Pyrrolo[2,1-c][1,4]benzodiazepine-β-glucuronide prodrugs with a potential for selective therapy of solid tumors by PMT and ADEPT strategies

Article abstract of DOI:10.1016/j.bmcl.2008.05.038

Pyrrolo[2,1-c][1,4]benzodiazepine-β-glucuronide prodrugs 15a-b, with a potential for selective therapy of solid tumors by PMT and ADEPT have been designed, synthesized and evaluated for selective cytotoxicity in the presence of the enzyme β-glucuronidase. The prodrugs have been found to possess reduced cytotoxicity compared to the parent moieties, and are excellent substrates for the enzyme, exhibiting cytotoxicity selectively in the presence of the enzyme. Enhanced water solubility and improved stability are the other important outcomes upon modifying these molecules as their prodrugs.

Full text of DOI:10.1016/j.bmcl.2008.05.038

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3769–3773
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Bioorganic & Medicinal Chemistry Letters
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Pyrrolo[2,1-c][1,4]benzodiazepine-b-glucuronide prodrugs with a potential
for selective therapy of solid tumors by PMT and ADEPT strategies
a
a
b
b
b
Ahmed Kamal a, , Venkatesh Tekumalla , P. Raju , V. G. M. Naidu , Prakash V. Diwan , Ramakrishna Sistla
*
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Division of Pharmacology, Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrrolo[2,1-c][1,4]benzodiazepine-b-glucuronide prodrugs 15a–b, with a potential for selective therapy
of solid tumors by PMT and ADEPT have been designed, synthesized and evaluated for selective cytotox-
icity in the presence of the enzyme b-glucuronidase. The prodrugs have been found to possess reduced
cytotoxicity compared to the parent moieties, and are excellent substrates for the enzyme, exhibiting
cytotoxicity selectively in the presence of the enzyme. Enhanced water solubility and improved stability
are the other important outcomes upon modifying these molecules as their prodrugs.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 26 March 2008
Revised 7 May 2008
Accepted 9 May 2008
Available online 16 May 2008
Keywords:
Pyrrolo[2,1-c][1,4]benzodiazepines
Prodrugs
b-Glucuronides
b-Glucuronidase
Selective therapy
PMT
ADEPT
Majority of the anticancer agents are cytotoxic in nature and
lack the ability to differentiate between a cancer and normal
healthy cell, to kill the cancer cells selectively without causing
any adverse effect to the non-target normal tissues.1 In general,
anticancer agents act preferentially on cells that are dividing. Con-
sequently, normal tissues that contain the most rapidly dividing
cells are also affected, such as the bone marrow, the gut mucosa
and hair follicles.
Enzyme-based therapies like antibody-directed enzyme pro-
drug therapy (ADEPT),2 gene directed enzyme prodrug therapy
(GDEPT),3 and prodrug monotherapy (PMT)4 could improve the
selectivity of the anticancer agents by transforming them into
non-toxic prodrugs that get selectively activated at the tumor site,
leading to localized toxicity. This kind of treatments could allow
the use of highly cytotoxic agents with simultaneous sparing of
the normal tissues.
Prodrug monotherapy is an approach, wherein an enzyme
which is intrinsically present in high concentrations in the malig-
nant tissues, is exploited to selectively activate a comparatively
non-toxic prodrug, leading to the formation of a toxic drug. The en-
zyme b-glucuronidase is reported to be present in high local con-
centrations in solid tumors compared to the normal tissues.5 The
liberation of lysosomal b-glucuronidase extracellularly leads to
the accumulation of the enzyme in the extracellular matrix of
the malignant tissues.6 Targeting the cancer tissues by exploiting
the presence of b-glucuronidase in high concentrations is advanta-
geous as the enzyme is not present in blood circulation.7 The en-
zyme is confined to lysosomes and therefore is not available for
activation of the prodrug in normal tissues. The enzyme is also re-
ported to be present in high concentrations in human breast can-
cers, which could be helpful for selective therapy of breast cancer.8
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs)9 are a class of
small molecules which bind to NH2 of the guanine in the minor
groove of DNA, leading to cytotoxicity. This class of molecules have
shown a potential to be developed as anticancer agents, and have
also been described to be more suitable for ADEPT strategy com-
pared to mustards, which are the most studied molecules for deliv-
ery by this strategy.10 The lack of selectivity towards cancer tissues
is one of the major drawbacks that is associated with anticancer
agents, including the PBDs that are in the process of development.
