Targeting the polyamine transport system with benzazepine- and azepine-polyamine conjugates

J. Med. Chem. 2010, 53, 7647–7663 7647

DOI: 10.1021/jm1007648

Targeting the Polyamine Transport System with Benzazepine- and Azepine-Polyamine Conjugates^†

‡

§

‡

ꢀ ꢀ

Sophie Tomasi, Jacques Renault, Benedicte Martin, Stephane Duhieu, Virginie Cerec, Myriam Le Roch, Philippe Uriac,

and Jean-Guy Delcros*^,§

^‡Produits Naturels-Syntheꢁses-Chimie Meꢀdicinale, Sciences Chimiques de Rennes, CNRS UMR6226, Faculteꢀde Pharmacie, UniversiteꢀRennes 1,

Universiteꢀ Europeꢀenne de Bretagne, Rennes Cedex, France, and ^§Groupe de Recherche en Theꢀrapeutique Anticanceꢀreuse,

Faculteꢀ de Meꢀdecine, Universiteꢀ Rennes 1, Universiteꢀ Europeꢀenne de Bretagne, Rennes Cedex, France

Received June 22, 2010

The polyamine transport system (PTS) whose activity is up-regulated in cancer cells is an attractive

target for drug design. Two heterocyclic (azepine and benzazepine) systems were conjugated to various

polyamine moieties through an amidine bound to afford 18 compounds which were evaluated for their

affinity for the PTS and their ability to use the PTS for cell delivery. Structure-activity relationship

studies and lead optimization afforded two attractive PTS targeting compounds. The azepine-

spermidine conjugate 14 is a very selective substrate of the PTS that may serve as a vector for

radioelements used for diagnoses or therapeutics in nuclear medicine. The nitrobenzazepine-spermine

conjugate 28 is a very powerful PTS inhibitor with very low intrinsic cytotoxicity, able to prevent the

growth of polyamine depleted cells in presence of exogenous polyamines.

Introduction

Polyamines are ubiquitous organic polycations present in

all living organisms (Figure 1). Although their precise func-

tions remain unknown, polyamines are essential in the regula-

tion of cell proliferation and differentiation. The remarkable

complexity of the mechanisms controlling their homeostasis

stresses their exceptional importance. All cells are equipped

witha multifaceted and highly regulatedenzymatic machinery

allowing polyamine synthesis, retroconversion, and degrada-

tion. Cells also possess active transport systems allowing

Figure 1. Structures of naturally occurring putrescine (1), spermi-

dine (2), and spermine (3).

drugs,^13-21but only a few of them display enhanced cytotoxicity

to cancer cells over their normal counterparts in vitro.^22,23The

import and export of polyamines. On a molecular level,

polyamine transport systems (PTS^a) controlling the import

of exogenous polyamines have been characterized in bacteria,

first successful design was reported recently by Barret and his

colleagues, who developed a spermine-epipodophyllotoxin

conjugate with selective uptake via the PTS and a wide therapeu-

in yeast,^1,2and in the protozoan parasites Leishmania and

Trypanosoma.^3,4In contrast, polyamine transport in mamma-

tic index, able to induce complete regression in human breast

lian cells remains a measurable import process^5-8still waiting

to be molecularly characterized.

tumor xenograft model after i.p. or oral administrations.²⁴In

addition, they also describe the development of fluorophor-

labeled polyamine probes to identify tumors expressing a highly

active PTS.^11,25,26Attempts have been also made to use poly-

amines as selective vectors of radioelements for tumor therapy

or imaging.^27-33

PTS have been recognized as potential targets for therapeutic

intervention in cancers.^8-10Many cancer cells exhibit elevated

polyamine import activity,^5,11,12probably due to their enhanced

need for these growth supporting factors. The elevated activity

of the PTS along with its broad structural tolerance which

allows the import of non-native polyamine conjugates, provide

an opportunity to selectively target cancer cells.^5,10The literature

reports many examples of polyamine conjugates with cytotoxic

Because of the high affinity of some polyamine conjugates for

the PTS, such structures have also been designed toward the

identification of polyamine transport inhibitors.^34-37Indeed the

ability of polyamine biosynthesis inhibitors (e.g., R-difluorome-

thylornithine, an ornithine decarboxylase inhibitor (DFMO))

to completely deplete internal polyamines and therefore inhibit

cancer cell growth, is overcome by the importation of poly-

amines from external sources, hence the need of potent PTS

inhibitors. In particular, spermine conjugates with amino acids

such as lysine seems to be very promising.^35,38,39

^†This work is dedicated to the memory of Professor Nikolaus Seiler

(1931-2006), an outstanding scientist and mentor and pioneer in the

ꢀ

polyamine field, and of Dr. Pierre Guenot (1948-2007).

*To whom correspondence should be addressed. Phone:

33.(0)4.78.78.59.71. Fax: 33.(0)4.78.78.28.87. E-mail: delcros@lyon.fnclcc.fr.

Address: Apoptosis Cancer & Development Laboratory, CNRS UMR5238,

ꢀ

Centre Leon Berard, 28 Rue Laennec, 69008 Lyon, France.

The lack of knowledge on the molecular nature of the

PTS still renders difficult a rational design of PTS-targeting

drugs. Therefore, to identify molecular recognition elements

and to delineate the structural tolerance accommodated by

^aAbbreviations: PTS, polyamine transport system; CHO, Chinese

hamster ovary; DFMO, R-difluoromethylornithine; CHO-MG, Chinese

hamster ovary cells polyamine transport deficient mutant; L1210, mouse

leukemia cells; PBS, phosphate buffered saline.

r

2010 American Chemical Society

Published on Web 10/06/2010

pubs.acs.org/jmc

7648 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Tomasi et al.

Figure 2. Structure and nomenclature of free (1-10) and conjugated (11-29) polyamines.

the PTS, systematic structure-activity relationship studies

were carried out on a series of polyamine conjugates with

complex systems such as arenes, azamacrocycles, or aro-

matic heterocycles^{16,23,32,40-44}All these conjugates were

screened in terms of their affinity for the PTS as well as for

their ability to be delivered in various cell lines possessing

or not an active and/or activated PTS. To further extend

our knowledge of the structure-activity relationship of

polyamine conjugates and based on our previous study

confirming the size of substituents as a limiting factor for

the conjugate selectivity on PTS,⁴³a similar approach was

carried out on two new series of benzazepine and azepine

heterocyclic systems coupled to various polyamine scaf-

folds by an amidine bond (Figure 2). This amidine function

leads to the rapid access of various chain moieties and

allows keeping of the positive charge in physiological con-

ditions in comparison to the amide one.

Results and Discussion

Synthesis. The final compounds 11-29 were polyamine

conjugates and were all evaluated as hydrochloride salts.

Their heterocyclic moiety, either an azepine or a benzazepine

ring, was linked to natural or synthetic polyamine chain,

diamines, triamines or tetramines, through an amidine bond

(Figure 2). Despite the various conjugation methods used, all

starting materials were identical and consisted of free or

suitably protected polyamines and lactams.

Preparation of Polyamines. Most polyamines used were

either commercial (putrescine 1, spermine 3, norspermidine

5) or known compounds (Figure 3): mono-Boc putrescine

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7649

Figure 3. Structures of protected polyamines (30-35).

Scheme 1^a

Scheme 2^a

^aReagents: (a) ethyl trifluoroacetate (1 equiv); (b) (Boc)₂O (3 equiv);

(c) K₂CO₃(5.2 equiv); (d) 4-bromobutyronitrile (1 equiv), KF/Celite;

(e) (Boc)₂O (1.5 equiv); (f) H₂, Raney Ni, NH₃/EtOH; (g) acrylonitrile

(1 equiv); (h) (Boc)₂O (1 equiv).

^aReagents: (a) Lawesson’s reagent, dioxane or toluol; (b) (CH₃)₂SO₄,

toluol; (c) (C₂H₅)₃OBF₄, CH₂Cl₂; (d) Br₂, CH₃COOH; (e) H₂SO₄,

HNO₃.

30,⁴⁵mono-Boc octanediamine 31,⁴⁶di-Boc spermidines

33,⁴⁷and 34,⁴⁸tri-Boc spermine 32,⁴⁹or protected dihydro-

xyspermine 35.⁵⁰

Adapted procedures were carried out to afford protected

triamines 36,⁴⁹37,⁴⁹and 39⁴⁹(Scheme 1) in a 39%, 44%, and

75% yields, respectively. The mono Boc protected com-

pound 41 resulted from a classical monoprotection of the

dimesitylhomospermine 40 (Scheme 1).⁵¹

(triethylamine or excessive polyamine) and mercury(II)

chloride (HgCl₂).⁶⁰This reaction produces HgS and HCl

that can be trapped by a base. Practical reasons prompted us

to carry out different procedures depending on the poly-

amine moiety (symmetrical or unsymmetrical) as well as on

the thiolactam lipophilicity. Our initial attempts consisted of

direct conjugation of a free polyamine (putrescine or sper-

mine) with the ε-caprothiolactam 43. Despite formation of

the expected amidine, the final isolation (extraction followed

by chromatographic purification) could not be achieved

because of the high hydrophilicity of the final compound.

Thus, conjugation of the ε-caprothiolactam (Scheme 3)

always involved the use of equimolar protected polyamines

whose lipophilic protective groups not only prevented the

formation of regioisomers (for unsymmetrical polyamines)

but also facilitated the isolation of the compounds 54-61.

The use of protective groups was also necessary to the

regioselective conjugation of unsymmetrical polyamines 33

and 35 to the thiolactam 49 (Scheme 4, syntheses of 66-67).

The free secondary amine of 35 did not notably react in our

conditions. Finally, the substituted lactams 50-52 were

coupled to the readily accessible tri-Boc spermine 32 and

furnished 69-71 in moderate to good yield.

In contrast, the direct coupling (Scheme 4) of lipophilic

thiolactam 49 to unprotected symmetrical chains such as

putrescine 1, 1,8-diaminooctane 4, norspermine 5, or sper-

mine 3 was successful for the preparation of 62-65. An

excess of the polyamine could be advantageously used

instead of triethylamine (TEA): it prevented the formation

of bis-conjugates and was easily removed by extraction in

aqueous medium. As we observed for the partially protected

chain 35, secondary amines did not react and the expected

amidines 62-65 could be isolated and purified by the usual

chromatographic methods.

Preparation of Lactams. The ε-caprolactam 42 is commer-

cially available. The benzazepinones 45 and 47 were prepared

from R-tetralone and 6-methoxy-1-tetralone, whose oxime

groups underwent Beckmann rearrangement.^52,53In the case

of 47, the Beckmann rearrangement resulted in the formation of

two regioisomers that could be separated. The substituted ana-

logues 46 and 48 were obtained from 45: its nitration was

achieved according to classical techniques^54,55to provide 48 in

good yield. The access to the bromo analogue 46 had been des-

cribed through the reduction of the nitro group to an amine

followed by a Sandmeyer reaction,^53,54but for this work, we

simply transposed chlorination conditions⁵⁶to the bromination

of 45 and directly obtained 46, the bromination site being

confirmed by ¹³C NMR spectroscopy.

