CA2106999A1 - Bis-naphthalimides containing amino-acid derived linkers as anticancer agents - Google Patents

Abstract

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

Description

W092/17453 2 1 0 6 9 9 9 PCT/US92/021~

11~

Bis-naphthalimides Containing ~mino-Acid Derived Linkers as Anticancer Agents .FI~:LD OF T~E INVENTION

This in~ention relates to bis-naphthalimides, including 2,2'-[1,2-ethanediylbislimino(1-methyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz~de]isoquinoline-1,3(2H)-dione] and 2,2'-11,2-ethanediylbi~[imino(2-methyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz~de]isoquinoline-1,3(2H~-dione], processes for their preparation, pharmaceutical compositions containing them, and methods of using them to trea~ cancer, particularly solid tumor carcinomas, in mammals.

Harnisch et al., U.S. Patent 4,841,052 issued June 20, 1989 describe naphthalic acid ~mides useful as charge-regulating substances in electrophotographic toners.

Brana et al.,U.S. Patent 4,874,863 issued October 17, 1989 discloses anticancer compounds of the formula:

WO92/17453 ~ t~ 9 PCT/US92/021~

;~ ,c.. , xl X3 ~N ~ ~ N~
~0` ~

wherein X1, X2, X3 and X4 are identical or different and are each H, NO2, NH2, C1-C6-alkylamino, di-Cl C6-alkylamino, OH, C1-C6-alkoxy, halogen, trihalomethyl, C1-C6 alkyl, formyl, C1-C~-alkylcaxbonyl, ureyl, C1-C6-alkylureyl and R
is a s~raight chain or branched C4-C1o-alkylene which is interrupted at one or two points in the 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.

There is provided by this invention bis-naphthalimide compounds ha~ing the formula ~

v ~ ~ R ~ O ~
X X' ~i) or enantiomeric or diastereomeric forms thereof, or mixtures of enantiomeric or diastereomeric forms W092/17453 2 1 0 ~ 9 ~ 9 PCT/US92/021~

thereof, or pharmaceutically acceptable salts thereof, wherein:

R1~ R2, R3, R4, R5, R~, and R7, independently, are H
or CH3;

X and X' are H or N02; and Y and Y' are H or N02, provided that at least one of X, X', Y, and Y' is N02.

Preferred compounds of the present invention include those compounds of formula (i) wherein:

Rl and R7 are CH3;

R2, R3, R4, R5, and R6 are H;

X and X' are N02, wherein the substituent is at the 3-position of the naphthalimide (i.e., the 5-pos~t~on of the lH-benzlde]isoquinoline-1,3(2H)-dione); and Y and Y' are H.
Preferred compounds of the pxesent invention also include those compounds of formula (i~ wherein:

R2 and R6 are CH3;
R1, R3, R4, RS, and R7 are H;

X and X' are N02, wherein the substituent is at the 3-position of the naphthalimide ~i.e., the 5-WO92~17453 .~ 1 ~ 6 ~ ~ ~ PCT~US92/02134 4 ~.

position of the lH-benz[de]isoquinoline-1,3(2H)-dione); and Y and Y' are H.S
Specifically preferred compounds of the present invention are the following:

~ StS)-2,2'-[1,2-ethanediylbis[imino~l-methyl-2,1 r ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-1,3t2H)-dione];

~ Racemic + ~eso)-2,2'-[1,2-ethanediylbistimino(l-methyl-2,1-ethanediyl)~]-bis[5-nitro-lH-benz[de]isoquinoline-1,3~2H)-dione];

(R,R)-2,2'-~1,2-ethanediylbis[iminotl-methyl-2,1-ethanediyl)]]-bis~5-nitro-lH-benz[de]isoquinoline-1,3(2H)-dione];
(Meso)-2,2'-11,2-ethanediylbistimino~l-methyl-2,1-ethanediyl)]]-bis~5-nitro-lH-benztde]isoquinoline-1,3(2H)-dione];

tS~s)-2~2~ 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~iminot2-methyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-1,3~2H)-dione];

tR~R)-2~2~-tl~2-ethanediylbis[imino(2-methyl-2 ethanediyl)]]-bist5-nitro-lH-benz~de]isoquinoline-1,3t2H)-dione]; or WO92/17453 2 1 0 G 9 9 9 P~T/US9~/021~

~ Meso)-2~2~-[l~2-ethanediylbisEimino(2-methyl-2 ethanediyl)]]-bis~5-nitro-lH-benz[de]isoquinoline-1,3~2H)-dione];
and pharmaceutically acceptable salts thereof.

Also provided by this invention are processes for the preparation of the compounds of 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 mammal.
Synthesis The present in~ention describes a series of bis-naphthalimides useful for treatin~ cancer containing linkers deri~ed from reduced peptides. Compounds of this invention are more soluble in aqueous media than prior art compounds not containing these reduced peptide linkers.
- Compound-q of this in~ention 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 produce 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 WO92/17453 2 1 0 6 ~ 9 ~ PCT/US92/021~
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 column chromatography before its salt can be prepared as described above.

Scheme y R1 R3 Rl R6 H2N ~ \N~

(ii) (iii) R3 R~ ~3 (i) W092/17~53 2 1 0 ~ 9 9 9 PCT/US92/02134 The parent anhydride (ii) is commercially available or can be prepared according to the procedures described by Hodgson et al., J~ Chem. Soc., 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'-carbonyldiimida~ole, followed by reaction withethylenediamine under standard conditions. Acid hydrolysis of the t-30C ~N-tert-butoxycarbonyl) protecting group in Ia was performed under standard conditions to afford IIa. The same procedure is utilized to produce compounds IIb or IIc by starting the sequence with the corresponding (R)-alanine ~compound b) or racemic alanine (compound c).
Reduction of IIa (dihydrochloride salt) with diborane in tetrahydrofuran at refluxing temperature afforded IIIa. IIIa was then neutralized with sodium ethoxide and the pure free base IVa was obtained by Kugelrohr distillation. IVa was reacted with the appropriate naphthalic anhydr$de in ethanol or tetrahydrofuran at reflux temperature to obtain Va.
Compounds Vb ~nd Vc are prepared similarly by using the corresponding IVb, or IVc, respectively.