The advancement of these molecules as anticancer drugs has been
delayed due to various problems relating to high toxicity and reac-
tivity, at different stages of their development.11 Another short-
coming that makes these compounds less effective in the
biological system is their low water solubility leading to decrease
in bioavailability.
In an effort to improve the selectivity of PBDs and overall devel-
opment of these molecules as novel anticancer agents, we have ini-
tiated investigations towards the development of PBD prodrugs. In
* Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.038

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3769–3773
this direction two new PBD-b-glucuronide prodrugs 15a–b (, Fig.
2) with a potential for selective therapy of solid tumors by PMT
and ADEPT strategies have been designed synthesized and evalu-
ated for selective toxicity in the presence of the enzyme b-glucu-
ronidase. Compound 15a is a prodrug of an imine-amide mixed
dimer 16a (Fig. 1) that has been developed in our laboratory and
has shown promising anticancer activity,12 whereas 15b is a pro-
drug of benzyl ether analogue of a naturally occurring PBD moiety,
DC-81 16b (Fig. 1). These prodrugs possess other additional fea-
tures like enhanced water solubility and stability, apart from their
ability to get activated by the enzyme b-glucuronidase.
The preparation of the PBD intermediate for the synthesis of
carbinolamine-amide glucuronide prodrug was carried out
employing the literature method,12 by coupling intermediate 2
with excess of 1,5-dibromopentane to obtain 3, which in turn
was coupled with 6, to yield the intermediate 4. Compound 4 upon
reduction with SnCl2, 2H2O afforded the desired precursor 5a,
which is utilized for the synthesis of the prodrug 15a. The prepara-
tion of DC-81 benzyl ether glucuronide prodrug was carried out
according to the literature method.13
The synthesis of the b-glucuronide promoiety was carried out
starting from glucurolactone (7),14 which was treated with cata-
lytic amount of NaOMe, followed by Ac2O and HClO4 to obtain
the intermediate 8. This upon treatment with HBr in acetic acid
resulted in the formation of compound 9, which on coupling with
4-hydroxy-3-nitrobenzaldehyde in presence of silver oxide in ace-
tonitrile provided the intermediate 10. Reduction of 10 with
NaBH4, in chloroform and isopropyl alcohol afforded the desired
b-glucuronide promoiety 11 (Scheme 1).
The key step of coupling the PBD intermediates 5a–b with the
glucuronide promoiety 11 using triphosgene and dibutyltin dilau-
rate to yield the carbamates 12a–b, which upon deprotection of
the diethylthioacetal group provided the carbinolamine intermedi-
ates 13a–b. The carbinolamines 13a–b were deacetylated using
NaOMe in methanol at 0–5 °C to provide the intermediates 14a–
b, followed by the hydrolysis of the carboxylic ester afforded the
desired PBD b-glucuronide prodrugs 15a–b15 (Scheme 2).
The prodrugs were found to possess visibly high water solubil-
ity than their corresponding drugs. The enzymatic activation stud-
ies showed the prodrugs 15a–b to be excellent substrates of the
O
H
N
HO
N
N
O
O
H
H
H
N
MeO
N
N
OMe
MeO
O
O
O
16a
16b
Figure 1. Pyrro[2,1-c][1,4]benzodiazepines: 16a imine-amide mixed dimer; 16b DC-81.
