Synthesis, metabolism and in vitro cytotoxicity studies on novel lavendamycin antitumor agents

Article abstract of DOI:10.1016/j.bmc.2010.01.037

A series of lavendamycin analogues with two, three or four substituents at the C-6, C-7 N, C-2′, C-3′ and C-11′ positions were synthesized via short and efficient methods and evaluated as potential NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor agents. The compounds were prepared through Pictet-Spengler condensation of the desired 2-formylquinoline-5,8-diones with the required tryptophans followed by further needed transformations. Metabolism and toxicity studies demonstrated that the best substrates for NQO1 were also the most selectively toxic to NQO1-rich tumor cells compared to NQO1-deficient tumor cells.

Full text of DOI:10.1016/j.bmc.2010.01.037

Bioorganic & Medicinal Chemistry 18 (2010) 1899–1909
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, metabolism and in vitro cytotoxicity studies on novel
lavendamycin antitumor agents
Wen Cai a, Mary Hassani b, Rajesh Karki a, Ervin D. Walter a, Katherine H. Koelsch b, Hassan Seradj a,
Jayana P. Lineswala a, Hamid Mirzaei a, Jeremy S. York a, Fatemeh Olang a, Minoo Sedighi a, Jennifer S. Lucas a,
Thomas J. Eads a, Anthony S. Rose a, Sahba Charkhzarrin a, Nicholas G. Hermann a, Howard D. Beall b,
,
*
Mohammad Behforouz a,
*
a Chemistry Department, Ball State University, Muncie, IN 47306, USA
b Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
a r t i c l e i n f o
a b s t r a c t
A series of lavendamycin analogues with two, three or four substituents at the C-6, C-7 N, C-20, C-30 and
C-110 positions were synthesized via short and efficient methods and evaluated as potential
NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor agents. The compounds were prepared
through Pictet–Spengler condensation of the desired 2-formylquinoline-5,8-diones with the required try-
ptophans followed by further needed transformations. Metabolism and toxicity studies demonstrated
that the best substrates for NQO1 were also the most selectively toxic to NQO1-rich tumor cells compared
to NQO1-deficient tumor cells.
Article history:
Received 10 November 2009
Revised 13 January 2010
Accepted 16 January 2010
Available online 25 January 2010
Keywords:
Lavendamycin analogues
Quinoline-5,8-diones
Antitumor
Ó 2010 Elsevier Ltd. All rights reserved.
NQO1
Cytotoxicity
1. Introduction
both were impractical, since they produced compound 3 in an
overall yield of less than 2% and in 9- and 20-steps, respectively.
Lavendamycin (1), a naturally occurring 7-aminoquinoline-5,8-
dione antitumor antibiotic, was first isolated from the fermenta-
tion broth of Streptomyces lavendulae by Balitz et al.1 and its struc-
ture was determined by Doyle et al.2 The latter study determined
that lavendamycin is a pentacyclic structure with two moieties
including quinoline-5,8-dione and indolopyridine (b-carboline)
(Chart).2 Lavendamycin is similar to another potent antibiotic anti-
tumor agent streptonigrin (2).1–5 The clinical use of both of these
antibiotics has been precluded because of their toxicity toward hu-
man cells.2,6–8 Initial structure–activity relationship (SAR) studies
have demonstrated that the essential moiety for the cytotoxic
activity of lavendamycin and streptonigrin is the 7-aminoquino-
line-5,8-dione moiety.7
Our group was able to develop two synthetic methods11,12 that
produced 3 in an overall yield of about 40% in only 5-steps. These
efficient methods, particularly those reported in 1996,12 made it
possible to prepare a large number of substituted lavendamycins
needed for various in vitro and in vivo screening tests.
O
A
O
A
4
CH3O
H2N
6
1'
N
B
N
B
6'
D
N
C
CO2H
CH3
CO2R
CH3
H2N
N
1
C
O
O
H2N
HO
H3CO
H3CO
4'
7'
12'
HN
1; R = H
3; R = CH3
2
D
E
9'
As it was thought that some analogues of the naturally occur-
ring antibiotic 1 might show activity toward human tumors, effi-
cient methods were needed to prepare these derivatives. There
were two previous reports on the synthesis of lavendamycin
methyl ester (3) (above and Table 1) by Kende9 and Boger10 but
We previously reported that a significant number of these
lavendamycin analogues have potent biological activity including
anti-HIV reverse transcriptase13 and antitumor activity.14–16 More
importantly, several screening tests on a number of lavendamycin
analogues have shown that a significant number of these com-
pounds possess low animal toxicity.13,17 The NCI in vivo studies
* Corresponding authors. Tel.: +1 406 243 5112; fax: +1 406 243 5228 (H.D.B.);
tel.: +1 765 760 007; fax: +1 765 285 6505 (M.B).
E-mail addresses: howard.beall@umontana.edu (H.D. Beall), mbehforo@bsu.edu
(M. Behforouz).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.037

1900
W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
Table 1
NQO1 can be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) and polycyclic aromatic hydrocarbons.21 Induction by pro-
carcinogens and the capacity of NQO1 for detoxifying reactive
metabolites suggest that changes in expression of NQO1 may occur
during carcinogenesis.22 Marked elevations in NQO1 activity and
mRNA content have been documented in both preneoplastic tissues
and established tumors. Tumors or cancer cell lines where increased
NQO1 expression has been observed include those from the lung, li-
ver, colon, andbreast.23 In addition, advanced, metastatictumorsap-
pear to express higher levels of NQO1 than non-metastatic tumors.24
Correlations between NQO1 activity in cancer cells and cytotox-
icity of antitumor quinones to those cells have been reported.25–27
In our previous studies with several series of indole- and quinolin-
equinone antitumor agents, including analogues of lavendamycin,
those compounds that were the best substrates for NQO1 were also
found to be selectively toxic to cell lines with high levels of NQO1
compared to cell lines that were deficient in NQO1.15,16,28–30 In this
report, we describe the synthesis, metabolism by recombinant
human NQO1 and anticancer activity of novel analogues of
lavendamycin with functional group substitutions on both the
quinoline-5,8-dione and indolopyridine moieties.
Structures of lavendamycins discussed in Sections 1 and 2
O
R2
N
R3
R4
R1NH
N
O
HN
No.
R1
R2
R3
R4
3
4
5
6
7
H
H
H
H
H
H
H
H
H
H
H
H
Cl
OCH3
H
CO2CH3
CONH2
CONH2
CO2C8H17-n
CO2(CH2)2CH(CH3)2
CO2CH3
CO2CH3
CONHC4H9-n
CH2OH
CO2C8H17-n
CO2CH3
CO2CH3
CO2CH3
CO2(CH2)2OH
CH3
H
H
H
H
CH3
H
H
H
H
CH3CO
H
CH3CO
CH3CO
CH3CO
2-Furyl-CO
CH3CO
H
H
8
9
10
11
12
13
14
15
16
CH3CO
H
H
H
H
CH3
CH3
H
2. Results and discussion
2.1. Synthetic chemistry
have shown that the maximum tolerated dose of three derivatives
3, 4 and 5 (Table 1) in mice is 400 mg/kg which is 31 and 1000
times higher than that for lavendamycin and streptonigrin, respec-
tively.13,17 The lavendamycin analogue 6 reduced tumor volume
(up to 80%) in mice bearing tumor xenografts at a daily dose of
300 mg/kg for 10 days without exhibiting drug-related weight loss
or lethality.17 A recent study also demonstrated that the normal rat
kidney epithelial cell line (NRK-52E) exhibited much less sensitiv-
ity to several lavendamycin analogues compared to the tumor cells
with the same origin.17 When lavendamycin analogues 7 and 6 at a
daily dose of 300 mg/kg and 8 at a daily dose of 100 mg/kg were
administered to nude mice for eight and seven days, respectively,
no drug-related deaths or toxicity were observed.17 Analogues 8,
7 and 6 (Table 1) inhibited tumor growth in nude mice by 69%,
88% and 78%, respectively, when administered at 100, 150 and
300 mg/kg for 7, 8 and 8 days, respectively.17
NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase) is a
two-electron reductase, characterized by its capacity for utilizing
either NADH or NADPH as reducing cofactors and by its inhibition
by dicoumarol.18 It is primarily located in the cytosol (>80%), but a
recent study has reported a substantial pool of NQO1 in the nuclei
of cancer cells.19 NQO1 is generally categorized as a detoxification
enzyme, and it can protect the cell from a broad range of chemi-
cally reactive metabolites including electrophiles and reactive oxy-
gen species.20 NQO1 can also function as an activating enzyme,
specifically for the reductive activation of antitumor quinones
and other bioreductive anticancer agents.21
Table 4 presents the structures of a total of 25 lavendamycins
that were the subject of our biological studies in this report. As
shown in Scheme 1, Pictet–Spengler (P–S) condensation of the qui-
nolinedione aldehydes (Table 2) with tryptophans (Table 3) yielded
the target lavendamycins 40–58 listed in Table 4. The amino lav-
endamycins 59–64 were prepared respectively by the acid hydro-
lysis of the corresponding 7-acylamino lavendamycins 40–45.
High resolution mass spectroscopy of 67 (Scheme 2) did not
produce a signal for the molecular ion M+, but rather gave one at
354.109. This corresponds to a molecular formula of C18H16N3O5
(Mꢀ2H2O+H)+, that may be due to the fragment shown in
Scheme 2, produced by the dehydration of 67 under HRMS
conditions.
Similar to our reported synthesis of b-methyltryptophan methyl
ester,31 the nitrobutanoate 71 (Scheme 3) and the precursor to the
ester 28, existed in two stereoisomers A and B in a ratio of 1/1 as
shown by NMR. However, upon recrystallization of the mixture,
stereoisomer B was completely converted and only the less soluble
isomer A was obtained in pure form. When 71A was reduced with
H2 in the presence of Ra–Ni the product was 5-benzyloxy b-meth-
yltryptophan methyl ester (73%).
