LV10045B - Bis-naphtalimides containing amino acid derived linkers as anticancer agents - Google Patents

Abstract

The invention relates to bis-naphthalimides, including to 2,2-1,2-ethanediyl (imino (1-methyl2,1) -etanediyl]] - bis- [5-nitro-1H-benz [de] isoquinoline-1,3 (2H) -dione] and 2,2'-1,2-ethanediyl-bis- [imine (2-methyl) 2,1-ethanedi] bis [5- nitro-1 H-benz [de] isoquinoline-1,3 (2H) -dione], processes for their preparation, pharmaceutical compositions containing them, and their use in the treatment of malignant tumors of mammals.

Description

1 LV 10045

5 TITLE l·

Bis-naphthalimides Containing Amino-Acid Derived Linkers as Anticancer Aģents 10

flEULOF. THE INVENTIOH

This invention relates to bis-naphthalimides, including 2,2'- [ 1,2-ethanediylbis [imino (l-methyl-2,1-15 ethanediyl) ] ] -bis (5-nitro-lH-benz [de 1 isoquinoline-1,3 (2H)-dione] and 2,2'-(l,2-ethanediylbis [imino(2-methyl-2, l-ethanediyl) ] J -bis[5-nitro-lH-benz [de] isoquinoline-l,3 (2H) -dione], processes for their preparation, pharmaceutical compositions containing 20 them, and methods of using them to treat cancer, particularly solid tumor carcinomas, in mammals.

BACKGROUND OF THE INVENTION 25 Harnisch et al., U.S. Patent 4,841,052 issued June 20, 1989 describe naphthalic acid imides useful as charge-regulating substances in electrophotographic toners. 30 Brana et al.,U.S. Patent 4,874,863 issued October 17, 1989 discloses anticancer compounds of the formula: 1 2

vherein X1, X*, χ3 and X4 are identical or different and are each Η, N02» NH2» Ci-C6-alkylamino, di-Cj.-C6-5 alkylamino, OH, Ci-C6-alkoxy, halogen, trihalomethyl, Ci-Cg alkyl, formyl, Ci-Cg-alkylcarbonyl, ureyl, Ci-C6-alkylureyl and R is a straight chain or branched C4-Cio-alkylene which is interrupted at one or two points in the 10 chain by a secondary or tertiary amino group, where 2 nitrogen atoms may additionally be bonded to one another by an alkylene group, or a salt with a physiologically tolerated acid.

15 DETAILED DESCRIPTION QF THE INVENTION

There is provided by this invention bis-naphthalimide compounds having the formula (i):

or enantiomeric or diastereomeric forms thereof, or mixtures of enantiomeric or diastereomeric forms - 20 3 LV 10045 thereof, or pharmaceutically acceptable salts thereof# wherein: R1, R2, R3, R4, R5/ R5, and R7, independently, are H or CH3; X and X1 are H or NO2; and γ and Y' are H or NO2, provided that at least one of X, X‘, Y, and Y' is NO2.

Preferred compounds of the present invention include those compounds of formula (i) wherein: R1 and R7 are CH3; R2, R3, R4, r5, and R6 are H; X and X' are NO2, vherein the substituent is at the 3-position of the naphthalimide (i.e./ the 5-position of the ΙΗ-benz(dejisoquinoline-l,3(2H)-dione)/ and Y and Y' are H.

Preferred compounds of the present invention also include those compounds of formula (i) wherein; R2 and R6 are CH3; R1, R3, R4, r5, and R7 are H; X and X' are NO2/ vherein the substituent is at the 3-position of the naphthalimide (L.e., the 5- 4 position of the ΙΗ-benz(dejisoquinoline-l,3(2H)- dione); and Y and Y' are H.

Specifically preferred compounds of the present invention are the folloving: (S,S) -2,2,-[l,2-ethanediylbis [imino(l-methyl-2,1-ethanediyl) ] ] -bis [5-nitro-lH-benz [de] isoquinoline-1,3(2H)-dione]; (Racemic + Meso)-2,2,-[l,2-ethanediylbis[imino(l-methyl-2, l-ethanediyl) J J -bis [5-nitro-lH-benz [de] isoquinoline-l, 3 (2H) -dione]; (R, R)-2,2'- [1,2-ethanediylbis [imino(l-methyl-2,1-ethanediyl) ] ] -bis [5-nitro-lH-benz [de] isoquinoline-1, 3(2H)-dione]; (Meso) -2,2 [1,2-ethanediylbis f imino (l-methyl-2,1- ethanediyl) ] ]-bis [5-nitro-lH-benz [de]isoquinoline-1,3(2H)-dione]; (S, S) -2,2 ’ - II, 2-ethanediylbis [imino (2-methyl-2,1-ethanediyl) ] ]-bis [5-nitro-lH-benz [de] isoquinoline-1,3(2H)-dione]; (Racemic + Meso) -2,2 ’ - [1,2-ethanediylbis [imino (2-methyl-2, l-ethanediyl) ] ]-bis [5-nitro-lH-benz [de] isoquinoline-l,3 (2H) -dione]; (R, R) -2,2' - [1,2-ethanediylbis [ imino (2-raethyl-2,1-ethanediyl) ]]-bis[5-nitro-lH-benz [de]isoquinoline-l,3(2H)-dione]; or 5 LV 10045 (Meso)-2,2[1/2-ethanediylbis[imino(2-methyl-2,1-ethanediyl) J ]-bis [5-nitro-lH-benz {de] isoquinoline-l,3(2H)-dioneJ; and pharmaceutically acceptable salts thereof.

Also provided by this invention are processes for the preparation of the compounds ©f formula (i), pharmaceutical compositions containing the compounds of formula (i), and methods of using these compounds for the treatment of cancer, particularly solid tumor carcinomas, in a mammai.

Synthgsla

The present invention describes a series of bis-naphthalimides useful for treating cancer containing linkers derived from reduced peptides. Compounds of * this invention are more soluble in aqueous media than prior art compounds not containing these reduced peptide linkers.

Compounds of this invention can be synthesized by reacting two equivalents of an anhydride of formula (ii) with one equivalent of a polyamine of formula (iii) in an inert solvent such as ethanol or dimethylformamide or tetrahydrofuran, for example, at a temperature ranging from ambient to the solvent's boiling temperature (Scheme A). The resulting suspension can then be filtered to give the free base of (i) or it can be acidified with the appropriate mineral or organic acid to producē a pharmaceutically acceptable salt, which can be obtained by filtration. Salts of the free base can also be prepared by acidifying a suspension of the free 6 base in ethyl alcohol or dichloromethane with the appropriate mineral or organic acid and collecting the formed solid by filtration. In some cases, the free base of (i) requires purification by colunvn 5 chromatography before its salt can be prepared as described above. 10

NHa (iii)

(i) * 7 LV 10045

The parent anhydride (ii) is commercially available or can be prepared according to the procedures described by Hodgson et al., J. Chem. Socļf p90 (1945). Reduced peptides of formula (iii) can be prepared according to the methods described below (Schemes I - IV).

The synthesis of compounds of formula Va, (see Scheme I below) can be accomplished by the reaction of t-BOC-(S)-alanine (compound a) with 1,1'-carbonyldiimidazole, followed by reaction with ethylenediamine under Standard conditions. Acid hydrolysis of the t-BOC (N-tert-butoxycarbonyl) protecting group in la was performed under Standard conditions to afford Ila. The same procedure is utilized to producē compounds Ilb or IIc by starting the sequence with the corresponding (R)-alanine (compound b) or racemic alanine (compound c).