WO92/17453 ~ 1 0 6 9 9 9 PCT/US92/021 . ~
` ` Scheme I

CH3NH2 ~CH2 ) 2NH2CH3 H o ( t -BOC ) HN ~ ~ ( t--30C ) HN ~ N ~N J~ NH ~ t -BOC ) a, S Ia, (S, S) b, R Ib, ~R,R) c, racemate Ic, d, 1 pair & meso H ~ H2N~N~NJ~rNH2 B2H6 ~' H2N~ ~N_l,NH2 2 HCl H CH3 2 HCl H CH3 Il III

NaOEt _~ H2N ~ N ~ NH2 EtOH, ~nhydride H CH3reflux IV

~N~N_N~ 2 C113S03N

Bis-naphthalimides of the formula ~XIX) where Rl~R3-R4-R5-R7~H, R2-R6=CH3 and X and Y are NO2 or H can be made by Scheme III. Reaction of ~S)-alanine methyl ester with oxalyl chloride in benzene at reflux temperature gave amide XVa. Amide XVa was converted to amide XVIa with ammonia in methanol under standard conditions. Amide XVIa was reduced with diborane in tetrahydrofuran to yield amine XVIIa as the tetrahydrochloride salt which was subsequently neu~ralized with sodium ethoxide to yield the free amine XVIIIa. Amine XVIIIa was condensed with the appropriate naphthalic anhydride to produce compound XIXa.
Compounds XIXb and XIXc could be prepared similarly by WO 9~/17453 2 1 0 ~ 9 ~ 9 PCT/US92/021~

using the corresponding compounds XVIIIb and XVIIIc, respectively. In addition, compounds XXa, XXVIIIb and XXIXb could be prepared by condensation of amine XVlIIb with appropriate naphthalic anhydrides.
Sçheme III

O benzene ~ O H CH3 NH~
H CoJ~NH2 ~ClrefluX H3COJ~ ~ ~OCH3 CH30H

a, s xv~, ~s, s b, R XVb, ~R, R~
c, racema~e xvc, ~racemate + meso~

H2N~ ~N~NE~2 ~ _~N ~,NH2 ~ lICl N oEt XVI XVII

H2N--~ N_ ~, NH EtOH, l~Et3 8~
c~3 H ~nhYdr~de, CH3 H O y , X--3 -N02 ~ Y--H ( S, S ) ~C--3-N02, Y--H ~R, R) ~, X-3-N02, Y-H ~rac~ mate ~ meao) .~a, X-Y--H ~ S, S ) XX~IIb, X--4-N02, Y--H (R, R) ~, X~3-N02, Y-6-N02 ~R, R) ~IY, X-3-N02, Y-H kacemate) WOg2/17453 % 1~ ~ PCT/IJS92~021~
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[de1isoquinoline-l,3(2H)-dione.
S 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 HCl salt with N-methylmorpholine), gave XXI. This was hydrogenated with palladium hydroxide in acetic acid to, yield XXII. Reaction of t-BOC-(R)-alanine with l,l'-carbonyldii'midazoLe, followed by addition of XX~I under standard conditions, afforded XXIII. Acid hydrolysis of 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 produce XXVII.

WO92/174~3 2 1 ~ ~ 9 9 9PCT/US92/021~

Sc~m~ IY

3 ~ OH ' ~C~3~2CHCH20COCl ~ N CN Pd~H)2 ~t-BOC)HN ~`~ ~ H2N ~ CN ~t-BOC)HN ~
o N-methylmorpholine O HOAc CH3 ,H IH

~t-BOC)HN ~ ~ NH2 XXII

~t-~OC)HN ~ 2. r~yIr tt-BOC~HN ~ N ~J~N ~ ., H~t BOC) ~meso), XXIII

H C~H3 H o 1. 3H3fTHF H ~ H
H2N~N NJ~"NH2- 2 HCl 2 o _ H2N~N N~,NH2~ 4 HCl o H CH3 H 3. HCl H CH3 H
~m~o), XXIV ~me o), xXv H CH3 H N ~ "~
H CH~ H
~m~o), XXVI

~ N ~ N - N ~ ~ 2 CH35C3H

O~N ~m-~o), XXVII N2 For compounds of the formula ~XXXX), the synthesis is shown in Scheme V (below). (t-BOC)-Alaninyl alanine was reacted with isobutyl chloroformate in methylene chloride at -10 to -15C in the presence of N-mothylmorpholine, followed by addition of (S)-alanine methylester hydrochlor~de 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 tetrahydrofuran yielded the polyamine tetrahydrochloride WO92/17453 PCT/US92/021~
210~999 12 XXXVIII, which was neutralized with sodium ethoxide to yield XXXIX. The amine XXXIX was condensed with the appropriate naphthalic anhydride to produce XXXX.

SshQms_~

(t-E~OC)HN~ ~OH ~ H N~OCH3 ~-- (t-BOC)HN~N~N~ocH3 o H H3o ~S,S.S) XXXV

NH3/M~OH CH3~H H CHJ~H NH HCI
(S-S-S)(t-80C)HN~ >~H~

X)(XVI
1. BH3/THF
H2N~N>~N~NH2 HCI 3 HCI C~H CH

(S,S.S) XXXVII ~S,S,S) XXXVIII

Na/EtOH CH3 y H CH3 ~H
H2N~N,~`N~NH2 (S,S,S) XXX~X

2 CH,SOJH

wo 92/17453 2 1 0 6 9 9 9 PCT/US92/021~
1~

Asymmetric bis-naphthalimides of the formula (XXXXVI) can be synthesized as shown in Scheme VI
~below). Reaction of XXII with l,l'-carbonyldiimidazole, followed by addition of (R)-benzyloxycarbonyl alanine, furnished XXXXI. Subsequentreaction with phosphorus pentasulfide in tetrahydrofuran gave the corresponding thioamide XXXXII. Treatment of XXXXII with Raney Nickel yielded amine XXXXIII. Amine XXXXIII was condensed with 3-nitro-l,8-naphthalic 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 produce non-symmetric bis-naphthalimides of the formula XXXXVI.

