Indian Journal of Chemistry Vol. 50B, August 2011, pp 1140-1144
Note Synthesis and anticancer activity of some new series of 1, 4-dihydropyridine derivatives R Surendra Kumarª, A Idhayadhullaª, A Jamal Abdul Nasser*a & K Muralib ªP. G. & Research Department of Chemistry, Jamal Mohamed College, Tiruchirappalli 620 020, India b
Department of Pharmaceutical Chemistry, C. L. Baid Metha College of Pharmacy, Chennai 600 097, India E-mail:
[email protected] Received 18 February 2010; accepted (revised) 13 April 2011
A series of 1,4-dihydropyridine derivatives 1a-c have been prepared from 4-substituted aromatic aldehyde, ethyl acetoacetate and ammonium hydroxide following Hantzsch method. The compounds 1a-c have been reacted with semicarbazide to give the compounds 2a-c. The compounds 1ac have been reacted with thiosemicarbazide to give the compounds 3a-c. The structures of synthesized compounds are confirmed by IR, ¹H and ¹³C NMR, mass spectrometry and elemental analyses. The newly synthesized compounds have been screened for preliminary anti-cancer activity against HepG2 (Liver), Hela (Cervical) and MCF-7 (Breast) cancer cells. The compound 2a is highly active against HepG2, MCF-7 and 3a is highly active against Hela (Cervical) and these have been selected for advanced preclinical development.
Results and Discussion A series of diethyl 2,6-dimethyl-4-substituted phenyl-1,4-dihydropyridine-3,5-dicarboxylate 1a-c were prepared by condensation with three compounds following Hantzsch synthesis10. The physicochemical characterization is presented in Table I. The compounds 1a-c had reacted with semicarbazide to give 2,2'-{[4-(4-substituted aromatic alcohols)-2,6-dimethyl-1,4-dihydropyridi ne-3,5-diyl]dicarbonyl}dihydrazinecarbothioamide 2a-c (Scheme I). The compounds 1a-c reacted with thiosemicarbazide to give the compounds 3a-c by hydrazinolysis method11,12 (Scheme II). Compounds 1a-c, 2a-c and 3a-c were found to be active in the preliminary anticancer screening studies. The results are summarized in Tables II, III and IV. The compounds were tested against HepG2 (Liver), Hela (Cervical), (Breast) cancer cell lines. Their GI50, TGI and LC50 values were determined. The result of the screening was expressed in terms of GI50 growth inhibitor concentration. Compound 2a is relatively more active than the other compounds against HepG2 (Liver) and MCF-7 (Breast). Likewise, compound 3a is relatively more active than the other compounds against Hela (Cervical) cancer cells. Experimental Section
Keywords: 1,4-Dihydropyridine, semicarbazide, thiosemicarbazide, condensation, anti-cancer activity
The 1,4-dihydropyridine derivatives are of interest because of their potential biological activities such as vasodilators, antihypertensive, anti-inflammatory agents¹, antihypoxic, antiischemic² and calcium channel modulators of the nifedipine type³. Amide group in the 3,5-position of 1,4-dihydropyridine derivatives enhanced pharmacological properties4-7. Thiosemicarbazide derivatives also significant of biological activities such as antitumour, fungicide, bactereocide, anti-inflammatory and antiviral8,9. On the basis of literature reports and considering the current series of thiosemicarbazide and semicarbazide containing 1,4-dihydropyridine derivatives 2a-c and 3a-c the anticancer activity of compounds 1a-c, 2a-c and 3a-c were tested in vivo.
Melting points were recorded in open capillary tubes and are uncorrected. The IR spectra (KBr) were recorded on a Shimadzu 8201pc (4000-400 cm-1) FT-IR spectrometer. The ¹H and ¹³C NMR were recorded on a Bruker DRX-400 MHz instrument. Mass spectra (EI) were recorded on a Jeol JMS D-300 spectro meter operating at 70 eV. The elemental analyses (C, H, N and S) were recorded using a Varian EL III elemental analyser. The homogeneity of the compounds was checked by thin layer chromatography (TLC) with silica gel plates. Synthesis of diethyl 4-(4-hydroxy-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydro pyridine-3,5dicarboxylate, 1a. A reaction mixture was made up of ethylacetoacetate (2.0 mol), 4-substitutedbenzaldehyde (1.0 mol) and ammonium hydroxide (1.0 mol) in methanol (20 mL). It was then heated and refluxed for 4 hr. The obtained solid was filtered off, washed with
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Table I — Physical and analytical characterization data of synthesized compounds 1a-c, 2a-c and 3a-c Compd
R
m.p. (°C)
Yield (%)
Mol.Wt.
