Design of quinolinedione-based geldanamycin analogues

Article abstract of DOI:10.1016/S0960-894X(03)00650-4

Quinoline-5,8-dione-based compounds were designed from the structure of the geldanamycin-bound Hsp-90 active site. The active site model predicted that aromatic substituents should be present at the 2-position, to take advantage of a hydrophobic pocket, a

Full text of DOI:10.1016/S0960-894X(03)00650-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3075–3078
Design of Quinolinedione-Based Geldanamycin Analogues
Robert Hargreaves,a Cynthia L. David,b Luke Whitesellb and Edward B. Skiboa,*
aDepartment of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA
bSteele Memorial Children’s Research Center, University of Arizona, Tucson, AZ 85724, USA
Received 1April 2003; accepted 29 April 2003
Abstract—Quinoline-5,8-dione-based compounds were designed from the structure of the geldanamycin-bound Hsp-90 active site.
The active site model predicted that aromatic substituents should be present at the 2-position, to take advantage of a hydrophobic
pocket, and amino substituents should be present at the 6- or 7-position. COMPARE analysis revealed that the LC50 profile of 2-
phenyl-6-(2-chloroethylamino)quinoline-5,8-dione has the highest geldanamycin-like activity (0.74 correlation coefficient).
# 2003 Elsevier Ltd. All rights reserved.
The term, ‘heat shock protein,’ comes from the initial
observation that this chaperone protein is elevated in
the presence of heat stress. Actually, any stress can
cause Hsp-90 elevation so as to process signaling pro-
teins important for cell division. The cancer cell is in
fact under a great deal of stress due to hypoxia, nutrient
deprivation, chemotherapy, and radiation. Thus, var-
ious histological cancers types in fact possess elevated
levels of Hsp-90 including leukemias, lymphomas, as
well as androgen- and estrogen-dependent cancers
(prostrate and breast).1À3 Heat shock protein (Hsp-90)
is a relatively new target for cancer chemotherpy and
therefore has been the subject of intense drug design
efforts.1,4,5 The prototype inhibitor of Hsp-90 is gelda-
namycin shown in the inset of Figure 1. This complex
natural product has been the subject of total synthesis6,7
and analogue development.8 In addition, the synthesis
and screening of purine libraries has afforded small
molecules capable of geldanamycin-like binding to
HSP-90.9À11 This laboratory approached HSP-90 inhi-
bitor design by molecular modeling and library syn-
thesis. The quinoline quinone analogue shown in Figure
1 was designed from the HSP-90 crystal structure.12
substituted 5,8-dimethoxyquinolines, to produce sub-
stituted quinoline-5,8-diones, was effected in high yields
with ceric ammonium nitrate.14 Reaction of the quino-
line-5,8-diones with aziridine in ethanol15 produced
both 6- and 7-aziridinylquinoline-5,8-diones, with the 6-
aziridinyl isomer being produced in much higher yield.16
Treatment of the quinoline-5,8-diones with aziridine for
long periods of time (several hours) could result in dia-
ziridination based of previous reports.17
The proton NMRs of the 6- and 7-aziridinyl isomers
were indistinguishable and the isomers had to be iden-
tified by HMBC and HMQC NMR experiments. The 6-
aziridinyl-isomer showed a HMBC 3-bond correlation
between both the 4- and 7-protons and the 5-carbon.
The 7-aziridinyl-isomer showed a HMBC 3-bond corre-
lation between the 4-proton and the 5-carbon and the 6-
proton and the 8-carbon. Therefore, the 6- and 7-iso-
mers can be distinguished by HMBC 3-bond correlation
The preparation of the target compounds was initiated
with the Friedlander quinoline synthesis, Scheme 1. Thus,
the reaction of 2-amino-3,6-dimethoxybenzaldehyde13
with the appropriate ketones afforded substituted-5,8-
dimethoxyquinolines. Oxidative demethylation of the
Figure 1. The colors shown in the quinoline structure and the corre-
sponding colors in the geldanamycin structure reflect overlapping
binding sites in the active site of Hsp-90.