Scheme VI
1. CDI
(S) CH,~,,,H H - --(1 EIOC)HN~o --NH2 HA ,CH~
XXII (CB~HN ~

(t-80C)HN>~ ~ J~NH(CBZ3 P4S ~ $N ~NJ~NH(CBZ) XXXXI XXXXII
O
~y O
Ralley Ni t:H, H H ~ ~
(t-BOC)HN>~ ~rNH, _ ~ o ~, H H CH~ (e.g., 3-NC~2) XXXXIII
o,. ~Y~
~,~ H ~ HCI
~t-80C)HN~N ~N~--Qx XXXXIV y~ o CH~H ~;~ 3 HCI

XXXXV

~N--N

The in~ention can be further understood by referring to the following Examples and Tables below.

FxamalQ 1 W09~/l7453 2 1 0 ~ 9 9 9 PCT/U592/021~

~S,S)-2,2'-[1,2-ethanediylbis~imino(1-methyl-2,1-ethanediyl)]]-bis[5~nitro-lH-benz[de]isoquinoline-1,3~2H)-dione3 methanesulfonate ~1:2) (Va) Part ~: ~IIIa) (S,S)-Nl,Nl'-1,2-ethanediylbis[1,2-propanediamine} tetrahydrochloride.
To a THF (80 ml) suspension of IIa (2.75 g, 10 mmol), there was added 200 ml of lM BH3-THF complex slowly. The mixture was stirred at room temperature for 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 of methanol and 5 ml of conc. HCl. The resulting white solid was collected on a filter under nitrogen to give 1.92 g (60%) of IIIa; mp 210-215C. lH-NMR ~D2O) ~ 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) mJe 175 ~M+l, free base). [a]D - 2.31 ~c~0.606, H20).

E~L~ IVa) (S,S)-Nl,N1 -1,2-ethanediylbis[1,2-propanediamine].
Sodium ~1.03 g, 45 mmol) was added to 50 ml of anhydrous ethanol; and the mixture stirred for l.S hours until all the sodium was dissolved. To this, there was added 3.2 g ~10 mmol) of IIIa. After stirring for 2 hours, the sodium chloride precipitate was removed by filtrat~on; and the solvent in the filtrate evaporated.
Kugelrohr distillation (120-140C at 1.4 mm) gave 1.29 g (74%) of IVa as a clear li~uid. lH-NMR ~CDCl~) ~ 2.96 ~m, 2H, 2 CH), 2.71 ~m, 4H, 2 CH2), 2.60 ~m, 2H, CH2), 2,40 ~m, 2H, CH2), 1.46 ~broa~, 6H, 2 NH2 and 2 NH) and 1.06 ~d, 6H, J-6.2 Hz, 2 CH3). MS tDCI) m/e 175 ~M~l).

WO 92/17453 PCr/US92/02134 2106999 ~ "

Part C: ~Va) (S,S)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-1,3~2H)-dione] methanesulfonate (1:2).
A mixture of 3-nitro-1,8-naphthalic anhydride (2.43 g, 10 mmol) and IVa ~0.87 g, 5 mmol) 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 chlor~de was added 0.26 g of methanesulfonic acid. After stirring at room temperature o~rernight, the solvent in the mixture was evaporated, and 45 ml of methanol was added to the res-idue. After refluxing for 2 hours. the yellow solid was collected on a filter, dried in vacuo at 78C for 2 hours to give 0.86 g (81%) of Va mp 212-213c (dec). lH-NMR ~DMSO-d6) ~ 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 proton~), 8.7-8.8 (broad, 4H, 2 NH2~), 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+1). Anal. Calcd for C32H28N68 2 C}~3S03H (MW 816.81): C, 50.00; H, q.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 30 characterized.

WO92/174532 1 ~ ~ 3 ~ ~ PCT/USg2/021~

Exam~l~ 2 (racemate + meso)-2,2'-[1,2-ethanediylbistimino~l-methyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz[de]isoquinoline-1,3(2H)-dione] methanesulfonate (1:2) ~Vc) Light yellow solid (18% yield); mp 220-221C (dec~.
lH-NMR of Vc is almost identical with that of Va except that Vc shows d of d at ~ 1.59 ppm due to the presence of dl pair and meso~ MS(DCI) m/e 625 (M+l). Anal.
Calcd for C32H2gN6O6 . 2 CH3SO3H H2O ~MW 834.83): C, , 48.92; 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.

E~3mL~s_~
(RtR~-2,2'-[1,2-ethanediylbis[imino~l-methyl-2,1-ethanediyl)~]-bisE5-nitro-lH-benz~de]~soquinoline-1,3(2H)-dione~ methanesulfonate (1:2~ (Vb) 20Light yellow solid (25% yield); mp 210-211C ~dec).
The lH-NMR of Vb is identical with that of Va. MS ~DCI) e 625 (M~1). Anal. Calcd for C32~2ON6O8 2 CH3SO3H
(MW 81~.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.
E~
~Meso)-2,2'-~1,2-ethanediylbis~imino(l-methyl-2,1-ethanediyl)l]-bis[5-nitro-lH-be~z~de~isoquinoline-1,3(2H)-dione] methanesulfonate (1:2) (XXVII) Part A: (XXI) 1,1-Dimethylethyl-~S)-~2-(cyanomethyl)amino~-1-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 TH~ was stirred fox 15 minutes. N-t-BOC-(S)-alanine (18.9 g, 100 ml) and N-methylmorpholine (10.1 g, 100 mmol) in lOO ~I of THF was stirred with an ice-salt bath. Isobutyl chloroformate (12.97 ml, 100 mmol) was added at such a rate that the temperature of the mixture won't exceed 10C and the mixture of the nitrile-N-methylmorpholine suspension was added after 2 minutes. 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), washed with aq. K2CO3 (2 x~
100 ml), H2O (1 x 100 ml). The layers were separated;
and the organic layer dried over anhydrous MgSO4, 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 99-100C. lH-NMR (CDC13) 8 7.32 ~broad, lH, NH), 5.12 ~d, lH, 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~
. :
E~LS_~: (XXII) l~l-dimethylethyl-(s)-t2-l(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 acld was hydrogenated (50 psi) in a Parr shaker for 1 1/2 hours. "
The catalyst in the mlxture was removed by ~iltration.
The acetic acld in the flltrate was removed by vacuum distillatlon to give a light yellow viscous liquid, which was added 15 ml of methanol and 1 g of sodium blcarbonate. `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, WO 92/174~3 2 1 ~ ~ 9 9 9 PCI`/US92/02134 45.2%) as a viscous liquid. lH-NMR (CDCl3) ~ 7.08 (broad, lH, NH), 5.28 (broad,- lH, 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) .
Part C: tXXIII) 1, 1-Dimethylethyl (meso)-[l, 2-ethanediylbislimino(1-methyl-2-oxo-2,1-ethanediyl)]]-bis(carbamate).
A mixture of N-t-BOC-(R)-alanine (0.74 g, 3.g mmol) and 1,1'-carbonyldiimidazole (0.64 g, 3.9 mmcl) 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 ~ol) in 10 ml of methylene chloride- with an ice bath cooling. The mixture was stirred at ambient temperature overnight. '~he product was collected on a filter to give XXIII (1.22 g, 77.9%) as a white solid.
lH-NMR (DMSO-d6) ~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, 10~, 6 CH3) and 1.18 ~d, 6H, 2 CH3). MS (CI) m/e 403 (M+1).