Reduction of Ila (dihydrochloride salt) with diborane in tetrahydrofuran at refluxing temperature afforded Illa. Illa was then neutralized with sodium ethoxide and the pure free base IVa was obtained by Kugelrohr distillation. IVa was reacted with the appropriate naphthalic anhydride in ethanol or tetrahydrofuran at reflux temperature to obtain Va. Compounds Vb and Vc are prepared similarly by using the corresponding IVb, or IVc, respectively. 8

žcherng-I CHļ NH2(CH2)2NH2

(t-BOOHN-Sf0" *"<t-®OC)HM 0 CKjH 0 NH(t-BOC) Ο H CH3 a, S la, <S,S) b, R Ib, <R,R) c, racemate Ic, d, 1 pair £ meso

CH3H O

HjN \ N ν«'/Νί 'Ντ NH2 °2 HC1 H CH3

Mi

THF

CHj H

2 HC1 H CH3 III

H

II

NaOEt ch3 h h,n*^n

EtOH, anhydridew reflux

2 CH3S03H 9 LV 10045 using the corresponding compounds XVIllb and XVIIIc, respectively. In addition, compounds XXa, XXVIIlb and XXIXb could be prepared by condensation of amiņe XVIIIb with appropriate naphthalic anhydrides. 5

Ichsmg III h3co\nh* CHja, s b, R c, racemat· 8C1 ♦ . ® bemen· - 0

Lr*flux Ο Η O CH3K °°Η3 CHS° H °

NJ!i_ CHjOH XVa, (S,S) XVb, (R, R) XVc, (racemat· + meso)

H2N XVI H CH, *

nh2 4 ICl

NaOEt

H CHj H

EtOH, NEtj anhydride, reflux

XVIII

XVII

XI Xa. X-3-N02, Υ-Η (S,S) XIXb. X-3-H02, Υ-Η (R,R) XI Xq, Χ-3-ΝΟ2# Υ-Η (racemate + meso) xxa, x-y-h (S/S) XXVITTb. Χ-4-ΝΟ2, Υ-Η (R,R) XXIXb. Χ-3-ΝΟ2, Υ-6-ΝΟ2 (R,R) XXXIVf X»3-N02, Y*H (racemate) 10 10

In Scheme III the position of X and Y is indicated using the numbering for the position in the corresponding naphthalic anhydride, and not the numbering for the lH-benz[de]isoquinoline-l,3(2H)-dione. 5 For compounds of the formula (XXVII), the synthesis is shown in Scheme IV. Reaction of t-BOC-(S)-alanine with isobutylchloroformate in the presence of N-methylmorpholine, followed by reaction with aminoacetonitrile (generated by neutralization of its 10 HC1 salt with N-methylmorpholine), gavē XXI. This was hydrogenated with palladium hydroxide in acetic acid to yield XXII. Reaction of t-BOC-(R)-alanine with 1,1'-carbonyldiimidazole, followed by addition of XXII under Standard conditions, afforded XXIII. Acid hydrolysis of 15 the t-BOC protecting group of XXIII was carried out under Standard conditions to furnish XXIV. Subsequent reduction with diborane in refluxing tetrahydrofuran yielded XXV, which was neutralized with sodium ethoxide to give XXVI. Amine XXVI was condensed with the appropriate naphthalic anhydride to producē XXVII. 20 LV 10045 Π

Scheme IV (S) (t-30C)

ch3 (t-BOC)HN

0 Η H NH, N-methylmorpholin«

Pd(0H)2 HOAc

XXI

0 XXII

Η H 0 (R) H*-'s ofj '* C"***(t-BOC) Hί^SfN'sx*sN(t'B0C)i o hch3vh (meao), XXIII

(t-BOC)HN

2. XXII HC1.

H?b H (2N^irNv^Ni\NH2·2,t:l 2. hCh3''h

1. 8H3/THF h.c;> h OH 3. HC1 • 4 HCl

hch3 h (mso), XXV

HiH*H

?Ν^Νν^Ν^ΝΗ2 H CH3 H

(m*JO), XXVI

Λ»· W> »chjH0 2 CHjSOjfl

0,N

(meso), XXVII NO, 5 For compounds of the formula (ΧΧΧΧ), the synthesis is shown in Scheme V (below). (t-BOC)-Alaninyl alanine was reacted with isobutyl chloroformate in methylene chloride at -10 to -15°C in the presence of N-methylmorpholine, followed by addition of (S)-alanine 10 methylester hydrochloride to give XXXV. The ester XXXV was converted to its amide XXXVI by bubbling ammonia in methanol. The t-BOC protecting group was removed by acid hydrolysis to furnish XXXVII. Subsequent diborane reduction of tripeptide amide XXXVII in refluxing 15 tetrahydrofuran yielded the polyamine tetrahydrochloride 12 XXXVIII/ vhich was neutrali2ed with sodium ethoxide to yield XXXIX. The amine XXXIX was condensed with the appropriate naphthalic anhydride to producē ΧΧΧΧ.

Scherng.-^

ch3 jh h o Ch, h

(t-BOC)HN'^V'N'^rVOH Ο H *CH, λ

NHj/MeOH

CH, H H O CH, H

X .NHj

HCI

(S,S,S) (t-BOC)HN^ Ύ γς 'N 0 H CH, H XXXVI 1. BIVTHF CH, H H 0 ch, H 2. MeOH • 2. 1 HCI 3· HCI r

HlN (S.S.S) χχχνιι CH, h h ch, hΧν.-’ν"' U Ī>U η , Ι|Λ|

H CH, (S.S.S) XXXVIII

4 HCI

Na/EtOH

CH, H H CH, H i/W H

h \:h, (S.S.S) XXXIX

ΧΧΧΧ

2 CHjSOaH LV 10045 13

Asymmetric bis-naphthalimides of the formula (XXXXVI) can be synthesized as shown in Scheme.VI (below) . Reaction of XXII with 1/1’-carbonyldiimidazole, followed by addition of (R)-5 benzyloxycarbonyl alanine, furnished ΧΧΧΧΙ. Subsequent reaction with phosphorus pentasulfide in tetrahydrofuran gavē the corresponding thioamide ΧΧΧΧΙΙ. Treatment of ΧΧΧΧΙΙ with Raney Nickel yielded amine ΧΧΧΧΙΙΙ. Amine ΧΧΧΧΙΙΙ was condensed with 3-nitro-l, 8-naphthalic 10 anhydride to yield naphthalimide XXXXIV. The (t-BOC) protecting group was hydrolyzed under Standard conditions to give XXXXV, which in turn can be condensed with another naphthalic anhydride to producē non-symmetric bis-naphthalimides of the formula XXXXVI. 14 (S) CH, Η Η

{t-BOC)HN v ‘NH,

0 XXII

Scherce VI 1. GDI

H C”, 2-lR) ,CBZ)HN>Y0H NH(CBZ) 'CH, CH, Η H 9 pg CH,.J* H . (t-BOC)HN^V'M * 1ί {<-ΒΟΟΗΝ^ΎΜ \*"\Ν

l H h^CH, 5 Η hAC ΧΧΧΧΙ

Raney Νί CH,» Η ΧΧΧΧΙΙ NH,

(t-BOC)HN'>^N'S>-'^N^'V'W1 Η H CHļ ΧΧΧΧΙΙΙ

CH, Η H

HCI

3 HCI

XXXXV vvv h ; Η H*

χχχχνι

The invention can be further understood by referring to the folloving Examples and Tables below. 10

Exairiple 1 15 LV 10045 (S, S) -2,2'- [1,2-ethanediylbis (imino (l-methyl-2,1-ethanediyl) ] ]-bis [5-nitro-lH-benz(de]isoquinoline-1,3(2H)-dione] methanesulfonate (1:2) (Va) 5 Part A: (Illa) (S,S)-N1,!*1’-!, 2-ethanediylbis[1, 2-propanediamine] tetrahydrochloride.

To a THF (80 ml) suspension of Ila (2.75 g, 10 mmol), there was added 200 ml of 1M BH3*THF complex slowly, The mixture was stirred at room temperature for 10 1 1/2 hours and then heated overnight to become a clear solution. After cooling to room temperature, the solution was carefully quenched with 100 ml of methanol, and then refluxed overnight. The solvent was evaporated, and to the remaining liquid was added 20 ml 15 of methanol and 5 ml of conc. HC1. The resulting white solid was collected on a filter under nitrogen to give 1.92 g (60%) of Illa; mp 210-215eC. 1H-NMR (D20) 5 3.60 (m, 2H, 2 CH), 3.40 (s, 4H, 2 CH2), 3.27 (m, 4H, 2 CH2) and 1.30 (d, 6H, J«6.9 Hz, 2 CH3). MS (DCI) m/e 175 20 (M+l, free base) . (α)0 - 2.31° (c-0.606, H20).