Amides 30, 31 and 32 were prepared as shown in Scheme 4. Al-
kyl esters 27, 33, 34 and 36 (Scheme 5) were prepared by the
Fischer esterification of the corresponding tryptophan or hydroxy-
tryptophan with the required alcohol. The preparation of 7-foram-
ido-2-formylquinoline-5,8-dione (20), necessary for the synthesis
of lavendamycin 53 is described in Section 4.
R3
R4
O
O
R2
R2
R1NH
R5
NH2
solvent
+
CHO
N
COR3
R4
R1NH
N
N
N
heat
O
O
H
HN
17-24
25-39
59 - 64
40-58
acid hydrolysis
R5
40 - 45
Scheme 1.

W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
1901
Table 2
Table 4
Structures of aldehydes for Scheme 1
Structures of lavendamycin analogues
O
O
O
R2
R2
R1NH
N
COR 3
R4
R1NH
N
CHO
N
O
HN
No.
R1
R2
17
18
19
20
21
22
23
24
CH3CO
H
H
H
H
H
H
H
Cl
See Refs. 11,12
See Ref. 17
See Ref. 17
CH3(CH2)2CO
(CH3)2CHCO
HCO
ClCH2CO
2-Furyl-CO
HO2C(CH2)2CO
H
R5
No. R1
CH3CO
R2 R3
R4
R5
See Ref. 15
See Ref. 16
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N[(CH2CH2)2]O
O(CH2)2CH(CH3)2
OCH3
N[(CH2)4]
NH(CH2)2OH
OCH3
H
H
H
OH
See Ref. 15
CH3CO
CH3CO
CH3CO
CH3CO
CH3(CH2)2CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
(CH3)2CHCO
HCO
ClCH2CO
ClCH2CO
2-Furyl-CO
HO2C(CH2)2CO
H
H
H
H
H
CH3 OH
See Ref. 15
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
See Ref. 15
See Ref. 17
OCH(CH3)CH2CH3
Table 3
NHCH2CH(OH)CH2OH
O(CH2)2CH3
N[(CH2CH2)2]NCH2Ph
OCH2CH3
OCH2CH3
OCH3
Structures of tryptophans for Scheme 1
COR3
NH2
R4
N
See Ref. 17
See Ref. 17
R5
OCH3
NH2
N[(CH2)4]
OCH3
OC4H9-n
H
No.
R3
R4
R5
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
OCH3
H
H
H
H
H
OH
OH
H
H
H
H
H
H
H
OH
H
H
See Ref. 15
See Ref. 15
Cl OCH2CH3
N[(CH2CH2)2]O
O(CH2)2CH(CH3)2
OCH3
H
H
H
H
H
H
N[(CH2CH2)2]O
O(CH2)2CH(CH3)2
OCH3
N[(CH2)4]
NH(CH2)2OH
OCH3
OH
CH3
H
CH3 OH
N[(CH2)4]
See Ref. 15
H
H
H
H
H
H
NHCH2CH2OH
NHCH2CH(OH)CH2OH
N[(CH2CH2)2]NCH2Ph
O(CH2)2CH3
OCH(CH3)CH2CH3
OCH2CH3
H
H
H
H
H
H
H
H
H
See Ref. 17
NQO1 substrates.15,16 Compounds 43, 47, 55, 61 and 64 displayed
good metabolism rates in the range of 28–43
NQO1 (Table 5).
OCH2CH3
OCH3
CH3
H
See Ref. 31
See Ref. 13
See Ref. 13
lmol/min/mg
OC4H9-n
NH2
H
H
Compound 52, however, with (CH3)2CHCONH and CO2CH3
groups at the R1 and R3 positions, respectively, exhibited the low-
est metabolism rate by NQO1 and ranked as the poorest substrate
(Table 5). The decreased reduction rate of 52 can be explained by
steric hindrance between the quinolinedione moiety of 52 and
the NQO1 active site due in part to the large and branched isobu-
tyramide group at R1. This steric interaction could result in unfa-
vorable positioning of 52 in the active site with subsequent poor
hydride ion reception and quinone reduction capability. Lavenda-
mycin analogue 56 also displayed a low metabolism rate by
NQO1 (Table 5) possibly due to the failure of the 2-furyl-CONH
group at the R1 position to intercalate between and form van der
Waals interactions with internal wall residues such as Trp-105
and Phe-106.16 A similar 2-furyl-CONH compound 9 (Table 1) with
H instead of CH3 at R4 was also ranked as a poor NQO1 substrate by
our recent molecular docking study.16 We previously demon-
strated that the presence of substituents at R2 also increases steric
interactions of lavendamycin ligands with the internal wall of the
active site.15,16 Accordingly, compound 58 with a Cl group at this
position was ranked as a poor substrate for NQO1 (Table 5).
2.2. Biological studies
2.2.1. Metabolism
Metabolism of the lavendamycin analogues by recombinant hu-
man NQO1 was examined. Reduction rates by NQO1 were mea-
sured using a spectrophotometric assay that employs cytochrome
c as the terminal electron acceptor15 and gives initial rates of lav-
endamycin analogue reduction (Table 5). The initial reduction rates
(lmol cytochrome c reduced/min/mg NQO1) were calculated from
the linear portion (0–30 s) of the reaction graphs.
Compound 59 with NH2 and CON[(CH2CH2)2]O groups at R1 and
R3 positions, respectively, displayed the highest metabolism rate
by NQO1 among the lavendamycin analogues (Table 5). This could
be due to the fact that the NH2 group, a small substituent, did not
produce steric hindrance with the internal wall of the NQO1 active
site resulting in favorable positioning of 59 for hydride ion recep-
tion from FAD and quinone reduction. Also, the morpholino group
at R3 could potentially form hydrogen bonding or van der Waals
interactions with the residues of the active site to increase 59 bind-
ing affinity in the active site. Our recent molecular modeling stud-
ies have demonstrated that lavendamycin analogues with a small
to medium size substituent at the R1 position and substituents at
R3 that are capable of hydrogen bonding or van der Waals
interactions with the key residues of the active site or FAD are good
2.2.2. In vitro cytotoxicity
Cytotoxicity studies were also performed on the lavendamycin
analogues with cell survival being determined by the MTT colori-
metric assay. We demonstrated an excellent positive linear corre-
lation between the IC50 values for clonogenic and MTT assays in
a previous study with lavendamycin analogues.15 We utilized the

1902
W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
NH3+Cl-
NHOC(CH2)2CO2H
NO2
b
a
+
O
N
N
H3N
N
CH3 HO2C(CH2)2COHN
O
N
CH3
O2N
N
CH3
Cl-
65
OH 66
OH 67
OH
c
O
N
CH3
d
OH
HO2C(CH2)2CONH
N
CHO
O
HO2C(CH2)2CONH
N
CH3
(M - 2H2O + H)+
HRMS fragment of 67
O
O
68
23
Scheme 2. Reagents and conditions: (a) see Ref. 16; (b) succinic anhydride, NaOAc, Na2SO3, DMF, Ar, 18 h, rt, 82%; (c) K2Cr2O7, CH3COOH, 20 h, rt, 44%; (d) SeO2, 1,4-dioxane,
H2O, 11 h, reflux, 57%.
CO2CH3
CO2CH3
NO2
H3C
H3C
NH
NO2
BnO
BnO
c
BnO
BnO
b
a
N
H
N
H
N
H
N
H
+
+ enantiomer
69
70
H3C
71A
71B
enantiomer
CO2CH3
NH2
HO
+ enantiomer
N
28
H
Scheme 3. Reagents and conditions: (a) CH3CH@N(CH3)2, CH3COOH, toluene, 4 days, 5 °C, 38%; (b) NaCH2CH3CO2CH3, Et3N, toluene, Ar, 1 h, rt, then 9 h at 103–105 °C, 97%;
(c) H2, Pd–C 10%, CF3COOH, 21 h, rt, 93%.
O
CO2N
NHZ
COR
NHZ
COR
NH2
O
a
b
N
N
N
H
H
H
72
Z = COOBn
73; R = NH(CH2)2OH, 79.5%
74; R = NHCH2CHOHCH2OH, 71% 31; R = NHCH2CHOHCH2OH, 85%
75; R = N(CH2CH2)2NBn, 92% 32; R = N(CH2CH2)2NBn, 52%
30; R = NH(CH2)2OH, 72%
Scheme 4. Reagents and conditions: (a) H2N(CH2)2OH for 73, H2NCH2CHOHCH2OH for 74, N(CH2CH2)2NBn for 75, CHCl3, Et3N, alcohol, Ar, 29 h for 73, 24 h for 74, and 48 h
for 75, rt; (b) HCOONH4, Pd–C 10%, isopropanol for 30, 32 and methanol for 31, Ar, 4.5 h for 30, 30 min for 31 and 32, rt.
COOH
NH2
COOR2
NH2
R1
R1
R2OH
Dry HCl, heat
N
N
H
H
R1 = H, OH
27; R1 = OH, R2 = CH2CH2CH(CH3)2; 91%
33; R1 = H, R2 = CH2CH2CH3; 88%
34; R1 = H, R2 = CH(CH3)CH2CH3; 69%
36; R1 = OH, R2 = CH2CH3; 29%
Scheme 5.
BE human colon adenocarcinoma cells stably transfected with hu-
man NQO1 cDNA.32 The BE cells had no measurable NQO1 activity
whereas activity in the transfected cells (BE-NQ) was 337 nmol/
min/mg total cell protein using dichlorophenolindophenol (DCPIP)
as the standard electron acceptor. In the present study, the cyto-
toxicity of the lavendamycin analogues (Table 6) was compared
using these cell lines.
Lavendamycin analogues such as 47, 55, 59 and 61 that were
good substrates for NQO1 (Table 5) were also more toxic to the
NQO1-rich BE-NQ cell line than the NQO1-deficient BE cell line
(Table 6). Compound 59, the best substrate for NQO1 (Table 1),
had the greatest differential toxicity with a selectivity ratio [IC50
(BE)/IC50 (BE-NQ)] of 9 (Table 6). Our previous studies also
determined that good lavendamycin substrates for NQO1 were

W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
1903
Table 5
Metabolism of lavendamycin analogues by recombinant human NQO1 monitored by spectrophotometric cytochrome c assay
O
R2
N
COR3
R4
R1NH
N
O
HN
R5
R4
No.
