Synthesis, in vitro and in vivo cytotoxicity of 6,7-diaryl-2,3,8,8a-tetrahydroindolizin-5(1H)-ones

Article abstract of DOI:10.1016/j.bmcl.2007.05.103

A 6,7-diaryl-2,3,8,8a-tetrahydroindolizin-5(1H)-one library was constructed and tested against the colon cancer cell line HCT-116 as an initial screen for cytotoxic properties. Of this library, the parent compound, in which the southern aromatic ring remains unsubstituted, and the northern aromatic ring carries a 4-methoxy group, exhibited the most potent cytotoxicity with an IC₅₀ value of 0.39 μM and displayed promising activity in vivo in the NCI's mouse hollow fiber assay.

Full text of DOI:10.1016/j.bmcl.2007.05.103

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4703–4707
Synthesis, in vitro and in vivo cytotoxicity of
6,7-diaryl-2,3,8,8a-tetrahydroindolizin-5(1H)-ones
F. Scott Kimball,a Ashok Rao Tunoori,a Samuel F. Victory,a Dinah Dutta,a
Jonathan M. White,a Richard H. Himesb and Gunda I. Georga,*
aDepartment of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045, USA
bDepartment of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA
Received 21 March 2007; revised 17 May 2007; accepted 17 May 2007
Available online 13 June 2007
Abstract—A 6,7-diaryl-2,3,8,8a-tetrahydroindolizin-5(1H)-one library was constructed and tested against the colon cancer cell line
HCT-116 as an initial screen for cytotoxic properties. Of this library, the parent compound, in which the southern aromatic ring
remains unsubstituted, and the northern aromatic ring carries a 4-methoxy group, exhibited the most potent cytotoxicity with an
IC50 value of 0.39 lM and displayed promising activity in vivo in the NCI’s mouse hollow fiber assay.
ꢀ 2007 Elsevier Ltd. All rights reserved.
As cancer cells adapt to various treatment modalities
and subsequently become resistant to modern and con-
ventional chemotherapy, the discovery and exploitation
of new chemical classes of compounds that avoid the
various cancer resistance mechanisms become an
increasingly crucial endeavor. Moreover, the discovery
of small molecules that exert cytotoxic properties via no-
vel mechanisms of action would be of paramount
importance. Such a discovery could also identify a novel
cancer target and thus provide a lead for the develop-
ment of a new class of anticancer agents. As such, our
continuing search for the discovery and development
of small molecule anticancer agents that operate
through an unknown mechanism of action led us to
our studies of the alkaloid tyloindicine family.
Although no total syntheses exist to date for tyloindicine
I, synthetic endeavors toward the related alkaloids ipal-
bidine3–13 and septicine11,14–24 have been utilizing syn-
thetic methods that have been incorporated into this
work.20,21 Research focusing on analogs related to tylo-
indicine I, ipalbidine, or septicine is sparse in the litera-
ture with the notable research in this area coming from
the laboratories of Sharma et al. and focusing on 6-aryl-
7-(4-(methylthio)phenyl)-2,3,8,8a-tetrahydroindolizin-
5(1H)-ones.25
OMe
MeO
Ar1
Ar2
HO
MeO
N
N
Tyloindicine I (1, Scheme 1) was isolated in 1991 in min-
ute quantities from the aerial parts of Tylophora indica
and falls into the family of seco-phenanthroindolizidine
alkaloids.1,2 Our interest in tyloindicine I is a result of its
potent nanomolar and cancer cell-selective cytotoxic
properties and therefore, we set out to synthesize 1
and related analogs to pursue additional biological
studies.2
XR
MeO
Tyloindicine I (1)
2 (X = O, S)
Ar1
Ar1
Ar2
7
O
5
N
N
6
Ar2
O
O
3
4
Ar1
Ar2
CO2H
CO2H
N
Keywords: Seco-phenanthroindolizidine alkaloids; COMPARE analy-
sis; Colon cancer; HCT-116; NCI mouse hollow fiber assay.
O
5
6
7
*
Corresponding author. Tel.: +1 612 626 6320; fax: +1 612 626
6318; e-mail: georg239@umn.edu
Scheme 1.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.103

