Pentamidine analogs: Syntheses, structures in solid state by 13C CP/MAS NMR spectroscopy, and X-ray crystallography and their preliminary biological screening against human cancer

Article abstract of DOI:10.1007/s00706-006-0515-z

The synthesis of four new 1,5-bis(4-amidinophenoxy)-3-oxapentane analogs is described. The structures of the obtained bis-amidines and bis-nitriles in the solid state are evaluated on the basis of ¹³C CP/MAS NMR spectra and theoretical calculat

Full text of DOI:10.1007/s00706-006-0515-z

Monatshefte f€ur Chemie 137, 1225–1240 (2006)
DOI 10.1007/s00706-006-0515-z
Pentamidine Analogs: Syntheses, Structures
13
in Solid State by C CP=MAS NMR
Spectroscopy, and X-Ray Crystallography
and their Preliminary Biological Screening
Against Human Cancer
1
1
Dorota Maciejewska1;ꢀ, Pawel Kazmierczak , Jerzy Zabinski ,
˙
´
´
Irena Wolska2, and Sylwia Popis1
1
Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Warsaw,
Warsaw, Poland
2
Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University,
´
Poznan, Poland
Received February 2, 2006; accepted March 7, 2006
Published online August 24, 2006 # Springer-Verlag 2006
Summary. The synthesis of four new 1,5-bis(4-amidinophenoxy)-3-oxapentane analogs is described.
The structures of the obtained bis-amidines and bis-nitriles in the solid state are evaluated on the basis
of 13C CP=MAS NMR spectra and theoretical calculations at DFT level. A single crystal X-ray
diffraction structure is presented for 1,5-bis(4-amidinophenoxy)-3-oxapentane. A preliminary anti-
cancer assay against three cell lines is also given.
Keywords. Amidines; NMR spectroscopy; X-Ray diffraction; Theoretical calculations.
Introduction
Aromatic bis-amidine compounds exhibit a wide range of activities against a num-
ber of pathogens [1], but only 1,5-bis(4-amidinophenoxy)pentane (pentamidine) is
in widespread clinical use against Pneumocystis carinii pneumonia (PCP) [2, 3].
Because of an increasing number of immunosuppressed patients that could be in-
fected by Pneumocystis carinii the current therapies against PCP need to be im-
proved. In a large number of patients undergoing treatment with pentamidine there
have commonly been drug-related toxicities demanding further search for new pent-
amidine analogs that have the desired potency and suitable bioavailability, but lost
ꢀ
Corresponding author. E-mail: domac@farm.amwaw.edu.pl

1226
D. Maciejewska et al.
their toxicity. Moreover, pentamidine and its analogs together with a number of other
diamidines were evaluated for their anticancer, antiviral, and anticoagulant activity
[4–8]. The importance of these compounds not only as the anti-PCP chemother-
apeutics but also in these other venues indicates an underdeveloped potentially
important class of compounds.
The biological activity of pentamidine and its analogs is closely related to their
interaction with DNA at the adenine-thymine rich region in the minor-groove.
Crystallographic studies of DNA-pentamidine complexes are useful tools for de-
signing analogs [9–11].
This paper reports the syntheses, structural data, and a preliminary anticancer
assay of the pentamidine analogs 1–4 (Fig. 1). Up to now, no systematic studies
of pentamidine analogs in the solid state have been performed. Thus, we used the
13C CP=MAS NMR technique and theoretical calculations of relevant shielding
constants at DFT level of theory to obtain information about the structure and
dynamic events in the solid state of the title compounds. The X-ray structure of
1,5-bis(4-amidinophenoxy)-3-oxapentane (1) is also presented and compared with
its DNA complex [10].
Fig. 1. Chemical formulae and atom numbering

Pentamidine Analogs
Results and Discussion
Syntheses
1227
The method of preparation of the diamidines generally followed the established
route [12–14], which involves the preparation of the dinitriles and their subsequent
conversion into diamidines. 1,5-Bis(4-amidinophenoxy)-3-oxapentane (1) was syn-
thesized according to previous reports [15]. The previously unknown N-alkyl
derivatives of 1,5-bis(4-amidinophenoxy)-3-oxapentane 2–4 were obtained in the
course of a three-step synthesis (Scheme 1). It involves O-alkylation of 4-hy-
droxybenzonitrile with bis(2-chloroethyl)ether, then conversion of the formed
1,5-bis(4-cyanophenoxy)-3-oxapentane (1b) [15] to 1,5-bis[4-(ethoxyiminoyl)phe-
noxy]-3-oxapentane (1c). The latter was treated with appropriate amines (butyl-
amine, cyclohexylamine, ethane-1,2-diamine) to yield the N-alkyl derivatives 2–4.
The new compounds 5b and 5 were prepared by modification of this procedure
(Scheme 2). 4-Fluorobenzonitrile was obtained from commercially available 4-fluoro-
benzaldehyde by refluxing its formic acid solution with hydroxylamine hydro-
chloride [16]. 1,5-Diamino-3-oxapentane was prepared from bis(2-chloroethyl)
ether by Gabriel synthesis [17]. The N,N-dialkylation of 1,5-diamino-3-oxapentane
with 4-fluorobenzonitrile resulted in the dinitrile 5b. It was converted to the di-
amidine 5 as described for 1.
X-Ray Structure of 1,5-Bis[4-amidinophenoxy]-3-oxapentane (1)
The crystal and molecular structures of 1 were determined by single crystal X-ray
diffraction. The crystallographic data, together with data collection and structure
Scheme 1