Part B: (IVa) (S,S)*-1,2-ethanediylbis[1,2-propanediamine],

Sodium (1.03 g, 45 mmol) was added to 50 ml of 25 anhydrous ethanol; and the mixture stirred for 1.5 hours until ali the sodium was dissolved. To this, there was added 3.2 g (10 mmol) of Illa. After stirring for 2 hours, the sodium chloride precipitate was removed by filtration; and the solvent in the filtrate evaporated. 30 Kugelrohr distillation (120-140°C at 1.4 mm) gavē 1.29 g (74%) of IVa as a clear liquid. 1H-NMR (CDCI3) δ 2.96 (m, 2H, 2 CH), 2.71 (m, 4H, 2 CH2), 2.60 (m, 2H, CH2), 2.40 (m, 2H, CH2), 1.46 (broad, 6H, 2 NH2 and 2 NH) and 1.06 (d, 6H, J-6.2 Hz, 2 CH3). MS (DCI) m/e 175 (M+l). 35 16

Part C: (Va) (S, S)-2,2'-11,2-ethanediylbis [imino (1-methyl-2, l-ethanediyl) ] ] -bis [5-nitro-lH-benz(de)isoquinoline-l, 3(2H)-dione] methanesulfonate (1:2) . A mixture of 3-nitro-l,8-naphthalic anhydride (2.43 g, 10 mmol) and IVa (0.87 g, 5 nunol) was stirred at room temperature for 4 hours, refluxed for 2 hours and then cooled to room temperature overnight. The solvent in the mixture was evaporated and the residue purified by column chromatography to give 0.87 g (27.9%) of a brown solid. The free base (0.84 g, 1.3 mmol) in 70 ml of methylene chloride was added 0.26 g of methanesulfonic acid. After stirring at room temperature overnight, the solvent in the mixture was evaporated, and 45 ml of methanol was added to the residue. After refluxing for 2 hours. the yellow solid was collected on a filter, dried in vacuo at 78®C for 2 hours to give 0.86 g (81%) of Va mp 212-213°c (dec) . *H-NMR (DMSO-de) δ 9.53 (d, 2H, J»2.2 Hz, aromatic protons), 8.96(d, 2H, J-2.2 Hz, aromatic protons), 8.83 (d, 2H, J*7.7 Hz, aromatic protons), 8.69 (d, 2H, J-7.3 Hz, aromatic protons), 8.7-8.8 (broad, 4H, 2 ΝΗ2Ί, 8.09 (t, 2H, J-7.9 Hz, aromatic protons), 5.46 (m, 2H, 2 CH), 3.86 (m, 2H), 3.2-3.5 (m, 6H), 2.20 (s, 6H, 2 CH3) and 1.57 (d, 6H, J*6.9 Hz, 2 CH3) . MS (DCI) m/e 625 (M+l), Anal. Calcd for C32H28N5O3 . 2 CH3SO3H (MW 816.81): C, 50.00; H, 4.44; N, 10.29; S, 7.85. Found: C, 49.88; H, 4.39; N, 10.14; S, 7.86.

Similarly, Examples 2 and 3 may be prepared and characterized. 17 LV 10045

Exarcple.2 (racemate + meso)-2,2,-[l,2-ethanediylbis[imino(l-methyl-2,l-ethanediyl)J]-bis[5-nitro-lH-benz[de]isoquinoline-l,3(2H)-dione] methanesulfonate 5 (1:2) (Vc)

Light yellow solid (18% yield); mp 220-221eC (dec). 1H-NMR of Vc is almost identical with that of Va except that Vc shows d of d at δ 1.59 ppm due to the presence 10 of dl pair and meso. MS(DCI) m/e 625 (M+l). Anal.

Calcd for Ca^aNgOa . 2 CH3SO3H · H20 (MW 834.83): C, 43.92; H, 4.59; N, 10.07; S, 7.68. Found: C, 49.10, 49.01; H, 4.45, 4.40; N, 10.02, 9.96; S, 7.39, 7.45. 15 Exampls 1 (R,R)-2,2’-(1,2-ethanediylbis[imino(l-methyl-2,1-ethanediyl)]J-bis(5-nitro-lH-benz[de]isoquinoline-1,3 (2H)-dione) methanesulfonate (1:2) (Vb) 20 Light yellow solid (25% yield); mp 210-211eC (dec).

The ijļ-NMR of Vb is identical with that of Va. MS (DCI) m/e 625 (M+l). Anal. Calcd for C32H28N6°8 ' 2 CH3SO3H (MW 816.81): C, 50.00; H, 4.44; N, 10.29; S, 7.85. Found: C, 49.98; H, 4.41; N, 10.14; S, 7.81. 25

Examole 26 (Meso)-2,2(1,2-ethanediylbis(imino(l-methyl-2,1-ethanediyl)])-bis(5-nitro-lH-benz[de] isoquinoline-1,3 (2H)-dione] methanesulfonate (1:2) (XXVII) 30

Part A: (XXI) 1,1-Dimethylethyl- (S)-[2-[(cyanomethyl)amino]-l-methyl-2-oxoethyl]carbamate. A mixture of aminoacetonitrile hydrochloride (9.25 g, 100 mmol) and N-methylmorpholine (10.1 g, 100 mmol) 35 in 100 ml of THF was stirred for 15 minūtes. N-t-BOC- 18 (S)-alanine (18.9 g, 100 ml) and N-methylmorpholine (10.1 g, 100 mmoi) in 100 ml of THF was stirred with an ice-salt bath. Isobutyl chloroformate (12.97 ml, 100 mmol) was added at such a rāte that the temperature of the mixture won't exceed 10°C and the mixture of the nitrile-N-methylmorpholine suspension was added after 2 minūtes. The mixture was stirred at room temperature overnight. The N-methylmorpholine hydrochloride was removed by filtration and the solvent in the filtrate was evaporated. The remaining liquid was diluted with methylene chloride (500 ml), vashed with aq. K2CO3 (2 x 100 ml), H2O (1 x 100 ml). The layers were separated; and the organic layer dried over anhydrous MgSOi, filtered and evaporated to give 15.85 g (69.7%) of the crude product. This was purified by column chromatography to give pure XXI (14.88 g, 65.5%), mp 99-100°C. 1H-NMR (CDCI3) δ 7.32 (broad, 1H, NH), 5.12 (d, 1H, J-5.8 Hz, NH), 4.15 (m, 3H, CH and CH2), 1.46 (s, 9H, 3 CH3) and 1.39 (d, 3H, J-7.0 Hz, CH3). MS (CI) m/e 228 (M+l).

Part B: (XXII) 1,l-dimethylethyl-(S)-[2-((2-aminoethyl)amino]-l-methyl-2-oxoethyl]carbamate. A mixture of XXI (2.0 g, 8.8 mmol) and palladium hydroxide (0.5 g) in 15 ml of acetic acid was hydrogenated (50 psi) in a Parr shaker for 1 1/2 hours. The catalyst in the mixture was removed by filtration. The acetic acid in the filtrate was removed by vacuum distillation to give a light yellow viscous liquid, which was added 15 ml of methanol and 1 g of sodium bicarbonate. The mixture was stirred at room temperature for several hours. The sodium acetate was removed by filtration; and the solvent in the filtrate evaporated to give the crude product. This was purified by column chromatography to give pure XXII (0.92 g, 19 LV 10045 45.2%) as a viscous liquid. lH-NMR (CDCI3) δ 7.08 (broad, 1H, NH), 5.28 (broad, 1H, NH), 3.28-3.26 (m, 2H, CH2), 2.84 (t, 2H, J=»5.9 Hz, CH2), 1.41 (S, 9H, 3 CH3) and 1.34 (d, 3H, J=6.9 Hz, CH3) . MS (CI) m/e 232 (M+l) . 5

Part C: (XXIII) 1,1-Diroethylethyl (meso)-[1,2-ethanediylbis{imino (l-methyl-2-oxo-2, l-ethanediyl) ] ] -bis(carbamate). A mixture of N-t-BOC-(R)-alanine (0.74 g, 3.9 nunol) 10 and 1,1'-carbonyldiimidazole (0.64 g, 3.9 mmol) in 20 ml of methylene chloride was stirred with an ice bath cooling for 1 1/2 hours. To this, there was added XXII (0.91 g, 3.9 mmol) in 10 ml of methylene chloride with an ice bath cooling. The mixture was stirred at ambient 15 temperature overnight. The product was collected on a filter to give XXIII (1.22 g, 77.9%) as a white solid. 1H-NMR (DMSO-dg) 6 7.83 (broad, 2H, 2 NH), 6.85 (m, 2H, 2 NH), 3.90 (m, 2H, 2 CH), 3.13 (m, 4H, 2 CH2), 1.40 (s, 10H, 6 CH3) and 1.18 (d, 6H, 2 CH3) . MS (CI) m/e 403 20 (M+l).

Part D: (XXIV) (Meso) -N,N'-1,2-ethanediylbis[2-aminopropanamide] dihydrochloride. A mixture of XXIII (1.18 g, 2.9 mmol) and 2.7 ml of 25 4.4 N HCl in 40 ml of dioxane was heated to reflux for 5 hours and then cooled to room temperature overnight.