R1
R2
R3
R5
Metabolism by NQO1 (lmol/min/mg)
43
44
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
(CH3)2CHCO
HCO
ClCH2CO
ClCH2CO
2-Furyl-CO
HO2C(CH2)2CO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
N[(CH2)4]
NH(CH2)2OH
OCH(CH3)CH2CH3
NHCH2CH(OH)CH2OH
O(CH2)2CH3
N[(CH2CH2)2]NCH2Ph
OCH2CH3
OCH2CH3
OCH3
OCH3
NH2
N[(CH2)4]
OCH3
OC4H9-n
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
43
2.2 0.7
3.9 2.0
7
36
6
6.8 3.5
8.7 1.5
3.6 0.3
21
6
0.04 0.08
20
13
34
5.8 1.7
5.2 2.6
6.9 1.1
103
2.9 1.4
31
35
26
28
8
2
5
OCH2CH3
H
H
H
H
H
H
N[(CH2CH2)2]O
O(CH2)2CH(CH3)2
OCH3
N[(CH2)4]
NH(CH2)2OH
OCH3
8
OH
OH
H
H
H
CH3
H
H
7
7
4
2
H
Table 6
Cytotoxicity of lavendamycin analogues toward BE (NQO1-deficient) and BE-NQ (NQO1-rich) human colon adenocarcinoma cell lines
O
R2
N
COR3
R4
R1NH
N
O
HN
R5
No.
R1
R2
R3
R4
R5
IC50
(lM)
Selectivity ratio
BE/BE-NQ
BE-NQ
BE
13
>50
33
>50
3.2 0.2
6.5 0.5
13
4.0 0.4
>50
14
12
8.0 1.6
>50
12
>50
18
>50
14
>50
43
44
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
(CH3)2CHCO
HCO
ClCH2CO
ClCH2CO
2-Furyl-CO
HO2C(CH2)2CO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
N[(CH2)4]
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
6.2 0.2
>50
40 10
1
8
2.1
—
NH(CH2)2OH
OCH(CH3)CH2CH3
NHCH2CH(OH)CH2OH
O(CH2)2CH3
N[(CH2CH2)2]NCH2Ph
OCH2CH3
OCH2CH3
OCH3
OCH3
NH2
N[(CH2)4]
OCH3
OC4H9-n
OCH2CH3
N[(CH2CH2)2]O
O(CH2)2CH(CH3)2
OCH3
N[(CH2)4]
NH(CH2)2OH
OCH3
0.8
>2.8
1.3
0.7
1.3
0.8
-
1.4
2.1
2.7
—
1.6
—
9.0
—
2.5
>1.1
1.0
2.0
18
2
2.4 0.1
9.8 0.8
10
1
1
4.8 0.2
>50
10
1
1
1
5.8 0.6
3.0 0.5
>50
7.6 0.2
>50
2.0 0.1
>50
5.7 0.6
1
1
1
H
H
H
H
H
H
OH
OH
H
H
H
45
1.1 0.1
22
3
1.1 0.1
45
3
3

1904
W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
selectively toxic towards BE-NQ versus BE cells.15,16 Recently stud-
ied lavendamycin analogues 10, 11, 12, 5, 3 and 16 (Table 1) exhib-
ited high selective toxicity toward BE-NQ cells (selectivity
ratios = 30, 11, 10, 9, 9 and 7, respectively).15,16 Four analogues,
8, 13, 14 and 15 (Table 1), suppressed the clonogenic survival of
A549 human lung carcinoma cells at concentrations less than
100 nM with 14 being the most potent analogue that inhibited
A549 colony growth by 70% at a concentration of 100 nM.14 Also,
the colony outgrowth of the cells was reduced by 70% at 10 nM
and by 100% at 100 nM concentration of the lavendamycin ana-
logue 5.14 Although the PC-3 human prostate cancer cell line is
rather resistant to antitumor effects of many anticancer agents,
these cells displayed sensitivity toward the cytotoxic properties
of 5 at a concentration as low as 10 nM.14 Compound 5 also exhib-
ited promising antiproliferative activities against cancer cell lines
of the National Cancer Institute (NCI) 60-cell line panel including
colon, ovarian and renal cells, and cancer cells in a hollow fiber
tumorigenesis assay.14
again washed with 15 mL of dichloromethane. The combined fil-
trates were evaporated and to the resulting solid, 50 mL of dichlo-
romethane was added and placed in a separatory funnel. The
solution was washed with sodium bicarbonate (2 ꢁ 50 mL), the or-
ganic layer was dried (MgSO4) and evaporated. The brown yellow
solid was dissolved in dichloromethane, and then concentrated
to a small volume. Addition of enough n-hexane gave the yellow
solid of pure 5 (78.6 mg, 30%): mp 192–195 °C; 1H NMR (CDCl3)
d 8.07 (s, 1H), 8.34 (d, 1H, J = 7.0), 8.60 (s, 1H), 8.64 (d, 1H,
J = 6.6), 8.80 (s, 1H), 10.31 (s, 1H); HRMS m/e calcd for
C11H6N2O4, 230.032; found, 230.032.
4.1.3. 7-N-(3-Carboxypropionyl)amino-2-formylquinoline-5,8-
dione (23)
Dione 68 (225.1 mg, 0.781 mmol) was added to 4 mL of dioxane
and 0.1 mL of water. To the mixture 167.9 mg (1.5 mmol) of sele-
nium dioxide was added and refluxed with stirring under argon
for 11 h. The reaction mixture was filtered hot and the filter cake
was returned to the flask and refluxed for 5 min with 5 mL of diox-
ane and then filtered. This process was repeated once more and the
combined filtrates were evaporated to produce 130 mg (55%) of the
yellow orange solid 23: mp 225.5 °C (dec); 1H NMR (DMSO-d6) d
2.50 (m, 2H), 2.88 (m, 2H), 7.8 (s, 1H), 8.28 (d, 1H, J = 8.0), 8.54
(d, 1H, J = 8.0), 10.16 (s, 1H), 10.23 (s, 1H), 12.17 (br s, 1H); HRMS
m/e C14H10N2O6, 303.061; found, 303.062.
Lavendamycin analogues 44, 46, 48, 49, 50, 52, 56, 57 and 58
that were poor substrates for NQO1 (Table 5) demonstrated no
selective toxicity toward BE-NQ cells or had no measurable cyto-
toxicity (IC50 >50 lM) (Table 6). Overall, our results suggest that
the best lavendamycin substrates for NQO1 were also the most
selectively toxic to the high-NQO1 BE-NQ cell line compared to
NQO1-deficient BE cells, consistent with our previous studies.15,16
4.1.4. 7-N-(3-Carboxypropionyl)amino-2-methylquinoline-5,8-
dione (68)
3. Conclusions
To a solution of potassium dichromate (1.10 g) in 29 mL of
water/glacial acetic acid mixture (13.4:15.6), 0.51 g (1.3 mmol) of
amide 67 was added and the reaction mixture was stirred at rt
for 20 h and then extracted with dichloromethane (5 ꢁ 50 mL).
The combined extracts were dried (MgSO4) and evaporated to give
0.26 g (69%) of the yellow solid 53: mp 144 °C (dec); 1H NMR
(DMSO-d6) d 2.73 (m, 7H), 7.71 (s, 1H), 7.73 (d, 1H, J = 8.3), 8.24
(d, 1H, J = 7.9), 9.98 (s, 1H); HRMS m/e calcd for C14H12N2O5,
288.075; found, 288.077.
Twenty-five novel lavendamycin analogues were synthesized,
characterized and evaluated as NQO1-directed antitumor agents.
In line with our earlier computational and structure–activity stud-
ies,15,16 analogues with small substituents at the R1 position and
groups capable of hydrogen bonding/van der Waals interactions
at the R3 position generally made the best substrates for NQO1.
Consequently, many of these compounds were also selectively
toxic to the cancer cells with elevated NQO1 activity. The best sub-
strate for NQO1 was 59, the 20-CON[(CH2CH2)2]O-7-NH2 derivative
that also was nine times more toxic to the high-NQO1 BE-NQ cells
than the NQO1-deficient BE cells. These data advance our under-
standing of the structure–activity relationships for NQO1-directed
antitumor quinones.
4.1.5. 5,7-Bis(3-carboxypropionylamino)-8-hydroxy-2-methyl-
quinoline (67)
5,7-Diamino-8-hydroxy-2-methylquinoline dihydrogen chlo-
ride salt16 (66, 1 g, 3.81 mmol) was added to stirred solution of so-
dium acetate (2 g), sodium sulfite (2 g) in 80 mL DMF under argon.
A solution of succinic anhydride (3.05 g) in 30 mL DMF was drop-
wise added and stirred for 18 h at rt. The reaction mixture was fil-
tered, water (3 mL) added to the filtrate and after1 h, 500 mL of
dichloromethane was added and kept in the refrigerator for 36 h.
The orange red solid was filtered and dried (1.2034 g, 81%): mp
180–180.6 °C; 1H NMR (DMSO-d6) d 2.54 (m, 4H), 2.64 (m, 4H),
2.70 (s, 3H), 7.35 (d, 1H, J = 8.8), 8.06 (s, 1H), 8.18 (d, 1H, J = 8.8),
9.65 (br s, 1H), 9.98 (br s, 1H); HRMS m/e calcd for C18H16N3O5
(Mꢀ2 H2O+H), 354.109; found, 354.108.
4. Experimental
4.1. Chemistry
4.1.1. General methods
For general methods, see Ref. 16. In nearly all of the experimen-
tal work-ups, solvents were evaporated under reduced pressure
and heat using a rotaevaporator. In each experiment, the progress
of the reaction was monitored by TLC and the reaction stopped
when TLC showed its completion. In some instances, no NMR sig-
nals were observed for active H’s, especially when DMSO-d6 was
the solvent of use.
