Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT

1690

J . Med. Chem. 2003, 46, 1690-1705

Self-Im m ola tive Nitr ogen Mu sta r d s P r od r u gs Clea va ble by Ca r boxyp ep tid a se

G2 (CP G2) Sh ow in g La r ge Cytotoxicity Differ en tia ls in GDEP T

Dan Niculescu-Duvaz, Ion Niculescu-Duvaz, Frank Friedlos, J an Martin, Panos Lehouritis, Richard Marais, and

Caroline J . Springer*

CR-UK Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK

Received October 17, 2002

Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been

synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2

(CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and

p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by

CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered

CPG2 (stCPG2(Q)3) or control â-galactosidase, are assessed. The effect of various structural

features on stability, kinetics of activation, and biological activity is discussed. The p-

methylamino prodrugs are the most stable compounds from this series, with the largest

cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent

compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-{N-[4′-Bis(2′′-iodoeth-

yl)aminophenyl]-N′-methylcarbamoyloxymethyl}phenylcarbamoyl-^L-glutamic acid, compound

39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing

â-galactosidase. An additional six compounds show better cytotoxicity differential than the

published N-{4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl}-^L-glutamic acid (CMDA) pro-

drug.

In tr od u ction

Prodrugs where the L-glutamic acid is linked direct-

ly onto the drug are already in clinical trials for

ADEPT.^20,21Self-immolative alkylating prodrugs, where

the L-glutamic acid is bonded through a linker to the

drug, were synthesized for GDEPT, cleavable by

CPG2.^22,23It was demonstrated that the two linkers, L1

and L2 (see Scheme 2), were substrates for CPG2 (K_M

) 1.7-3.1 µM and k_cat) 65.4-140 s^-1).²²The probable

mechanism of drug release from these self-immolative

prodrugs^24,25is shown in Scheme 1.

There are potential advantages of these self-immola-

tive prodrugs. The lipophilicity of the prodrugs and their

respective drugs may be altered with minimal effect on

the kinetics of activation by CPG2. Improvements can

be made in the event of unfavorable kinetics of activa-

tion due to unsuitable steric or electronic effects. Also

the range of drugs which can be converted to prodrugs

can be expanded, unrestricted by the structural sub-

strate requirements of CPG2.

A number of targeting strategies has been developed

in an attempt to increase the selectivity of anticancer

drugs. One approach uses antibodies conjugated with

enzymes to target tumor-associated antigens. The en-

zyme can activate a subsequently administered prodrug

in a therapy termed antibody-directed enzyme prodrug

therapy (ADEPT).^1,2Alternatively the gene for the

prodrug-activating enzyme is expressed in tumor cells

in a strategy called gene-directed enzyme prodrug

therapy (GDEPT). This methodology requires prodrugs

that are substrates for the expressed enzyme and which,

after activation, release cytotoxic drugs.³The targeting

of the genes is achieved by using viral^4,5or nonviral

delivery vectors.^6-9Several enzyme/prodrug systems

have been investigated and a number of clinical trials

using suicide gene therapy are ongoing.¹⁰

We have developed a GDEPT system based on the

enzyme carboxypeptidase G2 (CPG2, glutamate carboxy-

peptidase, EC 3.4.17.11) from Pseudomonas RS16.¹¹

CPG2 catalyzes the scission of the amidic,¹²urethanic

or ureidic,^13,14linkage between an aromatic nucleus and

L-glutamic acid. CPG2 has been expressed both intra-

cellularly (called CPG2*)¹⁵and tethered to the outer cell

surface (called stCPG2(Q)3)¹⁶in a range of tumor cell

lines. The extracellular tethering overcomes the need

for the prodrug to cross the tumor cell membrane for

activation. This method of expression is expected to

improve the bystander effect, whereby a prodrug is

activated to an active drug that kills neighboring tumor

cells not expressing the activating enzyme.^17-19

Ra tion a le of th e Design

Previous data suggested that the shape and bulk of

the cytotoxic drug moiety attached to the linker would

be of importance for the behavior of the self-immolative

prodrugs as substrates for CPG2 and, therefore, for their

biological activity.^22,23Modeling of prodrugs shows that

the shape and the van der Waals volumes of ortho- and

para-self-immolative nitrogen mustards are quite dif-

ferent. To compare these structural features, we report

here that both series of self-immolative alkylating

agents are synthesized and studied.

An important parameter for a GDEPT system is the

differential, or degree of activation, which is defined as

the ratio of IC_50,prodrugin cells that do not express CPG2

* To whom correspondence should be addressed. Tel: 020 8722 4214.

Fax: 020 8643 6902. E-mail: caroline@icr.ac.uk.

Published on Web 03/26/2003

Self-Immolative Nitrogen Mustards Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1691

Sch em e 1

Sch em e 2^a

a

(a) H₂, Pd/C 10%, THF; (b) N-benzyloxycarbonyloxysuccinimide, THF; (c) MeI, NaH, THF; (d) triphosgene, NEt₃, CH₂Cl₂, then L1A

or L2B, dibutyltin dilaurate, CH₂Cl₂; (e) activated L1A or L2B, DMA.

vs IC_50,prodrugin cells expressing CPG2.²⁶A large dif-

ferential indicates a good therapeutic window so that a

dose for in vivo use can be found that is toxic to the

tumor without damage to normal cells. We investigated

the relationship between the nature of the leaving

groups of the alkylating moiety and the cytotoxic

differential of the corresponding prodrugs. We are

interested in prodrugs able to release more cytotoxic

drugs. It was found that in both series the most effective

prodrugs are the bis-iodo nitrogen mustards, which

showed significant differentials (49-120-fold) between

cells expressing stCPG2(Q)3 and control cells.

1692 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9

Niculescu-Duvaz et al.

Sch em e 3^a

a

(a) NEt₃‚3HF, THF; (b) Mes₂O, NEt₃, DMAP, CH₂Cl₂; (c) NaI, acetone, reflux; (d) LiBr, THF, reflux; (e) LiCl, DMA; (f) NaCl, acetonitrile,

55 °C; (g) Pd(PPh₃)₄, morpholine, or pyrrolidine, CH₂Cl₂(for R₁) allyl) or HCOOH (for R₁) t-Bu).

Prodrugs releasing N-methylamino aniline mustards

were also investigated in comparison with the non-

methylated counterparts. It was anticipated that the

corresponding drugs would be more reactive as the

methylamino group is a stronger electron donor than

the amino group and thus a greater differential would

be expected.²⁷The methylamino prodrugs, as secondary

carbamates, are also potentially more stable.^28,29

The activity of these new prodrugs is assessed in the

colon carcinoma cell line WiDr expressing stCPG2(Q)3

or non-CPG2 expressing controls.

was protected as the benzyloxycarbonyl (Z) derivative

3b, methylated with methyl iodide and sodium hydride

to intermediate 4b. The Z protection has the purpose

of allowing selective monomethylation and increasing

the acidity of the N-H bond toward proton removal with

NaH. The methylamino compound 5b was obtained by

hydrogenolysis of benzyloxycarbonyl and coupled to the

activated L1A and L2B to yield the protected prodrug

intermediates 8b and 9b respectively.

The N,N-bis(2-hydroxyethyl)amino function was re-

stored in compounds 10-13 by treatment with triethyl-

amine trihydrofluoride in THF ³¹in good yield (Scheme

3). These compounds were transformed to the corre-

sponding bis-mesyl derivatives 14-17 using mesyl

anhydride/triethylamine in dichloromethane. These con-

ditions are preferable to the mesyl chloride/pyridine

method,¹²as the yield is improved and there is no risk

of formation of unwanted chloro products. The alkylat-

ing agents bis-iodo (18(a ,b), 21(a ,b), 25b, and 28b), bis-

bromo (19(a ,b), 22(a ,b), 26b, and 29b) and bis-chloro

(20a , 27b, and 30b) were obtained by exchanging the

mesyl leaving group for halogens with NaI/acetone,

LiBr/THF, or LiCl/dimethylacetamide, respectively. The

bis-chloro-nitrogen mustard, 23a , and the monomesyl-

mono-chloro derivative, 24a , were obtained as a mix-

ture, by reacting the compound 15a with NaCl in

acetonitrile. The reaction is slow at 55 °C, and conditions

could be adapted to obtain either of them as the main

product.

Resu lts a n d Discu ssion

Ch em istr y. Nineteen new self-immolative prodrugs

(compounds 31-44, Scheme 3) and their corresponding

drugs (compounds 57-63, Scheme 4), derived from o-

and p-amino and p-methylamino aniline nitrogen mus-

tards, were synthesized.

2- and 4-[Bis(2′-hydroxyethyl)amino]nitrobenzene were

used as starting materials and protected as 2- and 4-bis-

(2′-ter t-bu t yldim et h ylsilyloxyet h yl)a m in on it r oben -

zene, 1(a, ortho; b, para) (Scheme 2). After reduction to

the corresponding amines, 2a ,b, these were converted

to isocyanates and coupled with the linkers L1A and

L2B (Scheme 2) in the presence of catalytic amounts of

30

dibutyltin dilaurate leading to the protected prodrug

intermediates 6 (a , b; Z ) NH; R ) allyl) and 7 (a , b; Z

) O; R ) tert-butyl). To obtain the corresponding

methylamino analogues, the 4-amino intermediate 2b

Self-Immolative Nitrogen Mustards Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1693

Sch em e 4^a

a

(a) Boc₂O, THF; (b) NEt₃‚3HF, THF; (c) Mes₂O, NEt₃, DMAP, CH₂Cl₂; (d) NaI, acetone, reflux; (e) LiBr, THF, reflux; (f) NaCl,

acetonitrile, 55 °C; (g) LiCl, DMA; (h) HBr, AcOH.

The final deprotection, with formic acid for the

compounds deriving from L2B and Pd(0) with secondary

amines for the compounds deriving from L1A, led to the

desired self-immolative nitrogen mustard prodrugs 31-

44.

For the compounds 58b, 59a , 59b, 61b, and 62b which

proved too labile, a spectrophotometric method was

employed. The results are shown in Tables 1 and 2.

Difficulties were encountered in determining the half-

lives of the ortho nitrogen mustards. All the ortho

nitrogen mustard drugs (58a , 59a , and 60a ) have the

The access route to nine new corresponding active

drugs is summarized in Scheme 4. Silyl-protected o- and

p-phenylenediamines 2(a ,b) were converted to the cor-

responding Boc-derivatives 45(a ,b) (Scheme 4). The silyl

moieties were removed with triethylamine trihydro-

fluoride (NEt₃‚3HF) to obtain the hydroxyethyl-com-

pounds 46(a ,b). In an alternative procedure compounds

46(a ,b) were obtained directly from the 2- or 4-amino-

N-bis(2′-hydroxyethyl)anilines by treatment with Boc-

pyrocarbonate. The corresponding bis-hydroxy interme-

diate 47b for 4-methylamino aniline drugs was obtained

by desilylation of 3b. The Boc- or Z-protected 2-amino,

4-amino, and 4-methylamino alkylating agents 48-56

were obtained by a method similar to that used for the

corresponding protected prodrugs 14-30, namely me-

sylation of bis-hydroxy compounds 46(a ,b) and 47b and

subsequent halogen exchange.

1

correct microanalysis. However H NMR (in DMSO-d₆)

and LC-MS (in DMSO buffer) showed rapid cyclization

to the corresponding benzopiperidine derivative (see

Scheme 5), except for the bis(chloroethyl) derivative 60a .

The 4-amino aniline mustard prodrugs have shorter

half-lives in general than the N-methylated and their

2-amino counterparts (see Table 3). The half-lives of the

N-methylated compounds are consistently 3-4 times

longer than the corresponding nonmethylated ones,

irrespective of the halogen in the mustard moiety or the

linker1/linker2 origin. Similarly the o-amino analogues

have half-lives 2-3 times longer than p-amino prodrugs.

However, this was not the case for the correponding

drugs. The difference in reactivity between prodrugs and

their corresponding drugs varies in the range of 1-20-

fold (Table 1).

The active drugs 57-63 were obtained by deprotec-

tion with HBr/AcOH. These conditions have a dual

role: to remove the protecting groups tert-butyloxycar-

bonyl or benzyloxycarbonyl yielding the final products

and to trap the very reactive drugs as they formed as

hydrobromides, which otherwise will self-alkylate quickly

as free amines. In the salt form they are hygroscopic

but stable to storage.

P h ysicoch em ica l a n d Kin etic Da ta . The chemical

half-lives of the candidate prodrugs and parent drugs

were determined by HPLC (See Experimental Section).

The kinetics of activation of the self-immolative

prodrugs by CPG2 was measured for the bis(chloroethyl)

series, and the results are shown in Table 4. This series

was chosen in order to minimize the influence of the

nitrogen mustard moiety hydrolysis on the measured

kinetics.

All prodrugs showed K_m’s comparable with those of

linker 1 and linker 2.²²This indicates a good structural

fit for the CPG2 active site, even with the ortho

derivatives. However, the k_catremains low compared to

the directly linked prodrugs and the linkers alone.

32

1694 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9

Niculescu-Duvaz et al.

Ta ble 1. Prodrugs: Half-lives and Cytotoxicity Differentials between StCPG2(Q)3 and â-gal Expressing WiDr Cells

IC₅₀(µM)

T_1/2prodrug

T_1/2prodrug/

T_1/2drug

degree of

activation (fold)

compd no.

(min)

LacZ

stCPG2(Q)3

100 [(10]

CMDA

31a

31b

32a

32b

33a

34a

35a

35b

36a

36b

37a

38a

39b

40b

41b

42b

43b

44b

59

2.7

0.97

3.0

0.85

107.5

10.8

2.1

0.98

3.47

0.9

146.3

10.0

3.9

2.8

173.6

4.6

2.3

-

3230 [(120]

32.3

49.7

101.6

24.9

66.6

4.7

5.2

3.9

42.5

1.9

38.3

1.4

5.2

124.0

81.4

15.9

8.3

149.0 (80.7-275.0)

179.8 (71.7-449.4)

117.7 (83.8-165.2)

204.6 (108.6-384.6)

21.1 (12.0-36.9)

208.2 (86.4-500.7)

73.3 (45.3-119.8)

53.6 (36.9-78.0)

183.4 (83.8-400.4)

99.7 (19.1-520.6)

38.1 (19.1-76)

148.4 (65.1-338.2)

184.7 (105.4-324.4)

332.4 (222.6-496.3)

39.7 (27.0-58.4)

21.5 (16.2-28.4)

27.2 (14.1-52.5)

19.6 (15.0-25.7)

3.0 (2.1-4.4)

1.8 (1.0-3.2)

4.7 (7.8-27.8)

3.1 (1.8-5.3)

4.5 (2.9-7.1)

40.0 (22.7-70.3)

19.0 (11.7-30.9)

1.3 (0.8-2.0)

95.2 (57.1-158.5)

2.6 (1.2-6.0)

28.1 (11.1-43.6)

28.5 (17.5-47.5)

1.5 (0.8-2.6)

4.1 (2.5-6.7)

2.5 (1.6-3.9)

2.6 (1.7-4.1)

3.6 (2.2-6.0)

1.7 (1.1-2.5)

1.9

3.5

0.4

13.9

-

2.0

3.5

0.4

11.9

-

6.5

1.0

20.9

7.7

1.0

20.6

2.7

173.6

7.5

11.5

a

The round bracketed values indicate the 95% confidence intervals. For CMDA, the square bracketed values represent the standard

errors of the mean as published in ref 17.

Ta ble 2. Half-Lives and Cytotoxicities of Drugs in WiDr Cell

Line Expressing Either â-gal (control) or StCPG2(Q)3^a

Ta ble 3. Comparison of Half-Lives between 4-Amino,

4-Methylamino, and 2-Amino Aniline Mustard Prodrugs

compd. no T_1/2(min) IC₅₀(µM) LacZ IC₅₀(µM) stCPG2(Q)3

4-NH/4-NMe

compounds

compared

4-NH/2-NH

compounds

compared

58b

59a

59b

60a

61b

62b

63b

0.5

1.0

2.3

12.3

0.6

2.7

10.2 (4.8-18.3)

23.4 (12.4-44.1)

9.4 (5.7-18.5)

3.5 (2.1-5.7)

7.7 (4.1-14.5)

7.2 (3.6-14.4)

9.2 (5.7-15.1)

8.6 (4.2-14.0)

18.6 (10.7-32.2)

7.7 (5.0-14.8)

2.4 (1.3-4.4)

8.4 (4.6-15.5)

7.4 (3.5-15.5)

8.2 (5.3-12.5)

mustard type

T_1/2ratio

Linker-1 Prodrugs (Z ) NH, see Scheme 3)

bis-chloro

bis-bromo

bis-iodo

a/41b

32b/40b

31b/39b

0.28

0.30

0.25

a/33a

32b/32a

31b/31a

0.45

0.28

0.36

8.3

Linker-2 Prodrugs (Z ) O, see Scheme 3)

a

The bracketed values indicate the 95% confidence intervals.

bis-chloro

bis-bromo

bis-iodo

b/44b

36b/43b

35b/42b

0.30

0.33

0.21

b/37a

36b/36a

35b/35a

0.35

0.26

0.47

Sch em e 5

a

4-{[4′-(N,N-Bis(2′′-chloroethyl)amino)phenyl]carbamoyloxy-

methyl}phenylcarbamoyl-^L-glutamic acid.²⁰4-{[4′-(N,N-Bis(2′′-

b

chloroethyl)a mino)phenyl]ca rba moyloxymethyl}phenoxyca r-

bonyl-^L-glutamic acid.²⁰

Ta ble 4. Kinetics of the Linkers and the Bis(2-chloroethyl)

Prodrugs

compd no.

K_m(µM)

k_cat(s^-1

)

k_cat/K_m

Cytotoxicity Assa ys. The prodrugs and some of the

corresponding drugs were tested for cytotoxicity in WiDr

cells engineered for stable expression of (stCPG2(Q)3)

or, as a control, the non-prodrug activating enzyme

â-galactosidase (â-gal). For comparative purposes with

the directly linked prodrugs, the WiDr cell line was

treated with the N-{4-[(2-chloroethyl)(2-mesyloxyethyl)-

amino]benzoyl}-L-glutamic acid prodrug (CMDA), which

has undergone clinical trials in ADEPT.^20,21The results

are summarized in Table 1. The degree of activation

defined as the ratio of the IC₅₀value of the prodrug in

the â-gal expressing cell line to the IC₅₀value of the

prodrug in CPG2 expressing cell line was calculated for

the new compounds and for CMDA. The degree of

activation ranged between 1.4 and 124-fold (CMDA: 32-

fold¹⁹). Seven of the new prodrugs (31a , 31b, 32b, 35b,

36b, 39b, and 40b) give higher differentials than

CMDA.