Mol.Formula
1a
4-OH-3-OCH3-Ph
191
73
285.29
C16H15NO4
1b
4-OH-Ph
197
78
255.26
C15H13NO3
1c
4-OCH3-Ph
190
74
269.29
C16H15NO3
2a
4-OH-3-OCH3-Ph
201
62
433.41
C18H23N7O6
2b
4-OH-Ph
240
59
403.39
C17H21N7O5
2c
4-OCH3-Ph
191
65
417.41
C18H23N7O5
3a
4-OH-3-OCH3-Ph
220
61
465.54
C18H23N7O4S2
3b
4-OH-Ph
211
69
435.52
C17H21N7O3S2
3c
4-OCH3-Ph
210
66
449.55
C18H23N7O3S2
O
R
Calcd (Found) % H N
C 67.36 (67.30) 70.58 (70.55) 71.36 (71.30) 49.88 (49.81) 50.52 (50.50) 51.79 (51.75) 46.44 (46.47) 46.88 (46.83) 48.09 (48.12)
5.30 (5.31) 5.13 (5.17) 5.61 (5.68) 5.35 (5.31) 5.25 (5.29) 5.55 (5.51) 4.98 (4.92) 4.86 (4.88) 5.16 (5.19)
4.91 (4.87) 5.49 (5.49) 5.20 (5.20) 22.62 (22.62) 24.31 (24.31) 23.49 (23.49) 21.06 (21.06) 22.51 (22.54) 21.81 (21.80)
13.78 (13.80) 14.72 (14.76) 14.24 (14.20)
O O
EtO
OEt + H2N
H3 C
S
N H
H2N
NH
O
N H
EtOH NH2
CH3
R
H N NH
O O
H3C
HN CH3
N H
O NH2
1a-c
2a-c R= 4OH-3-OCH3-Ph, 4-OH-Ph, 4-OCH3-Ph Scheme I — Synthesis of compounds 2a-c
O
R
O O
EtO
OEt + H2N
H3C
N H
H2N
NH
N H
DMSO NH2
CH 3
R
O
H N NH
S S
H3C
N H
HN CH3
S NH2
1a-c
3a-c R= 4OH-3-OCH3-Ph, 4-OH-Ph, 4-OCH3-Ph Scheme II — Synthesis of compounds 3a-c
water and purified by recrystallization from absolute ethanol. The above procedure was followed the synthesis of compounds 1b and 1c. IR (KBr): 3342 (N-H str), 3024 (Ar-H), 2922 (C-H str of CH3 ), 1764 (C=O, ester), 1447 (C-OH), 814 cm -1 (Ar-H); 1H NMR (DMSO-d6): δ 9.47 (s, 1H,
C-OH), 8.41 (s, 1H, NH of pyridine ring), 6.34- 7.07 (m , 4H, Ph-ring), 4.67 (s, 1H, C4-H), 4.28 (q, 4H, C3-OCH2CH3 and C5-OCH2CH3, J= 7.2 Hz), 3.81 (s,3H, OCH3), 2.12 (s, 6H, C2-CH3 and C6-CH3), 1.28 (t, 6H, 5-COOCH2CH3 and 3-COOCH2CH3, J = 7.2 Hz).