4.1.6. 5-Hydroxytryptophan isoamyl ester (27)
5-Hydroxytryptophan (1.101 g, 5 mmol) in 60 mL anhydrous
isoamyl alcohol with 10 mL of hydrogen chloride ether solution
(1.0 M) was refluxed for 22 h. The reaction mixture was evapo-
rated, the solid salt was mixed with 50 mL of dichloromethane
and neutralized with a solution of 14% ammonium hydroxide.
The aqueous layer was extracted with EtOAc (3 ꢁ 75 mL) and the
combined organic extracts were dried (MgSO4) and then evapo-
rated to give 1.28 g (91%) of product 27 as a gel: 1H NMR
(DMSO-d6) d 0.89 (d, 6H, J = 6.2), 1.30 (m, 2H), 1.60 (m, 1H), 2.8
(m, 1H), 2.9 (m, 1H), 4.14 (t, 2H, J = 7.0), 6.55 (s, 1H), 6.75 (m,
1H), 6.95 (m, 1H), 7.10 (d, 1H, J = 8.8), 8.56 (m, 1H), 10.51 (s,
1H); HRMS m/e calcd for C16H22N2O3, 290.163; found, 290.162.
4.1.2. 7-N-Formamido-2-formylquinoline-5,8-dione (20)
In a 25 mL two-necked round-bottomed flask, equipped with a
stirring bar, a condenser and an argon filled balloon, 7-formamido-
2-methylquinoline-5,8-dione33 (7, 0.246 g, 1.14 mmol), selenium
dioxide (0.202 g, 1.82 mmol) were added to 18 mL of dried distilled
1,4-dioxane and 0.2 mL water and refluxed for 24 h. Solid black
selenium metal was allowed to settle and the supernatant was fil-
tered. The filter paper containing selenium was placed in a beaker,
10 mL of dioxane was added, and heated for a few minutes on a
steam bath to boiling. The mixture was filtered and the solid was

W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
1905
4.1.7. Methyl (2RS,3SR)-2-amino-3-[3-(5-hydroxyindolyl)]buta-
noate (28)
temperature was kept below 25 °C. Enough base was added to pro-
duce an oily layer at the top of the solution. Solidification of the oil
was facilitated by the addition of 5 mL methylcyclohexane and
scratching the side of the beaker inside the oil. The mixture was al-
lowed to stand in the refrigerator overnight, the resulting solid fil-
tered off, and washed thoroughly with cold water to remove any
remaining NaOH. The crystals of 70 were vacuum dried (1.61 g,
38%): mp 132–132.5 °C; 1H NMR (CDCl3) d 1.01 (d, 3H, J = 4.0),
1.04 (d, 3H, J = 4.0), 1.40 (br s, 1H), 1.46 (d, 3H, J = 6.6), 2.82 (m,
1H), 4.17 (q, 1H, J = 6.6), 5.10 (s, 2H), 6.92 (m, 1H), 7.06 (d, 1H,
J = 2.3), 7.22 (s, 2H), 7.37 (m, 5H), 7.89 (br s, 1H). Anal. Calcd for
C20H24N2O: C, 77.89; H, 7.84; N, 9.08. Found: C, 77.55; H, 7.74;
N, 8.86; HRMS m/e calcd, 308.193; found, 308.189.
In a 500 mL heavy-walled hydrogenation flask equipped with a
magnetic stirring bar, a cool solution of trifluoroacetic acid (0.96 g,
8.44 mmol) in 145 mL absolute ethanol was placed. To this, pow-
dered benzyloxy nitro ester 71A (1.03 g, 2.81 mmol) was added
and the mixture was stirred for 1 h for most of the solid to be dis-
solved. The magnetic bar was removed and 1.65 g of 10% Pd–C was
added. Using a Parr hydrogenator, the mixture was hydrogenated
at 40 psi at rt for 21 h, then filtered through a layer of Celite. The
filter cake was washed with absolute ethanol (3 ꢁ 15 mL) and
the filtrate was evaporated to dryness. To the residue, 60 mL ether,
2 mL water, 3 mL 14% ammonium hydroxide were added and the
yellow ether layer was separated. The aqueous layer was extracted
with ether (5 ꢁ 50 mL), then the combined ether extracts were
washed with 10% sodium chloride solution (2 ꢁ 10 mL), dried
(MgSO4) and evaporated to give a solid. The product was redis-
solved in some ether and evaporated. This process was repeated
nine more times and the off-white crystals were dried on a vacuum
pump, producing 0.65 g (93%) of 28: mp 88–95 °C (unable to
recrystallize 28 further): 1H NMR (CDCl3) d 1.28 (d, 3H, J = 7.1),
2.15 (br s, 2H), 3.57 (m, 1H), 3.65 (s, 3H), 3.89 (d, 1H, J = 3.8),
6.74 (m, 1H), 6.98 (d, 1H, J = 2.2), 7.03 (d, 1H, J = 2.5), 7.04 (s,
1H), 7.17 (d, 1H, J = 3.7), 7.95 (br s, 1H); HRMS m/e calcd for
C13H16N2O3; 248.115; found, 248.115.
4.1.10. Tryptophan 2-hydroxyethanamide (30)
In a 100 mL round-bottomed flask equipped with a magnetic
stirring bar and an argon filled balloon, a mixture of amide 73
(1.45 g, 3.82 mmol), isopropanol (66 mL), 10% Pd–C (1.785 g) and
dry ammonium formate (1.204 g, 19 mmol) was stirred at rt for
5 h. The reaction mixture was filtered and the filtrate was evapo-
rated to give 30 as a gel (774 mg, 82%); 1H NMR (CDCl3) d 3.02
(m, 2H), 3.39 (m, 2H), 3.65 (m, 2H), 3.67 (m, 1H) 7.09 (s, 1H),
7.15 (m, 1H), 7.21 (m, 1H), 7.37 (d, 1H, J = 8.0), 7.62 (br s, 1H),
7.83 (d, 1H, J = 7.3), 8.07 (br s, 1H); HRMS m/e calcd for
C13H17N3O2, 248.139; found, 248.139.
4.1.8. Methyl 3-[3-(5-benzyloxyindolyl)]nitrobutanoate (71)
In a 500 mL three-necked round-bottomed flask equipped with
a magnetic stirring bar, thermometer, condenser under argon flow
was placed indole 70 (1.72 g, 5.54 mmol) in 19.5 mL anhydrous tol-
uene. To this, 0.74 g (7.29 mmol) of freshly distilled triethylamine
and 1.0 g (8.42 mmol) of methyl nitroacetate were added. The
solution was stirred for 1 h at rt, then heated at 103–105 °C for
9 h and allowed to cool to rt. The solution was extracted with
a10% hydrochloric acid solution (3 ꢁ 24 mL) followed by water
(2 ꢁ 13 mL). The organic layer was dried (MgSO4), evaporated
and the solid was dried under a vacuum pump at 60 °C overnight
(1.98 g, 97%). NMR showed the product to be a mixture of the
two stereoisomers 71A and 71B. Careful recrystallization of this
product with a mixture of chloroform: hexane (60:40) produced
only 71A. An analytical sample was obtained by the recrystalliza-
tion of the product with 95% ethanol giving 71A as off-white crys-
tals: mp 110–111 °C; 1H NMR (CDCl3) d 1.53 (d, 3H, J = 3.5), 3.53 (s,
3H), 4.10 (m, 1H), 5.11 (s, 2H), 5.60 (d, 1H, J = 9.0), 6.94 (dd, 1H,
J = 9.0, 2.3), 7.05 (dd, 1H, J = 9.0, 2.5), 7.10 (m, 1H), 7.23 (s, 1H),
7.38 (m, 5H), 9.01 (br s, 1H). Anal. Calcd for C20H20N2O5: C,
65.21; H, 5.47; N, 7.60. Found: C, 65.04; H, 5.54; N, 7.57; HRMS
m/e calcd, 368.141; found, 368.137.
4.1.11. N-Carbobenzyloxytryptophan 2-hydroxyethanamide (73)
In a 25 mL round-bottomed flask equipped with a magnetic stir-
ring bar and an argon filled balloon, 544 mg (1.25 mmol) of succin-
imide ester 72, and 76.4 mg (1.25 mmol) of 2-hydroxyethanol
were added to a mixture of isopropyl alcohol (5.2 mL), chloroform
(5.2 mL) and triethylamine (0.8 mL). The reaction mixture was stir-
red at rt for 23 h and then mixed with 60 mL EtOAc. The organic
layer was washed with 10 mL portions of brine, 10% citric acid
and water, dried (MgSO4) and then evaporated to give tryptophan
hydroxyamide 73 as a gel (464.6 mg, 98%): 1H NMR (CDCl3) d 2.65
(br s, 2H), 3.40 (m, 1H), 3.48 (m, 1H), 3.67 (s, 2H), 5.10 (m, 5H), 5.43
(m, 1H), 7.08 (m, 1H), 7.15 (m, 1H), 7.29 (m, 7H), 7.55 (m, 1H), 8.41
(br s, 1H); HRMS m/e calcd for C21H24N3O4 (MH+), 382.176; found,
382.176.
4.1.12. Tryptophan 2,3-dihydroxypropanamide (31)
In a set up similar to that for 30 and in a 50 mL flask, amide 74
(411 mg, 1 mmol), anhydrous methanol (18 mL), 10% Pd–C
(315.3 mg) and anhydrous ammonium formate were mixed and
stirred at rt for 30 min. The mixture was filtered and the filtrate
evaporated to give 31 as a colorless gel (236 mg, 85%); 1H NMR
(DMSO-d6) d 2.80 (m, 1H), 2.97 (m, 2H), 3.11 (m, 1H), 3.25 (m,
3H), 3.46 (m, 2H), 3.56 (m, 1H), 6.98 (t, 1H, J = 8.1), 7.07 (t, 1H,
J = 8.1), 7.17 (s, 1H), 7.34 (d. 1H, J = 8.4), 7.58 (d, 1H, J = 8.4), 8.06
(br s, 1H), 8.29 (s, 1H), 10.89 (br s, 1H); HRMS m/e calcd for
C14H20N3O3 (MH+), 278.150; found, 278.150.