4704
F. S. Kimball et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4703–4707
In an attempted route to 1, for which the retrosynthesis
is shown in Scheme 1, we devised a synthesis starting
from b-keto acid 5, pyrrole 6, and aryl acetic acid 7.
These building blocks could be assembled to provide
intermediate pyrrolidine 4, which upon aldol condensa-
tion would provide bicyclic lactam 3. Alkylation of the
O- or S-enol amide of 3, followed by deoxygenation or
desulfurization of intermediate 2, could lead to the tar-
geted tyloindicine I. A model study was initiated as
shown in Scheme 2, and the synthesis of the desired lac-
tam intermediate 12 was accomplished as planned.
Saponification of the commercially available b-keto es-
ter 8 furnished the b-keto acid 9. A subsequent addition
of 9 to (di-(3,4-dihydro-2H-pyrrole))diiodozinc, fol-
lowed by decarboxylation, constructed the pyrrolidine
10.26 The amine in 10 was then acylated with phenylace-
tyl chloride to provide 11, which was subjected to KOH
in refluxing ethanol to provide the target indolizidine
lactam 12.27 The subsequent steps, the conversion of
12 to the corresponding tyloindicine I analog did not
proceed as planned and that route toward tyloindicine
I was abandoned.
activity of the test compounds. A value of 2 is assigned
for each compound dose that results in 50% or greater
reduction in viable cell mass either IP or SC. Com-
pounds with a combined IP and SC score of 20, an SC
score of 8, or a net cell kill of one or more cell lines
are referred to the NCI’s Biological Evaluation Commit-
tee for Cancer Drugs. Compound 12 received an SC
score of 8, demonstrating that 12 has promising activity
according to this point system. Considering that only
one colon cancer cell line was present in this assay panel,
the SC in vivo activity is encouraging.
Because of these promising test results, we set out to pre-
pare analogs of 12 so as to achieve an understanding of
the structure–activity relationship for this class of com-
pounds, to improve potency, and to prepare affinity ana-
logs that could be used to identify the biological target
or targets of 12. The analog library was generated utiliz-
ing the synthetic sequence shown in Scheme 2 from the
appropriately substituted b-keto and aryl acetic acids
and are shown in Table 1.
The analog library, along with compound 12, was tested
against the HCT-116 colon cancer cell line as the initial
screen to determine cytotoxicity (Table 1).
7-(4-Methoxyphenyl)-7-phenyl-2,3,8,8a-tetrahydroind-
olizin-5(1H)-one (12, NSC 707904) was submitted to the
NCI for screening in their panel of 60 cancer cell lines
and demonstrated selective cytotoxicity at sub-micro-
molar concentrations toward colon cancer and leukemia
cell lines. In one screen the concentration that inhibited
growth of the HCT-116 colon tumor cell line was
0.024 lM. In another screen it was 0.53 lM.
The results from the toxicity screen revealed that the
activity we observed in our laboratory with 12 is
Table 1. Cytotoxicity of 6,7-diaryl-2,3,8,8a-tetrahydroindolizin-5(1H)-
one analogs 12–25
The NCI’s COMPARE analysis of the 60 cell line assay
results indicated that 12 acts by a different mechanism of
action compared to all other anticancer agents in their
database including tyloindicine I.28,29
R1
N
O
As a result of these interesting findings, 12 (NSC
707904) was selected by the NCI for in vivo studies in
the NCI’s mouse hollow fiber assay.30,31 In this assay,
compounds are tested against a standard panel of 12 hu-
man cancer cell lines, including one colon cancer cell line
(COLO 205). A point system is used to assess the
R2
R4
R3
12-25
Analog
Northern aryl
substitution R1
Southern aryl
substitution
R2, R3, R4
HCT-116
cytotoxicity
IC50 (lM)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
O–LCBb
OMe
OH
H, H, H
Cl, H, H
0.39
13
3.2a
10
4
MeO
MeO
b
OMe, H, H
Me, H, H
Cl, Cl, H
c
CO2R
75%
65%
O
HN
O
H, Cl, H
8
10
8 (R = Et)
9 (R = H)
a, 50%
H, H, Cl
8
H, NH–LCBb, H
NH–LCBb, H, H
N3, H, H
>25
25
25
17
>25
9
MeO
MeO
H, H, H
d
H, NH2, H
H, H, H
H, H, H
O
N
N
80%
OBn
8.9
O
O
The cytotoxicity experiments were done as previously described32
except that the cells were incubated for 48 h after the addition of the
compounds.
11
12 (NSC707904)
a Assay conducted by National Cancer Institutes Developmental
Therapeutics Program.
Scheme 2. Reagents and condition: (a) 2.5% KOH(aq); (b) (di-(3,4-
dihydro-2H-pyrrole))diiodozinc, pH 7, phosphate buffer, MeOH; (c)
phenylacetyl chloride, TEA, DCM; (d) 5% KOH(EtOH), reflux.
b LCB = (+)-biotinyl-6-aminohexanoic acid.