The solvent in the mixture was evaporated to give XXIV (0.75 g, 94%) as a white solid. lH-NMR <DMS0-d6) 68.81 (broad, 2H, 2 NH), 8.33 (broad, 4H, 2 NH2), 3.83 (q, 2H, 30 J-6.9 Hz, 2 CH), 3.21 (broad s, 4H, 2 CH2) and 1.37 (d, 6H, J-6.9 Hz, 2 CH3) .

Part E: (XXV) (Meso)-N1,N1'-l,2-ethanediylbis[l,2-propanediamine]tetrahydrochloride 20 Το 30 ml or 1 M borane THF complex, there was added 0.75 g (2.7 mmol) of XXIV. The mixture was refluxed overnight. After cooling to room temperature, methanol (15 ml) was added slowly to the reaction mixture. The 5 mixture was then refluxed for 3 days. The solvents in the mixture were evaporated. To the residue was added 20 ml of methanol and 1.5 ml of conc. HC1. The mixture was stirred at room temperature for 4 hours, and then the solvent and excess HC1 evaporated to give XXV (0.81 10 g, 93.7%) as a white solid. ^-NMR (D20) 53.66 (m, 2H, 3.37 (s, 4H), 3.24 (m, 4H) and 1.29 (d, 6H, J-7.0 Hz, 2 CH3) . MS (CDI) m/e 175 (M+l) .

Part F; (XXVI) (Meso)-N1,N1 ’-l,2-ethanediylbis[1,ΣΙ 5 propanediamine]

To a freshly prepared solution of sodium ethoxide in ethanol (0.26 g of sodium in 40 ml of ethanol) was added 0.81 g of XXV. The mixture was stirred at room temperature for 1 hour. The sodium chloride was removed 20 by filtration; and the solvent in the filtrate evaporated. The product was isolated from the residue by Kugelrohr distillation (88-96°C at 0.4 mm) to give XXVI (0.32 g, 73.4%) as a clear liquid. 1H-NMR (CDCI3) δ 2.88 (m, 2H, 2 CH), 2.63 (m, 4H, 2 CH2), 2.48 (m, 2H, 25 CH2) / 2.29 (m, 2H, CH2), 1.54 (broad s, 6H, 2 NH2 and 2 NH) and 0.96 (d, 6H, J-6.2 Hz, 2 CH3). MS(CI) m/e 175 (M+l).

PartG: (XXVII) (Meso)-2,2 *-(1,2- 30 ethanediylbis[imino(l-methyl-2,l-ethanediyl)]]-bis{5-nitro-lH-benz(de]isoquinoline-l/3(2H)-dione] methanesulfonate (1:2). A mixture of 3-nitro-l,8-napthalic anhydride (0.78 g, 3.2 mmol) and XXVI (0.28 g, 1.6 mmol) in 25 ml 35 of ethanol was stirred at room temperature overnight and 21 LV 10045 then heated to reflux for 2 2/3 hours. The solvent in the mixture was evaporated to give the crude products. This was purified by column chromatography to give pure product (0.39 g, 39%) as its free base. This was 5 converted to its methanesulfonate salt, XXVII (0.33 g, 25.3%); mp 243-244.5°C (dec). 1H-NMR (DMSO-dg) 59.53 (s, 2H, aromatic protons), 8.96 (s, 2H, aromatic protons), 8.82 (d, 2H, J=8.0 Hz, aromatic protons), 8.77, 8.74 (broad, 4H, 2NH2+), 8.70 (d, 2H, J»6.9 Hz, 10 aromatic protons), 8.09 (t, 2H, J“7,7 Hz, aromatic protons), 5.47 (m, 2H, 2 CH), 3.87 (m, 2H), 3.43 (m, 2H), 3.27 (broad, 4H), 2.20 (s, 6H, 2 CH3SO3H) and 1,58 (d, 6H, J=6.6 Hz, 2 CH3). IR (KBr) 3445 (NH), 1770, 1668 (OO)citT1. MS (DCI) m/e 625 (M+l). Anal. Calcd 15 for C32H28N60e · 2 CH3SO3H · 1/2 H20 <MW 826.53); C, 49.41; H, 4.60; N, 10.17; S, 7.76. Found: C, 49.45, 49.41; H, 4.33, 4.29; N, 10.11, 10,21; S, 7.50, 7.58.

Exampl a.27. 20 (S,S)-2,2'-(l, 2-ethanediylbis [imino(2-methyl-2,1- ethanediyl)))-bis(5-nitro-lH-benz [de]isoquinoline-1,3(2H)-dione] methanesulfonate (1:2) (XIXa)

Part A: (XVa) Dimethyl N,N'-l,2-dioxo-l,2-25 ethanediyl) bis [S-alanine]. A mixture of (S)-alanine methyl ester hydrochloride (14 g, 100 mmol) and oxalyl chloride (4.9 ml, 55 mmol) in 150 ml of benzene was refluxed overnight. After cooling to roora temperature, the white solid was 30 collected on a filter to give 12.36 g (95%) of XVa; mp 167-170°C. 1H-NMR (DMSO-de) 5 9.15 (d, 2H, 2 NH) 4.40 (quintet, 2H, 2 CH), 3.67 (s, 6H, 2 OCH3) and 1.40 (d, 6H, 2 CH3). MS (DCI) m/e 261 (M+l). [a]D -65.32° (c-1.012, AcOH). 35 22

Part B; (XVIa) (S/S)-Ν,Ν'-bis(2-amino-l-methyl-2-oxoethyl)ethanediamine.

Compound XVa (5.21 g, 20 mmol) was added to a methanol solution saturated with ammonia with an ice 5 bath cooling. The mixture was further bubbled with ammonia for 1 hour; and then stirred at room temperature overnight. The white solid was collected on a filter to give 3.65 g (79%) of XVIa. *H-NMR (DMSO-dg) δ 8.50 (d, 2H, J-7.7 Hz, 2 NH), 7.50 (S, 2H, NH2), 7.21 (S, 2H, 10 nh2), 4.25 (quintet, 2H, J-7.3 Hz, 2 CH) and 1.31 <d, 6H, J-7.3 Hz, 2 CH3) . MS (DCI) m/e 231 (M+l).

[α)ο +69.14°(c-0.418, DMF) .

Part C: (XVIIa) (S,S)-N2,N2’-1,2-ethanediylbis (1,2-15 propanediamine] tetrahydrochloride.

To a mixture of XVIa (3.5 g, 15.2 mmol) in 100 ml of THF, there was added 150 ml of 1M BH3.THF complex. After refluxing overnight, the reaction mixture was cooled to room temperature and 80 ml of methanol was 20 added slowly. The mixture was refluxed overnight, and a small amount of precipitate present were removed by filtration. The solvents in the filtrate were evaporated; and to the remaining liquid was added 30 ml of methanol and 7.5 ml of conc. HC1 with an ice bath 25 cooling. After stirring for 1.5 hours, the mixture was triturated with ethyl ether; and the product was collected on a filter to give 3.92 g (81%) of XVIīa; 1H-NMR (D20) δ 3.54 (m, 2H, 2 CH), 3.31 (m, 6H), 3.06 (m, 2H) and 1.28 (d, 6H, J-6.6 Hz, 2 CH3). MS(DCI) m/e 175 30 (M+l, free base) . (a]0 +8.39® (c-0.632, H20) .

Part _D: (XVIIIa) (S,S)-N2,N2'-l,2-ethanediylbisH,2-propanediaminel.

Compound XVlla (3.2 g, 10 mmol) was added to a 35 freshly prepared sodiura ethoxide solution in ethanol LV 10045 <1.03 g of sodium in 50 ml of ethanol). After stirring at room temperature overnight, sodium chloride in the mixture was removed by filtration, and the solvent in the filtrate evaporated. The remaining mixture was purified by Kugelrohr distillation (110-124® C at 0.7 mm) to give 1.43 g (82%) of XVIIIa as a clear liquid. 1H-NMR (CDCI3) δ 2.8-2.4 (m, 10H, 4 CH2 and 2 CH), 1.57 (broad, 6H, 2 NH2 and 2 NH) and 0.91 (d, 6H, J-5.9 Hz, 2 CH3) . [α]ο +120® (c=0.310, benzene).

