Branched-chain and unsaturated 1,7-diaminoheptane derivatives as deoxyhypusine synthase inhibitors

Article abstract of DOI:10.1016/S0968-0896(97)10030-X

Deoxyhypusine synthase catalyzes the first step in the posttranslational biosynthesis of the unusual amino acid hypusine [Nε-(4-amino-2- hydroxybutyl)lysine] in eukaryotic translation initiation factor 5A (eIF- 5A). elF-5A and its single hypusine residue are essential for cell proliferation. Two series of 1,7-diaminoheptane derivatives were prepared and tested as inhibitors of human deoxyhypusine synthase. These include branched- chain saturated derivatives and both branched- and straight-chain unsaturated derivatives providing size and positional variation in branching and different torsional constraints. Of the branched-chain compounds, 7-amino-1- guanidinooctane (39) proved to be the most potent inhibitor in vitro (IC₅₀, 34 nM), while 1,7-diamino-trans-hept-3-ene (20a) displayed the greatest inhibition (IC₅₀, 0.7 μM) among the unsaturated compounds. Compound 39 also provided effective inhibition of hypusine production in Chinese hamster ovary cells in culture. Considerations of the in vitro inhibition data reported here, along with earlier findings, allowed some speculation concerning the conformation of the substrate spermidine during its productive interaction at the active site of deoxyhypusine synthase.

Full text of DOI:10.1016/S0968-0896(97)10030-X

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 253±270
Branched-chain and Unsaturated 1,7-Diaminoheptane
Derivatives as Deoxyhypusine Synthase Inhibitors
Young Bok Leey and J. E. Folk*
Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, NIH, Bethesda, MD 20892-4340, U.S.A.
Received 17 July 1997; accepted 6 October 1997
AbstractÐDeoxyhypusine synthase catalyzes the ®rst step in the posttranslational biosynthesis of the unusual amino
acid hypusine [Ne-(4-amino-2-hydroxybutyl)lysine] in eukaryotic translation initiation factor 5A (eIF-5A). eIF-5A and
its single hypusine residue are essential for cell proliferation. Two series of 1,7-diaminoheptane derivatives were pre-
pared and tested as inhibitors of human deoxyhypusine synthase. These include branched-chain saturated derivatives
and both branched- and straight-chain unsaturated derivatives providing size and positional variation in branching and
di€erent torsional constraints. Of the branched-chain compounds, 7-amino-1-guanidinooctane (39) proved to be the
most potent inhibitor in vitro (IC50, 34 nM), while 1,7-diamino-trans-hept-3-ene (20a) displayed the greatest inhibition
(IC50, 0.7 mM) among the unsaturated compounds. Compound 39 also provided e€ective inhibition of hypusine pro-
duction in Chinese hamster ovary cells in culture. Considerations of the in vitro inhibition data reported here, along
with earlier ®ndings, allowed some speculation concerning the conformation of the substrate spermidine during its
productive interaction at the active site of deoxyhypusine synthase. Published by Elsevier Science Ltd.
Introduction
that this protein and the hypusine modi®cation, which
marks its maturation, are vital for proliferation in all
eukaryotic species.2 Yeast cells are not viable if the two
genes for eIF-5A are inactivated4 and disruption of the
deoxyhypusine synthase gene in these cells results in
cessation of growth.5 E€ective inhibitors of deoxy-
hypusine synthase prevent the production of hypusine in
eIF-5A in cells6±8 and have been shown to arrest multi-
plication of mammalian cells, both normal and cancer,
in culture.7±9 Hence it was concluded that compounds
targeted speci®cally to inhibit deoxyhypusine synthase
could prove useful as novel antiproliferative agents.
In consideration of recent reports that eIF-5A may act
in HIV-1 replication as a Rev transactivator protein10
and may be implicated as a cofactor for Rex function in
HTLV replication,11 compounds that inhibit deoxy-
hypusine synthase may also bear potential as anti-HIV-
1 and anti-HTLV agents.
The amino acid hypusine (I) [Ne-(4-amino-2-hydroxy-
butyl)lysine], which is unique to eukaryotic translation
initiation factor 5A (eIF-5A), is formed post-
translationally from the polyamine spermidine (II).1,2
Transfer of the butylamine moiety of this polyamine to
the e-amino group of one particular lysine residue of the
eIF-5A precursor (Lys50 in human eIF-5A precursor) to
produce deoxyhypusine (III) [Ne-(4-aminobutyl)lysine]
is the ®rst step in hypusine biosynthesis and is catalyzed
by the enzyme deoxyhypusine synthase.1,2 eIF-5A and
the enzymes that catalyze its hypusine modi®cation are
abundant in all eukaryotic cells. Despite indication that
eIF-5A, unlike other initiation factors, is not required
for global protein synthesis,3 there is mounting evidence
Key words: Deoxyhypusine synthase inhibitors; branched chain
1,7-diaminoheptanes; unsaturated derivatives of 1,7-diamino-
heptane; diamines; diguanidines
*To whom reprint requests should be addressed at: Fax: 301-
402-0823; e-mail: jfolk@yoda.nidr.nih.gov
yCurrent address: Department of Anatomy and Neurobiology,
School of Medicine, University of Maryland at Baltimore, U.S.A.
OH
H
N
COOH
NH2
H2N
I
0968-0896/98/$19.00 Published by Elsevier Science Ltd.
PII: S0968-0896(97)10030-X