~t 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 2S 4.4 N HCl in 40 ml of dioxane wa 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 tDMSO-d6) ~8.81 ~broad, 2H, 2 NH), 8.33 ~broad~ ~H, 2 NH2), 3.83 ~q, 2H, J~6.9 Hz, 2 CH), 3.21 ~broad s, 4H, 2 C~2) and 1.37 (d, 6H, J-6.9 Hz, 2 CH3).

Part E: (XXV) (Meso)-N1,Nl -1,2-ethanediylbisll, 2-propanediamine~tetrahydrochloride WO92/17453 2 1 0 6 ~ 9 9 PCT/US92/02134 To 30 ml or 1 M borane THF complex, there was added O.75 g (2.7 mmol? of XXIV. The mixture was refluxed overnight. After cooling to room temperature, methanol (lS ml) was added slowly to the reaction mixture. The 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. HCl. The mixture was stirred at room temperature for 4 hours, and tnen the solvent and excess HCl evaporated to give XXV (0.81 g, 93.7%) as a white solid. lH-NMR (D20) ~3.66 (m, ~H, 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 -1,2-ethanediylbis[1,2-propanediaminei 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 by filtration; and the solvent in the filtrate evaporated. The product was isolated from the residue by Kugelrohr distillation (88-96C at 0.4 mm) t~o give XXVI ~0.32 g, 73.4%) as a clear liquid. lH-NMR ~CDC13) - 2.88 ~m, 2H, 2 CH), 2.63 ~m, 4H, 2 CH2), 2.48 ~m, 2H, CH2?, 2.29 (m, 2H, CH2), l.S4 (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).

Part G: (XXVII) ~Me~o)-2,2'-~1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]-bislS-nitro-lH-benztde]isoquinoline-1,3~2H)-dione~
methanesulfonate ~1:2).
A mixture of 3-nitro-1,8-napthalic anhydride 0.78 g, 3.2 mmol) and XXVI ~0.28 g, 1.6 mmol) in 25 ml of ethanol was stirred at room temperature overnight and W092/l7453 2 1 ~ 6 9 9 9 PCT/US92/02134 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 tO.39 g, 39%) as its free base. This was S converted to its methanesulfonate salt, XXVII (0.33 g, 25.3%); mp 243-244.5C (dec). 1H-NMR (DMSO-d6) ~9.53 ~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, 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 (C-O)cm~l. MS (DCI) m/e 625 (M+1). Anal. Calcd for C32H2gN6Og 2 CH3SO3H 1/2 H2O (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.

~am;~1~21 (S,S)-2,2'-[1,2-ethanediylbislimino(2-methyl-2,1-ethanediyl~3]-bis[5-nitro-lH-benztde]isoquinoline-1,3(2H)-dione] methanesulfonate (1:2) (XIXa) Part A: (XVa) Dimethyl N,N'-1,2-dioxo-1,2-ethanediyl)b~tS-alan$ne3.
A m~xture 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 room temperature, t~e white solid was collected on a filter to give 12.36 g (95%) of XVa; mp 167-170C. 1H-NMR ~DMSO-d6) ~ 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+1) . ta] D -65.32 (c~1.012, AcOH).

WO92/17453 2 1 0 ~ 9 9 9 PCT/US92/021~

~art B: (XVIa) ~S,S)-N,N'-bis(2-amino-1-methyl-2-oxoethyl)ethanediamine.
Compound XVa (5.21 g, 20 mmol) was added to a methanol solution saturated with ammonia with an ice bath cooling. The mixture was further bubbled with ammonia for 1 hour; and then stirred at room temperature o~ernight. The white solid was collected on a filter to give 3.65 g t79%) of XVIa. lH-NMR (DMSO-d6) 8 8.50 (d, 2H, J=7.7 Hz, 2 NH), 7.50 (s, 2H, NH2), 7.21 (s, ~H, NH2), 4.2S (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~
[a]D ~69.14(c=0.418, DMF).

Part C: (XVIIa) (S,S)-N2,N2 -1,2-ethanediyl~is[1,2 --propanediamine~ tetrahydrochloride.
To a mixture of XVIa (3.5 g, 15.2 mmol) in 100 ml of THF, there was added 150 ml of lM 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. HCl with an ice bath 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 XVIIa;
lH-NMR (D20) ~ 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 (M+1, free base). la]D +8.39 ~c-0.632, H20).

Part D: ~XVIIIa) ~S,S)-N2,N2'-1,2-ethanediylbisll,2-propanediamine].
Compound XVIIa ~3.2 g, 10 mmol) was added to a freshly prepared sodium ethoxide solution in ethanol WO92/17453 2 1 ~ 6 q 9 9 PCT/US92/021~
` 23 (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.
H-NMR (CDCl3) ~ 2.8-2.4 (m, lOH, 4 CH2 and ~ CH), 1.57 (broad, 6H, 2 NH2 and 2 NH) and 0.91 (d, 6H, J=5.9 Hz, 2 CH3). []D +120 (c=0.310, benzene).

