Synthesis and structure-activity relationship studies on tryprostatin A, an inhibitor of breast cancer resistance protein

Article abstract of DOI:10.1016/j.bmc.2008.02.050

Tryprostatin A is an inhibitor of breast cancer resistance protein, consequently a series of structure-activity studies on the cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These

Full text of DOI:10.1016/j.bmc.2008.02.050

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4626–4651
Synthesis and structure–activity relationship studies on
tryprostatin A, an inhibitor of breast cancer resistance protein
Hiteshkumar D. Jain,a Chunchun Zhang,a Shuo Zhou,a Hao Zhou,a Jun Ma,a
Xiaoxiang Liu,a Xuebin Liao,a Amy M. Deveau,a Christine M. Dieckhaus,b
Michael A. Johnson,b Kirsten S. Smith,b Timothy L. Macdonald,b
Hideaki Kakeya,c Hiroyuki Osadac and James M. Cooka,*
aDepartment of Chemistry, University of Wisconsin—Milwaukee, Milwaukee, WI 53201, USA
bDepartment of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22904, USA
cAntibiotics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Received 7 December 2007; revised 10 February 2008; accepted 11 February 2008
Available online 20 February 2008
Abstract—Tryprostatin A is an inhibitor of breast cancer resistance protein, consequently a series of structure–activity studies on the
cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These ana-
logues were assayed for their growth inhibition properties and their ability to perturb the cell cycle in tsFT210 cells. SAR studies
resulted in the identification of the essential structural features required for cytotoxic activity. The absolute configuration L-Tyr-L-
pro in the diketopiperazine ring along with the presence of the 6-methoxy substituent on the indole moiety of 1 was shown to be
essential for dual inhibition of topoisomerase II and tubulin polymerization. Biological evaluation also indicated the presence of
the 2-isoprenyl moiety on the indole scaffold of 1 was essential for potent inhibition of cell proliferation. Substitution of the indole
Na–H in 1 with various alkyl or aryl groups, incorporation of various L-amino acids into the diketopiperazine ring in place of
L-proline, and substitution of the 6-methoxy group in 1 with other functionality provided active analogues. The nature of the
substituents present on the indole Na–H or the indole C-2 position influenced the mechanism of action of these analogues.
Analogues 68 (IC50 = 10 lM) and 67 (IC50 = 19 lM) were 7-fold and 3.5-fold more potent, respectively, than 1 (IC50 = 68 lM)
in the inhibition of the growth of tsFT210 cells. Diastereomer-2 of tryprostatin B 8 was a potent inhibitor of the growth of three
human carcinoma cell lines: H520 (IC50 = 11.9 lM), MCF-7 (IC50 = 17.0 lM) and PC-3 (IC50 = 11.1 lM) and was equipotent with
etoposide, a clinically used anticancer agent. Isothiocyanate analogue 71 and 6-azido analogue 72 were as potent as 1 in the tsFT210
cell proliferation and may be useful tools in labeling BCRP.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
are highly promising new therapeutic agents against hu-
man cancers.
The cell cycle coordinates a variety of cellular functions
involved in the accurate replication of the genome and
cell division.1 These processes are tightly regulated pri-
marily at the G1/S and G2/M phase transitions by a
series of checkpoints. It has become clear that check-
point control defects in cancer cells contribute to tumor-
igenesis and are a significant reason for the increased
selectivity of tumors over normal cells towards
chemotherapy.2,3 Cell cycle inhibitors or modulators
With an increased understanding of the molecular biol-
ogy of cell cycle control it has become possible to devel-
op bioassays and screen for agents that specifically
interfere with these processes. One such method was
developed by Osada et al.4,5 which utilizes the synchro-
nous culture of the murine temperature-sensitive mutant
cell line, tsFT210, defective in the p34cdc2 gene. With this
assay a family of 2-isoprenylated diketopiperazine
indole alkaloids which effect cell cycle arrest at the
G2/M phase was isolated from the fermentation
broth of a marine fungal strain of Aspergillus fumigatus
BM939. It was found that tryprostatin A 1 (Chart 1) and
tryprostatin B 2 (Chart 1) completely inhibited cell cycle
Keywords: Tryprostatin A; Antimitotic; Anticancer; Breast cancer
resistance protein.
*
Corresponding author. Tel.: +1 414 229 5856; fax: +1 414 229
5530; e-mail: capncook@uwm.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.050

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4627
Chart 1.
progression of tsFT210 cells in the G2/M phase at a final
concentration of 50 lg/mL of 1 and 12.5 lg/mL of 2,
respectively.6–8 Since these indole alkaloids were isolated
only in small amounts, studies on the mechanism and
SAR were not reported earlier.
cytoplasmic network and spindle apparatus. Although
tryprostatin B 2 arrested cell cycle progression at a lower
concentration than 1, the inhibition was not due to inhi-
bition of the M phase. It was shown that 1 inhibited
microtubule assembly through a different type of mech-
anism than colchicine (Chart 1), vinblastine (Chart 1),
or maytansine–rhizoxin.13 Tryprostatin A 1 inhibited
microtubule assembly by interfering with the interaction
between microtubule-associated proteins (MAPs) and
the C-terminal domain of tubulin. Since 1 operated by
an entirely novel mechanism this may be important in
cancer chemotherapy, especially in multiple drug resis-
tance (MDR) cancers.
Tryprostatin A and B have previously been synthe-
sized,9–11 the aim of which was to study their mechanism
of action. The similarities in the structures of the try-
prostatins with etoposide (Chart 1) and azatoxin (Chart
1), a dual inhibitor of topoisomerase II (G2)/tubulin
polymerization (M), led to the investigation of the abil-
ity of the two tryprostatins to inhibit topoisomerase II
and tubulin polymerization. Biological evaluations12 of
1 and 2 indicated that both alkaloids were very weak
inhibitors of topoisomerase II in the topoisomerase
II assay; while only 1 had marginal activity in the
tubulin polymerization assay. This latter result was in
agreement with the data reported for 1 by Osada
et al.13 Osada et al.13 also reported 1 inhibited cell
cycle progression of rat normal fibroblast 3Y1 cells
specifically in the M phase. The concentration of 1 that
arrested cell cycle progression in the M phase corre-
sponded to that which induced a marked depolymeriza-
tion in situ of the microtubules containing both
Microtubules are hollow cylindrical tubes found in al-
most all eukaryotic cell types. They play an important
role in a variety of cellular functions, such as cell divi-
sion, cell movement, cell shape, and transport of organ-
elles inside the cell.14 Tubulin exists as a heterodimer of
a- and b-tubulin and is the major building block of
microtubules. Proteins such as the MAPs bind to and
modify microtubule properties.14,15 In the absence of
MAPs, a/b-tubulin heterodimers polymerize only by
treatment with high concentrations of glycerol or organ-
ic acids such as glutamate.16

4628
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
The discovery of numerous compounds from natural
sources which display a wide structural diversity and
are cytotoxic by perturbation of the dynamic instability
of microtubules has attracted much attention within the
last two decades.17–20 Microtubules have, therefore, be-
come an attractive pharmacological target for antican-
cer drug discovery.17–20 Almost all antimitotic agents
interact with the a/b-tubulin dimer, rather than microtu-
bule-associated proteins (MAPs) or other proteins in-
volved in microtubule functions. The Vinca alkaloids,
exemplified by vinblastine (Chart 1) and vincristine
(Chart 1), as well as the taxanes, such as paclitaxel
(Chart 1) and the semisynthetic analogue docetaxel
(Chart 1), are the most commonly used antimitotic
agents in the clinical treatment of cancer.21 Colchicine
is another important antimitotic agent; however, it has
limited medicinal utility due to its narrow therapeutic
index.
dures.17–20 Therefore, a pressing need to develop
simpler, more effective antitumor drugs still remains.
The development of MDR to chemotherapeutic agents
remains one of the primary obstacles in cancer treat-
ment. These arise from intrinsic or acquired mechanisms
of resistance. The overexpression of energy dependent
(ATP) transmembrane drug-efflux pumps, such as P-gly-
coprotein (MDR1), multidrug resistance protein
(MRP), and breast cancer resistance protein (BCRP),
have been shown to produce resistance to several com-
monly used chemotherapeutic agents. Breast cancer
resistance protein is a 72 kDa protein which probably
homodimerizes to form an active transport complex.29
BCRP was first identified in drug resistant MCF-7/
adrVp cells30 and has been recently reviewed.31–33 Like
other members of the ATP binding cassette family of
membrane transporters, such as MDR1 and MRP1,
BCRP is expressed in a variety of malignancies where
it may produce resistance to chemotherapeutic agents.
In addition, it has also been reported that BCRP expres-
sion may be a prognostic indicator in certain cancers
and is associated with poor response to chemother-
apy.34,35 Overexpression of BCRP has been reported in
a number of tumor types including: adenocarcinomas
(arising from the digestive tract, the endometrium, and
the lung), melanoma, soft tissue sarcomas,36 hematolog-
ical malignancies such as acute myeloid leukemia
(AML),37 and acute lymphoblastic leukemia (ALL).38
Elevated expression of BCRP results in resistance of
various cancer cell lines to antitumor drugs including:
topotecan, mitoxantrone, daunorubicin, doxorubicin,
and bisantrene.39 In addition, flavopiridol resistance is
mediated by BCRP.40
Additionally, the natural products combretastatin
A-4,22 curacin A,23 podophyllotoxin,24 epothilones A
and B,25 and dolastatin26 to cite just a few, are known
to be cytotoxic through binding interactions with
tubulin. Another antimitotic agent, estramustine
phosphate inhibits microtubule assembly by binding
to both microtubule-associated protein 2 (MAP2)
and tubulin,27 while 5,50-bis[8-(phenylamino)-1-naph-
thalenesulphonate] (bis-ANS) specifically inhibits
MAP-dependent microtubule assembly by interaction
with the C-terminal domain of the tubulin heterodi-
mer.28 These compounds may lead to useful cancer
therapeutic agents. Indeed, estramustine in combina-
tion with other antimicrotubule agents exhibits syner-
gistic cytotoxicity both in vitro and in vivo.27
However, no new tubulin polymerization inhibitor of
low molecular weight has reached clinical status, as
yet. Clinically available compounds such as paclitaxel
or vincristine face several disadvantages; principally:
(i) high toxicity, (ii) development of drug resistance in
patients, (iii) marginal oral bioavailability and poor sol-
ubility, and (iv) complex synthesis or isolation proce-
The clinical significance of BCRP along with its limited
expression in normal tissues makes BCRP a viable tar-
get for inhibition to reverse MDR. Several potent and
specific inhibitors of BCRP have been developed. This
has potentially opened the door to clinical applications
of BCRP inhibition. These inhibitors include the
O
H
O
O
O
O
H
H
N
N
H
N
H
HN
HN
HN
R
N
H
R
N
H
R
N
H
H
O
3
5
R = OMe
R = OMe
7 R = OMe
4 R = H
6 R = H
8
R = H
O
H
region D-aminoacid
variation
N
H
HN
region A-C6
variant
R1
N
R2
R3
O
region C-C2
variant
region B-N1
variant
Figure 1.

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4629
targeted agents: gefitinib and imatinib mesylate,41 as
well as the more specific inhibitors: fumitremorgin C,42
tryprostatin A,43 and GF120918.44 At concentrations
of 10–50 lM, tryprostatin A 1 was shown to reverse a
mitoxantrone-resistant phenotype and inhibited the cel-
lular BCRP-dependent mitoxantrone accumulation in
the human gastric carcinoma cell line EPG85-257RNOV
and the human breast cancer cell line MCF-7/AdrVp
(both exhibited acquired BCRP-mediated MDR). No
cytotoxicity was observed at effective concentrations.43
The lower cytotoxicity of 1 in comparison to 2, com-
bined with a unique mechanism for inhibition of tubulin
polymerization, as well as BCRP prompted investiga-
tion of the structure activity relationships (SAR) in 1
in order to enhance tubulin polymerization and/or
BCRP inhibition. To our knowledge, no SAR of try-
prostatin A has appeared in the literature, to date. The
SAR studies were designed to determine the minimum
structural requirements of tryprostatin A required to ex-
hibit potent and selective cytotoxic activity, in the search
for new anticancer agents. Several modifications, which
maintained the same backbone, were carried out as out-
lined in Figure 1: (A) substitution of the 6-position of
the aromatic ring, (B) alkylation of the indole NH, (C)
substitution of the 2-position of the indole moiety, and
(D) substitution of the L-proline residue in the diketopi-
perazine ring with other L-amino acids.
In the search for potent and selective antitumor agents,
the synthesis of a series of analogues (3–8, Fig. 1) of 1
and 2 was carried out in order to probe the importance
of the stereochemistry of the diketopiperazine ring on
the inhibition of topoisomerase II and/or tubulin poly-
merization. Tryprostatin analogues 1–8 were evaluated
for their ability to inhibit topoisomerase II and/or tubu-
lin binding protein as well as their tumor cell growth
inhibitory activity.12
2. Chemistry
More recently, Osada et al.13 reported the presence of
the 6-methoxy substituent on the indole moiety of 1 con-
ferred lower cytotoxicity to tryprostatin A and enhanced
the specificity for inhibition of microtubule assembly.
The synthesis of optically active 1 and 2 have been re-
ported.9–11 This method was also extended to the syn-
thesis of the enantiomers (3 and 4) and diastereomers
(5–8) of 1 and 2 (Scheme 1).11,12 The synthesis of 1–8
OEt
N
OEt
N
EtO
N
EtO
N
CH2Br
N
OEt
a
10
OEt
a
11
N
R
N
BOC
R
N
BOC
12
R
N
BOC
OEt
OEt
9a
R = OMe
9b R = H
14
R = OMe (92%)
R = OMe (91%)
13 R = H
(92%)
15 R = H
(91%)
b
b
EtO
N
EtO
N
N
N
R
N
BOC
R
N
BOC
OEt
OEt
16
R = OMe (85%)
18
R = OMe
17 R = H
(82%)
19 R = H
Fmoc
Fmoc
N
N
c
c
EtO
EtO
Cl
O
Cl
O
H
H
O
H
24
H
25
d
O
1
20
3
22
from
from
NH2
NH2
(82%)
(78%)
d
R
N
BOC
R
N
BOC
2 from 21
4 from 23
(81%)
(83%)
22
R = OMe (73%)
20
R = OMe (94%)
23 R = H (70%)
21 R = H
(93%)
Fmoc
Fmoc
N
N
Cl
O
Cl
d
d
H
H
O
25
24
7
20
5
22
from
(84%)
from
(84%)
8 from 21 (79%)
6 from 23 (86%)
Scheme 1. Reagents and conditions: (a) THF, n-BuLi, ꢀ78 ꢁC; (b) LDA (1.5 equiv), THF, isoprenylbromide (3 equiv); (c) 2 N aq HCl, THF, ꢀ78 ꢁC
to rt; (d) TEA, CHCl3; DEA, CH3CN, rt; xylene, reflux.

4630
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
(Scheme 1) began with indoles 9a and 9b which were
coupled with the anion of the Scho¨llkopf chiral auxiliary
11 (derived from L-valine), to afford the trans diastereo-
mers 14 and 15, respectively, with 100% diastereoselec-
tivity. The diastereoselectivity of the addition to the
Scho¨llkopf chiral auxillary was found to be 100% by
analysis of the 1H spectrum of the crude mixture of
the respective compounds. When indoles 14 and 15 were
treated with LDA at ꢀ78 ꢁC, followed by addition of
isoprenyl bromide, the 2-isoprenylpyrazine derivatives
18 and 19 were obtained, respectively. The pyrazine
moiety was removed from pyrazines 18 or 19 under
acidic conditions (aq HCl, THF) in 94% yield to afford
the 2-isoprenyl tryptophan 22 or 23, respectively. The
coupling of 2-isoprenyl tryptophan 22 or 23 with
Fmoc-D-proline 25 using triethylamine as the base was
followed by formation of the diketopiperazine ring.
The Boc protecting group was removed from the indole
N(H) function in refluxing xylene to afford 3 and 4,
respectively. Similarly, coupling of 2-isoprenyl trypto-
phan 22 or 23 with Fmoc-L-proline 24 afforded 5 or 6,
respectively. The natural products (1 and 2) were pre-
pared from the trans transfer of chirality from D-valine
(Scho¨llkopf) and from L-proline.9–11 The diastereomers
7 and 8 of 1 and 2, respectively, were prepared from
the trans transfer of chirality from D-valine (Scho¨llkopf)
and from D-proline, as outlined in Scheme 1.9–11
dium hydride afforded the indole Na-substituted ana-
logues 29–32 in 72–99% yields. Hydrolysis of pyrazines
29–32 with 2 N aq HCl in THF resulted in removal of
both the bislactim ether moiety and the silyl group. Try-
ptophans 33–36 were readily transformed into ana-
logues 37–40 of tryprostatin A under conditions
analogous to the steps in Scheme 1.
In order to synthesize the Na-substituted analogues (46
and 47) of 1, in region B in which the indole 2-position
carried the isoprenyl group, an analogous strategy was
employed. Thermal removal of the Boc protecting group
of 16 (Scheme 3) in refluxing xylene afforded pyrazine
41. Alkylation of 41 with benzyl bromide or allyl bro-
mide using sodium hydride afforded analogues 42 and
43 in 91% and 82% yields, respectively. These intermedi-
ates were readily transformed into analogues 46 and 47
of tryprostatin A under conditions analogous to the
steps in Scheme 1, as illustrated in Scheme 3.
To obtain analogues with different substitution at the
indole 2-position (region C) of 1, 2-bromo-indole 48
(Scheme 4) served as a common intermediate which
was easily obtained from indole 28 after sequential
treatment with NBS and Boc anhydride. As shown in
Scheme 4, lithium–halogen exchange, followed by treat-
ment with benzyl bromide or allyl bromide furnished the
2-substituted analogues 49 and 50. Analogues 49 and 50
were further transformed into 2-substituted indoles 55
and 56, respectively, under standard conditions (Scheme
4). For the synthesis of 51 (Scheme 4), the zinc reagent
was prepared through lithium–halogen exchange fol-
lowed by treatment with zinc chloride. Negishi cou-
pling47 of the zinc reagent with phenyl iodide using
Pd(OAc)2 in the presence of trifuryl phosphine afforded
the 2-phenyl indole 51 in 65% yield which was then
transformed to analogue 57 under conditions outlined
in Scheme 4. A variety of other conditions were at-
tempted to prepare the 2-phenyl indole 51, however
the Negishi coupling was the only method that was suc-
cessful in a practical sense.
Analogues 37–40 were intended as tryprostatin A mim-
ics in which the alkyl substituent was moved from the in-
dole 2-position to the indole NH. This was expected to
result in analogues that are more readily accessible than
the natural products which require methods for prenyl-
ation of tryptophan at C-2. As shown in Scheme 2, the
ortho-iodoaniline 26 was coupled with the internal al-
kyne10 27 in the presence of catalytic Pd(OAc)2 with
Na2CO3 as the base to provide indole 28 in 77%
yield.45,46
Alkylation of 28 with methyl iodide, isoprenyl bromide,
benzyl bromide or allyl bromide in the presence of so-
OEt
OEt
I
E t3S i
N
N
a
+
N
77%
N
MeO
NH2
MeO
MeO
N
H
TES
OEt
OEt
26
27
28
b
O
OEt
OEt
H
H
N
H
O
N
d
c
HN
NH2
N
MeO
N
R
N
R
MeO
N
R
TES
O
OEt
37
33
34
35
36
29
30
31
32
R =Me
(82%)
R =Me
(84%)
R =Me
(99%)
38
39
40
R =isoprenyl (80%)
R =isoprenyl (85%)
R =isoprenyl (75%)
R =benzyl
R =allyl
(74%)
(80%)
R =benzyl
R =allyl
(85%)
(85%)
R =benzyl
R =allyl
(72%)
(80%)
Scheme 2. Reagents and conditions: (a) Pd(OAc)2, LiCl, Na2CO3, DMF, 100 ꢁC, 77%; (b) NaH, DMF, RX; (c) 2 N aq HCl, EtOH, THF, ꢀ78 ꢁC to
rt; (d) 24, TEA, CHCl3; DEA, CH3CN, rt; xylene, reflux.