O2N
O2N
HOOC
HOOC
HO
O
O
O
O
HO
HO
HO
O
C
O
C
OH
OH
O
N
O
N
O
OH
OH
H
N
N
N
O
CH2
O
BnO
MeO
5
H
H
H
N
OMe MeO
O
O
O
15a
15b
Figure 2. Pyrro[2,1-c][1,4]benzodiazepine b-glucuronide prodrugs: 15a prodrug of imine-amide mixed dimer; 15b is prodrug of benzyl ether analogue of DC-81.
O
MeOOC
AcO
MeOOC
HO
O
O
O
i
ii
AcO
O
AcO
AcO
OH
OAc
OAc
OAc
Br
OH
8
9
7
D-(+)-Glucuronic acid-γ -lactone
iii
NO2
NO2
COOMe
COOMe
OAc
O
O
iv
O
OAc
OAc
OHC
O
OAc
HO
OAc
OAc
11
10
Scheme 1. Reagents and conditions: (i) a—methanol, NaOMe, 60 min, rt, b—Ac2O, HClO4, rt, overnight, 50%; (ii) 30% HBr/acetic acid, 0–5 °C, overnight, 62%; (iii) Ag2O,
4-hydroxy-3-nitrobenzaldehyde, acetonitrile, 4 h, 73%; (iv) NaBH4, CHCl3, (CH3)2CHOH, 0–5 °C, 3 h;78%.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3769–3773
3771
O2N
MeOOC
O
O
AcO
AcO
O
OAc
C
O
NH2
NH
RO
RO
MeO
CH(SEt)2
CH(SEt)2
i
N
N
MeO
O
O
12a: R = A
12b: R = B
5a: R = A
5b: R = B
ii
O2N
R2OOC
R1O
R1O
O
OR1
O
O
C
O
OH
N
RO
H
N
O
H
N
O
CH2
MeO
5
H
O
N
OMe
13a: R = A, R1 = OAc, R2 = Me
14a: R = A, R1 = OH, R2 = Me
15a: R = A, R1 = OH, R2 = H
iii
iv
O
A
13b: R = B, R1 = OAc, R2 = Me
14b: R = B, R1 = OH, R2 = Me
15b: R = B, R1 = OH, R2 = H
iii
iv
CH2
B
Scheme 2. Reagents and conditions: (i) a—Et3N, CO(COCl3)2, 25 min; b—Comp. 11, cat. dibutyltin dilaurate, 12a: 74%, 12b: 71%; (ii) HgCl2, CaCO3, CH3CN/H2O, 4:1, 13a: 77%,
13b: 84%; (iii) MeOH, MeONa, ꢀ15 °C, 14a: 74%, 14b: 79%; (iv) LiOH, H2O/MeOH/THF, 15–20 min, 0–5 °C, 15a: 76%, 15b: 82%.
Mechanism of activation
O2N
O2N
HOOC
O
O
H
O
HO
HO
O
C
O
C
OH
O
N
O
N
OH
OH
N
N
RO
RO
β- glucuronidase
H
H
MeO
MeO
O
O
15a-b
self immolation
of spacer
N
O
O2N
HO
RO
O
H
N
H
O
CH2
5
H
N
16a: R =
16b: R =
MeO
OH
N
OMe
spacer
O
PBD imine
16a-b
CH2
Scheme 3.

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A. Kamal et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3769–3773
enzyme b-glucuronidase. The two b-glucuronide prodrugs were
found to be highly stable when incubated at a pH of 7 and 37 °C
in a 0.02 M phosphate buffer. However, when these prodrugs were
incubated under similar conditions in the presence of E. coli b-glu-
curonidase (250 U), where hydrolyzed completely to form the par-
ent, PBD imine moieties (Scheme 3). The HPLC analysis16 of the
reaction mixture at fixed intervals showed the consumption of
the prodrugs and the simultaneous formation of the peaks corre-
sponding to the parent drug as well as 4-(hydroxymethyl)-2-nitro-
phenol self-immolative spacer. Prodrug 15a gets activated almost
completely in 60 min to form 16a (Fig. 3), while 15b gets activated
completely in 20 min to form 16b (Fig. 4).