~L~ (XIXa) (S,S)-2,2'-l1,2-ethanediylbis[imino(2 m~thyl-2,1-ethanediyl)]]-bis[5-nitro-lH-benz[de)isoquinoline-1,3(2H)-dione] methanesulfonate ~1:2).
A mixture of 3-nitro-1,8-naphthalic anhydride (1.46 g, 6.0 mmol) and XVIIIa (0.52 g, 3 mmol) in 30 ml of ethanol was stirred at room temperature overnight. To this, there wa~ added 0.63 g of methanesulfonic acid.
After stirring overnight, the product was isolated by filtration and pur~fied by heating in 50 ml of met~anol overnight to give 1.37 g ~55%) of XIXa; mp 254-255C
(dec~. lH-NMR (DMSO-d6~ ~ 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), B.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 C32H2gN6Og 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, 10.13s S, 7.83, 7.84.

W O 92/17453 PC~r/US92/02134 ~10~9~9 24 ~m~2~
(S,S)-2,2'-[1,2-ethanediylbislimino(2-methyl-2,1-ethanediyl)~]-bis[lH-benztde]isoquinoline-1,3(2H)-dione]
methanesulfonate (1:2) (XXa) By replacing 3-nitro-1 t 8-naphthalic anhydride with 1,8-naphthalic ~nhydride, compound XXa can be prepared.
White solid (78% yield); mp 289-290C ~dec). 1H-NMR (DMS0-d6) ~ 8.96 (broad, 2H, NH2~), 8.80 (broad, 2H, 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 ld, 6H, J~6.6 Hz, 2 CH3). MS (DCI) m/e 535 (M+1, free base).
Anal. Calcd for C32H30N404 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.

~22 (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 248-251C(dec).
lH-NMR ~DMS0-d6) ~ 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 (tr 2H, J~7.9 Hz, aromatic protons), 4.33-4.18 ~m, 4H, 2 CH2), 3.46 (m, 6H), 2.28 (s, 6H, 2 CH3) and 1.28 (m, 6H, 2 CH3). MS(Cl) m/e 625 (M~l). Anal. Calcd for C32H2gN60g 2 CH3S03H (MW 816.81 C, S0.00; H, 4.44; N, 10.09; S. 7.85. Found: C, 50.16;
H, 4.34; N, 10.19; S, 7.62.

WO92/17453 2 1~ G 9 9 9 PCT/US92/021~

le 30 ~Racemic + Meso)-2,2'-~1,2-ethanediylbistimino[2-methyl-2,1-ethanediyl]-bis[5-nitro-lH-benz[de]isoquinoline-1,3(2H)-dioneJ-methanesulfonate (1:2) (XIXc) Yellow solid (18.9% yield); mp 278-281C ~dec).
H-NMR (DMSO-d6) ~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, 7 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~1). Anal.
Calcd for C32H2gN6Og 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.

Exam~le 31 ~R,R)-2,2'-tl,2-ethanediylbistimino~2-methyl-2,1-ethanediyl]-bisl6-nitro-lH-benztde]~soquinoline-1,3~2H)-dione~ methanesulfonate ~1:2) ~XXVIIIb) Yellow solid (17.0% yield); mp 230-233C (dec) lH-NMR ~DMSO-d6) ~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), 2.2S (s, 5.4H, 1.8 CH3SO3H) and 1.35 ~d, 6H, 2 CH3). MS (CI) m/e 625 (M+1)~
Anal. Calcd for C32H2~N60~ 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.

WO 92/17453 2 1 0 ~ 9 9 9 PC~/llS92/02134 ~m;~

~R,R)-2,2'-[1,~-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 227-230C
(dec). lH-N~R (DMSO-d6) ~ 9.84 (d, 4H, J=1.5 Hz, 4 aromatic protons), 9.13 (d, 4H, J=1.4 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 CEI), 3.34 (broad, 4H, ~ CH2), 2.24 (s, 6H, 2 CH3) and 1.39 (d, 6H, J=4.7 Hz, ~ CH3). Anal. Calcd for C32H28N612 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.

~ac~le 42 (l~acemic)-2,2'-[1,2-ethanediylbislimino ~2-methyl-2,1-20 ethanediyl]-bis[5-nitro-lH-benz[de]isoquinoline-1,3(2H)-d~one] methanesulfonate (1:2) ~XXXIV) The compound was prepared by mixing equal amounts of the corresponding (S,S) and (R,R) enantiomers (i.e., 25 XIXa and XIXb, respectively) . Light brown solid (90%
yield); mp 254-255C (dec). N~ spectrum is identical with those of XIXa and XIXb. Anal. Calcd for C32H2gN6Og-2CH3S03H (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, 30 7.66.

WO92/17453 2 i ~ 6 9 ~ 9 PCT/US92/021~

~xamplQ 4~

~S,S,S)-5-nitro-2-[2-[[2-[~2-~5-nitro-1,3-dioxo-lH-benz[de]isoquinoline-2(3H)-yl)-l-methyle~hyl]amino]-1-5methylethyl]amino]-1-methylethyl]-lH-benz[de]isoquinoline-1,3~2H)-dione] methanesulfonate (1:2) (XXXX) 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 (3.88 g, 38.4 mmol), there was added isob-~tylchloroformate ~2.62 g, 19.2 mmol) dropwise to maintain the temperature between 10 to 15C. After addition was completed, the reaction mixture was stirred for another 15 minutes. 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 filtxate evaporated. The remaining liquid was diluted with methylene chloride (350 ml~, washed with 5 sodium bicarbonate ~2 x 150 ml), water (1 x 150 ml), 0.1 N HC1 tl x 150 ml), water 1 x 150 ml), brine (1 x 100 ml), dr~ed over anhydrous magnesium sulfate, filtered and evaporated to give 4.32 g of a white solid, XXXV (65.1% yield): mp 168-172C. lH-NMR (CDC13) ~ 6.70 (m, 2H, 2 NH), 4.98 (broad, lH), 4.48 (m, 2H, 2 CH), 3.70 Ss, 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. ta]25D ~
s0.3~ (C-0.600, CH2cl2)-Part B: tXXXVI) N-[(1,1-dimethylethoxy)carbonyl]-L-alanyl-L-alanyl-L-alaninamide.