1228
D. Maciejewska et al.
Scheme 2
refinement details are listed in Table 1, and hydrogen bonding parameters are given
in Table 2. A perspective view of the molecular conformation, together with the
atom numbering scheme, is shown in Fig. 2 (for drawing method see Ref. [18]).
1,5-Bis(4-amidinophenoxy)-3-oxapentane (ꢀ-oxapentamidine, 1)crystallizesfrom
water solution as the dihydrochloride dihydrate salt in the monoclinic space group
C2=c. The central O10 atom is located at two-fold crystallographic axis and the
asymmetric unit contains one half of the molecule, one chloride ion, and one water
molecule.
The crystal structures of only several aromatic diamidines derivatives related to
pentamidine have been reported [19–21]. Apart from the crystal structures of the
complexes of propamidine, pentamidine, ꢀ-oxapentamidine, berenil and its furan
derivatives and the DNA dodecamer d(CGCGAATTCGCG)2 have been determined
[9–11, 22]. These investigations indicate that the amidinium group of the drug could
form H-bonded interactions with acceptor atoms of either adenine or thymine bases
in the minor groove.
ꢀ-Oxapentamidine contains an ether linkage in place of the more sterically bulky
central methylene unit of pentamidine. All bond angles and distances within the struc-
ture of 1 are normal. Two aromatic rings are twisted by 19.45(5)ꢁ with respect to each
other, with the dihedral angle between the plane of the phenyl ring and its neighbouring
amidinium plane being 13.72(7)ꢁ as compared with 3–29ꢁ for related compounds.
The polyether linker of 1 adopts a folded conformation but in the crystal struc-
ture of the complex of ꢀ-oxapentamidine and DNA a stretch of the central chain
was found [10]. Such a conformation of 1 is probably due to molecular packing and
intermolecular C–Hꢂ ꢂ ꢂO hydrogen bonds. The extended conformation of the cen-
tral chain of ꢀ-oxapentamidine in the complex with DNA may be a major factor in
the positioning of the drug along the minor groove. So, our structure reveals a
considerable flexibility of the polyether chain.

Pentamidine Analogs
1229
Table 1. Crystal data, data collection, and structure refinement for 1
Compound
1
Empirical formula
Formula weight
T=K
C18H28Cl2N4O5
451.34
296(2)
˚
Wavelength=A
0.71073
Crystal system, space group
Unit cell dimensions
monoclinic, C 2=c
˚
a=A
˚
b=A
22.700(1)
10.2261(6)
9.6042(5)
101.265(4)
2186.5(2)
4, 1.371
˚
c=A
ꢁ=ꢁ
3
˚
Volume=A
Z, Dx=Mg=m3
ꢂ=mmꢃ1
F(000)
0.333
952
ꢃ-range for data collection=ꢁ
3.20–29.93
ꢃ31ꢄhꢄ29
ꢃ7ꢄkꢄ14
ꢃ13ꢄlꢄ13
hkl range
Reflections:
collected
10425
unique (Rint)
2924(0.022)
2497
observed (I>2ꢄ(I))
Data=restraints=parameters
Goodness-of-fit on F2
R(F) (I>2ꢄ(I))
wR(F2) (all data)
2924=0=151
1.068
0.0351
0.1000
3
˚
Max=min. ꢀꢅ (e=A )
0.326=ꢃ0.288
˚
Table 2. Hydrogen-bonding geometry (A and deg.) for 1
1
D–Hꢂ ꢂ ꢂA
d(D–H)
d(Hꢂ ꢂ ꢂA)
d(Dꢂ ꢂ ꢂA)
<(DHA)
OW–H2Wꢂ ꢂ ꢂCli
N11–H11Aꢂ ꢂ ꢂClii
N11–H11Bꢂ ꢂ ꢂCliii
N12–H12Bꢂ ꢂ ꢂCliii
N12–H12Aꢂ ꢂ ꢂOWiv
OW–H1Wꢂ ꢂ ꢂClv
C8–H8Bꢂ ꢂ ꢂOWi
C5–H5ꢂ ꢂ ꢂOWiv
0.82(2)
0.80(2)
0.88(2)
0.82(2)
0.84(2)
0.81(2)
0.97
2.38(2)
2.49(2)
2.50(2)
2.56(2)
2.05(2)
2.39(2)
2.68
3.204(1)
3.264(1)
3.313(1)
3.323(1)
2.865(2)
3.196(1)
3.509(2)
3.535(2)
3.610(1)
3.311(2)
177(2)
163(2)
155(2)
154(2)
164(2)
172(2)
144
0.93
2.67
154
C6–H6ꢂ ꢂ ꢂO(8)iv
C6–H6ꢂ ꢂ ꢂO10iv
0.93
2.69
170
0.93
2.68
126
Symmetry codes: (i) ꢃx; y; ꢃz ꢃ 1=2; (ii) x; ꢃy þ 1; z þ 1=2; (iii) ꢃx þ 1=2; ꢃy þ 3=2; ꢃz þ 1;
(iv) ꢃx; ꢃy þ 2; ꢃz; (v) ꢃx; ꢃy þ 1; ꢃz