Preparation of Thiolactams and Iminoethers. Thiolactams

43 and 49-52 were prepared in good yields (80%) by reaction of

lactams 42 and 45-48 with Lawesson’s reagent (Scheme 2).⁵⁷

The lactams 42 and 45 could also be converted to imi-

noethers (Scheme 2). Two methods were carried out. The

first method used dimethylsulfate as a methyl donor⁵⁸and

was applied to the lactam 42 to give 44 with a moderate yield

(35%). For the second method, triethyloxonium tetrafluoro-

borate and the benzazepinone 45 were solved together in

anhydrous dichloromethane to give the imidate 53, which

was not isolated.⁵⁹

Conjugation. The coupling of the heterocycles to the

polyamine chain was then carried out: the amidines 54-71

were obtained either from thiolactams 43 or 49-52 or from

iminoethers 44 or 53. Classically, thiolactams are reacted

with the polyamine moiety in the presence of a base

The second route to amidines used iminoethers to avoid

the presence of highly toxic mercury salts. Thus, in the first

attempt (Scheme 3), the iminoether 44 was isolated and

7650 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Tomasi et al.

Scheme 3^a

^aReagents: (a) protected PA R¹-H (1 equiv), TEA (4 equiv), HgCl₂, THF, Δ; (b) protected excessive PA R¹-H, Δ; (c) 3 M HCl, C₂H₅OH.

reacted with the protected putrescine 30 to give 54 that was

not isolated. Similarly (Scheme 4), the protected amine 41

was added to 53, and the desired compound 68 was obtained

in low yield (18% from the lactam 45).

The amidines 55-71 were purified by column chroma-

tography and characterized by usual techniques such as

IRFT and ¹H NMR spectroscopy.

with other polyamine conjugate systems.^16,40,43This suggests

that the heterocyclic moiety itself participates in the binding

of the conjugate to the PTS. We earlier postulated the exis-

tence of an hydrophobic pocket of set dimensions adjacent to

the PTS which serves as a docking site for the hydrophobic

cargo tethered to the polyamine chain.⁴²The fit binding of

the azepine or benzazepine moiety into this pocket could

participate in the high affinity of tetramine conjugates. In

addition, the polyamine conjugates with the bulkier benza-

zepine cargo had higher affinity for the PTS than their homo-

logues conjugated to azepine. The higher affinity of the

benzazepine conjugates may be related to their higher hydro-

phobicity, leading to a tighter anchorage to the hydrophobic

pocket.⁴²A similar trend was already observed in a series

of arenes tethered to polyamines, with the following trend

toward increasing transporter affinity: benzyl < naphtyl <

anthracenyl.²³

As observed for unsubstituted polyamines, the number of

nitrogen centers (e.g., 14 vs 12) as well as the tether (number

of CH₂spacer units) between the nitrogen centers (e.g., 13 vs

14 vs 16) had a dramatic effect on the K_ivalue of conjugated

polyamines. In the azepine-triamine series, a clear preference

for the aminobutyl spacer was evident (e.g., 16 vs 14 and 17).

The preference was also for a terminal aminobutyl moiety

(14 vs 15). The presence of hydrophilic hydroxyl groups on

the central aminobutyl chain of spermine was deleterious for

the affinity of both the free and conjugated polyamines.

Hydroxyl groups may cause steric hindrance or may hamper

hydrophobic interactions between the central methylene

chain and hydrophobic residues of the PTS. Such interac-

tions have been shown to be important to the recognition of

bacterial polyamine uptake systems.¹

The nitro group of 71 was reduced by H₂in the presence of

Pd/C to provide 72 quantitatively.

Last, the compounds 54-72 (with the exception of 68) were

deprotected using 2 M HCl in ethanol to afford the hydro-

chlorides 11-29. HBr was used for simultaneous deprotection

of the sulphonamide and Boc groups of 68,⁵¹which gave

the hydrochloride 25 after alkalinization in aqueous NaOH,

extraction, and final treatment with HCl.

All final compounds 11-29 were pure according to TLC,

HPLC, and elemental analysis criteria. They were fully analyzed

by usual techniques of IRFT, ¹H and ¹³C NMR and HRMS.

Affinity for the PTS. The intrinsic affinities of the con-

jugated and unsubstituted polyamine systems for the PTS (K_i

values) were determined in a competitive assay with radio-

labeled spermidine as previously reported.^16,41The follow-

ing trend toward increasing transporter affinity (lower K_i

value) was observed for both free or substituted polyamines:

diamines < triamines < tetramines. Although similar trends

were observed in a series of conjugated polyamines, the

conjugates with diamine or triamine chains generally dis-

played lower affinity (higher K_ivalues) than the unsubsti-

tuted homologues (e.g., 14 or 23 vs 2; 13 vs 5). In contrast, the

opposite trend was observed with the tetramine conjugates

(lower K_ivalues) (e.g., 18 or 22 vs 3; 25 vs 9). These observa-

tions are in total agreement with previous data collected

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7651

Scheme 4^a

^aReagents: (a) excessive R²-H, HgCl₂, THF, Δ; (b) R²-H (1 equiv), TEA (4 equiv), HgCl₂, THF, Δ; (c) 41 (0.9 equiv), TEA (0.9 equiv), CH₂Cl₂, 0 °C

then 40 °C; (d) 3 M HCl, C₂H₅OH; (e) 30% HBr in HOAc/PhOH/CH₂Cl₂then HCl; (f) H₂, Pd(OH)₂/C.

Transport and Selectivity. Two cellular models were cho-

sen to explore the selectivity of the conjugates toward the PTS:

(i) L1210 cells in absence or presence of DFMO, inhibition of

ornithine decarboxylase by DFMO leads to a significant increase

in polyamine uptake as a reaction to the depletion of intracellular

polyamine pools,⁶¹(ii) chinese hamster ovary (CHO) cells and

the polyamine transport deficient mutant CHO-MG cells.⁶²

These cells were challenged with the conjugates and intracellular

levels were monitored by HPLC. Polyamine conjugates that

selectively target the PTS should display an enhanced accumula-

tion in cells with greater PTS activity (e.g., CHO vs CHO-MG;

DFMO-treated L1210 vs L1210).

its accumulation in DFMO-treated cells may have several

explanations. There may be differences in the fine specificity

of the PTS in the different cellular models. In this context, it has

to be noted that while the amounts accumulated for most

analogues were similar in both cells, the accumulation of 15

was six times higher in CHO than in L1210 cells (Figure 4). It

has also been reported that free polyamines, formed as the result

of intracellular catabolism of some polyamine derivatives,

may prevent the up-regulation of the PTS activity by DFMO.

Such mechanism was proposed to explain the absence of

DFMO-enhanced accumulation of N⁴-benzylspermidine

derivatives.³²

First, the accumulation of the conjugates containing

naturally occurring polyamines was determined in L1210

and CHO cells after a 24 h exposure at various concentra-

tions (ranging from 0.1 to 100 μM). Both cell types accumu-

lated, in a concentration dependent manner, measurable

amounts of the conjugates (Figure 4A1-B1).

The accumulation of these derivatives was strongly depen-

dent on the PTS activity because their accumulation into CHO-

MG cells was significantly reduced when compared to CHO

(Figure 5). Co-treatment of L1210 cells with DFMO which

induces an upregulation of the PTS activity, enhanced the

accumulation of all conjugates but 19 and 23 (Figure 6).

However, the latter displayed a high CHO/CHO-MG accumu-

lation ratio (around 11), suggesting a high selectivity for the

PTS in CHO cells. The absence of observable enhancement of

The nature of the polyamine vector has a strong impact on

the accumulation of the conjugates. CHO and L1210 cells

accumulated higher amounts of spermidine than the spermine

or the putrescine conjugates (Figure 4). Although spermine

confers a much higher affinity of the conjugates for the PTS

(Table 1), it was not an efficient vector for their accumula-

tion into cells, a trend already reported for other spermine

conjugates.^40,43In addition, it also has to be noted that for a

given polyamine vector and despite their higher affinity for the

PTS cells accumulated lesser amounts of the benzazepine than

azepine conjugates (e.g., 14 vs 23). So the size and/or the

hydrophobicity of the cargo influence the interaction with the

PTS in two opposite ways. A bulkier and/or more hydrophobic

cargo confers more affinity for the PTS but reduces the amount

transported.

7652 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Tomasi et al.

Figure 4. Cellular uptake of naturally occurring polyamine conjugates (A) and triamine-azepine conjugates (B) in L1210 (1) and CHO (2)

cells cultured 24 h in presence of the compounds. Intracellular concentrations were determined by HPLC as described in the Experimental

Section. Results are the mean of triplicates. Bars, SD.

Altogether these observations confirm previous studies

indicating that spermidine is a better vector than putrescine

or spermine for cell delivery using the PTS.^32,40,43To deter-

mine the influence of the spermidine scaffold, we studied the

accumulation of azepine conjugates with various triamines:

homospermidine, norspermidine, N¹- or N⁸-tethered sper-

midine, and N³-aminopropyl-cadaverine. All triamine con-

jugates were actively taken up by a PTS dependent mecha-

nism in CHO and L1210 cells as suggested by their very high

CHO/CHO-MG accumulation ratio and their enhanced

accumulation in DFMO-treated L1210 cells. The terminal

aminobutyl motif appeared to be advantageous for accumu-

lation into L1210 and CHO cells. Indeed, both cells accu-

mulated higher amount of N¹-spermidine (14) and homo-

spermidine (16) conjugates than N⁸-spermidine (15) and

norspermidine (13) conjugates (Figure 4). The aminobutyl

motif seems to be the optimum size because the derivative

with a terminal aminopentyl (17) accumulated less than

the homologue with a terminal aminobutyl (14). In this

triamine series, there is a good correlation between the affinity

of the compounds for the PTS and their quantitative and

selective cell delivery. The more efficient delivery was observed

with compounds 14 and 16. Despite their slight difference in

affinity for the PTS (Table 1), they were both accumulated to

similar amounts in L1210 as well as in CHO cells. Both

compounds had an effect on cell growth as assessed using the

MTT assay (Figure 7). This effect was rather cytostatic than

cytotoxic because over 95% of the cells remains viable after a

48 h treatment with 100 μM of the compounds as determined

using a Trypan blue assay (data not shown). 16 had a stronger

impact on L1210 cells: for instance, 10 μM of 16 induced a 65%

reduction in cell growth as assessed using the MTT assay after

48 h culture (compared with 30% reduction with 14). A similar

assay performed in presence of DFMO demonstrated another

difference in the behavior of the two compounds: 16 partially

antagonized the cytostatic effect of DFMO. Because of its

lower impact on cell growth and its lack of antagonism toward

DFMO, 14 appears to be a more suitable vector for cell delivery

than 16. A time-course study shows that in presence of DFMO

the accumulation of 14 is rapid, with a plateau reached 8 h after

the beginning of exposure, time where DFMO treated cells

have accumulated around 4 times more compound 14 than the

control cells (Figure 8).

Its is of interest to identify PTS-selective structures with high

accumulation in cancer cells and devoid of cytotoxicity. Various

structures including unconjugated polyamines,^28-31,63-65

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7653

Figure 5. Cellular uptake of the conjugates in CHO and CHO-MG cells. Conjugates were added 24 h after seeding and collected 24 h later.