Part E; (XIXa) (S, S) -2,2’-[1,2-ethanediylbis [imino(2-methyl-2, l-ethanediyl) ] ]-bis [5-nitro-lH-benz[de]isoquinoline-l,3(2H) -dione] methanesulfonate (1:2) . A mixture of 3-nitro-l,8-naphthalic anhydride (1.46 g, 6.0 mmol) and XVIĪIa (0.52 g, 3 mmol) in 30 ml of ethanol was stirred at room temperature overnight. To this, there was added 0.63 g of methanesulfonic acid. After stirring overnight, the product was isolated by filtration and purified by heating in 50 ml of methanol overnight to give 1.37 g (55%) of XIXa; mp 254-255®C (dec). lH-NMR (DMSO-dg) 8 9.55 <d, 2H, J-1.9 Hz, aromatic protons), 9.0 (d, 2H, J-1.9 Hz, aromatic protons), 8.85 (d, 2H, J-8.1 Hz, aromatic protons), 8.73 (d, 2H, J=*7.0 Hz, aromatic protons), 8.11 (t, 2H, J*7.9 Hz, aromatic protons), 4.39-4.27 (m, 4H, 2 CH2), 3.75 (m, 2H, 2 CH), 2.26 (s, 6H, 2 CH3) and 1.35 (d, 6H, J-5.9 Hz, 2 CH3). MS (DCI) m/e 625 (M+l). Anal. Calcd for C32H28N608 * 2 CH3SO3H · H20 (MW 834.83): C, 48.92; H, 4.59; N, 10.07; S, 7.68. Found: C, 48.97, 48.84;H, 4.43, 4.46; N, 10.16, 10.13; S, 7.83, 7.84. 5 24

Example 28 (S, S) -2, 2*-(1, 2-ethanediylbis [imino(2-methyl-2,1-ethanediyl)]3-bis[ΙΗ-benz[de]isoquinoline-l,3(2H)-dione] methanesulfonate (1:2) (XXa)

By replacing 3-nitro-l,8-naphthalic anhydride with 1,8-naphthalic anhydride, compound XXa can be prepared.

White solid (78% yield); mp 289-290eC (dec) . XH-NMR (DMSO-dg) 5 8.96 (broad, 2H, NH2+), 8.80 (broad, 2H, 10 15 NH2+)/ 8.57-8.53 (m, 8H, aromatic protons)/ 7.94 (t, 4H, J=7.7 Hz, aromatic protons)/ 4.45-4.23 (m, 4H, 2 CH2)/ 3.81 (m, 2H, 2 CH), 2.28 (s, 6H, 2 CH3) and 1,36 (d, 6H, j=*6.6 Hz, 2 CH3) . MS (DCI) m/e 535 (M+l, free base) . Anal. Calcd for C32H3oN404 2 CH3S03H · H20 (MW 744.83): C, 54.83; H, 5.41; N, 7.42; S, 8.61. Found: C, 55.18, 55.29; H, 5.17, 5.24; N, 7.42, 7.47; S, 8.66, 8.64.

Examplg_2i 20 (R, R) -2,2'- (1,2-ethanediylbis (imino(2-methyl) -2,1- ethanediyl))-bis(5-nitro-lH-benz (de]isoquinoline-1,3(2H)-dione]-methanesulfonate (1:2) (XIXb)

Light brown solid (34.5% yield); mp 248-251°C (dec) . 25 lH-NMR (DMSO-dg) 6 9.54 (d, 2H, J»2.2 Hz, aromatic protons), 8.99 (d, 2H, J-1.9 Hz, aromatic protons), 8.83 (d, 2H, J=*8.0 Hz, aromatic protons), 8.72 (d, 2H, J**6.9 Hz, aromatic protons), 8.10 (t, 2H, J-7.9 Hz, aromatic protons), 4.33-4.18 (m, 4H, 2 CH2), 3.46 (m, 6H), 2.28 30 (s, 6H, 2 CH3) and 1.28 (m, 6H, 2 CH3). MS(C1) m/e 625 (M+l) . Anal. Calcd for C^HieNgOg · 2 CH3S03H (MW 816.81) C, 50.00; H, 4.44; N, 10.09; S. 7.85. Found: C, 50.16; H, 4.34; N, 10.19; S, 7.62. 25 LV 10045

Example 30 (Racemic + Meso)-2, 2*-(l, 2-ethanediylbis[imino[2-methyl-2, l-ethanediyl] -bis (5-nitro-lH-benz [de] isoquinoline-1,3 (2H)-dione]-methanesulfonate (1:2) (XIXc)

Yellow solid (18.9% yield); mp 278-281eC (dec). 1H-NMR (DMSO-dg) $9.58 (d, 2H, aromatic protons), 9.0 (d, 2H, aromatic protons), 8.85 (d, 2H, aromatic protons), 8,75 (d, 2H, aromatic protons), 8.13 (t, 2H, aromatic protons), 4.50-4.25 (m, 4H, 2 CH2), 3.80 (m, 2H, 2 CH), 3.40 (m, 4H, 2 CH2), 2.30 (s, 6H, 2 CH3) and 1.38 (d, 6H, 2 CH3). MS (CI) m/e 625 (M+l) . Anal.

Calcd for C32H28Ng08 · 2 CH3SO3H (MW 816.81) C, 50.00; H, 4.44; N, 10.09; S. 7.85, Found: C, 49.62; H, 4.42; N, 10.07; S, 7.80.

Esampls „31 (R,R)-2,2'-(1,2-ethanediylbis[imino(2-methyl-2,1-ethanediyl]-bis[6-nitro-lH-ben2(de]isoquinoline-l,3(2H)-dione] methanesulfonate (1:2) (XXVIIlb)

Yellow solid (17.0% yield); mp 230-233®C (dec). 1H-NMR (DMSO-d6) 5 9.03 (broad, 2H, NH2+), 8.93 (broad, 2H, NH2+), 8.76 (d, 2H, J*8.8 Hz, aromatic protons), 8.68-8.57 (m, 6H, aromatic protons), 8.13 (t, 2H, J*7.7 Hz, aromatic protons), 4.37-4.26 (m, 4H, 2 CH2), 3.77 (m, 2H, 2 CH), 3.36 (s, 4H, 2 CH2), 2.25 (s, 5.4H, 1.8 CH3SO3H) and 1.35 (d, 6H, 2 CH3). MS (CI) m/e 625 (M+l). Anal. Calcd for C32H28N6O3 *1.8 CH3SO3H (MW 797.59) C, 50.90; H, 4.45; N, 10.54; S. 7.24. Found: C, 50.65, 50.56; H, 4.36, 4.36; N, 10.22, 10.18; S, 6.96, 6.96. 26

Example 22 (R,R)-2, 2 *-(1,2-ethanediylbis[imino[2-methyl-2,1-ethanediyl)]-bis[5,8-dinitro-lH-benz[de]isoquinoline-1/3(2H)-dione] methanesulfonate (1:2) (XXIXb)

Light brown solid (21.1% yield); mp 227-230eC (dec) . ^-NMR (DMSO-dg) δ 9.84 (d, 4H, J-1.5 Hz, 4 aromatic protons), 9.13 (d, 4H, J31.4 Hz, aromatic protons), 9.02 (broad, 2H, NH2+), 8.86 (broad,,2H, NH2+), 4.50-4.25 (m, 4H, 2 CH2), 3.81 (m, 2H, 2 CH), 3.34 (broad, 4H, 2 CH2), 2.24 (s, 6H, 2 CH3) and 1.39 (d, 6H, J-4.7 Hz, 2 CH3) . Anal. Calcd for C32H28N6O12 · 2 CH3SO3H (MW 906.80) C, 45.03; H, 3.75; N, 12.36; S, 7.06. Found: C, 44.75; H, 3.69; N, 12.21; S, 6.87.

Example 42 (Racemic)-2,2(1,2-ethanediylbis[imino(2-methyl-2,1-ethanediyl]-bis[5-nitro-lH-benz [dej isoquinoline-l,3(2H)-dione] methanesulfonate (1:2) (XXXIV)

The compound was prepared by mixing equal amounts of the corresponding (S,S) and (R,R) enantiomers (i.e., XIXa and XIXb, respectively). Light brown solid (901 yield); mp 254-255eC (dec). NMR spectrum is identical with those of XIXa and XIXb. Anal. Calcd for ^32^28^6^8*2CH3SO3H (MW 816.81) C, 50.00; H, 4.44; N, 10.29; S, 7.85. Found: C, 49.69; H, 4.21; N, 10.19; S, 7.66. 27 LV 10045

Exflrpplg Λ1 (S, S,S) -5-nitro-2- [2- 1(2-(12- (5-nitro-l, 3-dioxo-lH-benz(de)isoquinoline-2(3H)-yl)-l-methylethyl]amino]-l-methylethyl]amino] -l-methylethyl] -1H-benz(de]isoquinoline-l,3(2H)-dione] methanesulfonate (1:2) (ΧΧΧΧ)

Part A: (XXXV) Methyl N-[N-(N-[(1,1- dimethylethoxy)carbonyl]-L-alanyl]-L-alanyl]-L-alanine.