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4631
Heck coupling48 of bromide 48 with methyl acrylate in
the presence of 5% Pd(PPh3)4 and Cy2NEt afforded
the coupled product 58 in 94% yield (Scheme 5). Ana-
logue 58 (Scheme 5) was then transformed into ester
Scheme 3. Reagents and conditions: (a) xylene, reflux, 80%; (b) NaH, DMF, RX; (c) 2 N aq HCl, EtOH, THF, ꢀ78 ꢁC to rt; (d) 24, TEA, CHCl3;
DEA, CH3CN, rt; xylene, reflux.
Scheme 4. Reagents and conditions: (a) NBS, CH3CN; (Boc)2O, DMAP, CH3CN, rt, 87%; (b) n-BuLi, THF, ꢀ78 ꢁC; RX; (c) n-BuLi, THF, ꢀ78 ꢁC;
ZnCl2; Pd(OAc)2, PhI, tri-2-furyl phosphine, rt, 65%; (d) 2 N aq HCl, EtOH, THF, ꢀ78 ꢁC to rt; (e) 24, TEA, CHCl3; DEA, CH3CN, rt; xylene,
reflux.
Scheme 5. Reagents and conditions: (a) Pd(PPh3)4, methyl acrylate, Cy2NMe, toluene, 95 ꢁC, 94%; (b) 2 N aq HCl, EtOH, THF, ꢀ78 ꢁC to rt, 80%;
(c) 24, TEA, CHCl3; DEA, CH3CN, rt; xylene, reflux.

4632
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
Scheme 6. Reagents and conditions: (a) 2 N aq HCl, EtOH, THF, ꢀ78 ꢁC to rt, 86%; (b) 24, TEA, CHCl3, DEA, CH3CN, rt; xylene, reflux; (c) NBS
or NCS, THF, ꢀ78 ꢁC to rt; (d) Fmoc-L-amino-acyl chloride, TEA, CHCl3; DEA, CH3CN, rt; xylene, reflux.
60, analogous to well-developed processes outlined pre-
viously in Scheme 1. It was well known in the literature
that the 6-methoxy group activated the C-2 position of
the indole nucleus towards electrophilic substitution.
As illustrated in Scheme 6, indole 28 was hydrolyzed
with aq 2 N HCl to afford the 6-methoxy-L-tryptophan
ethyl ester 61 in 86% yield. Ester 61 was further trans-
formed into the Na–H analogue 62 under conditions
similar to that described for 1 in Scheme 1. Analogue
62 was treated with NBS at ꢀ78 ꢁC to afford 2-bromo-
indole 63 in 80% yield. The corresponding 2-chloro ana-
logue 64 was prepared in 85% yield (from NCS) under
analogous conditions to those described above for the
preparation of 2-bromo-indole 63.
Scheme 7. Reagents and conditions: (a) Fmoc-L-amino-acylchloride, TEA, CHCl3; DEA, CH3CN, rt; xylene, reflux; (b) NaNO2, TFA, ꢀ78 ꢁC to
ꢀ20 ꢁC, 75%; (c) NH2NH2, FeCl3.6H2O, active C, MeOH, reflux, 91%; (d) CHCl3, ClC(S)Cl, 93%; (e) TfN3, aq CuSO4, Et3N, CH2Cl2/MeOH, 89%.

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4633
Two additional amino acids, other than proline, were
also incorporated into 1 (region D). As shown in
Scheme 6, 6-methoxytryptophan 61 was transformed
into the cyclic dipeptides 65 and 66 with L-valine and
L-phenylalanine, respectively, under conditions illus-
trated in Scheme 6. The 2-isoprenyl indole 20 was also
transformed to analogues 67 and 68, as shown in
Scheme 7.
The synthesis of C-6 substituted analogues of tryprost-
atin A modified in region A is depicted in Scheme 7.
The synthesis began with a highly regioselective pro-
cess for nitration of 2. Although several methods were
attempted to incorporate the nitro group at the de-
sired 6-position with only minimal success, this was
successfully carried out when 2 was treated with
NaNO2 in the presence of TFA49 at low temperature
to afford 69. To determine the regiochemistry, detailed
NMR analysis of the 6-nitro analogue 69 was carried
out. The coupling patterns of the aromatic ring pro-
tons could be employed to distinguish the 4- and 7-
substituted indoles from the 5- and 6-substituted
regioisomers, since one would not expect singlet pro-
tons in the spectrum of the 4- or 7-substituted indoles.
The 1H NMR spectrum of 69 clearly contained one
singlet (8.29 ppm) corresponding to one proton in
the aromatic region, consequently, the product of this
mononitration was either the 5- or 6-substituted regio-
isomer. In case of the 5-nitrosubstituted indole this
singlet would correspond to the proton at C(4),
whereas in the case of 6-nitrosubstituted indole this
singlet would correspond to the proton at C(7). The
6-nitro regioisomer would be expected to exhibit a
much stronger NOE signal between the indole N(H)
proton and the proton at C(7) than the one between
the indole N(H) proton and the proton at C(4). A
strong NOE signal was observed between this proton
singlet and the indole N(H) and vice versa. This fur-
ther ruled out the 5-nitro regioisomer. Reduction of
the nitro group in 69 (Scheme 7) with hydrazine in
the presence of FeCl3.H2O and activated carbon
in refluxing methanol45 furnished analogue 70
which was purified by column chromatography and
stored as the hydrochloride salt. Amine 70 was stirred
with thiophosgene in dry chloroform to afford the
6-isothiocyanate analogue 71 in high yield. Treatment
of amine 70 with triflyl azide (TfN3) in the presence
of copper sulfate afforded analogue 72 in 89%
yield.50,51
Figure 2. Representative agarose gel from the topoisomerase II-
mediated DNA relaxation assay. Data for all compounds are not
shown. Lane 1: DNA only; lane 2: DNA + topoisomerase II; lane 3:
DNA + topoisomerase II + m-AMSA (100 lM); lane 4: DNA + topo-
isomerase II + 1% DMSO; lane 5: DNA + topoisomerase II + 1
(100 lM); lane 6: DNA + topoisomerase II + 3 (100 lM); lane 7:
DNA + topoisomerase II + 2 (100 lM); lane 8: DNA + topoisomerase
II + 4 (100 lM).
phoresis experiment is presented in Figure 2. The
agent, m-AMSA, a known inhibitor of topoisomerase
II, was employed as the control (lane 3). The other
controls employed were no-enzyme (lane 1), enzyme
(lane 2), and 1% DMSO (lane 4). The gels were ana-
lyzed qualitatively by examination of the presence of
DNA bands that migrate farther down on the gel than
the negative controls. Topoisomerase II-mediated
relaxation of the DNA prevents the band from migrat-
ing down the gel as far as one that is still in a super-
coiled form. Therefore, DNA incubated with
topoisomerse II inhibitors will migrate farther on the
gel than the no-enzyme or DMSO controls. Lane 1
is DNA alone, existing in two forms- supercoiled
DNA and loosened DNA; lane 2 is topoisomerase II
together with DNA, and supercoiled DNA was re-
laxed by topoisomerase II completely. As illustrated
in Figure 2, 1 (lane 5) and 2 (lane 7) are both weak
inhibitors of topoisomerase II; however, the potency
cannot be determined from this data. The laddering
is evidence of inhibition of topoisomerase II. The
enantiomers of tryprostatin A 3 (lane 6) and B 4 (lane
8) were both inactive. The four diastereomers 5–8
(data not shown) were also found to be inactive as
topoisomerase II inhibitors in this assay. Tryprostatin
A 1 and B 2 are, therefore, weak inhibitors of topoiso-
merase II but their enantiomers (3 and 4) and diaste-
reomers (5–8) are not.
3.2. Effects of analogues 1–8 on tubulin polymerization
3. Biological evaluation and discussion
Tryprostatins 1–8 were also evaluated as inhibitors of
tubulin polymerization.13,54 Purified tubulin, containing
MAPs and GTP, was incubated at 37 ꢁC with either
DMSO (as a solvent control), colchicine (standard), or
analogues 1–8 and the change in absorbance was mea-
sured at 351 nM over 10 min. The concentration of the
standard (colchicine) and analogues (1–8) was varied
for different runs to obtain a delta absorbance versus
concentration curve. Illustrated in Figures 3 and 4 are
the results of the tubulin polymerization assay. Colchi-
cine (the positive control) strongly suppressed tubulin
3.1. Effects of analogues 1–8 on topoisomerase II
Tryprostatins 1–8 were evaluated as inhibitors of
topoisomerase II in the topoisomerase II-mediated
DNA relaxation assay.52,53 This assay measures the
ability of the compound to inhibit the ability of topo-
isomerase II to relax supercoiled DNA. The inhibitory
activities against topoisomerase II of compounds 1–8
were evaluated by agarose gel electrophoresis experi-
ments. The photopicture of 1–4’s agarose gel electro-

4634
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
Osada et al.13 recently reported that tryprostatin A 1
0.600
0.500
0.400
0.300
0.200
0.100
0.000
was a novel inhibitor of MAP-dependent microtubule
assembly and through disruption of the microtubule
spindle, specifically inhibited cell cycle progression at
the M phase. Thus biological evaluation of analogues
1–8 illustrated above indicated that 1 was a weak
inhibitor of both topoisomerase II and tubulin polymer-
ization, whereas 2 was only a weak inhibitor of topoiso-
merase II. The enantiomers (3 and 4) and diastereomers
(5–8) were inactive in both the tubulin polymerization
assay and topoisomerase II-mediated DNA relaxation
assay. In terms of the stereochemistry of the amino acids
present in the diketopiperazine ring, biological evalua-
tion indicated that ligands with the absolute configura-
tion L-Tyr-L-Pro (natural stereochemistry as in 1 and
2) were essential for inhibition of tubulin polymerization
and/or topoisomerase II. Modification of the absolute
configuration of the diketopiperazine ring from L-Tyr-
L-Pro (1 and 2) to D-Tyr-D-Pro (3 and 4), D-Tyr-L-Pro
(5 and 6), and L-Tyr-D-Pro (7 and 8) resulted in ana-
logues that were very poor inhibitors of topoisomerase
II and/or tubulin polymerization. Additionally, compar-
isons of analogues 1 and 2 indicated that presence of the
6-methoxy substituent in 1 resulted in analogues that are
dual inhibitors of microtubule assembly and topoiso-
merase II.
Tryprostatin A
Tryprostatin B
Colchicine
0
50
100
150
200
250
300
350
Concentration (micromolar)
Figure 3. Inhibition of tubulin polymerization by tryprostatin A 1 and
B 2, colchicine, a known tubulin polymerization inhibitor, was used as
a control.
0.350
0.325
0.300
0.275
0.250
0.225
0.200
0.175
0.150
Colchicine (IC50= 44.9)
In order to determine whether the absolute configura-
tion of L-Tyr and/or L-Pro in the diketopiperazine ring
was required to inhibit cell proliferation, analogues 1–8
were evaluated as inhibitors of three human lung
(H520), breast (MCF-7), and prostrate (PC-3) cancer
cell lines (Table 1).12 Analogues 1–7 were not potent
inhibitors (GI50 > 100 lM) of the growth of tumor cells
in the three human cancer cell lines evaluated. How-
ever, the diastereomer 8 of tryprostatin B 2 exhibited
potent cytotoxic activity at 100 lM against all three hu-
man cancer cell lines evaluated. It was found, in agree-
ment with Danishefsky et al.,55 that the inhibition of
tryprostatin B 2 against the growth of the three human
cancer cell lines evaluated occurred at higher concentra-
tions (GI50 > 100 lM) than that reported earlier6–8 for
isolated tryprostatin B 2. Danishefsky et al.55 have shed
some light on the apparent discrepancies in the cytotox-
icity of isolated 2 versus synthetic 2. Their studies55
Tryprostatin A-en (IC50= 755)
Tryprostatin B-en (IC50=787)
0
10
20
30
40
50
60
Concentration (micromolar)
Figure 4. Inhibition of tubulin polymerization by analogues 3 and 4.
Colchicine was employed as a control.
assembly (IC50 = 44.9 lM), while 1 (Fig. 3) caused a
moderate reduction in the rate of tubulin polymerization
(IC50 = 250 lM). Tryprostatin B 2 (Fig. 3) as well as the
enantiomers of tryprostatin A 3 and B 4 (Fig. 4)
were inactive in this assay (IC50s > 700 lM). Com-
pounds 5–8 (data not shown) were also found to be inac-
tive in this assay (less than 2% inhibition at 60 lM:
IC50s > 700 lM).
Table 1. Cell growth inhibition of tryprostatins 1–8 (at 10, 100 lM) on human lung (H520), breast (MCF-7) and prostate (PC-3) cancer cell linesa
Compound
Percent cell survivalb
H520
MCF-7
PC-3
10 lM
100 lM
10 lM
100 lM
10 lM
100 lM
1
2
3
4
5
6
7
8
80.1 4.1
77.6 3.6
81.7 3.9
>100
79.4 4.2
60.5 3.5
75.2 3.5
99.8 1.6
>100
>100
95.0 4.7
66.7 5.3
>100
99.2 4.2
95.5 2.8
>100
95.6 5.0
68.9 6.6
83.7 4.2
78.9 2.1
>100
88.2 5.8
>100
>100
>100
>100
95.8 1.3
>100
>100
>100
>100
>100
76.5 11.2
98.5 3.1
0.1 0.1
>100
99.0 4.6
0.0 0.0
97.3 5.9
>100
59.3 3.9
68.5 3.4
>100
0.2 0.0
99.3 1.8
88.3 8.4
>100
73.6 5.3
a Table 1 published in its entirety in Ref. 12.
b CellTiter 96TM AQueous non-radioactive cell proliferation assay (Promega) was used to determine growth inhibition. Percent inhibition values were
calculated versus control wells and were done in quadruplicate. Control wells contained 0.2% DMSO and the positive control was either etoposide
or m-AMSA (20 lM, 20 lM). Values are reported the standard deviation of the mean.

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4635
Table 2. Growth inhibition (GI50) in lM of human cancer cell lines by
8 and etoposide
If one examines the structures of tryprostatins (1–7) and
compares them with that of the active analogue 8, one
can generate the following conclusion: the L-Tyr unit
in the diketopiperazine ring was essential for potent
tumor cell growth inhibition since none of the other try-
prostatins (3–6), which contained the D-Tyr unit, exhib-
ited activity. Biological evaluation of analogue 8 also
indicated that the inhibition of the growth of human
cancer cells by analogue 8 was not due to the inhibition
of topoisomerase II or tubulin polymerization since ana-
logue 8 was inactive against these two molecular targets.
Further studies to identify the precise molecular targets
are required. The presence of the 6-methoxy group on 7
compared to 8 nearly eliminated the potent tumor cell
growth inhibitory activity against the three human can-
cer cell lines evaluated. The potent cytotoxic activity of
analogue 8 against human cancer cells led to the evalu-
ation of its activity against the growth of normal human
cell lines. In preliminary studies, 8 was found to be cyto-
toxic to normal human cell lines; however, further stud-
ies are required in this regard.
Compound
H520
MCF-7
PC-3
8a
15.8
11.9
8.7
15.9
17.0
55.6
11.9
12.3
11.1
8
Etoposide
a Data were obtained from NCI.
indicated that a DMSO solution of 2, upon standing in
air, undergoes slow transformation to a mixture of
products. The solutions of 2 containing detectable
byproducts were considerably more cytotoxic (ca. 50-
fold) than those containing apparently homogenous
tryprostatin. Growth inhibitory (GI50) potency of 8
was also compared to that of etoposide against the
growth of the three human lung (H520), breast
(MCF-7), and prostrate (PC-3) cancer cell lines (Table
2).12 Outlined in Table 2 are the results obtained from
the National Cancer Institute (NCI)56 screening of ana-
logue 8 on the same three human cancer cell lines. The
data obtained from the NCI for 8 were in complete
agreement with the data obtained for 8 in the present
study against all three human cancer cell lines evalu-
ated. Analogue 8 was 3-fold more potent than etopo-
side in inhibition of the growth of the MCF-7 human
cancer cell line. Also, analogue 8 was equipotent with
etoposide against the growth of H520 and PC-3 human
cancer cell lines.
Analogues 1–8 were selected by the NCI for evaluation
in its in vitro preclinical antitumor screening program.
The ability of compounds 1–8 to inhibit the growth of
tumor cells was measured as GI50 values, the concentra-
tion required to inhibit the growth of tumor cells in cul-
ture by 50%, as compared to a control (Table 3). In two
of the 60 tumor cell lines evaluated, tryprostatin A 1
showed GI50 values of 610ꢀ5 M. Again, tryprostatin B
Table 3. Cytotoxicity evaluation (GI50, lM) of compounds 1–8 against selected tumor cell lines56
Cell line
1
2
3
4
5
6
7
8
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
>100
11.3
2.73
ND
>100
>100
56.1
>25
>25
>25
>25
>25
>25
25.1
29.0
25.8
21.0
12.9
12.1
11.9
35.0
17.7
12.2
7.76
11.3
22.2
55.7
31.8
34.9
18.6
25.4
99.4
3.22
22.4
20.1
5.96
5.54
9.46
>100
>100
>100
92.0
MOLT-4
RPMI-8226
SR
>100
>100
50.6
37.1
5.68
76.9
Non-small cell lung cancer
HOP-92
EKVX
21.2
>100
43.1
>100
23.1
>25
ND
39.4
ND
>50
2.94
20.7
16.8
>100
1.70
6.90
Colon cancer
COLO 205
HT-29
>100
ND
>100
ND
>25
ND
21.2
37.5
12.4
16.1
39.3
ND
>100
40.3
17.7
5.25
Melanoma
LOX IMVI
>100
>100
>25
>50
33.5
17.8
>100
9.23
Ovarian
OVCAR-3
IGROV1
>100
90.5
>100
>100
>25
>25
38.2
16.0
>50
>50
28.7
32.3
85.5
50.0
9.69
11.5
Prostrate cancer
PC-3
DU-145
94.0
>100
>100
>100
>25
>25
21.2
>50
21.7
40.0
24.8
59.5
>100
>100
11.9
14.0
Breast cancer
MDA-MB-231/ATCC
BT-549
>100
79.7
>100
>100
58.2
>100
>25
>25
>25
11.9
7.21
40.0
13.0
9.77
25.5
49.6
26.9
25.9
>100
>100
>100
14.9
13.2
15.9
MCF-7
Renal cancer
UO-31
>100
>100
>25
12.6
27.3
45.7
>100
14.7