Intracellular levels of conjugates were determined by HPLC on perchloric extracts; mean values (SD) from three determinations. * p < 0.05,

significantly different from values determined in CHO cells. CHO/CHO-MG accumulation ratios are indicated as bold numbers over the

CHO-MG bars.

Figure 6. Effect of DFMO on intracellular accumulation of polyamine conjugates in L1210 cells. All cells were challenged with 10 µM

conjugates at the time of seeding in presence or absence of DFMO (5 mM). Cells were collected 48 h later, and intracellular levels of conjugates

were determined by HPLC as described in the Experimental Section. Values represent mean values (SD) from three determinations. * p < 0.05,

significantly different from values determined in control cells. DFMO/control accumulation ratios are indicated as bold numbers over the

DFMO bars.

conjugated polyamines such as N-benzylpolyamines,³²or poly-

amine analogues such as (Z)-1,4-diamino-2-butene³³have been

envisaged as vector of various isotopes such as boron, fluorine

or iodine for cancer therapy and imaging. But because of

their cytotoxicity, low selectivity, and/or low accumulation rate

in cells, their development was not pursued. The azepine-

spermidine conjugate 14 shares all properties required for an

effective PTS-targeting vector. In comparison of the behavior of

14 with that of N¹-benzylspermidine, a closely structurally

related compound,³²demonstrates its higher potential: its accu-

mulation in cells (e.g., CHO) is two times higher; 14 is a weak

cytostatic agent that can be used in combination of DFMO

(while N¹-benzylspermidine is cytotoxic in the 10 μM range, and

DFMO synergizes its cytotoxicity). Although we cannot predict

how isotopes carried by the azepine moiety will affect the

properties of 14, boron, iodine, and fluorine-substituted 14 are

currently being designed.

Polyamine Transport Inhibition. The importation of exo-

genous natural polyamines annihilates the growth-inhibitory

efficiency of DFMO-induced polyamine depletion in vivo.⁶⁶To

circumvent this problem, the systematic reduction of exogenous

polyamine sources using a combination of polyamine-deficient

diet with a decontamination of the gastrointestinal tract have

proved to be a viable strategy to recover the effectiveness of

DFMO but clearly lack tumor specificity.^67,68A most straight-

forward approach is the use of PTS inhibitors capable to prevent

the uptake of natural polyamines. A highly specific and selective

PTS inhibitor should be characterized by: (i) a high affinity for

7654 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Table 1. K_iValues for the Inhibition of Spermidine Transport by Free or Conjugated Polyamines in L1210 Cells

(cmpd) K_i^a(μM) {K_m/K_iratio}^b

Tomasi et al.

polyamine side chain

free polyamine

azepine conjugates

benzazepine conjugates

putrescine

(1) 208 ( 16.3 ^c{0.011}

(4) 24.1 ( 0.8 {0.098}

(5) 5.12 ( 0.21 {0.46}

(11) 763 ( 103 {0.003}

(12) 183 ( 12 {0.013}

(13) 161 ( 5.4 {0.015}

(14) 14.6 ( 0.9 {0.16}

(15) 44.7 ( 1.2 {0.053}

(16) 5.47 ( 0.19 {0.43}

(17) 45.9 ( 2.8 {0.051}

NA

(19) 35.1 ( 1.0 {0.067}

(20) 126 ( 10 {0.019}

NA^d

diaminooctane

norspermidine

spermidine

(23) 4.40 ( 0.27 {0.54}

NA

homospermidine

aminopropyl-diaminopentane

norspermine

(6) 2.30 ( 0.08 {1.03}

(7) 8.03 ( 0.50 {0.29}

(8) 1.51 ( 0.12 {1.57}

(3) 1.34 ( 0.31 {1.77}

NA

(21) 0.88 ( 0.04 {2.69}

(22) 0.15 ( 0.03 {15.8}

(26) 0.14 ( 0.01 {16.9}

(27) 0.23 ( 0.01 {10.3}

(28) 0.12 ( 0.02 {19.8}

(29) 0.19 ( 0.01 {12.5}

(25) 0.20 ( 0.01 {11.9}

(24) 2.25 ( 0.30 {1.05}

spermine

(18) 0.35 ( 0.01 {6.77}

homospermine

dihydroxy-spermine

(9) 0.73 ( 0.06 {3.25}

(10) 5.27 ( 0.41 {0.45}

NA

^aK_ivalues were calculated from the half-maximal inhibitory concentration (IC₅₀) estimated by iterative curve fitting for sigmoidal equations

describing polyamine uptake velocity in the presence of growing concentrations of antagonist. ^bThe K_mvalue for spermidine uptake (2.37 ( 0.45 μM)

was determined by Lineweaver-Burke analysis of transport velocity at increasing radiolabeled substrate concentrations. ^cData are expressed as mean

((SD) from three separate determinations. ^dNA: not available

Figure 7. Effect of the conjugates 14 and 16 on L1210 cell growth in the presence or absence of DFMO. Cells were cultured for 48 h with the

conjugates in the presence or absence of 5 mM DFMO. Cell growth rates were determined using the MTT assay. The relative cell growth rates

were calculated from the value of cell growth of the corresponding control cells cultured in the absence or in the presence of DFMO. Data are

mean of triplicates. Bars, SD.

the transporter, (ii) a very low cytotoxicity, the sole inhibition of

the PTS should not affect cell growth or viability in normal

conditions, (iii) an absence of DFMO antagonizing effect because

the major therapeutic application of PTS inhibitors is to be given

in association with this ODC inhibitor.

The substitution of the spermine chain by the analogues

norspermine (8) or homospermine (9) reduced to some extent

the affinity for the PTS (Table 1) and, in addition, both 21

and 25 were more cytotoxic on L1210 cells than 22 (data not

shown).

With its high affinity for the PTS (K_i= 0.15 μM), low uptake

(Figure4), andlimitedcytotoxicity (Figure 9), the benzazepine-

spermine conjugate 22 is an attractive lead. However, 22

antagonizes partially the cytostatic effect of DFMO (Figure 9).

Two strategies were followed for 22 optimization: (a) variations

on the spermine chain, (b) substitution on the benzazepine

moiety.

Four substitutions (-Br, -OCH₃, -NO₂, -NH₂) were

performed on the benzene ring of the benzazepine moiety to

generate new spermine derivatives. These substitutions did

not impair the affinity for the PTS (Table 1), but they greatly

affected the cytotoxicity of the compound and its behavior in

presence of DFMO (Figure 9). The bromo (26) or the

methoxy (27) derivatives were more cytotoxic than 22, and

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7655

they displayed a synergistic effect in presence of DFMO. The

amino derivative 29 had also more inhibitory effect on cell

growth and reversed very partially the effect of DFMO. In

contrast, the nitro derivative 28 up to 20 μM did not display

any effect on cell growth and did not show any synergistic or

antagonist effect in presence of DFMO. Therefore the nitro

derivative 28 is likely a potent and selective polyamine

transport inhibitor. We then investigated its ability to pre-

vent the reversion of DFMO-induced L1210 cell growth

inhibition by exogenous naturally occurring polyamines

(Figure 10).

Putrescine (1), spermidine (2), and spermine (3) are all

three able to antagonize the DFMO-induced cytostatic effect

in L1210 cells. The efficiency of 2 and 3 is much higher

because almost total reversion of the DFMO effect is

observed at 1 μM, while around 20 μM of 1 is required.

The coculture of L1210 cells with the derivative 28 at 20 μM

(a concentration that did not affect the growth of the cells)

prevented almost completely the reversion up to 100 μM of 1,

20 μM of 2, and 1 μM of 3, concentrations a lot higher than

those usually found in body fluids.^69-74

The nitrobenzazepine-spermine conjugate 28 appears to

be a potent polyamine transport inhibitor. It displays a very

low intrinsic cytotoxicity. DFMO-induced polyamine deple-

tion, which also translates into an activation of the PTS

activity, does not enhance the cytotoxicity of 28, as observed

with many polyamine analogues and derivatives.^9,10In

addition, 28 does not antagonize the cytostatic effect of

DFMO, demonstrating that 28 does not supply the cells

with their polyamine requirements (Figure 10). Such antago-

nist effect has been reported for derivatives acting as poly-

amine mimetics and for conjugates capable of releasing

polyamines after enzymatic cleavage.³²All these properties

may also be the consequence of a very low accumulation of

the compounds inside the cells. However, we could not check

Figure 8. Time course of the accumulation of 14 in L1210 cells

cultured in the presence or the absence of DFMO. Conjugate 14

(10 µM) and DFMO (5 mM) were added at the time of seeding. At

the indicated time, cells were collected and intracellular levels

of 14 were determined by HPLC as described in the Experimental

Section. Results are the mean of triplicates. Bars, SD.

Figure 9. Effect of the conjugates 22 and 26-29 on L1210 cell growth in the presence or absence of DFMO. Cells were cultured for 48 h with the

conjugates in the presence or absence of 5 mM DFMO. Cell growth rates were determined using the MTT assay. The relative cell growth rates

were calculated from the value of cell growth of the corresponding control cells cultured in the absence or in the presence of DFMO. Data are

mean of triplicates. Bars, SD.

7656 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Tomasi et al.

Figure 10. Effect of 28 on the reversion of the DFMO-induced cytostatic effect on L1210 cells by free polyamines. L1210 cells were seeded in

presence of 5 mM DFMO. Twenty-four hours later, cells were challenged with free polyamines (1, 2, or 3) in presence or absence of 28 (20 μM).

Cell growth was determined using a MTT assay 48 h later. Data are mean of triplicates. Bars, SD.

this aspect because 28 did not give rise, after derivatization

with o-phthalaldehyde, to a fluorescent derivative detectable

in our HPLC system.

A recent report has demonstrated that the combination of

DFMO with new lipophilic-spermine conjugates that are

highly potent polyamine transport inhibitors, is a valid

approach for cancer therapy in vivo.³⁸The intrinsic proper-

ties of 28 make it a novel lead for further polyamine-targeted

anticancer development.

0.22 mm). Solvent systems (expressed in volume percents)

and R_fare indicated in the text. The compounds were visua-

lized using UV light, ninhydrin, iodine, or alkaline solution of

KMnO₄. FTIR spectra were recorded on a Perkin-Elmer 16 PC

instrument (KBr pellets; ν: cm^-1). NMR spectra were recorded

on a Bruker DMX spectrometer at 500 MHz (¹H) or 125 MHz

(¹³C). TMS was used as the internal standard for NMR spectra

performed in CDCl₃. 3-(Trimethylsilyl)-1-propanesulfonic acid

(DSS) was used as the external standard for NMR spectra

1

recorded in D₂O. Attributions in H NMR of chemical shifts

were performed using selective decoupling experiments and

COSY spectra (for 17 and 23) recorded on a Bruker DMX

spectrometer at 500 MHz with a spectral window of 3004 Hz.