*Corresponding author. Tel.: +1-480-965-3581; fax: +1-480-965-
2747; e-mail: eskibo@asu.edu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00650-4

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R. Hargreaves et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3075–3078
Cancer Institute.18,19 The cytostatic parameters shown
in Table 2 include GI50 and TGI, which are the con-
centrations of drug required for 50% growth inhibition
and total growth inhibition, respectively. The cytotoxic
parameter is the LC50, which is the concentration required
for 50% cell kill. The log mean values for GI50, TGI,
and LC50 in 60 cell lines are provided in Table 2 along
with the log delta value (the maximum sensitivity in
excess of the mean) and the log range (the maximum
difference between the least sensitive and the most sen-
sitive cell lines). These parameters have provided
insights into selectivity and potency of antitumor agents
in previous studies.20 Large values of the delta and
range indicate high selectivity for some histological
cancers over others. Small negative median values (À5
to À6) along with small delta and range values indicate
an inactive compound.
Scheme 1. Preparation of aziridinated quinolinediones.
between the quinone proton of the product and either
one carbon (6-isomer) or two carbons (7-isomer).
The results shown in Table 2 led to the following
requirements for optimal activity: (1) Groups no bigger
than phenyl substituted at the 2-position. (2) Minimized
structures must have the 2-substituent planar with the
quinoline ring. (3) The aziridinyl group should be pres-
ent at the 6-position for optimal cytostatic activity. The
first generalization was made based on comparing the
cytostatic/cytotoxic parameters of 1–2 with those of 4–
5. The increase in size from phenyl to naphthyl results in
an increase in the median log GI50 and TGI parameters
(i.e., a decrease in potency). A decrease in cytostatic and
cytotoxic activity accompanies the replacement of the
phenyl of 1 with a 2-pyridyl substituent of 3. A mini-
mized structure of 3 revealed that repulsion between the
nitrogen lone pairs twists the 2-pyridyl ring out of the
plane of the quinazoline ring. In contrast, the phenyl
group of 1 is coplanar with the quinolinedione ring.
Finally, the data in Table 2 show that the 6-aziridinyl
analogues are consistently more active than 7-aziridinyl
analogues.
The aziridinyl group was present as a possible alkylating
center of the Hsp-90 active site as well as a starting
point for other analogues that could be obtained by ring
opening with a variety of nucleophiles. Currently,
libraries of a variety of 6- and 7-substituted quinoline-
dienes are being prepared and screened. In this report
the results for two aziridinyl ring opened analogues, 6-
(2-hydroxyethyl and 6-(2-chloroethyl), are presented. A
list of compounds discussed in this communication are
found in Table 1.
Cytotoxic and Cytostatic Evaluation
Provided in Table 2 are the cytostatic/cytotoxic para-
meters and cancer specificity for the compounds in
Table 1. These in vitro data were obtained under the
In Vitro Cell Line Screening Project at the National
Table 1. Quinolinedione analogues prepared and screened in this
study
The aziridinyl ring can undergo acid-catalyzed ring
opening utilizing either water or chloride present in the
buffered solution as nucleophiles.21 When 1 was
screened again ꢀ6 months later by the National Cancer
Institute, its cytostatic/cytotoxic parameters changed
substantially (new parameters listed under 7 in Table 2).
