeled Ab 38C2 (clAb or cl38C2), that possess both the catalytic
and the cell-targeting properties for a selective prodrug activation
at the tumor site.5 The other counterpart of this approach is a
prodrug that should be nontoxic and efficiently activated using
Ab 38C2 or the cl38C2 to afford the therapeutically effective cyto-
toxins for the desired outcome. Unfortunately, all prodrugs avail-
able so far lack these criteria, mainly due to a combination of the
drugs examined and that the prodrugs could not be activated effi-
ciently, and they cannot be advanced to preclinical studies.6,7
With new doxorubicin prodrugs, we have made some improve-
ments with respect to their toxicities and the activation rates,
but they might also not meet the desired criteria. We argue that
the desired prodrugs should (1) have sub- or low nanomolar
cytotoxic drug counterpart, (2) be at least 1000–10,000-fold less
toxic than the drug used, (3) have low nanomolar binding to
the catalyst, viz. cl38C2, and (4) be activated rapidly. With these
hypotheses, we are seeking new prodrugs from a highly potent
cytotoxin, such as CBI-TMI (1).8
duplex DNA at the N-3 position of adenines in the minor groove.11
Figure 1 also shows structures of several seco-CBI-TMI derivatives
(3–8), some of which were previously prepared and evaluated.12
These seco-CBI-TMIs are indeed the prodrug form of the corre-
sponding active CBI-TMI drugs, as the latter are produced in situ
before reacting with DNA. In fact, most duocarmycin analogs and
prodrugs were developed on this principle in that the cyclopropane
ring was generated by Winstein cyclization13 of the chloromethyl
group or an analogous reactive function and facilitated by the free
phenolic hydroxy or amine functional groups in 3–5. Similarly, the
isomeric compounds 6–8 could also undergo cyclization reaction
affording 1 or its corresponding imine analogs. Because these com-
OMe
OMe
OMe
OMe
OMe
N
N
N
H
N
H
OMe
MeO2C
O
O
N
H
O
O
CBI-TMI, 1
( IC50, 0.03 nM, L1210 cells)
Doucarmycin SA, 2
(IC50 = 0.01 nM, L1210 cells)
OMe
OMe
OMe
OMe
Cl
Cl
OMe
OMe
N
N
N
N
H
CBI-TMI (1) is a synthetic analog9 of the naturally occurring
antitumor antibiotic, duocarmycin SA (2)10 (Fig. 1). CBI (1,2,9,9a-
tetrahydro-cyclopropa[c]-benz[e]indole-4-one) derivatives are
both chemically stable, and also synthetically easily accessible.
Like the parent molecule, CBI-TMI selectively binds and alkylates
H
O
O
X
X
3: X = OH, IC50 (0.04 nM, L1210 cells)
4: X = NH2, IC50 (0.43 nM, AA8 cells)
5: X = NHMe, IC50 (0.17 nM, AA8 cells)
6: X = OH
7: X = NH2
8: X = NHMe
* Corresponding author. Tel.: +1 858 784 8512; fax: +1 858 784 8732.
E-mail address: subhash@scripps.edu (S.C. Sinha).
Figure 1. Structure of duocarmycin, its non-natural analog, CBI-TMI and its
precursors.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.205

L.-S. Li, S. C. Sinha / Tetrahedron Letters 50 (2009) 2932–2935
2933
OMe
OMe
OMe
the Ab 38C2-catalyzed activation. Like the previously studied pro-
drugs of doxorubicin, etoposide, camptothecin, and enediynes,
these prodrugs also possessed an ‘aldol-Michael’ linker, which on
treatment with Ab 38C2 would start the activation by the retro-al-
dol reaction. The resultant labile intermediates would undergo
b-elimination to give 6 from 6a. Other prodrugs would continue
subsequent transformation, including the decarboxylation reaction
and urea formation, under the physiologic conditions to give 6, 7,
or 8, which should serve as the precursor of compound 1 or its
imine analogs.
Cl
N
N
H
O
X
6a,b: X = a or b
7c: X = c
8d: X = d
O
O
N
O
We anticipated that prodrug 6a could be readily prepared by
modifying the previously described synthesis of 1 via a tricyclic
intermediate, 14a.18 However, we designed an alternative route
using the Grubbs ring-closing metathesis (RCM) reaction19 for
the synthesis of 14a (Scheme 1). Thus, the required diene precursor
13a for the RCM reaction was prepared starting with the readily
available phenol 10.20 The latter was synthesized from a commer-
cial compound, 1,3-dinaphthol, 9, in three steps, including reaction
of compound 9 with 4-methoxybenzyl amine (PMB-amine) to give
3(4-methoxybenzyl)amino-1-naphthol, deprotection of the latter
compound under acidic conditions using H2SO4 and TFA, and Boc
protection of the resulting free amine, 2-amino-1-naphthol, using
Boc anhydride, as described by Gieseg et al. Compound 10 was next
protected using benzylbromide and K2CO3 in the presence of tetra-
butyl-ammonium iodide, and the resulting product was iodinated
using NIS21 to yield the iodonaphthalene derivative 11. The latter
product underwent Pd-catalyzed Stille coupling reaction22 with
tributylvinylstannane to afford the vinylnaphthalene derivative
12. N-Alkylation of compound 12 with allylbromide using NaH in
DMF provided the requisite diene 13a for the RCM step that was
accomplished using the Grubbs catalyst I affording the tricyclic
compound 13a in good yield. Encouraged by this result, we exam-
ined whether the above RCM approach could be utilized to prepare
the ring expanded analogs of 14a (7-, 8-, and 9-membered cyclic
alkenes) and their corresponding prodrugs. Accordingly, dienes
13b–d were prepared by treatment of 13 with 4-iodo-1-butene,
5-iodo-1-pentene, and 6-iodo-1-hexene, respectively, and then
submitted to the RCM reactions using Grubbs catalysts I or II.