Broad band and gated decoupling ¹³C NMR spectra were

recorded, and the assignments were made using chemical shifts

and coupling constants (¹J and long-range coupling) and

HMQC, HMBC spectra (for 17 and 23). Values with an asterisk

(*) can be interchanged. Optical rotations were recorded with a

Perkin-Elmer 341 automatic polarimeter at 21.5 °C.

Conclusion

The structure-activity relationship studies of two families

of polyamine conjugates identified in each family a PTS-

targeting compound of interest. The azepine-spermidine

conjugate 14 is a very specific substrate of the PTS which

accumulates to high levels in cells equipped with an active

PTS. This compound could serve as a vector to accumulate

various isotopes for either cancer curing or tumor imaging.

The benzazepine series afforded a high affinity PTS inhibitor.

The nitrobenzazepine-spermine 28 prevents the reversion of

the DFMO-induced cytostatic effect by exogenous poly-

amines at physiological concentrations. This compound may

serve as an adjuvant in DFMO anticancer therapy which is

seriously impaired by the exogenous polyamines that are

imported into the cells via the PTS.

Electronic impact high resolution mass spectra (HRMS EI)

were recorded on a Varian MAT 311 double-focusing instru-

ꢀ

ment at the CRMPO (Centre Regional de Mesures Physiques de

l’Ouest, Rennes) with a source temperature of 140 °C, an ion

accelerating potential of 3 kV and ionizing electrons of 70 eV

and 300 μA. High resolution mass spectra determined by liquid

secondary ion mass spectrometry (HRMS LSIMS) were per-

formed on a ZabSpec Tof Micromass at the CRPMO with a

source temperature of 40 °C, an ion (Cs^þ) accelerating potential

of 8 kV, and mNBA (meta-nitrobenzylic alcohol) as matrix.

HPLC analyses were conducted on an Agilent 1100 series HPLC

system equipped with a 1200 series fluorimeter according to

versions of a previously described method.⁷⁵Polyamine derivatives

were determined by separation of the ion pairs formed with

n-octanesulfonic acid on a reversed-phase column (C18 column;

Experimental Section

Chemistry. Reagent-grade solvents were purchased from

chemical companies and used directly without further purifica-

tion unless otherwise specified. THF was dried under nitrogen

by distillation over sodium and benzophenone and diethylether

by distillation over LiALH₄. Dry ethanol was stored over 4 A

molecular sieves.

Merck Silica Gel 60 (70-230 mesh) was used as solid phase

for column chromatography. Thin-layer chromatographies

were performed on Merck Silica Gel 60 F254 (layer thickness:

Nucleosil 5, C18 AB, 100 mm^-5μm from Macherey-Nagel, Duren,

˚

€

Germany), reaction of the column effluent with o-phthalaldehyde

and N-acetylcysteine, and monitoring of fluorescence intensity

(excitation at 345 nm; emission at 455 nm) as detailed in the

Supporting Information.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7657

mixed and stirred in CH₃CN (18 mL) for 24 h at 45 °C. The

mixture was filtered, and the insoluble salts were washed with

CH₃CN. The filtrate was evaporated under reduced pressure.

The residue was dissolved in NaOH 1N (10 mL) and washed

with CH₂Cl₂. The organic layer was washed with brine, dried

over potassium carbonate, evaporated under reduced pressure,

and then chromatographed using CH₂Cl₂/CH₃OH/NH₄OH: 80/

20/0.5. The pure aminonitrile (0.370 g, 1.45 mmol, 1 equiv) was

dissolved in THF and reacted with di-tert-butyldicarbonate (1.5

equiv) at room temperature and stirred overnight. The diprotected

aminonitrile was purified by column chromatography with Et₂O.

This latter (0.460 g, 1.29 mmol) was dissolved in an ethanolic

solution of NH₃, and 1 g of Raney Nickel was added. The mixture

was stirred under hydrogen (5 bar) at 25 °C for 72 h. The Raney

nickel was removed by filtration, and the filtrate was evaporated

under reduced pressure. The residue was chromatographed using

CH₃OH/NH₄OH 95/5 and TLC was visualized using KMnO₄.

Oil, 44%; R_f0.49 (CH₃OH/NH₄OH 95/5).

Figure 11. Numbering assignment for heterocycle ring and poly-

amine chain.

Purities of compounds were >95% as determined by ele-

mental analyses performed by the Laboratoire de Microana-

ꢀ

lyses (Faculte de Pharmacie, Universite Paris XI, Chatenay-

Malabry). Purity of the derivatives was also determined using

analytical high voltage paper electrophoresis as >95%.⁷⁶

The compounds were numbered for the heterocyclic moiety

using IUPAC rules and for the polyamine moiety using letters

a-l (Figure 11).

Synthesis. Previously reported procedures were used for the

synthesis of compounds 30-35, 39, 40.

General Procedure A: Thionation. A solution of lactam and

Lawesson’s reagent (0.5-1 equiv) in dry dioxane or dry toluol

was refluxed for 6 h. After evaporation of the solvent under

reduced pressure, the residue was purified by column chroma-

tography.

General Procedure B: Coupling of Thiolactams to Protected

Polyamines. To a refluxing solution of thiolactam, suitably

protected polyamine (1 equiv) and triethylamine (4 equiv) in

dry THF (10-50 mL) was added mercury(II) chloride (HgCl₂,

1 equiv). A black precipitate of mercury sulfide HgS was

observed, and refluxing was continued for 1 h under stirring.

The THF was evaporated under reduced pressure, and the residue

was suspended in methanol. The mercury sulfide was removed by

filtration and washed with methanol. After evaporation of the

solvent under reduced pressure, the residue was dissolved in

CH₂Cl₂and then the organic layer was washed with a 0.2 M

aqueous solution of sodium thiosulfate Na₂S₂O₃. After drying over

K₂CO₃, the organic layer was evaporated under reduced pressure.

The residue was purified by column chromatography.

Data for N¹-tert-Butoxycarbonylpentane-1,5-diamine 38:⁴⁵

Oil, 75%; R_f0.35 (CH₃OH/NH₄OH 95/5).

N⁴,N⁹-Di-tert-butoxycarbonyl-4-azanonane-1,9-diamine 39.

A methanolic solution of acrylonitrile (0.46 g, 2.27 mmol,

1 equiv) was added dropwise for 1 h at 0 °C to a methanolic

solution of 39 (1 equiv). The mixture was stirred overnight. After

removal of MeOH, the residue was purified by column chro-

matography using CH₂Cl₂/CH₃OH/NH₄OH: 90/10/0.5. The

pure aminonitrile was dissolved in CH₂Cl₂and reacted with

di-tert-butyldicarbonate (1.5 equiv) at 0 °C over 3 h and then

stirred at room temperature overnight. The residue was chro-

matographed using Et₂O. The compound was dissolved in an

ethanolic solution of NH₃, and a spoon of Raney Nickel was

added. The mixture was put for 72 h under hydrogen (6 bar) at

25 °C. The residue was filtered and the precipitate was washed

with EtOH and the filtrate was evaporated under reduced

pressure. The residue was chromatographed using CH₃OH/

NH₄OH 95/5 and TLC was visualized using KMnO₄. Oil,

75%; R_f0.50 (CH₃OH/NH₄OH 95/5).

General Procedure C: Coupling Thiolactams to Free Polya-

mines. To a refluxing solution of thiolactam and free polyamine

(10-25 equiv) in 2 mL of dry THF was added mercury(II)

chloride HgCl₂(1 equiv). The mixture was refluxed for 1 h,

during which a black precipitate of HgS was observed. The THF

was removed under reduced pressure, and the residue was

suspended in CH₂Cl₂. The mercury sulfide (HgS) was removed

by filtration and then the organic layer was washed with a 0.2 M

aqueous Na₂S₂O₃. After drying over K₂CO₃, the organic layer

was evaporated under reduced pressure. The residue was pur-

ified by column chromatography.

Data for N¹-tert-Butoxycarbonyl-N⁵,N¹⁰-bis(mesitylenesul-

fonyl)homospermine 41. To a solution of N⁵,N¹⁰-bis(mesitylene-

sulfonyl)homospermine 40⁵¹(5.68 g, 9.56 mmol, 3 equiv) in

CH₂Cl₂was added dropwise at 0 °C a solution of di-tert-butyl

dicarbonate (1 equiv) in CH₂Cl₂. After stirring at 0 °C over 3 h

then at room temperature overnight, the solvent was evaporated

under reduced pressure. Column chromatography using CH₂Cl₂/

MeOH/NH₄OH: 70/10/1. Oil, 57%; R_f0.51 (CH₂Cl₂/MeOH/

NH₄OH 70/10/1).

Data for 1,3,4,5,6,7-Hexahydro-1-azepin-2-thione 43. Gen-

eral procedure A from 42 (2 g, 17.6 mmol) and Lawesson’s

reagent (0.5 equiv) in dry toluene (50 mL). Column chromatogra-

phy using CH₂Cl₂/Et₂O 95/5. White solid, 76%; R_f0.49 (CH₂Cl₂/

Et₂O 95/5).

General Procedure D: Removal of Boc Groups and Preparation

of Hydrochlorides. The amidine conjugate was stirred in a 2 M

solution of HCl gas in ethanol (1.2 equiv per amino group).

After evaporation of the ethanol, the residue was triturated in

anhydrous ether to give a white hygroscopic solid.

2-Methoxy-3,4,5,6-tetrahydro-2H-azepin-2-one 44. To

a

warmed solution of ε-caprolactam 42 (11.32 g, 0.1 mol, 1 equiv)

in toluol (35 mL) was added dropwise the dimethylsulfate

(1 equiv) for 45 min. The reaction mixture was stirred for 16 h

and then cooled at room temperature. A 50% aqueous solution

of K₂CO₃was added. When the CO₂release was finished,

stirring was continued for 90 min. The potassium methylsulfate

precipitate was eliminated by filtration and washed by diethy-

lether (3 ꢀ 10 mL). After decantation, the organic phase was

dried over K₂CO₃and concentrated under reduced pressure.

The product was purified by distillation. Liquid, 35%; R_f0.63

(CH₂Cl₂); Bp₁₀= 80 °C.

7-Bromo-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one 46. To a

solution of 45 (0.2 g, 1.24 mmol) in a mixture of AcOH (3 mL)

and water (1 mL) was added dropwise bromine (0.2 g, 1.30

mmol) dissolved in AcOH (1 mL) for 0.5 h. The solution was

stirred for 0.5 h and then cooled to 4 °C and water (20 mL) was

added to have a precipitate that was filtered off. Cream solid,

75%; R_f0.38 (CH₂Cl₂/EtOAc 80/20).

Data for (6R, 7S)-N¹,N⁴-Di-tert-butoxycarbonyl-(6,7-O-iso-

propylidene)-6,7-dihydroxyspermine 35.⁵⁰Oil, 21%; R_f0.44

(CH₃OH/NH₄OH 95/5).

N¹,N⁴-Di-tert-butoxycarbonylnorspermidine 36. A methano-

lic solution of norspermidine 5 (5 g, 38.1 mmol, 1 equiv) was

cooled to -78 °C, and ethyltrifluoroacetate (1 equiv) was added.