One notable change was the high selectivity toward leu-
kemia cell lines with cytostatic/cytotoxic parameters in
the range of <10À8 M. This observation prompted
screening of the ring opening of the aziridinyl ring
opened analogues 6 and 7. The absence of activity
Position
Compd
2
3
6
7
Phenyl
Phenyl
2-Pyridyl
2-Naphthyl
2-Naphthyl
Phenyl
H
H
H
H
H
H
H
Aziridinyl
H
Aziridinyl
Aziridinyl
H
H
Aziridinyl
1
2
3
4
5
6
7
H
H
Aziridinyl
NHCH2CH2OH
NHCH2CH2Cl
H
H
Phenyl
Table 2. Cytostatic and cytotoxic data obtained from the National Cancer Institute’s 60 cancer cell line pane
Compd
GI50
TGI
LC50
Median
Delta
Range
Median
Delta
Range
Median
Delta
Range
1
2
3
4
5
6
7
À7.28
À6.58
À6.19
À6.69
À5.46
À5.53
À7.56
0.72
1.42
1.28
1.31
1.34
2.47
0.44
2.18
2.87
1.87
2.38
2.11
4.00
2.07
À6.29
À5.75
À5.610.88
À5.97
À4.97
À4.70
À6.49
1.71
1.82
4.00
3.27
À5.39
1.56
1.36
2.98
2.02
4.95
À5.010.72 .73
À5.25
2.50
1
1.47
1.08
1.49
1.51
2.22
2.05
2.20
2.47
0.88
0.95
1.32
2.314.00
2.13
1.34
1.53
À4.39
À4.22
À5.69

R. Hargreaves et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3075–3078
3077
observed for the 6-(2-hydroxyethyl) derivative 6 indi-
cates that an alkylating center, either aziridinyl or
chloroethylamino, is required for optimal activity.
not all geldanamycin analogues, such as NSC-255105 in
Figure 2, target Hsp-90. The molecular targets of 1 and
7 are as yet unknown, but the cdks are the likely targets
based on COMPARE studies.
COMPARE analysis18 revealed that the LC50 profile of
7 correlates well with that of a geldanamycin analogue,
NSC-255105, shown in Figure 2 (0.74 correlation coef-
ficient). COMPARE analysis of the LC50, GI50, and
TGI profiles of 7 against the ‘molecular targets’ data-
base revealed good correlations with a variety of tyr-
osine kinases including cdk2 and cdk5. There was no
correlation with levels of the two-electron reducing
enzyme DT-diaphorase in the cancer panel indicating
reductive activation of the quinone system is not neces-
sary. This enzyme is involved in the reductive activation
of aziridinyl quinones as alkylating agents.20,22 Thus,
the structure of NSC-255105 reflects some aspects of the
quinolinedione structure–activity relationship outlined
above. Thus a planar ‘phenyl group’ is present in NSC-
255105. The COMPARE results merely indicate that
both compounds share a common cytotoxic mechanism.
The next section addresses the role of Hsp-90 inhibition
in quinolinedione cytotoxicity.
Acknowledgements
The authors gratefully acknowledge support from the
National Science Foundation,The Arizona Disease
Control Research Commission (to E.B.S. and L.W.),
and the National Institutes of Health (CA091056). The
MALDI-TOF was purchased with funds from NSF
grant no. CHE-0131222.
References and Notes
1. Neckers, L. Trends Mol. Med. 2002, 8, S55.
2. Everett, S. A.; Swann, E.; Naylor, M. A.; Stratford,
M. R. L.; Patel, K. B.; Tian, N.; Newman, R. G.; Vojnovic,
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3. Jiang, B.; Xiong, W. N.; Yang, C. G.; Jiang, H. L.; Cheng,
F.; Chen, K. X. Bioorg. Med. Chem. 2002, 10, 2775.
4. Neckers, L.; Schulte, T. W.; Mimnaugh, E. Invest. New
Drugs 1999, 17, 361.
Hsp90/Chaperone Assays
5. Neckers, L.; Schulte, T. W.; Mimnaugh, E. Invest. New
Drugs 1999, 17, 361.
6. Andrus, M. B.; Meredith, E. L.; Simmons, B. L.; Sekhar,
B. B. V. S.; Hicken, E. J. Org. Lett. 2002, 4, 3549.
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8. Nimmanapalli, R.; O’Bryan, E.; Bhalla, K. Cancer Res.
2001, 61, 1799.
9. Chiosis, G.; Lucas, B.; Shtil, A.; Huezo, H.; Rosen, N.
Bioorg. Med. Chem. 2002, 10, 3555.