254
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
1a,13 1b,14 1d,15 and 1e (racemic and meso)16 and 1c (bp
H
N
115±118 ꢀC/0.5 Torr, prepared in 80% yield from 3-
methyl-1,5-dibromopentane17 by treatment with NaCN
in DMSO18) to the diamines 2a±e was achieved by
reduction with borane/THF. Reaction of 2c and 2d with
equivalent amounts of S-methylisothiouronium sulfate
yielded monoguanylated diamines 4c and 4d, respec-
tively; treatment of 2a±e with excess amounts of the
reagent gave the diguanidines 3a±e.
NH2
H2N
II
H
N
COOH
NH2
H2N
III
Scheme 2 outlines the preparation of the diamines
and diguanidines of Table 2 with unsaturated bonds at
the 2 position. 7-(2-Tetrahydropyranyloxy)-2-heptyn-1-ol
(5) was prepared in 85% yield by reaction of the
Grignard derivative of 1-(2-tetrahydropyranyloxy)-5-
hexyne19 with formaldehyde in a 1:1 mixture of THF
and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone
(DMPU). A less satisfactory yield of 5 (50±60%) was
obtained by Ferdinandi and Just20 using a di€erent
procedure. Removal of the THP protecting group of 5
a€orded diol 6. Our overall yield of 6 (65%) is
superior to that (38%) reported by Crombie et al.21 for
6 prepared directly from 5-hexyn-1-ol. Reaction of 6
with mesyl chloride in methylene chloride provided 7,
which was converted to diazide 8 by addition of NaN3
in DMSO. Selective reduction of the azido groups of 8
to amino groups without e€ect on the alkyne function-
ality was accomplished with triphenylphosphine in aqu-
eous THF22 to yield 9. Transformation of 9 to trans
diaminoalkene 10 was conducted by reduction with
LiAlH4 in THF and conversion of 9 to cis double-bon-
ded amine 11 by hydrogenation using Lindlar catalyst
(Pd-PbO-CaCO3). Guanylation of 9, 10, and 11 with O-
methylisourea hydrogen sulfate a€orded the unsatu-
rated guanidines 12, 13, and 14, respectively.
In our search for inhibitors of deoxyhypusine synthase,
attention was focused toward compounds structurally
related to the substrate spermidine.6,7 The mono-
guanylated
diamine,
1-amino-7-guanidinoheptane
(GC7), was found to display ecient inhibition in vitro
through competition with spermidine for binding to
enzyme.6 In Chinese hamster ovary cells, GC7 was a
remarkable inhibitor causing complete inhibition of
hypusine biosynthesis and curtailment of growth at con-
centrations as low as 1 mM in the medium.8 Growth
arrest without signi®cant reduction in cellular spermi-
dine or eIF-5A precursor underlines the critical role of
hypusine in cell proliferation.8 The ecient entry of
GC7 into cells by way of the polyamine transport sys-
tem8 is an additional advantage arising from its struc-
tural relationship to spermidine.
Besides their value as exogenous regulators of cellular
function, deoxyhypusine synthase inhibitors that act as
a consequence of their structural similarity to spermi-
dine o€er promise for further understanding of enzyme
mechanism. Early ®ndings with compounds similar in
structure to spermidine led to speculation on features of
the spermidine-binding site of the enzyme.6 The crystal
structure of a complex of deoxyhypusine synthase with
bound NAD12 recently determined by X-ray analysis
has opened the way to a comprehensive computer-
assisted study of this site and of the enzyme's catalytic
mechanism. In conjunction with this study, as well as in
a continuing e€ort to ®nd more potent compounds to
test as antiproliferative drugs, and because of the simple
structure and potent inhibitory activity of GC7, we were
prompted to synthesize and test a number of modi®ed
1,7-diaminoheptanes. Here we describe the work lead-
ing to the identi®cation of 7-amino-1-guanidinooctane
(39) as a new lead compound.
The acetylenic and ole®nic diamines and diguanidines of
Table 2, in which unsaturation is at the 3 or 4 position,
were accessible through ring opening of oxiranes with
the lithio derivative of 1-(2-tetrahydropyranyloxy)-4-
pentyne23 in mixtures of THF and a dipolar aprotic
cosolvent. Addition of ethylene oxide to a 50% DMPU/
THF solution of the pentynyllithium yielded the achiral
7-(2-tetrahydropyranyloxy)-3-heptyn-1-ol (15a) in 75%
yield. A less satisfactory yield of 15a (45%) was repor-
ted by Grob and Moesch24 with the use of ethylene
oxide and the Grignard derivative of 1-(2-tetra-
hydropyranyloxy)-4-pentyne. Using the racemic forms
of propylene oxide and 1,2-epoxybutane in 13% hexa-
methylphosphoramide (HMPA)/THF solutions yields
of 68% and 40% were realized for the racemic octynol
and nonynol derivatives, 15b and 15c, respectively. In
order to de®ne the speci®city of enzyme-inhibitor inter-
action, which will be discussed later, we employed
optically pure (S)-(-)-propylene oxide to prepare the
(S)-enantiomer of 15b, (S)-15b, in 65% yield as a
Chemistry
The saturated branched-chain diamines and their gua-
nylated derivatives of Table 1, with the exception of
those with a branch on the same carbon atom as one of
the basic groups, were prepared as outlined in Scheme 1.
Conversion of the known branched-chain pimelonitriles