The temperature was maintained at -78 °C for 1 h and then

allowed to reach 0 °C for 1 h. A methanolic solution of di-tert-

butyldicarbonate (3 equiv) was then added at room tempera-

ture. The mixture was stirred for 1 h, made alkaline with K₂CO₃

(5.2 equiv), and left overnight. After filtration of insoluble salts,

the filtrate was evaporated and chromatographed using

CH₃OH/NH₄OH 95/5. Colorless oil, 39%; R_f0.43 (CH₃OH/

NH₄OH 95/5).

N¹,N⁵-Di-tert-butoxycarbonylhomospermidine 37. N¹-tert-

Butoxycarbonylputrescine 30 (0.500 g, 2.7 mmol, 1 equiv),

4-bromobutyronitrile (1 equiv), and KF on Celite (6 equiv) were

7658 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Tomasi et al.

Data for 7-Methoxy-1,3,4,5-tetrahydro-2H-1-benzazepin-2-

one 47.⁵³White solid, 15%; R_f0.70 (acetone).

60-70 °C at 15-20 mmHg for 5 h. To remove the excess of imidate,

the mixture was warmed at 60 °C under reduced pressure (10^-2

mmHg). Column chromatography using CH₃OH/NH₄OH 90/10

(n). Oil, 85%; 0.35 (CH₃OH/NH₄OH 90/10).

7-Nitro-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one 48. To a

mixture of concentrated sulfuric acid (6.75 mL) and fuming

nitric acid (5.4 mL) was added 45 (0.9 g, 5.58 mmol). The

suspension was heated to 90 °C for 5 min. Then the mixture

was cooled to 0 °C and the precipitate was filtered off and

crystallized in a mixture of diethyl ether-hexane (80/20). Yel-

low solid, 77%; R_f0.36 (CH₂Cl₂/Et₂O 80/20).

Data for 1,3,4,5-Tetrahydro-2H-1-benzazepin-2-thione 49. Gen-

eral procedure A from 45 (2 g, 12.4 mmol) and Lawesson’s reagent

(1 equiv) in dry toluene (50 mL). Column chromatography using

CH₂Cl₂. Yellow oil, 80%; R_f0.60 (CH₂Cl₂).

7-Bromo-1,3,4,5-tetrahydro-2H-1-benzazepin-2-thione 50. Gen-

eral procedure A from 46 (0.3 g, 1.25 mmol) and Lawesson’s

reagent (0.7 equiv) in dry toluene (20 mL). Column chromatogra-

phy using CH₂Cl₂. White solid, 60%; R_f0.19 (CH₂Cl₂/Et₂O 80/20).

7-Methoxy-1,3,4,5-tetrahydro-2H-1-benzazepin-2-thione 51.

General procedure A from 47 (0.35 g, 1.84 mmol) and

Lawesson’s reagent (0.5 equiv) in dry toluene (20 mL) without

further purification. White solid, 50%; R_f= 0.70 (CH₂Cl₂).

7-Nitro-1,3,4,5-tetrahydro-2H-1-benzazepin-2-thione 52. Gen-

eral procedure A from 48 (0.47 g, 2.28 mmol) and Lawesson’s

reagent (0.5 equiv) in dry toluene (25 mL). Column chromatogra-

phy using Et₂O/pentane 80/20. White solid, 70%; R_f0.71 (Et₂O/

pentane 80/20).

Data for N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)putrescine

62. General procedure C from 49 (0.500 g, 2.82 mmol, 1 equiv)

and putrescine 1. Column chromatography using CH₃OH/

NH₄OH: 90/10. Oil, 40%; R_f0.20 (CH₃OH/NH₄OH 90/10).

Data for N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)octane-1,8-

diamine 63. General procedure C from 49 (0.300 g, 1.69 mmol,

1 equiv) and 1,8-diaminooctane 4. Column chromatography

using CH₃OH/NH₄OH: 90/10. Oil, 74%; R_f0.38 (CH₃OH/

NH₄OH 90/10).

Data for N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)norspermine 64.

General procedure B from 49 (0.47 g, 2.65 mmol, 1 equiv) and

norspermine 8. Column chromatography using MeOH/NH₄OH:

50/50. Oil, 32%; R_f0.41 (MeOH/NH₄OH 50/50).

Data for N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)spermine 65.

General procedure B from 49 (0.24 g, 1.36 mmol, 1 equiv) and sper-

mine 3. Column chromatography using isopropylamine/CH₃OH/

CHCl₃: 2/4/4. Oil, 60%; R_f0.11 (isopropylamine/CH₃OH/CHCl₃2/

4/4).

Data for N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)-N⁴,N⁸-di-

tert-butoxycarbonyl spermidine 66. General procedure B from

49 (0.150 g, 0.85 mmol, 1 equiv) and N⁴,N⁸-di-tert-butoxycar-

bonylspermidine 33. Column chromatography using CH₂Cl₂/

CH₃OH/NH₄OH: 90/10/0.5. Oil, 80%; R_f0.36 (CH₂Cl₂/

CH₃OH/NH₄OH 90/10/0.5).

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁴-tert-butoxycarbonylputres-

cine 54. To a solution of N-mono-tert-butoxycarbonylputrescine 30

(0.36 g, 1.91 mmol, 1 equiv) in CH₂Cl₂was added an excess of

imidate 44 (5 equiv). The mixture was allowed to warm at 60-70 °C

at 15-20 mmHg for 5 h. To remove the excess of imidate, the

mixture was warmed at 60 °C under reduced pressure (10^-2mmHg).

Oil, 91%, R_f0.65 (isopropylamine/CH₃OH/CHCl₃2/4/4).

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁸-tert-butoxycarbonyloc-

tane-1,8-diamine 55. General procedure B from 43 (0.275 g,

2.13 mmol, 1 equiv) and 31 (1 equiv). Column chromatography

using isopropylamine/CH₃OH/CHCl₃: 1/4/4. Oil, 72%; R_f

0.35 (isopropylamine/CH₃OH/CHCl₃1/4/4), 0.18 (CH₃OH/

NH₄OH 90/10).

(6R,7S)-N¹²-(4,5-Dihydro-3H-1-benzazepin-2-yl)-N¹,N⁴-di-

tert-butoxycarbonyl-(6,7-O-isopropylidene)-6,7-dihydroxysper-

mine 67. General procedure B from 49 (0.090 g, 0.51 mmol,

1 equiv) and (6R,7S)-N¹,N⁴-di-tert-butoxycarbonyl-(6,7-O-

isopropylidene)-6,7-dihydroxyspermine 35. Column chroma-

tography using CH₂Cl₂/CH₃OH/NH₄OH: 90/10/0.7. Oil,

71%; R_f0.48 (CH₂Cl₂/CH₃OH/NH₄OH 90/10/0.7).

Data for N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)-N¹⁵-tert-

butoxycarbonyl-N⁵,N¹⁰-bis(mesitylenesulfonyl)homospermine 68.

To a 1 M solution of triethyloxonium tetrafluoroborate⁶⁰(1.42

mL, 1.5 equiv) in anhydrous CH₂Cl₂was added dropwise at 0 °C

under nitrogen atmosphere a solution of 45 (0.163 g, 0.943 mmol,

1 equiv) in anhydrous CH₂Cl₂. After stirring overnight at room

temperature, a solution in anhydrous CH₂Cl₂of 41 (0.555 g, 0.9

equiv) and of Et₃N (0.085 g, 0.118 mL, 0.9 equiv) was added

dropwise at 0 °C. The mixture was stirred at room temperature

during 4 h and warmed at 40 °C during 15 mn. After addition of

5 mL of water, an extraction with CH₂Cl₂(2 ꢀ 5 mL) was

performed. The organic layers were mixed, washed with KOH

20% (5 mL), and then with H₂O/NaCl, dried over K₂CO₃,

filtered, and concentrated under reduced pressure. Column chro-

matography using CH₃OH/NH₄OH: 99/1. Oil, 18%; R_f0.39

(CH₃OH/NH₄OH 99/1).

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁴,N⁷-di-tert-butoxycarbonyl-

norspermidine 56. General procedure B from 43 (0.201 g, 1.55 mmol,

1 equiv) and 36 (1 equiv). Column chromatography using isopro-

pylamine/CH₃OH/CHCl₃: 1/4/4. Oil, 74%; R_f0.37 (isopropylamine/

CH₃OH/CHCl₃1/4/4), 0.11 (CH₃OH/NH₄OH 90/10).

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁴,N⁸-di-tert-butoxycarbonyl-

spermidine 57. General procedure B from 43 (0.200 g, 1.55 mmol,

1 equiv) and 33 (1 equiv). Column chromatography using isopro-

pylamine/CH₃OH/CHCl₃: 0.5/4/4. Oil, 81%; R_f0.35 (CH₃OH/

NH₄OH 90/10).

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁵,N⁸-di-tert-butoxycarbonyl-

spermidine 58. General procedure B from 43 (0.135 g, 1.04 mmol,

1 equiv) and 34 (1 equiv). Column chromatography using isopro-

pylamine/CH₃OH/CHCl₃: 1/4/4. Oil, 72%; R_f0.38 (isopropylamine/

CH₃OH/CHCl₃1/4/4).

N¹-(7-Bromo-4,5-dihydro-3H-1-benzazepin-2-yl)-N⁴,N⁹,N¹²-tri-

tert-butoxycarbonyl-spermine 69. General procedure B from 50

(0.256 g, 0.59 mmol, 1 equiv) and N⁴,N⁹,N¹²-tri-tert-butoxycarbo-

nyl-spermine 32. Column chromatography using CH₂Cl₂/CH₃OH:

95/5.

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁵,N⁹-di-tert-butoxycarbonyl-

homospermidine 59. General procedure B from 43 (0.169 g, 1.31

mmol, 1 equiv) and 37 (1 equiv). Column chromatography using

isopropylamine/CH₃OH/CHCl₃: 1/4/4. Oil, 65%; R_f0.44 (iso-

propylamine/CH₃OH/CHCl₃1/4/4), 0.13 (CH₃OH/NH₄OH

90/10).

Oil, 68%; R_f0.25 (CH₂Cl₂/CH₃OH 95/5).

N¹-(7-Methoxy-4,5-dihydro-3H-1-benzazepin-2-yl)-N⁴,N⁹,N¹²-

tri-tert-butoxycarbonyl-spermine 70. General procedure B from 51

(0.100 g, 0.48 mmol, 1 equiv) and N⁴,N⁹,N¹²-tri-tert-butoxycarbonyl-

spermine 32. Column chromatography using CH₃OH/NH₄OH: 99/

1. Oil, 49%; R_f0.31 (CH₃OH/NH₄OH 99/1).

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁴,N⁹-di-tert-butoxycarbonyl-

4-azanonane-1,9-diamine 60. General procedure B from 43 (0.176 g,

1.36 mmol, 1 equiv) and 39 (1 equiv). Column chromatography

using isopropylamine/CH₃OH/CHCl₃: 1/4/4. Oil, 77%; R_f0.46

(isopropylamine/CH₃OH/CHCl₃1/4/4), 0.13 (isopropylamine/

CH₃OH/CHCl₃0.5/4/4).