1230
D. Maciejewska et al.
Fig. 2. A perspective view of the molecular conformation of 1 together with the atom numbering
scheme
The crystal structure of 1 is stabilized by a number of hydrogen bonds (Table 2)
involving the ꢀ-oxapentamidine, water molecules, and chloride ions. The molecu-
lar packing and hydrogen bonding scheme in the crystal structure are depicted in
Fig. 3. Thus, within the crystal, the molecules of ꢀ-oxapentamidine are linked via
C6–H6ꢂ ꢂ ꢂO8 and C6–H6ꢂ ꢂ ꢂO10 interactions (Table 2) to form columns along the
z axis. Also, the water molecules and the chloride ions, via OW–H1Wꢂ ꢂ ꢂCl and
Fig. 3. Perspective drawing of the packing arrangement of 1 along the b-axis with dashed lines
indicating the hydrogen bonding scheme

Pentamidine Analogs
1231
OW–H2Wꢂ ꢂ ꢂCl hydrogen bonds, create infinite chains along both sides of the
columns. Adjacent columns and chains are further linked by N11–H11Aꢂ ꢂ ꢂCl,
N12–H12Aꢂ ꢂ ꢂOW hydrogen bonds and weaker interactions C5–H5ꢂ ꢂ ꢂOW and
C8–H8Bꢂ ꢂ ꢂOW and in consequence wide bands are formed. The three-dimensional
supramolecular structure results from combination of the bands via N11–H11Bꢂ ꢂ ꢂCl
and N12–H12Bꢂ ꢂ ꢂCl hydrogen bonds.
13C CP=MAS NMR Spectra in the Solid State
The 13C CP=MAS NMR spectroscopy in the solid state is a complementary tool to
the X-ray diffraction method. Of special interest is its application to the investiga-
tion of polymorphism and dynamic events of biologically active compounds in the
solid state [23]. It was also of interest to obtain more structural information on
those molecules for which we have no XRD data. The notation used in the dis-
cussion of the NMR results is illustrated in Fig. 1. Assignments of 13C resonances
in solid state were made from comparison with their solution spectra, and with the
spectra of structurally related para-substituted benzenes. Theoretical shielding
constants computed for geometries optimized at DFT level of theory for bis-ami-
dines 1–5, as well as for bis-nitriles 1b and 5b, and for X-ray derived atomic
coordinates of 1 were used as an aid to the analysis of the more complicated
spectral pattern. The 13C CP=MAS NMR spectra of 1–5, and of 1b and 5b are
shown in Figs. 4 and 5, and the most probable assignments of signals are given in
Table 3.
The central O ether atom incorporated in 1–5 formally dissects these molecules
into two halves. For equivalent pairs of atoms from both parts of the molecule we
could expect a different pattern of resonances in the solid state spectra depending
on conformational equilibria. Surprisingly, not many differences were found as we
compared 13C atom signals of the aliphatic groupings in solutions and the solid
state (i.e. there are single, and=or partially broadened ones). We have observed
more significant differences only for aromatic carbons: the asymmetric non-aver-
aged resonances in the spectra of bis-diamidines 1, 5, and bis-nitriles 1b, 5b. The
separations of resonances of the ortho pairs C2, C6 (C20, C60) atoms and meta pairs
C3, C5 (C30, C50) atoms are equal to 5–11 ppm and 1–2 ppm. It has been reported
previously [24, 25] that benzene rings may exhibit different degrees of mobility
dependent on intermolecular interactions and the nature of substituents present at C
atoms. Both carbon atoms ortho to alkoxy or amino linkers are shielded in different
ways: lone electron pairs of oxygen or nitrogen atoms pointing to the correspond-
ing C atoms result in a high field shift of their resonances [26, 27]. The separations
of signals of meta carbons are much smaller indicating that amidine and nitrile
moieties are almost coplanar with the benzene rings in 1, 1b, and 5b. Besides, in
the spectrum of 5 we observed different resonances of C atoms in both benzene
rings indicating that the ring planes are slightly twisted relative to all another. All
those facts could indicate the lack of benzene and chain linker motions of 1, 5, 1b,
and 5b in solid state.
In the spectra of 2–4 the aromatic C2, C6 and C3, C5 pairs of peaks broaden and
become indistinct. Similarly, in the spectra of 3 and 4 the alkyl C8 (C80), C9 (C90)
atoms resonances broadened. This was interpreted with a reorientation of those