4.1.9. 3-(Isopropylaminoethylidene)-5-benzyloxyindole (70)31,34
A 250 mL three-necked round-bottomed flask equipped with a
magnetic stirring bar, thermometer, dropping funnel, Claisen head,
a condenser with flowing ice-water (using an ice-water circulator)
and a calcium chloride drying tube, was immersed in an ice-salt
bath. A solution of 5-benzyloxyindole 69 (3.06 g, 0.014 mol) in
50 mL of glacial acetic acid was placed in the flask and then freshly
distilled ethylideneisopropylamine34 (1.31 g, 0.015 mol) in 23 mL
anhydrous toluene was dropwise added over an h, maintaining
the mixture temperature below 15 °C. The reaction mixture was
stirred at this temperature for 30 more minutes and then kept in
the refrigerator for four days. With vigorous stirring, the mixture
was added to 115 mL of ice-water, 38 mL of ether and then the
dark upper ether layer was separated from the aqueous layer.
The ether solution was extracted with 1 M potassium hydrogen
sulfate (3 ꢁ 23 mL) and all the aqueous solutions were combined
and basified with 10 M NaOH to pH 11 or higher, while the solution
4.1.13. N-Carbobenzyloxytryptophan 2,3-dihydroxypropan-
amide (74)
In a similar procedure to that of 73, succinimide ester 72
(1.74 g, 4 mmol), 2,3-dihydroxypropanamine (364 mg, 4 mmol)
were added to a mixture of 8 mL of isopropyl alcohol, 16 mL of
chloroform and 1.12 mL of triethylamine and stirred at rt for
24 h. To the mixture, 120 mL EtOAc was added and the solution
was washed consecutively with 10 mL portions of brine, 10% citric
acid and then water. The organic layer was dried (Mg SO4) and
evaporated to give 1.66 g (71%) of 74 as a gel: 1H NMR (DMSO-
d6) d 3.10 (m, 5H), 4.25 (m, 2H), 4.50 (m, 1H), 4.72 (m, 1H), 5.17
(m, 4H), 7.00 (m, 2H), 7.13 (s, 1H), 7.35 (m, 5H), 7.60 (m, 1H),

1906
W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
7.98 (br s, 1H), 10.80 (br s, 1H); HRMS m/e calcd for C22H26N3O5
The mixture was extracted with dichloromethane (4 ꢁ 40 mL),
washed with 40 mL of brine, dried (MgSO4) and then evaporated
to yield 0.88 g (69%) of 34 as a light brown gel. 1H NMR (CDCl3) d
0.90 (m, 3H), 1.20 (m, 3H), 1.60 (m, 4H), 3.00 (m, 1H), 3.30 (m,
1H), 3.80 (m, 1H), 4.87 (m, 1H), 7.07 (s, 1H), 7.20 (m, 2H), 7.35
(d, 1H, J = 7.7), 7.65 (d, 1H, J = 7.7), 8.22 (br s, 1H); HRMS m/e calcd
for C15H20N2O5, 260.152; found, 260.151.
MH+), 412.187; found, 412.187.
4.1.14. Tryptophan N-benzylpiperazine amide (32)
In a setup similar to that for 30, and in a 50 mL flask, a mixture
of amide 75 (250 mg, 0.5 mmol), ammonium formate (158 mg,
2.5 mmol) and 10% Pd–C (790 mg) were added to 20 mL anhydrous
isopropyl alcohol and stirred at rt for 35 min. The reaction mixture
was filtered and the filter cake was washed with 20 mL isopropa-
nol. The solid obtained upon the evaporation of the filtrate was
suspended in 3 mL of dichloromethane and introduced on top of
a 4 in diameter flash chromatography column. The column was
first eluted with a mixture of dichloromethane/methanol (10:1)
to remove the unreacted starting materials and then the product
was eluted with methanol/dichloromethane (5:1). Evaporation of
the eluant gave a yellowish white solid that was dried under a vac-
uum pump at 70–80 °C over 48 h, yielding 95 mg (52%) of the
white solid 32: mp 86–87 °C; 1H NMR (DMSO-d6) d 1.70 (m, 2H),
2.16 (m, 2H), 2.80 (m, 1H), 2.90 (m, 1H), 3.29 (m, 8H), 3.91 (m,
1H), 6.95 (m, 1H), 7.01 (m, 1H), 7.12 (s, 1H), 7.27 (m, 5H), 7.35
(d, 1H, J = 8.0), 7.47 (d, 1H, J = 7.8), 10.85 (s, 1H); HRMS m/e calcd
for C22H27N4O (MH+), 363.217; found, 363.218.
4.1.18. 5-Hydroxytryptophan ethyl ester (36)
In
a 100 mL round-bottomed flask, 5-hydroxtrypyophan
(1.101 g, 5 mmol), ethanol (50 mL) and 10 mL of hydrogen chloride
ether solution (1.0 M) was heated in an oil bath at 88 °C for 24 h.
The reaction mixture was evaporated, the solid salt was mixed
with 50 mL of dichloromethane and neutralized with a solution
of 14% ammonium hydroxide. The aqueous layer was extracted
with EtOAc (3 ꢁ 75 mL) and the combined organic extracts were
dried (MgSO4) and then evaporated to give 367 mg (29%) of ester
36 as a gel: 1H NMR (CDCl3) d 1.22 (t, 3H, J = 7.2), 1.80 (br s, 2H),
2.02 (s, 1H), 3.00 (m, 1H), 3.22 (m, 1H), 3.77 (m, 1H), 4.15 (q, 2H,
J = 7.2), 6.72 (m, 1H), 7.01 (m, 2H), 7.22 (m, 1H), 7.95 (br s, 1H);
HRMS m/e calcd for C13H16N2O3, 248.115; found, 248.115.
4.1.19. 7-N-Acetyl-110-hydroxydemethyllavendamycin isoamyl
ester (41)
4.1.15. N-Carbobenzyloxytryptophan N-benzylpiperazine
amide (75)
In a 250 mL three-necked round-bottomed flask, equipped with
a magnetic stirring bar and a condenser under an argon flow, iso-
amyl ester 27 (84 mg, 0.3 mmol) in 10 mL anhydrous DMF and
100 mL anhydrous anisole were placed. To this solution, aldehyde
17 (73.2 mg, 0.3 mmol) was added and heated to 150 °C over 3 h
and then the heat was maintained for an additional 5 h. The golden
yellow solution was allowed to cool to rt and then evaporated to
give an orange solid that was washed with 3 mL of acetone fol-
lowed by 3 mL of ether. The filtrate also gave some product as a
brown solid. Total weight of the product 41 was 80.2 mg (53%):
mp >290 °C; Rf = 0.23 (0.2:5 MeOH/CH2Cl2); 1H NMR (DMSO-d6) d
1.01 (d, 6H, J = 6), 1.70 (m, 1H), 2.30 (m, 2H), 2.42 (s, 3H), 4.38 (t,
2H, J = 6.4), 7.20 (m, 1H), 7.35 (d, 1H, J = 8.8), 7.65 (d, 1H, J = 1.4),
7.71 (s, 1H), 8.40 (d, 1H, J = 8.1), 8.72 (d, 1H, J = 8.1), 8.79 (s, 1H),
9.45 (s, 1H), 10.12 (br s, 1H), 11.48 (br s, 1H); HRMS m/e calcd
for C28H24N4O6 512.170; found, 512.169.
In a similar set up to that for 73, succinimide ester 72 (435 mg,
1 mmol), N-benzylpiperazine (176 mg, ꢂ 0.18 mL, 1 mmol) were
added to 5 mL chloroform, absolute ethanol (3 mL), and 0.14 ml
(1 mmol) of triethylamine and was stirred at rt for 48 h. The mix-
ture was evaporated and the solid was dissolved in 90 mL dichlo-
romethane and the solution was washed with 30 mL of water
followed by 2 ꢁ 30 mL of 10% citric acid. The aqueous layer was ex-
tracted with 2 ꢁ 10 mL dichloromethane and the combined organ-
ic extracts were washed with 30 mL of 1 M sodium bicarbonate
solution, followed by 5 mL of water, dried (MgSO4) and evaporated
to dryness to give a gel. This material was further dried under a
vacuum pump at 60 °C overnight to produce the white solid of
75 (455 mg, 92%): mp = 134–136 °C; 1H NMR (CDCl3) d 1.32 (m,
4H), 2.01 (m, 4H), 3.16 (m, 2H), 3.30 (s, 2H), 4.96 (m, 1H), 5.11
(s, 2H), 5.76 (m, 1H), 7.25 (m, 14H) 7.64 (d, 1H, J = 7.7), 8.08 (s,
1H); HRMS m/e calcd for C30H32N4O3, 496.247; found, 496.247.
4.1.20. 7-N-Acetyl-110-hydroxylavendamycin methyl ester (42)
In a set up similar to that for 41, in a 500 mL flask, aldehyde 17
(180.7 mg, 0.58 mmol), 5-hydroxy-b-methyltryptophan methyl es-
ter (28, 198.8 mg, 0.8 mmol) in 390 mL of anhydrous anisole were
slowly heated to 162 °C over a period of 3 h and then allowed to
continue at this temperature for 26 h. In the last 2 h of the reaction,
argon flow was replaced by an oxygen flow. The reaction mixture
was evaporated to give a dark red solid (201 mg, 59%): mp 180–
181 °C (dec); Rf = 0.49 (0.4:5 MeOH/CH2Cl2); 1H NMR (DMSO-d6)
d 2.30 (s, 3H), 2.96 (s, 3H), 3.94 (s, 3H), 7.16 (d, 1H, J = 8.8), 7.38
(d, 1H, J = 8.8), 7.61 (br s, 1H), 7.71 (s, 1H), 8.36 (d, 1H, J = 8.2),
8.69 (d, 1H, J = 8.3), 9.42 (s, 1H), 9.42 (s, 1H), 10.12 (s, 1H), 11.56
(br s, 1H); HRMS m/e calcd for C25H18N4O6, 470.122; found,
470.121.