The quinolinediones listed in Table 1 were evaluated as
Hsp90/chaperone active agents using the following
assays: (1) Competition for binding to C-terminal ATP-
binding site on Hsp-90 using immobilized novobiocin.23
(2) Competition for binding to N-terminal ATP-binding
site on Hsp-90 using immobilized geldanamycin.24 3.
Heat shock response in fibroblasts stable transfected
with GFP reporter construct.25 4. Inhibition of lucifer-
ase re-folding in rabbit reticulocyte.26 None of the
compounds shown in Table 1 were active in these assays
when tested at concentrations of 20 and 200 mM.
10. Chiosis, G.; Timaul, M. N.; Lucas, B.; Munster, P. N.;
Zheng, F. F.; Sepp-Lorenzino, L.; Rosen, N. Chem. Biol.
2001, 8, 289.
11. Lucas, B.; Rosen, N.; Chiosis, G. J. Comb. Chem. 2001, 3,
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12. Stebbins, C. E.; Russo, A. A.; Schneider, C.; Rosen, N.;
Hartl, F. U.; Pavletich, N. P. CELL 1997, 89, 239.
13. Showalter, H. D. H.; Pohlmann, G. OPPI Briefs 1992, 24,
484.
14. To a stirred solution of 2-substituted-5,8-dimethoxy-
quinoline (2.83 mmol) in acetonitrile (50 mL) was added drop
wise a solution ceric ammonium nitrate (12.0 mmol) in water
(50 mL). After 45 min the reaction mixture was extracted with
dichloromethane (3Â50 mL). The combined organic layers
were washed with water (50 mL), dried (Na2SO4) and the sol-
vent removed in vacuo to yield a yellow solid.
15. To a stirred slurry ofthe quinolinedione (0.85 mmol) in
methanol (10 mL) was added aziridine (2.54 mmol). After 2 h
the solvent was removed in vacuo and the resulting solid was
purified by flash chromatography (SiO2, chloroform) to yield a
yellow solid. This mixture of the two isomers was separated by
prep-TLC (SiO2, chloroform).
The rational design of Hsp-90 inhibitors using the
structure of ATP-active-site-bound geldanamycin affor-
ded two cytostatic/cytotoxic compounds 1 and 7. Both
compounds possess an alkylating center at the 6-posi-
tion, but do not require reductive activation of the quin-
one ring for activity. Although COMPARE analysis
revealed geldanamycin-like cytostatic and cytotoxic
activity, none of the compounds prepared in this study
actually interact with Hsp-90 or interfere with protein
renaturation. An explanation for this observation is that
16. Giorgi-Renault, S.; Renault, J.; Gebel-Servolles, P.;
Baron, M.; Paoletti, C.; Cros, S.; Bissery, M. C.; Lavelle, F.;
Atassi, G. J. Med. Chem. 1991, 34, 38.
17. Paull, D. K.; Shoemaker, R. H.; Hodes, L.; Monks, A.;
Scudiero, D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. J.
Natl. Cancer Inst. 1989, 81, 1088.
18. Boyd, M. R. Status of the NCI Preclinical Antitumor
Drug Discovery Screen. In Principles Practices Oncol. (PPO
Figure 2. Structure of NSC 255105, a geldanamycin analogue in the
National Cancer Institute’s archives.

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R. Hargreaves et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3075–3078
updates), DeVita, V. T., Hellman, S., Rosenberg, S. A., Eds.;
J. B. Lippincott, USA, 1989, 3.
19. Xing, C.; Skibo, E. B.; Dorr, R. T. J. Med. Chem. 2001,
44, 3545.
20. Skibo, E. B.; Xing, C. Biochemistry 1998, 37, 15199.
21. Suleman, A.; Skibo, E. B. J. Med. Chem. 2002, 45, 1211.
22. Marcu, M. G.; Schulte, T. W.; Neckers, L. J. J. Natl.
Cancer Inst. 2000, 92, 242.
23. Whitesell, L.; Mimnaugh, E. G.; DeCosta, B.; Myers,
C. E.; Neckers, L. M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
8324.