1232
D. Maciejewska et al.
Fig. 4. The 13C CP=MAS NMR spectra of 1–4 in the solid state
parts of molecules. The resonances of C atoms of both butyl chains (C10–C13 and
C100–C130) in 2 are sharp singlets, thus demonstrating similarities in the chain
conformations.

Pentamidine Analogs
1233
Fig. 5. The 13C CP=MAS NMR spectra of 1,5-bis[(4-amidinophenyl)amino]-3-oxapentane dihydro-
chloride 5, 1b, and 5b in the solid state

1234
D. Maciejewska et al.
Table 3. Assignments of resonances in the 13C CP=MAS NMR spectra of 1–5, 1b, and 5b
No.
13C chemical shifts in solid state=ppm
C1, C10 C2, C20 C3, C30 C4, C40 C5, C50 C6, C60 C7, C70 C8, C80 C9, C90 C10, C100; C11, C110;
C12, C120; C13, C130;
C14, C140; C15, C150
1
2
3
163.4
163.9
163.4
121.8
114.3
114.6
129.9
130.8
131.4
117.5
118.7
118.8
128.9
130.8
131.4
110.4
114.3
114.6
163.4
163.9
160.5
66.2
68.6
68.5
70.8
69.6
70.8
–
42.3; 31.2; 20.4; 13.1
53.0; 31.9; 24.6; 26.6;
24.8; 31.9
4
5
163.3
151.2
113.2
116.1;
112.1
120.9
113.8
131.8
128.9
118.9
105.9;
105.6
105.9
97.0
131.8
128.9
113.2
108.3;
112.1
112.5
108.6
163.3
162.7
68.5
40.0
68.5
69.0
45.2; 45.2
–
1b
5b
162.4
151.3
135.4
135.4
136.7
133.1
119.5
120.9
68.7
44.5
70.7
68.4
–
The resonances of C8 (C80) in 5, and imidazoline C11, C12 (C110, C120) atoms
proximal to N atoms are broadened and=or splitted into unequal doublets (or quar-
tets) due to residual coupling to quadrupolar 14N [28]. In the spectra of bis-nitriles 1b
and 5b the resonances of C atoms from nitrile groups are broadened. The water
molecules, which were detected by X-ray measurement and elemental analyses,
might influence the mobility of more hydrated molecules in the solid state.
Computation Results
The DFT method was used to study the geometries and NMR resonances of
compounds 1–5, 1b, and 5b. We have analyzed folded and extended ‘zig-zag’
Fig. 6. Hypothetical conformations in solid state (folded and extended) shown for 1b

Pentamidine Analogs
1235
conformations of the ether linkage (see examples shown in Fig. 6), and we found
that for bis-amidines the folded (bent) conformations are slightly more stable. For
1b the extended conformation is slightly preferred by 10.5 kJ=mol, but for 5b, the
folded one is more stable by 7.5 kJ=mol. These differences are not significant and
could not be decisive for the preferred structure in the solid state where the inter-
molecular interactions play a meaningful role. Thus, we decided to compute the
NMR shielding constants for the atomic coordinates corresponding to both folded
and extended conformations, and to compare them with the experimental chemical
shifts of C atoms in the solid state NMR spectra. The calculated shielding constants
for equivalent pairs of C atoms were the same in extended conformations, but
slightly different for folded ones (on average by 0.2 ppm for the ether linkage,
and 1 ppm for the aminoether). Next, we analyzed the correlation coefficients r2
for the correlations between the calculated shielding constants ꢄ (for both confor-
mations of the linker) and the experimental chemical shifts ꢆ. We obtained linear
correlations with r2 values between 0.982 to 0.997. The correlation coefficients
were higher for the structures with folded linker for 1–5, and with the extended one
for 1b and 5b. Simultaneously, using the same method we computed the shielding
constants for the crystallographic coordinates of 1. The calculated shielding con-
stants for the X-ray geometry of 1 are slightly different from those for the DFT
geometry of 1 with folded linker, but in both cases linear correlations were found
(see Fig. 7). The above finding is good evidence that linear correlations between
the computed shielding constants for DFT geometries and the experimental che-
mical shifts could be used to characterize the molecular structure of the studied
compounds. However, the resonances in the spectra of 2–5 are broad and this gave
rise to some uncertainty. We ventured a guess of the favored solid-state conforma-
tions only for 1b and 5b, the spectra of which are sharp: for both we propose
extended ‘zig–zag’ conformations (see Fig. 6).
Fig. 7. Linear correlations between calculated shielding constants ꢄ and experimental chemical
shifts ꢆ for 1: - - - - - for DFT structure, r2 ¼ 0.997; — for X-ray structure, r2 ¼ 0.996