L1-OH^(a)

L2-OH^(a)

L1-4-NHPhN(CH₂CH₂Cl)₂

L2-4-NHPhN(CH₂CH₂Cl)₂

33a

37a

41b

44b

3.1

1.7

<5

0.55

1.03

6.05

2.43

65.4

140

10-50

<10

21.1

82.3

-

(b)

(c)

-

2.92

4.27

5.50

4.32

5.31

4.14

0.91

1.78

a

L1-OH, L2-OH: for structure see Scheme 2.^{20 b}L1-4-NHPhN-

(CH₂CH₂Cl)₂) 4-{[4′-(N,N-bis(2′′-chloroethyl)amino)phenyl]car-

bamoyloxymethyl}phenylcarbamoyl-^L-glutamic acid.²⁰^cL2-4-

NHPhN(CH₂CH₂Cl)₂) 4-{[4′-(N,N-bis(2′′-chloroethyl)amino)phen-

yl]carbamoyloxymethyl}phenoxycarbonyl-^L-glutamic acid.²⁰

Discu ssion

The self-immolative prodrugs²⁴

(pro-prodrugs) have

a number of advantages. The possibility of modifying

the activation kinetics by using suitable linkers and the

broad range of anticancer agents that can be converted

to prodrugs is emphasized.^33-35

Our strategy to improve the biological efficacy of the

self-immolative prodrugs has three approaches:

1. Using more effective leaving groups than chlorine

in the nitrogen mustard moiety, such as iodine or

bromine.¹³

Despite possessing a smaller k_catcompared to CMDA,

the bis(iodoethyl) and bis(bromoethyl) prodrugs proved

to be highly active against the CPG2-expressing WiDr

cell line. This could be due to the higher potency of the

released drugs (IC₅₀in the range of 0.5-2.3 µM)

compared to the drug derived from CMDA (120 µM).¹⁹

Self-Immolative Nitrogen Mustards Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1695

As a general observation, prodrugs incorporating

linker 1 are more effective than those containing linker

2. The five most effective prodrugs in terms of dif-

ferential are all linker 1 based (Table 1).

The differential obtained in tumor cells transfected

with CPG2 seems to be dependent on an optimal

chemical reactivity of the prodrugs and the correspond-

ing drugs. This is in good agreement with previous

observation on the in vitro and in vivo behavior of the

direct prodrugs in CPG2-based GDEPT systems. ³⁶The

increased lipophilicity of these prodrugs and drugs could

also be important for their improved biological activity.

QSAR was attempted on the synthesized self-immo-

lative prodrugs, using the available in vitro data on

WiDr. One important parameter determining the anti-

tumor activity of the prodrugs is the kinetics of activa-

tion. However, despite significant differences between

ortho and para nitrogen mustard prodrugs in terms of

activity, the kinetics of activation is very similar for both

classes (see Table 3). Therefore, other parameters

should be considered to rationalize the differences in

activity between the synthesized prodrugs.

Another parameter important in explaining the range

of differentials between CPG2-expressing and nonex-

pressing cells is assumed to be the chemical reactivity

of the released active drugs. For nitrogen mustards, this

is known to parallel their half-lives.^37,38An equation

linking the cytotoxicity differentials of the prodrugs 31b,

32b, 39b, 40b, 41b with the half-lives of the corre-

sponding released drugs 58b, 59b, 61b, 62b, 63b has

been computed, which supports our hypothesis (Figure

1),

F igu r e 1.

2. Employing ortho nitrogen mustards in order to

improve the kinetics by a different positioning of the

self-immolative prodrug in the active site of the enzyme.

3. Synthesizing N-methylated self-immolative pro-

drugs in order to improve the stability of the carbamate

linkage between the linker and the drug moiety and to

increase the basicity and therefore the chemical reactiv-

ity of the released mustard.

The use of I and Br instead of Cl as leaving groups in

the nitrogen mustards led to drugs with shorter half-

lives and increased potency (IC₅₀) 0.5-2.7 µM). The

corresponding prodrugs also exhibited shorter half-lives,

but for many of the prodrugs the differentials in the

WiDr cell lines are better than those of CMDA (31a ,

31b, 32b, 35b, 36b, 39b, and 40b). The two most

effective prodrugs in terms of differential are the iodo

derivatives 31b and 39b.

The prodrugs belonging to the ortho series are less

effective than their para counterparts. However, even

in the ortho series, the I and Br nitrogen mustard

prodrugs are the most active and significant differen-

tials were obtained in the transfected WiDr cell line (50

and 25-fold for 31a and 32a , respectively). The ortho

prodrugs had lower K_m’s with respect to the linkers and

the para series, which is of potential benefit in in vivo

situations.

The N-methylation has a beneficial effect on their

biological activity despite having no benefit on the

kinetics. The prodrugs show lower chemical reactivity

than the nonmethylated counterparts, presumably due

to increased stability of the secondary carbamate. This

effect is minimal at the drug level.

log ∆ ) 2.1212 - 0.1094T_1/2

n ) 5, r ) 0.986, r²) 0.970, s ) 0.068, F ) 103.5

where ∆ ) cytotoxicity differential and T_1/2) half-life

of drug.

No other significant QSAR could be determined for

this set of compounds. This equation also point out to

the limitations for this type of prodrug, since bis-iodo

derivatives are the most reactive nitrogen mustard

synthesized.

Con clu sion s

Nineteen new self-immolative prodrugs and their

corresponding drugs derived from aniline nitrogen

mustard have been synthesized. The new prodrugs

behave as substrates for CPG2 and yielded significant

IC₅₀differential between stCPG2(Q)3 expressing and

control â-gal-expressing WiDr cells. The differential

ranged between 1.4 and 124-fold. Seven prodrugs (31a ,

31b, 32b, 35b, 36b, 39b, and 40b) were more active

than CMDA. The released drugs were also 5.4-41.7-

fold more potent than the 4-[(2-mesyloxyethyl)(2-chlo-

roethyl)amino]benzoic acid drug released from CMDA.

This is the first example of self-immolative prodrugs

cleavable by CPG2 that show better differentials than

CMDA in a GDEPT system.

Exp er im en ta l Section

All starting materials, reagents, and anhydrous solvents

(i.e., THF packed under N₂) were purchased from Aldrich,

unless otherwise stated. Kieselgel 60 (0.043-0.060) was used

1696 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9

Niculescu-Duvaz et al.

in gravity columns (Art 9385 and 15111, Merck). TLC was

performed on precoated sheets of Kieselgel 60 F₂₅₄(Art 5735,

Merck). Melting points were determinated on a Kofler hot-

stage (Reichert Thermovar) melting point apparatus and are

uncorrected. Low resolution EI and FAB spectra were per-

formed on a VG-2AB-SE double focusing magnetic sector

mass spectrometer (Fisons Instruments, Warrington, Manches-

ter, UK), operating at a resolution of 1000. High-resolution

accurate mass spectra were determined on the same system,

but with a resolution set to 8000-10000. Masses are measured

chromatography (cyclohexane:ethyl acetate 1:1) to afford 3b

(4.65 g, 95%) as an oil. ¹H NMR δ_H: -0.01 (s, 12H, SiCH₃),

0.84 (s, 18H, Si-t-Bu), 3.41 (t, 4H, NCH₂, J ) 5.86 Hz), 3.67 (t,

4H, CH₂OSi), 5.09 (s, 2H, PhCH₂), 6.58 (d, 2H, H_arom3+5, J )

8.89 Hz), 6.82 (d, 2H, H_arom2+6), 7.28-7.40 (m, 5H, H_{arom benzyl}),

9.28 (s, 1H, NH). MS m/z: 558 (M⁺, 35), 423 (M⁺- PhCH₂

-

CO₂, 25); acc. mass: (C₃₀H₅₀N₂O₄Si₂) calcd 558.3309, found

558.3330. Anal. (C₃₀H₅₀N₂O₄Si₂) C, H; N required 5.01, found

4.60%

4-(N′-Ben zyloxyca r bon yl-N′-m eth yla m in o)-N,N-bis[(2′-

(ter t-bu tyld im eth ylsilyloxy)eth yl]a n ilin e (4b). 3b (5.2 g,

9.3 mmol) was dissolved in dry THF (60 mL), and NaH (60%

in mineral oil, 0.6 g, 15 mmol) was added. After 40 min stirring

at room temperature under argon, methyl iodide (586 µL, 9.3

mmol) was added and the stirring continued for 12 h. The

solvent was evaporated and the residue redissolved in ethyl

acetate (100 mL) and extracted with distilled water (100 mL).

The organic layer was dried and evaporated to afford 4b (5.34

by peak matching the unknown with

a mass of known

composition. Reported spectra are by FAB unless otherwise

stated. NMR spectra were determined in Me₂SO-d₆on a

Brucker AC250 spectrometer (250 MHz) at 30 °C (303 K)

unless otherwise stated. IR spectra (film) were recorded on a

Perkin-Elmer 1720X FT-IR spectrometer. Elemental analysis

were determined by Butterworth Laboratories Ltd. (Tedding-

ton, Middlesex, UK) and are within 0.4% of theory except when

stated. The chemical stability of the prodrugs and their

propensity to behave as substrates for CPG2 were determined

by HPLC.

1

g, 100%) as an oil. H NMR δ_H: 0.00 (s, 12H, SiCH₃), 0.88 (s,

18H, Si-t-Bu), 3.21 (s, 3H, NCH₃), 3.45 (t, 4H, NCH₂, J ) 6.47

Hz), 3.71 (t, 4H, CH₂OSi), 5.10 (s, 2H, PhCH₂), 6.60 (d, 2H,

4-Nit r o-b is[2′-(ter t-b u t yld im et h ylsilyloxy)et h yl]a n i-

lin e (1b). 4-Nitro-[bis(2′-hydroxyethyl)]aniline (5.0 g, 22.1

mmol) and tert-butyldimethylsilyl chloride (7.5 g, 50 mmol)

were dissolved in 20 mL of DMF; imidazole (4.76 g, 70 mmol)

was added, and the solution was stirred at room temperature

for 20 h. The solution was then concentrated and purified by

column chromatography (cyclohexane:AcOEt 1:1) to afford 1a

H

_arom3+5, J ) 9.08 Hz), 6.99 (d, 2H, H_arom2+6, J ) 7.25 Hz), 7.20-

7.35 (m, 5H, H_aro_{m benzyl}). MS m/z: 572 (M⁺, 50), 595 (M⁺

+

Na, 7), 437 (M⁺- PhCH₂- CO₂, 45); acc. mass: (C₃₁H₅₂N₂O₄-

Si₂) calcd 572.3466, found 572.3485.

4-Meth ylam in o-N,N-bis[(2′-(ter t-bu tyldim eth ylsilyloxy)-

eth yl]a n ilin e (5b). 4b (2.9 g, 5.06 mmol) was dissolved in

ethyl acetate (120 mL), and Pd/C 10% catalyst (1.6 g) was

added. The suspension was stirred under H₂atmosphere for

3 h. The catalyst was filtered off, and the filtrate was

1

(8.9 g, 89%) as a yellow oil. H NMR δ_H(ppm): -0.03 (s, 12H,

SiCH₃), 0.81 (s, 18H, Si-t-Bu), 3.64 (t, 4H, NCH₂, J ) 5.19 Hz),

3.78 (t, 4H, CH₂OSi), 6.83 (d, 2H, H_arom2+6, J ) 9.37 Hz), 8.00

(d, 2H, H_arom3+5); MS m/z: 455 (M⁺+ 1, 88), 477 (M⁺+ 23, 5),

439 (M⁺- Me, 53), 397 (M⁺- t-Bu, 30); acc. mass: (C₂₂H₄₃N₂O₄-

Si₂) calcd 455.2761, found 455.2767. Anal. (C₂₂H₄₃N₂O₄Si₂) C,

H, N.

1

evaporated to afford 5b (2.23 g, 100%) as an oil. H NMR δ_H:

-0.01 (s, 12H, SiCH₃), 0.84 (s, 18H, Si-t-Bu), 2.58 (s, 3H,

NCH₃), 3.22-3.32 (t, 4H, NCH₂), 3.63 (t, 4H, CH₂OSi, J ) 6.19

Hz), 4.87 (s, 1H, NH), 5.10 (s, 2H, PhCH₂), 6.43 (d, 2H, H_arom3+5

,

J ) 8.92 Hz), 6.65 (d, 2H, H_arom2+6), 7.20-7.35 (m, 5H, H_arom

_benzyl). MS m/z: 438 (M⁺, 100), 451 (M⁺+ Na, 25); acc. mass:

(C₂₃H₄₆N₂O₂Si₂) calcd 438.3098, found 438.311500. Anal.

(C₂₃H₄₆N₂O₂Si₂) C, H; N required 6.38, found 5.88.

The same procedure affords: 2-Nitr o-bis[2′-(ter t-bu tyld i-

m eth ylsilyloxy)eth yl]a n ilin e (1a ), yellow oil (95.1%). Puri-

fied by column chromatography (eluent: cyclohexane:AcOEt

1

3:1); H NMR δ_H(ppm): -0.03 (s, 12H, SiCH₃), 0.80 (s, 18H,

Dia llyl 4-{[4′-Bis(2′′-ter t-bu tyld im eth ylsilyloxyeth yl)

a m in op h en yl]ca r ba m oyloxym eth yl}p h en ylca r ba m oyl-^L-

glu ta m a te 6b (Z ) NH). A 2.0 g (4.7 mmol) amount of 2b

was dissolved in 30 mL of DCM, triethylamine (1.24 mL, 9.5

mmol) was added and then triphosgene (0.48 g, 1.57 mmol) in

one portion with vigorous stirring. After 15 min, the solvent

was evaporated and the residue taken up in THF. The

undissolved solid was filtered off, and the filtrate was evapo-

rated to dryness. The corresponding isocyanate was obtained

as an oil which was redissolved in 20 mL of CH₂Cl₂and mixed

with a solution of diallyl N-[4-(hydroxymethyl)phenylcarbam-

oyl]-L-glutamate (linker1, L1A) (1.76 g, 4.7 mmol) in 30 mL of

CH₂Cl₂.²²Dibutyltin dilaurate (120 µL) was added and the

stirring continued for 20 h at room temperature. After the

evaporation of the solvent the residue was purified by pre-

parative HPLC (cyclohexane:AcOEt, 3:1) to yield 6b (3.10 g,

80%) as a foamy solid, mp 50-53 °C. ¹H NMR, δ_H, (ppm): -0.01

(s, 12H, SiCH₃), 0.84 (s, 18H, Si-t-Bu), 1.80-2.15 (2m, 2H, CH₂-

CH(NH)), 2.45 (t, 2H, CH₂CO₂, J ) 7.29 Hz), 3.42 (t, 4H, NCH₂,

J ) 5.31 Hz), 3.68 (t, 4H, CH₂OSi), 4.25-4.35 (m, 1H, CH(NH)-

CH₂), 4.55 (d, 2H, CH₂O allyl, J ) 5.39 Hz), 4.60 (d, 2H, CH₂O

allyl, J ) 5.28 Hz), 4.99 (s, 2H, PhCH₂), 5.15-5.37 (m, 4H,

CH₂d allyl), 5.80-6.05 (m, 2H, CHd allyl), 6.58 (d, 2H,

Si-t-Bu), 3.27 (t, 4H, NCH₂, J ) 5.87 Hz), 3.65 (t, 4H, CH₂-

OSi), 6.98 (dt, 1H, H₅, J ) 7.58 Hz), 7.37 (dd,1H, H₃, J ) 8.43

Hz), 7.48 (dt,1H, H₄, J ) 7.80 Hz), 7.67 (dd, 1H, H₆, J ) 8.05

Hz); MS m/z: 455 (M⁺+ 1, 32), 397 (M⁺- t-Bu, 8), 309 (M⁺

- SiTBDM, 100); acc. mass: (C₂₂H₄₃N₂O₄Si₂) calcd 455.2761,

found 455.2745. Anal. (C₂₂H₄₃N₂O₄Si₂) C, H; N required 6.17,

found 6.94%.

4-Am in o-bis[2′-(ter t-bu tyld im eth ylsilyloxy)eth yl]a n i-

lin e (2b). 5.50 g (12.1 mmol) of 1b was dissolved in 90 mL of

THF, 1.5 g Pd/C 10% was added and the suspension was

stirred under H₂atmosphere for 6 h. The catalyst was then

filtered off, the solvent evaporated and the residue submitted

to purification by column chromatography (cyclohexane:AcOEt

3:1). 2b (4.90 g, 95%) resulted as an oil. ¹H NMR δ_H(ppm):

0.00 (s, 12H, SiCH₃), 0.85 (s, 18H, Si-t-Bu), 3.30 (t, 4H, NCH₂,

J ) 6.20 Hz), 3.63 (t, 4H, CH₂OSi), 4.34 (s, 2H, NH₂), 6.46 (s,

4H, H_arom); MS m/z: 424 (M⁺, 70); acc. mass: (C₂₂H₄₄N₂O₂Si₂)

calcd 424.2941, found 424.2950. Anal. (C₂₂H₄₄N₂O₂Si₂): H, N;

C required 62.21, found 62.63%.

2-Am in o-bis[2′-(ter t-bu tyld im eth ylsilyloxy)eth yl]a n i-

lin e (2a ) was synthesized in a similar way. An oil was

obtained (4.60 g, 99%). ¹H NMR, δ_H(ppm): 0.04 (s, 12H,

SiCH₃), 0.85 (s, 18H, Si-t-Bu), 3.02 (t, 4H, NCH₂, J ) 6.19 Hz),

3.56 (t, 4H, CH₂OSi), 6.49 (dt, 1H, H₅, J ) 7.51 Hz), 6.63 (dd,

1H, H₃, J ) 7.92 Hz), 6.78 (dt, 1H, H₄, J ) 7.54 Hz), 7.00 (dd,

H

_{arom3′+5′}, J ) 8.99 Hz), 6.66 (d, 1H, NH-G, J ) 7.99 Hz), 7.19

(d, 2H, H_{arom2′+6′}), 7.27 (d, 2H, H_arom2+6, J ) 8.46 Hz), 7.39 (d,

2H, H_arom3+5), 8.69 (s, 1H, PhNH), 9.26 (s, 1H, Ph′NH); MS

m/z: 827 (M⁺+ 1, 5), 849 (M⁺+ Na, 55); acc. mass:

1H, H₆, J ) 7.81 Hz); MS m/z: 425 (M⁺+ 1, 28), 367 (M⁺

-

(C₄₂H₆₇N₄O₉Si₂) calcd 827.4447, found 827.4440. Anal. (C₄₂H₆₆

N₄O₉Si₂): C, H, N.

-

t-Bu + 1, 15); acc. mass: (C₂₂H₄₅N₂O₂Si₂) calcd 425.3020, found

425.3034.