4636
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
2 (GI50 > 40 lM) was considerably less active against
the growth of the 60 tumor cell lines evaluated. In 9 of
the 60 tumor cell lines evaluated, the most active ana-
logue 8 showed GI50 values of 610ꢀ5 M. Analogue 5,
the diastereomer of tryprostatin A, 1 was more active
than 1 in the inhibition of the growth of tumor cells in
most of the tumor cell lines evaluated. Analogues 3
and 7 were both considerably less active than 1 in inhi-
bition of the growth of the tumor cells in the NCI
screening program. However, analogue 4, the enantio-
mer of tryprostatin B 2, as well as both of the diastereo-
mers 6 and 8 were more active than 2 in inhibition of the
growth of tumor cells in most of the tumor cell lines
evaluated. It is noteworthy that compounds 4, 5, 6
and 8 were not general cell toxins but showed selectivity
both within a type of tumor cell line and across different
tumor cell lines, with inhibitory values, which in some
instances, differed by 100-fold.
A 30 lM concentration of 1 arrested cell cycle progres-
sion in the M phase, as previously reported.8,13 Many
of these analogues were found to have similar activity
as tryprostatin A against tsFT210 cell proliferation.
Analogue 38, which closely resembled tryprostatin A,
was inactive. Substitution of the 2-isoprenyl moiety in
38 with a smaller methyl substituent (37) also resulted
in an inactive analogue. Replacement of the Na-isopre-
nyl group in analogue 38 with an allyl group (40) re-
sulted in an analogue that was equipotent to 1 in the
inhibition of cell proliferation. Similarly, replacement
of the Na-isoprenyl group in 38 with a Na-benzyl group
39 also resulted in an analogue that was equipotent to 1
in inhibiting cell proliferation. However, analogues 39
and 40 inhibited cell cycle progression at the G1 phase.
The biological data of analogues 39 and 40 indicated
substitution of the indole N(H) with a benzyl moiety
or allyl moiety was highly conducive for inhibition of
cell proliferation and caused cell cycle arrest in the G1
phase. Tryprostatin A 1 analogues in which the indole
NH was substituted with a benzyl moiety 46 or allyl
moiety 47 also afforded active analogues that were equi-
potent with 1 in inhibition of the growth of tsFT210
cells. However, this inhibition was not cell cycle depen-
dent. Removal of the 2-isoprenyl group in 1 afforded
analogue 62 which was inactive. Similarly, removal of
the 2-isoprenyl group in 1 and substitution of it with a
bromine atom 63 or chlorine atom 64 also resulted in
3.3. Structure–activity relationships of tryprostatin A
analogues
Because 1 was an inhibitor of BCRP, the tryprostatin
A-related analogues (37–40, 46, 47, 55, 56, 60, and 62–
72) were evaluated in vitro for the ability to disrupt
the cell cycle and to inhibit tsFT210 cell prolifera-
tion.4,13,58 The inhibitory potency (IC50) values are listed
in Table 4 and compared with tryprostatin A 1.
Table 4. Effect of tryprostatin A-related analogues on cell cycle progression and tsFT210 cell proliferation
Compound
R1
R2
R3
R4
R5
IC50 (lM)a
Effect on cell cycle arrestb
1
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
NO2
NH2
NCS
N3
H
Me
Isoprenyl
H
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
Isopropyl
Benzyl
68
M phase at 30 lM
No effectc
No effect
37
38
39
40
46
47
55
56
60
62
63
64
65
66
67
68
69
70
71
72
>100
>100
46
Isoprenyl
H
Benzyl
Allyl
Benzyl
Allyl
H
H
G1 phase at 100 lM
G1 phase at 100 lM
No effect
H
62
55
Isoprenyl
Isoprenyl
Benzyl
Allyl
75
>100
60
No effect
No effect
H
G1, G2/M phase at 100 lM
No effect
No effect
H
>100
>100
96
H
H
Br
H
No effect
No effect
No effect
H
Cl
H
>100
>100
100
19
H
H
H
H
H
H
H
M phase at 100 lM
G1, G2/M phase at 100 lM
No effect
H
Isoprenyl
Isoprenyl
Isoprenyl
Isoprenyl
Isoprenyl
Isoprenyl
Isopropyl
Benzyl
H
10
H
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
>100
>100
50
No effect
No effect
H
H
G1, G2/M phase at 100 lM
G1, G2/M phase at 100 lM
H
60
a Exponentially growing tsFT210 cells were treated with test compounds at 32 ꢁC for 48 h. Cell viability was measured using the color reagent, WST-
8TM
.
b Exponentially growing tsFT210 cells were treated with test compounds at 32 ꢁC for 18 h. Then, flow cytometric analysis and nuclei staining were
carried out, as described in Section 5.
c No effect even at 100 lM.

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4637
inactive analogues. Comparison of the analogues 62–64
with the activity of the active analogue 1 indicated the
lipophilic 2-isoprenyl group in 1 played an important
role in the inhibition of cell proliferation. The lipophilic
2-isoprenyl moiety may play an important role in the
interaction with the molecular target and/or may in-
crease the lipophilicity of the molecule thereby facilitat-
ing passive diffusion into the cells. Substitution of the
2-isoprenyl group of 1 with a 2-benzyl group 55 or
2-methyl acrylate moiety 60 afforded inactive analogues.
However, a 2-allyl substituted analogue 56 of 1 was
found to be equipotent to 1 in the inhibition of cell pro-
liferation. Analogue 56 also arrested cell cycle progres-
sion at the G1, G2/M phase. Substitution of the
L-proline residue in the diketopiperazine ring of 1 with
an L-valine residue (67) afforded a 3.5-fold more potent
inhibitor of the growth of tsFT210 cells than 1. Similarly
replacement of the L-proline residue in 1 with an L-phe-
nyl alanine residue (68) resulted in an analogue that was
7-fold more potent than 1 in the inhibition of the growth
of tsFT210 cells, but this inhibition was not cell cycle
dependent. The biological data of analogues 67 and 68
indicated substitution of the L-proline residue in the
diketopiperazine ring of 1 with other L-amino acids
was highly conducive for inhibition of cell proliferation.
Removal of the 2-isoprenyl group from analogue 67
afforded 65 which slightly inhibited (IC50 > 100 lM) cell
proliferation. Again, removal of the 2-isoprenyl moiety
from analogue 68 afforded analogue 66 which was 10-
fold less potent than 68 in the cell proliferation assay
again indicating the importance of the 2-isoprenyl moi-
ety in the inhibition of cell proliferation. Analogue 66
also arrested cell cycle progression in the M phase at
100 lM. Replacement of the 6-methoxy group in 1 with
a nitro group (69) or amino group (70) resulted in ana-
logues that were poor (IC50 > 100 lM) inhibitors of the
growth of tsFT210 cells. However, substitution of the 6-
methoxy group in 1 with an isothiocyanate group 71 or
azide group 72 resulted in analogues that were equipo-
tent with 1 in the inhibition of the growth of tsFT210
cells. Both compounds 71 and 72 inhibited the cell cycle
progression of tsFT210 cells at the G1, G2/M phase.
nism of action of the tryprostatins indicate that tryprost-
atin A 1 is a weak inhibitor of topoisomerase II and
tubulin polymerization, whereas tryprostatin B 2 is only
a weak inhibitor of topoisomerase II. The absolute con-
figuration of L-Tyr-L-pro in the diketopiperazine ring of
the tryprostatins was shown to be essential for inhibition
of tubulin polymerization and/or topoisomerase II. The
6-methoxy substituent in 1 was shown to promote inhi-
bition of both topoisomerase II and tubulin polymeriza-
tion in in vitro assays. Biological evaluation indicated
that the presence of the 2-isoprenyl moiety on the indole
scaffold in 1 was essential for inhibition of cell prolifer-
ation. Removal of the 2-isoprenyl group in 1 and substi-
tution of the indole NH with a benzyl group or allyl
group also afforded analogues that inhibited cell prolif-
eration. The 6-methoxy substituent in 1 could be re-
placed with various groups to afford active analogues.
Various L-amino acids other than L-proline could be
incorporated into the diketopiperazine ring of 1 to
afford active analogues. The nature of the substituent
present on the indole NH or at the C-2 position influ-
enced the mechanism of action of the analogue and
highlights the versatility of the tryprostatin skeleton as
a template for drug discovery. Analogue 8 was more po-
tent than etoposide (a clinically used anticancer drug)
against the three human cancer cell lines evaluated.
However, preliminary biological evaluation against nor-
mal cells indicated that it was toxic which may limit its
potential use in this regard. More work is required to de-
fine this. In the NCI preclinical screening program ana-
logue 5, the diastereomer of tryprostatin A 1, was more
active than 1 in the inhibition of growth of tumor cells in
most of the tumor cell lines evaluated. Similarly, ana-
logue 4, the enantiomer of tryprostatin B 2 as well as
both the diastereomers 6 and 8, were more active than
2 in inhibition of the growth of tumor cells in most of
the tumor cell lines evaluated.
5. Experimental
All melting points were determined using a Thomas-
Hoover capillary melting point apparatus or an Electro-
thermal model IA8100 digital melting point apparatus
and are uncorrected. Reagents and starting materials
were obtained from commercial suppliers and used with-
out further purification unless otherwise indicated. Un-
less specified otherwise, solvents were freshly distilled
prior to use: tetrahydrofuran (THF), benzene, toluene,
dioxane, and diethyl ether were distilled under nitrogen
from sodium metal utilizing benzophenone as an indica-
tor; MeOH and EtOH were distilled over Mg metal and
I2; dichloromethane was dried over MgSO4 and then
distilled over P2O5; triethylamine was dried over KOH
and then distilled over KOH. Flash column chromatog-
raphy was carried out on silica gel purchased from E. M.
Laboratories (grade 60). HPLC grade solvents were
used for all chromatography. Analytical thin-layer chro-
matography (TLC) was conducted on precoated plates:
silica gel 60F-254, 0.25 mm thickness, manufactured by
E. Merck & Co., Germany. Indoles were visualized with
Dragendorf’s reagent or a saturated solution of ceric
ammonium sulfate in 50% H2SO4. Ketones or aldehydes
Turner and Sullivan et al.57 have recently shown that
tryprostatin A 1 is a specific and potent inhibitor of
BCRP (breast cancer resistance protein), which further
indicates the potential of analogues of tryprostatin A 1
synthesized in the present study in the potential inhibi-
tion of BCRP. Some of the analogues are currently
being evaluated as inhibitors of BCRP and will be a to-
pic of future communication. The isothiocyanate ana-
logue 71 and the 6-azido analogue 72 may be excellent
irreversible inhibitors in studies of BCRP.
4. Conclusion
In summary, the first structure–activity investigation
into the cell cycle inhibitory effects of the tryprostatin
A 1 analogues has been carried out. The SAR of try-
prostatin A 1 suggests that the search for a potent and
selective antitumor agent, in the tryprostatin series, still
looks promising. Studies on elucidation of the mecha-

4638
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
were visualized with an aqueous solution of 2,4-dinitro-
phenylhydrazine in 30% H2SO4. The H NMR spectra
1H, J = 7.6 Hz). 13C NMR (62.90 MHz, CDCl3) d
14.40, 16.66, 19.01, 28.21, 29.26, 31.60, 31.73, 56.05,
60.56, 60.64, 83.11, 114.89, 116.73, 119.60, 121.97,
123.96, 124.19, 131.42, 135.13, 149.72, 162.25, 163.50.
CIMS m/e (relative intensity) 442 (M+, +1). HRMS
Calcd for C25H35N3O4m/z = 441.2628, found m/z =
441.2536. This material was used directly in a later step.
1
were recorded on a Bruker 300-MHz or 500-MHz multi-
ple-probe instrument. Infrared spectra were recorded on
a Nicolet Dx FTIR DX V5.07 spectrometer or a Perkin
Elmer 1600 Series FT-IR spectrometer. Low resolution
mass spectral data (EI/CI) were obtained on a Hew-
lett-Packard 5985B gas chromatography-mass spec-
trometer. High resolution mass spectral data were
taken on a VG autospectrometer (Double Focusing
High Resolution GC/Mass Spectrometer, UK). Optical
rotations were measured on a JASCO DIP-370 polarim-
eter. Microanalyses were performed on a CE Elantech
EA1110 elemental analyzer.
5.3. (3R,6S)-3-[(1-tert-Butyloxycarbonyl-6-methoxy)-3-
indoyl]methyl-3,6-dihydro-6-isopropyl-2,5-diethoxypyr-
azine (14)
Indole 14 (5.5 g) was prepared in 92% yield from 9a
(4.4 g, 12.7 mmol) and 11 (3.06 g,14.5 mmol), analogous
to the procedure described for the synthesis of 12. 14:
27
5.1. (3S,6R)-3-[(1-tert-Butyloxycarbonyl-6-methoxy)-3-
indoyl]methyl-3,6-dihydro-6-isopropyl-2,5-diethoxypyr-
azine (12)
½aꢁ ꢀ25.1ꢁ (c 0.8, CHCl3); IR mmax (NaCl) 2970,
D
1
1730, 1690 cmꢀ1; H NMR (250 MHz, CDCl3) d 0.63
(d, 3H, J = 6.8, Hz), 0.92 (d, 3H, J = 6.9 Hz), 1.21 (t,
3H, J = 7.1 Hz), 1.29 (t, 3H, J = 7.1 Hz), 1.62 (s, 9H),
2.15 (m, 1H), 3.13 (d, 2H, J = 4.8 Hz), 3.53 (t, 1H,
J = 3.4 Hz), 3.83 (s, 3H), 3.94–4.16 (m, 4H), 4.25 (dd,
1H, J = 3.8 Hz), 6.80 (dd, 1H, J = 2.2 and 8.6 Hz),
7.21 (s, 1H), 7.42 (d, 1H, J = 8.6 Hz), 7.67 (br, s, 1H).
13C NMR (62.90 MHz, CDCl3) d 14.43, 16.68, 19.04,
28.23, 29.37, 31.72, 55.58, 56.13, 60.42, 60.51, 60.67,
82.93, 99.03, 111.49, 116.74, 120.10, 122.83, 125.28,
136.10, 149.81, 157.67, 162.29, 163.49. CIMS m/e (rela-
tive intensity) 472 (M+, +1). HRMS Calcd for
C26H37N3O5 m/z = 471.2733, found m/z = 471.2739.
This material was used directly in a later step.
To a solution of 10 (3.41 g, 16.1 mmol) in dry THF
(60 mL) under nitrogen, n-BuLi (2.5 M, 7.08 mL,
17.7 mmol) was added dropwise at ꢀ78 ꢁC. The solution
which resulted was stirred at ꢀ78 ꢁC for 30 min and
treated slowly with a solution of crude 3-bromo-methyl-
indole 9a (4.79 g, 14.1 mmol) in THF (30 mL). The mix-
ture which resulted was stirred at ꢀ78 ꢁC for 20 h, and
then allowed to slowly warm to rt. The solution was
concentrated under reduced pressure and diluted with
a saturated aqueous solution of NaHCO3. The aqueous
layer was extracted with CH2Cl2 (3 · 20 mL). The com-
bined organic layers were washed with brine (30 mL)
and dried (K2CO3). After removal of solvent under re-
duced pressure, the residue was purified by flash chro-
5.4. (3R,6S)-3-(1-tert-Butyloxycarbonyl-3-indoyl)methyl-
3,6-dihydro-6-isopropyl-2,5-diethoxypyrazine (15)
matography (silica gel, hexane/EtOAc, 10:1) to afford
27
D
12 as an oil (6.04 g, 91%): ½aꢁ +24.7ꢁ (c 0.9, CHCl3);
Indole 15 (6.6 g) was prepared in 91% yield from 9b and
11, analogous to the procedure described for the synthe-
sis of 12. 15: 1H NMR (300 MHz, CDCl3) d 0.66 (d, 3H,
J = 6.8, Hz), 0.95 (d, 3H, J = 6.9 Hz), 1.23 (t, 3H,
J = 7.1 Hz), 1.32 (t, 3H, J = 7.1 Hz), 1.64 (s, 9H), 2.18
(m, 1H), 3.19 (m, 2H), 3.56 (t, 1H, J = 3.4 Hz), 3.96–
4.21 (m, 4H), 4.29 (dd, 1H, J = 4.3 Hz), 7.15–7.29 (m,
2H), 7.36 (s, 1H), 7.59 (d, 1H, J = 7.5 Hz), 8.08 (d,
1H, J = 7.6 Hz). 13C NMR (62.90 MHz, CDCl3) d
14.40, 16.66, 19.01, 28.21, 29.26, 31.60, 31.73, 56.05,
60.56, 60.64, 83.11, 114.89, 116.73, 119.60, 121.97,
123.96, 124.19, 131.42, 135.13, 149.72, 162.25, 163.50.
CIMS m/e (relative intensity) 442 (M+, +1). HRMS
Calcd for C25H35N3O4 m/z = 441.2628, found m/z
= 441.2634. This material was used directly in a later
step.
IR mmax (NaCl) 2970, 1730, 1690 cmꢀ1
;
1H NMR
(250 MHz, CDCl3) d 0.63 (d, 3H, J = 6.8 Hz), 0.92 (d,
3H, J = 6.9 Hz), 1.21 (t, 3H, J = 7.1 Hz), 1.29 (t, 3H,
J = 7.1 Hz), 1.62 (s, 9H), 2.15 (m, 1H), 3.13 (d, 2H,
J = 4.8 Hz), 3.53 (t, 1H, J = 3.4 Hz), 3.84 (s, 3H),
3.94–4.16 (m, 4H), 4.25 (dd, 1H, J = 3.8 Hz), 6.80 (dd,
1H, J = 2.2 and 8.6 Hz), 7.21 (s, 1H), 7.42 (d, 1H,
J = 8.6 Hz), 7.67 (br, s, 1H). 13C NMR (62.90 MHz,
CDCl3) d 14.43, 16.68, 19.04, 28.23, 29.37, 31.72,
55.58, 56.13, 60.42, 60.51, 60.67, 82.93, 99.03, 111.49,
116.74, 120.10, 122.83, 125.28, 136.10, 149.81, 157.67,
162.29, 163.49. EIMS m/e (relative intensity) 471 (M+,
47), 261 (21), 212 (100), 169 (67), 141 (20), 57 (51). Anal.
Calcd for (C26H37N3O5) C, H, N. This material was
used directly in a later step.
5.2. (3S,6R)-3-(1-tert-Butyloxycarbonyl-3-indoyl)methyl-
3,6-dihydro-6-isopropyl-2,5-diethoxypyrazine (13)
5.5. (3S,6R)-3-[(1-tert-Butyloxycarbonyl-2-isoprenyl-6-
methoxy)-3-indoyl]-methyl-3,6-dihydro-6-isopropyl-2,5-
diethoxypyrazine (16)
Indole 13 (6.7 g) was prepared in 92% yield from 9b
(5.1 g, 16.4 mmol) and 10 (3.7 g, 17.6 mmol) analogous
to the procedure described for the synthesis of 12. 13:
To a solution of pyrazine 12 (1.94 g, 4.11 mmol) in dry
THF (30 mL) at ꢀ78 ꢁC under nitrogen, a solution of
lithium diisopropylamide (LDA, 1.5 M in THF,
4.2 mL, 6.17 mmol) was added dropwise. The mixture
which resulted was stirred at ꢀ78 ꢁC for 60 min. The
dry (HBr free) 4-bromo-2-methylbutene (1.03 g,
6.91 mmol) was then added dropwise at ꢀ78 ꢁC. The
mixture was stirred at ꢀ78 ꢁC for 1 h and allowed to
1H NMR (300 MHz, CDCl3)
d
0.66 (d, 3H,
J = 6.8, Hz), 0.95 (d, 3H, J = 6.9 Hz), 1.23 (t, 3H,
J = 7.1 Hz), 1.32 (t, 3H, J = 7.1 Hz), 1.64 (s, 9H), 2.18
(m,1H), 3.19 (m, 2H), 3.56 (t, 1H, J = 3.4 Hz), 3.96–
4.21 (m, 4H), 4.29 (dd, 1H, J = 4.3 Hz), 7.15–7.29 (m,
2H), 7.36 (s, 1H), 7.59 (d, 1H, J = 7.5 Hz), 8.08 (d,