Somewhat surprisingly, only the dimerization products 15b–d
were obtained in good yields under the attempted conditions pre-
sumably due to the increased ring strain in 7- to 9-membered tri-
cyclic olefin products.
O
N
N
HO
O
HO
O
O
O
O
a
b
H
N
O
HO
HO
O
O
O
c
d
Figure 2. Structure of the proposed prodrugs of CBI-TMI precursors that may be
activated using antibody 38C2-catalyzed retro-aldol and b-elimination reactions.
pounds could cause indiscriminate toxicity to normal cells, we
decided to explore a prodrug approach for their delivery.14 In this
strategy, a prodrug functionalized with a linker can be selectively
activated using tumor-associated proteases (TAPs)15 or a non-
endogenous enzyme, including catalytic Ab.16 Here, we report
our preliminary study toward the synthesis of a CBI-TMI prodrug
6a and its activation using Ab 38C2.17
Figure 2 shows structures of the CBI-TMI prodrug, 6a, as well as
several designed analogs, 6b, 7c, and 8d, as the viable candidates of
I
NHBoc
OH
NHBoc
a, 72%
Ref. 7
OBn
OH
OH
9
10
11
NHBoc
NBoc
BrCH2CH=CH2
c, 88%
b, 95%
OBn
OBn
With the intermediate 14a in hand, synthesis of the desired pre-
cursor 25 of the prodrug 6a progressed as shown in Scheme 2, via a
coupling of amine 20 with the acid chloride 23. Here, amine 20 was
prepared from the intermediate 14a and the acid chloride 23 was
obtained from the commercially available aldehyde 21. Thus, inter-
mediate 14a was hydroborated using BH3ÁSMe2 and the hydrobo-
rated product was oxidized with H2O2/NaOH giving alcohol 16.
The free hydroxyl group in 16 was protected as TBDPS ether, and
the benzyl group was removed by the Pd-catalyzed hydrogenolysis
affording intermediate 17. Compound 17 was alkylated with tosyl-
ate 18 and cesium carbonate in the presence of 18-Crown-6 to give
compound 19. The latter product underwent OsO4-catalyzed
dihydroxylation and subsequent oxidative cleavage of the resulting
diol with Pb(OAc)4 to afford an aldol product. The Boc group in the
latter compound was removed using TFA/CH2Cl2 to give a free
amine compound 20. Separately, chlorotrimethoxyindolecarboxy-
late, 23, was prepared by a usual acyl chloride forming reaction
of the readily available acid 22 (prepared from aldehyde 21)23 with
oxalyl chloride. Next amine 20 was coupled with acid chloride 23
affording amide 24, and the TBDPS protecting group in the latter
product was removed using HFÁPy giving the desired alcohol pre-
cursor, 25, of the prodrug 6a. We expected that compound 25
should be converted to 6a using CCl4 and PPh3, however to our sur-
prise, no such reaction took place.
12
13a
I(CH2)nCH=CH2
(n = 2, 3, 4)
c
PhCH=Ru(PCy3)2Cl2 (I) d, 80%
( )n
NBoc
NBoc
OBn
OBn
13b: n = 2, 80%
13c: n = 3, 85%
13d: n = 4, 88%
14a
PhCH=Ru(PCy3)2Cl2 (I), or
PhCH=Ru(PCy3)[cyclo-CH(NMes-CH2)2] (II)
d or e
OBn
Boc
( ) n
Boc
N
N
15b: n = 1, 60%
15c: n = 2, 70%
15d: n = 3, 60%
( )n
BnO
Scheme 1. Synthesis of tricyclic intermediate (14a) by RCM approach. Reagents
and conditions: (a) (i) BnBr, K2CO3, Bu4NI, DMF, (ii) NIS, p-TsOH, THF. MeOH; (b)
Bu3SnCH@CH2, Pd(PPh3)4 (5 mol %), 2,6-di-tert-butyl-4-methyl phenol, toluene; (c)
NaH, DMF; (d) for compound 14a: Grubbs cat
compounds 15b–d: Grubbs cat II (10 mol %), CH2Cl2.