INDIAN J. CHEM., SEC B, AUGUST 2011
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Table II — Anticancer HepG2 (Liver), activity of the synthesized compounds 1a-c, 2a-c and 3a-c Compd GI50 (µm) 16.2 15.2 17.2 31.2 28.8 27.5 23.5 27.8 28.5 0.01
1a 1b 1c 2a 2b 2c 3a 3b 3c Doxorubicin(standard)
HepG2 (Liver) TGI LC50 (µm) (µm) 29.1 25.2 30.2 62.5 57.8 54.1 47.8 51.2 56.5 0.13
88.3 81.5 >100 72.1 88.8 92.1 >100 82.3 >100 0.58
Table III — Anticancer Hela (Cervical) activity of the synthesized compounds 1a-c, 2a-c and 3a-c Compd GI50 (µm) 1a 1b 1c 2a 2b 2c 3a 3b 3c Doxorubicin (standard)
22.6 20.5 18.8 18.2 20.8 22.5 31.1 25.3 20.5 0.05
Hela (Cervical) TGI (µm) LC50 (µm) 48.4 33.5 30.8 48.3 51.8 55.3 64.3 51.2 47.8 0.41
>100 82.8 88.5 86.2 >100 >100 71.8 >100 88.3 0.88
Diethyl 4-(4-hydroxyphenyl)-2,6-dimethyl-1,4dihydropyridine-3,5-dicarboxylate, 1b. IR (KBr): 3352 (N-H str), 3026 (Ar-H), 2961 (C-H str of CH3),1742 (C=O, ester), 1438 (C-OH), 823 cm -1 (ArH); 1H NMR (DMSO-d6): δ 9.31 (s,1H, C-OH), 8.21 (s,1H, NH of pyridine ring), 6.86- 7.17 (m, 5H, Phring), 4.69 (s,1H,C4-H), 4.23 (q, 4H, C3-OCH2CH3 and C5OCH2 CH3, J =7.2 Hz), 2.23 (s, 6H, C2-CH3 and C6-CH3), 1.30 (t, 6H, 3-COOCH2CH3 and 5-COOCH2CH3, J= 7.2 Hz). Diethyl4-(4-methoxyphenyl)-2,6-dimethyl-1,4dihydropyridine-3,5-dicarboxylate, 1c. IR (KBr): 3344(N-H str),3012(Ar-H), 2952 (C-H str of CH3 ), 1731(C=O, ester), 834 cm -1 (Ar-H) ; 1H NMR (DMSO-d6): δ 8.27 (s,1H, NH of pyridine ring), 6.867.17 (m, 5H, Ph-ring), 4.60 (s,1H,C4-H), 4.20 (q, 4H, C3-OCH2CH3 and C5-OCH2CH3, J= 7.2 Hz), 3.84 (s,3H,-OCH3), 2.38 (s, 6H, C2-CH3 and C6-CH3),1.39
Table IV — Anti-cancer MCF-7(Breast) activity of the synthesized compounds 1a-c, 2a-c and 3a-c Compd GI50 (µm) 1a 1b 1c 2a 2b 2c 3a 3b 3c Doxorubicin (standard)
22.9 19.2 18.3 32.3 18.2 20.1 27.1 30.2 19.8 0.02
MCF-7( Breast) TGI (µm) LC50 (µm) 46.8 41.3 43.2 58.8 41.2 43.8 51.5 50.2 32.3 0.21
>100 88.5 84.1 74.8 >100 >100 89.2 >100 >100 0.74
(t, 6H, 3-COOCH2 CH3 and 5-COOCH2CH3 J= 7.2 Hz). Synthesis of 2,2'-{[4-(4-hydroxy-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydro pyridine-3,5-diyl]dicarbonyl}dihydrazinecarboxamide, 2a. A reaction mixture was made up of compound 1a (0.1 mol), semicarbazide dissolved in ethanol (30 mL). It was then heated under reflux for 10 hr. The solid was obtained, after cool and poured into crushed ice. The solid was collected by filtration, washed with water and purified by recrystallization from ethanol. The above procedure was followed for the synthesis of compounds 2b and 2c. IR (KBr): 3342 (NH), 3220 (NH2), 3192 (NHC=O), 3028 (Ar-H), 2941 (C-H str of CH3), 1717 (C=O), 1472 (C-OH), 1091 cm-1 (N-C-N); 1H NMR (DMSO-d6): δ 9.71(s, 2H, NH2), 9.41(s,1H, OH), 8.64 (bs,1H,NH of pyridine ring), 8.01 (d, 1H, C3 - CONH and C5 - CONH, J = 