N¹-(7-Nitro-4,5-dihydro-3H-1-benzazepin-2-yl)-N⁴,N⁹,N¹²-

tri-tert-butoxycarbonyl-spermine 71. General procedure B from

52 (0.200 g, 0.90 mmol, 1 equiv) and N⁴,N⁹,N¹²-tri-tert-butox-

ycarbonyl-spermine 32. Column chromatography using CH₂Cl₂/

CH₃OH: 95/5. Oil, 49%; R_f0.32 (CH₂Cl₂/CH₃OH 95/5).

N¹-(7-Amino-4,5-dihydro-3H-1-benzazepin-2-yl)- N⁴,N⁹,N¹²-tri-

tert-butoxycarbonyl-permine 72. Pearlman’s catalyst (0.8 equiv)

was added to 71 (0.200 g, 0.34 mmol, 1 equiv) dissolved in EtOH.

N¹-(4,5-Dihydro-3H-azepin-2-yl)-N⁴,N⁹N¹²-tri-tert-butoxycar-

bonylspermine 61. To a solution of N¹,N⁴,N⁹-tri-tert-butoxycarbo-

nylspermine 32 (0.500 g, 0.99 mmol, 1 equiv) in DMF was added an

excess of imidate 44 (8 equiv). The mixture was allowed to warm at

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7659

The mixture was stirred for 60 h under an atmosphere of H₂at room

temperature. The mixture was then filtered, washed with EtOH, and

concentrated under reduced pressure. Oil, 100%; R_f0.50 (CH₃OH/

NH₄OH 95/5).

24.15, 24.17 (C-b, C-g), 27.72 (C-6), 29.22 (C-5), 32.32 (C-3), 39.05

(C-h), 41.38 (C-a), 44.22 (C-7), 47.12, 47.30 (C-d, C-e) 169.79 (C-2).

HRMS (LSIMS) (m/z) calcd for C₁₄H₃₁N₄(M þ H)^þ255.2549;

found 255.2547. Anal. (C₁₄H₃₀N₄3HCl 2H₂O) C, H, N.

3

N¹-(4,5-Dihydro-3H-azepin-2-yl)putrescine Hydrochloride.

11 was prepared from 54 using 0.9 M HClg in AcOEt. A

recrystallization was performed with an isopropyl alcohol/

EtOH 50/50 mixture. White solid, 37%. FTIR 2400 to 3600 þ

2057 (NH^þ), 1657 (CdN). ¹H NMR (500 MHz, D₂O) δ 1.66 (m,

2H, H-6), 1.74-1.81 (m, 8H, H-b, H-c, H-4, H-5), 2.67 (m, 2H,

H-3), 3.04 (t, 2H, J = 6.7 Hz, H-d), 3.26 (t, 2H, J = 6.2 Hz, H-a),

3.47 (t, 2H, J = 5 Hz, H-7). ¹³C NMR (125 MHz, D₂O) δ 23.57

(C-4), 24.26* (C-b), 24.56* (C-c), 27.90 (C-6), 29.40 (C-5), 32.43

(C-3), 39.40 (C-d), 41.59 (C-a), 44.58 (C-7), 169.93 (C-2). HRMS

(LSIMS) (m/z) calcd for C₁₀H₂₂N₃(M þ H)^þ183.1735; found

183.1743. Anal. (C₁₀H₂₁N₃.2HCl.0.25H₂O) C, H, N.

N¹-(4,5-Dihydro-3H-azepin-2-yl)-azanonane-1,9-diamine

Hydrochloride 17. General procedure D from 60, white solid,

91%. FTIR 2200 to 3650 þ 2034 (NH^þ), 1646 (CdN). ¹H NMR

(500 MHz, D₂O) δ 1.49 (m, 2H, H-f), 1.68-1.81 (m, 10H, H-e,

H-g, H-4, H-5, H-6), 2.08 (m, 2H, H-b), 2.70 (m, 2H, H-3), 3.04

(t, 2H, J = 7.5 Hz, H-h), 3.11 (t, 2H, J = 8 Hz, H-d), 3.16 (t, 2H,

J = 8 Hz, H-c), 3.36 (t, 2H, J = 7 Hz, H-a), 3.52 (m, 2H, H-7).

¹³C NMR (125 MHz, D₂O) δ 23.16 (C-f), 23.48 (C-4), 24.19

(C-b), 25.49 (C-e), 26.64 (C-g), 27.77 (C-6), 29.37 (C-5), 32.53

(C-3), 39.30 (C-a), 39.55 (C-h), 44.48 (C-7), 45.26 (C-c), 47.85

(C-d) 170.22 (C-2). HRMS (LSIMS) (m/z) calcd for C₁₄H₃₁N₄

(M þ H)^þ255.2549; found 255.2551. Anal. (C₁₄H₃₀N₄3HCl

3

N¹-(4,5-Dihydro-3H-azepin-2-yl)octane-1,8-diamine Hydro-

chloride 12. General procedure D from 55, white solid, 79%.

R_f0.14 (CH₃OH/NH₄OH 50/50). FTIR 2350 to 3650 þ 2020

(NH^þ), 1654 (CdN). ¹H NMR (500 MHz, D₂O) δ 1.37 (m, 8H,

H-c, H-d, H-e, H-f), 1.63-1.70 (m, 6H, H-b, H-g, H-6), 1.73 (m,

2H, H-4), 1.80 (m, 2H, H-5), 2.68 (m, 2H, H-3), 3.02 (t, 2H, J =

0.1H₂O) C, H, N.

N¹-(4,5-Dihydro-3H-azepin-2-yl)spermine Hydrochloride 18.

18 was prepared from 61 using 0.9 M HClg in AcOEt. White

solid, 26%. FTIR 2500 to 3600 þ 2058 (NH^þ), 1658 (CdN). ¹H

NMR (500 MHz, D₂O) δ 1.67 (m, 2H, H-6), 1.73 (m, 2H, H-4),

1.80-1.82 (m, 6H, H-e, H-f, H-5), 2.05-2.13 (m, 4H, H-b, H-i),

2.69 (m, 2H, H-3), 3.11-3.20 (m, 10H, H-c, H-d, H-g, H-h, H-j),

3.34 (t, 2H, J = 7 Hz, H-a), 3.51 (t, 2H, H-7, J = 5 Hz, H-7). ¹³C

NMR (125 MHz, D₂O) δ 23.16 (C-e, C-f), 23.46 (C-4), 24.13*

(C-b), 24.17* (C-i), 27.76 (C-6), 29.36 (C-5), 32.52 (C-3), 36.95

(C-j), 39.26 (C-a), 44.48 (C-7), 44.94* (C-c), 45.34* (C-h), 47.38*

(C-d), 47.40* (C-g), 170.23 (C-2). HRMS (LSIMS) (m/z) calcd

for C₁₆H₃₆N₅(M þ H)^þ297.2892; found 297.2826. Anal.

(C₁₆H₃₅N₅4HCl 2.5H₂O) N, C; H calcd 9.08, found 8.47.

7.5 Hz, H-h), 3.22 (t, 2H, J = 7 Hz, H-a), 3.49 (m, 2H, H-7). ¹³

C

NMR (125 MHz, D₂O) δ 23.65 (C-4), 25.88, 26.33, 27.00, 27.06,

28.42 (C-b, C-c, C-d, C-e, C-f, C-g), 28.02 (C-6), 29.40 (C-5),

32.42 (C-3), 39.92 (C-h), 42.17 (C-a), 44.27 (C-7), 169.70 (C-2).

HRMS (LSIMS) (m/z) calcd for C₁₄H₃₀N₃(M þ H)^þ240.2440;

found 240.2440. Anal. (C₁₄H₂₉N₃2HCl 0.4H₂O) C, H, N.

3

N¹-(4,5-Dihydro-3H-azepin-2-yl)norspermidine Hydrochlor-

ide 13. General procedure D from 56, white solid, 93%. FTIR

2310 to 3400 þ 2001 (NH^þ), 1654 (CdN). ¹H NMR (500 MHz,

D₂O) δ 1.67 (m, 2H, H-6), 1.74 (m, 2H, H-4), 1.80 (m, 2H, H-5),

2.05-2.16 (m, 4H, H-b, H-e), 2.70 (m, 2H, H-3), 3.14 (t, 2H, J =

7.8 Hz, H-f), 3.17-3.22 (m, 4H, H-c, H-d), 3.35 (t, 2H, J = 7 Hz,

H-a), 3.51 (m, 2H, H-7). ¹³C NMR (125 MHz, D₂O) δ 23.45

(C-4), 24.15 (C-b, C-e), 27.76 (C-6), 29.34 (C-5), 32.51 (C-3),

36.92 (C-f), 39.21 (C-a), 44.47 (C-7), 45.06* (C-c), 45.47* (C-d),

170.23 (C-2). HRMS (EI) (m/z) calcd for C₁₂H₂₆N₄M^þ226.2158;

found 226.2148. Anal. (C₁₂H₂₆N₄3HCl 0.1H₂O) C, H, N.

3

N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)putrescine Hydro-

chloride 19. General procedure D from 62, white solid, 89%;

R_f0.20 (CH₃OH/NH₄OH 90/10). FTIR 2400 to 3600 þ 2049

1

(NH^þ), 1668 (CdN). H NMR (500 MHz, D₂O) δ 1.76-1.93

(m, 4H, H-b, H-c), 2.31 (m, 2H, H-4), 2.51 (t, 2H, J = 7.2 Hz,

H-3), 2.79 (t, 2H, J = 7.3 Hz, H-5), 3.04 (t, 2H, J = 7.0 Hz, H-d),

3.54 (t, 2H, J = 6.5 Hz, H-a), 7.23 (d, 1H, J = 7.6 Hz, H-9),

7.35-7.43 (m, 3H, H-6, H-7, H-8). HRMS (LSIMS) (m/z) calcd

for C₁₄H₂₂N₃(M þ H)^þ232.1814; found 232.1810. Anal.

(C₁₄H₂₁N₃2HCl 0.5H₂O) C, H, N.