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4639
warm to rt overnight. The solvent was removed under
reduced pressure. The residue was taken up in CH2Cl2
and washed with a 5% aqueous solution of NaHCO3.
The aqueous layer was extracted with CH2Cl2
(3 · 50 mL). The combined organic layers were dried
(K2CO3). After removal of the solvent under reduced
pressure, the residue was separated by flash chromatog-
raphy (silica gel, hexane/EtOAc, 15:1) to provide 16
(1.89 g, 85%) as an oil: IR mmax (NaCl) 2970, 1730,
J = 5.0 and 14.2 Hz), 3.68 (d, 2H, J = 5.1 Hz), 3.70 (m,
1H), 3.83 (s, 3H), 4.13 (qd, 2H, J = 2.1 and 7.1 Hz),
5.16 (t, 1H, J = 5.1 Hz), 6.82 (dd, 1H, J = 2.3 and
8.5 Hz), 7.33 (d, 1H, J = 8.5 Hz), 7.69 (d, 1H,
J = 2.3 Hz); 13C NMR (62.90 MHz, CDCl3) d 14.04,
18.02, 25.48, 26.03, 28.05, 30.35, 55.62, 60.85, 83.47,
100.31, 111.32, 113.98, 118.46, 122.28, 123.66, 131.70,
136.56, 136.95, 150.35, 157.41, 175.02; EIMS m/e (rela-
tive intensity) 430 (M+, 3), 272 (36), 228 (100). HRMS
Calcd for C24H34N2O5 m/z = 430.2468, found m/z
= 430.2481. This material was used directly in a later
step.
1690 cmꢀ1 1H NMR (250 MHz, CDCl3) d 0.61 (d,
,
3H, J = 6.8 Hz), 0.94 (d, 3H, J = 6.8 Hz), 1.18 (t, 3H,
J = 7.1 Hz), 1.31 (t, 3H, J = 7.1 Hz), 1.61 (s, 3H), 1.63
(s, 9H), 1.70 (s, 3H), 2.19 (m, 1H), 2.88 (dd, 1H,
J = 7.4 and 14.2 Hz), 3.23 (dd, 1H, J = 3.9 and
14.3 Hz), 3.56 (t, 1H, J = 3.4 Hz), 3.69 (d, 2H,
J = 6.0 Hz), 3.83 (s, 3H), 3.94–4.22 (m, 5H), 5.16 (t,
1H, J = 5.8 Hz), 6.78 (dd, 1H, J = 2.3 and 8.5 Hz),
7.37 (d, 1H, J = 8.6 Hz), 7.65 (d, 1H, J = 2.3 Hz). EIMS
m/e (relative intensity) 539 (M+, 65), 439 (11), 328 (16),
272 (58), 228 (100), 212 (55), 169 (31), 141 (16), 57 (48);
Anal. Calcd for (C31H45N3O5) C, H, N. This material
was used directly in a later step.
5.8. (S)-1-tert-Butyloxycarbonyl-2-isoprenyltryptophan
ethyl ester (21)
Ester 21 (1.1 g) was prepared in 93% yield from 17 as de-
26
scribed above for the preparation of 20. 21: ½aꢁ +19.7ꢁ
D
(c 0.8, CH3OH); 1H NMR (300 MHz, CDCl3) d 1.23 (t,
3H, J = 7.1 Hz), 1.66 (s, 9H), 1.68 (s, 3H), 1.74 (s, 3H),
1.80 (br s, 2H), 2.89 (dd, 1H, J = 8.9 and 14.2 Hz), 3.19
(dd, 1H, J = 5.0 Hz and 14.3 Hz), 3.73–3.77 (m, 3H),
4.13–4.19 (m, 2H), 5.19 (t, 1H, J = 1.4 Hz), 7.20–7.25
(m, 2H), 7.49 (dd, 1H, J = 1.0 and 7.0 Hz), 8.08 (dd,
1H, J = 1.5 Hz and 8.8 Hz); 13C NMR (62.90 MHz,
CDCl3) d 14.11, 18.15, 25.60, 25.99, 28.09, 30.27,
54.94, 61.02, 83.72, 114.04, 115.38, 118.09, 121.91,
122.43, 123.66, 129.68, 132.11, 136.00, 138.03, 150.33,
175.09. CIMS m/e (relative intensity) 401 (M+ +1). This
material was used directly in a later step.
5.6. (3S,6R)-3-[(1-tert-Butyloxycarbonyl-2-isoprenyl)-3-
indoyl]-methyl-3,6-dihydro-6-isopropyl-2,5-diethoxypyr-
azine (17)
Indole 17 (3.4 g) was prepared in 82% yield from 13 un-
der the conditions described above for the preparation
1
of 16. 17: H NMR (300 MHz, CDCl3) d 0.59 (d, 3 H,
J = 6.8 Hz), 0.91 (d, 3 H, J = 6.9 Hz), 1.15 (t, 3 H,
J = 7.1 Hz), 1.28 (t, 3 H, J = 7.1 Hz), 1.60 (s, 9 H),
1.62 (s, 3 H), 1.68 (s, 3 H), 2.09 (m, 1 H), 2.92 (m,
1H), 3.23 (m, 1 H), 3.56 (t, 1 H, J = 3.4 Hz), 3.71 (d, 2
H, J = 6.1 Hz), 3.98–4.20 (m, 5 H), 5.14 (t, 1 H,
J = 6.0 Hz), 7.10–7.15 (m, 2 H), 7.49 (dd, 1H, J = 2.1
and 6.9 Hz), 7.99 (dd, 1H, J = 1.3 and 7.2 Hz). 13C
NMR (62.90 MHz, CDCl3) d 14.34, 14.36, 16.53,
18.13, 19.05, 25.60, 26.10, 28.09, 29.33, 31.41, 56.86,
60.36, 60.40, 60.69, 83.29, 114.98, 115.27, 119.04,
121.80, 122.40, 123.15, 130.54, 131.47, 135.94, 137.83,
150.47, 162.78, 163.23. CIMS m/e (relative intensity)
510 (M+ +1). Anal. Calcd for (C30H43N3O40.25H2O)
C, H, N. This material was used directly in a later step.
5.9. (R)-1-tert-Butyloxycarbonyl-2-isoprenyl-6-methoxy-
tryptophan ethyl ester (22)
Ester 22 (0.86 g) was prepared in 73% yield from 14 as
27
D
described above for the preparation of 20. 22: ½aꢁ
ꢀ15.9ꢁ (c 0.9, CHCl3); All spectroscopic data were iden-
tical to that for 20 (the enantiomer of 22) reported in the
previous experiment except the optical rotation was
opposite in sign. This material was used directly in a la-
ter step.
5.10. (R)-1-tert-Butyloxycarbonyl-2-isoprenyltryptophan
ethyl ester (23)
5.7. (S)-1-tert-Butyloxycarbonyl-2-isoprenyl-6-methoxy-
tryptophan ethyl ester (20)
Ester 23 (0.77 g) was prepared in 70% yield from 15 as
27
D
described above for the preparation of 20. 23: ½aꢁ
ꢀ19.9ꢁ (c 0.9, CH3OH); All spectroscopic data were
identical to that for 21 (the enantiomer of 23) reported
in the previous experiment except the optical rotation
was opposite in sign. This material was used directly
in a later step.
To a solution of 2-prenylpyrazine 16 (1.27 g, 2.36 mmol)
in THF (30 mL) at 0 ꢁC was added an aqueous solution
of 2 N HCl (10 mL). The reaction mixture was allowed
to warm to rt and stirred for 1.5 h. A cold aqueous solu-
tion of 15% NH4OH was added. The solution was con-
centrated under vacuum and diluted with CH2Cl2. The
aqueous layer was extracted with CH2Cl2. The com-
bined organic layers were dried (K2CO3) and the solvent
was removed under vacuum. The residue was separated
5.10.1. Tryprostation A (1). Fmoc-L-proline (126 mg,
0.374 mmol) was dissolved in thionyl chloride (1 mL).
The solution which resulted was stirred overnight at rt.
Excess thionyl chloride was removed under reduced
pressure. The Fmoc-L-proline chloride 24 which resulted
was dissolved in dry CHCl3 (1 mL). This solution was
added dropwise at 0 ꢁC to a solution of 20 (107 mg,
0.249 mmol) and triethylamine (63.0 mg, 0.623 mmol)
in dry CHCl3 (6 mL). The mixture which resulted was
stirred at 0 ꢁC for 0.5 h and then at rt overnight. After
by flash chromatography (silica gel, EtOAc) to provide
27
D
20 (0.95 g, 94%) as an oil: ½aꢁ +15.2ꢁ (c 0.92, CHCl3);
IR mmax (NaCl) 2975, 1730, 1615 cmꢀ1
;
1H NMR
(250 MHz, CDCl3) d 1.22 (t, 3H, J = 7.1 Hz), 1.46–
1.55 (m, 2H), 1.63 (s, 9H), 1.66 (s, 3H), 1.71 (s, 3H),
2.82 (dd, 1H, J = 8.8 and 14.2 Hz), 3.12 (dd, 1H,

4640
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
removal of solvent under reduced pressure, a solution of
diethylamine (DEA, 2.5 mL) in acetonitrile (2.5 mL)
was added in the same flask. The reaction mixture was
stirred at rt for 2 h [monitored by TLC (silica gel) until
the disappearance of starting material]. Acetonitrile and
excess DEA were removed under reduced pressure. Xy-
lene (25 mL) was added into the same reaction vessel
and the solution degassed. The reaction mixture was
stirred at reflux for 2 d at which time examination by
TLC (silica gel) indicated the disappearance of starting
material. After removal of xylene under reduced pres-
sure, the residue was subjected to flash chromatography
5.10.4. Enantiomer of tryprostatin B (4). Enantiomer 4
(80 mg) was prepared in 83% yield from 23 as described
above for the preparation of tryprostatin A 1. The start-
ing material used here was D-tryptophan derivative 23
26
D
and Fmoc-D-Pro-Cl 25. 4: ½aꢁ 71.9ꢁ (c 1.1, CHCl3).
Anal. Calcd for (C21H25N3O2Æ3/4H2O) C, H, N. All
spectroscopic data were identical to that for 2 (the enan-
tiomer of 4) reported in the previous experiment except
the optical rotation was opposite in sign. All spectro-
scopic data were identical to that reported for 2 (the
enantiomer of 4) in a previous experiment except the
optical rotation was opposite in sign.
(silica gel, CHCl3/CH3OH, 95:5) to provide tryprostatin
27
D
A 1 (78 mg, 82%) as a solid: ½aꢁ ꢀ65.9ꢁ (c 0.97, CHCl3)
5.10.5. Diastereomer-1 of tryprostatin A (5). Indole 5
(105 mg) was prepared in 84% yield from 22 as described
above for the preparation of tryprostatin A 1. The start-
27
D
[lit. [7] ½aꢁ
ꢀ69.7ꢁ (c 0.70, CHCl3)]; 1H NMR
(250 MHz, CDCl3) d 1.73 (s, 3H), 1.76 (s, 3H), 1.85–
2.07 (m, 3H), 2.27–2.34 (m, 1H), 2.89 (dd, 1H,
J = 11.4 and 15.0 Hz), 3.41 (d, 2H, J = 7.2 Hz), 3.53–
3.72 (m, 3H), 3.81 (s, 3H), 4.05 (dd, 1H, J = 6.9 and
7.7 Hz), 4.32 (dd, 1H, J = 2.7 and 11.1 Hz), 5.28 (dd,
1H, J = 5.8 and 8.6 Hz), 5.61 (s, 1H), 6.74 (dd, 1H,
J = 2.2 and 8.6 Hz), 6.81 (d, 1H, J = 2.1 Hz), 7.32 (d,
1H, J = 8.6 Hz), 7.80 (br s, 1H); 13C NMR
(62.90 MHz, CDCl3) d 17.92, 22.63, 25.07, 25.69,
25.71, 28.32, 45.38, 54.56, 55.73, 59.23, 94.87, 104.38,
109.27, 118.33, 119.98, 122.27, 135.14, 135.27, 136.25,
156.31, 165.80, 169.37. EIMS m/e (relative intensity)
381 (M+,4), 228 (100), 212 (14), 198 (9). HRMS Calcd
for C22H27N3O3m/z = 381.2052, found m/z = 381.2044.
Anal. Calcd for C22H27N3O3Æ1/3H2O (C, H, N). The
spectral data for 1 were identical to that reported by
Osada et al.7 in the literature.
ing material used here was D-tryptophan derivative 22
26
and Fmoc-L-Pro-Cl 24. 5: ½aꢁ ꢀ20.0ꢁ (c 0.12, CHCl3),
D
1
IR mmax (NaCl) 3269, 2971, 1673, 1650 cmꢀ1; H NMR
(300 MHz, CDCl3) d 1.18–1.43 (m, 2H), 1.60–1.71 (m,
1H), 1.74 (s, 3H), 1.78 (s, 3H), 1.99–2.08 (m, 1H), 2.67
(dd, 1H, J = 6.3, 10.7 Hz), 3.08 (dd, 1H, J = 4.5,
14.7 Hz), 3.16 (dd, 1H, J = 2.5, 9.5 Hz), 3.35 (d, 1H,
J = 5.0 Hz), 3.39 (d, 2H, J = 7.7 Hz), 3.47–3.57 (m, 1H),
3.81 (s, 3H), 4.23 (dd, 1H, J = 4.3, 8.5 Hz), 5.26 (tt, 1H,
J = 1.3, 6.0 Hz), 6.26 (d, 1H, J = 3.6 Hz), 6.74 (dd, 1H,
J = 2.3, 8.6 Hz), 6.78 (d, 1H, J = 2.1 Hz), 7.37 (d, 1H,
J = 8.6 Hz), 7.92 (br s, 1H), 13C NMR (62.90 MHz,
CDCl3) d 17.88, 21.51, 24.85, 25.74, 28.96, 29.33, 45.03,
55.62, 57.70, 58.62, 94.31, 104.20, 109.08, 118.95,
119.80, 122.70, 135.20, 135.34, 135.73, 155.97, 165.65,
169.23. EIMS m/e (relative intensity) 381 (M+, 15), 228
(100). Anal. Calcd for (C22H27N3O3Æ3/4H2O) C, H, N.
5.10.2. Tryprostatin B (2). Indole 2 (300 mg) was prepared
in81% yield underconditionsdescribedabovefortheprep-
5.10.6. Diastereomer-1 of tryprostatin B (6). Indole 6
(77 mg) was prepared in 86% yield from 23 as described
above for the preparation of tryprostatin A 1. The start-
26
D
aration of tryprostatin A 1. 2: ½aꢁ ꢀ70.9ꢁ (c 0.80, CHCl3)
27
{lit.7 ½aꢁ ꢀ71.1 (c 0.63, CHCl3)}; IR mmax (NaCl) 3303,
D
ing material used here was D-tryptophan derivative 23
1
2971, 1678, 1661 cmꢀ1; H NMR (300 MHz, CDCl3) d
1.76 (s, 3H), 1.79 (s, 3H), 1.75–2.03 (m, 3H), 2.32 (m,
1H), 2.96 (dd, 1H, J = 11.4 and 15.9 Hz), 3.46–3.49 (m,
2H), 3.59–3.72 (m, 3H), 4.06 (dd, 1H, J = 7.5, 8.0 Hz),
4.36 (dd, 1H, J = 3.5, 11.0 Hz), 5.31 (dd, 1H, J = 6.5,
7.0 Hz), 5.62 (br s, 1H), 7.09–7.18 (m, 2H), 7.31 (d, 1H,
J = 7.7 Hz), 7.48 (d, 1H, J = 7.7 Hz), 8.00 (br s, 1H), 13C
NMR (62.90 MHz, CDCl3) d 18.37, 23.03, 25.50, 25.98,
26.13, 28.74, 45.80, 54.95, 59.66, 105.03, 111.17, 118.13,
120.07, 120.30, 122.26, 128.37, 135.82, 135.91, 136.80,
166.183, 169.74. CIMS m/e (relative intensity) 352
(M++1, 100), 198 (28). Anal. Calcd for (C21H25N3O2Æ1/
4H2O) C, H, N. The spectral data for 2 were identical to
that reported by Osada et al.7 in the literature.
26
D
and Fmoc-L-Pro-Cl 24. 6: ½aꢁ ꢀ41.9ꢁ (c 0.45, CHCl3).
Anal. Calcd for (C21H25N3O2Æ1/5H2O) C, H, N. All
spectroscopic data were identical to that reported for 8
except the optical rotation was opposite in sign.
5.10.7. Diastereomer-2 of tryprostatin A (7). Indole 7
(48 mg) was prepared in 84% yield from 20 as described
above for the preparation of tryprostatin A 1. The start-
ing material used here was L-tryptophan derivative 20
27
D
and Fmoc-D-Pro-Cl 25. 7: ½aꢁ 21.0ꢁ (c 0.32, CHCl3).
Anal. Calcd for (C22H27N3O3Æ3/8H2O) C, H, N. All
spectroscopic data were identical to that reported for 5
in a previous experiment except the optical rotation
was opposite in sign.
5.10.3. Enantiomer of tryprostatin A (3). Enantiomer 3
(75 mg) was prepared in 78% yield from 22 as described
above for the preparation of tryprostatin A 1. The start-
5.10.8. Diastereomer-2 of tryprostatin B (8). Diastereo-
mer 8 (104 mg) was prepared in 79% yield from 21 as de-
scribed above for the preparation of tryprostatin A 1.
ing material used here was D-tryptophan derivative 22
The starting material used here was L-tryptophan deriv-
28
D
26
D
and Fmoc-D-Pro-Cl 25. 3: ½aꢁ 70.3ꢁ (c 1.0, CHCl3).
ative 21 and Fmoc-D-Pro-Cl 25. 8: ½aꢁ 42.8ꢁ (c 0.65,
CHCl3). IR mmax (NaCl) 3266, 2977, 1666 cmꢀ1
.
1H
Anal. Calcd for (C22H27N3O3Æ3/5H2O) C, H, N. All
spectroscopic data were identical to that reported for 1
(the enantiomer of 3) in a previous experiment except
the optical rotation was opposite in sign.
NMR (300 MHz, CDCl3) d 1.30–1.44 (m, 1H), 1.58–
1.71 (m, 2H), 1.75 (s, 3H), 1.79 (s, 3H), 2.00–2.09 (m,
1H), 2.69 (dd, 1H, J = 6.3, 16.8 Hz), 3.08–3.19 (m,
2H), 3.37–3.43 (m, 1H), 3.44 (d, 2H, J = 6.3 Hz), 3.48–