4-(N′-Ben zyloxyca r b on yla m in o)-N,N-b is[(2′-(ter t-b u -

tyld im eth ylsilyl-oxy)eth yl]a n ilin e (3b). 4-Amino-N,N-bis-

[(2′-(tert-butyldimethylsilyl-oxy)ethyl]-aniline 2b (3.74 g, 8.8

mmol) was dissolved in THF (100 mL), and N-(benzyloxycar-

bonyloxy)succinimide (2.25 g, 9.0 mmol) was added. The

solution was stirred at room temperature for 16 h. The solvent

was evaporated, and the residue was purified by column

Using a similar procedure the following compounds were

obtained: d ia llyl 4-{[2′-bis(2′′-ter t-bu tyld im eth ylsilyloxy-

et h yl)a m in op h en yl]ca r ba m oyloxyben zyl}ca r b a m oyl-^L-

glu ta m a te 6a (Z ) NH) was obtained as an oil (1.99 g; 65.6%)

after purification by preparative HPLC (cyclohexane:AcOEt

7:1). H NMR, δ_H, (ppm): -0.06 (s, 12H, SiCH₃), 0.79 (s, 18H,

Si-t-Bu), 1.90-2.20 (2m, 2H, CH₂CH(NH)), 3.00 (t, 4H, NCH₂,

1

Self-Immolative Nitrogen Mustards Prodrugs

) 5.66 Hz), 3.49 (t, 4H, CH₂OSi), 4.29-4.32 (m, 1H,

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1697

J

4-Bis(2′-ter t-bu tyld im eth ylsilyloxyeth yl)a m in o-N-ter t-

bu tyloxyca r bon yla n ilin e (45b). A 1.0 g (2.35 mmol) amount

of 2b was dissolved in THF (30 mL), and tert-butyl pyrocar-

bonate (565 mg, 2.6 mmol) was added. The solution was stirred

at room temperature for 16 h. The solvent was evaporated,

and the residue was purified by preparative HPLC (cyclohex-

ane:AcOEt 6:1) to afford 45b (1.23 g, 100%) as an oil. ¹H NMR,

δ_H, (ppm): 0.00 (s, 12H, SiCH₃), 0.85 (s, 18H, Si-t-Bu), 1.47

(s, 9H, O-t-Bu), 3.41 (t, 4H, NCH₂, J ) 5.93 Hz), 3.69 (t, 4H,

CH₂OSi), 6.57 (d, 2H, H_arom3+5, J ) 8.87 Hz), 7.18 (d, 2H,

CH₂CH(NH)), 4.57 (dd, 4H, CH₂O-allyl, J ₁)10.08 Hz, J ₂)5.26

Hz), 5.03 (s, 2H, PhCH₂), 5.16-5.36 (m, 4H, CH₂d allyl), 6.60

(d, 1H, NH-G, J ) 8.02 Hz), 7.01 (dt, 1H, H_arom4(5)′, J _o) 7.66

Hz, J _m) 1.34 Hz), 7.12 (dd, 1H, H_arom5(4)′), 7.25 (d, 2H, H_arom3+5

J ) 8.53 Hz), 7.37 (d, 2H, H_arom2+6), 7.95 (dd, 1H, H_arom6′, J _o

,

)

8.53), 8.43 (s, 1H, PhNH, J ) 7.87 Hz), 8.64 (s, 1H, Ph′NH);

MS m/z: 827 (M⁺+ 1, 5), 849 (M⁺+ 23, 55); acc. mass:

(C₄₂H₆₆N₄O₉Si₂Na) calcd: 849.4266, found 849.4240. Anal.

(C₄₄H₇₃N₃O₁₀Si₂), C, H, N.

H

_arom2+6), 8.80 (d, 1H, NH); MS m/z: 524 (M⁺, 23), 469 (M⁺

-

Di-tert-butyl4-{[2′-bis(2′′-(tert-butyldimethylsilyloxyethyl)-

a m in op h en yl]ca r ba m oyloxym eth yl}p h en yloxyca r bon yl-

L-glu ta m a te 7a (Z ) O) was obtained from L2B as an oil (0.97

g; 47.5%) after purification by preparative HPLC (cyclohexane:

t-Bu, 15), 423 (M⁺- t-Bu - CO₂, 18); acc. mass: (C₂₇H₅₂N₂O₄-

Si₂) calcd: 524.3466, found 524.3480. Anal. (C₂₇H₅₂N₂O₄Si₂):

C, H, N.

1

2-Bis(2′-ter t-bu tyld im eth ylsilyloxyeth yl)a m in o-N-ter t-

bu tyloxyca r bon yla n ilin e (45a ) was obtained by the same

method as an oil (4.27 g, 91.4%) after purification by prepara-

tive HPLC (cyclohexane:AcOEt 4:1). ¹H NMR, δ_H, (ppm): -0.13

(s, 12H, SiCH₃), 0.83 (s, 18H, Si-t-Bu), 1.46 (s, 9H, CO₂-t-Bu),

3.01 (t, 4H, NCH₂, J ) 5.56 Hz), 3.49 (t, 4H, CH₂OSi), 6.99

(dt, 1H, H_arom4(5), J _o) 7.57, J _m) 1.20 Hz), 7.11 (dd, 1H,

AcOEt 7:1). H NMR, δ_H, (ppm): -0.06 (s, 12H, SiCH₃), 0.79

(s, 18H, Si-t-Bu), 1.40 (s, 9H, CO₂-t-Bu), 1.42 (s, 9H, CO₂-t-

Bu), 2.31-2.37 (m, 2H, CH₂CO₂), 3.01 (t, 4H, NCH₂, J ) 5.70

Hz), 3.50 (t, 4H, CH₂OSi), 5.12 (s, 2H, PhCH₂), 7.01-7.12(m,

4H, H_{arom3+5, 3′, 5′}), 7.30 (dd, 1H, H_arom4′, J _o) 7.15 Hz, J _m) 1.24

Hz), 7.39 (d, 2H, H_arom3+5, J ) 7.39 Hz),), 7.79 (dd, 1H, H_arom6′

,

J _o) 8.16 Hz, J _m) 1.18 Hz), 8.07 (d, 1H, NH-G, J ) 7.87 Hz),

8.49 (s, 1H, Ph′NH); MS m/z: 860 (M⁺+ 1, 37), 882 (M⁺+ 23,

12); acc. mass: (C₄₄H₇₄N₃O₁₀Si₂) calcd: 860.4913, found

860.4900. Anal. (C₄₄H₇₃N₃O₁₀Si₂) C, H, N.

H

_arom5(4), J _o) 7.44 Hz), 7.29 (dd, 1H, H_arom3, J _o) 8.66, J _m)

1.23 Hz), 7.94 (d, 1H, H_arom6, J _o) 8.17 Hz), 8.13 (s, 1H, PhNH);

MS m/z: 525 (M⁺+ 1, 15), 469 (M⁺- t-Bu + 1, 15); acc.

mass: (C₂₇H₅₃N₂O₄Si₂) calcd: 525.3544, found 525.3560. Anal.

(C₂₇H₅₂N₂O₄Si₂) H, N; C required 61.78, found 62.19.

Di-tert-butyl4-{[4′-bis(2′′-tert-butyldimethylsilyloxyethyl)-

a m in op h en yl]ca r ba m oyloxym eth yl}p h en yloxyca r bon yl-

L-glu ta m a te 7b (Z ) O) was obtained from L2B as an oil (3.73

Dia llyl 4-{[4′-(N,N-Bis(2′′-h yd r oxyeth yl)a m in o)p h en yl]-

ca r ba m oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m a te 10b

(Z ) NH). A 2.0 g (2.42 mmol) amount of 6b (Z ) NH) was

dissolved in 60 mL of THF, 6.0 mL of triethylamine trihydro-

fluoride was added, and the solution was stirred at room

temperature for 7 h. The solvent was evaporated, and the

residue was diluted with AcOEt, extracted with H₂O (200 mL),

saturated aqueous NaHCO₃(200 mL), and again with H₂O

(200 mL), dried (MgSO₄), and evaporated to yield 10b (1.4 g,

97%) as a foamy solid, mp 79-81 °C. ¹H NMR, δ_H, (ppm):

1.85-2.10 (2m, 2H, CH₂CH(NH)), 2.46 (t, 2H, CH₂CO₂, J )

8.14 Hz), 3.30-3.40 (m, 4H, NCH_2,under H₂O), 3.45-3.55 (m,

4H, CH₂OH), 4.25-4.35 (m, 1H, CH(NH)CH₂), 4.55 (d, 2H,

CH₂O allyl, J ) 5.37 Hz), 4.60 (d, 2H, CH₂O allyl, J ) 5.26

Hz), 4.72 (t, 2H, OH, J ) 5.21 Hz), 4.99 (s, 2H, PhCH₂), 5.15-

5.40 (m, 4H, CH₂d allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.59

(d, 2H, H_{arom3′+5′}, J ) 8.76 Hz), 6.64 (d, 1H, NH-G, J ) 8.22

Hz), 7.19 (d, 2H, H_{arom2′+6′}), 7.27 (d, 4H, H_arom2+6, J ) 8.56 Hz),

7.38 (d, 2H, H_arom3+5), 8.67 (s, 1H, PhNH), 9.25 (s, 1H, Ph′NH);

MS, m/z: 599 (M⁺+ 1, 35), 621 (M⁺+ Na, 7); acc. mass:

1

g, 66.7%). H NMR, δ_H, (ppm): 0.00 (s, 12H, SiCH₃), 0.85 (s,

18H, Si-t-Bu), 1.40 (s, 9H, CO₂-t-Bu), 1.42 (s, 9H, CO₂-t-Bu),

1.70-2.00 (2m, 2H, CH₂CH(NH)), 2.34 (t, 2H, CH₂CO₂, J )

7.81 Hz), 3.42 (t, 4H, NCH₂, J ) 5.03 Hz), 3.69 (t, 4H, CH₂-

OSi), 3.95-4.05 (m, 1H, CH(NH)CH₂), 5.08 (s, 2H, PhCH₂),

6.59 (d, 2H, H_{arom3′+5′}, J ) 8.86 Hz), 7.10 (d, 2H, H_arom2+6, J )

8.44 Hz), 7.19 (d, 2H, H_{arom2′+6′}), 7.41 (d, 2H, H_arom3+5), 8.09 (d,

1H, NH-G, J ) 7.75 Hz), 9.24 (s, 1H, Ph′NH); MS m/z: 860

(M⁺+ 1, 37); acc. mass: (C₄₄H₇₄N₃O₁₀Si₂) calcd: 860.4913,

found 860.4900. Anal. (C₄₄H₇₃N₃O₁₀Si₂): C, H, N.

Dia llyl 4-{N-[4′-Bis(2′′-ter t-bu tyld im eth ylsilyloxyeth -

yl)a m in op h en yl]-N-m eth ylca r ba m oyloxym eth yl}p h en yl-

ca r ba m oyl-^L-glu ta m a te 8b. Activated L1A, diallyl N-(4-{[4-

n it r oph en oxyca r bon yloxy]m et h yl}ph en ylca r ba m oyl)-^L-

glutamate (2.1 g, 3.9 mmol), prepared as described previ-

ously,²²and 5b (2.2 g, 5 mmol) were dissolved in DMA (50

mL) and stirred for 5 days at room temperature. The solvent

was evaporated and the residue purified by column chroma-

tography (CH₂Cl₂:AcOEt 9:1) to yield 8b (0.92 g, 28%) as an

oil. ¹H NMR, δ_H, (ppm): 0.01 (s, 12H, SiCH₃), 0.83 (s, 18H,

Si-t-Bu), 1.80-2.10 (2m, 2H, CH₂CH(NH)), 2.44 (t, 2H, CH₂-

CO₂, J ) 8.25 Hz), 3.11 (s, 3H, NCH₃), 3.46 (t, 4H, NCH₂, J )

5.58 Hz), 3.69 (t, 4H, CH₂OSi), 4.25-4.35 (m, 1H, CH(NH)-

CH₂), 4.53 (d, 2H, CH₂O allyl, J ) 5.45 Hz), 4.59 (d, 2H, CH₂O

allyl, J ) 5.38 Hz), 4.94 (s, 2H, PhCH₂), 5.14-5.38 (m, 4H,

CH₂d allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.60 (d, 3H,

H_{arom3′+5′}+ NH-G, J ) 8.70 Hz), 6.99 (d, 2H, H_{arom2′+6′}), 7.15 (d,

2H, H_arom2+6), 7.32 (d, 2H, H_arom3+5, J ) 8.20 Hz), 8.60 (s, 1H,

PhNH); MS m/z: 841 (M⁺+ 1, 5), 864 (M⁺+ Na, 3), 437 (M⁺

- L1ACH₂OCO, 100); acc. mass: (C₄₃H₆₉N₄O₉Si₂) calcd

841.4603, found 841.4630. Anal. (C₄₃H₆₈N₄O₉Si₂): C, H; N

required 6.66, found 6.22.

(C₃₀H₃₉N₄O₉) calcd 599.2717, found 599.2707. Anal. (C₃₀H₃₈

N₄O₉): H, N; C required 60.66, found 60.19.

-

The following compounds were prepared according to the

same procedure:

Dia llyl 4-{[2′-[N,N-bis(2′′-h yd r oxyeth yl)a m in o)p h en yl]-

ca r ba m oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m a te 10a

(Z ) NH), obtained as a white foamy solid (1.70 g, 63.5%).¹H

NMR, δ_H, (ppm): 1.84-2.15 (2m, 2H, CH₂CH(NH)), 2.47 (t,

2H, CH₂CO₂, J ) 8.20 Hz), 2.98 (t, 4H, NCH_2,J ) 5.68 Hz),

3.33 (t, 4H, CH₂OH), 4.30-4.40 (m, 1H, CH(NH)CH₂), 4.54-

4.66 (m, 4H, CH₂O allyl), 5.07 (s, 2H, Ph-CH₂), 5.21-5.38 (m,

4H, CH₂d allyl), 5.87-6.05 (m, 2H, CHd allyl), 6.63 (d, 1H,

NH-G, J ) 7.98 Hz), 6.99-7.09 (m, 2H, H_{arom4′(5′),}H_arom3′,J )

8.76 Hz), 7.26-7.30 (t, 1H, H_{arom5′(4′)}) 7.30 (d, 2H, H_arom3+5, J )

8.58 Hz), 7.40 (d, 2H, H_arom2+6), 7.95 (dd, 1H, H_arom6′, J _o) 8.67

Hz, J _m) 1.40 Hz), 8.65 (s, 1H, Ph-NH), 9.02 (s, 1H, Ph′-NH);

MS, m/z: 599 (M⁺+ 1, 75), 621 (M⁺+ Na, 72); acc. mass:

(C₃₀H₃₉N₄O₉) calcd 599.2717, found 599.2740. Anal. (C₃₀H₃₈N₄O₉‚

0.7 H₂O): C, H, N.

Using a similar procedure, d i-ter t-bu tyl 4-{N-[4′-bis(2′′-

t er t -b u t y ld i m e t h y ls i ly lo x y e t h y l)a m i n o p h e n y l]-N -

m e t h y lc a r b a m o y lo x y m e t h y l}p h e n o x y c a r b o n y l-L -

glu ta m a te 9b was obtained from activated L2B, di-tert-butyl

N-(4-{[4-nitrophenoxycarbonyloxy]methyl}phenoxycarbonyl)-

L-glutamate as an oil (3.8 g, 92.5%). ¹H NMR, δ_H, (ppm): 0.00

(s, 12H, SiCH₃), 0.85 (s, 18H, Si-t-Bu), 1.40 (s, 9H, CO₂-t-Bu),

1.42 (s, 9H, CO₂-t-Bu), 1.75-2.05 (2m, 2H, CH₂CH(NH)), 2.33

(t, 2H, CH₂CO₂), 3.14 (s, 3H, NCH₃), 3.46 (t, 4H, NCH₂, J )

5.03 Hz), 3.69 (t, 4H, CH₂OSi), 3.90-4.05 (m, 1H, CH(NH)-

CH₂), 5.03 (s, 2H, PhCH₂), 6.62 (d, 2H, H_{arom3′+5′}, J ) 9.03 Hz),

7.03 (d, 4H, H_{arom2′+6′}+ H_arom3+5, J ) 8.90 Hz), 7.27 (d, 2H,

Di-ter t-bu tyl 4-{[2′-(N,N-bis(2′′-h yd r oxyeth yl)a m in o)-

p h e n yl]ca r b a m oyloxym e t h yl}p h e n yloxyca r b on yl-^L-

glu ta m a te 11a (Z ) O) was obtained as a white foamy solid

1

(2.35 g, 99%), mp 57-60 °C. H NMR, δ_H, (ppm): 1.40 (s, 9H,

CO₂-t-Bu), 1.41 (s, 9H, CO₂-t-Bu), 1.70-2.0o (2m, 2H, CH₂-

CH(NH)), 2.30-2.37 (m, 2H, CH₂CO₂-t-Bu), 2.96 (t, 4H, NCH₂,

J ) 5.74 Hz), 3.20 (t, 4H, CH₂OH), 3.97-4.04 (m, 1H, CH(NH)-

CH₂), 4.64 (t, 2H, N(CH₂CH₂OH)₂, J ) 5.14 Hz), 5.13 (s, 2H,

PhCH₂), 6.98-7.05 (m, 2H, H_{arom3′+5′}, J ) 8.87 Hz), 7.10 (d,

H

_arom2+6), 8.09 (d, 1H, NH-G, J ) 7.83); MS m/z: 875 (M⁺+ 1,

38), 897 (M⁺+ Na, 12), 819 (M⁺- t-Bu, 3); acc. mass:

(C₄₅H₇₆N₃O₁₀Si₂) calcd: 874.5040, found 874.5069.

1698 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9

2H, H_arom3+5, J ) 8.52 Hz), 7.27 (dd, 1H, H_arom4′, J _o) 7.75, J _m

Niculescu-Duvaz et al.

4-(N′-Ben zyloxyca r bon yl-N′-m eth yla m in o)-N,N-b is(2′-

h yd r oxyeth yl)a n ilin e (47b) was obtained by desilylation of

3b, as described previously for compound 10b, as a wax (1.2

) 1.53 Hz), 7.43 (d, 2H, H_arom2+6), 7.94 (dd, 1H, H_arom6′, J _o

)

8.05, J _m) 1.53 Hz), 8.09 (d, 1H, NH-G, J ) 7.89 Hz), 9.09 (s,

1

1H, Ph′-NH); MS m/z: 632 (M⁺+ 1, 100); acc. mass: (C₃₂H₄₆

-

g, 88%). H NMR δ_H: 3.14 (s, 3H, NCH₃), 3.38 (t, 4H, NCH₂),

3.45-3.55 (t, 4H, CH₂OH), 4.71 (t, 2H, OH, J ) 5.16 Hz), 5.04

(s, 2H, PhCH₂), 6.62 (d, 2H, H_arom3+5, J ) 8.17 Hz), 7.02 (d,

2H, H_arom2+6), 7.26-7.36 (m, 5H, H_{arom benzyl}). MS m/z: 344 (M⁺,

100), 367 (M⁺+ Na, 20); acc. mass: (C₁₉H₂₄N₂O₄) calcd

344.1746, found 344.1736. Anal. (C₁₉H₂₄N₂O₄) C, H, N.

N₃O₁₀) calcd 632.3183, found 632.3170. Anal. (C₃₂H₄₅N₃O₁₀) C,

H, N.

Di-t-butyl4-{[4′-(N,N-Bis(2′′-hydroxyethyl)amino)phenyl]-

ca r b a m oyloxym et h yl}p h en yloxyca r b on yl-^L-glu t a m a t e

11b (Z ) O). The compound was obtained from 7b (Z ) O) by

1

Diallyl 4-{[4′-(N,N-Bis(2′′-m esyloxyeth yl)am in o)ph en yl]-

car bam oyloxym eth yl}ph en ylcar bam oyl-^L-glu tam ate (14b).