1236
D. Maciejewska et al.
Table 4. The percent of test cell growth relative to control cell growth (T=C ꢂ 100%) for 1–4
Compounds
(c ¼ 1 ꢂ 10ꢃ4 M)
Human tumor cell lines
NCI-H460
MCF-7
SF-298
1
2
3
4
57
52
32
49
55
45
46
72
48
50
114
112
Preliminary Anticancer Assay
Compounds 1–5 were accepted for evaluation by the Division of Cancer Treatment
and Diagnosis, National Cancer Institute (Bethesda, USA) in a 3-cell line panel,
consisting of the MCF7 (breast), NCI-H460 (lung), and SF-268 (CNS) cells. The
details are available at the web site http:==dtp.nci.nih.gov. The results are reported
in Table 4 as the percentage of growth of the treated cells compared with untreated
control cells. Substitution of the amidino groups of 1 by alkyl or 4,5-dihydroimi-
dazoline moieties gave compounds that were less potent relative to the unsubsti-
tuted analog. Up to now, only 1 was scheduled for evaluation against the full panel
of 60 tumor cell lines. It inhibits 50% of growth of human leukemia CCRF-CEM
cell line at a concentration of 1 ꢂ 10ꢃ6 M. Its cytotoxicity against the remaining
cancer cell lines (non-small cell lung, colon, CNS, melanoma, ovarian, renal, pros-
tate, breast) was much lower. Compound 5 is still in testing.
Conclusions
The reported series of pentamidine analogs was synthesized in sufficient yields. In
the 13C CP=MAS NMR spectra of bis-amidines 1 and 5 and in both bis-nitriles 1b
and 5b we observed distinct resonances of aromatic carbons, which could be evi-
dence of the absence of intramolecular motions in solid state. That was not the case
for N-alkylated bis-amidines 2–4. We have not observed the resonance pattern
(e.g. double signals), which could be explained in terms of polymorphism.
In the crystal the molecules of 1 are organized into a three-dimensional network
by different intermolecular interactions. The crystal structural analysis lends support
to the role of hydrogen bonds in influencing the conformation of the ꢀ-oxapent-
amidine and the crystal packing.
The bis-amidines1–3 at1 ꢂ 10ꢃ4 M reducethegrowth of MCF7 (breast), NCI-H460
(lung), and SF-268 (NCS) cells on average by 48%. Compound 1 inhibits 50% of
growth of a human leukemia CCRF-CEM cell line at 1 ꢂ 10ꢃ6 M.
Experimental
All chemicals were purchased from the major chemical suppliers as high or highest purity grade and
used without any further purification. Melting points were determined with a Digital Melting Point
1
13
Apparatus 9001. H and C NMR spectra in solution were recorded at 25ꢁC with a Varian Unity
plus-200, and standard Varian software was employed. Several (as indicated) 1H NMR and 13C NMR