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
255
Table 1. Physical and deoxyhypusine synthase inhibitory properties of branched-chain derivatives of 1,7-diaminoheptane
Compound
Structurea
Yield %
(methods)b
mp ꢀC
Recryst.c
solvent
Formula
Anal.d
IC50, mM
90e(O)
87f(O)
74g(O)
44(A)
60(A)
55(A)
163±4
A
A
A
A
A
A
C,H,N,Cl
2.5‹0.2
13.2‹1.0
431‹137
69.8‹5.7
46.9‹9.6
>1000
.
C8H20N2 2HCl
CH3
CH3
25b
H2N
H2N
NH2
NH2
.
C8H20N2 2HCl
(S)-25b
25c
2a
197±8
148
C,H,N,Cl
C,H,N,Cl
C,H,N,Cl
C,H,N,Cl
C,H,N,Cl
.
C9H22N2 2HCl
C2H5
H2N
H2N
NH2
NH2
.
C8H20N2 2HCl
150
CH3
.
C8H20N2 2HCl
CH3
2b
225
H2N
H2N
NH2
NH2
.
C8H20N2 2HCl
2c
248
CH3
.
C9H22N2 2HCl
2d
2e
66(A)
65(A)
236
A
B
C,H,N,Cl
C,H,N,Cl
>1000
>1000
H2N
NH2
H3C
CH3
.
C9H22N2 2HCl
CH3 CH3
233±4
H2N
NH2
NH2
.
C9H22N2 2HCl
CH3
meso-2e
68(A)
196
A
C
C,H,N,Cl
>1000
H2N
CH3
.
C10H24N6 H2SO4
CH3
26b
40(K)
33(K)
40(K)
45(B)
284±5
glass
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
0.41‹0.08
1.3‹0.1
1.1‹0.2
R
R
R
R
.
C10H24N6 H2SO4
CH3
(S)-26b
26c
.
C11H26N6 H2SO4
C2H5
240±4 dec
295 dec
C
R
R
R
R
.
C10H24N6 H2SO4
3a
D
48.5‹3.1
CH3
(continued)

256
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
Table 1Ðcontd
Compound
Structurea
Yield %
(methods)b
mp ꢀC
Recryst.c
solvent
Formula
.
Anal.d
IC50, mM
CH3
3b
50(B)
44(B)
40(B)
25(B)
38(B)
307±8 dec
C
C
C
E
C
C10H24N6 H2SO4 C,H,N,S,
24.6‹1.9
R
R
R
R
.
C10H24N6 H2SO4
3c
294 dec
305 dec
C,H,N,S
C,H,N,S
C,H,N,S
C,H,N,S
723‹160
706‹126
>1000
CH3
.
C11H26N6 H2SO4
3d
R
R
R
H3C
CH3
.
C11H26N6 H2SO4
3e
CH3 CH3
295 dec
R
R
.
C11H26N6 H2SO4
CH3
meso-3e
272±4 dec
>1000
R
CH3
h
.
C9H22N4 2HCl
4c
4d
43(C)
27(C)
140
oil
C,H,N,Cl
C,H,N,Cl
17.2‹3.0
17.8‹1.0
H2N
R
CH3
.
C10H24N4 2HCl
H2N
R
H3C
CH3
8i
269±70
glass
C
C,H,N,S 0.034‹0.006
.
C9H22N4 H2SO4
39
40
CH3
H2N
R
13i
C,H,N,Cl
5.8‹1.2
j
.
C9H22N4 2HCl
CH3
R
NH2
aR=-NHC(=NH)NH2.
bThe methods of preparation designated by the letters refer to general methods given in the Experimental.
cA, ethanol±ether; B, abs. Ethanol; C, water±ethanol; D, water-methanol; E, water.
dAnalytical results were within 0.4% of the theoretical value.
eYield from 19b (Table 2).
fYield from (S)-19b (Table 2).
gYield from 19c (Table 2).
hMonohydrate.
iOverall yield from 15b (Experimental).
precursor of (S)-19b, (S)-22b, (S)-25b, and (S)-26b.
(2R)-8-(2-Tetrahydropyranyloxy)-4-octyn-2-ol, the (R)-
enantiomer of 15b, was prepared by Hillis and Ronald25
in 69% yield by this procedure. Our [a]D of+9.9ꢀ for
(S)-15b is consistent with that of 10ꢀ reported for the
(R)-form of compound 15b.25 The partially blocked
alkyne diols 15a±c were converted to the diaminoalk-
ynes 19a±c by employing the same series of procedures
described above in Scheme 2 for transformation of
alcohol 5 to amine 9. Re¯uxing of 19a with LiAlH4 in

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
257
Table 2. Physical and deoxyhypusine synthase inhibitory properties of unsaturated derivatives of 1,7-diaminoheptane
Compound
Structurea
Yield %
(methods)b
mp ꢀC Recryst.c
solvent
Formula
Anal.d
IC50, mM
.
C7H14N2 2HCl
9
22e (D,E,F,G,H) 204±5
A
A
A
C,H,N,Cl 621‹14
H2N
NH2
NH2
.
C7H14N2 2HCl
19a
19b
31f(L,E,F,G,H)
23f (L,E,F,G,H)
192
C,H,N,Cl
C,H,N,Cl
1‹0.05
21‹3
H2N
.
C8H16N2 2HCl
H3C
H2N
126±7
NH2
NH2
NH2
22f (L,E,F,G,H)
9f (L,E,F,G,H)
156
150
A
A
C,H,N,Cl 12.3‹0.6
C,H,N,Cl 200‹44
.
C8H16N2 2HCl
H3C
H2N
(S)-19b
.
C9H18N2 2HCl
C2H5
H2N
19c
.
C9H18N6 H2SO4 C,H,N,S
12
40 (K)
41 (K)
83 (N)
66 (N)
268 dec
280 dec
glass
B
B
248‹14
5.6‹0.4
31‹1
R
R
R
.
C9H18N6 H2SO4 C,H,N,S
22a
R
g
.
C10H20N6 2HCl
H3C
22b
C,H,N,Cl
C,H,N,Cl
R
R
R
g
.
C10H20N6 2HCl
H3C
(S)-22b
glass
24‹2
R
.
C7H16N2 2HCl
10
30 (I)
22 (I)
243
238
oil
A
A
C,H,N,Cl 41.9‹2.4
C,H,N,Cl 0.73‹0.06
H2N
NH2
NH2
.
C7H16N2 2HCl
20a
20b
H2N
.
C8H18N2 2HCl
CH3
24 (M)
C,H,N,Cl
8‹0.5
H2N
NH2
.
C9H20N6 H2SO4 C,H,N,S
13
23
54 (K)
77(K)
325 dec
>350
B
B
25‹1.6
R
R
R
R
.
C9H20N6 H2SO4 C,H,N,S
6.4‹0.4
(continued)