4.1.16. DL-Tryptophan n-propyl ester (33)
Using a similar procedure to that for ester 27, ester 33 was
obtained in 88% yield by the reaction of n-propyl alcohol with
DL-tryptophan in the presence of anhydrous HCl in ether as a thick
honey colored paste. TLC showed the product to be pure: Rf = 0.58
(3:2 acetone/EtOAc); 1H NMR (CDCl3) d 0.90 (m, 3H), 1.6 (s, 2H),
1.75 (m, 2H), 3.04 (m, 1H), 3.27 (m, 1H), 3.83 (m, 1H), 4.05 (m,
2H), 7.06 (s, 1H), 7.12 (m, 1H), 7.20 (m, 1H), 7.35 (d, 1H, J = 8.1),
7.63 (d, 1H, J = 8.1), 8.30 (br s, 1H).
4.1.17.
L-Tryptophan sec-butyl ester (34)
L-Tryptophan (1 g, 4.9 mmol) and sec-butyl alcohol (60 mL)
were placed in a 100 mL round-bottomed flask equipped with a
magnetic stirring bar, a condenser and an oil bubbler. The flask
was cooled in an ice-bath for 10 min and then 12 mL of an anhy-
drous HCl–ether solution was added via a syringe through a rubber
septum at the top of the condenser. The flask was removed from
the ice-bath, placed in an oil bath and then refluxed for 22 h. Three
drops of concd sulfuric acid was added and the solution was re-
fluxed for an additional 2 h. When thin layer chromatography indi-
cated the completion of the reaction, the mixture was allowed to
cool to rt to provide a solid residue. Dichloromethane (50 mL)
was added to the residue, then placed in a separatory funnel and
treated with a 5% solution of sodium bicarbonate until pH ꢂ8.
4.1.21. 7-N-Demethyllavendamycin 2-hydroxyethanamide (44)
Using a similar set up as that for 41, in a 500 mL flask, trypto-
phan (2-hydroxyethan)amide (30, 125.8 mg, 0.5 mmol) was dis-
solved in
a mixture of 10 mL anhydrous DMF and 220 mL
anisole, heated to 84 °C and to this, 122 mg (0.5 mmol) of 7-acet-
amido-2-formylquinoline-5,8-dione (17) was added. The reaction
mixture was gradually heated to 150–155 °C over 3 h and kept at
this temperature for another 3 h. The mixture was evaporated,
the solid was washed consecutively with 5 mL portions of acetone,
ether and finally petroleum ether to give 131 mg of a product. NMR

W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
1907
showed that this product contained some of the 5,8-dihydroxy
4.1.25. 7-N-Acetyldemethyllavendmycin N-benzylpiperazine
amide (49)
derivative that was converted to 44 by treating with 63.65 mg of
DDQ in 23 mL THF and allowed to stir at rt for 20.5 h. The solid
was consecutively washed with 3 mL portions of THF, acetone
and petroleum ether yielding 124 mg (53%) of 44 as an orange so-
lid: mp >280 °C; Rf = 0.47 (0.2:5 MeOH/CH2Cl2); 1H NMR (DMSO-
d6) d 2.32 (s, 3H), 3.53 (m, 2H), 3.62 (m, 2H), 4.86 (m, 1H), 7.42
(dd, 1H, J = 7.0, 7.0), 7.67 (m, 2H), 7.84 (s, 1H), 8.54 (m, 2H), 9.07
(s, 1H), 9.09 (m, 1H), 9.42 (d, 1H, J = 8.4), 10.32 (br s, 1H), 11.98
(br s, 1H); HRMS m/e calcd for C25H19N5O5, 469.138; found,
469.137.
In a set up similar to that for 41, in a 100 mL flask, aldehyde 17
(122 mg, 0.5 mmol) in 40 mL anhydrous anisole was stirred and
heated to 70 °C until it was dissolved. A solution of N-benzylpiper-
azine amide of tryptophan (32, 180 mg, 0.5 mmol) in 5 mL dried
and freshly distilled pyridine was introduced to the reaction flask
using a syringe. The reaction mixture was heated to reflux
(30 min) and then maintained at this temperature for 6 h. The mix-
ture was allowed to cool to rt, the yellow lemon precipitate was
filtered off and the filter cake was washed with THF (3 mL), fol-
lowed by petroleum ether (3 mL). The solid was vacuum dried,
suspended in 10 mL THF and then treated with DDQ (94 mg,
0.41 mmol), stirred at rt for 24 h in order to oxidize any 5,8-dihy-
droxy lavendamycin present in the mixture to 49. The brown
reaction mixture was filtered off, the filter cake washed with THF
(3 mL) followed by petroleum ether (3 mL) and dried on a vacuum
pump giving 115 mg (40%): mp >260 °C; Rf = 0.88 (1:7 MeOH/
CH2Cl2); 1H NMR (DMSO-d6) d 2.32 (s, 3H), 3.30 (m, 4H), 3.57 (s,
2H), 3.76 (m, 4H), 7.32 (m, 6H), 7.70 (m, 2H), 7.81 (s, 1H), 8.47
(d, 1H, J = 7.7), 8.55 (d, 1H, J = 8.2), 8.70 (s, 1H), 8.86 (d, 1H,
J = 8.2), 10.30 (br s, 1H), 11.85 (br s, 1H); HRMS m/e calcd for
C34H28N6O4; 584.217; found, 584.219.
4.1.22. N-Acetyldemethyllavendamycin sec-butyl ester (46)
In a set up similar to that for 41, anhydrous and freshly distilled
xylene (145 mL) was placed in the flask and to this, 7-acetamido-2-
formylquinoline-5,8-dione (17, 0.2401 g, 1 mmol) was added and
heated to 100 °C. To the mixture, a solution of L-tryptophan sec-bu-
tyl ester (34, 0.2568 g, 1 mmol) in 12 mL of anhydrous and freshly
distilled xylene under argon was added via a syringe and then
heated for 16 h at 130 °C until the reaction was completed (TLC).
The mixture was filtered off to produce a greenish solid and the fil-
trate was kept in the refrigerator overnight to give more product
(total, 0.2785 g, 57%). An analytical sample of 46 was obtained by
recrystallization with DMSO: mp = 268 °C; Rf = 0.46 (1.7:10
MeOH/CH2Cl2); 1H NMR (CDCl3) d 1.03 (t, 3H, J = 7.7), 1.42 (d, 3H,
J = 6.2), 1.8 (m, 1H), 2.30 (s, 3H), 5.2 (m, 1H), 7.34 (t, 1H, J = 8.1),
7.62 (t, 1H, J = 8.1), 7.69 (d, 1H, J = 8.4), 7.93 (m, 2H), 8.21 (d, 1H,
J = 7.7), 8.35 (br s, 1H), 8.52 (d, 1H, J = 8.4), 8.88 (s, 1H), 9.17 (d,
1H, J = 8.4), 11.23 (br s, 1H); HRMS m/e cacld for C27H25N4O5 (M
+ 3H)+, 485.182; found, 485.183.
4.1.26. 7-N-Acetyl-110-hydroxydemethyllavendamycin ethyl
ester (51)
Using a similar set up to that for 41, in a 100 mL flask, 5-
hydroxytryptophan ethyl ester (36, 29.76 mg, 0.12 mmol) in 4 mL
of DMF was added to 40 mL anhydrous anisole and then with
29.8 mg (0.12 mmol) of 7-acetamido-2-formylquinoline-5,8-
dione9,10 (17) was gradually heated to 160 °C over 3 h and kept
at this temperature for 23.5 h. The orange solution was evaporated
to give a dark solid. The crude product was washed with 3 mL ace-
tone and dried under vacuum producing the dark violet 51 (27 mg,
48%): mp >280 °C; Rf = 0.69 (0.2:5 MeOH/ CH2Cl2); 1H NMR
(DMSO-d6) d 1.44 (m, 3H), 2.30 (s, 3H), 4.40 (m, 2H), 7.17 (m,
1H), 7.40 (m, 1H), 7.67 (br s, 1H), 7.74 (s, 1H), 8.44 (d, 1H,
J = 8.4), 8.78 (d, 1H, J = 8.4), 8.84 (s, 1H), 9.45 (s, 1H), 10.15 (br s,
1H), 11.53 (br s, 1H); HRMS m/e calcd for C25H18N4O6, 470.122;
found, 470.121.
4.1.23. 7-N-Acetyldemethyllavendamycin 2,3-dihydroxypropan-
amide (47)
In a set up similar to that for 41, tryptophan 2,3-dihydroxy-
propanamide (31, 114.8 mg, 0.44 mmol) was dissolved in 40 mL
anhydrous DMF and 240 mL anhydrous anisole and heated to
80 °C in a 500 mL flask. To this, 7-acetamido-2-formylquino-
line-5,8-dione (17, 97.6 mg, 0.44 mmol) was added and the mix-
ture was gradually heated to 150–154 °C over a period of 3 h,
and then continued at this temperature for five additional hours.
The reaction mixture was concentrated to a small volume pro-
ducing 47 as orange crystals. The crystals were washed with
ethyl ether and dried (104.2 mg, 52%): mp 214–215 °C (dec);
Rf = 0.4 (0.3:5 MeOH/CH2Cl2); 1H NMR (DMSO-d6) d 2.30 (s,
3H), 3.37 (m, 1H), 3.45 (m, 2H), 3.60 (m, 1H), 4.75 (m, 1H),
5.00 (m, 1H), 7.45 (m, 1H), 7.74 (m, 2H), 7.84 (s, 1H), 8.55
(m, 2H), 9.07 (s, 1H), 9.12 (br s, 1H), 9.34 (d, 1H, J = 8.4),
11.97 (br s, 1H); HRMS m/e calcd for C26H21N5O6, 499.149;
found, 499.147.