8.5 Hz), 7.33- 7.27 (m,5H, Phring), 6.20 (s,2H, -OCNH2), 6.01 (s,1H, -NHCO), 5.11 (s, 2H,C4-H), 3.83 (s, 1H, O CH3), 2.25 (s, 6H, C2-CH3 and C6-CH3); ¹³C NMR (DMSO-d6): δ 164.2 (CONH), 157.2 (CONH2), 148.3 (2, 6-C in pyridine ring), 147.1 (C-OCH3), 147.7 (C-OH), 134.1(C4-Ph), 122.7 (Ph- C), 116.8 (Ph-C), 103.2 (3,5-C-CO-NH), 56.7(C-OCH3), 19.0 (2,6-CH3); EI-MS: m/z 449.48 (M+ + 1, 28.10%), 419.45 (28.10%), 347.36 (10.43%), 317.73 (100%), 287.29 (17%), 201.26 (21%), 185.26 (33%), 157.21 (70%). 2,2'-{[4-(4-hydroxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-diyl]dicerbonyl}dihydra zinecarboxamide, 2b. IR (KBr): 3349 (N-H str), 3212 (NH2), 3180 ( NH-C=O ), 3024(Ar-H), 2922 (C-H str of CH3 ), 1764 (C=O, ester), 1447(C-OH), 1087(N-C-
NOTES
N), 814 cm -1 (Ar-H); 1H NMR (DMSO-d6): δ 9.78 (s, 2H, NH2), 9.47 (s, 1H, C-OH), 8.41 (s, 1H, NH of pyridine ring), 8.04(d,1H, C3 - CONH and C5CONH, J=8.5Hz), 7.34-7.07 (m, 4H, Ph-ring), 6.24 (s,2H, -OCNH2), 6.12 (s,1H, -NHCO), 4.67 (s, 1H, C4-H), 2.12 (s, 6H, C2-CH3 and C6-CH3 ); ¹³C NMR (DMSO-d6): δ 163.8 (CONH), 156.3 (CONH2), 147.2 (2,6-C in pyridine ring), 147.9 (C-OH), 136.8 (C4Ph), 130.8 (Ph-C ), 114.9 (Ph-C), 102.6 (3,5-C-CONH), 18.1 (2,6-CH3); EI-MS: m/z 403.49 (M+ + 1,10.43%), 317.34 (100%), 287.31 (17%), 257.31 (20%), 201.26 (21%), 173.21 (13%), 157.21 (33%). 2,2'-{[4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-diyl]dicarbonyl}dihydra zinecarboxamide, 2c. IR (KBr): 3352 (N-H str), 3218(NH2), 3173 (NH-C=O), 3026(Ar-H), 2961(C-H str of CH3), 1742 (C=O, ester), 1072(N-C-N), 823 cm -1 (Ar-H); 1H NMR (DMSO-d6): δ 9.68 (s, 2H, NH2), 8.21 (s, 1H, NH of pyridine ring), 8.13 (d,1H, C3 - CONH and C5- CONH, J=8.7 Hz), 7.17- 6.86 (m, 4H, Ph-ring), 6.28 (s, 2H, -OCNH2), 6.08 (s, 1H, -NHCO), 4.65 (s, 1H, C4-H), 3.89 (s, 1H, OCH3), 2.12 (s, 6H, C2-CH3 and C6-CH3); ¹³C NMR (DMSO-d6): δ 162.4 (CONH), 155.7 (CONH2), 157.1 (C-OCH3), 147.2 (2,6-C in pyridine ring), 136.1 (C4 -Ph), 130.3 (Ph-C),114.2 (Ph-C), 103.0 (3,5-C-CO-NH), 56.1 (C-OCH3), 18.5 (2,6-CH3); EI-MS: m/z 417.41 (M + + 1, 28.10 %), 387.38 (10.43%), 331.73 (100%), 271.31(17%), 243.25 (13%),215.6 6(21%), 187.20 (33 %),157.84 (22%). Synthesis of 2,2'-{[4-(4-hydroxy-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydro pyridine-3,5-diyl]dicarbonyl}dihydrazinecarbothioamide, 3a. A reaction mixture was made up of compound 1a (0.1 mol), thiosemicarbazide dissolved in ethanol (30 mL) and a few drops of DMSO. It was then heated under reflux for 10 hr. The solid was obtained after cool and poured into crushed ice. The solid was collected by filtration, washed with water and purified by recrystallisation from ethanol. The above procedure was followed for the synthesis of compounds 3b and 3c. IR (KBr): 3342 (NH), 3220 (NH2), 3028 (Ar-H), 3192 (NH-C=O), 1242 (C=S), 1717 (C=O), 1472 (C-OH), 1091 cm -1 (N-C-N); 1H NMR (DMSO-d6 ): δ 9.71 (s, 2H, NH2), 9.41(s, 1H, OH), 8.64 (bs, 1H, NH of pyridine ring ), 8.01 (d,1H, C3 - CONH and C5- CONH, J=8.4Hz), 7.33- 7.27 (m, 5H, Ph-ring), 5.11(s, 2H, C4-H), 2.25 (s, 6H, C2-CH3 and C6-CH3), 2.02 (d, 1H, -NHCS); ¹³C NMR (DMSO-d6): δ 184.7 (-SC- NH-), 165.8 (-CO-NH-), 147.7 (2,6 -C in pyridine ring), 146.8 (-C- OH),133.3 (Ph-C), 114.7