The pyrrolobenzodiazepine-b-glucuronide prodrugs 15a and
15b were evaluated for their selective cytotoxicity using MTT as-
120
15a
spacer
16a
100
80
60
40
20
0
0
20
40
60
80
100
Incubation time (min)
Figure 3. A graph showing the consumption (a decrease in the percentage peak
area) of the prodrug (15a) with simultaneous formation (an increase in the perc-
entage peak area) of the corresponding active parent PBD moiety (16a) and the
spacer during the HPLC analysis of activation reaction using b-glucuronidase.
120
15b
Spacer
16b
100
80
60
40
20
0
0
20
40
60
80
Incubation time (min)
Figure 4. A graph showing the consumption (a decrease in the percentage peak
area) of the prodrug (15b) with simultaneous formation (an increase in the perc-
entage peak area) of the corresponding active parent PBD moiety (16b) and the
spacer during the HPLC analysis of activation reaction using b-glucuronidase.
Table 1
Selective cytotoxicity of the prodrugs in HT 29 cell line
Prodrug IC50
IC50 (lmol) in the
Activation
IC50 (lmol) of
(lmol) presence of b-
factor (QIC50
)
the parent PBD
glucuronidase
15a
15b
2690
5280
12.88
41.68
208.85
126.67
8.12
30.19
Figure 5. Morphology of HT-29 cells (adenocarcinoma) treated with prodrugs and
drugs under microscope after 2 h incubation.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3769–3773
3773
say,17 in the presence of the enzyme b-glucuronidase using HT 29
colon cancer (adenocarcinoma) cell lines. They were found to pos-
sess highly reduced cytotoxicity compared to the parent imines
(Table 1). The prodrug 15a was found to kill 50% of cells (IC50) at
a concentration of 2690 lmol, while its parent drug (16a) showed
an IC50 of 8.12 lmol. The prodrug when incubated with the HT 29
cells in the presence of the enzyme b-glucuronidase exhibited an
IC50 of 12.88 lmol, which is similar to that of the drug. The IC50 val-
ues of the prodrugs and their corresponding parent moieties
shown in Table 1 clearly indicate that the toxicity of the prodrug
in the presence of the enzyme is similar to that of the parent drug.
2. (a) Bagshawe, K. D. Br. J. Cancer 1987, 56, 531; (b) Bagshawe, K. D. Drug Dev. Res.
1995, 34, 220; (c) Denny, W. A. Cancer Invest. 2004, 22, 604.
3. (a) Springer, C. J.; Niculescu-Duvaz, I. Adv. Drug Deliv. Rev. 1996, 22, 351; (b)
Niculescu-Duvaz, I.; Springer, C. J. Mol. Biotechnol. 2005, 30, 71.
4. (a) Bosslet, K.; Czech, J.; Hoffman, D. Tumor Target 1995, 1, 45; (b) Sinhababu, A.
K.; Thakker, D. R. Adv. Drug Deliv. Rev. 1996, 19, 241; (c) Bosslet, K.; Straub, R.;
Blumrich, M.; Czech, J.; Gerken, M.; Sperker, B.; Kroemer, H. K.; Gesson, J.-P.;
Koch, M. C. Cancer Res. 1998, 58, 1195.
5. (a) Bosslet, K.; Czech, J.; Hoffman, D. Tumor Target. 1995, 1, 45; (b) Bosslet, K.;
Straub, R.; Blumrich, M.; Czech, J.; Gerken, M.; Sperker, B.; Kroemer, H. K.;
Gesson, J.-P.; Koch, M.; Monneret, C. Cancer Res. 1998, 58, 1195.
6. Murdter, T.; Sperker, B.; Kivisto, K.; McClellan, M.; Fritz, P.; Friedel, G.; Linder,
A.; Bosslet, K.; Toomes, H.; Diekesmann, R.; Kroemer, H. Cancer Res. 1997, 57,
2440.