F. S. Kimball et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4703–4707
4705
Table 2. Cytotoxicity of tylophorine analogs 26–28
consistent with the higher of the two values obtained in
the NCI screen. A Topliss decision tree approach was
applied toward the selection of the initial analogs 13–
16 with the lead compound 12 being at the top of the
schematic Topliss tree.33 With respect to structural mod-
ifications, the 4-chloro analog 13 was less active than 12
suggesting that the southern aryl ring has either strin-
gent steric requirements or is controlled by either -r
or -p factors. In attempts to further define the southern
aryl ring’s role, continuing down the Topliss tree to the
4-methoxy analog 14 revealed that it too was less active
than the lead compound 12. This information indicates
that the steric requirements of the southern ring may
supercede any favorable or negative influences the -r
or -p factors may have been contributing. The other
analogs in the Topliss series, 15–18, further suggested
that regardless of the lipophilicity or the chlorine substi-
tution patterns, overall changes in the southern ring are
not well tolerated. Notwithstanding these results, one
photoaffinity analog (21) and three biotinylated analogs
(19, 20, 22) were generated in attempts to aid in the iso-
lation of biological targets and allow for the determina-
tion of the mechanism of action of this class of
compounds.34 These efforts were not successful with
affinity analogs 19–22 showing poor cytotoxicity. Fur-
thermore, aniline 23 had poor activity. These results fur-
ther illustrate the steric sensitivity of the southern
aromatic ring.
R2
MeO
R1
N
O
MeO
OMe
26-28
Analog
R1, R2
HCT-116 cytotoxicity IC50 (lM)
26
27
28
H, OMe
H, H
OBn, OMe
>10
5.0a
3.3
a Assay conducted by the National Cancer Institute’s Developmental
Therapeutics Program.
well. It would seem that for this series, the electronics
of the aryl rings might have a larger influence than pre-
viously anticipated.
Overall, our results exhibit trends complementary to
those outlined by Sharma et al. in which a 6,7-diaryl-
2,3,8,8a-tetrahydroindolizin-5(1H)-one scaffold was em-
ployed with variously substituted northern and southern
aromatic rings.25 Notably, Sharma found that more
highly substituted northern rings led to a decrease in
activity, whereas the southern ring did retain moderate
activity with mono- and di-substituted systems similar
to analogs 13–18, 27, and 28. Although a southern
ortho- and/or para-substituted methoxy group did not
significantly affect activity, replacement with a hydroxyl
group led to a significant decrease in activity suggesting
H-bonding moieties were detrimental to activity similar
to our aniline analog 23. Activities were tested against 8
cell lines, not including the HCT-116 cell line, thereby
making direct comparisons with our results difficult.
With respect to the northern ring, the phenol 24 as well
as the benzyl protected analog 25 were prepared and
tested. These compounds also showed reduced cytotox-
icity. Phenol 24 was prepared in quantitative yield from
12 by treatment with BCl3, n-Bu4NI, CH2Cl2 (À78 ꢁC to
0 ꢁC) for 2 h. Analog 25 was synthesized as shown in
Scheme 2, using the O-benzyl analog of 8. Thus, the
northern aromatic ring appears to favor the 4-methoxy
substitution over the 4-hydroxy substitution thereby
suggesting that the electronic or lipophilic character of
the northern ring affects the biological activity of these
analogs.
In summary, a short synthesis was designed to construct
6,7-diphenyl-2,3,8,8a-tetrahydroindolizin-5(1H)-one (12)
as well as the subsequent analogs 13–28. The lead
compound 12 was shown to exhibit the most potent
cytotoxicity toward the HCT-116 colon cancer cell line
and showed promising activity in the mouse hollow fiber
assay. There appear to be stringent steric requirements
controlling the degree of aryl substitution in the
southern and northern aromatic rings. Overall, the low
molecular weight, potent and selective cytotoxicity
toward colon cancer cell lines, and potential of
ultimately determining a novel mechanism of action
make this collection of compounds an interesting avenue
toward the discovery of a new class of anticancer agents
worthy of further examination and scientific scrutiny.
Further efforts to investigate the steric and electronic
parameters for the southern aryl ring were inspired by
the nanomolar cytotoxicity of the related alkaloids
tyloindicine I (Scheme 1)1,2 and tylophorine,24 that carry
two methoxy groups on each aromatic ring. The
cytotoxicity of these natural products suggests that
electronic factors may serve to override the steric sensi-
tivity of the southern aromatic ring inherent in analogs
13–21 and 23. As such, the more highly substituted ana-
logs 26–28 were prepared following the synthesis shown
in Scheme 2, using the appropriate b-keto esters and
acyl chlorides. The IC50 values for analogs 26–28 are
listed in Table 2. With the introduction of a second
methoxy group at the southern ring, a comparison of
27 and 12 further confirms that substitution of the
southern ring is detrimental to activity. In addition,
the inferior cytotoxicity of the tri- and tetra-substituted
northern ring analogs 26 and 28, relative to the sub-
micromolar activity of the parent compound 12, sug-
gests that increasing the steric encumbrance around
the northern aromatic ring has detrimental effects as
Acknowledgments
These studies were supported by the National Cancer
Institute (CA 90602 and N01-CM-67259). The authors
thank Dr. Ravi Varma (NCI, DTP) for carrying out
the COMPARE analysis for analog 12 (NSC 707904)