WO92/17453 2 1 0 6 Y Y ~ 28 PCT/US92/021~

To 80 mi of methanol saturated with ammonla, there was added 2.75 g (7.96 mmol) of XXXV. The reaction mixture was cooled with an ice bath, and then further reacted with ammonia for 10 minutes. The reaction was stirred at ambient temperature overnight. The solvent in the solution was evaporated to give a yellow solid, XXXVI ~2.45 g, 93% yield); mp 202-208C. lH-NHR (DMSO-d6) ~ 7.83-7.95 ~m, 2H, 2 NH), 7. 25 ~broad, lH, NH), 7.01 (broad, 2H, NH2), 4.2 (m, 2H, 2 CH), 3.83 (m, lH, CH) 1.38 (s, 9H, 3 CH3) and 1.20 (m, 9H, 3 CH3). MS
(DCl) m/e 331 (M+1). [a]25D - 6.00 (c=0.0600, DMSO).

(XXXvII) L-alanyl-L-alanyl-L-alaninamide hydrochloride.
A mixture of XXXVI ( 1. 95 g, 5 . 9 mmol ) and 4.4 M HCl (2.68 ml) in dioxane was added to 40 ml of dioxane at 0C 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). lH-NMR (D20) ~ 4.2-4.05 (m, 2H 2 CH), 2.95-3.8~ (m, lH, CH) and 1.25 ~m, 9H, 3 CH3). MS
(Cl) m/e 231 (M + 1). IR (KBr) 3438, 3294 lNH2), 1676, 1639 (C-0) cm~1. la]25D -19.96 (c-0.606, CH30H).

~L$_~: (XXXVIII~ ~S,S,S)-Nl-(2-amino-1-methylethyl)-N2-(2-aminopropyl)-1,2-propanediamine tetrahydrochloride.
To a mixture of XXXVII (1.70 g, 6.37 mmol) in 150 ml of THF, there was added 58 ml of 1 ~ B~3~THF complex.
The mixture was refluxed overnight to become a clear solution. The solution was cooled in an ice bath and quenched dropwise with 50 ml of methanol. The solution was refluxed overnight. The solvents in the reaction solution were removed by xotary evaporation. To the remaining liquid was added methanol ~50 ml) and it was WO92/17453 ~ 10~ 9 9 ~ PCT/US92/021 evaporated again to remove trimethylborate. The liquid was diluted with methanol (25 ml) followed by the addition of 4.0 ml of conc HCl. After stirring for 2 hr, this was triturated with ethyl ether, filtered to give 1.lO g (51.7~ yield~ of XXXVIII as a white solid;
mp 277-278C (dec). MS (DCl) m/e 189 (M+l).. IR (KBr) 3436 (NH, NH2) cm~l.

E~t E: (XXXIX) (S,S,S)-Nl-(2-amino-l-methylethyl)-N2-(2-aminopropyl)-l,2-propanediamine.
To a freshly prepared sodium ethoxide solution (0.32 g of sodium of 20 ml of ethanol), there was added l.05 g ~3.1 mmol) of XX~III. The mixture was stirred at r~om 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 - 104C) to give 0.41 g of XXXIX as a light yellow liquid ~70.2% yield) lH
NMR (CDC13) ~ 2.90 ~m, lH, CH), 2.80 (m, 2H, 2 CH), ~.53-2.30 ~m, 6H, 3 CH2), 1.58 ~broad, 6H, 2 NH2 and 2 NH) and 0.98 ~m, 9H, 3 C~3).

~art F: (XXXX) (S,S,S)-5-nitro-2-~2-~[2-~12-(5-nitro-l,3-dioxo-lH-benz~de]isoquinoline-2(3H)-yl)-l-methylethyl]amino]-l-methylethyl]amino]l-methylethyl]-lH-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 ~0.4l g, 2.2 mmol) in 25 ml of ethanol was stirred at room temperature overnight and then heated to reflux for l l/4 hours. The solvent in the mixture was e~aporated, 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 ~ ' WO92/17453 2 I O ti 9 ~ 9 PCT/US92/02134 methanesulfonate, XXXX ~0.65 g; 35.6% yield); mp 189-192C (shrink). lH NMR (CDC13) ~ 9.28 (d, lH, J=1.9 Hz, aromatic proton), 9.25 (d, lH, J=2.2 Hz, aromatic proton), 9.15 (d, lH, J=2.2 ~z, aromatic proton), 9.12 (d, lH, J=2.2 Hz, aromatic proton, 8.81 (d, lH, J=7.3 Hz, aromatic proton, 8.76 (d, lH, J=7.3 Hz, aromatic proton), 8.46 (d, lH, J=8.1 Hz, aromatic proton), 8.42 (d, lH, J=8.4 Hz, aromatic proton), 7.98 ~t, lH, J=7.7 Hz, aromatic proton), 7.94 (t, lH, J=7.92 Hz, aromatic proton), 5.76 (m, lH, CH), 4.59 (m, lH), 4.36 (m, 2H), 404 (m, lH), 3.91 (m, lH~, 3.96 (m, lH), 3.39 (m, 2H), ' 1.99 ~s, ~H, 2 CH3), l.S4 ~d, 3H, J-7.0 Hz, CH3), 1.54 ~d, 3H, J=6.6 Hz, CH~) and 1.48 ~d, 3H, J=6.6 Hz, C~
MS (DCl) m/e 639 (M+l). Anal. Calcd for C33H30N6Og-2CH3SO3H (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.

W092/174532 1 ~ 6 9 ~ 9 PCT/U~9~/~2134 Tab1e 1 ' R~ R4 R~

(i) ~2, R3, R4, RS, and R6 s H

E~. ~ Y. Y' sl, B7 ~tereochemi~try 3--N02 H CH3 ~S) 2 3 -N02 H CH3 ~ racemic + me~o 3 3-N02 ~ CH3 (R) 5 3-No2 6-N02 ~ ~3 ~S) 6 ~ -~2 H CH3 ~ S ) 7 3-N02 6-N02 CH3 ~R) 8 4--N02 H CH3 ~R) 2 6 3 -N02 H CH3 ~ o ) In Table l the position of X and Y is indicated using the number~ng for the position in the corresponding naphthalic anhydride, and not the numbering for ~he lH-benztde]isoquinoline-l,3~2H)-dione.