7. Angenault, S.; Thirot, S.; Schmidt, F.; Monneret, C.; Pfeiffer, B.; Renard, P. Bioorg.
Med. Chem. Lett. 2003, 13, 947.
The prodrug 15b also exhibited
a reduction in toxicity
(IC50 = 5280 lmol) when compared to the parent imine (16b),
which showed an IC50 of 30.19 lmol. In the presence of the enzyme
15b exhibited an IC50 value (41.68 lmol) similar to that of the par-
ent moiety.
Both the prodrugs were found to possess highly reduced toxic-
ity and were also good substrates for the enzyme b-glucuronidase.
The activity of the prodrugs upon activation by the enzyme ap-
proached that of the imines; indicating the complete activation
ability of the prodrugs.
The cells after incubating for 2 h with the drug 16b showed
shrinkage (Fig. 5b), on the other hand its prodrug 15b at the same
concentration did not show any shrinkage (Fig. 5c) and the mor-
phology was similar to that of the control cells (Fig. 5a). The pro-
drug 15b in the presence of the enzyme b-glucuronidase
exhibited cell morphology (shrinkage) similar to that of the cells
treated with the drug 16b (Fig. 5d).
In conclusion, the enzymatic activation and selective cytotoxic-
ity studies of the prodrugs reveal that these molecules possess
good activation ability and are potential candidates for use in
selective therapy of solid tumors. Compounds 15a and 15b possess
a potential for use in PMT of solid tumors that over express the en-
zyme b-glucuronidase.18 The prodrugs can also be used for anti-
body-directed enzyme prodrug therapy (ADEPT)19 of cancer. The
other important consequence of these glucuronide prodrugs was
highly improved water solubility provided to the drug by the glu-
curonide functionality.20
8. Albin, N.; Massaad, L.; Toussaint, C.; Mathieu, M.-C.; Morizet, J.; Parise, O.;
Gouyette, A.; Chabot, G. G. Cancer Res. 1993, 53, 3541.
9. Kamal, A.; Rao, M. V.; Laxman, N.; Ramesh, G.; Reddy, G. S. K. Curr. Med. Chem.,
Anticancer Agents 2002, 2, 215.
10. (a) Masterson, L. A.; Spanswick, V. J.; Hartley, J. A.; Begent, R. H.; Howard, P. W.;
Thurston, D. E. Bioorg. Med. Chem. Lett. 2006, 16, 252; (b) Bagshawe, K. D. Expert
Rev. Anticancer Ther. 2006, 6, 1421.
11. Walton, M. I.; Goddard, P.; Kelland, L. R.; Thurston, D. E.; Harrap, K. R. Cancer
Chemother. Pharmacol. 1996, 38, 431.
12. Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.;
Kondapi, A. K.; Sreenu, V. B.; Nagarajaram, H. A. J. Med. Chem. 2002, 45, 4679.
13. (a) Langley, D. R.; Thurston, D. E. J. Org. Chem. 1987, 52, 91; (b) Courtney, S. M.;
Thurston, D. E. Tetrahedron Lett. 1993, 34, 5327; (c) Bose, D. S.; Jones, G. B.;
Thurston, D. E. Tetrahedron 1992, 48, 751.
14. Bollenback, G. N.; Long, J. W.; Benjamin, D. G.; Lindquist, J. A. J. Am. Chem. Soc.
1955, 77, 3312.