258
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
Table 2Ðcontd
Compound
Structurea
Yield %
(methods)b
mp ꢀC Recryst
solventc
Formula
Anal.d
IC50, mM
.
C7H16N2 2HCl
NH2
NH2
11
72(J)
73 (J)
50(J)
190±2
204±6
128±32
C
C
A
C,H,N,Cl 16.8‹2.5
C,H,N,Cl 20.8‹3.0
C,H,N,Cl 107‹21
H2N
H2N
.
C7H16N2 2HCl
21a
21b
.
C8H18N2 2HCl
CH3
NH2
H2N
.
C9H20N6 H2SO4 C,H,N,S
R
14
24
74(K)
315 dec
315 dec
B
B
37‹3
23‹3
R
.
C9H20N6 H2SO4 C,H,N,S
82 (K)
R
R
aR=-NHC(=NH)NH2.
bThe methods of preparation designated by the letters refer to general methods given in Experimental.
cA, ethanol±ether; B, water±ethanol; C, methanol±ether.
dAnalytical results were within 0.4% of the theoretical value.
eOverall yield from THP-hexyne.
fOverall yield from THP-pentyne.
gHemihydrate.
THF provided a moderate yield of trans diaminoalkene
20a, whereas no 20b was produced even upon prolonged
treatment of 19b under these conditions. Use of the
reducing agent in diglyme at re¯ux temperature gave the
target compound 20b in acceptable yield. Hydrogena-
tion of 19a and 19b using Lindlar catalyst yielded the cis
double-bonded diamines 21a and 21b, respectively.
Reaction of the unsaturated 1,7-diaminoheptanes 19a,
20a, and 21a with O-methylisourea hydrogen sulfate
a€orded diguanidines 22a, 23, and 24, whereas use of
this reagent with 1,7-diaminooctynes 19b and (S)-19b
gave only low yields of impure products; their treatment
with the more reactive guanylating reagent 1H-pyrazole-
1-carboxamidine. HCl in DMF containing 2 equivalents
of diisopropylethylamine a€orded 22b and (S)-22b in
good yields (83 and 66%, respectively).
The racemic form of 7-amino-1-guanidinooctane (39)
and that of 1-amino-7-guanidinooctane (40) (Table 1)
were prepared as outlined in Scheme 5 starting with
racemic 8-(2-tetrahydropyranyloxy)-4-octyn-2-ol (15b).
The key intermediate, 1-amino-7-tert-butoxycarbonyl
(t-Boc)aminooctane (34), was obtained through trans-
formation of 15b to 1-(2-tetrahydropyranyloxy)-7-amino-
octane (29) by a series of reactions involving formation
of mesylate 27, its conversion to azide 28 and simulta-
neous reduction of the multiple bond and production of
amine by catalytic hydrogenation. After protection of
the amino group of 29 by reaction with di-tert-butyldi-
carbonate to form 30, selective removal of the THP
blocking group to produce 31 was followed by another
series of reactions involving conversion of the alcohol to
the mesylate 32, its transformation to the azide 33 and
®nally catalytic hydrogenation to yield the mono
blocked diamine 34. Conversion of its free amino
group to a guanidino group to form 35 by the use of
O-methylisourea hydrogen sulfate was followed by
removal of the t-Boc group with TFA yielding one of the
target compounds 39. Alternative amino group blocking
allowed selective modi®cation of the other amino
group and hence synthesis of the second target com-
pound. Introduction of the benzyloxycarbonyl group
on the free amino group of 34 upon its treatment with
Scheme 4 describes the preparation of the 8- and 9-car-
bon saturated 1,7-diamines and diguanidines of Table 1.
Catalytic hydrogenation of the diaminoalkynes 19b and
19c using Adams catalyst (PtO2) a€orded 1,7-diamino-
octane (25b) and 1,7-diaminononane (25c), respectively,
through complete reduction of the triple bonds. Gua-
nylation of these diamines with O-methylisourea
hydrogen sulfate provided the guanidine compounds
26b and 26c.

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
259
R1
R3 R4
NC
CN
R2
R5
i.
R1
1a-e
R3 R4
H2N
NH2
R2
R5
2a-e
ii.
R1
R3
R2
R4
R5
H2NC(=HN)HN
R'
3a-e, R' = NHC(=NH)NH2
4c,d, R' = NH2
a, R1 = CH3, R2 = R3 = R4 = R5 = H
b, R2 = CH3, R1 = R3 = R4 = R5 = H
c, R3 = CH3, R1 = R2 = R4 = R5 = H
d, R3 = R4 = CH3, R1 = R2 = R5 = H
e, R2 = R5 = CH3, R1 = R3 = R4 = H
.
Scheme 1. (i) BH3, THF, ~; (ii) [CH3SC(=NH)NH2]2 H2SO4, H2O, ~.
OH
i
O
O
O
O
5
ii
R
OH
iii, iv, v
R
7. R = Ms
8. R = N3
9. R = NH2
HO
6
vii
vi
NH2
H2N
viii
11
H2N
NH2
H2NC(=NH)HN
NHC(=NH)NH2
viii
10
12
viii
NHC(=NH)NH2
H2NC(=NH)NH
14
H2NC(=NH)NH
NHC(=NH)NH2
13
Scheme 2. (i) C2H5MgBr, THF, ~, DMPU, HCHO; (ii) PPTS, EtOH; (iii) CH3SO2Cl, TEA, CH2Cl2; (iv) NaN3, DMSO; (v) Ph3P,
.
THF, H2O; (vi) LiAlH4, THF, ~; (vii) H2, Pd (Lindlar Catalyst), EtOH; (viii) CH3OC(=NH)NH2 H2SO4, MeOH, TEA.