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(Ph-C), 104.8 (3,5-C in pyridine ring), 55.9 (C-OCH3), 43.5 (4C in pyridine ring), 19.3 (2,6 -C-CH3 in pyridine ring); EI-MS: m/z 465.54 (M ++1, 33.25%), 347.36 (17.43%), 317.53 (100%) , 287.29 (17%), 201.26 (41%), 185.16 (43%), 156.22 (22%). 2,2'-{[4-(4-hydroxy-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-diyl]dicarbo nyl}dihydrazinecarbothioamide, 3b. IR (KBr): 3348 (NH), 3200 (NH-C=O), 3217 (NH2), 3076 (Ar-H), 1701 (C=O), 1461 (C-OH), 1240 (C=S), 1087 cm-1 (N-C-N); 1H NMR (CDCl3): δ 9.70 (s, 2H, NH2), 9.48(s, 1H, OH), 8.64 (s,1H, NH of pyridine ring ), 8.11(d, 1H, C3-CONH and C5- CONH,), 7.30 - 7.42 (m , 5H, Ph-ring), 5.17 (s, 2H, C4-H), 2.31 (s, 6H, C2CH3 and C6-CH3), 2.12 (d, 1H, -NHCS); ¹³C NMR (DMSO-d6): δ 183.9 (-SC-NH-), 165.6 (-CO-NH-), 148.1 (2, 6 -C in pyridine ring), 130.4 (Ph-C), 114.0 (Ph-C), 104.9 (3,5-C in pyridine ring), 44.1 (4C in pyridine ring), 19.4 (2, 6 -C-CH3 in pyridine ring); EIMS: m/z 435.52 (M + + 1, 28.10%), 420.50 (28.10%), 405.49 (10.43%), 317.34 (100%), 287.31 (17%), 257.31 (35.73%), 201.26 (21%), 173.21(33%), 157.21 (70%). 2,2'-{[4-(4-hydroxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-diyl]dicarbonyl}dihydra zinecarbothioamide, 3c. IR (KBr): 3323 (NH), 3251 (NH-C=O), 3231(NH2),3034(Ar-H), 1717(C=O), 1251(C=S), 1095 (N-C-N), 808 cm-1 (Ar-H); 1H NMR (DMSO-d6): δ 9.82 (s, 2H, NH2), 8.57 (bs,1H, NH of pyridine ring), 8.05 (d, 1H, C3 - CONH and C5- CONH), 7.33- 7.27 (m, 5H, Ph-ring), 5.21 (s, 2H,C4-H), 3.81 (s,3H,-OCH3), 2.10 (d,1H,-NHCS), 2.20 (s, 6H, C2-CH3 and C6 - CH3); ¹³C NMR (DMSO-d6): δ 181.7 (-SC-NH-), 166.2 (-CO-NH-), 148.6 (2, 6 -C in pyridine ring), 132.1 (Ph-C), 115.2 (Ph-C), 105.3 (3,5 – C in pyridine ring), 56 .7 (-O CH3), 44.7 (4C in pyridine ring), 18.8(2, 6 -C- CH3 in pyridine ring); EI-MS: m/z 449.55 (M+ +1,11.02%), 331.36 (100%), 270.66 (16.81%), 243.15 (17.09%), 215.67 (20.36%), 187.19 (37.43%), 157.80 (20%). Anti-cancer activity The newly synthesized compounds 2a-c were screened for their anticancer activity according to the procedure suggested13. Compounds 1a-c, 2a-c and 3a-c were submitted for the three cell lines with one dose of primary anticancer assay with a concentration of 100 µm for 48 hr (MTT anticancer assay). The three cell lines used in the present investigation were HepG2 (Liver), Hela (Cervical), and MCF-7(Breast).