4.1.27. 7-N-Formyldemethyllavendamycin methyl ester (53)
In a similar set up as that for 41, 7-formamido-2-formylquino-
line-5,8-dione (20, 50 mg, 0.22 mmol), and tryptophan methyl es-
ter (46 mg, 0.21 mmol) were dissolved in 125 mL of anhydrous
anisole in a 250 mL flask, heated to 130 °C over an hour and contin-
ued for another 1.5 h. Anisole was evaporated and the solid residue
was washed with 5 mL of acetone. The brownish solid was filtered
off and washed with a few mL acetone and dried. The product 53
weighed 18.45 mg (19.6%): mp >300 °C; Rf = 0.79 (0.2:2.3 MeOH/
EtOAC/CH2Cl2); 1H NMR (CDCl3) d 4.1 (s, 3H), 7.42 (m, 1H), 7.69
(m, 1H), 7.78 (d, 1H, J = 8.1), 8.02 (s, 1H), 8.26 (d, 1H, J = 8.1),
8.53 (br s, 1H), 8.61 (d, 1H, J = 8.4), 8.76 (br s, 1H), 9.02 (s, 1H),
9.26 (d, 1H, J = 8.4), 11.85 (br s, 1H); HRMS m/e calcd for
C23H14N4O5, 426.099; found, 426.096.
4.1.24. 7-N-Acetyldemethyllavendamycin n-propyl ester (48)
In a set up similar to that of 41, in a 500 mL flask, 7-acetamido-
2-formylquinoline-5,8-dione (17, 149 mg, 0.61 mmol), tryptophan
n-propyl ester (33, 162 mg, 0.66 mmol) in 90 mL anhydrous and
distilled xylene were heated to 100 °C over 1 h and then allowed
to reflux for 3 h. While hot, some solid impurities were filtered
and the filtrate was evaporated to a small volume and allowed to
cool giving an orange red solid. The product was dried on a vacuum
pump, producing 80 mg (30%) of 48: mp 265 °C (dec): Rf = 0.48
(0.1:5 MeOH/CH2Cl2); 1H NMR (CDCl3) d 1.13 (t, 3H, J = 7.3), 1.94
(m, 2H), 2.36 (s, 3H), 4.46 (t, 2H, J = 7.4), 7.40 (t, 1H, J = 8.1), 7.67
(t, 1H, J = 6.9), 7.75 (d, 1H, J = 8.0), 8.00 (s, 1H), 8.26 (d, 1H,
J = 7.6), 8.44 (s, 1H), 8.58 (d, 1H, J = 8.4), 8.97 (s, 1H), 9.22 (d, 1H,
J = 7.1), 11.83 (br s, 1H); HRMS m/e calcd for C26H20N4O5;
468.143; found, 468.144.
4.1.28. 7-N-Chloroacetyldemethyllavendamycin amide (54)
In a set up similar to that of 53, 7-N-chloroacetyl-2-formylquin-
oline-5,8-dione15 (21, 82.7 mg, 0.3 mmol), tryptophan amide13 (39,
61 mg, 0.3 mmol), and anhydrous anisole (120 mL) were mixed,
stirred and heated to 155 °C over a period of 3 h. Upon the comple-
tion of the reaction after 20 h, the mixture was allowed to cool to
room temperature and the yellow solid was filtered off. The prod-
uct was washed with petroleum ether and dried on a vacuum
pump overnight to give 68 mg (49%) of pure 54: mp >280 °C;
Rf = 0.35 (0.25:5 MeOH/CH2Cl2). 1H NMR (DMSO-d6) d 4.55 (s,

1908
W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
2H), 7.39 (t, 1H, J = 8.4), 7.70 (t, 1H, J = 8.4), 7.82 (d, 1H, J = 8.0), 8.49
(s, 1H), 8.49 (d, 1H, J = 8.1), 8.61 (d, 1H, J = 8.8), 9.02 (s, 1H), 9.12 (d,
1H, J = 9.2), 9.75 (s, 1H), 9.93 (s, 1H), 10.19 (s, 1H), 12.26 (br s, 1H);
HRMS m/e calcd for C23H14ClN5O4, 462.097; found, 462.097.
CH2Cl2); 1H NMR CDCl3) d 1.57 (m, 3H), 4.57 (q, 2H, J = 7.2), 5.86
(br s, 2H), 7.42 (m, 1H), 7.68 (m, 1H), 7.77 (d, 1H, J = 7.3), 8.27 (d,
1H, J = 7.8), 8.67 (d, 1H, J = 8.2), 9.00 (s, 1H), 9.22 (d, 1H, J = 8.3),
11.86 (br s, 1H); HRMS m/e calcd for C23H16ClN4O4 (MH+),
447.086; found, 447.086.
4.1.29. 7-N-Chloroacetyldemethyllavendamycin pyrrolidine
amide (55)
4.1.33. Demethyllavendamycin morpholine amide (59)
In a similar set up to that of 53, quinolinedione aldehyde 21
In a 25 mL two-necked round-bottomed flask, equipped with a
magnetic stirring bar, a condenser and an argon filled balloon, 7-N-
acetyldemethyllavendamycin morpholine amide15 (40, 163 mg,
0.33 mmol) was placed and kept for a few min in an ice-bath. To
this 15.8 mL of 70% sulfuric acid was dropwise added until the so-
lid was dissolved. The mixture was heated at 61 °C for 6 h. To the
reaction mixture, 10 mL water was added and neutralized to a
pH of 8.5 with a saturated solution of sodium carbonate. The mix-
ture was evaporated to give a solid material, that was finely ground
by mortar and pestle and extracted with 160 mL of chloroform in a
Soxhlet extractor overnight. The resulting solution was evaporated
and dried to give 91 mg (63%) of the red orange 59: mp 275–
276 °C; Rf = 0.39 (0.01:5 MeOH/CH2Cl2); 1H NMR (DMSO-d6) d
3.74 (m, 8H), 5.95 (s, 1H), 7.39 (t, 1H, J = 8.1), 7.48 (br s, 2H),
7.70 (m, 2H), 8.47 (d, 1H, J = 8.1), 8.49 (t, 1H, J = 8.1), 8.71 (s, 1H),
8.84 (d, 1H, J = 8.4) 11.87 (br s, 1H). HRMS m/e calcd for
C25H20N5O4 (MH+), 454.154; found, 454.151.
(42.5 mg, 0.15 mmol), tryptophan pyrrolidine amide15 (29, 39 mg,
0.15 mmol) and 55 mL anhydrous anisole were mixed in
a
100 mL flask. The mixture was gradually heated to 155 °C over a
period of 3 h and then heated at this temperature for an additional
18 h. The reaction mixture was filtered off hot and the filter cake
was washed with 4 mL of dichloromethane followed by 4 mL of
chloroform. The filtrate was evaporated, the crude product was
introduced on a small column of silica gel and eluted with ethyl
acetate to produce 11 mg (21%) of pure 55 as a yellow solid: mp
264 °C (dec); Rf = 0.77 (0.25:5 MeOH/CH2Cl2). 1H NMR (DMSO-d6)
d 1.96 (m, 4H), 3.64 (m, 2H), 3.98 (m, 2H), 4.66 (s, 2H), 7.41 (t,
1H, J = 8.1), 7.71 (m, 2H), 7.81 (s, 1H), 8.48 (d, 1H, J =8.1), 8.57 (d,
1H, J = 8.1), 8.88 (s, 1H), 8.94 (d, 1H, J = 8.4), 10.65 (s, 1H), 11.87
(br s, 1H); HRMS m/e calcd for C27H20ClN5O4, 513.124; found,
513.120.
4.1.30. 7-N-(2-Furyl)carbonyllavendamycin methyl ester (56)
Using a similar set up to that of 53, in a 50 mL flask, a mixture
of 7-furoylamino-2-formylquinoline-5,8-dione16 (22, 29.7 mg,
0.10 mmol), b-methyltryptophan methyl ester31 (37, 23.1 mg,
0.10 mmol) and 32 mL freshly distilled anhydrous xylene was
gradually heated to 145 °C over a 3 h period. The heat was continued
for an additional 19 h and then the mixture was evaporated to
dryness. The dark orange solid was washed with a small amount of
chloroform producing the light orange solid 56 (30.11 mg, 60%):
mp 327–328 °C; Rf = 4.1 (0.1:5 MeOH/CH2Cl2); 1H NMR (CDCl3) d
3.21 (s, 3H), 4.09 (s, 3H), 6.68 (m, 1H), 7.41 (m, 2H),7.68 (m, 2H),
7.82 (m, 1H), 8.06 (m, 1H), 8.36 (m, 1H), 8.53 (d, 1H, J = 8.4), 9.12
(d, 1H, J = 8.4), 9.36 (s, 1H), 11.92 (br s, 1H). HRMS m/e calcd for
C28H18N4O6, 506.122; found, 506.122.
4.1.34. 110-Hydroxydemethyllavendamycin isoamyl ester (60)
In a procedure similar to that of 59, 7-N-acetyldemethyllaven-
damycin isoamyl ester (41, 25.1 mg, 0.05 mmol) and 2.4 mL of sul-
furic acid (70%) was heated at 60 °C for 3.5 h. The reaction mixture
was carefully neutralized with a saturated solution of sodium
bicarbonate to pH 8, and then extracted with chloroform
(4 ꢁ 100 mL). The organic extracts were washed with water fol-
lowed by brine and then dried (MgSO4). Evaporation of the solvent
produced 16 mg (70%) of the orange product 60: mp > 280 °C;
Rf = 0.23 (0.2:5 MeOH/CH2Cl2); 1H NMR (DMSO-d6) d 1.01(d, 6H,
J = 6.2), 1.74 (m, 3H), 4.43 (m, 2H), 5.95 (s, 1H), 7.25 (d, 1H,
J = 8.8), 7.61 (d, 1H, J = 8.8), 7.78 (br s, 1H), 8.54 (d, 1H, J = 8.4),
8.90 (d, 1H, J = 8.4), 8.97 (s, 1H), 9.46 (s, 1H), 11.82 (br s, 1H); HRMS
m/e calcd for C26H22N4O5, 471.167; found, 471.169.