260
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
i, ii
R
O
O
O
O
OH
iii
15a-c
R
HO
iv, v, vi
R
R'
OH
16a-c
R'
17a-c, R' = Ms
18a-c, R' = N3
19a-c, R' = NH2
viii
vii
NH2
H2N
R
R
H2N
NH2
21a,b
20a,b
ix
ix
ix
NHC(=NH)NH2
H2NC(=NH)HN
R
H2NC(=NH)HN
NHC(=NH)NH2
24
23
H2NC(=NH)HN
NHC(=NH)NH2
22a,b
a, R = H; b, R = CH3; c, R = C2H5
Scheme 3. (i) nBuLi, THF; (ii) (a) DMPU, ethylene oxide; (b) HMPA, propylene oxide, (c) HMPA, 1,2-epoxybutane; (iii) PPTS,
EtOH; (iv) CH3SO2Cl, TEA, CH2Cl2; (v) NaN3, DSMO; (vi) Ph3P, THF, H2O; (vii) (a) LiAlH4, THF, (b) LiAlH4, diglyme; (viii) H2,
.
Pd (Lindlar Catalyst), EtOH; (ix) (a) CH3OC(=NH)NH2 H2SO4, MeOH, TEA, (b) 1H-pyrazole-C(=NH)NH2, DIEA, DMF.
N-(benzyloxycarbonyloxy)succinimide to give 36 was
followed by selective removal of the t-Boc blocking group
with TFA a€ording 37. Guanylation of this alternatively
blocked diamine with 1H-pyrazole-1-carboxamidine to
produce 38 and removal of the benzyloxycarbonyl
group by catalytic hydrogenation provided 40.
inhibitors of deoxyhypusine synthase. For comparison,
Table 3 gives the inhibitor properties of 1,7-diamino-
heptane (C7) and its mono and bis guanylated deriva-
tives, GC7 and GC7G, respectively, determined in
parallel with those of the compounds of Tables 1 and 2.
The competitive-type inhibition exerted by C7, GC7,and
GC7G against spermidine, together with their simila-
rities in structure to this polyamine substrate, led us to
the earlier conclusion that binding of each of these
compounds occurs at the same site on the enzyme.6,7
Because the present compounds also bear strong struc-
tural resemblance to spermidine, it is assumed that they
too interact with deoxyhypusine synthase at the site
normally occupied by this substrate.
Results and Discussion
Table 1 lists the properties of the branched-chain satu-
rated 1,7-diaminoheptane derivatives and Table 2 those
of both straight- and branched-chain unsaturated 1,7-
diaminoheptane derivatives prepared and evaluated as

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
261
R
R
H2N
i
H2N
NH2
NH2
19b,c
25b,c
ii
R
H2NC(=NH)HN
NHC(=NH)NH2
26b,c
b, R = CH3; c, R = C2H5
.
Scheme 4. (i) H2, PtO2 (Adams Catalyst), EtOH; (ii) CH3OC(=NH)NH2 H2SO4, MeOH, TEA.
CH3
O
i,ii
O
OH
15b
CH3
R
O
O
27. R = Ms
28. R = N3
iii, iv
CH3
R
CH3
NHBOC
v
O
O
HO
31
29. R=NH2
30. R=NHBOC
CH3
CH3
NHBOC
vii, viii, ix
ZNH
R
R
32. R = Ms
33. R = N3
34. R = H2N
36. R = NHBOC
37. R = NH2
38. R = NHC(=NH)NH2
CH3
R
x
H2NC(=NH)HN
39
CH3
35. R = NHBOC
39. R = NH2
H2N
NHC(=NH)NH2
40
Scheme 5. (i) CH3SO2Cl, CH2Cl2, TEA; (ii) NaN3, DMSO; (iii) H2, PtO2 (Adams Catalyst), EtOH; (iv) (BOC)2O, DMF; (v) PPTS,
EtOH; (vi) CH3OC(=NH)NH2 H2SO4, MeOH, TEA; (vii) ZOSu, CHCl3; (viii) TFA, 0 ꢀC; (ix) 1H-pyrazole-C(=NH)NH2, DIEA,
DMF; (x) H2, Pd black, MeOH.
.
Certain branched-chain and unsaturated derivatives of
spermidine, speci®cally 1-methylspermidine [N-(3-amino-
deoxyhypusine synthase substrates. Others, namely
1,1-dimethylspermidine2 and N-(3-aminopropyl)-1,4-
diamino-2-butyne,27 do not act as substrates, but were
shown to display some degree of inhibition. Hence, the
compounds listed in Tables 1 and 2 were designed with
butyl)-1,4-diaminobutane],26
N-(3-aminopropyl)-1,4-
diamino-cis-2-butene,27 and N-(3-aminopropyl)-1,4-di-
amino-trans-2-butene,27 were reported to function as