I (10 mol %), CH2Cl2; (e) for

2934
L.-S. Li, S. C. Sinha / Tetrahedron Letters 50 (2009) 2932–2935
OH
TBDPSO
TBDPSO
TsO
HO
NBoc
NBoc
NBoc
NBoc
HO
18
a, 88%
b, 64%
c, 85%
OBn
OBn
OH
O
14a
17
16
19
TBDPSO
OMe
OMe
OMe
OMe
OMe
OMe
e
NH
d, 80%
O
O
Ref. 23
OHC
+
N
H
N
H
Cl
HO
OMe
OMe
OMe
O
23
22
21
20
HO
O
f, 60%
OMe
OMe
OMe
OMe
OMe
OMe
OH
OTBDPS
N
N
O
g, 84%
N
H
N
H
O
O
O
HO
25
HO
O
O
24
OMe
OMe
OMe
OMe
OMe
OMe
OH
OH
N
N
Ab 38C2, pH 7.4
PBS, pH 7.4
N
H
N
H
25
O
O
– [acetone]
– [MVK]
O
OH
26
27
O
Scheme 2. (A) Synthesis of an immediate precursor of the CBI-TMI prodrug 6a. Reagents and conditions: (a) BH3.SMe2, THF, 0 °C then H2O2, NaOH, 60 °C; (b) (i) TBDPSCl,
imidazole, DMF, 0 °C to rt, (ii) H2, Pd/C (10%), MeOH, rt; (c) Cs2CO3, 18-crown-6, CH3CN, rt; (d) (i) OsO4, NMO, acetone, H2O, rt, (ii) Pb(OAc)4, CH2Cl2, rt; (iii) TFA, CH2Cl2, 0 °C;
(e) (COCl)2, DMF (cat.), benzene; (f) Et3N, THF, 0 °C to rt; (g) HFÁPy, THF, 0 °C to rt. (B) Attempted Ab 38C2-catalyzed activation of the prodrug analog 25.
While we were yet to develop a suitable method for the conver-
sion of 25 to 6a, we examined the 38C2-catalyzed activation of the
former as a model to give phenol 27 via a Michael-type adduct, 26.
These observations suggested that new CBI prodrugs equipped
with longer or more active linker should be conceived for an effec-
tive release of the free drug when antibody 38C2 will be used as a
catalyst. In particular, the linker should be conjugated to parent
molecule 6 through the carbamate function rather than the ethe-
real function. Therefore, now we are focusing on new prodrugs of
6–8, such as 6b, 7c, and 8d, which are more likely to be activated
by Ab 38C2.
In conclusion, we have developed an efficient RCM approach to
the synthesis of the CBI skeleton, which can be applied to synthe-
size a wide variety of CBI analogs and CBI-derived prodrugs. In our
approach, the prodrugs would possess appropriate linkers that are
susceptible to aldolase antibodies or a tumor-associated protease.
The preliminary activation study with the precursor of our CBI-
TMI prodrug showed that the ethereal linker was not suitable for
an effective release of the drug using Ab 38C2. Further study along
this line as well as development of new prodrugs that can be acti-
vated using Ab 38C2 or TAPs are in progress and will be described
in a due course.
Thus, compound 25 (100
amount of antibody 38C2 (5
l
l
M) was incubated with a catalytic
M) at 37 °C overnight, and was then
analyzed by LCMS analysis. The LCMS showed the formation of the
retro-aldol intermediate 26 along with the remaining 25 in
approximately 3:2 ratio. However, the subsequent b-elimination
of 26 to give 27 was very slow, which was surprising because the
b-ketoalkylether of a phenolic drug was reported to undergo b-
elimination reaction to produce free drug under the physiologic
conditions. Moreover, antibody 38C2 was also known to catalyze
b-elimination reaction of b-alkoxy-ketones and aldehydes to give
free alcohol or phenol and the
a,b-unsaturated aldehydes or ke-
tones. Whereas there is no clear explanation why intermediate
26 does not undergo b-elimination reaction to produce 27 at pH
7.4 (PBS buffer), the inertness of the former intermediate to anti-
body 38C2 can be due to the steric hindrance of the substrate. Pre-
sumably, the ketone linker in intermediate 26 is very close to the
bulky drug molecule, and out of the reach of the reactive lysine res-
idues in the antibody 38C2 binding sites. Therefore the antibody
could not catalyze b-elimination reaction in intermediate 26. This
result may not be surprising, because a compound bearing a longer
linker is activated faster than one coupled to an analogous shorter
linker, as found in our recent studies on doxorubicin prodrugs.
Acknowledgments
We thankfully acknowledge the Skaggs Institute for Chemical
Biology and National Cancer Institute (R01CA120289) for the
financial support.

L.-S. Li, S. C. Sinha / Tetrahedron Letters 50 (2009) 2932–2935
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Supplementary data
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Supplementary data (spectroscopic data and experimental pro-
cedures) associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2009.03.205.
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