3

N¹-(4,5-Dihydro-3H-azepin-2-yl)spermidine Hydrochloride

14. General procedure D from 57, white solid, 81%. FTIR

2300 to 3600 þ 2077 (NH^þ), 1654 (CdN). ¹H NMR (500 MHz,

D₂O) δ 1.68 (m, 2H, H-6), 1.74 (m, 2H, H-4), 1.80-1.82 (m, 6H,

H-e, H-f, H-5), 2.08 (m, 2H, H-b), 2.70 (m, 2H, H-3), 3.07 (t, 2H,

J = 7 Hz, H-g), 3.13-3.18 (m, 4H, H-c, H-d), 3.35 (t, 2H, J = 7

Hz, H-a), 3.51 (m, 2H, H-7). ¹³C NMR (125 MHz, D₂O) δ 23.16

(C-e), 23.46 (C-4), 24.16* (C-b), 24.30* (C-f), 27.76 (C-6), 29.36

(C-5), 32.51 (C-3), 39.19* (C-a), 39.25* (C-g), 44.48 (C-7), 45.32

(C-c), 47.44 (C-d) 170.23 (C-2). HRMS (LSIMS) (m/z) calcd for

3

N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)octane-1,8-diamine

Hydrochloride 20. General procedure D from 63, white solid,

87%; R_f0.38 (CH₃OH/NH₄OH 90/10). FTIR 2400 to 3400 þ

2054 (NH^þ), 1663 (CdN). ¹H NMR (500 MHz, D₂O) δ

1.40-1.48 (m, 8H, H-c, H-d, H-e, H-f), 1.68* (m, 2H, H-b),

1.77* (m, 2H, H-g), 2.30 (m, 2H, H-4), 2.48 (t, 2H, J = 7 Hz,

H-3), 2.77 (t, 2H, J = 7.2 Hz, H-5), 3.01 (t, 2H, J = 7.5 Hz, H-h),

3.49 (t, 2H, J = 7 Hz, H-a), 7.24 (d, 1H, J = 7.6 Hz, H-9),

7.35-7.43 (m, 3H, H-6, H-7, H-8). ¹³C NMR (125 MHz, D₂O) δ

25.90, 26.28, 27.07, 27.12, 28.45 (C-b, C-c, C-d, C-e, C-f, C-g),

28.87 (C-5), 29.14 (C-3), 29.73 (C-4), 39.90 (C-h), 42.89 (C-a),

123.87 (C-9), 128.26 (C-8), 128.31 (C-7), 130.26 (C-6), 135.19*

(C-5a), 135.49* (C-9a), 166.40 (C-2). HRMS (LSIMS) (m/z)

calcd for C₁₈H₃₀N₃(M þ H)^þ288.2440; found 288.2443. Anal.

C₁₃H₂₉N₄(M

þ

H)^þ241.2392; found 241.2395. Anal.

(C₁₃H₂₈N₄3HCl 0.45H₂O) C, H, N.

3

N⁸-(4,5-Dihydro-3H-azepin-2-yl)spermidine Hydrochloride

15. General procedure D from 58, white solid, 83%. FTIR

2312 to 3600 þ 2043 (NH^þ), 1654 (CdN). ¹H NMR (500 MHz,

D₂O) δ 1.67 (m, 2H, H-6), 1.73-1.81 (m, 8H, H-b, H-c, H-4,

H-5), 2.12 (m, 2H, H-f), 2.68 (m, 2H, H-3), 3.13 (t, 4H, J = 7.8

Hz, H-d, H-e), 3.18 (t, 2H, J = 8 Hz, H-g), 3.27 (t, 2H, J = 6.7

Hz, H-a), 3.50 (m, 2H, H-7). ¹³C NMR (125 MHz, D₂O) δ 23.40

(C-4), 23.53, 24.13, 24.26 (C-b, C-c, C-f), 27.86 (C-6), 29.36

(C-5), 32.46 (C-3), 36.94 (C-g), 41.50 (C-a), 44.36 (C-7), 44.90

(C-e), 47.61 (C-d) 169.94 (C-2). HRMS (LSIMS) (m/z) calcd for

(C₁₈H₂₉N₃2HCl 0.1H₂O) C, H, N.

3

N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)norspermine Hydro-

chloride 21. General procedure C from 64, white solid, 75%;

R_f0.13 (isopropylamine/CH₃OH/CHCl₃2/4/4), 0.46 (CH₃OH/

NH₄OH: 50/50). FTIR 2450 to 3300 þ 2066 (NH^þ), 1657

1

(CdN). H NMR (500 MHz, D₂O) δ 2.10-2.23 (m, 6H, H-b,

H-e, H-h), 2.33 (m, 2H, H-4), 2.53 (t, 2H, J = 7 Hz, H-3), 2.80 (t,

2H, J = 7.3 Hz, H-5), 3.12-3.28 (m, 10H, H-c, H-d, H-f, H-g,

H-i), 3.63 (t, 2H, J = 7 Hz, H-a), 7.26 (d, 1H, J = 8 Hz, H-9),

7.37-7.45 (m, 3H, H-6, H-7, H-8). ¹³C NMR (125 MHz, D₂O) δ

23.11 (C-e), 24.13* (C-b), 24.32* (C-h), 28.80 (C-5), 29.27 (C-3),

29.80 (C-4), 36.93 (C-i), 39.98 (C-a), 45.01, 45.11, and 45.52

(C-c, C-d, C-f, C-g), 123.96 (C-9), 128.27 (C-8), 128.54 (C-7),

130.29 (C-6), 135.28 (C-5a, C-9a), 167.06 (C-2). HRMS (LSIMS)

(m/z) calcd for C₁₉H₃₄N₅(M þ H)^þ332.2814; found 332.2812.

Anal. (C₁₉H₃₃N₅4HCl 2H₂O) C, H, N.

C₁₃H₂₉N₄(M

þ

H)^þ241.2392; found 241.2395. Anal.

(C₁₃H₂₈N₄3HCl 0.6H₂O) C, H, N.

3

N¹-(4,5-Dihydro-3H-azepin-2-yl)homospermidine Hydrochloride

16. General procedure D from 59, white solid, 98%. FTIR 2336

to 3610 þ 2045 (NH^þ), 1662 (CdN). ¹H NMR (500 MHz, D₂O) δ

1.66 (m, 2H, H-6), 1.71-1.79 (m, 12H, H-b, H-c, H-f, H-g, H-4,

H-5), 2.67 (m, 2H, H-3), 3.05 (t, 2H, J = 7.2 Hz, H-h), 3.08-3.12

(m, 4H, H-d, H-e), 3.25 (t, 2H, J = 6.5 Hz, H-a), 3.49 (m, 2H, H-7).

¹³C NMR (125 MHz, D₂O) δ 22.99, 23.26 (C-c, C-f), 23.39 (C-4),

3

7660 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21

Tomasi et al.

N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)spermine Hydrochloride

22. General procedure D from 65, white solid, 73%; R_f0.16 (isopro-

pylamine/CH₃OH/CHCl₃2/4/4), 0.43 (CH₃OH/NH₄OH 50/50).

FTIR 2500 to 3350 þ 2054 (NH^þ), 1657 (CdN). ¹H NMR (500

MHz, D₂O) δ 1.80-1.83 (m, 4H, H-e, H-f), 2.10 (m, 2H, H-i), 2.19

(m, 2H, H-b), 2.32(m, 2H, H-4), 2.52 (t, 2H, J =7 Hz, H-3), 2.79(t,

2H, J = 7.3 Hz, H-5), 3.10-3.19 (m, 8H, H-d, H-g, H-h, H-j), 3.24

(t, 2H, J = 8.1 Hz, H-c), 3.61 (t, 2H, J = 7 Hz, H-a), 7.24 (d, 1H,

J= 7.5 Hz, H-9), 7.36-7.44 (m, 3H, H-6, H-7, H-8). ¹³CNMR(125

MHz, D₂O) δ 23.24* (C-e), 23.27* (C-f), 24.19* (C-b), 24.40* (C-i),

28.87 (C-5), 29.35 (C-3), 29.87 (C-4), 37.05 (C-j), 40.11 (C-a), 45.01*

(C-c), 45.41* (C-h), 47.49 (C-d, g), 124.04, 128.35, 128.59, 130.36

(C-6, 7, 8, 9), 135.34 (C-5a, C-9a), 167.11 (C-2). HRMS (LSIMS)

(m/z) calcd for C₂₀H₃₆N₅(M þ H)^þ346.2971; found 346.2980.

Anal. (C₂₀H₃₅N₅4HCl 1.5H₂O) C, H, N.

H-3), 2.77 (t, 2H, J = 7.2 Hz, H-5), 3.12-3.22 (m, 8H, H-d, H-g,

H-h, H-j), 3.24 (t, 2H, J = 8 Hz, H-c), 3.62 (t, 2H, J = 7.2 Hz, H-a),

7.17 (d, 1H, J = 8.3 Hz, H-9), 7.56-7.59 (m, 2H, H-6, H-8). ¹³

C

NMR (125 MHz, D₂O) δ 25.71* (C-e), 25.75* (C-f), 26.68* (C-b),

26.85* (C-i), 31.22 (C-4), 31.71 (C-3), 32.00 (C-5), 39.49 (C-j),

42.63 (C-a), 47.48* (C-c), 47.85* (C-h), 49.94* (C-d), 49.95* (C-g),

123.54 (C-7), 128.16 (C-9), 133.63 (C-8), 135.51 (C-6), 137.14*

(C-5a), 139.94* (C-9a), 169.54 (C-2). HRMS (LSIMS) (m/z) calcd

for C₂₀H₃₅N₅Br (M þ H)^þ424.2076; found 424.2068. Anal.

(C₂₀H₃₄N₅Br 4HCl 2H₂O) C, H, N.

3

N¹-(7-Methoxy-4,5-dihydro-3H-1-benzazepin-2-yl)spermine

Hydrochloride 27. General procedure D from 70, white solid,

75%; R_f0.22 (isopropylamine/CH₃OH/CHCl₃2/4/4), 0.44

(CH₃OH/NH₄OH 50/50). FTIR 2300 to 3650 þ 2067 (NH^þ),

1

1654 (CdN). H NMR (500 MHz, D₂O) δ 1.83 (m, 4H, H-e,

3

N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)spermidine Hydrochlo-

ride 23. General procedure D from 66, white solid, 88%; R_f0.45

(isopropylamine/CH₃OH/CHCl₃2/4/4), 0.62 (CH₃OH/NH₄OH

50/50). FTIR 2480 to 3280 þ 2052 (NH^þ), 1663 (CdN). ¹H NMR

(500MHz, D₂O) δ1.75-1.84 (m, 4H, H-e, H-f), 2.19 (m, 2H, H-b),

2.28 (m, 2H, H-4), 2.47 (t, 2H, J = 7.1 Hz, H-3), 2.75 (t, 2H, J =

7.3 Hz, H-5), 3.05 (t, 2H, J = 7.2 Hz, H-g), 3.15 (t, 2H, J = 7.4 Hz,

H-d), 3.25 (t, 2H, J = 8 Hz, H-c), 3.60 (t, 2H, J = 7.1 Hz, H-a),

H-f), 2.12 (m, 2H, H-i), 2.19 (m, 2H, H-b), 2.31 (m, 2H, H-4),

2.51 (t, 2H, J = 7 Hz, H-3), 2.77 (t, 2H, J = 7 Hz, H-5),

3.12-3.21 (m, 8H, H-d, H-g, H-h, H-j), 3.23 (t, 2H, J = 8 Hz,

H-c), 3.60 (t, 2H, J = 7 Hz, H-a), 3.87 (s, 3H, OCH₃), 7.00-7.02

(m, 2H, H-6, H-8), 7.20 (d, 1H, J = 8.6 Hz, H-9). ¹³C NMR (125

MHz, D₂O) δ 23.23* (C-e), 23.32* (C-f), 24.16* (C-b), 24.37*

(C-i), 29.03 (C-5), 29.48 (C-3), 29.55 (C-4), 37.00 (C-j), 39.97

(C-a), 44.97* (C-c), 45.38* (C-h), 47.44 (C-d, C-g), 113.37 (C-8),

115.37 (C-6), 125.31 (C-9), 128.44 (C-9a), 136.92 (C-5a), 158.54

(C-7), 166.82 (C-2). HRMS (LSIMS) (m/z) calcd for C₂₁H₃₈N₅O

(M þ H)^þ376.3076; found 376.3077. Anal. (C₂₁H₃₇N₅O 4HCl

7.23 (d, 1H, J = 7 Hz, H-9), 7.32-7.41 (m, 3H, H-6, H-7, H-8). ¹³

C

NMR (125 MHz, D₂O) δ22.81 (C-e), 23.95 (C-b, C-f), 28.42 (C-5),

28.88 (C-3), 29.42 (C-4), 38.84 (C-g), 39.66 (C-a), 44.96 (C-c), 47.12

(C-d), 123.58 (C-9), 127.89 (C-8), 128.07 (C-7), 129.85 (C-6),

134.88 (C-5a, C-9a), 166.65 (C-2). HRMS (LSIMS) (m/z) calcd

for C₁₇H₂₉N₄(M þ H)^þ289.2392; found 289.2390. Anal.