262
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
Table 3. Deoxyhypusine synthase inhibitory properties of 1,7-diaminoheptane and its guanylated derivatives
Compound
C7
Structurea
IC50, mM
4.5‹0.5
H2N
NH2
GC7G
GC7
3.6‹0.2
R
R
0.05‹0.0006
R
NH2
aR=NHC(=NH)NH2.
the knowledge that the enzyme possesses tolerance at its
spermidine binding site for a degree of carbon chain
branching and unsaturation.
provides it additional binding strength28 so that its
association with enzyme may, indeed, be as strong as, or
stronger than, that of the ammonium group. Thus, the
dramatic increase in the e€ectiveness of inhibition
experienced with diguanidine 26c over diamine 25c may
be explained by a change in carbon chain alignment
caused by guanidinium group binding. The adjustment
in chain position resulting from charge delocalization
and/or size of the two basic centers could allow ready
accommodation of the ethyl group of 26c at that region
of the enzyme that accepts the methyl group of diamine
25b even though the ethyl group might be only partially
positioned within this region. Basic center binding
could, in a similar way, cause the increases in inhibition
seen with the other branched chain diguanidines over
their parent diamines (Table 1), although in cases other
than 25b versus 26b it is unlikely that there are speci®c
sites for interaction of inhibitor methyl branches with
enzyme. The comparable degrees of inhibition given by
the unbranched compounds C7 and GC7G are in accor-
dance with this suggestion and indicate that the binding
force exerted by guanidinium groups at both terminal
positions in heptane derivatives approximates that
exerted by ammonium groups at these positions.
Comparison of the inhibitory properties of the com-
pounds of Table 1 with those of C7 and its guanyl deri-
vatives (Table 3) reveals that diamines 2a, 2b, and 2c
with methyl branches at positions 2, 3, and 4, respec-
tively, of the 1,7-diaminoheptane carbon chain, 2d with
2 methyl groups at the 4 position and 2e and meso-2e
with one methyl group at position 3 and one at position
5 display either no signi®cant degree of inhibition or
much poorer inhibition than unbranched 1,7-diamino-
heptane (C7). The same relationship is seen with diguani-
dines 3a±e versus 1,7-diguanidinoheptane (GC7G),
although in each case, except 3e and meso-3e, the guani-
dines provide better inhibition than their amine counter-
parts. On the other hand, 1,7-diaminooctane (25b),
the methyl group of which is on the same carbon as one
of its amino groups, is a better inhibitor than the parent
compound, C7. The same relationship is seen with
diguanidines 26b and GC7G. This, together with the fact
that the racemic compound, 25b, and the enantiomer,
(S)-25b, di€er signi®cantly in inhibitory properties, as
do 26b and (S)-26b, is evidence of a speci®c interaction
of the methyl branch at this position with the active site
of the enzyme. The very restricted nature of this inter-
action is de®ned by the poor anity of 25c, which con-
tains an ethyl group in place of the methyl group of 25b.
Thus, that region of the enzyme's active site that e€ec-
tively accommodates the methyl branch of 25b is of
insucient size to allow ready access of an ethyl group.
This exclusion of the ethyl group is relaxed, however,
when it is oriented at the same relative position in
diguanidine 26c; this compound provides excellent inhi-
bition. The size of the guanidinium group is greater than
that of the ammonium group and its charge is more
delocalized.28 For this reason the guanidinium group
binds less strongly to anionic ligands. However, its
ability to form pairs of zwitterionic hydrogen bonds
In earlier studies with straight chain inhibitors of deox-
yhypusine synthase it was found that with those in
which separation of the two required terminal basic
centers was most favorable for inhibition, that is, by an
unbranched 7- or 8-carbon chain or by a straight 7-
member chain in which the central carbon atom was
replaced by O, S, or N, the combination of one guani-
dinium group and one ammonium group provided by
far the most potent inhibition.6,7 It was concluded on
the basis of this ®nding that, even though both sites on
the enzyme that normally bind the primary amino
groups of spermidine can accommodate guanidino
groups, one of these sites favors a guanidino groups
over an amino group, while the other strongly prefers
the amino group.6 This preference is borne out here in