To a THF solution (50ml) of (t-BOC)-Ala-Ala-OH (5.0 g, 19.2 mmol) and N-methylmorpholine (3.Θ8 g, 38.4 mmol), there was added isobutylchloroformate (2.62 g, 19.2 mmol) dropwise to maintain the temperature between 10 to 15°C. After addition was completed, the reaction mixture was stirred for another 15 minūtes. To this, there was added 2.68 g (19.2 mmol) of (s)-alanine methyl ester hydrochloride. After stirring at ambient temperature overnight/ the white N-methylmorpholine hydrochloride was removed by filtration; and the solvent in the filtrate evaporated. The remaining liquid was diluted with methylene chloride (350 ml)# vashed with 5% sodium bicarbonate (2 x 150 ml), water (1 x 150 ml), 0.1 N HC1 (1 x 150 ml), water 1 x 150 ml), brine (1 x 100 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to give 4.32 g of a white solid, XXXV (65.11 yield): mp l68-172eC. 1h-NMR (CDCI3) 5 6.70 (m, 2H, 2 NH), 4.98 (broad, 1H), 4.48 <m, 2H, 2 CH), 3.70 (s, 3H, CH3), 1.38 (s, 18H, 6 CH3) and 1.30 (m, 6H, 2 CH3). MS (Cl) m/e 346 (M+l). IR (KBr) 3391, 3319, 3275 (NH), 1742, 1710, 1674, 1638 (C-0) cm*l. (α)25ρ -50.33° (C-0.600, CH2CI2).

Part B: (XXXVI) N-((1,l-dimethylethoxy)carbonyl]-L-alanyl-L-alanyl-L-alaninamide. 28 Το 80 ml of methanol saturated with ammonia, there was added 2.75 g (7.98 mmol) of XXXV. The reaction mixture was cooled with an ice bath, and then further reacted with ammonia for 10 minūtes. The reaction was stirred at ambient temperature overnight. The solvent in the solution was evaporated to give a yellow solid, XXXVI (2.45 gf 93% yield); mp 202-208°C. ^H-NHR (DMS0-dg) δ 7.83-7.95 (m, 2H, 2 NH)f 7.25 (broad, 1H, NH), 7.01 (broad, 2H, NH2)t 4.2 (m, 2H, 2 CH), 3.83 (m, 1H, CH) 1.38 (s, 9H, 3 CH3) and 1.20 (m, 9H, 3 CH3). MS (DC1) m/e 331 (M+l) . [CC]25d - 6.00° (c=0.0600, DMSO) .

Part C; (XXXVII) L-alanyl-L-alanyl-L-alaninamide hydrochloride. A mixture of XXXVI (1.95 g, 5.9 mmol) and 4.4 M HC1 (2.63 ml) in dioxane was added to 40 ml of dioxane at 0eC for 2 hr, and then warmed to room temperature overnight. The solvent in the mixture was evaporated to dryness to give 1.77 g of XXXVII as an off-white solid; mp 237-240°C (dec) . 1H-NMR (D2O) 5 4.2-4.05 (m, 2H 2 CH), 2.95-3.85 (m, 1H, CH) and 1.25 (m, 9H, 3 CH3), MS (Cl) m/e 231 (M+l). IR (KBr) 3438, 3294 <NH2>, 1676, 1639 (CO) cm"!. [a]25D -19.96° (c-0.606, CH3OH) .

Part D: (XXXVIII) (S, S, S)-N1-(2-amino-l-methylethyl) -N2- (2-aminopropyl) -1# 2-propanediamine tetrahydrochloride.

To a mixture of XXXVII (1.70 g, 6.37 mmol) in 150 ml of ΤΗΓ, there was added 58 ml of 1 M BH3»THF complex.

The mixture was refluxed overnight to become a clear solution. The solution was cooled in an ice bath and guenched dropwise with 50 ml of methanol. The solution was refluxed overnight. The solvents in the reaction solution were removed by rotary evaporation. To the remaining liquid was added methanol (50 ml) and it was 29 LV 10045 evaporated again to remove trimethylborate. The liquid was diluted with methanol (25 ml) followed by the addition of 4.0 ml of conc HC1. After stirring for 2 hr, this was triturated with ethyl ether, filtered to give 1.10 g (51.71 yield) of XXXVIII as a vhite solid; mp 277-27 8°C (dec) . MS (DC1) m/e 189 (M+l) . IR (KBr) 3436 (NH, NH2) cm"!.

Part E; (XXXIX) (S, S, S)-N1-(2-amino-l-methylethyl) -N2-(2-aminopropyl) -1,2-propanediamine.

To a freshly prepared sodium ethoxide solution (0.32 g of sodium of 20 ml of ethanol), there was added 1.05 g (3.1 mmol) of XXXVIII. The mixture was stirred at room temperature overnight. The sodium chloride was removed by filtration; and the solvent in the filtrate evaporated. The remaining liquid was purified by Kugelrohr distillation (0.4 mm, 94 - 104eC) to give 0.41 g of XXXIX as a light yellow ļiquid (70.2% yield) *H NMR (CDCI3) δ 2.90 (m, 1H, CH), 2.80 (m, 2H, 2 CH), 2.53-2.30 (m, 6H, 3 CH2), 1.58 (broad, 6H, 2 NH2 and 2 NH) and 0.98 (m, 9H, 3 CH3).

Part F: (ΧΧΧΧ) (S,S,S)-5-nitro-2-(2-[ (2-({2-(5-nitro- 1,3-dioxo-lH-benz[de]isoquinoline-2(3H)-yl)-l-methylethyl]amino]-l-methylethyl]amino)l-methylethyl]-ΙΗ-benz(de]isoquinoline-l,3(2H)-dione] methanesulfonate (1:2) . A mixture of 3-nitro-l,8-naphthalic anhydride (1.06 g, 4.4 mmol) and XXXIX (0.41 g, 2.2 mmol) in 25 ml of ethanol was stirred at room temperature overnight and then heated to reflux for 1 1/4 hours. The solvent in the mixture was evaporated, and the remaining dark residue was purified by column chromatography to give 0.83 g of the free base as a light brown solid (59.1% yield), which was then converted to its 30 methanesulfonate, ΧΧΧΧ (0.65 g; 35.6% yield); mp 189-192°C (shrlnJc). *H NMR (CDCI3) δ 9.28 {d, 1H, J-1.9 Hz, aromatic proton), 9.25 (d/ 1H, J*2.2 Hz, aromatic proton), 9.15 <d, 1H, J»2.2 Hz, aromatic proton), 9.12 5 (d, 1H, J*2.2 Hz, aromatic proton, 8.81 (d, 1H, J»7.3

Hz, aromatic proton, 8.76 (d, 1H, J-7.3 Hz, aromatic proton), 8.46 (d, 1H, J=8.1 Hz, aromatic proton), 8.42 (d, 1H, J=8.4 Hz, aromatic proton), 7.98 (t, 1H, J-7.7

Hz, aromatic proton), 7.94 (t, 1H, J-7.92 Hz, aromatic 10 proton), 5.76 (m, 1H, CH), 4.59 (m, 1H), 4.36 (m, 2H), 404 (m, 1H), 3.91 (m, 1H), 3.96 (m, 1H), 3.39 (m, 2H), 1.99 (s, 6H, 2 CH3), 1.64 (d, 3H, J-7.0 Hz, CH3), 1.54 (d, 3H, J=6.6 Hz,' CH3) and 1.48 (d, 3H, J-6.6 Hz, CH3) . MS (DC1) m/e 639 (M+l). Anal. Calcd for 15 C33H3oN$08»2CH3S03H (MW 830.44) c, 50.60; H, 4.61; N, 10.12; S, 7.72. Found: C, 50.70; H, 4.61; N, 9.98; S, 7.69. 31 LV 10045

Table 1

(')

R2, R3, R4, R5/ and R6 » H £x. 1^1’ B1, B7 (3teceochemistry> 1 3-N02 H CH3 (S) 2 3-N02 H CH3 (racemic ♦ meso) 3 3-N02 H CH3 (R) 5 3-N02 6-NO2 CH3 (S) 6 4-N02 H CH3 (S) 7 3-N02 6-NO2 CH3 (R) 8 4-N02 H CH3 (R) 26 3-N02 H CH3 (meso)