Pentamidine Analogs
1237
spectra in solution were recorded with a Bruker Avance DMX 400. The solid state 13C CP=MAS NMR
spectra were measured using a Bruker Avance DMX 400. A powdered sample was spun at 8 kHz.
Contact time of 4 ms, repetition time of 10 s, and spectral width of 24kHz were used for accumulation
of 3000 scans. Chemical shifts ꢆ=ppm were referenced to TMS. Elemental analyses were averaged
from two independent determinations and agreed favourably with the calculated values. Prefabricated
silica gel sheets (Merck Kieselgel 60 F254) were used for TLC.
1,5-Bis(4-cyanophenoxy)-3-oxapentane (1b) [15]
4-Hydroxybenzonitrile (1a, 1.20 g, 10mmol) and 0.72g bis(2-chloroethyl)ether (5 mmol) were added
to 15 cm3 1-methyl-2-pyrrolidone together with 2.08g anh. K2CO3 (15 mmol). The mixture was
allowed to stand at 130ꢁC for 2 h while stirring. The resulting hot solution was added to 200 cm3 cold
H2O. The formed solid was filtered off and washed with H2O. The precipitate was recrystallized from
absolute ethanol to give 1.18 g (78%) of sandy crystals. Mp 136–137ꢁC (Ref. [15]: 134–135ꢁC);
1H NMR (400MHz, CDCl3): ꢆ ¼ 3.94 (t, J ¼ 4.8 Hz, 9-H2, 90-H2), 4.19 (t, J ¼ 4.8 Hz, 8-H2, 80-H2),
6.94–6.96 (m, 2-H, 20-H, 6-H, 60-H), 7.56–7.58 (m, 3-H, 30-H, 5-H, 50-H) ppm; 13C NMR (100 MHz,
CDCl3): ꢆ ¼ 67.90 (8-C, 80-C), 69.87 (9-C, 90-C), 104.46 (4-C, 40-C), 115.47 (2-C, 20-C, 6-C, 60-C),
119.31 (7-C, 70C), 134.18 (3-C, 30-C, 5-C, 50-C), 162.14 (1-C, 10-C) ppm; IR (KBr): ꢇꢁ¼ 2218, 1604,
1504, 1257 cmꢃ1
.
1,5-Bis[(4-cyanophenyl)amino]-3-oxapentane (5b, C18H18N4O)
A 0.78g (51%) of yellowish crystals from absolute ethanol (mp 121.6–122.8ꢁC) was obtained from
1.21g 5a (10 mmol) and 0.885 g 1,5-diamino-3-oxapentane dihydrochloride (5mmol) with 5.53 g anh.
K2CO3 (40 mmol) by the procedure described above. 1H NMR (400 MHz, CDCl3): ꢆ ¼ 3.35 (q,
J ¼ 4.8 Hz, 8-H2, 80-H2), 3.71 (t, J ¼ 4.8Hz, 9-H2, 90-H2), 4.69 (br s, NH), 6.55–6.57 (m, 2-H, 20-H,
6-H, 6-H0), 7.38–7.40 (m, 3-H, 30-H, 5-H, 50-H) ppm; 13C NMR (100MHz, CDCl3): ꢆ ¼ 42.54 (8-C,
80-C), 69.09 (9-C, 90-C), 98.44 (4-C, 40-C), 112.20 (2-C, 20-C, 6-C, 60-C), 120.45 (7-C, 70C), 133.54
(3-C, 30-C, 5-C, 50-C), 151.20 (1-C, 10-C) ppm; IR (KBr): ꢇꢁ¼ 3360, 2210, 1605, 1524 cmꢃ1
.
1,5-Bis[4-(ethoxyiminoyl)phenoxy]-3-oxapentane dihydrochloride (1c, C22H28N2O5 ꢂ 2HCl)
To 25 cm3 absolute ethanol saturated with dry HCl 1.54 g 1,5-bis(4-cyanophenoxy)-3-oxapentane
(5mmol) were added. The solution was kept at ambient temperature for 24 h in a sealed vessel.
Anhydrous ether was then added until complete precipitation was attained. The precipitate was quickly
filtered off (avoiding prolonged suction) and dried under reduced pressure giving the very hygroscopic
HCl salt in nearly quantitative yield. The crude product was used in the next step without additional
purification.
1,5-Bis(4-amidinophenoxy)-3-oxapentane dihydrochloride (1) [15]
The crude 1c was added to 25cm3 absolute ethanol saturated with anh. NH3, and the mixture was
stirred at ambient temperature for 24h in a sealed vessel. The solvent was evaporated under reduced
pressure and the residue was stirred with aqueous NaOH (1g NaOH in 30cm3 H2O) for 0.5 h. The
precipitated white solid was filtered off and washed with cold H2O. The free base was converted to its
HCl salt by refluxing with an ethanolic solution of HCl (20 cm3 absolute ethanol and 2.5cm3 17.7%
aqu. solution of HCl in absolute ethanol) until complete dissolution of the solid was achieved. Anhydrous
ether (40 cm3) was added to this clear solution at ambient temperature. The precipitate was filtered
off, washed with ether, and dried at 60ꢁC for 2 h to give 1.20 g white solid (57%). Mp 258–260ꢁC
(Ref. [15]: 256–258ꢁC); 1H NMR (200MHz, DMSO-d6): ꢆ ¼ 3.86 (br s, 9-H2, 90-H2), 4.26 (br s, 8-H2,
80-H2), 7.14–7.19 (m, 2-H, 20-H, 6-H, 60-H), 7.88–7.93 (m, 3-H, 30-H, 5-H, 50-H), 9.22 (br s, NH2),
9.32 (br s, NH2) ppm; 13C NMR (50.3 MHz, DMSO-d6): ꢆ ¼ 67.6 (8-C, 80-C), 68.7 (9-C, 90-C), 114.7
(2-C, 20-C, 6-C, 60-C), 119.4 (4-C, 40-C), 130.1 (3-C, 30-C, 5-C, 50-C), 162.7 (1-C, 10-C), 164.6 (7-C,
70-C) ppm. IR (KBr): ꢇꢁ¼ 3418, 3325, 3151.5, 1682, 1663, 1609, 1489, 1273 cmꢃ1
.