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
263
the case of the easily prepared monoguanylated dia-
c
mines 4c and 4d, which are far superior as inhibitors to
diamines 2c and 2d and diguanidines 3c and 3d. Thus,
we were prompted to compare the inhibition given by
the monoguanylated forms of 1,7-diaminooctane, 39
and 40. Synthesis of these isomers, albeit somewhat
involved, was undertaken with the assurance of reveal-
ing that site on the enzyme at which speci®c interaction
with inhibitor methyl branch occurs. Because 25b and
26b are stronger inhibitors than are C7 and GC7G,
respectively, we also hoped to obtain an inhibitor
superior to GC7. From the di€erence in IC50 values
between 39 and 40 it is evident that methyl-branch
interaction occurs in close vicinity to the site on the
enzyme that prefers the amino group, as depicted in
Figure 1. We speculated earlier that this is the site with
which the amino group at the aminopropyl end of sper-
midine interacts during its productive binding.6 Strong
support for this binding mode comes from a recent
report that 1-methylspermidine, the methyl branch of
which is on the same carbon as the primary amino
group at its aminopropyl end, is a substrate for deox-
yhypusine synthase.26
a
b
NH2
H2N
N
H
Spermidine
CH3
NH2
H2N
N
H
1-Methylspermidine
CH3
H2N
NHC(=NH)NH2
39
H2N
H2N
N
H
NH2
N-(3-Aminopropyl)-
1,4-diamino-2-butyne
Careful testing showed 39 to be a somewhat better
inhibitor than GC7. Although its increase in potency of
inhibition over GC7 as re¯ected in the IC50 values was
not as pronounced as hoped for, 39 did give noticeably
greater reductions in hypusine biosynthesis in cultured
Chinese hamster ovary cells. Reductions of 20 and 60%
were realized after 18 h at levels of 0.1 and 0.3 mM,
respectively, in the medium as compared to 0 and 8%
for GC7 at the same respective levels; essentially com-
plete inhibition of hypusine production was seen after
18 h at a 1 mM level of either inhibitor.
NH2
NH2
9
19a
H2N
Figure 1. Proposed orientation of representative compounds at
the spermidine binding site of deoxyhypusine synthase. a and b
represent those portions of the site (depicted above the struc-
tures of the compounds) that bind the primary amino groups of
spermidine; the amino group at the aminopropyl end of sper-
midine interacts with a, which prefers an amino group, whereas
the aminobutyl moiety of spermidine is directed toward b,
which favors a guanidino group. c represents that portion of
the binding site that interacts speci®cally with a methyl group
on the same carbon of a compound as (one of) its primary
amino group(s).
Examination of the IC50 values of Table 2 shows several
striking features of the inhibition given by unsaturated
forms of 1,7-diaminoheptane and its derivatives. Per-
haps the most notable of these are the pronounced
e€ects on inhibition of the position of the unsaturated
bond in the carbon chain and of carbon chain methyl
branching. The unsaturated bond in each of these com-
pounds is either in the 2 or in the 3 position from a basic
center and each compound is either a diamine or a
diguanidine. Therefore, if these compounds, as believed,
inhibit by binding in the position normally occupied by
spermidine at the active center of deoxyhypusine syn-
thase, it seems obvious that they can be oriented in
either of two directions (i.e., either with their unsatu-
rated bond within the active site region in which the
aminopropyl portion of spermidine binds or within that
region in which the aminobutyl part binds). Spermidine
derivatives with double or triple bonds at the C-6-7
position act as substrates or inhibitors for deoxy-
hypusine synthase.27 Since the unsaturated bond in each
of these spermidine derivatives is located at position 2
from the aminobutyl end, one might presume that each
of those compounds of Table 2 with unsaturation at
position 2 (i.e., compounds 9, 12, 10, 13, 11, and 14) can
bind to enzyme with its unsaturated bond in the ami-
nobutyl region of the spermidine binding site, as depic-
ted for compound 9 in Figure 1. Indeed, the IC50 values
for these inhibitors are not inconsistent with the
approximate relative inhibitory properties reported for
the unsaturated spermidine derivatives. The poorest of
the inhibitors with unsaturation at position 2 are those
with the triple bonds, (i.e., 9 and 12) while the poorest
inhibition by the unsaturated spermidine derivatives is