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4641
1
3.58 (m, 1H), 4.24 (dd, 1H, J = 4.4, 8.6 Hz), 5.30 (tt, 1H,
J = 1.4, 7.3 Hz), 6.09 (d, 1H, J = 3.4 Hz), 7.07–7.13 (m,
2H), 7.07–7.13 (m, 2H), 7.23–7.28 (m, 1H), 7.51 (dd, 1H,
J = 2.1, 6.8 Hz), 8.00 (br s, 1H). 13C NMR (62.90 MHz,
CDCl3) d 17.94, 21.53, 24.91, 25.76, 28.99, 29.27, 45.08,
57.74, 58.66, 104.46, 110.37, 118.27, 119.52, 119.67,
121.56, 128.33, 134.97, 135.55, 136.66, 165.59, 169.09.
EIMS m/e (relative intensity) 351 (M+, 13), 198 (100).
Anal. Calcd for (C21H25N3O2Æ1/8H2O) C, H, N.
2945, 1688, 1613 cmꢀ1; H NMR (300 MHz, CDCl3) d
0.63 (d, 3H, J = 6.8 Hz), 0.95 (m, 15H), 0.98 (d, 3H,
J = 6.9 Hz), 1.14 (t, 3H, J = 7.1 Hz), 1.23 (t, 3H,
J = 7.1 Hz), 2.23 (m, 1H), 2.80 (dd, 1H, J = 14.04 Hz
and 4.53 Hz), 3.45 (dd, 1H, J = 14.02 and 3.51 Hz),
3.73 (s, 3H), 3.84 (s, 3H), 3.85 (m, 1H), 3.90–4.15 (m,
5H), 6.65 (m, 2H), 7.50 (d, 1H, J = 9.2 Hz); 13C NMR
(75.5 MHz, CDCl3) d 4.8, 7.6, 14.3, 14.4, 16.7, 19.1,
31.6, 31.9, 33.1, 55.7, 59.2, 60.4, 60.5, 60.7, 91.9, 108.2,
121.2, 124.2, 124.6, 132.3, 140.6, 156.7, 162.7, 163.9.
MS (CI, CH4) m/e (relative intensity) 500 (M++1,
100), 470 (16), 386 (14), 288(21). Anal. Calcd for
(C28H45N3O3Si) C, H, N. This material was used di-
rectly in a later step.
5.11. (5R,2S)-3,6-Diethoxy-5-[6-methoxy-2-(triethyl-silyl)-
3-indolyl]methyl-2,5-dihydropyrazine (28)
To a three-neck flask (3 L) equipped with an overhead
stir were added iodoaniline derivative 26 (150 g),
Scho¨llkopf derivative 27 (265 g), LiCl (2.55 g), Na2CO3
(159 g), palladium (II) acetate (1.75 g) and anhydrous
DMF (2 L). The mixture was then degassed with a vac-
uum pump three times at rt with Ar. The suspension
which resulted was heated for 36 h at 100 ꢁC under an
atmosphere of Ar. At this point TLC (silica gel) indi-
cated 26 had been consumed and the reaction mixture
was cooled to rt and the DMF was removed under vac-
uum (aspirator). Methylene chloride (2 L) was added to
the residue and the suspension which resulted was fil-
tered to remove unwanted salts. After removal of the
CH2Cl2, the crude product was purified by flash chro-
matography (silica gel, 2% EtOAc in hexane) to give
77% of the desired 6-methoxy substituted indole 28.
5.13. Na-Isoprenyl-(2S,3R)-3,6-diethoxy-5-[6-methoxy-3-
indolyl]methyl-2,5-dihydropyrazine (30)
Indole 30 (2.1 g) was prepared in 75% yield from 28
and isoprenyl bromide under conditions described
above for the preparation of 29. 30: 1H NMR
(300 MHz, CDCl3) d 0.64 (d, 3H, J = 6.78 Hz), 0.92
(d, 3H, J = 6.87 Hz), 1.32 (m, 7H), 1.81 (d, 6H,
J = 12.21 Hz), 2.06 (s, 2H), 3.24 (dd, 3H, J = 3.24 Hz
and 2,34 Hz), 3.87 (s, 3H), 4.14 (m, 3H), 4.30 (s,
1H), 4.55 (d, 2H, J = 6.93 Hz), 5.34 (s, 1H), 6.74 (m,
2H), 7.50 (d, 1H, J = 2.64 Hz). This material was used
directly in a later step.
IR mmax (NaCl) 3388, 2944, 1683 cmꢀ1
;
1H NMR
5.14. Na-Benzyl-(2S,5R)-3,6-diethoxy-5-[6-methoxy-2-
(triethyl-silyl)-3-indolyl]methyl-2,5-dihydropyrazine (31)
(300 MHz, CDCl3) d 0.67 (d, 3H, J = 6.8 Hz), 0.85–
1.05 (m, 18H), 1.20 (t, 3H, J = 7.1 Hz), 1.30 (t, 3H,
J = 7.1 Hz), 2.25 (m, 1H), 2.80 (dd, 1H, J = 13.5 Hz
and J = 10.6 Hz), 3.46 (dd, 1H, J = 14.1 Hz and
J = 3.1 Hz), 3.84 (s, 3H), 3.88 (t, 1H, J = 3.9), 4.01–
4.21 (m, 5H), 6.70 (dd, 1H, J = 8.7 Hz and
J = 2.2 Hz), 6.82 (d, 1H, J = 2.1 Hz), 7.60 (d, 1H,
J = 8.7 Hz), 7.77 (s, br, 1H); 13C NMR (75.5 MHz,
CDCl3) d 4.1, 7.9, 14.7, 14.8, 17.1, 19.5, 32.1, 32.5,
56.0, 59.3, 60.9, 61.0, 61.1, 93.9, 109.3, 121.8, 124.4,
124.7, 130.5, 139.5, 157.0, 163.1, 164.2. MS (CI, CH4)
m/e (relative intensity) 486 (M++1, 100), 456 (13), 372
(51), 274 (27). HRMS Calcd for C27H43N3O3Sim/
z = 485.3074, found m/z = 485.3055. This material was
used directly in a later step.
Indole 31 (1.65 g) was prepared from 28 and benzyl bro-
mide in 72% yield under conditions described above for
1
the preparation of 29. 31: H NMR (300 MHz, CDCl3)
d 0.88–0.96 (m, 13H), 1.09 (d, 3H, J = 6.87 Hz), 1.21–
1.36 (m, 9H), 2.09 (s, 1H), 2.30–2.40 (m, 1H), 3.02
(dd, 1H, J = 14.22 Hz and 9.21 Hz), 3.59 (dd, 1H,
J = 16.61 Hz and 5.46 Hz), 3.88 (s, 3H), 3.93 (t, 1H,
J = 3.33 Hz), 4.05–4.34 (m, 5H), 5.48 (br, 2H), 6.51 (d,
1H, J = 2.1 Hz), 6.74 (dd, 1H, J = 8.67 Hz and
2.19 Hz), 6.95 (d, 2H, J = 6.96 Hz), 7.20–7.30 (m, 2H),
7.63 (d, 1H, J = 8.67 Hz); 13C NMR (75.5 MHz, CDCl3)
d 4.57, 7.52, 14.17, 14.30, 16.62, 19.13, 20.94, 31.34,
31.40, 31.53, 34.62, 49.57, 55.46, 58.71, 60.38, 60.67,
92.98, 108.38, 121.01, 124.67, 125.01, 125.66, 126.80,
127.55, 127.70, 128.40, 128.94, 132.48, 138.49, 140.41,
156.67, 162.76, 163.95. This material was used directly
in a later step.
5.12. Na-Methyl-(2S,5R)-3,6-diethoxy-5-[6-methoxy-2-
(triethyl-silyl)-3-indolyl]methyl-2,5-dihydropyrazine (29)
Sodium hydride (60% in mineral oil, 0.2 g) in several
portions was added to a mixture of 28 (1.5 g,
3.08 mmol), CH3I (0.65 g, 4.55 mmol) and anhydrous
DMF (20 mL) at 0 ꢁC. After this mixture was stirred
for 2 h, analysis by TLC (silica gel) indicated the absence
of starting material. The reaction solution was quenched
with water (1 mL) and then was neutralized with an
aqueous solution of NH4Cl after which it was extracted
with EtOAc (3 · 20 mL). The combined organic layers
were washed with brine (2 · 30 mL) and dried
(K2CO3). The solvent was removed under reduced pres-
sure and the residue was subjected to a short wash col-
umn (silica gel, EtOAc/hexane, 1:4) to provide the
pyrazine 29 (1.6 g, 99%). Mp 91–92 ꢁC; IR mmax (NaCl):
5.15. Na-Allyl-(2S,5R)-3,6-diethoxy-5-[6-methoxy-3-
indolyl]methyl-2,5-dihydropyrazine (32)
Indole 32 (1.9 g) was prepared from 28 and allyl bro-
mide in 80% yield under conditions described above
for the preparation of 29. 32: 1H NMR (300 MHz,
CDCl3) d 0.64 (d, 4H, J = 6.78 Hz), 0.92 (d, 3H,
J = 6.87 Hz), 1.33 (m, 6H), 2.16 (t, 1H, J = 3.60 Hz),
3.32 (m, 3H), 3.86 (s, 3H), 4.16 (m, 3H), 4.60 (m, 2H),
5.02 (s, 1H), 5.18 (d, 1H, J = 1.35), 5.31 (s, 1H), 5.96
(m, 1H), 6.72 (m, 3H), 7.50 (d, 1H, J = 8.58). EIMS
m/e (relative intensity) 411(M+, 46). This material was
used directly in a later step.

4642
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
5.16. Na-Methyl-6-methoxy-L-tryptophan ethyl ester (33)
5.20. 3-(6-Methoxy-1-methyl-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (37)
Ester 33 (700 mg) was prepared in 84% yield from 29
(1.5 g, 3 mmol) as described above for the preparation
of 20. 33: IR mmax (NaCl) 3374, 3311, 2980, 1736,
Indole 37 (280 mg) was prepared as described above for
the preparation of 1 in 82% yield. The starting material
used here was L-tryptophan derivative 33 (360 mg,
1.3 mmol) and Fmoc-L-pro-Cl 24 (690 mg, 2.05 mmol).
1
1623 cmꢀ1; H NMR (300 MHz, CDCl3) d 1.30 (t, 3H,
J = 7.1 Hz), 1.62 (s, br, 2H), 2.99 (dd, 1H, J = 14.4 Hz
and 7.7 Hz), 3.24 (dd, 1H, J = 14.3 Hz and 4.7 Hz),
3.65 (s, 3H), 3.79 (dt, 1H, J = 12.6 Hz and 7.4 Hz),
3.89 (s, 3H), 4.18 (q, 2H, J = 7.1 Hz), 6.75–6.83 (m,
3H), 7.48 (d, 1H, J = 8.6 Hz); 13C NMR (75.5 MHz,
CDCl3) d 14.1, 30.6, 32.5, 55.2, 55.8, 60.6, 93.1, 108.9,
109.8, 119.6, 122.6, 126.5, 137.8, 156.6, 175.0. EIMS
m/e (relative intensity) 276 (M+, 4), 174 (100), 159
(11). Anal. Calcd for (C15H20N2O3) C, H, N. This mate-
rial was used directly in a later step.
37: IR mmax (NaCl) 3430, 1610, 1550, 1390 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) d 2.02–2.05 (m, 4H), 2.89
(dd, 1H, J = 15.03 Hz and 10.92 Hz), 3.59–3.74 (m,
5H), 3.88 (s, 3H), 4.08 (dd, 1H, J = 14.8 Hz and
7.14 Hz), 4.32 (d, 2H, J = 10.92 Hz), 5.78 (br, 1H),
6.77–6.91 (m, 3H), 7.48 (d, 1H, J = 14.0 Hz); 13C
NMR (75.5 MHz, CDCl3) d 22.54, 26.66, 28.20, 32.66,
45.29, 54.44, 55.65, 59.13, 93.01, 108.25, 109.36,
119.20, 121.37, 126.71, 138.18, 156.77, 165.49, 169.21.
EIMS m/e (relative intensity) 327 (M+, 14), 174 (100).
Anal. Calcd for (C18H21N3O3) C, H, N.
5.17. Na-Isoprenyl-6-methoxytryptophan ethyl ester (34)
5.21. 3-(6-Methoxy-1-(3-methyl-but-2-enyl)-1H-indol-3-
ylmethyl)-hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione (38)
Ester 34 (850 mg) was prepared from 30 in 85% yield un-
der conditions described above for the preparation of
20. 34: H NMR (300 MHz, CDCl3) d 1.24–1.30 (m,
4H), 1.83 (t, 3H, J = 18.21 Hz), 2.06 (s, 3H), 3.04 (dd,
1H, J = 14.37 Hz and 7.68 Hz), 3.22 (d, 1H,
J = 4.8 Hz), 3.87 (s, 3H), 4.13–4.21 (m, 4H), 5.78 (br,
1H), 4.59 (d, 2H, J = 6.54 Hz), 5.37 (s, 1H), 6.82 (m,
3H), 7.48 (d, 1H, J = 8.04 Hz). This material was used
directly in a later step.
1
Indole 38 (190 mg) was prepared from 34 in 80% yield
under conditions described above for the preparation
1
of 1. 38: H NMR (300 MHz, CDCl3) d 1.76–1.85 (m,
3H), 2.00–2.03 (s, 3H), 2.05–2.14 (s, 3H), 2.32 (d, 1H,
J = 6.84 Hz), 2.87 (dd, 1H, J = 15.09 Hz and
11.04 Hz), 3.58–3.70 (m, 3H), 3.88 (s, 3H), 4.08 (dd,
1H, J = 14.80 Hz and 7.14 Hz), 4.32 (d, 2H,
J = 10.92 Hz), 4.61 (d, 1H, J = 6.87 Hz), 5.38 (t, 1H,
J = 1.38 Hz), 5.78 (br, 1H), 6.80–6.82 (m, 2H), 6.90 (s,
1H), 7.45 (d, 1H, J = 6.33 Hz); 13C NMR (75.5 MHz,
CDCl3) d 17.99, 22.53, 25.59, 26.82, 28.22, 44.00,
45.29, 53.34, 54.52, 55.63, 59.13, 93.47, 109.22, 119.20,
119.46, 121.66, 125.27, 136.50, 137.47, 156.55, 165.50,
169.19. EIMS m/e (relative intensity) 381 (M+, 16), 160
(100), 228 55. Anal. Calcd for (C22H27N3O3) C, H, N.
5.18. Na-Benzyl-6-methoxytryptophan ethyl ester (35)
Ester 35 (1.0 g) was prepared from 31 in 85% yield un-
der conditions described above for the preparation of
1
20. 35: H NMR (300 MHz, CDCl3) d 1.16–1.26 (m,
3H), 1.87 (s, 1H), 2.01 (s, 1H), 3.02 (dd, 1H,
J = 14.28 Hz and 7.32 Hz), 3.18–3.25 (m, 1H), 3.87 (s,
3H), 4.06–4.18 (m, 3H), 5.17 (s, 1H), 6.71 (s, 1H),
6.77–6.80 (m, 1H), 6.87 (s, 1H), 7.08 (d, 1H,
J = 7.38 Hz), 7.22–7.28 (m, 2H), 7.50 (d, 1H,
J = 8.61 Hz); 13C NMR (75.5 MHz, CDCl3) d 14.1,
30.6, 32.5, 55.2, 55.8, 60.6, 93.1, 108.9, 109.8, 119.6,
122.6, 126.5, 137.8, 156.6; 13C NMR (75.5 MHz,
CDCl3) d 13.27, 20.06, 29.87, 48.89, 54.26, 54.66,
59.41, 59.92, 92.54, 108.13, 109.53, 118.81, 121.82,
124.99, 125.88, 126.64, 127.82, 136.55, 136.63, 155.62,
174.25. Anal. Calcd for (C21H24N2O3H2O) C, H, N.
This material was used directly in a later step.
5.22. Na-Benzyl-3-(6-methoxy-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (39)
Indole 39 (130 mg) was prepared from 35 in 74% yield
under conditions described above for the preparation of
1
1. 39: mp 124–126 ꢁC; H NMR (300 MHz, CDCl3) d
1.70–1.81 (m, 4H), 2.03–2.06 (m, 2H), 2.82 (dd, 1H,
J = 10.74 Hz and 6.69 Hz), 3.11–3.18 (m, 2H), 3.40 (dd,
1H, J = 14.55 Hz and 5.67 Hz), 3.51–3.61 (m, 1H), 3.82
(s, 3H), 4.22–4.27 (m, 1H), 5.21 (s, 1H), 6.01 (d, 1H,
J = 3.09 Hz), 6.72 (s, 1H), 6.80 (d, 1H, J = 8.70 Hz),
6.91 (s, 1H), 7.13–7.16 (m, 2H), 7.28–7.33 (m, 2H), 7.49
(d, 1H, J = 8.70Hz); 13C NMR (75.5 MHz, CDCl3) d
12.41, 28.74, 30.68, 44.97, 49.98, 55.54, 57.77, 58.44,
93.29, 108.48, 109.34, 119.82, 122.03, 126.77, 126.89,
127.69, 128.77, 137.24, 156.53, 165.31, 169.14. Anal.
Calcd for (C24H25N3O30.5H2O) C, H, N.
5.19. Na-Allyl-6-methoxytryptophan ethyl ester (36)
Ester 36 (640 mg) was prepared from 32 in 85% yield
under conditions described above for the preparation
of 20. 36: 1H NMR (300 MHz, CDCl3) d 1.23–1.29
(m, 4H), 1.72 (s, 2H), 2.06 (s, 1H), 3.04 (d, 1H,
J = 7.56 Hz), 3.87 (s, 3H), 4.12–4.18 (m, 2H), 4.63 (d,
1H, J = 1.5 Hz), 5.18 (dd, 2H, J = 17.2 Hz and
8.7 Hz), 5.98–6.00 (m, 1H), 6.75–6.87 (m, 3H), 7.50
(d, 1H, J = 8.43 Hz). 13C NMR (75.5 MHz, CDCl3) d
14.09, 30.80, 48.55, 55.02, 55.60, 60.45, 93.24, 108.85,
110.06, 117.09, 119.56, 122.53, 125.39, 133.27, 137.10,
156.32, 175.19. This material was used directly in a la-
ter step.
5.23. Na-Allyl-3-(6-methoxy-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (40)
Indole 40 (220 mg) was prepared from 36 in 80% yield
under conditions described above for the preparation
1
of 1. 40: mp 84–86 ꢁC; H NMR (300 MHz, CDCl3) d
1.20–1.25 (m, 3H), 2.07 (d, 1H, J = 7.56 Hz), 2.25 (d,