1238
D. Maciejewska et al.
1,5-Bis[4-(N-butylamidino)phenoxy]-3-oxapentane dihydrochloride
(2, C26H38N4O3 ꢂ 2HCl ꢂ ½H2O)
A suspension of 1c and 1.60g butylamine (22 mmol) was stirred in 25cm3 absolute ethanol at ambient
temperature for 72 h. Workup was the same as described above to give 1.42g white solid (55%). Mp
1
241–242ꢁC; H NMR (200MHz, DMSO-d6): ꢆ ¼ 0.92 (t, J ¼ 7.4 Hz, 13-H3, 130-H3), 1.29–1.47 (m,
12-H2, 120-H2), 1.55–1.70 (m, 11-H2, 110-H2), 3.38–3.51 (m, a signal of water included, 10-H2,
100-H2), 3.83–3.88 (m, 9-H2, 90-H2), 4.22–4.26 (m, 8-H2, 80-H2), 7.12–7.16 (m, 2-H, 20-H, 6-H, 60-H),
7.79–7.83 (m, 3-H, 30-H, 5-H, 50H), 9.08 (br s, NH), 9.44 (br s, NH), 9.82 (br s, NH) ppm; 13C NMR
(50.3MHz, DMSO-d6): ꢆ ¼ 13.5 (13-C, 130-C), 19.4 (12-C, 120-C), 29.4 (11-C, 110-C), 42.1 (10-C,
100-C), 67.6 (8-C, 80-C), 68.8 (9-C, 90-C), 114.5 (2-C, 20-C, 6-C, 60-C), 120.5 (4-C, 40-C), 130.1 (3-C,
30-C, 5-C, 50-C), 161.7 (7-C, 70-C), 162.2 (1-C, 10-C) ppm; IR (KBr): ꢇꢁ¼ 3044, 2932, 2874, 1674.1,
1609, 1508, 1269 cmꢃ1
.
1,5-Bis[4-(N-cyclohexylamidino)phenoxy]-3-oxapentane dihydrochloride
(3, C30H42N4O3 ꢂ 2HCl ꢂ 2½H2O)
A white solid (0.98 g, 32%) was obtained from 1c and 1.98 g cyclohexylamine (20 mmol) by the
1
procedure described above. Mp 291–292ꢁC (dec); H NMR (200MHz, DMSO-d6): ꢆ ¼ 1.10–1.88
(m, cyclohexyl protons), 3.85 (br s, 9-H2, 90-H2), 4.23 (br s, 8-H2, 80-H2), 7.10–7.14 (m, 2-H, 20-H, 6-
H, 60-H), 7.75–7.79 (m, 3-H, 30-H, 5-H, 50-H) ppm; 13C NMR (50.3MHz, DMSO-d6): ꢆ ¼ 24.2 (13-C,
130-C), 24.7 (12-C, 120-C, 14-C, 140-C), 31.1 (11-C, 110-C, 15-C, 150-C), 51.6 (10-C, 100-C), 67.6 (8-C,
80-C), 68.8 (9-C, 90-C), 114.4 (2-C, 20-C, 6-C, 60-C), 121.2 (4-C, 40-C), 130.2 (3-C, 30-C, 5-C, 50-C),
160.7 (7-C, 70-C), 162.0 (1-C, 10-C) ppm; IR (KBr): ꢇꢁ¼ 3036, 2932, 2858, 1670, 1609, 1512,
1261 cmꢃ1
.
1,5-Bis[4-(4,5-dihydro-2-imidazoyl)phenoxy]-3-oxapentane dihydrochloride
(4, C22H22N4O3 ꢂ 2HCl ꢂ 3H2O)
A suspension of 1c and 1.20 g ethane-1,2-diamine (20 mmol) was refluxed in 25cm3 absolute ethanol
for 24 h. After workup described for 1 2.10 g cream solid (80%) were obtained. Mp 89–90ꢁC; 1H NMR
(200 MHz, DMSO-d6): ꢆ ¼ 3.36 (s, 10-H2, 100-H2, 11-H2, 110-H2), 3.83–3.87 (m, 9-H2, 90-H2), 4.24–
4.28 (m, 8-H2, 80-H2), 7.17–7.22 (m, 2-H, 20-H, 6-H, 6-H0), 8.02–8.07 (m, 3-H, 30-H, 5-H, 50-H), 10.60
(s, NH) ppm; 13C NMR (50.3 MHz, DMSO-d6): ꢆ ¼ 44.0 (10-C, 100-C, 11-C, 110-C), 67.7 (8-C, 80-C),
68.7 (9-C, 90-C), 114.0 (4-C, 40-C), 115.0 (2-C, 20-C, 6-C, 60-C), 130.9 (3-C, 30-C, 5-C, 50-C), 163.1
(7-C, 70-C), 163.8 (1-C, 10-C) ppm;. IR (KBr): ꢇꢁ¼ 3317, 3128, 1651, 1612, 1504cmꢃ1
.
1,5-Bis[(4-amidinophenyl)amino]-3-oxapentane dihydrochloride (5, C18H24N6O ꢂ 2HCl ꢂ ½H2O)
A 1.29 g (61% yield) beige solid (mp 288–292ꢁC (dec)) was obtained from 5b by the procedure
1
described for 1. H NMR (400 MHz, DMSO-d6): ꢆ ¼ 3.30 (t, J ¼ 5.0 Hz, 8-H2, 80-H2), 3.61 (t,
J ¼ 5.0 Hz, 9-H2, 90-H2), 5.16 (br s, NH), 6.73–6.75 (m, 2-H, 20-H, 6-H, 6-H0), 7.70–7.73 (m, 3-H,
30-H, 5-H, 50-H), 8.75 (s, NH), 8.91 (s, NH) ppm;13C NMR (100MHz, DMSO-d6): ꢆ ¼ 42.18 (8-C,
80-C), 68.60 (9-C, 90-C), 111.20 (2-C, 20-C, 6-C, 60-C), 112.10 (4-C, 40-C), 129.73 (3-C, 30-C, 5-C,
50-C), 153.56 (1-C, 10-C), 164.29 (7-C, 70C) ppm; IR (KBr): ꢇꢁ¼ 3317, 3128, 1651, 1612, 1504 cmꢃ1
.
X-Ray Diffraction Studies
An X-ray quality crystal of 1,5-bis(4-amidinophenoxy)-3-oxapentane (ꢀ-oxapentamidine, 1) was
obtained from water solution as the dihydrochloride dihydrate salt by slow evaporation. Diffraction
data were collected on an Oxford Diffraction KM4CCD diffractometer [29] at 296 K, using graphite-
monochromated MoKꢈ radiation. A total of 782 frames were measured in six separate runs. The
!-scan was used with a step of 0.75ꢁ, two reference frames were measured after every 50 frames, they
did not show any systematic changes either in peaks’ positions or in their intensities. The unit cell
parameters were determined by least-squares treatment of setting angles of 5749 highest-intensity