In Table 1 the position of X and Y is indicated uslng the numbering for the position in the corresponding naphthalic anhydride, and not the numbering for the lH-benz(de]isoquinoline-l,3(2H)-dione. 32

Iaklg-2

R1, and R5 H Eju ī*_r β2 β6 β4 fi7 (stereochemistry) 27 3-N02 H CH3 (S) H H 28 H H CH3 (S) H H 29 3-N02 H CH3 (R) H H 30 3-N02 H CH3 (racemic + meso) H H 31 4-N02 H CH3 (R) H H 32 3-N02 6- •NO2 CH3 (R) H H 42 3-N02 K CH3 (racemic) H H 43 3-N02 H Rj-CH3 (S); Re-H CH3 (S) CH3 (S) 10 In Table 2 the position of X and Y is indicated using the numbering for the position in the corresponding naphthalic anhydride, and not the numbering for the ΙΗ-benz[de]isoquinoline-l,3(2H)-dione. 33 LV 10045

Ut.il i£g

Γη vitro Growth Inhibi-torv ActivitY 5 10 L1210 celis were maintained in RPMI-1640 a medium supplemented with 10% heat inactivated fetal bovlne serum and 50 mL mercaptoethanol/liter medium (RPMI-L) . B16 celis were maintained in RPMI-1640 medium supplemented with 15% heat inactivated fetal bovine serum and antiobiotics (RPMI-C).

Exponentially groving murine leukemia L1210 celis (lxl03 celis) in 0.1 mL medium were seeded on day 0 in a 15 96-well microtiter plate. On day 1, 0.1 μΐ aliquot of medium containing graded concentrations of tēst analogs was added to the initial volume. After incubation at 37°C in a humidified incubator for 3 days, the plates were centrifuged briefly and 100 mL of the growth medium 20 was removed. Celi cultures were incubated with 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT; 1 mg/ml in Dulbecco's phosphate buffer saline) for 4 hours at 37°C. The resulting purple formazan precipitate was solubilized with 200 μL of 0.04 25 N HC1 in isopropyl alcohol. Absorbance was read in a Titertek Multiskan MCC scaning well spectrophotometer (Flow Laboratories) at a tēst vavelength of 570 nm and a reference vavelength of 630 nm.

The ID50 values were determined by a Computer 30 program that fit ali of the data (8 determinations per concentration and 12 concentrations per tēst analog) to the following equation: Y- {(Am - Ao)/(l+(X/ID5Q)n))+Ao where: Am - absorbance of the control celis; Ao -35 absorbance of the celis in the presence of highest drug 34 concentration; Υ » observed absorbance; X - drug concentration; ID50 » dose of drug that inhibits the growth of celis to one half that of the control celis. 5 Results of the in vitro L1210 growth inhibition testing are shown in Table 3.

Iable_2 . v.q,+ ΙΰΐΔ. mg/ml), 1 0.047 2 0.062 3 0.051 26 0.074 27 0.0025 28 0.21 29 <0.01 30 <0.01 31 0.19 32 0.057 42 <0.001 43 0.04 in.Vivo Tumor Modeļa

Representative compounds of the present invention 15 have been extensively tested in a variety of pre-clinical tests of anti-cancer activity which are indicative of clinical utility. For example, the presently claimed compounds show striking in vivo efficacy against three types of human tumors xenografted 20 in nude mice, namely DLD-2 human colon carcimonui, ΜΧ-1 human mammary carcinoma, and LX-1 human lung carcinoma. 35 LV 10045

In addition, a representative compound of the present invention is active against several murine tumors, including murine M16c mammary adenocarcinoma and murine C51 colon adenocarcinoma. In addition, a representative compound of the invention is active against murine mammary tumors in transgenic mice containing the v-Ha-ras oncogene.

The methods used in the testing of compounds in the in vivo human tumor xenograft models are described below.

In Vivo Human Tumor Xenoaraft Models

The DLD-2 human colon tumor, ΜΧ-1 human mammary carcinoma, and LX-1 human lung tumor were originally obtained from a surgically removed primary colon carcinoma, breast tumor, and non-small lung carcinoma, respectively. The human tumor lines were maintained by serial passage in athymic nude mice. The ΜΧ-1 human mammary carcinoma and LX-1 human lung tumor are established tumors used by the NCI. The DLD-2, ΜΧ-.1, and LX-1 tumor models have been well characterized.

The mice used in these experiments were outbred Swiss mice or BALB/c mice bearing the nude (nu/nu) gene. On day 0 male and female mice veighing 22-30 g are inoculated with 0.2 mL of a 25% tumor mince. This mince is prepared by mincing fresh tumor tissue, grown subcutaneously in passage mice, in sterile physiological saline. Palpable tumors veighing approximately 50 mg appear in the mice vithin 7-10 days after inoculation.

The mice are pair matched by tumor veight and sex into groups of ten each and the tēst compounds and vehicle control are administered intravenously (i.v.) once daily for nine consecutive days. A >20% decrease in body 36 veight on day 5 folloving compound administration is considered an indication of toxicity. Tumor measurements and body weights are recorded once a veek. Fifteen to 18 days after the initial injection the mice are 5 veighed, sacrificed and the tumors excised and veighed.

The efficacy of the tēst compounds is determined by the extent of tumor growth inhibition in treated versus vehicle-treated control mice. Initial tumor weights 10 (mg) are calculated from the tumor dimensions (mm) measured from caliper measurements, using the formula for a prolate ellipsoid (mg of tumor veight (length x width2)/2). Net tumor weights are calculated for each of the treated groups and the vehicle-treated control 15 group by subtracting the initial tumor veight from the final tumor veight on day 15. Results are expressed as a percentage decrease relative to the mean tumor veight for the control vehicle-treated group. 20

Tumor Grovth Inhibition 1 - mean tumor veight of treated mean tumor veight of control x 100

Activity Crlteria

The criteria of the National Cancer Institute (NCI) 25 for activity in the in vivo cancer models vere used.

Tumor grovth inhibition of 58-89% in the DLD-2 assay is considered moderate activity and inhibition of £90% is condidered good to excellent activity. Actual tumor regressions (IR * incomplete regression; FR - full 30 regression) indicate excellent to outstanding activity. Compounds demonstrating <58% grovth inhibition are considered inactive. 37 LV 10045

The compounds of Examples 1# 2, 3, 26/ 27/ and 30 exhibited excellent to outstanding activity against DLD-2 human colon tumors. Example 29 exhibited good to excellent activity against DLD-2 human colon tumors.

In addition, the compounds of Examples 2, ,3/ 27, 29/ and 30 exhibited excellent to outstanding activity in the ΜΧ-1 human breast tumor modei. Example 1 exhibited good to excellent activity against ΜΧ-1 human breast tumor.

Examples 2,3, 27, and 29 exhibited good to excellent activity against LX-1 human lung tumors.

Activit/ Against Mammary Carcinomas_in Transoenic Mice Containino the ras Oncogene

Transgenic mice carrying the v-Ha-ras oncogene linked to the MMTV promoter were constructed originally in the laboratory of Professor Phil Leder at Harvard University (Sinn et al. (1987) Celi 49: 465-475). The female transgenic animals develop mammary tumors, and thus can be used to evaluate aģents for activity against these breast tumors. The grovth characteristics of these tumors has been characterized extensively (Diamond and Dexter (1991) Proc. Amer. Assoc. Cancer Res. 32: 299) and mimic the clinical situation.

Female ras-containing transgenic mice with breast tumors were treated i.v. with saline Controls or with the tēst compound administered daily for 9 days and the group mean tumor growth rāte for each tēst compound was monitored.

The demonstrated effectiveness of the compounds of the present invention in the human colon, breast, and lung tumor xenograft models indicate that the compounds of the present invention may be useful for the treatment 38 of a broad spectrum of solid tumors in man, and, in particu'lar, tumors of the colon, breast, and lung. This conclusion is further supported by published analyses correlating pre-clinical tēst results with clinical efficacy of anti-cancer aģents. For example, see:

Goldin and Venditti (1980) Recent Results Cancer Research 76: 176-191; Goldin et al. (1981) Eur. J.

Cancer 17: 129-142; Mattern et al. (1988) Cancer and Metastasis Reviev 7: 263-284; Jackson et al. (1990) Cancer Investigations 8: 39-47. Based on these published analyses, the broad spectrum and exceptional high Ievel of antitumor activity exhibited by the presently claimed compounds provide strong evidence that the compounds claimed in present invention may have important therapeutic utility in the treatment of cancer in man.