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4643
1H, J = 22 Hz), 3.24 (d, 2H, J = 5.70 Hz), 3.70 (dd, 1H,
J = 13.92 Hz and 7.01 Hz), 3.83 (s, 3H), 4.06–4.13 (m,
3H), 4.60 (m, 3H), 5.18 (d, 1H, J = 3.10 Hz), 5.89–5.94
(m, 1H), 6.70–6.83 (m, 3H), 7.42 (d, 1H, J = 5.61 Hz).
Anal. Calcd for (C20H23N3O3) C, H, N.
107.16, 107.67, 115.57, 119.70, 122.45, 123.18, 131.67,
133.62, 136.12, 136.71, 155.46, 162.78, 163.26. EIMS
m/e (relative intensity) 479 (M+, 13), 268 (100). This
material was used directly in a later step.
5.27. Na-Benzyl-2-isoprenyl-6-methoxytryptophan ethyl
ester (44)
5.24. (2S,5R)-3,6-Diethoxy-2-isopropyl-5-[6-methoxy-3-
indolyl]methyl-2,5-dihydropyrazine (41)
Ester 44(330 mg) was preparedin 87% yield from 42, asde-
scribed above for the preparation of 20. 44: IR mmax (KBr)
3054, 2305, 1733, 1422, 1265 cmꢀ1; 1H NMR (300 MHz,
CDCl3) d 1.18–1.32 (m, 3H), 1.60 (d, 8H, J =15.30 Hz),
3.01 (t, 1H, J = 9.30), 3.40 (d, 2H, J = 6.30 Hz), 3.80 (s,
3H), 4.17 (m, 2H), 5.04 (s, 1H), 5.26 (s, 1H), 6.66 (s, 1H),
6.80 (d, 1H, J = 3.96 Hz), 6.92 (d, 2H, J = 7.17 Hz), 7.25
(d, 3H, J = 7.50 Hz), 7.48 (d, 1H, J = 8.61 Hz); 13C
NMR (75.5 MHz, CDCl3) d 14.06, 17.86, 24.01, 25.40,
30.64, 46.54, 55.52, 55.67, 60.78, 93.51, 106.85, 108.49,
118.89, 121.57, 122.43, 125.72, 126.98, 128.53, 132.68,
136.34, 137.40, 137.92, 155.99, 175.33. EIMS m/e (relative
intensity) 420 (M+, 13), 318 (100). This material was used
directly in a later step.
A solution of 16 (500 mg, 0.93 mmol) in xylene was stir-
red at reflux for 3 d at which time examination by TLC
(silica gel) indicated the disappearance of starting mate-
rial. After removal of xylenes under reduced pressure,
the residue was subjected to flash chromatography (sil-
ica gel, hexane/EtOAc, 10:1) to provide 41 (330 mg,
80%): 1H NMR (300 MHz, CDCl3) d 0.61 (d, 3H,
J = 6.75 Hz), 0.91 (d, 3H, J = 6.87 Hz), 1.22 (t, 3H,
J = 7.08 Hz), 1.35 (t, 4H, J = 7.14 Hz), 1.77 (d, 5H,
J = 7.92), 2.15 (s, 1H), 3.24 (m, 3H), 3.42 (d, 2H,
J = 7.20 Hz), 3.83 (s, 3H), 3.98–4.30 (m, 4H), 5.29 (s,
1H), 6.73 (m, 2H), 7.42 (t, 1H, J = 8.61 Hz), 7.65 (s,
1H); 13C NMR (75.5 MHz, CDCl3) d 14.29, 16.32,
17.71, 18.99, 25.00, 25.67, 28.88, 30.82, 55.58, 57.23,
59.92, 60.24, 60.45, 93.97, 106.77, 108.10, 119.53,
120.94, 123.99, 125.28, 127.67, 134.16, 135.57, 155.45,
162.74, 163.41. EIMS m/e (relative intensity) 439 (M+,
13), 212 (54), 169 (100). Anal. Calcd for (C26H37N3O3)
C, H,N. This material was used directly in a later step.
5.28. Na-Allyl-2-isoprenyl-6-methoxytryptophan ethyl
ester (45)
Ester 45 (88 mg) was prepared in 86% yield from 43
(130 mg, 0.27 mmol) as described above for the prepa-
ration of 20. 45: IR mmax (NaCl) 3365, 1730,
5.25. Na-Benzyl-(2S,5R)-3,6-diethoxy-2-isopropyl-5-[6-
methoxy-3-indolyl]methyl-2,5-dihydropyrazine (42)
1625 cmꢀ1 1H NMR (300 MHz, CDCl3) d 1.26 (m,
;
3H), 1.78 (t, 10H, J = 10.44), 2.94 (dd, 1H, J = 8.46
and J = 5.82), 3.20 (d, 1H, J = 5.04), 3.44 (d, 1H,
J = 6.03), 3.86 (s, 3H), 4.16 (m, 2H), 4.61 (t, 2H,
J = 2.43), 4.84 (d, 1H, J = 15.93), 5.12 (m, 2H), 5.90
(s, 1H), 6.75 (t, 2H, J = 6.33), 7.44 (d, 1H, J = 8.55);
13C NMR (75.5 MHz, CDCl3) d 14.05, 17.91, 23.86,
25.49, 30.54, 45.44, 55.47, 55.71, 60.78, 93.50, 106.40,
108.28, 115.91, 118.81, 121.72, 122.40, 132.54, 133.40,
136.18, 136.95, 155.84, 175.30. EIMS m/e (relative
intensity) 370 (M++1, 12), 268 (100). Anal. Calcd for
(C22H30N2O3) C, H, N. This material was used directly
in a later step.
Indole 42 (550 mg) was prepared in 91% yield from 41
and benzyl bromide as described above for the prepa-
1
ration of 29. 42: H NMR (300 MHz, CDCl3) d 0.62
(d, 3H, J = 6.69 Hz), 0.93 (d, 3H, J = 6.84 Hz), 1.17–
1.22 (m, 7H), 1.62 (t, 7H, J = 9.4 Hz), 2.10 (m, 1H),
3.21–3.39 (m, 4H), 3.77 (s, 3H), 4.10 (m, 4H), 5.04
(s, 1H), 5.22 (s, 1H), 6.60 (s, 1H), 6.70 (d, 1H,
J = 8.55 Hz), 6.88 (d, 2H, J = 6.87 Hz), 7.27 (m, 3H),
7.48 (d, 1H, J = 8.55 Hz); 13C NMR (75.5 MHz,
CDCl3) d 14.28, 16.33, 17.80, 19.08, 24.02, 25.57,
28.07, 29.14, 30.77, 46.43, 55.59, 57.35, 59.96, 60.17,
60.44, 92.95, 107.46, 107.91, 119.77, 122.23, 123.16,
125.67, 126.82, 128.47, 131.87, 136.55, 137.08,
138.30, 155.61, 162.67, 163.3. This material was used
directly in a later step.
5.29. Na-Benzyl-2-isoprenyl-3-(6-methoxy-1H-indol-3-
ylmethyl)-hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione (46)
Indole 46 (125 mg) was prepared in 87% yield from 44
as described above for the preparation of 1. 46: IR mmax
5.26. Na-Allyl-(2S,5R)-3,6-diethoxy-2-isopropyl-5-[6-
methoxy-3-indolyl]methyl-2,5-dihydropyrazine (43)
(KBr) 3010, 2408, 1735 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) d 1.75 (dd, 3H, J = 7.14 Hz and J = 7.41 Hz),
1.55 (s, 2H), 1.64 (d, 4H, J = 11.82 Hz), 2.06 (d, 2H,
J = 2.52), 2.28 (d, 2H, J = 2.70 Hz), 2.75 (m, 1H),
3.00 (m, 1H), 3.38 (m, 2H), 3.82 (s, 3H), 4.14 (m,
3H), 5.15 (d, 1H, J = 7.47 Hz), 5.27 (t, 1H,
J = 8.88 Hz), 6.60 (s, 1H), 6.86 (m, 2H), 6.91 (d, 2H,
J = 6.18 Hz), 7.05 (d, 2H, J = 2.70 Hz), 7.30 (s, 1H);
13C NMR (75.5 MHz, CDCl3) d 13.93, 17.83, 21.31,
23.87, 24.32, 25.38, 28.28, 46.44, 55.69, 59.19, 61.22,
64.48, 84.64, 93.94, 108.52, 118.68, 121.60, 126.09,
126.93, 127.21, 127.88, 128.46, 128.68, 129.40, 137.99,
155.72, 169.02. EIMS m/e (relative intensity) 472
(M+, 5), 318 (48) 91 (100). Anal. Calcd for
(C29H33N3O3H2O) C, H, N.
Indole 43 (135 mg) was prepared in 82% yield from 41
(150 mg, 0.34 mmol) and allyl bromide (50 mg,
0.40 mmol) as described above for the preparation of
29. 43: 1H NMR (300 MHz, CDCl3) d 0.60 (t, 3H,
J = 6.75 Hz), 0.91 (t, 3H, J = 6.84 Hz), 1.31 (m, 7H),
1.69 (t, 7H, J = 19.7 Hz), 2.10 (m, 1H), 3.14–3.27 (m,
3H), 3.42 (d, 1H, J = 6.45 Hz), 3.85 (s, 3H), 4.12 (m,
4H), 4.58 (s, 2H), 4.78 (d, 1H, J = 8.67 Hz), 5.09 (d,
2H, J = 9.12 Hz), 5.85 (m, 1H), 6.70 (m, 2H), 7.45 (d,
1H, J = 8.58 Hz); 13C NMR (75.5 MHz, CDCl3) d
14.29, 16.32, 17.83, 18.98, 19.56, 23.87, 25.45, 29.17,
30.79, 45.31, 55.65, 57.41, 59.94, 60.18, 60.43, 92.96,

4644
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
5.30. Na-Allyl-2-isoprenyl-3-(6-methoxy-1H-indol-3-ylme-
thyl)-hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione (47)
3H, J = 3.75 Hz), 0.98 (d, 3H, J = 6.87 Hz), 1.20 (t,
3H, J = 7.11 Hz), 1.29 (m, 6H), 1.41 (s, 10H), 2.87 (s,
1H), 3.64 (d, 1H, J = 3.36), 3.88 (s, 3H), 4.00 (m, 1H),
4.56 (s, 2H), 6.89 (d, 1H, J = 8.58 Hz), 7.01 (d, 2H,
J = 7.08 Hz), 7.20 (m, 3H), 7.52 (d, 1H, J = 8.58 Hz),
7.75 (s, 1H). EIMS m/e (relative intensity) 561 (M+,
14), 461 (11), 212 (100). Anal. Calcd for (C33H43N3O5)
C, H, N. This material was used directly in a later step.
Indole 47 (80 mg) was prepared as described above for the
preparation of 1. The starting material used here was L-
tryptophan derivative 45 (85 mg, 0.23 mmol) and Fmoc-
1
L-pro-Cl 24 (126 mg, 0.37 mmol) in 82% yield. 47: H
NMR (300 MHz, CDCl3) d 1.27 (s, 1H), 1.51–1.76 (m,
9H), 2.37 (t, 1H, J = 3.60 Hz), 3.00 (s, 1H), 3.29 (t, 1H,
J = 4.20 Hz), 3.40 (m, 1H), 3.60 (d, 1H, J = 5.70 Hz),
3.92(s, 3H), 4.23 (d, 1H, J = 9.60 Hz), 4.70 (d, 1H,
J = 10.2 Hz), 5.14 (s, 1H), 5.54 (s, 1H), 6.95 (d, 1H,
J = 7.71 Hz), 7.18 (s, 1H), 7.24 (d, 1H, J = 5.70 Hz).
EIMS m/e (relative intensity) 421 (M+, 14), 268 (100).
+TOF MS HRMS Calcd for (C25H31N3O3 + Li)+m/z
= 428.2525, found m/z = 428.2519.
5.33. (2S,5R)-3,6-Diethoxy-2-allyl-5-[1-tert-butyloxycar-
bonyl-6-methoxy-3-indolyl]methyl-2,5-dihydropyrazine (50)
A solution of n-BuLi (2.5 M in hexane, 0.22 mL,
0.54 mmol) was added dropwise to a solution of 2-bromo-
pyrazine 29 (250 mg, 0.46 mmol) in dry THF (8 mL) at
ꢀ78 ꢁC under nitrogen. The mixture which resulted was
stirred at ꢀ78 ꢁC for 30 min and then warmed to 0 ꢁC for
10 min. Then allyl bromide (82.8 mg, 0.69 mmol) was
added quickly at 0 ꢁC. The mixture was stirred at 0 ꢁC
for 1 h and allowed to warm to rt overnight. The solvent
was removed under reduced pressure. The residue was ta-
ken up in CH2Cl2 and washed with a 5% aqueous solution
ofNaHCO3. Theaqueous layer wasextractedwithCH2Cl2
(3 · 20 mL). The combined organic layers were dried
(Na2SO4).Afterremovalofthesolventunderreducedpres-
sure, the residue was separated by flash chromatography
(silica gel, hexane/EtOAc, 15/1) to provide 50 (198 mg,
85%) as an oil. 50: IR mmax (KBr) 3054, 2306, 1733,
5.31. (5S,2R)-3,6-Diethoxy-2-bromo-5-[1-tert-butyloxy-
carbonyl-6-methoxy-3-indolyl]methyl-2,5-dihydropyrazine
(48)
A solution of NBS (183 mg, 1.03 mmol) which had been
dissolved in acetonitrile (10 mL) was syringed into a solu-
tion of 28 (500 mg, 1.03 mmol) in acetonitrile (40 mL) at
0 ꢁC. The reaction mixture was allowed to stir at 0 ꢁC for
30 min at which time analysis by TLC (silica gel) indicated
the absence of starting material. To this solution was then
added
4-dimethylaminopyridine
(DMAP,
7 mg,
0.057 mmol) and di-tert-butyl-dicarbonate (450 mg,
2.06 mmol) at rt. After the reaction solution was stirred
for another 1 h, analysis by TLC (silica gel) indicated the
disappearance of the intermediate. The solvent was re-
moved under reduced pressure, and the residue was parti-
tioned between CH2Cl2 (100 mL) and H2O (100 mL).
The organic layer was separated, and the aqueous layer
was extracted with CH2Cl2 (2 · 80 mL). The combined or-
ganic layers were dried (Na2SO4) and the solvent was re-
moved under reduced pressure. The residue was purified
by flash chromatography (silica gel, EtOAc/hexanes, 4:
96) to afford 48 (492 mg) as an oil in 87% yield. 48: IR mmax
1
1422 cmꢀ1; H NMR (300 MHz, CDCl3) d 0.65 (d, 3H,
J = 6.78 Hz), 0.97 (d, 3H, J = 6.90 Hz), 1.23 (t, 10H,
J = 6.54 Hz), 1.66 (d, 8H, J = 7.17 Hz), 2.20 (m, 1H),
3.31 (d, 1H, J = 3.30 Hz), 3.64 (s, 1H), 3.88 (s, 3H), 4.07
(m, 3H), 4.21 (m, 2H), 4.94 (d, 2H, J = 6.36 Hz), 5.98 (m,
1H), 6.84 (d, 1H, J = 2.34 Hz), 7.70 (d, 1H, J = 2.25 Hz),
7.98 (s, 1H); 13C NMR (75.5 MHz, CDCl3) d 14.23,
16.46, 18.97, 28.08, 29.19, 30.59, 31.33, 55.54, 60.32,
83.25, 98.73, 99.77, 110.94, 114.75, 119.42, 136.23,
136.70, 157.07. EIMS m/e (relative intensity) 511 (M+,
10), 244 (50), 200 (100). +TOF MS HRMS Calcd for
(C29H41N3O5+
= 512.3126. This material was used directly in a later step.
H)+m/z = 512.3124,
found
m/z
(NaCl) 2970, 1735, 1690 cmꢀ1 1H NMR (250 MHz,
;
CDCl3) d 0.62 (d, 3H, J = 6.8 Hz), 0.97 (d, 3H,
J = 6.8 Hz), 1.17 (t, 3H, J = 7.1 Hz), 1.28 (t, 3H,
J = 7.1 Hz), 1.67 (s, 9H), 2.20 (m, 1H), 2.92 (dd, 1H,
J = 8.1 and 13.9 Hz), 3.28 (dd, 1H, J = 4.6 and 13.9 Hz),
3.68 (t, 1H, J = 3.3 Hz), 3.84 (s, 3H), 3.95–4.25 (m, 5H),
6.80 (dd, 1H, J = 2.2 and 8.6 Hz), 7.38 (d, 1H,
J = 8.6 Hz), 7.63 (d, 1H, J = 2.1 Hz); 13C NMR
(62.90 MHz, CDCl3) d 14.31, 14.35, 16.59, 19.09, 28.24,
31.04, 31.46, 55.62, 55.78, 60.49, 60.74, 84.46, 99.59,
107.81,111.61, 119.69, 120.59, 123.66, 137.34, 149.35,
157.65, 162.77, 163.37. EIMS m/e (relative intensity) 551
(M+, 17), 549 (M+, 17), 470 (64), 414 (61), 370 (58), 341
(32), 240 (49), 238 (49), 212 (72), 169 (100), 141 (39). Anal.
Calcd for(C26H36N3O5Br) C, H, N. This material was used
directly in a later step.
5.34. (5R,2S)-3,6-Diethoxy-5-[6-methoxy-2-phenyl-3-
indolyl]methyl-2,5-dihydropyrazine (51)
A solution of n-BuLi (2.5 M in hexane, 0.27 mL,
0.68 mmol) was added dropwise to a solution of 2-bromo-
pyrazine 29 (250 mg, 0.46 mmol) in dry THF (10 mL) at
ꢀ78 ꢁC under nitrogen. The mixture which resulted was
stirred at ꢀ78 ꢁC for 30 min and then warmed to 0 ꢁC for
10 min. Then dry anhydrous pure zinc chloride (0.68 mL,
0.69 mmol) was added quickly at 0 ꢁC. The mixture which
resulted was stirred at 0 ꢁC for 1 h and iodobenzene was
added and this was followed by addition of tri-2-furyl
phosphine (21 mg, 0.1 mmol) and Pd(OAc)2 (12 mg,
0.05 mmol). The mixture which resulted was then stirred
overnight. The solvent was removed under reduced pres-
sure. The residue was taken up in CH2Cl2 and washed with
a 5% aqueous solution ofNaHCO3. The aqueous layer was
extracted with CH2Cl2 (3 · 20 mL). The combined organic
layers were dried (Na2SO4). After removal of the solvent
under reduced pressure, the residue was separated by flash
chromatography (silica gel, hexane/EtOAc, 15/1) to pro-
5.32. (2S,5R)-3,6-Diethoxy-2-5-[1-tert-butyloxycarbonyl-
2-benzyl-6-methoxy-3-indolyl]methyl-2,5-dihydropyrazine
(49)
Indole 49 (800 mg) was prepared in 85% yield from 29
and benzyl bromide, as described below for the prepara-
1
tion of 50. 49: H NMR (300 MHz, CDCl3) d 0.65 (d,