68.86; H, 4.04; N, 15.06. Found: C, 68.72; H, 3.99; N, 15.07.
6-Aziridinyl-2-(2-naphthyl)quinoline-5,8-dione (4): mp 179–
180 ꢁC dec.; TLC (ethyl acetate/hexanes [1:1]) Rf 0.25; IR
(KBr disc) 1638, 1598, 1578, 1305, 1142, 1006 cmÀ1; 1H NMR
(500 MHz, CDCl3) d 8.69 (s, 1H), 8.47 (d, J=8.5 Hz, 1H), 8.31
(dd, 1H), 8.23 (d, J=8 Hz, 1H), 7.99 (m, 2H), 7.89 (m, 1H),
7.56 (m, 2H), 6.38 (s, 1H), 2.37 (s, 4H); MS 326 (M+), 325,
298, 241, 152. Anal. calcd (C21H14N2O2): C, 77.29; H, 4.32; N,
8.58. Found: C, 77.04; H, 4.27; N, 8.45.
7-Aziridinyl-2-(2-naphthyl)quinoline-5,8-dione (5): mp 206–
207 ꢁC dec.; TLC (ethyl acetate/hexanes [1:1]) Rf 0.18; IR
(KBr disc) 1676, 1578, 1310, 1264, 1133, 1099 cmÀ1; 1H NMR
(500 MHz, CDCl3) d 8.71(s, 1H), 8.51(d, J=8 Hz, 1H), 8.30
(dd, 1H), 8.21 (d, J=8.5 Hz, 1H), 8.01 (m, 1H), 7.98 (d, J=9
Hz, 1H), 7.89 (m, 1H), 7.55 (m, 2H), 6.50 (s, 1H), 2.36 (s, 4H);
MS 326 (M+), 299, 271, 242, 227, 203, 152. Anal. calcd
(C21H14N2O2): C, 77.29; H, 4.32; N, 8.58. Found: C, 77.17; H,
4.26; N, 8.42.
24. Bagatell, R.; Paine-Murrieta, G. D.; Taylor, C. W.; Pul-
cini, E. J.; Akinaga, S.; Benjamin, I. J.; Whitesell, L. Clin.
Cancer Res. 2000, 6, 3312.
25. Hartson, S. D.; Thulasiraman, V.; Huang, W.; Whitesell,
L.; Matts, R. L. Biochemistry 1999, 38, 3837.
26. Physical properties. 6-Aziridinyl-2-phenylquinoline-5, 8-
ꢁ
dione (1): mp 1 83 C dec.; TLC (chloroform/methanol [19:1])
Rf 0.68; IR (KBr disc) 1676, 1654, 1578, 1468, 1445, 1382,
6-(2-Hydroxyethylamino)-2-phenylquinoline-5,8-dione (6) was
prepared by treating the 2-phenylquinoline-5,8-dione with
ethanolamine in ethanol: mp 218–219 ꢁC dec.; TLC (chloro-
form/methanol [9:1]) Rf 0.26; IR (KBr disc) 3405, 3333, 1677,
1
1309, 1273, 1133, 1094 cmÀ1; H NMR (500 MHz, CDCl3) d
8.46 (d, J=8.5 Hz, 1H), 8.17 (m, 2H), 8.05 (d, J=8.5 Hz, 1H),
7.52 (m, 3H), 6.47 (s, 1H), 2.34 (s, 4H); 13C NMR (125 MHz,
CDCl3) d 183.01, 181.15, 162.04, 157.33, 147.89, 137.47,
135.33, 130.68, 128.97, 127.80, 126.93, 123.32, 120.11, 27.76;
MS 276 (M+), 249, 221, 193, 182, 153, 102. Anal. calcd
(C17H12N2O2): C, 73.90; H, 4.38; N, 10.14. Found: C, 73.61;
H, 4.46; N, 10.09.