Over a solution of 10b (Z ) NH) (0.50 g, 0.83 mmol), 4-N-

(dimethylamino)pyridine (25 mg, 0.2 mmol), and NEt₃(364 µL,

2.6 mmol) in CH₂Cl₂(10 mL), mesyl anhydride (0.435 g, 2.5

mmol) dissolved in CH₂Cl₂(10 mL) was added. After stirring

at room temperature for 2.5 h, the solution was diluted with

CH₂Cl₂to 50 mL, extracted with 10% aq citric acid (2 × 50

mL), aq NaHCO₃(50 mL), and distilled water (50 mL), dried

over MgSO₄, and evaporated to afford 14b (0.62 g, 99%) as a

a similar method, as a solid (2.73 g, 99%), mp 47-50 °C. H

NMR, δ_H, (ppm): 1.40 (s, 9H, CO₂-t-Bu), 1.42 (s, 9H, CO₂-t-

Bu), 1.70-2.05 (2m, 2H, CH₂CH(NH)), 2.30-2.40 (m, 2H, CH₂-

CO₂-t-Bu), 3.35 (t, 4H, NCH₂, J ) 5.75 Hz), 3.50 (t, 4H,

CH₂OH), 3.95-4.05 (m, 1H, CH(NH)CH₂), 4.63 (t, 2H, OH, J

) 5.34 Hz), 5.08 (s, 2H, PhCH₂), 6.60 (d, 2H, H_{arom3′+5′}, J )

8.87 Hz), 7.10 (d, 2H, H_arom2+6, J ) 8.42 Hz), 7.19 (d, 2H,

H

_{arom2′+6′}), 7.41 (d, 2H, H_arom3+5), 8.10 (d, 1H, NH-G, J ) 7.74

Hz), 9.20 (s, 1H, Ph′NH); MS, m/z: 632 (M⁺+ 1, 100); acc.

mass: (C₃₂H₄₆N₃O₁₀) calcd 632.3183, found 632.3170. Anal.

(C₃₂H₄₅N₃O₁₀): C, H; N required 6.65, found 6.19.

1

solid, mp 55-58 °C. H NMR δ_H, (ppm): 1.80-2.10 (2m, 2H,

CH₂CH(NH)), 2.46 (t, 2H, CH₂CO₂, J ) 7.92 Hz), 3.15 (s, 6H,

CH₃SO₃), 3.67 (t, 4H, NCH₂, J ) 5.45 Hz), 4.27 (t, 5H, CH(NH)-

CH₂+ CH₂OMes), 4.55 (d, 2H, CH₂O allyl, J ) 5.42 Hz), 4.61

(d, 2H, CH₂O allyl, J ) 5.26 Hz), 5.01 (s, 2H, PhCH₂), 5.15-

5.40 (m, 4H, CH₂) allyl), 5.85-6.00 (m, 2H, CHd allyl), 6.64

(d, 1H, NH-G, J ) 7.95 Hz), 6.74 (d, 2H, H_{arom3′+5′}, J ) 8.97

Hz), 7.28 (d, 4H, H_arom2+6+ H_{arom2′+6′}, J ) 8.53 Hz), 7.39 (d,

2H, H_arom3+5, J ) 8.49 Hz), 8.68 (s, 1H, PhNH), 9.38 (s, 1H,

Ph′NH); MS, m/z: 754 (M⁺, 17), 777 (M⁺+ Na, 28); acc. mass:

(C₃₂H₄₂N₄O₁₃S₂Na) calcd 777.2088, found 777.2080. Anal.

(C₃₂H₄₂N₄O₁₃S₂): C, H, N.

Dia llyl 4-{N-[4′-bis(2′′-h yd r oxyeth yl)a m in op h en yl]-N-

m eth ylca r ba m oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta -

m a te 12b was obtained from 8b by a similar method as a gum

(0.58 g, 93.6%). ¹H NMR, δ_H, (ppm): 1.75-2.15 (2m, 2H, CH₂-

CH(NH)), 2.44 (t, 2H, CH₂CO₂, J ) 8.54 Hz), 3.12 (s, 3H,

NCH₃), 3.38 (t, 4H, NCH_2,J ) 5.50 Hz), 3.50 (t, 4H, CH₂OH),

4.25-4.35 (m, 1H, CH(NH)CH₂), 4.53 (d, 2H, CH₂O allyl, J )

5.31 Hz), 4.59 (d, 2H, CH₂O allyl, J ) 5.28 Hz), 4.71 (t, 2H,

OH, J ) 5.45 Hz), 4.94 (s, 2H, PhCH₂), 5.15-5.38 (m, 4H,

CH₂d allyl), 5.85-6.00 (m, 2H, CHd allyl), 6.61 (d, 3H,

H_{arom3′+5′}+NH-G, J ) 8.90 Hz), 6.99 (d, 2H, H_{arom2′+6′}), 7.17 (d,

4H, H_arom2+6), 7.33 (d, 2H, H_arom3+5, J ) 8.09 Hz), 8.62 (s, 1H,

PhNH); MS, m/z: 613 (M⁺+ 1, 45); acc. mass: (C₃₁H₄₁N₄O₉)

calcd 613.2874, found 613.2853. Anal. (C₃₁H₄₀N₄O₉): C, H, N.

Using the same procedure the following compounds were

synthesized:

Diallyl 4-{[2′-(N,N-Bis(2′′-m esyloxyeth yl)am in o)ph en yl]-

car bam oyloxym eth yl}ph en ylcar bam oyl-^L-glu tam ate (14a)

was obtained (1.33 g, 57%) as an oil, purified by preparative

HPLC (cyclohexane:AcOEt 1:1). ¹H NMR δ_H, (ppm): 1.82-2.12

(2m, 2H, CH₂CH(NH)), 2.45 (t, 2H, CH₂CO₂, J ) 8.00 Hz), 3.05

(s, 6H, CH₃SO₃), 3.29 (t, 4H, NCH₂, J ) 5.25 Hz), 4.10 (t, 4H_,

CH₂OMes), 4.25-4.35 (m, 1H, CH₂CH(NH)), 4.55 and 4.61 (dd,

2H, CH₂O allyl, J ) 5.26 Hz), 5.05 (s, 2H, PhCH₂), 5.20-5.37

(m, 4H, CH₂d allyl), 5.83-6.00 (m, 2H, CHd allyl), 6.62 (d,

1H, NH-G, J ) 7.97 Hz), 7.06 (dt, 1H, H_{arom4′(5′)}, J _o) 7.66 Hz,

J _m) 1.38 Hz), 7.19 (dt, 1H, H_{arom4′(5′)}), 7.29 (d, 2H, H_arom3+5, J

) 8.54 Hz), 7.38 (d, 2H, H_arom2+6), 7.95 (d. 1H, H_arom6′J ) 8.14

Hz), 8.68 (s, 1H, PhNH), 8.64 (s, 1H, Ph′NH); MS, m/z: 755

(M⁺+ 1, 1), 777 (M⁺+ Na, 5); acc. mass: (C₃₂H₄₂N₄O₁₃S₂Na)

calcd 777.2088, found 777.2055. Anal. (C₃₂H₄₂N₄O₁₃S₂): C, H,

N.

Di-ter t-bu tyl 4-{[2′-(N,N-Bis(2′′-m esyloxyeth yl)a m in o)-

p h e n yl]ca r b a m oyloxym e t h yl}p h e n yloxyca r b on yl-^L-

glu ta m a te (15a ) resulted as a foamy solid (2.20 g, 91.9%),

mp 43-44 °C. ¹H NMR, δ_H, (ppm): 1.40 (s, 9H, CO₂-t-Bu), 1.41

(s, 9H, CO₂-t-Bu), 1.75-2.03 (2m, 2H, CH₂CH(NH)), 2.50 (m,

2H, CH₂CO₂-t-Bu), 3.04 (s, 6H, CH₃SO₃), 3.29 (t, 4H, NCH₂, J

) 5.24 Hz), 3.95-4.05 (m, 1H, CH(NH)CH₂), 4.10 (t, 4H, CH₂-

OMes), 5.13 (s, 2H, PhCH₂), 7.07-7.11 (m, 3H, H_arom3+5, H_arom

_4′(5′)), 7.19 (t, 1H, H_{arom5′(4′),}J _o) 8.47 Hz, J _m) 1.36 Hz), 7.45

(d, 3H, H_{arom2+6, 3′}, J ) 8.69 Hz), 7.69 (q, 1H, H_arom6′, J _o) 8.01

Hz, J _m) 1.04 Hz), 8.15 (d, 1H, NH-G, J ) 7.88 Hz), 8.44 (s,

1H, Ph′NH); MS, m/z: 788 (M⁺+ 1, 35), 810 (M⁺+ 23, 100);

acc. mass: (C₃₄H₅₀N₃O₁₄S₂) calcd: 788.2734, found: 788.2760.

Anal. (C₃₄H₄₉N₃O₁₄S₂): C, H, N.

Di-ter t-bu tyl 4-{N-[4′-bis(2′′-h yd r oxyeth yl)a m in op h e-

n yl]-N-m eth ylca r ba m oyloxym eth yl}p h en yloxyca r bon yl-

L-glu ta m a te 13b was obtained from 9b by a similar method

as a glass (2.29 g, 91%). ¹H NMR, δ_H, (ppm): 1.40 (s, 9H, CO₂-

t-Bu), 1.42 (s, 9H, CO₂-t-Bu), 1.70-2.00 (2m, 2H, CH₂CH(NH)),

2.34 (t, 2H, CH₂CO₂), 3.14 (s, 3H, NCH₃), 3.39 (t, 4H, NCH₂),

3.48-3.55 (m, 4H, CH₂OH), 3.90-4.05 (m, 1H, CH(NH)CH₂),

4.72 (t, 2H, OH, J ) 5.28 Hz), 5.04 (s, 2H, PhCH₂), 6.63 (d,

2H, H_{arom3′+5′}, J ) 8.85 Hz), 7.03 (d, 2H, H_{arom2′+6′}), 7.06 (d, 4H,

H

_arom2+6, J ) 8.00 Hz), 7.29 (d, 2H, H_arom3+5), 8.11 (d, 1H,

NH-G, J ) 7.85 Hz); MS, m/z: 645 (M⁺, 30), 668 (M⁺+ Na,

10); acc. mass: (C₃₃H₄₇N₃O₁₀) calcd 645.3240, found 645.3261.

Anal. (C₃₃H₄₇N₃O₁₀): C, H, N.

4-Bis(2′-h yd r oxyet h yl)a m in o-N-ter t-b u t yloxyca r b on -

yla n ilin e (46b) (0.39 g, 69%) was obtained from 45b by a

similar method as a gum. ¹H NMR, δ_H, (ppm): 1.45 (s, 9H,

CO₂-t-Bu), 3.34 (t, 4H, NCH₂, J ) 6.25 Hz), 3.45-3.55 (m, 4H,

CH₂OH), 4.65 (t, 2H, OH, J ) 5.06 Hz), 6.58 (d, 2H, H_arom3+5

,

J ) 8.69 Hz), 7.18 (d, 2H, H_arom2+6), 8.77 (d, 1H, NH-G); MS,

m/z: 296 (M⁺, 85), 319 (M⁺+ Na, 7), 240 (M⁺- t-Bu, 52; acc.

mass: (C₁₅H₂₄N₂O₄) calcd 296.1736, found 296.1750.

In an alternative procedure, 4-amino-N-bis(2-hydroxyethyl)-

aniline (2.6 g, 13.3 mmol) was dissolved in THF (80 mL), tert-

butyl pyrocarbonate (2.9 g, 13.5 mmol) was added, and the

reaction mixture was stirred at room temperature for 16 h.

The solvent was evaporated and the residue was purified by

preparative HPLC (AcOEt:ethanol 9:1) to afford 46b (3.0 g,

76%).

2-Bis(2′-h yd r oxyet h yl)a m in o-N-ter t-b u t yloxyca r b on -

yla n ilin e (46a ) was obtained as an oil (7.2 g, 63.8%). By

reacting tert-butyl pyrocarbonate with 2-amino-N-bis(2-hy-

droxyethyl)aniline the same intermediate was obtained (7.10

g, 100%). The compound was purified by preparative HPLC

Di-ter t-bu tyl 4-{[4′-(N,N-Bis(2′′-m esyloxyeth yl)a m in o)-

p h e n yl]ca r b a m oyloxym e t h yl}p h e n yloxyca r b on yl-^L-

glu ta m a te (15b), was obtained as an solid (0.80 g, 95%), mp

41-46 °C. ¹H NMR, δ_H, (ppm): 1.41 (s, 9H, CO₂-t-Bu), 1.43

(s, 9H, CO₂-t-Bu), 1.70-2.05 (2m, 2H, CH₂CH(NH)), 2.30-2.40

(m, 2H, CH₂CO₂-t-Bu), 3.14 (s, 3H, CH₃SO₃), 3.15 (s, 3H, CH₃-

SO₃), 3.67 (t, 4H, NCH₂), 3.95-4.05 (m, 1H, CH(NH)CH₂), 4.30

1

(AcOEt). H NMR, δ_H, (ppm): 1.45 (s, 9H, CO₂-t-Bu), 2.96 (t,

4H, NCH₂, J ) 5.96 Hz), 4.49 (t, 4H, CH₂OH), 6.92-7.08 (m,

2H, H_arom4+5), 7.24 (dd, 1H, H_arom3, J _o) 7.88, J _m) 1.60 Hz),

(t, 4H, CH₂OMes), 5.12 (s, 2H, PhCH₂), 6.76 (d, 2H, H_{arom3′+5′}

J ) 8.05 Hz), 7.12 (d, 2H, H_arom2+6, J ) 7.16 Hz), 7.30 (d, 2H,

_{arom2′+6′}), 7.44 (d, 2H, H_arom3+5), 8.09 (d, 1H, NH-G), 9.36 (s,

1H, Ph′NH); MS, m/z: 788 (M⁺+ 1, 45), 810 (M⁺+ 23, 10);

,

7.90 (d, 1H, H_arom6), 8.65 (s, 1H, PhNH); MS m/z: 297 (M⁺

+

1, 25), 241 (M⁺- t-Bu + 1, 30); acc. mass: (C₁₅H₂₅N₂O₄) calcd

H

297.1814; found 297.1830. Anal. (C₁₅H₂₄N₂O₄) C, H, N.

Self-Immolative Nitrogen Mustards Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1699

acc. mass: (C₃₄H₅₀N₃O₁₄S₂) calcd: 788.2734, found: 788.2720.

Anal. (C₃₄H₄₉N₃O₁₄S₂): C, H, N; S required 8.14, found 7.71.

819 (M⁺+ 1, 100); acc. mass: (C₃₀H₃₇N₄O₇I₂) calcd: 819.0752,

found: 819.0740. Anal. (C₃₀H₃₆N₄O₇I₂): C, H, N.

Using the same procedure, the following compounds have

been obtained:

Dia llyl 4-{N-[4′-bis(2′′-m esyloxyeth yl)a m in op h en yl]-N-

m e t h y lc a r b a m o y lo x y m e t h y l}p h e n y lc a r b a m o y l-^L-

glu ta m a te (16b) was obtained as a gum (0.73 g, 100%). ¹H

NMR, δ_H, (ppm): 1.85-2.20 (2m, 2H, CH₂CH(NH)), 2.44 (t,

2H, CH₂CO₂, J ) 7.77 Hz), 3.14 (s, 9H, CH₃SO₃, NCH₃), 3.71

(t, 4H, NCH₂), 4.29 (t, 5H, CH₂OMes + CH(NH)CH₂, J ) 4.95

Hz), 4.54 (d, 2H, CH₂O allyl, J ) 5.40 Hz), 4.60 (d, 2H, CH₂O

allyl, J ) 5.30 Hz), 4.96 (s, 2H, PhCH₂), 5.15-5.37 (m, 4H,

CH₂d allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.61 (d, 1H,

NH-G, J ) 7.99 Hz), 6.75 (d, 2H, H_{arom3′+5′}, J ) 9.00 Hz), 7.08

Diallyl4-{[2′-(N,N-bis(2′′-iodoethyl)amino)phenyl]carbam-

oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m a te (18a ) as a

solid, purified by preparative HPLC (eluent: cyclohexane:

AcOEt, 3:1), mp 95-6 °C, (0.134 g, 68.7%). ¹H NMR, δ_H,

(ppm): 1.82-2.16 (2m, 2H, CH₂CH(NH)), 2.45 (t, 2H, CH₂-

CO₂, J ) 7.38 Hz), 3.21 (m, 8H, N(CH₂CH₂I)₂), 4.28-4.34 (m,

1H, CH(NH)CH₂), 4.53-4.62 (m, 4H, CH₂O allyl, 5.09 (s, 2H,

PhCH₂), 5.10-5.36 (m, 4H, CH₂d allyl), 5.86-6.09 (m, 2H,

CHd allyl), 6.61 (d, 1H, NH-G, J ) 7.98 Hz), 7.04 (dt, 1H,

(d, 2H, H_{arom2′+6′}), 7.18 (d, 2H, H_arom2+6), 7.33 (d, 2H, H_arom3+5

,

J ) 8.24 Hz), 8.61 (s, 1H, PhNH). MS m/z: 769 (M⁺+ 1, 4);

acc. mass: (C₃₃H₄₅N₄O₁₃S₂) calcd 769.2425, found 769.2456.

Anal. (C₃₃H₄₄N₄O₁₃S₂): C, H, N, S.

H

_{arom4′(5′)}, J _o) 7.65 Hz, J _m) 1.55 Hz), 7.17 (dt, 1H, H_{arom5′(4′)},

J _o) 7.90 Hz, J _m) 1.30 Hz), 7.28 (d, 2H, H_arom3+5, J ) 8.55

Hz), 7.38 (d, 2H, H_arom2+6), 7.65 (dd, 1H, H_arom6′, J _o) 8.10 Hz,

J _m) 1.30 Hz), 8.51 (s, 1H, PhNH), 8.64 (s, 1H, Ph′NH); MS,

m/z: 819 (M⁺+ 1, 1.5), 841 (M⁺+ Na, 6.5); acc. mass:

(C₃₀H₃₆N₄O₇I₂Na) calcd: 841.0571, found: 841.0555. Anal.

(C₃₀H₃₆N₄O₇I₂): H, N, I; C required 44.03, found 44.46.

Di-ter t-bu tyl 4-{N-[4′-bis(2′′-m esyloxyeth yl)a m in op h e-

n yl]-N-m eth ylca r ba m oyloxym eth yl}p h en yloxyca r bon yl-

L-glu ta m a te (17b) was obtained as a glass (2.84 g, 100%).