(C₁₇H₂₈N₄3HCl 0.5H₂O) C, H, N.

3

2H₂O) C, H, N.

N¹-(7-Nitro-4,5-dihydro-3H-1-benzazepin-2-yl)spermine

Hydrochloride 28. General procedure D from 71, yellow solid, 84%;

R_f0.15 (isopropylamine/CH₃OH/CHCl₃2/4/4), 0.45 (CH₃OH/

NH₄OH 50/50). FTIR 2300 to 3600 þ 2059 (NH^þ), 1661

3

(6R,7S)-N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)-6,7-dihydroxy-

spermine Hydrochloride 24. General procedure D from 67, white

solid, 90%; [R]_D^21.5= þ0.57° (c 11.7 mmol, H₂O); R_f0.10 (isopro-

pylamine/CH₃OH/CHCl₃2/4/4), 0.60 (CH₃OH/NH₄OH 50/50).

FTIR 2300 to 3600 þ 2031 (NH^þ), 1657 (CdN). ¹H NMR (500

MHz, D₂O) δ 2.14 (m, 2H, H-i), 2.26 (m, 2H, H-b), 2.32 (m, 2H,

H-4), 2.53 (t, 2H, J = 7.2 Hz, H-3), 2.81 (t, 2H, J = 7.3 Hz, H-5),

3.16 (t, 2H, J = 7.8 Hz, H-j), 3.21* (m, 2H, H-d), 3.27 (t, 2H, J = 8

Hz, H-h), 3.33 (t, 2H, J = 8.1 Hz, H-c), 3.46* (m, 2H, H-g), 3.65 (t,

2H, J = 7 Hz, H-a), 3.95-4.00 (m, 2H, H-e, H-f), 7.27 (d, 1H, J =

7.6 Hz, H-9), 7.35-7.45 (m, 3H, H-6, H-7, H-8). ¹³C NMR (125

MHz, D₂O) δ 24.02* (C-i), 24.21* (C-b), 28.81 (C-5), 29.29 (C-3),

29.81 (C-4), 36.96 (C-j), 40.04 (C-a), 45.23* (C-c), 45.65* (C-h),

49.94 (C-d, C-g), 68.65 (C-e, C-f), 123.96 (C-9), 128.29 (C-8),

128.55 (C-7), 130.32 (C-6, 7, 8, 9), 135.29 (C-5a, 9a), 167.09 (C-2).

HRMS (LSIMS) (m/z) calcd for C₂₀H₃₆N₅O₂(M þ H)^þ378.2869;

found 378.2864. Anal. (C₂₀H₃₅N₅O₂.4HCl.0.5H₂O) C, H, N.

N¹-(4,5-Dihydro-3H-1-benzazepin-2-yl)homospermine Hydro-

chloride 25. The deprotection step of 68 was previously

reported.⁵¹White solid, 60%; R_f0.17 (isopropylamine/CH₃OH/

NH₄OH), 0.40 (CH₃OH/NH₄OH). FTIR 2300 to 3650 þ 2078

(NH^þ), 1658 (CdN). ¹H NMR (500 MHz, D₂O) δ 1.79-1.87 (m,

12H, H-b, H-c, H-f, H-g, H-j, H-k), 2.33 (m, 2H, H-4), 2.52 (t, 2H,

J = 7 Hz, H-3), 2.80 (t, 2H, J = 7.3 Hz, H-5), 3.05-3.16 (m, 10H,

H-d, H-e, H-h, H-i, H-l), 3.58 (m, 2H, H-a), 7.25 (d, 1H, J = 7.6

Hz, H-9), 7.37-7.45 (m, 3H, H-6, H-7, H-8). ¹³C NMR (125

MHz, D₂O) δ 23.14, 23.20, and 23.47 (C-c, C-f, C-g, C-i), 24.31*

(C-b), 24.44* (C-k), 28.82 (C-5), 29.20 (C-3), 29.74 (C-4), 39.18

(C-l), 42.25 (C-a) 47.62, 47.30, and 47.47 (C-d, C-e, C-h, C-i),

123.91 (C-9), 128.26 (C-8), 128.43 (C-7), 130.27 (C-6), 135.26

(C-5a, C-9a), 166.75 (C-2). HRMS (LSIMS) (m/z) calcd for

1

(CdN), 1521, 1355 (NO₂). H NMR (500 MHz, D₂O) δ 1.84

(m, 4H, H-e, H-f), 2.13 (m, 2H, H-i), 2.23 (m, 2H, H-b), 2.39 (m,

2H, H-4), 2.60 (t, 2H, J = 7.2 Hz, H-3), 2.92 (t, 2H, J = 7.3 Hz,

H-5), 3.12-3.22 (m, 8H, H-d, H-g, H-h, H-j), 3.26 (t, 2H, J = 8

Hz, H-c), 3.68 (t, 2H, J = 7.2 Hz, H-a), 7.45 (d, 1H, J = 8.6 Hz,

H-9), 8.17 (dd, 1H, J = 8.8 Hz, J = 2.6 Hz, H-8), 8.30 (d, 1H,

J = 2.6 Hz, H-6). ¹³C NMR (125 MHz, D₂O) δ 23.18* (C-e),

23.22* (C-f), 24.14* (C-b), 24.28* (C-i), 29.04 (C-5), 29.34 (C-3),

29.39 (C-4), 36.94 (C-j), 40.42 (C-a), 44.94* (C-c), 45.30* (C-h),

47.44 (C-d, C-g), 123.70 (C-8), 124.86 (C-6), 125.75 (C-9), 136.76

(C-5a), 141.55 (C-9a), 146.61 (C-7), 167.42 (C-2). HRMS

(LSIMS) (m/z) calcd for C₂₀H₃₅N₆O₂(M þ H)^þ391.2821;

found 391.2817. Anal. (C₂₀H₃₄N₆O₂4HCl 0.9H₂O) C, H, N.

3

N¹-(7-Amino-4,5-dihydro-3H-1-benzazepin-2-yl)spermine hydro-

chloride 29. General procedure D from 72, cream solid, 59%; R_f

0.13 (isopropylamine/CH₃OH/CHCl₃2/4/4), 0.37 (CH₃OH/

NH₄OH 50/50). FTIR 2300 to 3650 þ 2071 (NH^þ), 1662 (CdN).

¹H NMR (500 MHz, D₂O) δ 1.82 (m, 4H, H-e, H-f), 2.11 (m, 2H,

H-i), 2.21 (m, 2H, H-b), 2.34 (m, 2H, H-4), 2.55 (t, 2H, J = 7.1 Hz,

H-3), 2.83 (t, 2H, J = 7.1 Hz, H-5), 3.11-3.21 (m, 8H, H-d, H-g,

H-h, H-j), 3.24 (t, 2H, J = 8 Hz, H-c), 3.62 (t, 2H, J = 7.2 Hz, H-a),

7.33-7.37 (m, 3H, H-6, H-7, H-8). ¹³C NMR (125 MHz, D₂O) δ

23.18* (C-e), 23.18* (C-f), 24.14* (C-b), 24.29* (C-i), 28.85 (C-5),

29.18 (C-3), 29.40 (C-4), 36.96 (C-j), 40.20 (C-a), 44.94* (C-c),

45.33* (C-h), 47.41 (C-d, C-g), 135.84* (C-5a), 137.53* (C-9a),

167.18 (C-2). HRMS (LSIMS) (m/z) calcd for C₂₀H₃₇N₆(M þ H)^þ

361.3080; found 361.3078. Anal. (C₂₀H₃₆N₆5HCl 1.3H₂O) H, N;

3

C calcd 42.42, found 43.79.

Biological Studies. Unless otherwise stated, usual laboratory

chemicals were purchased from Merck (Darmstadt, Germany)

or Sigma (St Louis, MO, USA). DFMO was obtained from Ilex

Oncology (San Antonio, TX, USA) and [¹⁴C]spermidine trihy-

drochloride from Amersham (Les Ulis, France). All data are

given as mean values of three or more experiments. Compar-

isons between means were made using the Student’s t test

assuming significance at p < 0.05.

C₂₂H₄₀N₅(M þ H)^þ373.3205; found 373.3293. Anal. (C₂₂H₃₉N₅

3

4HCl 3.5H₂O) N; H calcd 8.65, found 7.98; C calcd 45.37, found

3

45.94.

N¹-(7-Bromo-4,5-dihydro-3H-1-benzazepin-2-yl)-spermine

Hydrochloride 26. General procedure D from 69, white solid, 82%;

R_f0.16 (isopropylamine/CH₃OH/CHCl₃2/4/4), 0.45 (CH₃OH/

NH₄OH 50/50). FTIR 2300 to 3650 þ 2054 (NH^þ), 1657 (CdN).

¹H NMR (500 MHz, D₂O) δ 1.83 (m, 4H, H-e, H-f), 2.13 (m, 2H,

H-i), 2.21 (m, 2H, H-b), 2.31 (m, 2H, H-4), 2.52 (t, 2H, J = 7.2 Hz,

Cell Culture. Murine leukemia L1210, CHO, and CHO-MG

cells were grown in RPMI 1640 medium supplemented with

10% fetal calf serum, glutamine (2 mM), penicillin (100 U/mL),

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7661

References

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described.⁴³To prevent artifactual results due to the oxidation

of the conjugates by the serum amine oxidase present in fetal calf

serum (Supporting Information Figure 1),⁷⁷cell culture medium

was supplemented with aminoguanidine (2 mM).

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ꢀ

^

(LNCC-Comites d’Ille-et-Vilaine, du Morbihan et des Cotes

d’Armor) for financial support and for fellowships to S.T. and

B.M. (LNCC). EU COST 922 is acknowledged for support.

Our sincere thanks are also due to A. Bondon (CNRS UMR

6509, Rennes, France) for performing HMBC and HMQC

ꢀ

spectra and to the late Dr. P. Guenot (Centre Regional de

Mesures Physiques de l’Ouest, Rennes, France) for mass

measurements.

Supporting Information Available: Spectroscopic and purity

data for target compounds, HPLC analysis, and additional

information. This material is available free of charge via the

Internet at http://pubs.acs.org.

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Article Doi

Targeting the polyamine transport system with benzazepine- and azepine-polyamine conjugates

DOI: 10.1021/jm1007648

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Full text of DOI:10.1021/jm1007648

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