Pentamidine Analogs
1239
reflections chosen from the whole experiment. Intensity data were corrected for Lorentz and
polarization effects [30]. The structures were solved by direct methods with the SHELXS-97 pro-
gram [31] and refined with full-matrix least-squares by the SHELXL-97 program [32]. The
2
2
2
2
2
function ꢂwðjFoj ꢃ jFcj Þ was minimized with wꢃ1 ¼ ½ꢄ2ðFoÞ þ ð0:0511PÞ þ 1:1072Pꢅ, where
2
2
P ¼ ðFo þ 2Fc Þ=3. All non-hydrogen atoms were refined with anisotropic thermal parameters. The
coordinates of the hydrogen atoms involved in hydrogen bonds N–Hꢂ ꢂ ꢂCl, N–Hꢂ ꢂ ꢂO, O–Hꢂ ꢂ ꢂCl,
namely H11A, H11B, H12A, H12B, H1W, and H2W were found in difference Fourier synthesis and
their positional parameters were refined. The positions of the other hydrogen atoms were generated
geometrically and refined as a riding model. Thermal parameters of all hydrogen atoms were calcu-
lated as 1.2 times Ueq of the respective carrier carbon atom. An empirical ꢃex1=ti4nction correction was
0
2
also applied according to the formula Fc ¼ kFc½1 þ ð0:001ꢉFc ꢊ3=sin2ꢃÞꢅ
[32], and the extinc-
tion coefficient ꢉ was equal to 0.0039(6). Crystallographic data were deposited at the Cambridge
Crystallographic Data Center as supplementary publications No CCDC 600091.
Molecular Modelling
Both crystallographic and optimized atom coordinates for 1 and only optimized for 2–5, 1b, and 5b
were used for computation of shielding constants ꢄ=ppm of 13C atoms to assign the resonances in
solid-state NMR spectra. We have employed the DFT method with B3LYP=6-311 þ (d,p) hybrid
functional for structure optimization of cations of 1–5, and B3LYP=6-31G(d,p) for neutral molecules
1b and 5b, and the CHF–GIAO approach for the NMR shielding constants computations using the
Gaussian 98 program [33].
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¨
Verleger: Springer-Verlag GmbH, Sachsenplatz 4–6, 1201 Wien, Austria. – Herausgeber: Osterreichische Akademie der Wissen-
¨
schaften, Dr.-Ignaz-Seipel-Platz 2, 1010 Wien, und Gesellschaft Osterreichischer Chemiker, Eschenbachgasse 9, 1010 Wien,
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