¹H NMR, δ_H, (ppm): 1.40 (s, 9H, CO₂-t-Bu), 1.42 (s, 9H, CO₂-

t-Bu), 1.70-2.00 (2m, 2H, CH₂CH(NH)), 2.34 (t, 2H, CH₂CO₂),

3.15 (s, 6H, CH₃SO₃), 3.17 (s, 3H, NCH₃), 3.73 (t, 4H, NCH₂),

3.90-4.05 (m, 1H, CH(NH)CH₂), 4.29 (t, 4H, CH₂OMes, J )

5.54 Hz), 5.05 (s, 2H, PhCH₂), 6.77 (d, 2H, H_{arom3′+5′}, J ) 8.90

Hz), 7.05 (d, 2H, H_arom3+5), 7.12 (d, 2H, H_{arom2′+6′}), 7.29 (d, 2H,

Di-ter t-bu tyl 4-{[2′-(N,N-bis(2′′-iodoeth yl)am in o)ph en yl]-

ca r b a m oyloxym et h yl}p h en yloxyca r b on yl-L-glu t a m a t e

(21a ) resulted as a foamy solid, purified by preparative HPLC

(cyclohexane:AcOEt, 3:1), (0.109 g, 67%), mp 39-40 °C. ¹H

NMR, δ_H, (ppm): 1.41 (s, 9H, CO₂-t-Bu), 1.42 (s, 9H, CO₂-t-

Bu), 1.75-2.10 (2m, 2H, CH₂CH(NH)), 2.31-2,38 (m, 2H, CH₂-

CO₂), 3.20-3.25 (m, 8H, N(CH₂CH₂I)₂), 3.90-4.10 (m, 1H,

CH(NH)CH₂), 5.18 (s, 2H, PhCH₂), 7.05 (dt, 1H, H_{arom5′(4′)}, J _o

) 7.55 Hz, J _m) 1.24 Hz), 7.10 (d, 2H, H_arom3+5, J ) 8.44 Hz),

7.18 (t, 1H, H_{arom4′(5′)}), 7.34 (d, 1H, H_arom3′, J ) 7.34 Hz), 7.43

(d, 2H, H_arom2+6), 7.93 (d, 1H, H_arom6′J ) 7.57 Hz), 8.10 (d, 1H,

NH-G, J ) 7.93 Hz), 8.57 (s, 1H, Ph′-NH). MS, m/z: 852 (M⁺

+ 1, 40), 874 (M⁺+ Na, 37); acc. mass: (C₃₂H₄₄N₃O₈I₂) calcd

852.1218; found 852.1240. Anal. (C₃₂H₄₃N₃O₈I₂): C, H, N.

H

_arom2+6), 8.11 (d, 1H, NH-G, J ) 7.26 Hz). MS m/z: 801 (M⁺,

45), 824 (M⁺+ Na, 10); acc. mass: (C₃₅H₅₁N₃O₁₄S₂) calcd

801.2843, found 801.2812.

2-Bis(2′-m esyloxyeth yl)a m in o-N-ter t-bu tyloxyca r bon -

yla n ilin e (48a ) resulted as an oil (2.01 g, 88%). Purified by

preparative HPLC (CH₂Cl₂:AcOEt 5:1). ¹H NMR, δ_H, (ppm):

1.46 (s, 9H, CO₂-t-Bu), 3.13 (s, 6H, CH₃SO₃), 3.27 (t, 4H, NCH₂,

J ) 5.03 Hz), 4.08 (t, 4H, CH₂OMes), 7.03 (t, 1H, H_arom4(5), J _o

) 7.97 Hz), 7.18 (t, 1H, H_arom5(4)), 7.40 (dd, 1H, H_arom3, J _o

)

Di-ter t-bu tyl 4-{[4′-(N,N-bis(2′′-iodoeth yl)am in o)ph en yl]-

ca r b a m oyloxym et h yl}p h en yloxyca r b on yl-^L-glu t a m a t e

(21b) resulted as a gum, purified by preparative HPLC

(cyclohexane:AcOEt 3:1) (0.2 g, 94%). ¹H NMR, δ_H, (ppm): 1.41

(s, 9H, CO₂-t-Bu), 1.42 (s, 9H, CO₂-t-Bu), 1.75-2.10 (2m, 2H,

CH₂CH(NH)), 2.34 (t, 2H, CH₂CO₂, J ) 7.37 Hz), 3.25-3.40

(t, 4H, NCH₂, under H₂O), 3.68 (t, 4H, CH₂I), 3.95-4.05 (m,

8.93 Hz), 7.99 (d, 1H, H_arom6, J _o) 9.08 Hz), 8.13 (s, 1H, PhNH);

MS, m/z: 453 (M⁺+ 1, 30), 475 (M⁺+ 23, 55), 397 (M⁺- t-Bu

+ 1, 100); acc. mass: (C₁₇H₂₉N₂O₈S₂) calcd: 453.1365, found:

453.1350. Anal. (C₁₇H₂₉N₂O₈S₂): H, N, S; C required 45.12,

found 45.63%.

4-Bis(2′-m esyloxyeth yl)a m in o-N-ter t-bu tyloxyca r bon -

yla n ilin e (48b) was obtained after purification by preparative

1H, CH(NH)CH₂), 5.10 (s, 2H, PhCH₂), 6.64 (d, 2H, H_{arom3′+5′}

,

1

HPLC (CH₂Cl₂:AcOEt 3:1) as an oil (155 mg, 57%). H NMR,

J ) 8.51 Hz), 7.10 (d, 2H, H_arom2+6, J ) 8.21 Hz), 7.28 (d, 2H,

δ_H, (ppm): 1.45 (s, 9H, CO₂-t-Bu), 3.13 (s, 6H, CH₃SO₃), 3.66

(t, 4H, NCH₂, J ) 5.67 Hz), 4.27 (t, 4H, CH₂OMes), 6.72 (d,

2H, H_arom3+5, J ) 8.93 Hz), 7.27 (d, 2H, H_arom2+6), 8.93 (s, 1H,

H

_{arom2′+6′}), 7.42 (d, 2H, H_arom3+5), 8.07 (broad s, 1H, NH-G), 9.33

(s, 1H, Ph′-NH). MS, m/z: 851 (M⁺, 92), 874 (M⁺+ Na, 8);

acc. mass: (C₃₂H₄₃N₃O₈I₂) calcd 851.1140; found 851.1150.

NH). MS, m/z: 452 (M⁺, 41), 396 (M⁺- t-Bu, 44), 357 (M⁺

-

Diallyl 4-{N-[4′-bis(2′′-iodoeth yl)am in oph en yl]-N-m eth -

ylca r b a m oyloxym et h yl}p h en ylca r b a m oyl-^L-glu t a m a t e

(25b), purified by column chromatography (cyclohexane:AcOEt

1:1), resulted as a solid (0.165 g, 60%), mp 138-140 °C. ¹H

NMR, δ_H, (ppm): 1.80-2.15 (2m, 2H, CH₂CH(NH)), 2.44 (t,

2H, CH₂CO₂), 3.13 (s, 3H, NCH₃), 3.25-3.35 (t, 4H, NCH₂),

3.70 (t, 4H, CH₂I, J ) 6.17 Hz), 4.25-4.40 (m, 1H, CH(NH)-

CH₂), 4.54 (d, 2H, CH₂O allyl, J ) 6.27 Hz), 4.60 (d, 2H, CH₂O

allyl, J ) 4.42 Hz), 4.95 (s, 2H, PhCH₂), 5.10-5.37 (m, 4H,

CH₂d allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.62 (d, 3H,

H_{arom3′+5′}+NH-G, J ) 8.67 Hz), 7.09 (d, 2H, H_{arom2′+6′}), 7.18 (d,

2H, H_arom2+6), 7.33 (d, 2H, H_arom3+5, J ) 7.72 Hz), 8.61 (s, 1H,

PhNH). MS m/z: 833 (M⁺+ 1, 7), 855 (M⁺+ Na, 14); acc.

mass: (C₃₁H₃₈N₄O₇I₂Na) calcd 855.0728, found 855.0755. Anal.

(C₃₁H₃₈N₄O₇I₂): H, N; C required 44.73, found 45.24%.

Di-ter t-bu tyl 4-{N-[4′-bis(2′′-iod oeth yl)a m in op h en yl]-

N-m et h ylca r b a m oyloxym et h yl}p h en yloxyca r b on yl-^L-

glu ta m a te (28b), purified by column chromatography (cyclo-

hexane:AcOEt 1:1), resulted as a glass (0.165 g, 79.2%), mp

40-41 °C. ¹H NMR, δ_H, (ppm): 1.40 (s, 9H, CO₂-t-Bu), 1.42

(s, 9H, CO₂-t-Bu), 1.75-2.05 (2m, 2H, CH₂CH(NH)), 2.34 (t,

2H, CH₂CO₂), 3.16 (s, 3H, NCH₃), 3.25-3.35 (t, 4H, NCH₂),

3.72 (t, 4H, CH₂I, J ) 7.76 Hz), 3.90-4.05 (m, 1H, CH(NH)-

CH₂), 5.05 (s, 2H, PhCH₂), 6.65 (d, 3H, H_{arom3′+5′}, J ) 8.86 Hz),

7.06 (d, 2H, H_arom3+5, J ) 8.29 Hz), 7.12 (d, 2H, H_{arom2′+6′}), 7.30

(d, 2H, H_arom2+6), 8.11 (d, 1H, NH-G, J ) 7.78 Hz). MS m/z:

865 (M⁺, 57), 888 (M⁺+ Na, 26); acc. mass: (C₃₃H₄₅N₃O₈I₂)

calcd 865.1310, found 865.1296.

CH₃SO₃, 48); acc. mass: (C₁₇H₂₈N₂O₈S₂) calcd: 452.1287,

found: 452.1270.

4-(N′-Ben zyloxyca r bon yl-N′-m eth yla m in o)-N,N-b is(2′-

m esyloxyeth yl)a n ilin e (53b) was obtained as an oil (1.96

g, 100%). ¹H NMR δ_H: 3.15 (s, 6H, CH₃SO₃), 3.18 (s, 3H,

NCH₃), 3.73 (t, 4H, NCH₂, J ) 5.63 Hz), 4.30 (t, 4H, CH₂OMes),

5.06 (s, 2H, PhCH₂), 6.77 (d, 2H, H_arom3+5, J ) 8.93 Hz), 7.12

(d, 2H, H_arom2+6), 7.25-7.37 (m, 5H, H_{arom benzyl}). MS m/z: 500

(M⁺, 100), 523 (M⁺+ Na, 15); acc. mass: (C₂₁H₂₈N₂O₈S₂) calcd

500.1287, found 500.1260.

Diallyl4-{[4′-(N,N-Bis(2′′-iodoethyl)amino)phenyl]carbam-

oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m a te (18b). A so-

lution of 14b (0.12 g, 0.13 mmol) and NaI (0.22 g, 1.5 mmol)

in 8 mL of acetone was stirred at reflux for 4 h. The solvent

was evaporated, and the residue was retaken up in 30 mL of

AcOEt, washed with 20 mL of H₂O, dried (MgSO₄), and

evaporated. The residue was purified by column chromatog-

raphy (cyclohexane: AcOEt 1:1) to afford 18b (0.10 g, 94%)

1

as a brown solid, mp 138-141 °C. H NMR, δ_H, (ppm): 1.85-

2.15 (2m, 2H, CH₂CH(NH)), 2.45 (t, 2H, CH₂CO₂, J ) 7.79 Hz),

3.20-3.35 (t, 4H, NCH_2,under H₂O peak), 3.67 (t, 4H, CH₂I),

4.25-4.40 (m, 1H, CH(NH)CH₂), 4.55 (d, 2H, CH₂O allyl, J )

5.43 Hz), 4.61 (d, 2H, CH₂O allyl, J ) 5.23 Hz), 5.02 (s, 2H,

PhCH₂), 5.15-5.40 (m, 4H, CH₂d allyl), 5.85-6.05 (m, 2H,

CHd allyl), 6.58-6.65 (m, 3H, NH-G + H_{arom3′+5′}), 7.28 (d, 4H,

H_arom2+6+ H_{arom2′+6′}, J ) 8.44 Hz), 7.39 (d, 2H, H_arom3+5, J )

8.33 Hz), 8.62 (s, 1H, PhNH), 9.29 (s, 1H, Ph′NH); MS, m/z:

1700 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9

Niculescu-Duvaz et al.

2-Bis(2′-iod oe t h yl)a m in o-N -t er t -b u t yloxyca r b on yl-

a n ilin e (49a ) was obtained from the corresponding bis-mesyl

derivative as an oil (0.121 g, 78.6%) and was purified by

preparative HPLC (cyclohexane:AcOEt, 3:1). ¹H NMR, δ_H,

(ppm): 1.46 (s, 9H, CO₂-t-Bu), 3.29 (m, 8H, N(CH₂CH₂I)₂), 7.01

(dt, 1H, H_arom4(5), J _o) 7.97, J _m) 1.35 Hz), 7.15 (t, 1H, H_arom5(4)),

7.32 (dd, 1H, H_arom3, J _o) 7.83, J _m) 1.35 Hz), 7.90 (dd, 1H,

NH-G, J ) 7.97 Hz), 7.05 (t, 1H, H_{arom4′(5′)}, J ) 7.67 Hz), 7.19

(t, 1H, H_{arom5′(4′)}), 7.26 (d, 2H, H_arom3+5, J ) 8.55 Hz), 7.38 (d,

2H, H_arom2+6), 7.95 (d, 1H, H_arom6′, J ) 8.05 Hz), 8.63 (s, 1H,

PhNH), 8.66 (s, 1H, Ph′NH); MS, m/z: 725 (M⁺+ 1, 1), 747

(M⁺+ Na, 4), 645 (M⁺- HBr + 1, 1); acc. mass: (C₃₀H₃₆N₄O₇-

Br₂Na) calcd 745.0848, found 745.0830. Anal.(C₃₀H₃₆N₄O₇Br₂)

C, H, N, Br.

H

_arom6, J _o) 8.21, J _m) 1.35 Hz), 8.25 (s, 1H, PhNH). MS, m/z:

Di-ter t-b u t yl 4-{[2′-(N,N-b is(2′′-b r om oet h yl)a m in o)-

p h e n yl]ca r b a m oyloxym e t h yl}p h e n yloxyca r b on yl-^L-

glu ta m a te (22a ) was obtained as a solid and purified by

preparative HPLC (cyclohexane:AcOEt 3:1), (0.103 g, 86.5%),

mp 32-35 °C. ¹H NMR, δ_H, (ppm): 1.41 (s, 9H, CO₂-t-Bu), 1.42

(s, 9H, CO₂-t-Bu), 1.87-2.10 (2m, 2H, CH₂CH(NH)), 2.34 (t,

2H, CH₂CO₂), 3.33 (t, 4H, NCH₂), 3.44 (t, 4H, CH₂Br), 3.93-

4.10 (m, 1H, CH(NH)CH₂), 5.17 (s, 2H, PhCH₂), 7.05-7.40 (m,

4H, H_arom3+5+ H_{arom4′(5′)}+ H_{arom5′(4′)}), 7.42 (d, 2H, H_arom2+6, J )

8.57 Hz), 7.94 (d, 1H, H_arom6′, J ) 8.00 Hz), 8.10 (broad d, 1H,

NH-G), 8.64 (s, 1H, Ph′-NH). MS, m/z: 758 (M⁺+ 1, 78), 780

(M⁺+ Na, 100), 678 (M⁺+ 1 - HBr, 25); acc. mass:

517 (M⁺+ 1, 40), 389 (M⁺- HI + 1, 55); acc. mass:

(C₁₅H₂₃N₂O₂I₂) calcd 516.9849; found 516.9870. Anal. (C₁₅H₂₂

N₂O₂I₂): C, H, N.

-

4-Bis(2′-iod oe t h yl)a m in o-N -t er t -b u t yloxyca r b on yl-

a n ilin e (49b) was obtained by the same method as a solid,

mp 121-125 °C (0.25 g, 53%). ¹H NMR, δ_H, (ppm): 1.45 (s,

9H, O-t-Bu), 3.20-3.35 (t, 4H, NCH₂), 3.67 (t, 4H, CH₂I, J )

7.57 Hz), 6.61 (d, 2H, H_arom3+5, J ) 8.87 Hz), 7.26 (d, 2H,

H

_arom2+6), 8.93 (s, 1H, NH). MS, m/z: 516 (M⁺, 75), 460 (M⁺

-

t-Bu, 100); acc. mass: (C₁₅H₂₂N₂O₂I₂) calcd 515.9771; found

515.9750.

(C₃₂H₄₄N₃O₈Br₂) calcd 756.1495; found 756.1470. Anal. (C₃₂H₄₃

-

4-(N′-Ben zyloxyca r bon yl-N′-m eth yla m in o)-N,N-b is(2′-

iod oeth yl)a n ilin e (54b) was obtained as a solid (0.40 g,

N₃O₈Br₂‚0.1toluene) C, H, N.

Dia llyl 4-{N-[4′-b is(2′′-b r om oet h yl)a m in op h en yl]-N-

m e t h y lc a r b a m o y lo x y m e t h y l}p h e n y lc a r b a m o y l-^L-

glu ta m a te (26b) was obtained from 16b as a solid, (0.16 g,

64%), mp 104-107 °C. ¹H NMR, δ_H, (ppm): 1.80-2.15 (2m,

2H, CH₂CH(NH)), 2.44 (t, 2H, CH₂CO₂), 3.13 (s, 3H, NCH₃),

3.56 (t, 4H, NCH₂, J ) 6.74 Hz), 3.75 (t, 4H, CH₂Br), 4.25-

4.35 (m, 1H, CH(NH)CH₂), 4.54 (d, 2H, CH₂O allyl, J ) 5.52

Hz), 4.59 (d, 2H, CH₂O allyl, J ) 5.21 Hz), 4.95 (s, 2H, PhCH₂),

5.10-5.37 (m, 4H, CH₂d allyl), 5.80-6.00 (m, 2H, CHd allyl),

6.61 (d, 1H, NH-G, J ) 8.34 Hz), 6.68 (d, 2H, H_{arom3′+5′}, J )

8.89 Hz), 7.09 (d, 2H, H_{arom2′+6′}), 7.18 (d, 2H, H_arom2+6), 7.33 (d,

2H, H_arom3+5, J ) 8.26 Hz), 8.61 (s, 1H, PhNH). MS m/z: 738

(M⁺, 5), 761 (M⁺+ Na, 15); acc. mass: (C₃₁H₃₈N₄O₇Br₂) calcd

759.1005, found 759.1021. Anal. (C₃₁H₃₈N₄O₇Br₂): C, H, N.

1

70.9%), mp 93-95 °C. H NMR δ_H: 3.16 (s, 3H, NCH₃), 3.30

(t, 4H, NCH₂, J ) 5.63 Hz), 3.72 (t, 4H, CH₂I, J ) 7.67 Hz),

5.06 (s, 2H, PhCH₂), 6.65 (d, 2H, H_arom3+5, J ) 8.78 Hz), 7.13

(d, 2H, H_arom2+6), 7.25-7.37 (m, 5H, H_{arom benzyl}). MS m/z: 564

(M⁺, 100), 587 (M⁺+ Na, 12); acc. mass: (C₁₉H₂₂N₂O₂I₂) calcd

563.9771, found 563.9750. Anal. (C₁₉H₂₂N₂O₂I₂): C, H, N.

Di-ter t-b u t yl 4-{[4′-(N,N-Bis(2′′-b r om oet h yl)a m in o)-

p h e n yl]ca r b a m oyloxym e t h yl}p h e n yloxyca r b on yl-^L-

glu ta m a te (22b). To a THF solution (15 mL) of 15b (0.18 g,

0.23 mmol) was added LiBr (0.44 g, 5.0 mmol). After 1.5 h

stirring at reflux, the solvent was evaporated, and the residue

was retaken up in CH₂Cl₂(25 mL) and extracted with H₂O

(25 mL), and the organic layer was dried (MgSO₄) and

evaporated to dryness. Purification was achieved by prepara-

tive HPLC (cyclohexane:AcOEt 3:1), when product 22b (0.12

Di-ter t-bu tyl 4-{N-[4′-bis(2′′-br om oeth yl)a m in op h en yl]-

N-m et h ylca r b a m oyloxym et h yl}p h en yloxyca r b on yl-^L-

glu ta m a te (29b) was obtained from 17b as a solid, (0.67 g,

1

g, 69%) was obtained as a solid, mp 48-50 °C. H NMR, δ_H,

(ppm): 1.41 (s, 9H, CO₂-t-Bu), 1.42 (s, 9H, CO₂-t-Bu), 1.75-

2.10 (2m, 2H, CH₂CH(NH)), 2.34 (t, 2H, CH₂CO₂), 3.55 (t, 4H,

NCH₂), 3.72 (t, 4H, CH₂Br), 3.95-4.05 (m, 1H, -CH(NH)CH₂),

5.10 (s, 2H, PhCH₂), 6.68 (d, 2H, H_{arom3′+5′}, J ) 8.72 Hz), 7.10

(d, 2H, H_arom2+6, J ) 8.00 Hz), 7.28 (d, 2H, H_{arom2′+6′}), 7.42 (d,

2H, H_arom3+5), 8.06 (broad s, 1H, NH-G), 9.32 (s, 1H, Ph′-NH).