4.1.31. 7-N-(3-Carboxypropionyl)demethyllavendamycin n-butyl
ester (57)
4.1.35. 110-Hydroxylavendamycin methyl ester (61)
Using the same set up as that of 53, 60 mL of anhydrous anisole
was placed in a 100 mL flask and then tryptophan n-butyl ester13
(38, 39.5 mg, 0.15 mmol) was added followed by 40 mg
(0.13 mmol) of 7-N-(3-carboxypropionyl)-2-formylquinoline-5,8-
dione (23). With stirring, this mixture was heated at 120 °C for
19.5 h. The reaction mixture was filtered to remove the solid impu-
rity and the filtrate was evaporated. The amorphous solid was
recrystallized from 10 mL ethyl acetate to give 22 mg (29%) of 57
as a red crystalline solid: mp 232.4 °C (dec); Rf = 0.83 (2.5:3.5
MeOH/CHCl3). 1H NMR (DMSO-d6) d 1.01 (t, 3H, J = 7.3), 1.54 (m,
2H), 1.83 (m, 2H), 2.50 (m, 2H), 2.90 (m, 2H), 4.50 (m, 2H), 7.50
(m, 1H), 7.70 (m, 2H), 7.80 (s, 1H), 8.54 (d, 1H, J = 8.0), 8.59 (d,
1H, J = 8.4), 8.93 (d, 1H, J = 8.4), 9.09 (s, 1H), 10.35 (br s, 1H),
11.97 (br s, 1H); HRMS m/e calcd for C29H25N4O7 (MH+), 541.172;
found, 541.172.
In a similar set up and procedure to that of 59, acetyllavenda-
mycin ester 42 (98.4 mg, 0.21 mmol) was slowly added to 15 mL
of a 70% solution of sulfuric acid at 0 °C and then heated at 60 °C
for 3 h. The reaction mixture was treated with a saturated solution
of sodium carbonate to pH 8 and extracted with 8 ꢁ 75 mL of ethyl
acetate, dried and evaporated to give 61 as red crystals. The aque-
ous layer was evaporated to dryness, the solid was ground and ex-
tracted with 150 mL ethyl acetate for 24 h in a Soxhelet extractor.
Evaporation of the solution afforded more of 61 (total weight
33.7 mg, 38%): mp >280 °C; Rf = 0.72 (0.4:5 MeOH/CH2Cl2); 1H
NMR (DMSO-d6) d 2.85 (s, 3H), 3.90 (s, 3H), 5.85 (s, 1H), 7.06 (d,
1H, J = 8.8), 7.30 (d, 1H, J = 8.8), 7.41 (br s, 2H), 7.49 (s, 1H), 8.23
(d, 1H, J = 8.1), 8.50 (d, 1H, J = 8.1), 9.30 (s, 1H), 11.36 (s, 1H); HRMS
m/e calcd for C23H16N4O5, 428.112; found, 428.113.
4.1.36. Demethyllavendamycin pyrrolidine amide (62)
In a similar procedure to that for 59, 51 mg (0.11 mmol) of 7-N-
acetyldemethyllavendamycin pyrrolidine amide15 (43), was hydro-
lyzed with 5.2 mL of 70% sulfuric acid to give 24 mg (50%) of the
orange red product 62: mp 300 °C; Rf = 0.22 (0.1:5 MeOH/CH2Cl2);
1H NMR (DMSO-d6) d 1.92 (m, 4H), 3.63 (m, 2H), 3.98 (m, 2H), 5.95
(s, 1H), 7.39 (m, 2H), 7.77 (m, 3H), 8.45 (m, 2H), 8.84 (m, 2H), 11.89
(br s, 1H); HRMS m/e calcd for C25H20N5O3 (MH+) 438.156; found
438.156.
4.1.32. 6-Chlorodemethyllavenamycin ethyl ester (58)
In a 500 mL flask with similar set up to that of 53, a mixture
of 7-amino-6-chloro-2-formylquinoline-5,8-dione (24, 47.4 mg.
0.2 mmol) and tryptophan ethyl ester17 (35, 46.4 mg, 0.2 mmol)
and 120 mL anhydrous anisole was slowly heated to 130 °C over
3 h. The reaction mixture was refluxed for 1 h and then allowed
to cool to rt. The solvent was evaporated to dryness, the solid
washed with acetone (20 mL), filtered and dried under vacuum
giving 47 mg (49%) of 58: mp >270 °C; Rf = 0.26 (0.1:5 MeOH/

W. Cai et al. / Bioorg. Med. Chem. 18 (2010) 1899–1909
1909
4.1.37. Demethyllavendamycin 2-hydroxyethanamide (63)
Lavendamycin 44 (70.5 mg, 0.15 mmol) was added slowly to
7.2 mL of 70% sulfuric acid and heated at 60 °C for 3.5 h. The mix-
ture was treated with a saturated solution of sodium carbonate to
pH 8 and evaporated to dryness. The residue was extracted with
70 mL of methanol/dichloromethane (10:60) and then with
35 mL of the same mixture. The extracts were concentrated to a
small volume and the red crystals of 63 were filtered off
(18.9 mg, 30%): mp >280 °C; Rf = 0.31 (0.3:5 MeOH/CH2Cl2); 1H
NMR (DMSO-d6) d 3.83 (m, 2H), 4.40 (m, 2H), 5.02 (br s, 2H),
5.96 (s, 1H), 7.40 (m, 1H), 7.73 (m, 2H), 7.78 (d, 1H, J = 8.0), 8.5
(d, 1H, J = 8.0), 8.85 (d, 1H, J = 8.1), 8.92 (d, 1H, J = 8.1), 9.12 (s,
1H), 12.01 (br s, 1H); HRMS m/e calcd for C23H17N5O4, 427.128;
found, 427.130.
humidified atmosphere containing 5% CO2 for 3–5 days. MTT
(50 g) was added and the cells were incubated for another 4 h.
Medium/MTT solutions were removed carefully by aspiration, the
MTT formazan crystals were dissolved in 100 L DMSO, and absor-
bance was determined on a plate reader at 560 nm. IC50 values
(concentration at which cell survival equals 50% of control) were
determined from semi-log plots of percent of control versus con-
centration. Selectivity ratios were defined as the IC50 value for
the BE cell line divided by the IC50 value for the BE-NQ cell line.
l
l
Acknowledgments
The financial support by the National Institutes of Health is
greatly appreciated (NIH Grants: R15CA74245, M.B.; R15CA78232
and P20RR017670, H.D.B.). We also thank professor David Wil-
liams and the staff at the mass spectroscopy laboratory at Indiana
University for their help in obtaining mass spectral data.
4.1.38. Demethyllavendamycin methyl ester (64)
Using a similar set up as that for 59, 7-N-butyryldemethyllaven-
damycin methyl ester17 (45, 22 mg, 0.05 mmol) was slowly added
to 2 mL of 70% sulfuric acid in a 5 mL flask with stirring. The mix-
ture was heated at 60 °C for 4 h, then carefully neutralized with a
saturated solution of sodium carbonate to pH 8 and extracted with
chloroform (2 ꢁ 100 mL and 50 mL). The extracts were evaporated
to give 64 as a red solid (17 mg, 85%): mp 220 °C (dec); Rf = 0.35
(0.1:5 MeOH/CH2Cl2); 1H NMR (CDCl3) d 4.00 (s, 3H), 5.35 (br s,
2H), 6.15 (s, 1H), 7.41 (m, 1H), 7.70 (m, 2H), 7.81 (d, 1H, J = 8.0),
8.27 (d, 1H, J = 8.0), 8.60 (d, 1H, J = 8.3), 9.20 (d, 1H, J = 8.3), 12.00
(br s, 1H); HRMS m/e calcd for C22H14N4O4, 398.101; found,
398.099.
References and notes
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4.2. Biology
4.2.1. Cell culture
BE human colon adenocarcinoma cells and stably NQO1-trans-
fected BE-NQ cells32 were a gift from Dr. David Ross (University
of Colorado-Denver, Denver, CO). Cells were grown in a minimum
essential medium (MEM) with Earle’s salts, non-essential amino
acids, L-glutamine and penicillin/streptomycin, and supplemented
with 10% fetal bovine serum (FBS), sodium bicarbonate and HEPES.
Cell culture medium and supplements were obtained from Gibco,
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37 °C under a humidified atmosphere containing 5% CO2.
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4.2.2. Spectrophotometric cytochrome c assay
Lavendamycins analogue reduction was monitored using a
spectrophotometric assay in which the rate of reduction of cyto-
chrome c was quantified at 550 nm. Briefly, the assay mixture con-
tained cytochrome c (70
NQO1 (0.1–3 g) (gift from Dr. David Ross, University of Colorado-
Denver, Denver, CO) and lavendamycins (25 M) in a final volume
lM), NADH (1 mM), recombinant human
l
l
of 1 mL Tris–HCl (25 mM, pH 7.4) containing 0.7 mg/mL BSA and
0.1% Tween-20. Reactions were carried out at room temperature
and started by the addition of NADH. Rates of reduction were cal-
culated from the initial linear part of the reaction curve (0–30 s)
and results were expressed in terms of
lmol of cytochrome c re-
duced/min/mg of NQO1 using a molar extinction coefficient of
21.1 mMꢀ1 cmꢀ1 for cytochrome c. All reactions were carried out
at least in triplicate.
29. Swann, E.; Barraja, P.; Oberlander, A. M.; Gardipee, W. T.; Hudnott, A. R.; Beall,
H. D.; Moody, C. J. J. Med. Chem. 2001, 44, 3311.
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4.2.3. Cell viability assay
Growth inhibition was determined using the MTT colorimetric
assay. Cells were plated in 96-well plates at
10,000 cells/mL and allowed to attach overnight (16 h). Lavenda-
mycin analogue solutions were applied in medium for 2 h. Laven-
damycin analogues solutions were removed and replaced with
fresh medium, and the plates were incubated at 37 °C under a
a density of