25
15. 15a. mp 163–164 °C; ½aꢁD þ 83:33 (c = 0.3, CH3OH); 1H NMR (CD3OD,
500 MHz): d 1.32 (t, 2H, J = 7.18 Hz), 1.76–1.91 (m, 4H), 1.96–2.20 (m, 8H),
3.39–3.78 (m, 10H), 3.85 (s, 3H), 3.87 (s, 3H), 3.96–4.17 (m, 4H), 4.80–5.01 (br
s, 1H), 5.02–5.11 (m, 1H), 5.26 (d, 1H, J = 12.20 Hz), 5.68 (d, 1H, J = 10.05 Hz),
6.71 (s, 1H), 6.76 (s, 1H), 7.19 (s, 1H), 7.36 (s, 1H), 7.48 (s, 2H), 7.65 (s, 1H), 8.56
(s, 1H, NH); ESI-MS: m/z 964 [MꢀH]ꢀ; HRMS: [MꢀH]ꢀ calcd for C45H50N5O19
m/z 964.3100, found (ESI) m/z 964.3083; IR (KBr) (mmax cmꢀ1): 3422, 2925,
2855, 1693, 1609, 1526, 1382, 1435, 1061, 1016; Anal. Calcd for: C45H51N5O19
:
C, 55.96; H, 5.32; N, 7.25%. Found: C, 55.75; H, 5.50; N, 7.03%; 15b: mp 151–
25
152 °C; ½aꢁD þ 6:66 (c = 0.3, CH3OH); 1H NMR (CD3OD, 500 MHz): d 1.97–2.19
(m, 4H), 3.38–3.69 (m, 6H), 3.76–3.80 (m, 1H, J = 9.33 Hz), 3.89 (s, 3H), 4.96–
5.16 (m, 5H), 5.66 (d, 1H, J = 10.05 Hz), 6.88 (s, 1H), 7.22 (s, 1H), 7.24–7.30 (m,
1H), 7.31–7.36 (m, 2H), 7.37–7.45 (m, 4H), 7.51–7.70 (br s, 1H); ESI-MS: m/z
724 [MꢀH]ꢀ; HRMS: [MꢀH]ꢀ calcd for C34H34N3O15 m/z 724.1989, found (ESI)
m/z 724.2014; IR (KBr) (mmax cmꢀ1): 3424, 2924, 2854, 1673, 1613, 1532,
1460, 1322, 1126, 1021; Anal. Calcd for C34H35N3O15: C, 56.28; H, 4.86; N,
5.79%. Found: C, 56.36; H, 4.62; N, 5.98%.
16. The enzymatic reaction mixtures were analyzed using Phenomenex C18
reverse phase column at 254 nm and 30:70 Acetonitrile–H2O as eluent system
at 1 ml/min flow. The compound 15b with a retention time of 3.01 min, 4-
(hydroxymethyl)-2-nitrophenol spacer 6.35 min and 16b 22.87 min. 15a
retention time was 3.13 min and 16a was 22.40 min.
Acknowledgments
Authors V.T. and V.G.M.N. are thankful to CSIR, New Delhi and
P.R. is thankful to UGC for awarding research fellowships.
17. (a) Edmondson, J. M.; Armstrong, L. S.; Martinez, A. O. J. Tissue Cult. Meth. 1988,
11, 15; (b) http://www.atcc.org/common/documents/pdf/30-1010k.pdf.
18. Connors, T. A.; Whisson, M. E. Nature 1966, 210, 866.
19. Bosslet, K.; Straub, R.; Blumrich, M.; Czech, J.; Gerken, M.; Sperker, B.; Kroemer,
H. K.; Gesson, J.-P.; Koch, M.; Monneret, C. Cancer Res. 1998, 58, 1195.
20. (a) Gesson, J. P.; Jacquesy, J.-C.; Mondon, M.; Petit, P.; Renoux, B.;
Andrianomenjanahary, S.; Dufat-Trinh Van, H.; Koch, M.; Michel, S.;
Tillequin, F.; Florent, J.-C.; Monneret, C.; Bosslet, K.; Czech, J.; Hoffmann, D.
Anti-Cancer Drug. Res. 1994, 9, 409; (b) Goldstein, J. A.; Faletto, M. B. Environ.
Health Perspect. 1993, 100, 169.
References and notes
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1996, 22, 269.