1

69.7%), mp 36-39 °C. H NMR, δ_H, (ppm): 1.41 (s, 9H, CO₂-

t-Bu), 1.42 (s, 9H, CO₂-t-Bu), 1.75-2.05 (2m, 2H, CH₂CH(NH)),

2.34 (t, 2H, CH₂CO₂), 3.16 (s, 3H, NCH₃), 3.58 (t, 4H, NCH₂,

J ) 7.00 Hz), 3.77 (t, 4H, CH₂Br), 3.90-4.05 (m, 1H, CH(NH)-

CH₂), 5.05 (s, 2H, PhCH₂), 6.70 (d, 2H, H_{arom3′+5′}, J ) 8.89 Hz),

7.06 (d, 2H, H_arom3+5, J ) 8.34 Hz), 7.12 (d, 2H, H_{arom2′+6′}), 7.29

(d, 2H, H_arom2+6), 8.11 (d, 1H, NH-G, J ) 7.86 Hz). MS m/z:

771 (M⁺, 75); acc. mass: (C₃₃H₄₅N₃O₈Br₂) calcd 769.1573, found

769.1550. Anal. (C₃₃H₄₅N₃O₈Br₂): C, H, N.

MS, m/z: 758 (M⁺+ 1, 100), 713 (M⁺- CO₂, 40), 679 (M⁺

+

1 - Br, 25); acc. mass: (C₃₂H₄₃N₃O₈Br₂) calcd 756.1495; found

756.1480. Anal. (C₃₂H₄₃N₃O₈Br₂): C, H, N.

Using a similar procedure the following compounds were

obtained:

2-Bis(2′-b r om oet h yl)a m in o-N-ter t-b u t yloxyca r b on yl-

a n ilin e (50a ) was obtained from 48a as an oil (0.79 g, 85.3%)

and was purified by preparative HPLC (cyclohexane:AcOEt

Dia llyl 4-{[4′-(N,N-Bis(2′′-br om oeth yl)a m in o)p h en yl]-

car bam oyloxym eth yl}ph en ylcar bam oyl-^L-glu tam ate (19b)

was obtained after purification by column chromatography

(cyclohexane:AcOEt 1:1) as a solid, mp 121-123 °C (0.25 g,

1

3:1). H NMR, δ_H, (ppm): 1.45 (s, 9H, CO₂-t-Bu), 3.30 (t, 4H,

NCH₂, J ) 5.96 Hz), 3.45 (t, 4H, CH₂Br), 7.02 (dt, 1H, H_arom4(5)

,

J _o) 7.88, J _m) 1.36 Hz), 7.16 (t, 1H, H_arom5(4)), 7.36 (dd, 1H,

1

43%). H NMR, δ_H, (ppm): 1.80-2.10 (2m, 2H, CH₂CH(NH)),

H

_arom3, J _o) 7.85, J _m) 1.33 Hz), 7.91 (dd, 1H, H_arom6, J _o

)

+

8.12, J _m) 1.28 Hz), 8.35 (s, 1H, PhNH); MS, m/z: 423 (M⁺

2.45 (t, 2H, CH₂CO₂, J ) 8.19 Hz), 3.55 (t, 4H, NCH₂, J )

7.15 Hz), 3.71 (t, 4H, CH₂Br, J ) 7.19 Hz), 4.25-4.35 (m, 1H,

CH(NH)CH₂), 4.54 (d, 2H, CH₂O allyl, J ) 5.41 Hz), 4.60 (d,

2H, CH₂O allyl, J ) 5.29 Hz), 5.00 (s, 2H, PhCH₂), 5.15-5.40

(m, 4H, CH₂d allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.64 (d,

1H, NH-G, J ) 7.41 Hz), 6.66 (d, 2H, H_{arom3′+5′}, J ) 9.00 Hz),

7.27 (d, 4H, H_arom2+6+ H_{arom2′+6′}, J ) 8.59 Hz), 7.38 (d, 2H,

1, 40), 367 (M⁺- t-Bu + 1, 100), 341 (M⁺- Br, 80); acc. mass:

(C₁₅H₂₃N₂O₂Br₂) calcd 421.0126, found 421.0140. Anal. (C₁₅H₂₂

N₂O₂Br₂): H, N, Br; C required 42.68, found 43.20%.

-

4-Bis(2′-b r om oet h yl)a m in o-N-ter t-b u t yloxyca r b on yl-

a n ilin e (50b) was obtained by the same method, as a solid,

mp 91-92 °C (0.18 g, 73.5%). ¹H NMR, δ_H, (ppm): 1.45 (s,

9H, O-t-Bu), 3.55 (t, 4H, NCH₂, J ) 6.93 Hz), 3.67 (t, 4H, CH₂-

Br), 6.65 (d, 2H, H_arom3+5, J ) 8.92 Hz), 7.27 (d, 2H, H_arom2+6),

8.92 (s, 1H, NH). MS, m/z: 422 (M⁺, 95), 366 (M⁺- t-Bu, 100);

acc. mass: (C₁₅H₂₂N₂O₂Br₂) calcd 420.0048; found 420.0060.

Anal. (C₁₅H₂₂N₂O₂Br₂): H, N; C required 42.68, found 43.13%.

4-(N′-Ben zyloxyca r bon yl-N′-m eth yla m in o)-N,N-bis(2′-

br om oet h yl)a n ilin e (55b) was obtained as an oil (0.32 g,

63.9%). ¹H NMR δ_H: 3.17 (s, 3H, NCH₃), 3.58 (t, 4H, NCH₂, J

) 7.12 Hz), 3.72 (t, 4H, CH₂Br), 5.06 (s, 2H, PhCH₂), 6.70 (d,

2H, H_arom3+5, J ) 8.98 Hz), 7.12 (d, 2H, H_arom2+6), 7.25-7.37

(m, 5H, H_aro_{m benzyl}). MS m/z: 470 (M⁺, 100), 493 (M⁺+ Na, 7);

acc. mass: (C₁₉H₂₂N₂O₂Br₂) calcd 648.0048, found 648.0030.

Anal. (C₁₉H₂₂N₂O₂Br₂) H, N; C required 48.53, found 49.01%.

H

_arom3+5, J ) 8.62 Hz), 8.68 (s, 1H, PhNH), 9.38 (s, 1H, Ph′NH);

MS, m/z: 724 (M⁺, 3), 747 (M⁺+ Na, 2), 645 (M⁺- Br, 2);

acc. mass: (C₃₀H₃₆N₄O₇Br₂Na) calcd 745.0848, found 745.0860.

Anal. (C₃₀H₃₆N₄O₇Br₂): C, H, N, Br.

Dia llyl 4-{[2′-(N,N-bis(2′′-br om oeth yl)a m in o)p h en yl]-

car bam oyloxym eth yl}ph en ylcar bam oyl-^L-glu tam ate (19a)

was purified by column chromatography (cyclohexane:AcOEt

3:1) to yield 19a (0.197 g, 85%) as a solid, mp 120-122 °C. ¹H

NMR, δ_H, (ppm): 1.87-2.08 (2m, 2H, CH₂CH(NH)), 2.45 (t,

2H, CH₂CO₂, J ) 7.38 Hz), 3.30 (t, 4H, NCH₂, J ) 5.99 Hz),

3.44 (t, 5H, CH₂Br), 4.26-4.35 (m, 1H, CH(NH)CH₂), 4.53-

4.62 (dd, 4H, CH₂O allyl), 5.07 (s, 2H, PhCH₂), 5.16-5.37 (m,

4H, CH₂d allyl), 5.82-5.99 (m, 2H, CHd allyl), 6.63 (d, 1H,

Self-Immolative Nitrogen Mustards Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1701

Dia llyl 4-{N-[4′-Bis(2′′-ch lor oet h yl)a m in op h en yl]-N-

m e t h y lc a r b a m o y lo x y m e t h y l}p h e n y lc a r b a m o y l-^L-

glu ta m a te (27b). 16b (0.245 g, 0.32 mmol) was dissolved in

DMA (25 mL). Lithium chloride (0.21 g, 5 mmol) was added

to the reaction mixture, and it was stirred for 24 h at room

temperature. The solvent was evaporated, and the residue was

taken up in AcOEt (50 mL) and extracted with distilled water

(2 × 50 mL). The organic layer was separated, dried (MgSO₄),

and evaporated. The residue was purified by column chroma-

tography (eluent AcOEt:cyclohexane 1:1) to afford 27b (0.105

g, 51%) as a gum. ¹H NMR, δ_H, (ppm): 1.80-2.20 (2m, 2H,

CH₂CH(NH)), 2.44 (t, 2H, CH₂CO₂, J ) 8.26 Hz), 3.13 (s, 3H,

NCH₃), 3.70 (s, 8H, N(CH₂CH₂Cl)₂), 4.25-4.40 (m, 1H, CH(NH)-

CH₂), 4.54 (d, 2H, CH₂O allyl, J ) 5.44 Hz), 4.59 (d, 2H, CH₂O

allyl, J ) 5.35 Hz), 4.95 (s, 2H, PhCH₂), 5.10-5.35 (m, 4H,

CH₂d allyl), 5.80-6.00 (m, 2H, CHd allyl), 6.61 (d, 1H,

NH-G, J ) 8.35 Hz), 6.70 (d, 2H, H_{arom3′+5′}, J ) 8.86 Hz), 7.08

(s, 2H, PhCH₂), 7.02-7.10 (m, 3H, H_arom3+5+ H_{arom5′(4′)}), 7.12

(q, 1H, H_{arom4′(5′)}, J _o) 7.75 Hz, J _m) 1.20 Hz), 7.40 (d, 2H,

H

_arom2+6, J ) 8.61 Hz), 7.96 (d, 1H, H_arom6′, J ) 8.18 Hz), 8.10

(d, 1H, NH-G, J ) 7.91 Hz), 8.67 (s, 1H, Ph′-NH). MS, m/z:

668 (M⁺+ 1, 100), 690 (M⁺+ Na, 50), 632 (M⁺- Cl, 55); acc.

mass: (C₃₂H₄₄N₃O₈Cl₂Na) calcd 690.2325; found 690.2350.

Anal. (C₃₂H₄₃N₃O₈Cl₂): C, H, N.

Di-ter t-bu tyl 4-{[2′-(N,N-(2′′-m esyloxyeth yl)(2′′-ch lor o-

et h yl)a m in o)p h en yl]ca r b a m oyloxym et h yl}p h en yloxy-

ca r bon yl-^L-glu ta m a te (24a ) resulted as a glassy solid (0.065

g, 20.5%), mp 36-37 °C, as the second fraction from the HPLC

1

separation. H NMR, δ_H, (ppm): 1.41 (s, 9H, CO₂-t-Bu), 1.42

(s, 9H, CO₂-t-Bu), 1.80-2.05 (2m, 2H, CH₂CH(NH)), 2.34 (m,

2H, CH₂CO₂), 3.04 (s, 6H, CH₃SO₃), 3.28 (t, 4H, NCH₂), 3.55

(t, 2H, CH₂Cl, J ) 6.02 Hz), 3.95-4.02 (m, 1H, -CH(NH)CH₂),

4.12 (t, 2H, CH₂OMes, J ) 5.34 Hz), 5.15 (s, 2H, PhCH₂), 7.03-

7.11 (m, 3H, H_arom3+5+ H_{arom5′(4′)}), 7.20 (t, 1H, H_{arom4′(5′)}, J )

(d, 2H, H_{arom2′+6′}), 7.18 (d, 2H, H_arom2+6), 7.33 (d, 2H, H_arom3+5

,

7.75 Hz), 7.43 (d, 2H, H_arom2+6, J ) 8.47 Hz), 7.96 (q, 1H, H_arom6′

,

J ) 8.56 Hz), 8.61 (s, 1H, PhNH). MS m/z: 671 (M⁺+ Na,

20); acc. mass: (C₃₁H₃₈N₄O₇Cl₂Na) calcd 671.2015, found

671.2037. Anal. (C₃₁H₃₈N₄O₇Cl₂): C, H, N Cl.

J _o) 8.15 Hz, J _m) 1.27 Hz), 8.10 (d, 1H, NH-G, J ) 7.87 Hz),

8.53 (s, 1H, Ph′-NH). MS, m/z: 728 (M⁺+ 1, 30), 750 (M⁺

+

-

Na, 100), 691 (M⁺+ 1-HCl, 20); acc. mass: (C₃₃H₄₆N₃O₁₁

SClNa) calcd 750.2439; found 750.2460. Anal. (C₃₃H₄₆N₃O₁₁

SCl): C, H, N.

Di-ter t-bu tyl 4-{N-[4′-bis(2′′-ch lor oeth yl)a m in op h en yl]-

N-m et h ylca r b a m oyloxym et h yl}p h en yloxyca r b on yl-^L-

glu ta m a te (30b) was obtained from 17b as a solid, (0.60 g,

4-Bis(2′-ch lor oet h yl)a m in o-N-ter t-b u t yloxyca r b on yl-

a n ilin e (51a ) a n d 2-[(2′-ch lor oeth yl)(2′-m esyloxyeth yl)-

a m in o]-N-ter t-bu tyloxyca r bon yla n ilin e (52a ) were ob-

tained by the same method. After HPLC separation (cyclohex-

ane:AcOEt 1.5:1) 51a resulted from the first fraction as an oil

(0.195 g, 26.4%). ¹H NMR, δ_H, (ppm): 1.45 (s, 9H, O-t-Bu), 3.25

(m, 4H, NCH₂, J ) 5.85 Hz), 3.55 (t, 4H, CH₂Cl), 7.02 (t, 1H,

1

95.2%), mp 35-39 °C. H NMR, δ_H, (ppm): 1.40 (s, 9H, CO₂-

t-Bu), 1.42 (s, 9H, CO₂-t-Bu), 1.75-2.05 (2m, 2H, CH₂CH(NH)),

2.39 (t, 2H, CH₂CO₂), 3.16 (s, 3H, NCH₃), 3.72 (s, 8H,

N(CH₂CH₂Cl)₂), 3.90-4.05 (m, 1H, CH(NH)CH₂), 5.05 (s, 2H,

PhCH₂), 6.72 (d, 2H, H_{arom3′+5′}, J ) 8.96 Hz), 7.06 (d, 2H,

H

_arom3+5, J ) 8.25 Hz), 7.11 (d, 2H, H_{arom2′+6′}), 7.30 (d, 2H,

_arom2+6), 8.12 (d, 1H, NH-G). MS m/z: 681 (M⁺, 28), 704 (M⁺

H

_arom4(5), J ) 7.90 Hz), 7.17 (t, 1H, H_arom5(4)), 7.36 (d, 1H, H_arom3,

+ Na, 20); acc. mass: (C₃₃H₄₅N₃O₈Cl₂) calcd 681.2584, found

681.2560. Anal. (C₃₃H₄₅N₃O₈Cl₂): C, H, N, Cl.

J ) 7.87 Hz), 7.92 (d, 1H, H_arom6, J ) 8.00 Hz), 8.34 (s, 1H,

PhNH); MS, m/z: 333 (M⁺+ 1, 40), 355 (M⁺+ Na, 3), 296

(M⁺- Cl, 40), 277 (M⁺- t-Bu + 1, 90); acc. mass: (C₁₅H₂₃N₂O₂-

Cl₂) calcd 333.1137, found 333.1150. Anal. (C₁₅H₂₂N₂O₂Cl₂) C,

H, N.

Dia llyl 4-{[2′-(N,N-bis(2′′-ch lor oeth yl)a m in o)p h en yl]-

car bam oyloxym eth yl}ph en ylcar bam oyl-^L-glu tam ate (20a)

was obtained from 14a . The product purified by HPLC

(cyclohexane:AcOEt 2:1) resulted as a solid (0.054 g, 50%), mp

From the second fraction 52a resulted as an oil (0.164 g,

1

118-9 °C, H NMR, δ_H, (ppm): 1.84-2.13 (2m, 2H, CH₂CH-

19.1%). H NMR, δ_H, (ppm): 1.45 (s, 9H, O-t-Bu), 3.13 (s, 3H,

(NH)), 2.45 (t, 2H, CH₂CO₂, J ) 8.00 Hz), 3.25 (t, 4H, NCH₂,

J ) 5.80 Hz), 3.54 (t, 4H, CH₂Cl), 4.26-4.35 (m, 1H, CH(NH)-

CH₂), 4.53-4.61 (dd, 4H, CH₂O allyl, J ) 5.34 Hz), 5.06 (s,

2H, PhCH₂), 5.16-5.37 (m, 4H, CH₂O) allyl), 5.82-5.99 (m,

2H, CHd allyl), 6.65 (d, 1H, NH-G, J ) 8.00 Hz), 7.05 (t, 1H,

CH₃SO₃), 3.29 (t, 4H, NCH₂), 3.55 (t, 2H, CH₂Cl, J ) 5.76 Hz),

4.10 (t, 2H, CH₂OMes, J ) 5.08 Hz), 7.03 (t, 1H, H_arom4(5), J _o

)

7.35 Hz), 7.17 (t, 1H, H_arom5(4)), 7.36 (d, 1H, H_arom3, J _o) 7.76

Hz), 7.91 (dd, 1H, H_arom6, J _o) 8.05 Hz), 8.35 (s, 1H, PhNH).

MS, m/z: 415 (M⁺+ 23, 25), 393 (M⁺+ 1, 65), 356 (M⁺+ 1 -

HCl, 20); acc. mass: (C₁₆H₂₆N₂O₅SCl) calcd 393.1251; found

393.1260. Anal. (C₁₆H₂₆N₂O₅SCl): C, H, N, Cl; S required 8.16,

found 7.70%.

H

_{arom4′(5′)}, J ) 7.00 Hz), 7.20 (t, 1H, H_{arom5′(4′)}, J ) 7.90 Hz),

7.25 (d, 2H, H_arom3+5, J ) 8.48 Hz), 7.37 (d, 2H, H_arom2+6), 7.97

(d, 1H, H_arom6′, J ) 7.82 Hz), 8.64 (s, 1H, PhNH), 8.67 (s, 1H,

Ph′NH); MS, m/z: 635 (M⁺+ 1, 9), 657 (M⁺+ Na, 18), 598

(M⁺- Cl, 6); acc. mass: (C₃₀H₃₆N₄O₇Cl₂Na) calcd 657.1859,

found 657.1880. Anal.(C₃₀H₃₆N₄O₇Cl₂): C, H, N, Cl.