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INDIAN J. CHEM., SEC B, AUGUST 2011
In this current protocol, each cell line was preincubated on micro titer plate. The results for each test are reported as percentage of the growth of the treated cells when compared to the untreated control cells. The compounds that reduce the growth any one of the cell lines to approximately 32% or less were evaluated as having anti tumor activity. 0.1 mL of the cell suspension (containing 5 × 106 cells/100 µL) and 0.1 mL of the test solution (6.25 µg-100 µg in 1% DMSO such that the final concentration of DMSO in media was less than 1%) were added to the 27 well plates and kept in a 5% CO2 incubator at 37°C for 72 hr. The blank contained only cell suspension and control wells contained 1% DMSO and cell suspension. After 72 hr, 20 µL of MTT was added and kept in the CO2 incubator for 2 hr followed by addition of 100 µL propanol. The plate was covered with aluminum foil to protect it from light. Then the 27 well plates were kept in a rotary shaker for 10-20 min. After 10-20 min, the 27 well plates were processed on an ELISA reader for absorption at 562 nm. Conclusion A new series of 1,4-dihydropyridine derivatives 2a-c and 3a-c have been synthesized. The synthesized compounds 1a-c, 2a-c and 3a-c were found to be active against three cancer cell lines. The compound 2a had highly active against HepG2 (Liver), MCF7(Breast) and 3a is highly active against Hela (Cervical). It is concluded that there is ample scope for further study in developing these compounds as anticancer agents.
Acknowledgement The authors wish to thank C. L. Baid Metha College of Pharmacy Chennai, India for the studies on anti-cancer activity. The authors sincerely thank principal and management of Jamal Mohamed College, for providing laboratory facility and financial support. References 1 Godfraid T, Miller R & Wibo M, Pharmacol Rev, 38, 1986, 321. 2 Khadilkar B & Borkar S, Synth Commun, 28, 1998, 207. 3 Schnell B, Krenn W, Faber K & Kappe C O, J Chem Soc Perkin Trans 1, 2000, 4382. 4 Pattan S R, Rasal V B, Venkatramana N, Khade A B, Butle S R, Jadhav S G, Desai B & Manvi F V, Indian J Chem, 46B, 2007, 698. 5 Suresh T, Swamy S K & Reddy V M, Indian J Chem, 46B, 2007,115. 6 Bhavik D, Dinesh S, Naliapara A Y, Shah A & Saxena A K, Bioorg Med Chem, 9, 2001, 1993. 7 Amini M, Navidpour L & Shafiee A, Daru, 16, 2008, 9. 8 Nandi A K, Chaudhri S, Mazumdar S K & Ghosh S, J Chem Soc Perkin Trans 2, 11, 1984, 1729. 9 Ali M A, Chowdhary M A & Naziruddin M, Polyhedron, 3, 1984, 595. 10 Hadizadeh F, Shaficee A, Kazemi R & Mohammadi M, Indian J Chem, 41B, 2002, 2679. 11 Srivastava S K, Srivastava S & Srivastava S D, Indian J Chem, 41B, 2002, 2357. 12 Ojha S, Ameta U, Dhakar N & Talesara G L, Indian J Chem, 46B, 2007, 860. 13 Scudiero D A, Shoemaker R H & Paul K D, Cancer Res, 48, 1988, 4827.