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4645
1
vide 51 (170 mg, 65%) as an oil. H NMR (300 MHz,
CDCl3) d 0.95 (d, 3H, J = 3.75 Hz), 1.22 (m, 3H), 1.33
(m, 6H), 1.41 (s, 10H), 1.65 (m, 3H), 2.81 (s, 1H), 3.34
(m, 3H), 3.90 (s, 3H), 6.88 (m, 2H), 7.40 (m, 3H), 7.56 (d,
2H, J = 8.58 Hz), 7.71 (s, 1H), 7.52 (d, 1H, J = 2.10 Hz),
7.75 (s, 1H). EIMS m/e (relative intensity) 547 (M+, 56),
236 (100). Anal. Calcd for (C32H41N3O5H2O) C, H, N.
This material was used directly in a later step.
(50 mL) and purged with argon. Toluene (10 mL) was
added and this was followed by methyl acrylate
(0.16 mL) and dicyclohexylmethylamine (0.09 mL,
0.43 mmol). The reaction was heated to 95 ꢁC for 48 h,
cooled and then filtered through celite. The solvent
was removed under reduced pressure and the crude
product was purified by flash chromatography to afford
1
58 (190 mg, 94%). 58: H NMR (300 MHz, CDCl3) d
0.77 (d, 3H, J = 6.78 Hz), 0.89 (t, 3H, J = 3.48 Hz),
1.07 (d, 3H, J = 6.87 Hz), 1.25 (m, 6H), 1.67 (s, 9H),
2.18 (m, 1H), 2.89 (t, 1H, J = 9.27 Hz), 3.46 (d, 1H,
J = 10.74 Hz), 3.81 (s, 3H), 3.90 (s, 3H), 4.14 (m, 3H),
6.50 (d, 1H, J = 11.46 Hz), 6.86 (d, 1H, J = 2.64 Hz),
7.54 (d, 1H, J = 8.67 Hz), 7.73 (s, 1H), 8.02 (d, 1H,
J = 13.53 Hz); 13C NMR (75.5 MHz, CDCl3) d 14.00,
14.15, 14.28, 16.58, 18.97, 22.54, 28.06, 30.30, 31.48,
31.67, 51.34, 55.48, 56.57, 60.53, 60.70, 60.85, 84.17,
99.08, 112.27, 118.04, 119.50, 122.68, 124.06, 131.18,
137.94, 150.24, 158.86, 162.31, 163.29, 167.39. EIMS
m/e (relative intensity) 555 (M+, 64), 455 (37), 412
(22), 288 (18), 244 (100), 212 (92). Anal. Calcd for
(C30H41N3O7) C, H, N. This material was used directly
in a later step.
5.35. (S)-1-tert-Butyloxycarbonyl-2-benzyl-6-methoxy-
tryptophan ethyl ester (52)
Ester 52 (185 mg) was prepared in 87% yield from 49 as
described above for the preparation of 20. 52: 1H NMR
(300 MHz, CDCl3) d 1.22 (m, 3H), 1.39 (s, 9H), 1.67 (s,
3H), 2.96 (d, 1H, J = 8.28 Hz), 3.18 (d, 1H, J = 5.46 Hz),
3.89 (s, 3H), 4.10 (m, 2H), 4.49 (s, 2H), 6.90 (d, 1H,
J = 2.34 Hz), 7.02 (d, 2H, J = 6.93 Hz), 7.23 (m, 3H),
7.44 (d, 1H, J = 8.58 Hz), 7.80 (s, 1H); 13C NMR
(75.5 MHz, CDCl3) d 14.10, 27.66, 28.09, 30.13, 32.20,
54.86, 55.58, 64.16, 83.61, 100.01, 111.64, 115.22,
119.38, 122.52, 125.81, 127.44, 128.03, 130.13, 136.79,
139.84, 150.13, 157.60, 174.92. This material was used
directly in a later step.
5.39. 2-Methyl acrylyl-6-methoxytryptophan ethyl ester
(59)
5.36. 2-Benzyl-3-(6-methoxy-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (55)
Indole 59 (180 mg) was prepared in 87% yield from 58 as
described above for the preparation of 20. 59: 1H NMR
(300 MHz, CDCl3) d 1.23 (m, 3H), 1.58 (s, 2H), 1.67 (m,
10H), 3.04 (d, 1H, J = 8.67 Hz), 3.23 (d, 1H,
J = 4.83 Hz), 3.82 (s, 3H), 3.90 (s, 3H), 4.17 (d, 2H),
6.38 (d, 1H, J = 15.0 Hz), 6.91 (d, 1H, J = 2.16 Hz),
7.48 (d, 1H, J = 8.67 Hz), 7.75 (s, 1H), 8.01 (d, 1H,
J = 15.09 Hz); 13C NMR (75.5 MHz, CDCl3) d 13.96,
22.09, 28.06, 30.68, 51.55, 55.04, 55.52, 61,13, 84.54,
99.34, 112.66, 118.63, 120.19, 120.88, 123.40, 131.37,
136.29, 137.77, 150.06, 158.99, 164.46, 167.17, 174.88.
EIMS m/e (relative intensity) 446 (M+, 35), 244 (100).
This material was employed directly in the next step.
Indole 55 (95 mg) was prepared in 81% yield from 52, as
described above for the preparation of 1. 55: H NMR
1
(300 MHz, CDCl3) d 1.26 (t, 2H, J = 5.16 Hz), 1.64 (s,
1H), 2.01 (m, 2H), 2.33 (m, 1H), 3.03 (t, 1H,
J = 11.25 Hz), 3.58–3.73 (m, 2H), 3.83 (s, 3H), 4.09 (m,
2H), 4.32 (d, 1H, J = 10.80 Hz), 5.65 (s, 1H), 6.78 (s,
2H), 7.18 (d, 2H, J = 6.90 Hz), 7.28 (t, 2H, J = 5.70 Hz),
7.40 (d, 2H, J = 9.60 Hz), 7.80 (s, 1H); 13C NMR
(75.7 MHz, CDCl3) d 22.53, 25.65, 28.24, 32.20, 45.32,
54.54, 55.64, 59.15, 94.79, 105.76, 109.56, 118.46,
122.03, 126.48, 128.43, 128.70, 128.89, 136.49, 138.03,
156.53, 165.60, 169.24. EIMS m/e (relative intensity) 403
(M+, 25), 335 (8), 250 (100), 218 (6). Anal. Calcd for
(C24H25N3O3) C, H, N.
5.40. 2-Methyl acrylyl-3-(6-methoxy-1H-indol-3-yl-methyl)-
hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione (60)
5.37. 2-Allyl-3-(6-methoxy-1H-indol-3-ylmethyl)-hexahy-
dro-pyrrolo[1,2-a]pyrazine-1,4-dione (56)
Compound 60 (110 mg) was prepared in 87% yield from
59 as described above for the preparation of 1. 60: mp
135–137 ꢁC; 1H NMR (300 MHz, CDCl3) d 2.24 (m,
4H), 3.54 (m, 2H), 3.80 (s, 3H), 3.91 (s, 3H), 4.14 (m,
2H), 6.02 (s, 1H), 6.70 (d, 1H, J = 2.82 Hz), 7.08 (d,
1H, J = 2.79 Hz), 7.38 (s, 1H), 7.68 (d, 1H,
J = 12.76 Hz), 8.25 (s, 1H); 13C NMR (75.5 MHz,
CDCl3) d 14.12, 21.24, 24.35, 28.36, 44.27, 51.64,
55.36, 61.41, 64.01, 93.64, 102.72, 111.40, 119.39,
121.00, 127.26, 128.53, 138.36, 138.61, 159.00, 167.32,
175.08. EIMS m/e (relative intensity) 397 (M+, 34), 324
(77), 293 (100). HRMS Calcd for C21H23N3O5m/z
= 397.1638, found m/z = 397.1657.
Indole 56 (88 mg) was prepared in 81% yield from 53, as
described above for the preparation of 1. 56: H NMR
1
(300 MHz, CDCl3) d 1.22 (m, 2H), 1.76 (s, 1H), 2.02
(m, 2H), 2.34 (s, 1H), 3.01 (t, 1H, J = 5.73 Hz), 3.48 (t,
1H, J = 6.30 Hz), 3.67 (s, 2H), 4.05–4.11 (m, 3H), 4.32
(d, 1H, J = 9.60 Hz), 5.15 (m, 1H), 5.88 (m, 1H), 6.78
(t, 2H, J = 3.30 Hz), 7.38 (m, 1H), 8.08 (s, 1H). EIMS
m/e (relative intensity) 353 (M+, 8), 200 (100). +TOF
MS HRMS Calcd for (C20H23N3O3 + Li)+m/z =
360.1899, found m/z = 360.1896.
5.38. (2S,5R)-3,6-Diethoxy-2-isopropyl-5-[1-tert-butyl-
oxycarbonyl-2-methyl-acrylate-6-methoxy-3-indolyl]
methyl-2,5-dihydropyrazine (58)
5.41. 6-Methoxy-L-tryptophan ethyl ester (61)
Ester 61 (700 mg) was prepared in 86% yield from 28
(1.5 g, 31 mmol) as described above for the preparation
Indole 29 (200 mg, 0.36 mmol) and Pd(PPh3)4 (21 mg,
0.018 mmol) were placed in a round-bottomed flask
1
of 20. 61: IR mmax (NaCl) 3365, 1730, 1625 cmꢀ1; H

4646
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
NMR (300 MHz, CDCl3) d 1.23 (t, 3H, J = 7.1 Hz), 1.60
(s, br, 2H), 3.0 (dd, 1H, J = 14.3 Hz and 7.7 Hz), 3.22
(dd, 1H, J = 14.4 Hz and 4.8 Hz), 3.78 (m, 1H), 3.81
(s, 3H), 4.15 (q, 2H, J = 7.2 Hz), 6.80 (m, 2H), 6.90 (d,
1H, J = 2.0 Hz), 7.45 (d, 1H, J = 8.5 Hz), 8.15 (s, br,
1H); 13C NMR (75.5 MHz, CDCl3) d 14.0, 30.7, 54.5,
54.9, 60.8, 94.7, 109.2, 111.0, 119.1, 121.8, 121.9,
136.5, 156.1, 175.2. MS (CI) m/e 263 (M++1, 100). Anal.
Calcd for (C14H18N2O3) C, H, N. This material was
used directly in a later step.
3.52–3.60 (m, 1H), 3.82 (s, 3H), 4.26 (t, 1H,
J = 4.2 Hz), 6.43 (br, 1H), 6.74–6.84 (m, 2H), 7.35 (d,
1H, J = 9.4 Hz), 9.08 (s, br, 1H); 13C NMR
(75.5 MHz, CDCl3) d 18.30, 21.49, 28.96, 45.13, 55.54,
58.00, 58.32, 94.44, 105.26, 109.94, 118.96, 121.12,
121.42, 135.13, 156.57, 165.40, 169.11. EIMS m/e (rela-
tive intensity) 347 (M+, 14), 194 (100), 154 (91). +TOF
MS HRMS Calcd for (C17H18N3O3 + Na)+m/z
= 370.0934, found m/z = 370.0935.
5.45. 3-Benzyl-6-(6-methoxy-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (66)
5.42. 3-(6-Methoxy-1H-indol-3-ylmethyl)-hexahydro-
pyrrolo[1,2-a]pyrazine-1,4-dione (62)
Indole 66 (78 mg) was prepared from 61 in 73% yield un-
der conditions described above for the preparation of 1.
66: H NMR (300 MHz, CDCl3) d 1.99–2.05 (m, 2H),
2.60–2.63 (m, 2H), 3.23–3.29 (m, 3H), 3.50 (d, 1H,
J = 6.80 Hz), 3.82 (s, 3H), 4.14–4.16 (m, 2H), 4.92 (s,
1H), 6.74–6.83 (m, 3H), 7.22–7.29 (m, 2H), 7.76 (s,
1H), 8.42 (br, 1H). MS (EI) m/e (relative intensity) 363
(M+, 100), 287(62.5). Anal. Calcd for (C21H21N3O30.5-
H2O) C, H, N.
Indole 62 (280 mg) was prepared as described above for
the preparation of 1 in 82% yield. The starting material
used here was L-tryptophan derivative 61 (360 mg,
1.37 mmol) and Fmoc-L-pro-Cl 24 (690 mg, 2.05 mmol).
mp 133–135 ꢁC; IR mmax (KBr) 3100, 1700, 1400,
1
1050 cmꢀ1 1H NMR (300 MHz, CDCl3) d 1.98–2.06
;
(m, 3H), 2.29–2.36 (m, 1H), 2.94 (dd, 1H, J = 15.01
and 10.93 Hz), 3.55–3.77 (m, 3H), 3.87 (s, 3H), 4.10–
4.15 (m, 1H), 4.36 (d, 1H, J = 11.8 Hz), 5.76 (br, 1H),
6.83 (dd, 1H, J = 8.6 and 2.2 Hz), 6.90 (d, 1H,
J = 2.0 Hz), 6.98 (s, 1H), 7.47 (d, 1H, J = 8.6 Hz), 8.03
(br, 1H); 13C NMR (75.5 MHz, CDCl3) d 21.41, 28.80,
30.54, 44.94, 55.52, 57.76, 58.19, 94.58, 109.01, 109.58,
119.48, 121.40, 123.12, 136.93, 156.42, 165.66, 169.80.
EIMS m/e (relative intensity) 313 (M+, 14), 160 (100).
Anal. Calcd for (C17H19N3O3) C, H, N. This material
was used directly in a later step.
5.46. (3S,6S)-3-Isopropyl-6-[6-methoxy-2-(3-methylbut-
2-enyl)-1H-indol-3-ylmethyl]piperazine-2,5-dione (67)
Indole 67 (63 mg) was prepared from 20 in 85% yield un-
der conditions described above for the preparation of 1.
1
67: H NMR (300 MHz, CDCl3) d 0.95–0.97 (d, 3H,
J = 6.8 Hz), 1.06–1.08 (d, 3H, J = 7.1 Hz), 1.77–1.80
(d, 6H, J = 9.8 Hz), 2.42 (m, 1H), 2.91–2.99 (dd, 1H,
J = 11.0 and 14.4 Hz), 3.43–3.46 (d, 2H, J = 7.2 Hz),
3.58–3.64 (dd, 1H, J = 3.3 and 14.5 Hz), 3.85 (s, 3H),
3.92 (s, 1H), 4.25–4.29 (d, 1H, J = 9.4 Hz), 5.29–5.32
(t, 1H, J = 8.2 Hz), 5.79 (s, 1H), 6.00 (s, 1H), 6.77–
6.81 (dd, 1H, J = 8.6 and 2.2 Hz), 6.84 (d, 1H,
J = 2.1 Hz), 7.38–7.41 (d, 1H, J = 8.6 Hz), 7.83 (br,
1H). MS (EI) m/e (relative intensity) 383 (M+). +TOF
MS HRMS Calcd for (C22H29N3O3 + H)+m/z =
384.2287, found m/z = 384.2282.
5.43. 3-(2-Bromo-6-methoxy-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (63)
A solution of NBS (28.5 mg) which had been dissolved
in THF (1 mL) was syringed into a solution of 62
(50 mg, 0.16 mmol) in THF (10 mL) at ꢀ78 ꢁC. The
reaction mixture which resulted was allowed to stir at
rt overnight at which time analysis by TLC (silica gel)
indicated the absence of starting material. The solvent
was removed under reduced pressure. The residue was
purified by preparative TLC (silica gel, 5% EtOH/
CH2Cl2) to afford 63 as a powder in 80% yield. 1H
NMR (300 MHz, CDCl3) d 1.76–1.89 (m, 2H), 2.11
(dd, 2H, J = 16.2 Hz and 8.8 Hz), 2.97 (dd, 1H,
J = 10.44 and 6.6 Hz), 3.12–3.37 (m, 3H), 3.52–3.60
(m, 1H), 3.83 (s, 3H), 4.27 (d, 1H, J = 4.2 Hz), 6.37
(br, 1H), 6.75–6.84 (m, 2H), 7.39 (d, 1H, J = 8.6 Hz),
8.92 (br, 1H); 13C NMR (75.5 MHz, CDCl3) d 21.52,
28.95, 29.77, 45.18, 55.54, 57.78, 58.06, 94.32, 108.61,
108.70, 110.00, 118.88, 121.30, 136.71, 156.59, 165.33,
168.96. EIMS m/e (relative intensity) 391 (M+, 18), 393
(M+, 18), 154 (100), 240 (78).
5.47. (3S,6S)-3-Benzyl-6-[6-methoxy-2-(3-methylbut-2-
enyl)-1H-indol-3-ylmethyl]piperazine-2,5-dione (68)
Indole 68 (45 mg) was prepared from 20 in 75% yield un-
der conditions described above for the preparation of
1.68: 1H NMR (300 MHz, CDCl3) d 1.81 (d, 6H,
J = 9.8 Hz), 1.90–1.98 (m, 1H), 3.12–3.26 (m, 2H),
3.41–3.43 (d, 2H, J = 7.5 Hz), 3.85 (s, 3H), 4.05–4.08
(d, 1H, J = 10.1 Hz), 4.25 (m, 2H), 5.32–5.35 (t, 1H,
J = 8.4 Hz), 5.53 (s, 1H), 5.75 (s, 1H), 6.78–6.85 (m,
4H), 7.25–7.28 (m, 3H), 7.43–7.46 (d, 1H, J = 9.3 Hz),
7.83 (br, 1H). MS (EI) m/e (relative intensity) 431
(M+, 100). +TOF MS HRMS Calcd for (C26H29
N3O3+ H)+m/z = 432.2287, found m/z = 432.2292.
5.44. 3-(2-Chloro-6-methoxy-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (64)
5.48. 2-Isoprenyl-3-(6-nitro-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (69)
Indole 64 (55 mg) was prepared in 85% yield from 62 un-
der conditions described above for the preparation of
63. 64: H NMR (300 MHz, CDCl3) d 1.81–1.90 (m,
2H), 2.15 (dd, 2H, J = 16.2 Hz and 8.8 Hz), 2.97 (dd,
1H, J = 10.44 Hz and 6.6 Hz), 3.17–3.32 (m, 3H),
Tryprostatin B 2 (20 mg, 0.056 mmol) was dissolved in
anhydrous THF (5 mL) and trifluoroacetic acid (2 mL)
was added, after which the mixture was cooled to
ꢀ78 ꢁC. A solution of NaNO2 (20 mg, 0.28 mmol) in
1