4 -{[4 ′-(N ,N -B i s (2 ′′-b r o m o e t h y l )a m i n o )p h e n y l ]-

ca r ba m oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m ic Acid

(32b). 19b (75 mg, 0.1 mmol) and Pd tetrakis(triphenylphos-

phine) (5 mg, 5 µmol) were dissolved in 4 mL of CH₂Cl₂.

Pyrrolidine (30 µL, 0.36 mmol) was added in one portion. After

30 min stirring, the solution was diluted with AcOEt. The

pyrrolidine salt of the deprotected carboxylic acid precipitated

at once. The reaction mixture was partially evaporated, and

the remaining solvent was diluted with AcOEt and concen-

trated to remove CH₂Cl₂selectively. The precipitate left in the

flask after discarding the solvent was washed twice with

AcOEt, dried, dissolved in 8 mL of methanol, and eluted

through a column loaded with 40 cm³IRC50 resin (H form)

previously washed with MeOH. After evaporation the elute

4-(N′-Ben zyloxyca r bon yl-N′-m eth yla m in o)-N,N-b is(2′-

ch lor oeth yl)a n ilin e (56b) was obtained as an oil (0.245 g,

62.5%). ¹H NMR δ_H: 3.17 (s, 3H, NCH₃), 3.72 (s, 8H, N(CH₂CH₂-

Cl)₂), 5.06 (s, 2H, PhCH₂), 6.72 (d, 2H, H_arom3+5, J ) 9.01 Hz),

7.12 (d, 2H, H_arom2+6), 7.25-7.37 (m, 5H, H_{arom benzyl}). MS m/z:

380 (M⁺, 100), 403 (M⁺+ Na, 5); acc. mass: (C₁₉H₂₂N₂O₂Cl₂)

calcd 380.1058, found 380.1040. Anal. (C₁₉H₂₂N₂O₂Cl₂) H, N;

C required 59.85, found 60.26%.

Di-ter t-b u t yl 4-{[2′-(N,N-b is(2′′-ch lor oet h yl)a m in o)-

p h en yl] ca r b a m oyloxy m et h yl}-p h en yl-oxyca r b on yl-^L-

glu ta m a te (23a ) a n d d i-ter t-bu tyl 4-{[2′-(N,N-(2′′-m esyl-

oxyeth yl)(2′′-ch lor oeth yl)a m in o)p h en yl]ca r ba m oyloxy-

m eth yl}ph en yloxycar bon yl-^L-glu tam ate (24a). To a stirred

solution of 0.348 g (0.44 mmol) 15a in 10 mL of acetonitrile

heated at 50-60 °C (in the bath), 0.234 g (4.0 mmol) of NaCl

(finely grounded) were added at once. After 48 h the reaction

was stopped, the solvent evaporated under vacuum. The oily

residue containing a mixture of 23a and 24a was dissolved in

10 mL of AcOEt, washed, dried and evaporated again. The

product was purified by preparative HPLC (cycloxexane:AcOEt

3:1). The first separated fraction afforded 23a as a glassy solid

(0.149 g, 50%), mp 40-41 °C. ¹H NMR, δ_H, (ppm): 1.40 (s,

9H, CO₂-t-Bu), 1.41 (s, 9H, CO₂-t-Bu), 1.78-2.03 (2m, 2H, CH₂-

CH(NH)), 2.34 (t, 2H, CH₂CO₂), 3.26 (t, 4H, NCH₂, J ) 6.07

Hz), 3.55 (t, 4H, CH₂Cl), 3.95-4.01 (m, 1H, -CH(NH)CH₂), 5.15

1

yield 32b (56 mg, 84%) as a white solid, mp 105-107 °C. H

NMR, δ_H, (ppm): 1.70-2.10 (2m, 2H, CH₂CH(NH)), 2.25-2.35

(m, 2H, CH₂CO₂), 3.52 (t, 4H, NCH₂, J ) 7.50 Hz), 3.71 (t,

4H, CH₂Br), 4.10-4.25 (m, 1H, CH(NH)CH₂), 5.00 (s, 2H,

PhCH₂), 6.48 (d, 1H, NH-G, J ) 7.95 Hz), 6.66 (d, 2H, H_{arom3′+5′}

,

J ) 8.83 Hz), 7.28 (d, 4H, H_arom2+6+ H_{arom2′+6′}, J ) 8.41 Hz),

7.39 (d, 2H, H_arom3+5, J ) 8.17 Hz), 8.68 (s, 1H, PhNH), 9.38

(s, 1H, Ph′NH). MS, m/z: 665 (M⁺+ Na, 15); acc. mass:

(C₂₄H₂₈N₄O₇Br₂Na) calcd 665.0222, found 665.0211.

Using this deprotection procedure the following compounds

were synthesized:

4-{[4′-(N,N-Bis(2′′-iod oet h yl)a m in o)p h en yl]ca r b a m -

oyloxym et h yl}p h en ylca r b a m oyl-^L-glu t a m ic a cid (31b )

was obtained from diallyl ester 18b by a similar procedure

1702 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9

Niculescu-Duvaz et al.

but using morpholine instead of pyrrolidine as allyl scavenger.

31b (0.045 g, 61%) resulted as a solid, mp 110-112 °C. ¹H

NMR, δ_H, (ppm): 1.75-2.15 (2m, 2H, CH₂CH(NH)), 2.20-2.35

(m, 2H, CH₂CO₂), 3.25-3.35 (t, 4H, NCH₂, under H₂O), 3.67

(t, 4H, CH₂I), 4.15-4.30 (m, 1H, CH(NH)CH₂), 5.00 (s, 2H,

J ) 8.45 Hz), 8.74 (s, 1H, PhNH). MS m/z: 658 (M⁺, 8), 681

(M⁺+ Na, 15); acc. mass: (C₂₅H₃₀N₄O₇Br₂Na) calcd 679.0379,

found 679.0386.

4-{N-[4′-Bis(2′′-ch lor oet h yl)a m in op h en yl]-N-m et h yl-

ca r ba m oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m ic a cid

(41 b) was obtained as a solid using pyrrolidine as allyl

PhCH₂), 6.48 (d, 1H, NH-G, J ) 8.53 Hz), 6.62 (d, 2H, H_{arom3′+5′}

,

1

J ) 8.55 Hz), 7.28 (d, 4H, H_arom2+6+ H_{arom2′+6′}, J ) 8.38 Hz),

7.39 (d, 2H, H_arom3+5, J ) 8.09 Hz), 8.68 (s, 1H,PhNH), 9.38 (s,

1H, Ph′NH); MS, m/z: 738 (M⁺, 20), 761 (M⁺+ Na, 25); acc.

mass: (C₂₄H₂₈N₄O₇I₂Na) calcd: 760.9945, found: 760.9960.

scavanger, (0.075 g, 90%), mp 70-72 °C. H NMR, δ_H, (ppm):

1.75-2.00 (2m, 2H, CH₂CH(NH)), 2.27 (t, 2H, CH₂CO₂), 3.14

(s, 3H, NCH₃), 3.71 (s, 8H, N(CH₂CH₂Cl)₂), 4.10-4.20 (m, 1H,

CH(NH)CH₂), 4.95 (s, 2H, PhCH₂), 6.49 (d, 1H, NH-G), 6.70

(d, 2H, H_{arom3′+5′}, J ) 8.60 Hz), 7.09 (d, 2H, H_{arom2′+6′}), 7.18 (d,

2H, H_arom2+6), 7.34 (d, 2H, H_arom3+5, J ) 8.14 Hz), 8.71 (s, 1H,

PhNH). MS m/z: 568 (M⁺, 25), 591 (M⁺+ Na, 100); acc.

mass: (C₂₅H₃₀N₄O₇Cl₂Na) calcd 591.1389, found 591.1371.

4-{[4′-(N,N-Bis(2′′-m esyloxyet h yl)a m in o)p h en yl]ca r -

ba m oyloxym eth yl}p h en yl-oxyca r bon yl-^L-glu ta m ic Acid

(34b). Di-tert-butyl ester 15b (0.10 g, 0.13 mmol) was dissolved

in formic acid (5 mL) and stirred under argon at room

temperature for 48 h. The formic acid was evaporated under

vacuum (pump), and the residue was evaporated five times

with CH₂Cl₂and toluene to yield 34b as a foamy solid (0.078

g, 89%), mp 92-95 °C. ¹H NMR, δ_H, (ppm): 1.75-2.15 (2m,

2H, CH₂CH(NH)), 2.37 (t, 2H, CH₂CO₂, J ) 7.81 Hz), 3.13 (s,

6H, CH₃SO₃), 3.67 (t, 4H, NCH₂, J ) 5.22 Hz), 4.00-4.10 (m,

1H, CH(NH)CH₂), 4.28 (t, 4H, CH₂OMes), 5.10 (s, 2H, PhCH₂),

6.75 (d, 2H, H_{arom3′+5′}, J ) 6.62 Hz), 7.11 (d, 2H, H_arom3+5, J )

8.37 Hz), 7.28 (d, 2H, H_{arom2′+6′}), 7.42 (d, 2H, H_arom2+6), 8.04 (d,

1H, NH-G, J ) 6.40 Hz), 9.33 (s, 1H, Ph′NH). MS, m/z: 676

(M⁺+ 1, 100); acc. mass: (C₂₆H₃₄N₃O₁₄S₂), calcd 676.1482,

found: 676.1470. Anal. (C₂₆H₃₃N₃O₁₄S₂): C, H, N.

4-{[2′-(N,N-Bis(2′′-iod oet h yl)a m in o)-p h en yl]-ca r b a m -

oyloxym eth yl}-p h en yl-ca r ba m oyl-^L-glu ta m ic a cid (31a )

was obtained from diallyl ester 18a by a similar procedure

using morpholine. A solid (0.100 g, 93.4%) resulted, mp 96-

98 °C. ¹H NMR, δ_H, (ppm): 1.72-2.08 (2m, 2H, CH₂CH(NH)),

2.29 (t, 2H, CH₂CO₂, J ) 6.25 Hz), 3.16-3.26 (m, 8H,

N(CH₂CH₂I)₂), 4.18-4.22 (m, 1H, CH(NH)CH₂), 5.09 (s, 2H,

PhCH₂), 6.45 (d, 1H, NH-G, J ) 7.98 Hz), 7.04 (dt, 1H,

H

_{arom4′(5′)}, J _o) 7.62, J _m) 1.61 Hz), 7.17 (dt, 1H, H_{arom5′(4′)}, J _o)

7.78, J _m) 1.39 Hz), 7.28 (d, 1H, H_arom3+5, J ) 8.60 Hz), 7.38

(d, 2H, H_arom2+6, J ) 8.40 Hz), 7.92 (dd, 1H, H_arom6′, J _o) 8.16,

J _m) 1.32 Hz), 8.51 (s, 1H, NH-Ph), 8.65 (s, 1H, Ph′NH); MS,

m/z: 739 (M⁺+ 1, 30), 761 (M⁺+ Na, 100), 611 (M⁺- HI +

1, 25); acc. mass: (C₂₄H₂₈N₄O₇I₂Na) calcd: 760.9945, found:

760.9969. Anal. (C₂₄H₂₈N₃O₈I₂.0.8AcOEt): C, H, N.

4-{[2′-(N,N-Bis(2′′-br om oeth yl)a m in o)p h en yl]ca r ba m -

oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m ic Acid (32a ).

The previous described procedure using pyrrolidine led to 32a

1

(0.068 g, 95.6%) as a solid, mp 94-95 °C. H NMR, δ_H, (ppm):

1.84-2.03 (2m, 2H, CH₂CH(NH)), 2.27 (t, 2H, CH₂CO₂, J )

7.18 Hz), 3.30 (t, 4H, NCH₂, J ) 5.69 Hz), 3.42 (t, 4H, CH₂-

Br), 4.13-4.21 (m, 1H, CH(NH)CH₂), 5.06 (s, 2H, PhCH₂), 6.47

(d, 1H, NH-G, J ) 7.84 Hz), 7.04 (dt, 1H, H_{arom4′(5′)}, J _o) 8.68,

J _m) 1.56 Hz), 7.18 (dt, 1H, H_{arom5′(4′)}, J _o) 7.78, J _m) 1.43

Hz), 7.25 (d, 1H, H_arom3+5, J ) 8.64 Hz), 7.37 (d, 2H, H_arom2+6),

7.94 (dd, 1H, H_arom6′, J ) 8.16, J _m) 1.24 Hz), 8.61 (s, 1H,

NHPh), 8.69 (s, 1H, Ph′NH). MS, m/z: 645 (M⁺+ 1, 6), 667

(M⁺+ Na, 55), 611 (M⁺- Br, 16); acc. mass: (C₂₄H₂₈N₄O₇-

Br₂Na) calcd: 760.9945, found: 760.9969. Anal. (C₂₄H₂₇N₃O₈-

Br₂): C, H; N required 8.70, found 8.16.

The following compounds were prepared by the same

procedure:

4-{[4′-(N,N-Bis(2′′-iod oet h yl)a m in o)p h en yl]ca r b a m -

oyloxym eth yl}p h en yloxyca r bon yl-^L-glu ta m ic a cid (35b)

was obtained from di-tert-butyl ester 21b as a solid (0.065 g,

73%), mp 117-120 °C, by a similar procedure. ¹H NMR, δ_H,

(ppm): 1.80-2.10 (2m, 2H, CH₂CH(NH)), 2.37 (t, 2H, CH₂CO₂,

J ) 6.80 Hz), 3.20-3.40 (t, 4H, NCH₂, under H₂O), 3.72 (t,

4H, CH₂I), 4.00-4.10 (m, 1H, CH(NH)CH₂), 5.10 (s, 2H,

PhCH₂), 6.64 (d, 2H, H_{arom3′+5′}, J ) 8.77 Hz), 7.12 (d, 2H,

H

_arom2+6, J ) 8.29 Hz), 7.28 (d, 2H, H_{arom2′+6′}), 7.42 (d, 2H,

_arom3+5), 8.06 (d, 1H, NH-G, J ) 7.45 Hz), 9.35 (s, 1H, Ph′NH).

4-{[2′-(N,N-Bis(2′′-ch lor oeth yl)a m in o)p h en yl]ca r ba m -

oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m ic a cid (33a )

was obtained as a solid (0.054 g, 69.5%), mp 93-94 °C using

morpholine as allyl scavanger. ¹H NMR, δ_H, (ppm): 1.77-2.00

(2m, 2H, CH₂CH(NH)), 2.28 (t, 2H, CH₂CO₂, J ) 7.49 Hz), 3.25

(t, 4H, NCH₂, J ) 5.82 Hz), 3.55 (t, 4H, CH₂Cl), 4.14-

4.22 (m, 1H, CH(NH)CH₂), 5.07 (s, 2H, PhCH₂), 6.48 (d, 1H,

NH-G, J ) 7.77 Hz), 7.05 (t, 1H, H_{arom4′(5′)}, J _o) 7.56 Hz), 7.19

(t, 1H, H_{arom5′(4′)}), 7.25 (d, 1H, H_arom3+5, J ) 8.53 Hz), 7.37 (d,

2H, H_arom2+6), 7.94 (d, 1H, H_arom6′, J ) 8.10 Hz), 8.62 (s, 1H,

NH-Ph), 8.71 (s, 1H, Ph′NH). MS, m/z: 577 (M⁺+ Na, 100);

acc. mass: (C₂₄H₂₈N₄O₇Cl₂Na) calcd: 577.1254, found: 577.1254.

Anal. (C₂₄H₂₇N₃O₈Cl₂‚0.6H₂O): C, H, N.

MS, m/z: 740 (M⁺+ 1, 100), 762 (M⁺+ Na, 45), (M⁺- I, 30);

acc. mass: (C₂₄H₂₈N₃O₈I₂) calcd 739.9966; found 739.9980.

Anal. (C₂₄H₂₇N₃O₈I₂): C, H, N.

4-{[4′-(N,N-Bis(2′′-br om oeth yl)a m in o)p h en yl]ca r ba m -

oyloxym eth yl}p h en yloxyca r bon yl-^L-glu ta m ic a cid (36b)

was obtained from di-tert-butyl ester 22b as a solid (0.065 g,

73%), mp 73-76 °C, by a similar procedure. ¹H NMR, δ_H,

(ppm): 1.80-2.10 (2m, 2H, CH₂CH(NH)), 2.37 (t, 2H, CH₂-

CO₂), 3.55 (t, 4H, NCH₂), 3.72 (t, 4H, CH₂Br), 4.00-4.15 (m,

1H, CH(NH)CH₂), 5.10 (s, 2H, PhCH₂), 6.69 (d, 2H, H_{arom3′+5′}

J ) 8.55 Hz), 7.12 (d, 2H, H_arom2+6, J ) 8.28 Hz), 7.29 (d, 2H,

_{arom2′+6′}), 7.42 (d, 2H, H_arom3+5), 8.02 (d, 1H, NH-G), 9.32 (s,

,

H

4-{N-[4′-Bis(2′′-iod oeth yl)a m in op h en yl]-N-m eth ylca r -

bam oyloxym eth yl}ph en ylcar bam oyl-^L-glu tam ic acid (39b)

was obtained as a solid using pyrrolidine as allyl scavanger,

1H, Ph′-NH). MS, m/z: 646 (M⁺+ 1, 100), 668 (M⁺+ Na, 55);

acc. mass: (C₂₄H₂₈N₃O₈Br₂) calcd 644.0243; found 644.0230.

Anal. (C₂₄H₂₇N₃O₈Br₂): H, N; C required 44.67, found 45.09%.

4-{[2′-(N,N-Bis(2′′-m esyloxyeth yl)am in o)ph en yl]car bam -

oyloxym eth yl}p h en yloxyca r bon yl-^L-glu ta m ic a cid (34a )

resulted as a foamy solid (0.063 g, 99%), mp 56-57 °C. ¹H

NMR, δ_H, (ppm): 1.80-2.04 (2m, 2H, CH₂CH(NH)), 2.37 (t,

2H, CH₂CO₂, J ) 7.96 Hz), 3.04 (s, 6H, CH₃SO₃), 3.30 (t, 4H,

NCH₂, J ) 5.21 Hz), 4.00-4.05 (m, 1H, CH(NH)CH₂), 4.11 (t,

4H, CH₂OMes), 5.13 (s, 2H, PhCH₂), 7.03-7.19 (m, 4H,

1

(0.105 g, 77.8%), mp 103-105 °C. H NMR, δ_H, (ppm): 1.75-

2.00 (2m, 2H, CH₂CH(NH)), 2.28 (t, 2H, CH₂CO₂, J ) 7.43 Hz),

3.14 (s, 3H, NCH₃), 3.30 (t, 4H, NCH₂), 3.72 (t, 4H, CH₂I, J )

7.64 Hz), 4.10-4.25 (m, 1H, CH(NH)CH₂), 4.96 (s, 2H, PhCH₂),

6.46 (d, 1H, NH-G, J ) 7.48 Hz), 6.64 (d, 2H, H_{arom3′+5′}, J )

8.85 Hz), 7.10 (d, 2H, H_{arom2′+6′}), 7.17 (d, 2H, H_arom2+6, J ) 7.49

Hz), 7.34 (d, 2H, H_arom3+5), 8.68 (s, 1H, PhNH). MS m/z: 752

(M⁺+ 1, 10), 775 (M⁺+ Na, 35); acc. mass: (C₂₅H₃₀N₄O₇I₂Na)

calcd 775.0102, found 775.0088.