Dosaoe and.Formulation

The antitumor compounds (active ingredients) of this invention can be administered to inhibit tumors by any means that producēs contact of the active ingredient with the agent’s site of action in the body of a mammai. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients.

They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and Standard pharmaceutical practice.

The dosage administered will be a tumor-inhibiting amount of active ingredient and will, of course, vary depending upon known factors such as the pharmacodynamic 39 LV 10045 characteristics of the particular active ingredient, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment, 5 and the effect desired. Usually a daily dosage of active ingredient can be about 5 to 400 milligrams per kilogram of body veight. Ordinarily, 10 to 200, and preferably 10 to 50, milligrams per kilogram per day given in divided doses 2 to 4 times a day or in 10 sustained release form is effective to obtain desired results.

Dosage forms (compositions) suitable for internai administration contain from about 1.0 milligram to about 500 milligrams of active ingredient per unit. In these 15 pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total veight of the composition.

The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and 20 povders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.

Gelatin capsules contain the active ingredient and povdered carriers, such as lactose, sucrose, mannitol, 25 starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication 30 over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere or enteric coated for selective disintegration in the gastrointestinal tract. 40

Liquid dosage forms for oral ādministration can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar Solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral Solutions.

Solutions for parenteral administration contain preferably a water soluble salt of the active ingredient, suitable stabilizing aģents, and if necessary, buffer substances. Antioxidizing aģents such as sodium bisulfite, sodium sulfite, or ascorbic acid either alone or combined are suitable stabilizing aģents. Also used are citric acid and its salts and sodium EDTA. In addtion, parenteral Solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Suitable pharmaceutical carriers are described in Remlngton1s Pharmaceutical Sciences, Mack Publishing Company, a Standard reference text in this field.

Useful pharmaceutical dosage forms for administration of the compounds of this invention can be illustrated as follows.

Capsules: Capsules are prepared by filling Standard two-piece hard gelatin capsules each with 100 milligrams of powdered active ingredient, 175 milligrams of lactose, 24 milligrams of talc, and 6 milligrams magnesium stearate.

Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are vashed and dried. 41 LV 10045

Tablets: Tablets are prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient# 0.2 milligrams of colloidal Silicon 5 dioxide# 5 milligrams of magnesium stearate# 275 milligrams of microcrystalline cellulose. 11 milligrams of cornstrach and 98.8 milligrams of lactose.

Appropriate coatings may be applied to increase palatability or delay absorption. 10

Injectable; A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made 15 isotonic with sodium chloride and sterilized.

Suspension: An aqueous suspension is prepared for oral administration so that each 5 milliliters contain 100 milligrams of finely divided active ingredient# 200 20 milligrams of sodium carboxymethyl cellulose# 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution# U.S.P.# and 0.025 milliliters of vanillin.

In the present disclosure it should be understood 25 that the specified materiāls and conditions are important in practicing the invention but that unspecified materiāls and conditions are not excluded so long as they do not prevent the benefits of the invention from being realized. 30 42 42 LV 10045 CLAIMS. WHAT IS CLAIMED IS: 1. A compound, or a pharmaceutically acceptable salt thereof, which is selected from the group consisting of: (S,S)-2,2'-[1,2-ethanediylbis Iimino(l-methyl-2,1-ethanediyl)]J-bis[5-nitro-lH-benz[de]isoquinoline-1,3(2H)-dione]; (Racemic + Meso)-2,2’-[1,2-ethanediylbis(iminotiniet hy 1-2, l-ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-l,3(2H)-dione]; (R, R)-2,2'-[1,2-ethanediylbis [imino(l-methyl-2/1-ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-1/ 3(2H)-dione]; (Meso)-2,2[1,2-ethanediylbis [imino(l-methyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz [de]isoquinoline-1/ 3(2H)-dione]; (S, S)-2,2'-(l,2-ethanediylbis [imino(2-methyl-2,1-ethanediyl) ] ] -bis [5-nitro-lH-benz [de] isoquinoline-1, 3(2H)-dione]; (Racemic + Meso)-2,2'-[l,2-ethanediylbis[imino(2-methyl-2,l-ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-l, 3(2H)-dione]; (R,R)-2,2'-[1,2-ethanediylbis (imino <2-methyl-2,1-ethanediyl) ] ] -bis [5-nitro-lH-benz [de] isoquinoline-1,3 (2H)-dione]; 43 (Meso)-2,2il,2-ethanediylbis [imino{2-methyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-l,3<2H)-dione]. 2. The compound, or a pharmaceutically acceptable / salt thereof, which is (R,R)-2,2'-[l,2- ethanediylbis[imino(l-methyl-2, l-ethanediyl) ] ]-bis [5-nitro-lH-benz[de]isoquinoline-l/3(2H)-dione]. 3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of Claim 1. 4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of Claim 2. 5. A method of treating a solid tumor carcinoma in a mammai comprising administering to a mammai bearing such a tumor, a tumor-inhibiting amount of a compound of Claim 1. 6. A method of treating a solid tumor carcinoma in a mammai comprising administering to a mammai bearing such a tumor, a tumor-inhibiting amount of a compound of Claim 2. LV 10045 \

5 TITLE

Bis-naphthalimides Containing Amino-Acid Derived Linkers as Anticancer Aģents

ABSTRACT OF THE INVENTIOM

This invention relates to bis-naphthalimides, including 2,2[1,2-ethanediylbis[imino (l-methyl-2,1-15 ethanediyl) ] ] -bis [5-nitro-lH-benz [de]isoquinoline-1,3(2H)-dione] and 2,2'-{l,2-ethanediylbislimino(2-methyl-2,l-ethanediyl)]]-bis[5-nitro-lH-benz(de]isoquinoline-l,3(2H)-dioneJ, processes for their preparation, pharmaceutical compositions containing 20 them, and methods of using them to treat cancer in mammals,

Claims (6)

Formula 1. A compound or a pharmaceutically acceptable salt thereof selected from the group consisting of: (5.5) -2,2 '- [1,2-ethanediyl] The compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of: (5.5) -2,2' - [1,2-ethanediyl] [imino (1-methyl-1,2-ethanediyl)] - bis [5-nitro-1H-benz [de] isoquinoline-1,3 (2H) -dione], (Racemic + Meso) -2.2 ' - [1,2-ethanediyl] imino (1-methyl-2,1-ethanediyl)] bis [5-nitro-1H-benz [de] isoquinoline-1,3 (2H) -dione], R) -2,2 '- [1,2-Ethanediyl] imino (1-methyl-1,2-ethanediyl)] bis [5-nitro-1H-benz [de] isoquinoline-1,3 (2H) -dione], (Meso) -2,2 '- [1,2-ethanediyl] imino (1-methyl-1,2-ethanediyl)] - bis [5-nitro-1H-benz [de] isoquinoline-1 , 3 (2H) -dione], (5.5) -2,2 '- [1,2-ethanediyl] imino (2-methyl-1,2-ethanediyl)] - bis [5-nitro-1H-benzo] de] isoquinoline-1,3 (2H) -dione], (Racemic + Meso) -2,2 '- [1,2-ethanediyl] imino (2-methyl > 2, l-ethanediyl)] - bis [5 -Nitro-1H-benzo [de] zinolin-1,3 (2H) -dione], (R, R) -2,2 '- [1,2-ethanediyl] imino (2-methyl-2, l -etāndii 1)]] - bis [5-nitro-1H-benz [de] isoquinoline-1,3 (2H) -dione], (Meso) -2,2 '- [1,2-ethanediyl] imino (2-methyl) -2,1-ethanediyl]] - bis [5-nitro-1H-benz [de] isoquinoline-1,3 (2H) -dione], A compound or a pharmaceutically acceptable salt thereof according to the invention. f. 1p, which is (R, R) -2,2 '- [1,2-ethanediyl] imino (1-methyl-2,1-ethanediyl)] - bis [5-nitro-1H-benz [de] isoquinoline) -1.3 (2H) -dione]. 2 3. A pharmaceutical composition comprising a therapeutically acceptable carrier and a therapeutically effective amount of a compound according to the invention. f. l.p. Medicament composition according to claim 3, characterized in that it contains a therapeutically acceptable carrier and a therapeutically effective amount of the compound according to the invention. f. 2.p. A method of treating solid carcinoma of mammals, characterized in that mammals of such a tumor are given a tumor-inhibiting amount of the compound of the invention according to the invention. Method according to claim 5, characterized in that the mammal is given a tumor-inhibiting amount of a compound according to the invention of f.2.p.