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4647
TFA (2 mL) was slowly added to the cooled solution via
a syringe over a 10 min period. The reaction mixture was
stirred for an additional 30 min and then allowed to
warm to ꢀ20 ꢁC for 30 min, after which CH2Cl2
(10 mL) and cold aq NH4OH (14%) were added until
pH 8. The layers were separated and the aqueous layer
was washed with CH2Cl2 (3 · 10 mL). The combined or-
ganic layers were washed with brine (10 mL) and dried
(Na2SO4). After removal of the solvent under reduced
pressure, the residue was purified by flash chromatogra-
phy (silica gel, CH2Cl2/ethanol 20:1) to provide 69
(17.5 mg, 76%) as a yellow powder. 69: IR mmax (KBr)
mixture was stirred for 4 h and then the solution was
treated with triethylamine (2 mL). After the solvent
was removed under reduced pressure, the residue was
purified by a short wash column (silica gel, CH2Cl2/eth-
anol, 20:1) to provide 71 as an oil (20.0 mg, 91%). 71: IR
m
max (KBr) 2961, 2120, 1615 cmꢀ1; 1H NMR (300 MHz,
CDCl3) d 1.59 (s, 3H), 1.75 (s, 3H), 1.82 (s, 3H), 2.34 (m,
1H), 2.97 (dd, 1H, J = 10.86 Hz and J = 11.16 Hz), 3.50
(s, 2H), 3.63 (m, 3H), 4.14 (s, 1H), 4.32 (d, 1H,
J = 6.96 Hz), 5.31 (s, 1H), 5.61 (s, 1H), 7.03 (d, 1H,
J = 6.72 Hz), 7.28 (s, 1H), 7.42 (d, 1H, J = 8.52 Hz),
8.07 (s, 1H); 13C NMR (75.5 MHz, CDCl3) d 17.95,
22.53, 25.13, 25.49, 25.68, 28.31, 29.62, 45.36, 54.49,
59.19, 105.33, 108.07, 118.41, 118.50, 118.94, 124.68,
127.27, 134.81, 136.20, 138.83, 165.37, 169.26. EIMS
m/e (relative intensity) 408 (M+, 18), 255 (100). Anal.
Calcd for (C22H24N4O2S0.3H2O) C, H, N.
1
3249, 2923, 1650, 1540, 1451, 1304.5 cmꢀ1; H NMR
(300 MHz, CDCl3) d 1.60 (s, 3H), 1.76 (s, 3H), 1.84–
2.06 (m, 3H), 2.34 (s, 2H), 3.04 (dd, 1H, J = 10.86 Hz
and J = 4.32 Hz), 4.12 (m, 2H), 4.35 (d, 1H, 8.74 Hz),
5.34 (s, 1H), 5.53 (s, 1H), 7.53 (d, 1H, J = 8.82 Hz),
8.03 (d, 1H, J = 6.00 Hz), 8.29 (s, 1H), 8.53 (s, 1H);
13C NMR (75.5 MHz, CDCl3) d 14.08, 17.96, 22.47,
24.30, 25.66, 28.28, 45.38, 54.57, 59.15, 95.47, 106.08,
107.60, 115.52, 117.38, 118.34, 128.05, 129.42, 132.82,
143.15, 165.78, 169.19. EIMS m/e (relative intensity)
396 (M+, 22), 355 (10), 381 (23), 243 (100). Anal. Calcd
for (C21H24N4O4) C, H, N.
5.51. 2-Isoprenyl-3-(6-azido-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (72)
6-Aminotryprostatin B 70 (15 mg, 0.041 mmol) was
dissolved in CH2Cl2 (2 mL). Triethylamine (0.1 mL,
0.72 mmol) and a solution of CuSO4 (2.0 mg, 0.014
mmol) in H2O (0.05 mL) were added to the reaction
mixture. A freshly prepared solution of TfN3 (21 mg,
0.12 mmol) in CH2Cl2 (1 mL) was then added, and the
solution which resulted was brought to homogeneity
by adding MeOH (1 mL). The solution which resulted
was stirred at rt for 2 h. The reaction solution was then
poured into a saturated solution of aq NaHCO3 (5 mL)
and extracted with CH2Cl2 (3 · 10 mL). The combined
organic layers were washed with brine (10 mL) and dried
(Na2SO4). After the solvent was removed under reduced
pressure, the residue was purified by a short wash col-
umn (silica gel, CH2Cl2/EtOH, 20:1) to provide 72 as
an oil (14.5 mg, 90%). 72: IR mmax (KBr) 2960, 2112.2,
5.49. 2-Isoprenyl-3-(6-amino-1H-indol-3-ylmethyl)-hexa-
hydro-pyrrolo[1,2-a]pyrazine-1,4-dione (70)
Iron (III) chloride hexahydrate (25 mg, 0.063 mmol) and
active carbon (2 mg, 0.16 mmol) were added to a solu-
tion of 69 in MeOH (4 mL). After the reaction mixture
was heated to reflux, hydrazine monohydrate (0.1 mL)
was added dropwise. The reaction mixture was allowed
to stir at reflux until analysis by TLC (silica gel) indi-
cated the disappearance of starting material (3 h). The
reaction was then cooled to rt and the catalysts were re-
moved by filtration through a pad of celite. The CH3OH
was removed under vacuum and the residue was dis-
solved in CH2Cl2. The organic layer was then washed
with water, brine, and dried (Na2SO4). After the
CH2Cl2was removed under reduced pressure, the residue
was purified by a short wash column (silica gel, CH2Cl2/
ethanol, 20:1) to provide 70 as an oil (20.6 mg, 88%). 70:
1H NMR (300 MHz, CDCl3) d 1.77 (m, 4H), 1.99–2.06
(m, 5H), 2.33 (s, 1H), 2.89 (t, 1H, J = 14.04 Hz), 3.41
(m, 3H), 3.59–3.67 (m, 4H), 4.12 (s, 1H), 4.34 (d, 1H,
J = 9.03 Hz), 5.29 (dd, 1H, J = 4.80 Hz and
J = 7.20 Hz), 5.67 (s, 1H), 6.55 (d, 1H, J = 6.60 Hz),
6.64 (s, 1H), 7.28 (d, 1H, J = 3.60 Hz), 7.69 (s, 1H);
13C NMR (75.5 MHz, CDCl3) d 17.85, 22.55, 24.94,
25.63, 27.47, 28.26, 45.29, 54.46, 59.17, 96.49, 104.42,
110.30, 118.34, 120.04, 121.27, 134.02, 134.90, 136.74,
142.04, 165.78, 169.20. EIMS m/e (relative intensity)
366 (M+, 10), 198 (62), 165 (100). Anal. Calcd for
(C21H26N4O2H2O) C, H, N. This material was con-
verted into the hydrochloride salt for storage purposes.
1615 cmꢀ1 1H NMR (300 MHz, CDCl3) d 1.77 (m,
;
6H), 2.06 (s, 3H), 2.34 (s, 1H), 3.00 (t, 1H, J = 8.67),
3.47 (s, 2H), 3.60 (m, 3H), 4.12 (t, 1H, J = 7.20), 4.33
(d, 1H, J = 9.63), 5.32 (s, 1H), 5.61 (s, 1H), 6.83 (d,
1H, J = 6.60), 6.99 (s, 1H), 7.38 (d, 1H, J = 12.00),
7.96 (s, 1H). EIMS m/e (relative intensity) 392 (M+,
28). HRMS Calcd for C21H24N6O2m/z = 392.1961,
found m/z = 392.1957.
5.52. Topoisomerase II-mediated DNA relaxation
assay52,53
The DNA relaxation assay tests the ability of a drug to
inhibit the Topo II-mediated relaxation of supercoiled
DNA. The assay was performed in a total volume of
10 lL and contained 62.5 lg of plasmid pUC18 DNA
(from Escherichia coli) and 100 lM drug in an incuba-
tion assay buffer (0.05 M Tris–HCl, pH 8.0, 0.12 M
KCl, 0.01 M MgCl2, 0.5 mM ATP, 0.5 mM DTT,
30 mg/mL BSA). Stock solutions of drug were made
up in either DMSO or EtOH and the total percentage
of these in the assay mixture was kept to less than 1%.
The mixture was allowed to warm to 37 ꢁC and the reac-
tion was initiated by the addition of 2.0 U of Topo II
(TopoGEN, Inc.). The reaction was allowed to proceed
for 30–45 minutes before being stopped by addition of
5.50. 2-Isoprenyl-3-(6-isothiocyanato-1H-indol-3-ylmethyl)-
hexahydro-pyrrolo[1,2-a]pyrazine-1,4-dione (71)
6-Aminotryprostatin B 70 (20 mg, 0.055 mmol) was dis-
solved in CHCl3 (4 mL) and thiophosgene (0.2 mL,
0.003 mmol) was added dropwise at rt. The reaction

4648
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
2.5 lL decatenation buffer (5% sarkosyl, 25% glycerol,
and 0.0025% bromophenol blue). The drugs were then
extracted from the incubation with 10 lL of 24:1 CHCl3:
isoamyl alcohol and the samples loaded onto a 1% aga-
rose gel to run for 90 min at 90 V. The gel was stained
with ethidium bromide and destained with H2O. The
DNA bands were detected on a UV light box and pho-
tographed with Polaroid 525 film. Controls were no-en-
zyme, enzyme, 100 lM m-AMSA, and 1% DMSO.
After incubation with drug for 72 h, the CellTiter 96TM
AQueous Non-Radioactive Cell Proliferation Assay (Pro-
mega) was used to evaluate cell survival. Cells were trea-
ted with a solution of MTS [3-(4,5-dimethylthiozol-2-yl)-
5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tet-
razolium] and an electron coupling reagent, PMS (phen-
azine methosulfate) diluted in RPMI-1640 medium.
MTS (Owen’s reagent)60 was bioreduced by viable cells
into formazan, and the amount of formazan present
can be measured by reading the absorbance at
490 nm.61 The amount of formazan present was propor-
tional to the number of living cells in culture. Vehicle
control lanes were assumed to have 100% cell survival
and the percentage of cells remaining in the drug-treated
wells was calculated as a percentage of these control
wells. The absorbance of wells containing only the
MTS reagent (the plate blank) was subtracted from all
wells.
The gels were analyzed qualitatively by looking for the
presence of DNA bands that migrate farther down on
the gel than the negative controls. Topo II-mediated
relaxation of the DNA prevents the band from migrat-
ing down the gel as far as one that is still in a supercoiled
form. Therefore, DNA incubated with Topo II inhibi-
tors will migrate farther on the gel than the no-enzyme
or DMSO controls.
5.53. Microtubule assembly assay13,54
5.55. Cell culture
Tubulin, containing MAPs (microtubule-associated pro-
teins), was prepared as described in the literature.59 The
tubulin polymerization assay was run at 37 ꢁC by adding
to 240 lL of PME buffer (100 mM PIPES, pH 6.9,
2 mM EGTA, 1 mM MgSO4), 8 lL of GTP (50 mM),
32 lL of drug in DMSO, and 120 lL of tubulin (added
last). The change in absorbance was measured at 351 nm
over 10 min. The sample cuvette was zeroed against a
reference cuvette containing 360 lL TBE buffer, 8 lL
GTP (50 mM), and 32 lL DMSO. The concentration
of the drug solution was varied for different runs to ob-
tain a delta absorbance versus concentration curve.
Standard curves were prepared on each batch of sepa-
rately prepared tubulin using colchicine as the standard.
Polymerization assays were conducted on the tryprosta-
tins (1–8) and similar derivative (colchicine).
A temperature-sensitive cdc2 mutant cell line, tsFT210,
which was isolated from the mouse mammary carci-
noma cell line FM3A, was a kind gift from Dr. F. Han-
aoka (RIKEN).4 The tsFT210 cells were maintained in
RPMI 1640 with 10% fetal calf serum at the permissive
temperature of 32 ꢁC.
5.56. Cell cycle analysis
In a synchronous-culture assay, cells were seeded at a
density of 1 · 105 cells/mL in 0.5 mL into a 24-well plate
and were preincubated at 32 ꢁC for 1 h. Then, 5 lL of
each sample solution was added, and the cells were incu-
bated at 32 ꢁC for 18 h. After incubation, morphological
characteristics of the cells were examined by microscopic
observation. The cells were subjected to flow cytometric
analysis as described below to confirm the DNA con-
tents in cells.
5.54. Cytotoxicity assay
Three human cell lines were purchased from American
Type Culture Collection (ATCC) and used in all cyto-
toxicity assays. The MCF-7 breast adenocarcinoma
cells, NCI-H520 lung squamous cell carcinoma cells
and PC-3 prostate adenocarcinoma cells were main-
tained at 37 ꢁC in a humidified atmosphere containing
5% CO2. Cells were subcultured twice a week in
RPMI-1640 medium supplemented with 10% fetal bo-
vine serum (FBS), 2 mM L-glutamine, and 0.1 mM
non-essential amino acids.
Flow cytometric analysis was performed essentially as
described by previous reports.4,58 The harvested cells
were stained with solution containing 50 lg/mL propidi-
um iodide, 0.1% sodium citrate, and 0.2% NP-40 and
analyzed for DNA contents using a flow cytometer
(Coulter Co., Hialeah, FL).
5.57. Cell staining
Carnoy fixation and staining were performed with slight
modification. Cells were treated with 0.55% of KCl for
20 min, fixed in Carnoy’s solution and dropped onto a
wet glass slide. The chromosomes and intact nuclei were
stained with 1 mg/mL of Hoechst 33258, and examined
by using fluorescent microscopy (Olympus, Tokyo,
Japan).
Normally growing cells were plated at 1 · 104 cells/well
into 96-well plates and incubated for 24 h at 37 ꢁC. After
24 h, the cells were drugged for initial screening with
100 lM, 50 lM, and 10 lM drug dissolved in a DMSO
or EtOH vehicle (less than 1% in culture medium). Any
drug showing <50% cell survival at 100 lM was further
tested using appropriate drug concentrations to deter-
mine its growth inhibition-50% (GI50). Drugs were run
in quadruplicate or greater and control wells contained
an appropriate percentage of DMSO or EtOH, usually
0.2%. Positive controls were either etoposide (ETOP)
or amsacrine (m-AMSA).
5.58. Proliferation assay
Exponentially growing tsFT210 cells were treated with
test compounds at 32 ꢁC for 48 h. The cell number
was evaluated by the subsequent color reaction. The
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-

H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
4649
disulfophenyl)-2H tetrazolium, mono-sodium salt,
Acknowledgments
WST-8TM (Nakalai Tesque, Kyoto, Japan) was added,
and the cells were further incubated for 4 h at 37 ꢁC.
The absorbance (A450) of each well was measured by a
Wallac 1420 multilabel counter (Amersham Biosciences,
Piscataway, NJ).
We thank the National Institute of Mental Health
MH46851 in part, and the Research Growth Initiative
of the University of Wisconsin—Milwaukee for support
of this work.
Appendix A
Elemental analyses
Compound
Formula
Calculated (%)
Found (%)
H
C
H
N
C
N
1
2
3
4
5
6
7
8
C22H27N3O3Æ1/3H2O
C21H25N3O2Æ1/4H2O
C22H27N3O3Æ3/5H2O
C21H25N3O2Æ3/4H2O
C22H27N3O3Æ3/4H2O
C21H25N3O2Æ1/5H2O
C22H27N3O3Æ3/8H2O
C21H25N3O2Æ1/8H2O
C26H37N3O5
68.19
70.86
67.36
69.11
66.90
71.04
70.41
71.31
66.22
68.99
70.08
67.29
65.18
68.11
66.04
69.27
69.90
67.97
71.04
71.32
71.02
56.73
70.56
67.96
71.44
64.85
64.12
65.16
67.74
63.62
65.62
63.86
7.20
10.84
11.81
10.71
11.51
10.64
11.84
11.73
11.88
8.91
7.79
8.17
8.41
10.14
7.58
12.84
11.02
10.19
11.89
9.56
7.56
8.57
7.63
7.48
68.21
70.88
67.33
69.11
66.92
71.06
70.44
71.34
66.23
68.69
70.10
67.49
64.96
67.71
65.80
69.03
69.54
68.20
71.39
70.90
70.61
56.97
70.23
67.51
71.09
65.22
63.96
64.93
67.36
63.96
65.20
63.52
7.16
7.25
7.26
7.28
7.26
7.24
7.26
7.21
7.90
8.66
8.56
9.16
7.36
6.68
6.75
7.28
5.89
6.28
8.01
7.81
6.75
6.47
7.98
7.24
6.56
7.10
6.98
6.36
5.49
5.73
14.26
5.54
10.85
11.85
10.75
11.55
10.67
11.79
11.75
11.85
8.55
7.40
8.15
8.34
10.24
7.81
13.06
11.29
9.87
11.63
9.82
7.89
8.28
7.48
7.14
7.94
7.22
7.25
7.32
7.27
7.21
7.25
7.20
7.91
8.40
8.53
9.08
7.30
7.03
6.47
7.13
6.31
6.56
8.48
8.16
7.14
6.59
7.72
7.61
6.25
7.44
6.87
6.11
5.91
6.10
7.29
5.95
12
16
17
29
33
35
37
38
39
40
41
45
46
48
49
51
55
58
61
62
66
69
70
71
C31H45N3O5
C30H43N3O4Æ1/4H2O
C28H45N3O3Si
C15H20N2O3
C21H24N2O3ÆH2O
C18H21N3O3
C22H27N3O3
C24H25N3O3Æ0.5H2O
C20H23N3O3
C26H37N3O3
C22H30N2O3
C29H33N3O3ÆH2O
C26H36N3O5Br
C33H43N3O5
C32H41N3O5ÆH2O
C24H25N3O3
C30H41N3O7
C14H18N2O3
C17H19N3O3
C21H21N3O3Æ0.5H2O
C21H24N4O4
7.43
10.41
7.56
10.69
13.41
11.29
14.13
14.58
13.55
10.05
7.98
10.54
13.68
11.73
14.47
15.02
13.13
C21H26N4O2ÆH2O
C22H24N4O2SÆ0.3H2O
High resolution mass spectra (HRMS) (EI)
Compound
Formula
Calculated mass
Found Mass
1
C22H27N3O3
C25H35N3O4
C26H37N3O5
C25H35N3O4
C24H34N2O5
381.2052
441.2628
471.2733
441.2628
430.2468
428.2525
381.2044
441.2536
471.2739
441.2634
430.2481
13
14
15
20
47
C25H31N3O3 + Li+
428.2519
(continued on next page)

4650
H. D. Jain et al. / Bioorg. Med. Chem. 16 (2008) 4626–4651
Appendix A (continued)
Compound
Formula
Calculated mass
Found Mass
50
56
60
64
67
68
72
C29H41N3O5 + H+
C20H23N3O3 + Li+
C21H23N3O5
C21H23N3O5 + Na+
C22H29N3O3 + H+
C26H29N3O3 + H+
C21H24N6O2
512.3124
360.1899
397.1638
370.0934
384.2287
432.2287
392.1961
512.3126
360.1896
397.1657
370.0935
384.2282
432.2292
392.1957
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