H

_arom3+5, H_arom4′, H_arom5′), 7.44 (d, 2H, H_arom2+6, J ) 8.56 Hz),

7.95 (d, 2H, H_arom6′, J ) 7.96 Hz), 8.10 (d, 1H, NH-G, J ) 8.10

Hz), 8.42 (s, 1H, Ph′NH). MS, m/z: 676 (M⁺+ 1, 18), 698 (M⁺

+ 23, 10); acc. mass: (C₂₆H₃₄N₃O₁₄S₂), calcd 676.1482, found:

676.1460. Anal. (C₂₆H₃₃N₃O₁₄S₂.0.8 HCO₂H): C, H, N.

4-{N-[4′-Bis(2′′-b r om oet h yl)a m in op h en yl]-N-m et h yl-

ca r ba m oyloxym eth yl}p h en ylca r ba m oyl-^L-glu ta m ic a cid

(40b) was obtained as a solid using pyrrolidine as allyl

1

scavanger, (0.10 g, 80%), mp 75-77 °C. H NMR, δ_H, (ppm):

1.80-1.95 (m, 2H, CH₂CH(NH)), 2.28 (t, 2H, CH₂CO₂, J ) 7.33

Hz), 3.14 (s, 3H, NCH₃), 3.71 (s, 8H, N(CH₂CH₂Cl)₂), 4.10-

4.20 (m, 1H, CH(NH)CH₂), 4.95 (s, 2H, PhCH₂), 6.48 (d, 1H,

NH-G, J ) 7.17 Hz), 6.68 (d, 2H, H_{arom3′+5′}, J ) 8.74 Hz), 7.09

4-{[2′-(N,N-Bis(2′′-iod oet h yl)a m in o)p h en yl]ca r b a m -

oyloxym eth yl}p h en yloxyca r bon yl-^L-glu ta m ic a cid (35a )

was obtained from di-tert-butyl ester 21a as a solid (0.096 g,

93.2%), mp 56-58 °C, by a similar procedure. ¹H NMR, δ_H,

(ppm): 1.86-2.11 (2m, 2H, CH₂CH(NH)), 2.36 (t, 2H, CH₂-

(d, 2H, H_{arom2′+6′}), 7.18 (d, 2H, H_arom2+6), 7.34 (d, 2H, H_arom3+5

,

Self-Immolative Nitrogen Mustards Prodrugs

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9 1703

CO₂, J ) 7.02 Hz), 3.20-3.26 (m, 8H, N(CH₂CH₂I)₂), 3.97-

4.10 (m, 1H, CH(NH)CH₂), 5.17 (s, 2H, PhCH₂), 7.05-7.18 (m,

4H, H_arom2+6+ H_{arom4′(5′)}+ H_{arom4′(5′)}), 7.34 (d, 1H, H_arom3′, J )

4-{N-[4′-Bis(2′′-ch lor oet h yl)a m in op h en yl]-N-m et h yl-

car bam oyloxym eth yl}ph en yloxycar bon yl-^L-glu tam ic acid

(44b) was obtained from di-tert-butyl ester 30b as a solid

(0.355 g, 81.5%), mp 60-63 °C, by a similar procedure. ¹H

NMR, δ_H, (ppm): 1.80-2.10 (2m, 2H, CH₂CH(NH)), 2.36 (t,

2H, CH₂CO₂, J ) 7.95 Hz), 3.16 (s, 3H, NCH₃), 3.72 (s, 8H,

N(CH₂CH₂Cl)₂), 3.95-4.10 (m, 1H, CH(NH)CH₂), 5.05 (s, 2H,

PhCH₂), 6.72 (d, 2H, H_{arom3′+5′}, J ) 9.02 Hz), 7.08 (d, 2H,

7.80 Hz), 7.43 (d, 2H, H_arom2+6, J ) 8.40 Hz), 7.93 (d, 1H, H_arom6′

,

J ) 8.13 Hz), 8.06 (d, 1H, NH-G, J ) 7.99 Hz), 8.57 (s, 1H,

Ph′NH). MS, m/z: 740 (M⁺+ 1, 100), 762 (M⁺+ Na, 45), (M⁺

- I, 100); acc. mass: (C₂₄H₂₈N₃O₈I₂) calcd 739.9966; found

739.9990. Anal. (C₂₄H₂₇N₃O₈I₂+0.2C₇H₈): C, H, N.

H

_arom3+5, J ) 9.72 Hz), 7.11 (d, 2H, H_{arom2′+6′}), 7.31 (d, 2H,

4-{[2′-(N,N-Bis(2′′-br om oeth yl)a m in o)p h en yl]ca r ba m -

oyloxym eth yl}p h en yloxyca r bon yl-^L-glu ta m ic a cid (36a )

was obtained from di-tert-butyl ester 22a as a solid (0.081 g,

92%), mp 63-64 °C, by a similar procedure. ¹H NMR, δ_H,

(ppm): 1.80-2.10 (2m, 2H, CH₂CH(NH)), 2.36 (t, 2H, CH₂-

CO₂H, J ) 7.33 Hz), 3.32 (t, 4H, NCH₂, J ) 5.95 Hz), 3.44 (t,

4H, CH₂Br), 3.97-4.10 (m, 1H, -CH(NH)CH₂), 5.16 (s, 2H,

PhCH₂), 7.06 (t, 1H, H_{arom4′(5′)}, J ) 7.56 Hz), 7.10 (d, 2H,

_arom2+6), 8.06 (d, 1H, NH-G). MS m/z: 569 (M⁺, 25), 592 (M⁺

+ Na, 30); acc. mass: (C₂₅H₂₉N₃O₈Cl₂) calcd 569.1352, found

569.1332.

4-Bis(2′-m esyloxyet h yl)a m in oa n ilin e H yd r ob r om id e

(57b). To a solution containing 0.145 g (0.32 mmol) 48b in

AcOH (1 mL) was added 45% HBr in AcOH solution (2 mL),

and the reaction mixture was stirred at room temperature for

10 min. The reaction mixture was added dropwise in dry Et₂O

under stirring. A white precipitate was formed, which was

centrifugated. The pellet was resuspended in fresh Et₂O (40-

45 mL) and centrifugated again. The procedure was repeated

five times. 57b‚2HBr was obtained as a hygroscopic solid (0.12

g, 73%).¹H NMR, δ_H, (ppm): 3.15 (s, 6H, CH₃SO₃), 3.75 (t, 4H,

NCH₂, J ) 5.55 Hz), 4.32 (t, 4H, CH₂OMes), 6.89 (d, 2H,

H

_arom3+5, J ) 7.99 Hz), 7.19 (t, 1H, H_{arom5′(4′)}, J ) 7.63 Hz), 7.41

(d, 2H, H_arom2+6), 7.95 (d, 1H, H_arom6′, J ) 8.08 Hz), 8.03 (d,

1H, NH-G, J ) 7.68 Hz), 8.66 (s, 1H, Ph′NH). MS, m/z: 646

(M⁺+ 1, 35), 668 (M⁺+ Na, 50), 566 (M⁺- Br, 45); acc. mass:

(C₂₄H₂₈N₃O₈Br₂) calcd 644.0243; found 644.0220. Anal. (C₂₄H₂₇

-

N₃O₈Br₂+0.3C₇H₈): C, H, N.

4-{[2′-(N,N-Bis(2′′-ch lor oeth yl)a m in o)p h en yl]ca r ba m -

oyloxym eth yl}p h en yloxyca r bon yl-^L-glu ta m ic a cid (37a )

was obtained from di-tert-butyl ester 23a as a solid (0.111 g,

74%), mp 55-56 °C, by a similar procedure. ¹H NMR, δ_H,

(ppm): 1.70-2.07 (2m, 2H, CH₂CH(NH)), 2.37 (t, 2H, CH₂-

CO₂), 3.27 (t, 4H, NCH₂, J ) 6.00 Hz), 3.56 (t, 4H, CH₂Cl),

4.00-4.07 (m, 1H, -CH(NH)CH₂), 5.16 (s, 2H, PhCH₂), 7.02-

H

_arom3+5, J ) 9.07 Hz), 7.18 (d, 2H, H_arom2+6), 9.57 (s, 3H, NH₃⁺).

Anal. (C₁₂H₂₀N₂O₆S₂.1.8HBr): C, H, N.

The following compounds were obtained by the same

procedure:

4-Bis(2′-iod oeth yl)a m in oa n ilin e h yd r obr om id e (58b)

was obtained as a solid (0.105 g, 94%), mp 155-158 °C. ¹H

NMR, δ_H, (ppm): 3.31 (t, 4H, NCH₂, J ) 7.72 Hz), 3.74 (t, 4H,

CH₂I), 6.77 (d, 2H, H_arom3+5, J ) 8.87 Hz), 7.19 (d, 2H, H_arom2+6),

9.62 (s, 3H, NH₃⁺). MS, m/z: 417 (M⁺+ 1, 100); acc. mass:

7.12 (m, 3H, H_arom3+5, H_{arom4′(5′)}), 7.20 (dt, 1H, H_{arom5′(4′)}, J _o

7.76 Hz, J _m) 1.47 Hz), 7.40 (d, 2H, H_arom2+6,), 7.96 (dd, 2H,

)

H

_arom6′, J _o) 7.96 Hz, J _m) 1.27 Hz), 8.08 (d, 1H, NH-G, J )

8.08 Hz), 8.67 (s, 1H, Ph′NH). MS, m/z: 556 (M⁺+ 1, 30), 578

(M⁺+ 23, 10); acc. mass: (C₂₄H₂₈N₃O₈Cl₂) calcd 556.1253;

found 556.1270. Anal. (C₂₄H₂₇N₃O₈Cl₂): C, H, N.

(C₁₀H₁₅N₂I₂) calcd 416.9325; found 416.9340. Anal. (C₁₀H₁₄

N₂I₂.1.8HBr.0.2CH₃COOH): C, H, N.

-

4-Bis(2′-br om oeth yl)a m in oa n ilin e h yd r obr om id e (59b‚

2HBr ) was obtained as a solid (0.16 g, 100%), mp > 230 °C

(decomp).¹H NMR, δ_H, (ppm): 3.59 (t, 4H, NCH₂, J ) 6.99 Hz),

3.79 (t, 4H, CH₂Br), 6.82 (d, 2H, H_arom3+5, J ) 7.87 Hz), 7.19

(d, 2H, H_arom2+6), 9.63 (s, 3H, NH₃⁺). MS, m/z: 323 (M⁺+ 1,

100); acc. mass: (C₁₀H₁₅N₂Br₂) calcd 320.9602; found 320.9587.

Anal. (C₁₀H₁₄N₂Br₂‚1.6HBr‚0.2CH₃COOH): C, H, N.

2-Bis(2′-iod oeth yl)a m in oa n ilin e h yd r obr om id e (58a )

was obtained as a solid (0.195 g, 67.8%), mp < 50 °C. MS,

m/z: 417 (M⁺+ 1, 65); acc. mass: (C₁₀H₁₅N₂I₂) calcd 416.9325;

found 416.9316. Anal. (C₁₀H₁₄N₂I₂‚HBr): C, H, N.

4-{[2′-(N,N-(2′′-Ch lor oeth yl)(2′′-m esyloxyeth yl)a m in o)-

p h e n yl]ca r b a m oyloxym e t h yl}p h e n yloxyca r b on yl-^L-

glu ta m ic a cid (38a ) was obtained from di-tert-butyl ester 24a

as a solid (0.058 g, 94.1%), mp 61-63 °C, by a similar

1

procedure. H NMR, δ_H, (ppm): 1.80-2.05 (2m, 2H, CH₂CH-

(NH)), 2.36 (t, 2H, CH₂CO₂, J ) 8.01 Hz), 3.03 (s, 3H, CH₃-

SO₂), 3.28 (t, 4H, NCH₂, J ) 5.37 Hz), 3.54 (t, 2H, CH₂Cl, J )

5.92 Hz), 3.95-4.02 (m, 1H, -CH(NH)CH₂), 4.11 (t, 2H, CH₂-

OMes), 5.14 (s, 2H, PhCH₂), 7.02-7.22 (m, 4H, H_arom3+5, H_arom4′

,

H

_arom5′), 7.42 (d, 2H, H_arom2+6, J ) 8.40 Hz), 7.95 (d, 2H, H_arom6′

2-Bis(2′-br om oeth yl)a m in oa n ilin e h yd r obr om id e (59a )

was obtained as a solid (0.102 g, 32%), mp 150-151 °C. MS,

m/z: 323 (M⁺+ 1, 100); acc. mass: (C₁₀H₁₅N₂Br₂) calcd

320.9602; found 320.9594. Anal. (C₁₀H₁₄N₂Br₂.HBr): C, H, N.

J ) 7.88 Hz), 8.08 (d, 1H, NH-G, J ) 7.98 Hz), 8.67 (s, 1H,

Ph′NH). MS, m/z: 615 (M⁺, 100), 638 (M⁺+ Na, 17); acc.

mass: (C₂₅H₃₀N₃O₁₁ClS) calcd 615.1275, found 615.1290. Anal.

(C₂₅H₃₀N₃O₁₁ClS): C, H, N.

2-Bis(2′-ch lor oeth yl)a m in oa n ilin e h yd r obr om id e (60a )

was obtained as a solid (0.124 g, 72.1%), mp 147-148 °C. H

NMR, δ_H, (ppm): 3.36 (t, 4H, NCH₂, J ) 6.70 Hz), 3.60 (t, 4H,

CH₂Cl), 7.25-7.53 (m, 4H, H_arom); MS, m/z: 223 (M⁺+ 1, 100),

195 (M⁺- Cl, 10); acc. mass: (C₁₀H₁₅N₂Cl₂) calcd 233.0612;

found 233.0624. Anal. (C₁₀H₁₄N₂Cl₂.HBr): C, H, N.

The following compounds were prepared by the same

method, except that the reaction time was 1.5 h instead of 10

min:

4-{N-[4′-Bis(2′′-iod oeth yl)a m in op h en yl]-N-m eth ylca r -

ba m oyloxym et h yl}p h en yloxyca r b on yl-^L-glu t a m ic a cid

(42b) was obtained from di-tert-butyl ester 28b as a solid (0.53

1

g, 83.9%), mp 70-72 °C, by a similar procedure. H NMR, δ_H,

(ppm): 1.80-2.15 (2m, 2H, CH₂CH(NH)), 2.37 (t, 2H, CH₂-

CO₂, J ) 9.00 Hz), 3.16 (s, 3H, NCH₃), 3.25-3.35 (t, 4H,

NCH₂), 3.72 (t, 4H, CH₂I, J ) 7.62 Hz), 4.00-4.10 (m, 1H,

CH(NH)CH₂), 5.05 (s, 2H, PhCH₂), 6.65 (d, 2H, H_{arom3′+5′}, J )

8.95 Hz), 7.07 (d, 2H, H_arom3+5, J ) 8.46 Hz), 7.12 (d, 2H,

H

_{arom2′+6′}), 7.29 (d, 2H, H_arom2+6), 8.10 (d, 1H, NH-G, J ) 7.81

4-Bis(2′-iod oeth yl)a m in o-N-m eth yla n ilin e h yd r obr o-

m id e (61b) was obtained as a solid (0.19 g, 86.5%), mp 140-

Hz). MS m/z: 753 (M⁺, 43), 776 (M⁺+ Na, 43); acc. mass:

1

(C₂₅H₂₉N₃O₈I₂) calcd 753.0022, found 753.0044.

142 °C. H NMR, δ_H, (ppm): 2.89 (s, 3H, NCH₃), 3.30 (t, 4H,

NCH₂, J ) 7.18 Hz), 3.74 (t, 4H, CH₂I), 6.80 (d, 2H, H_arom3+5

,

4-{N-[4′-Bis(2′′-b r om oet h yl)a m in op h en yl]-N-m et h yl-

car bam oyloxym eth yl}ph en yloxycar bon yl-^L-glu tam ic acid

(43b) was obtained from di-tert-butyl ester 29b as a solid

(0.437 g, 85.3%), mp 64-67 °C, by a similar procedure. ¹H

NMR, δ_H, (ppm): 1.80-2.10 (2m, 2H, CH₂CH(NH)), 2.37 (t,

2H, CH₂CO₂, J ) 8.05 Hz), 3.16 (s, 3H, NCH₃), 3.58 (t, 4H,

NCH₂, J ) 6.84 Hz), 3.77 (t, 4H, CH₂Br), 4.00-4.10 (m, 1H,

CH(NH)CH₂), 5.05 (s, 2H, PhCH₂), 6.70 (d, 2H, H_{arom3′+5′}, J )

9.04 Hz), 7.07 (d, 2H, H_arom3+5, J ) 8.50 Hz), 7.12 (d, 2H,

J ) 8.90 Hz), 7.33 (d, 2H, H_arom2+6), 10.28 (s, 3H, NH₂⁺). MS,

m/z: 431 (M⁺+ 1, 100), 453 (M⁺+ Na, 2); acc. mass:

(C₁₁H₁₇N₂I₂) calcd 430.9481; found 430.9470. Anal. (C₁₁H₁₆

N₂I₂.2HBr): H, N; C required 22.32, found 22.78%.

-

4-Bis(2′-b r om oet h yl)a m in o-N-m et h yla n ilin e h yd r o-

br om id e (62b) was obtained as a solid (0.134 g, 90.7%), mp

1

191-194 °C. H NMR, δ_H, (ppm): 2.89 (s, 3H, NCH₃), 3.59 (t,

4H, NCH₂, J ) 6.93 Hz), 3.81 (t, 4H, CH₂Br), 6.85 (d, 2H,

H

_{arom2′+6′}), 7.29 (d, 2H, H_arom2+6), 8.10 (d, 1H, NH-G, J ) 8.07

H

_arom3+5, J ) 8.79 Hz), 7.33 (d, 2H, H_arom2+6), 10.29 (s, 3H,

Hz). MS m/z: 659 (M⁺, 90), 682 (M⁺+ Na, 95); acc. mass:

NH₂⁺). MS, m/z: 337 (M⁺+ 1, 100); acc. mass: (C₁₁H₁₇N₂Br₂)

(C₂₅H₂₉N₃O₈Br₂) calcd 657.0321, found 657.0340.

calcd 334.9758; found 334.9770.

1704 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 9

Niculescu-Duvaz et al.

4-Bis(2′-ch lor oet h yl)a m in o-N-m et h yla n ilin e h yd r o-

br om id e (63b) was obtained as a solid (0.088 g, 80.8%), mp

179-182 °C. H NMR, δ_H, (ppm): 2.89 (s, 3H, NCH₃), 3.74 (s,

8H, N(CH₂CH₂Cl)₂), 6.87 (d, 2H, H_arom3+5, J ) 8.55 Hz), 7.32

(d, 2H, H_arom2+6), 10.23 (s, 3H, NH₂⁺). MS, m/z: 247 (M⁺+ 1,

100), 269 (M⁺+ Na, 5); acc. mass: (C₁₁H₁₇N₂Cl₂) calcd

247.0769; found 247.0750. Anal. (C₁₁H₁₆N₂Cl₂.2HBr): C, H, N.

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J M020462I

Article Doi

Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT

DOI: 10.1021/jm020462i

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