4706
F. S. Kimball et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4703–4707
into ether (50 mL). The aqueous layer was extracted with
ether (2 · 20 mL). The combined ether extracts were dried
over Na2SO4 and concentrated under reduced pressure,
keeping the water bath at 20 ꢁC. The crude acid (semi-
solid) was used immediately in the next step without any
purification (0.970 g, 50%).
and for his support of this project. We also wish to
thank the NCI’s DTP for carrying out cytotoxicity as-
says for compounds 12 (NSC 707904), 14 (NSC
707907), and 27 (NSC 707909). The complete cancer
screening data for these compounds are available on
the DTP website.
Synthesis of 10. To a solution of 9 (4.0 g, 20 mmol) in
MeOH (10 mL) were added 1 M phosphate buffer (5 mL)
to a pH ꢀ7.5 and a freshly prepared solution of (di-(3,4-
dihydro-2H-pyrrole))diiodozinc (4.57 g, 10 mmol in water
(60 mL). The reaction mixture was stirred for 3 days at
room temperature and then cooled to 0 ꢁC and acidified
using Congo red as the indicator. The reaction mixture
was extracted with ether and the ether layer was discarded.
The water layer was cooled to 0 ꢁC and basified carefully
to pH ꢀ8 using K2CO3. The water layer was extracted
with CHCl3 (3 · 20 mL). The combined extracts were
dried over Na2SO4 and concentrated to provide 10 as oil.
The crude product (3 g, 75%) was used in the next step
without additional purification.
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ethanolic KOH (5%, 20 mL). After refluxing this reaction
for 2 h, the solvent was removed under reduced pressure.
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with CH2Cl2 (2 · 50 mL), dried over Na2SO4, and con-
centrated under reduced pressure. Column chromatogra-
phy on silica gel, using EtOAc/hexanes (1:1), provided 12
(2.7 g, 80%, mp = 179–182 ꢁC) as colorless crystals. 1H
NMR (400 MHz, CDCl3) d 1.75 (m, 1H), 1.88 (m, 1H),
2.06 (m, 1H), 2.29 (m, 1H), 2.82 (m, 2H), 3.63–3.72 (m,
2H), 3.75 (s, 3H), 3.95 (m, 1H), 6.67 (d, J = 7.4 Hz, 2H),
6.98 (d, J = 8.6 Hz, 2H), 7.19 (m, 5H); 13C NMR
(100 MHz, CDCl3) d 23.59, 34.22, 37.96, 45.21, 55.54,
56.02, 113.69, 127.04, 127.96, 130.29, 131.54, 132.77,
136.75, 145.10, 159.23, 164.53. HRMS (ES+) m/z calcd
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reaction mixture was stirred at room temperature for 40 h.
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the ether layer was discarded. The aqueous layer was
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34. Synthesis of 19. With gentle heating and stirring, LC-
biotin (0.070 g, 0.20 mmol) was solubilized in DMF
(2 mL) after which was added EDCI (0.050 g, 0.26 mmol).
With the addition of DMAP (0.070 g, 0.57 mmol) the

F. S. Kimball et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4703–4707
4707
reaction mixture turned orange in color and the solution
was stirred for 30 min. at ambient temperature. In a
separate flask was dissolved aniline 23 (0.021 g,
0.063 mmol) in DMF (2 mL) and the LC-biotin mixture
was subsequently added. The reaction was allowed to
progress over 24 h after which it was quenched with the
addition of water. EtOAc was added and used to extract
the product into the organic phase. Equal amounts of
water and EtOAc were added and the organic product was
extracted into the EtOAc layer. The aqueous and organic
phases were separated and the aqueous phase was
extracted an additional four times with EtOAc. The
combined organic fractions were washed four times with
water to remove excess DMF then once with brine. The
organic phase was dried over sodium sulfate then
condensed under reduced pressure. The biotinylated
product 19 was purified via flash column chromatography
using silica gel (9% MeOH/CH2Cl2) in 29% yield (0.029 g):
1H NMR (400 MHz, CDCl3) d 1.20–2.35 (m, 20H), 2.60–
2.95 (m, 4H), 3.10–3.35 (m, 3H), 3.60–3.73 (m, 2H), 3.73
(s, 3H), 4.00 (m, 1H), 4.28 (m, 1H), 4.42 (m, 1H), 6.36 (d,
J = 19.58 Hz, 1H), 6.51 (d, J = 7.55 Hz, 1H), 6.65 (d,
J = 7.56 Hz, 2H), 6.76 (d, J = 9.56 Hz, 1H), 6.96 (m, 4H),
7.39 (d, J = 9.73 Hz, 1H), 7.61 (m, 1H), 9.03 (d,
J = 11.84, 1H); MS (FAB+) [M+H]+ C37H48N5O5S: m/z
674.