W O 92/17453 PCT/Us92/02134 ~ 1 0 ~

\N/~

(i) ~1, R3, and R5 = H

E~ Y, ~ ~2, B6 B4 ~7 (~tereochemi~try) 27 3-NO2 H CH3 ~S) H H
28 H H CH3 ~5) H H
29 3-NO2 H CH3 (R) H H
3-NO2 H CH3 ~racemlc ~ me~o) H
31 4-NO2 H CH3 (R) H H
32 3-NO2 6-NO2 CH3 ~R) H
42 3-NO2 H CH3 ~racemic) H H
43 3-NO2 H R2-CH3 (S); R6-H CH3 (S) CH3 ~S) 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 lH-benzlde]isoquinoline-1,3(2H)-dione.

wo 92/174~3 2 1 ~ 6 9 9 g PCT/US92/02134 Utility In vi~rQ Growth In~ihi~Q~y_~ctivltY
Ll210 cells were maintained in RPMI-1~40 a medium supplemented with 10% heat inactivated fetal bovine serum and 50 mL mercaptoethanol/l~ter medium (RPMI-L).
Bl6 cells were maintained in RPMI-1640 medium supplemented with lS~ heat inactivated fetal bovine serum and antiobiotics (RPMI-C).

Exponentially growing murine leukemia Ll2l0 cells (lx103 cells) in 0.l mL medium were seeded on day 0 in a 96-well microtiter plate. On day l, 0.l ~L aliquot of medium containing graded concentrations of test analogs was added to the initial ~olume. After incubation at 37C in a humldified incubator for 3 days, the plates were centrifuged briefly and l00 mL of the growth medium was removed. Cell cultures were incubated with 50 ~L of 3-~4,5-dimethylthiazol-2-yl)-2,S- diphenyltetrazolium bromide ~MTT; 1 mg/ml in Dulbecco's phosphate buffer saline) for 4 hours at 37C. The resulting purple formazan precipitate was solubilized with 200 ~L of 0.04 N HCl in i~opropyl alcohol. Absorbance was read in a Titertek ~ultiskan MCC scaning well spectrophotometer (Flow Laboratories) at a test wavelength o~ 570 nm and a reference wavelength of 630 nm.
The IDso values were determined by a computer program that fit all of the data (8 determinations per concentration and 12 concentrations per test analog) to the follow~ng e~uation:
Y ~ ~(Am - Ao)/~l+(X~ID~o)n))+Ao where: Am 8 absorbance of the control cells; Ao -absorbance of the cells in the presence of highest drug ~10~
WO9~/174~3 ~ PCT/US92/021 concentration; Y = observed absorbance; X - drug concentration; ID50 - dose of drug that inhibits the --growth of cells to one half that of the control cells.

Results of the in vitro L1210 growth inhibition testing are shown in Table 3.

Takle 3 8__~2~ I~Q /~/ml 1 0.047 ~ 0.062 3 O.OS1 26 0.074 27 0.00~5 28 0.21 29 <0.01 C0.01 31 0.19 32 0.0~7 42 <0.001 43 0.04 ' Representative compounds of the present invention have been extensively tested in a variety of pre-cl~nical test~ of anti-cancer activity which ~re indicative of clinical utility. For example, the presently claimed compounds show str~king ~n vlvo efficacy Against three types of human tumors xenografted in nude mice, namely DLD-2 human colon carcimoma, MX-l human mammary carcinoma, and LX-1 human lung carcinoma.

W092/17453 21 0 ~ 9 9 9 PCT/US92/021~

In addition, a representative compound of the present invention is active against several murine tumors, including murine Ml6c 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 ~ivo human tumor xenograft models are described below.

The DLD-2 human colon tumor, MX-l human mammary carcinoma, and LX-l humzn lung tumor were originally obtained from a surgically removed primary colon carcinoma, breast tumor, and non-small lung carcinom~, respectively. The human tumor lines were maintained by serial passage in athymic nude mice. The MX-l human mammary carcinoma and LX-l human lung tumor are established tumor8 used by the NCI. The DLD-2, MX-l, and LX-l tumor models have been well characterized.
The mice used in these experiments were outbred Sw~ss mice or BALB/c mice bearing the nude (nu/nu) gene.
On day O male and female mice weighing 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 s~erile physiological saline. Palpable tumors weighing approximately 50 mg appear in the mice within 7-lO days after inoculatlon.
The mice are pair matched by tumor weight and sex into groups of ten each and the test compoundq and vehicle control are administered intravenously (i.v.) once daily for nine consecutive days. A ~20% decrease in body WO92/17453 2 1 ~ 6 9 9 9 PCT/US92/02134 36 ,~

weiqht on day 5 following compound administration is considered an indication of toxicity. Tumor measurements and body weights are recorded once a week. Fifteen to 18 days after the initial injection the mice are S weighed, sacrificed and the tumors excised and weighed.

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

% Tumor Growth ~1 -,mp~n-~L~nr-~e~ ~t of trea~ x 100 Inhibition l mean tumor weight of controlJ

l~ctivity Criter; a The criteria of the National Cancer Institute (NCI) for activity in the in v~vo cancer models were,used.
Tumor growth 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 regression) indicate excellent to outstanding activity.
Compounds demonstrating <58% growth inhibition are considered inactive.

WO92/17453 2 1 0 ~ 9 3 9 PCT/US92/021~

The compounds of Examples l, 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 MX-l human breast tumor model. Example 1 exhibited good to excellent activity against MX-l human breast tumor.
Examples 2,3, 27, and 29 exhibited good to excellent activity against LX-l human lung tumors.

Activity A~ainst Mam~L~_~,L

Transgenic mice carrying the v-Ha-r~s oncogene linked to the MMTV promoter were constructed originally in the laboratory of Professor Phil Leder at HarYard University ~Sinn et al. (1987) Cell 49: 465-47S). The female transgenic animals develop mammary tumors, and thus can be used to evaluate agents for activity against these breast tumors. The growth characteristics of these tumors has been characterized extensively ~Diamond and Dexter (l99l) 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 test compound administered daily for 9 days and the group mean tumor growth rate for each test 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 WO92/174~3 ~ 1 0 6 9 ~ ~ ` PCT/US92/02134 of a broad spectrum of solid tumors in man, and, in particular, tumors of the colon, breast, and lung. This conclusion is further supported by published analyses correlating pre-clinical test results with clinical efficacy of anti-cancer agents. 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 Review 7: 263-284; Jackson et al. ~1990) Cancer Investigations 8: 39-47. Based on these published analyses, the broad spectrum and exceptional high level of antitumor activity exhibited by the presently claimed compounds prov~de strong evidence that the compounds claimed in present invention may have important therapeutic utili.ty in the treatment of cancer in man.