1607, 1573, 1524, 1338, 1251, 1129, 1066 cmÀ1 1H NMR
;
(300 MHz, DMSO) d 8.39 (d, J=8.4 Hz, 1H), 8.29 (d, J=8.4
Hz, 1H), 8.26 (m, 3H), 7.56 (m, 2H), 7.42 (t, J=5.4 Hz, 1H),
5.90 (s, 1H), 4.88 (t, J=5.7 Hz, 1H), 3.62 (m, 2H), 3.28 (m,
2H); MS (MALDI TOF) 295 (M+H+). Anal. calcd
ꢁ
.
7-Aziridinyl-2-phenylquinoline-5,8-dione (2): mp 1 85 C dec.;
(C17H14N2O3 0.25H2O): C, 68.33; H, 4.89; N, 9.37. Found: C,
68.42; H, 4.74; N, 9.39.
IR (KBr disc) 1676, 1654, 1578, 1309, 1273, 1133, 1094 cmÀ1
;
1H NMR (500 MHz, CDCl3) d 8.44 (d, J=8.2 Hz, 1H), 8.18
(m, 2H), 8.08 (d, J=8.2 Hz, 1H), 7.52 (m, 3H), 6.36 (s, 1H),
2.36 (s, 4H); 13C NMR (125 MHz, CDCl3) d 183.67, 179.89,
161.34, 158.38, 147.32, 137.47, 135.09, 130.57, 129.01, 127.67,
127.63, 124.09, 118.67, 27.89; MS 276 (M+), 275, 248, 193,
102. Anal. calcd (C17H12N2O20.25H2O): C, 72.72; H, 4.49; N,
9.98. Found : C, 72.48; H, 4.40; N, 9.88.
6-Aziridinyl-2-(2-pyridyl)quinoline-5,8-dion (3). (130 mg,
38%); mp 202–204 ꢁC dec.; TLC (chloroform/methanol [19:1])
Rf 0.61; IR (KBr disc) 1672, 1602, 1582, 1318, 1264, 1131
cmÀ1; 1H NMR (500 MHz, CDCl3) d 8.79 (d, J=8.2 Hz, 1H),
8.71(m, 2H), 8.53 (d, J=8.2 Hz, 1H), 7.88 (dt, 1H), 7.40 (m,
1H), 6.49 (s, 1H), 2.35 (s, 4H); 13C NMR (125 MHz, CDCl3)
182.97, 181.15, 160.55, 157.43, 154.20, 149.36, 147.55, 137.18,
6-(2-chloroethylamino)-2-phenylquinoline-5,8-dione (7). To a
solution of 6-aziridinyl-2-phenylquinoline-5,8-dione (20 mg,
0.072 mmol) in ethanol (10 mL) was added four drops of 4 M
HCl. The solution was sonicated for one min and then stirred
for 10 min. The solution was then filtered and the solid washed
with ice-cold ethanol. The solid was then crystallized from
chloroform/hexanes to yield dark red crystals: (16 mg, 71%);
mp 197–198 ꢁC dec.; TLC (chloroform/ethyl acetate [6:4]) Rf
0.38; IR (KBr disc) 3195, 3018, 1675, 1608, 1577, 1512, 1458,
1
1344, 1292, 1246, 1116 cmÀ1; H NMR (300 MHz, CDCl3) d
8.43 (d, J=8.2 Hz, 1H), 8.20 (m, 2H), 8.02 (d, J=8.2 Hz, 1H),
7.52 (m, 3H), 6.17 (t, J=6 Hz, 1H), 5.98 (s, 1H), 3.78 (t,
J=5.7 Hz, 2H), 3.62 (q, J=6 Hz, 2H); 13C NMR (125 MHz,
CDCl3); MS (MALDI TOF) 313, 315 (M+H+). Anal. calcd
(C17H13ClN2O2): C, 65.29; H, 4.19; N, 8.96. Found: C, 65.25;
H, 4.16; N, 8.94.
135.48, 128.20, 125.12, 124.15, 122.95, 120.13, 27.79; MS 277
+
.
(M ), 248, 222, 194, 179. Anal. calcd (C16H11N3O2 0.1H2O): C,