The antitumor compounds (active ingredients) of this invention can be admini~tered to inhibit tumors by any means that produces contact of the active ingredient with the agent 15 site of action in the body of a mammal.
They can be admini-~tered by any conventional means available for use in con~un~tion with pharmaceu~`-als, either as lndividual 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 pract~ce.
- The dosage administered will be a tumor-inhibiting amount of active ingredient and will, of course, vary depending upon Xnown factors such as the pharmacodynamic W092/17453 2 1 0 6 .9 9 9 PCT/U$92/021~

characteristics of the particular active ing edient, and its mode and route of administrationi age, health, and weight o~ the recipien~; nature and extent of symptoms;
kind of concurrent treatment, frequency of treatment, and the effect desired. Usually a daily dosage of active ingredient can be about 5 to 400 milligrams per kilogram of ~ody weight. Ordinarily, l0 to 200, and preferably l0 to 50, milligrams per kilogram per day given in divided doses 2 to 4 times a day or in sustained release form is effective to obtain desired results.
Dosage forms tcompositions) s~itable for internal adminis~ration ccntain from about l.0 milligram to about 500 milligrams of active ingred-ent per unit. In these pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
The active ingredient can be administered orally în solid dosage forms, such as capsules, tablets, and powders, 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 powdered carriers, such as lactose, sucrose, mannitol, 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 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.

W092/l74~3 2 1 0 6 9 ~ 9 PCT/USg2/02134 Liquid dosage forms for oral administration 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 p-opylene 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 agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid either alone or combined are suitable stabilizing agents. 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 Remin~ton's Pharma~ytical Sci~es, 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.

S~U~LL1~: 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 in~ected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules are washed and dried.

W~ g2/17453 2 1 0 6 9 3 9 PCT/US92/021~

Tablets: Tablets are prepared by conventional procedures so that the dosage unit is lO0 milligrams of active ingredient, 0.2 milligrams of colloidal silicon S dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose. ll milligrams of cornstrach and 98.8 milligrams of lactose.
Appropriate coatin~s may be applied to i~crease palatability or delay absorption.
l~is~hl~: A parenteral composition suitable for administration by in jection is prepared by stirring 1. 5%
by weight of active ingredient in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.

5~a2Q~alQn: An aqueous suspension is prepared for oral administration so that each 5 milliliters contain lO0 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of so~ium benzoate, l.0 grams of sorbitol solution, U.S.P., and 0.025 milliliters of vanillin.

In the present disclosure it should be understood that the specified materials and conditions are important in practicing the invention but that unspecified materials and condi~ions are not excluded so long as they do not prevent the benefits of the invention from being realized.

Claims (10)

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[imino(1-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione];

(Racemic + Meso)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione];

(R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione];

(Meso)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione);

(S,S)-2,2'-[1,2-ethanediylbis[imino(2-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione];

(Racemic + Meso)-2,2'-[1,2-ethanediylbis[imino(2-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione];

(R,R)-2,2'-[1,2-ethanediylbis[imino(2-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione];

(Meso)-2,2'-[1,2-ethanediylbis[imino(2-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione].

2. The compound, or a pharmaceutically acceptable salt thereof, which is (R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2,1-ethanediyl)]]-bis[5-nitro-1H-benz[de]isoquinoline-1,3(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 mammal comprising administering to a mammal 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 mammal comprising administering to a mammal bearing such a tumor, a tumor-inhibiting amount of a compound of Claim 2.

7. A method for preparing a compound of the formula:

IV
said method comprising: .
(a) reacting t-BOC-alanine with 1,1' carbonyldiimidazole followed by reaction with ethylenediamine to yield a compound of the formula:

(I); and (b) removing the t-BOC group of (I) with mineral acid followed by reduction of the dihydrochloride salt with diborane in refluxing tetrahydrofuran followed by neutralization of the acid salt with sodium ethoxide to yield a compound of the formula (IV).

8. A method for preparing a compound of the formula:

XVIII
said method comprising:
(a) reacting alanine methyl ester with oxalyl chloride followed by treatment with ammonia in methanol to yield a compound of the formula:

XVI ; and (b) reduction of the amide functionality with diborane in tetrahydrofuran followed by neutralization of the tetrachloride salt with sodium ethoxide to yield a compound of the formula (XVIII).

9. A method for preparing a compound of the formula:

(meso), XXVI

said method comprising:
(a) reacting t-BOC-(S)-alanine with isobutyl-chloroformate in the presence of N-methylmorpholine, followed by reaction with aminoacetonitrile to yield a compound of the formula:

XXI ; and (b) reducing the nitrile of (XXI) with palladium hydroxide in acetic acid to yie1d a compound of the formula:

XXII ; and (c) reacting t-BOC-(R)-alanine with 1,1'-carbonyldiimidazole followed by reaction with amine (XXII) to yield a compound of the formula:

(meso), XXIII ; and (d) removing the t-BOC group with mineral acid followed by reduction of the amide function with diborane followed by neutralization of the tetrahydrochloride salt with sodium ethoxide to yield a compound of the formula (XXVI).

10. A method for preparing a compound of the formula:
(S,S,S) XXXIX

said method comprising:
(a) reacting (t-BOC)-alaninyl alanine with isobutylchloroformate in the presence of N-methyl morpholine, followed by reaction with (S)-alanine methyl ester to yield a compound of the formula:
(S,S,S) XXXV ; and (b) reacting compound (XXXV) with ammonia in methanol to yield a compound of the formula:

(S,S,S) XXXVI ; and (c) reduction of the amide functionality with diborane in tetrahydrofuran followed by neutralization of the tetrahydrochloride salt with sodium ethoxide to yield a compound of the formula (XXXIX).