264
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
that given by the derivative with the triple bond at the
C-6-7 position.27
mesh). 1H NMR spectra were recorded on a Varian
Gemini 300 spectrometer. Chemical shifts are expressed
relative to internal standard TMS or HOD at 4.8 ppm
(D2O). Mass spectra were obtained on a JEOL SX 102
spectrometer (FAB). The optical rotations were recor-
ded at 20ꢀ using a Perkin-Elmer 341 polarimeter with a
1 dm cuvette. Elemental analyses were performed by
Atlantic Microlabs, Inc., Norcross, Georgia, and were
within 0.4% of the theoretical values.
It is more dicult to speculate as to the mode of binding
of those inhibitors that contain unsaturated bonds at
the 3 position. A methyl branch on the terminal carbon
atom nearest the unsaturated bond in each case causes a
pronounced loss in inhibitory potency, for example in
19b versus unbranched 19a and in 20b versus unbran-
ched 20a. This is in contrast to the enhancement of
inhibition caused by a methyl branch at a terminal car-
bon position in the saturated inhibitors (Table 1) and
may signify opposite modes of binding for the saturated
and unsaturated inhibitors. The evidence cited above for
speci®c interaction of the methyl groups at the terminal
carbon atoms of the saturated inhibitors with the ami-
nopropyl end of the enzyme's spermidine binding site,
as indicated by the orientation of compound 39 in
Figure 1, suggests that, like inhibitors with unsaturated
bonds in the 2 position (i.e., 9 in Figure 1) those with
unsaturation at the 3 position may bind to enzyme with
their unsaturated areas within the aminobutyl region of
the spermidine binding site, as shown for 19a in Figure 1.
2-Methyl-1,7-diaminoheptane
dihydrochloride
(2a).
Method A. A solution of pimelonitrile 1a (2.04 g,
15 mmol) in 20 mL of THF was added slowly to BH3-
THF (1 M, 75 mL, 75 mmol) at room temperature under
argon. The reaction was heated at re¯ux for 2 h and
then cooled and carefully hydrolyzed with 6 N HCl
(50 mL). Most of the THF was distilled away and the
solution was cooled, basi®ed (solid NaOH), and extrac-
ted with CHCl3 (5Â20 mL). The combined extracts were
dried over Na2SO4, concentrated and distilled to a€ord
ꢀ
0.95 g (44%) of free amine (bp 60 C/0.9 Torr). A solu-
tion of this amine (2 mmol) in 5 mL of ethanol was
treated with 2 N HCl in ethanol (2.2 mL) and the solvent
removed under vacuum. The remaining solid was
ꢀ
Interestingly, of the unsaturated 1,7-diaminoheptane
derivatives (Table 2), the 3-alkynes 19a and 22a and the
3-trans alkenes 20a and 23 are the most ecient inhibi-
tors. Those derivatives with cis double bonds in the 3
position, 21a and 24, are signi®cantly less e€ective. The
restricted rotations resulting from unsaturation between
carbon atoms confers on these compounds certain spa-
tial characteristics. Thus the con®gurations of the 3-cis
alkenes, 21a and 24, are such that the distances between
carbon atoms adjoining their doubly bonded carbons
are considerably less than those between the carbons
adjoining the triply bonded carbons of 19a and 22a and
the trans doubly bonded carbons of 20a and 23. Since
the latter distances, those in the more e€ective inhibi-
tors, approximate that between terminal carbons of a
saturated 4-carbon chain in its fully extended con-
formation, it seems reasonable to suggest that during
productive binding a sizable portion of the spermidine
molecule is oriented in its binding site in a fully, or
almost fully, extended conformation and that this con-
formation extends to, or includes its central nitrogen
atom.
recrystallized from ethanol-ether to give 2a: mp 150 C,
1H NMR (D2O) d 0.98±1.00 (d, 3H), 1.38±1.50 (m, 6H),
1.67±1.75 (m, 2H), 1.80±1.87 (m, 1H), 2.96±3.03 (m,
4H); MS (FAB, t-glyc) 145 (M+H). Similarly prepared
were 2b (bp of free amine, 58±60 ꢀC/0.6 Torr): 1H NMR
(D2O) d 0.91±0.93 (d, 3H), 1.37±1.45 (m, 4H), 1.50±1.56
(m, 1H), 1.62±1.69 (m, 4H), 2.98±3.06 (m, 4H); MS
(FAB, t-glyc) 145 (M+H); 2c (bp of free amine, 59±60
ꢀC/0.6 Torr): 1H NMR (D2O) d 0.90±0.92 (d, 3H), 1.21±
1.29 (m, 2H), 1.36±1.45 (m, 2H), 1.49±1.53 ( m, 1H),
1.65±1.72 (m, 4H), 2.99 (t, 4H); MS (FAB, t-glyc) 145
(M+H); 2d (bp of free amine, 66±68 ꢀC/0.7 Torr): 1H
NMR (D2O) d 0.90 (s, 6H), 1.25±1.30 (m, 4H), 1.62±1.66
(m, 4H), 2.98 (t, 4H); MS (FAB, t-glyc) 159 (M+H); 2e
(bp of free amine, 65 ꢀC/0.4 Torr): 1H NMR (D2O) d
0.89±0.91 (d, 6H), 1.18 (t, 2H), 1.50±1.55 (m, 2H), 1.60±
1.69 (m, 4H), 3.01±3.07 (m, 4H); MS (FAB, t-glyc) 159
ꢀ
(M+H); meso-2e (bp of free amine, 67±72 C/0.5 Torr):
1H NMR (D2O) d 0.91±0.93 (d, 6H), 1.12±1.20 (m, 1H),
1.23±1.32 (m, 1H), 1.41±1.48 (m, 2H), 1.65±1.73 (m,
4H), 2.97±3.10 (m, 4H); MS (FAB, t-glyc) 159 (M+H).
2-Methyl-1,7-diguanidinoheptane sulfate (3a). Method B.
A mixture of diamine 2a (free base, 0.217 g, 1.5 mmol)
and S-methylisothiuronium sulfate (0.46 g, 3.3 mmol) in
2.5 mL of water was heated at re¯ux for 1 h. After
cooling, 20 mL of absolute ethanol was added. The oil
that formed crystallized upon trituration with ethanol.
Recrystallization from aqueous methanol gave 0.22 g
(45%) of 3a: mp 295 C dec.; H NMR (D2O) d 0.92±
0.94 (d, 3H), 1.33±1.45 (m, 6H), 1.54±1.62 (m, 2H),
1.74±1.80 (m, 1H), 3.02±3.08 (m, 2H), 3.16±3.21 (m,
Experimental
Melting points were determined on a Thomas-Hoover
apparatus in open capillary tubes and are uncorrected.
TLC was performed on Merck silica gel 60 F254 analy-
tical plates visualized with UV, tolidine-Cl2, ninhydrin,
Sakaguchi and/or aqueous KMnO4. Flash chromato-
graphy was conducted on Merck silica gel 60 (230±400
ꢀ
1

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
265
1
2H); MS (FAB, t-glyc) 229 (M+H). Similarly prepared
were: 3b: 1H NMR (D2O) d2 0.89±0.92 (d, 3H), 1.34±
1.47 (m, 5H), 1.56±1.60 (m, 4H), 3.16±3.25 (m, 4H); MS
(FAB, t-glyc) 229 (M+H); 3c: H NMR (D2O) d 0.88±
0.90 (d, 3H), 1.18±1.26 (m, 2H), 1.32±1.42 (m, 2H),
oil: H NMR (CDCl3) d 1.52±1.75 (m, 10H), 2.24±2.29
(m, 2H), 3.38±3.54 (m, 2H), 3.66±3.89 (m, 2H), 4.25 (m,
2H), 4.59 (t, 1H).
1
Hept-2-yn-1,7-diol (6). Method E. A mixture of 5
(15.9 g, 75 mmol) and pyridinium-p-toluene sulfonate
(1.89 g, 7.5 mmol) in 600 mL of 95% ethanol was stirred
at 53±58 ꢀC for 5 h. Following evaporation of solvent,
the residue was ¯ash chromatographed (1/1, acetone/
hexane) to give 7.3 g (76%) of 6 as a colorless oil: 1H
NMR (CDCl3) d 1.55±1.71 (m, 4H), 2.26±2.30 (m, 2H),
3.69 (t, 2H), 4.25 (s, 2H).
1.45±1.51 (m, 1H), 1.54±1.66 (m, 4H), 3.17 (t, 3H); MS
1
(FAB, t-glyc) 229 (M+H); 3d: H NMR (D2O) d 0.87
(s, 6H), 1.21±1.27 (m, 4H), 1.51±1.57 (m, 4H), 3.16 (t,
4H); MS (FAB, t-glyc) 243 (M+H); 3e: 1H NMR
(D2O) d 0.88±0.90 (d, 6H), 1.15 (t, 2H), 1.40±1.48 (m,
2H), 1.55±1.67 (m, 4H), 3.17±3.30 (m, 4H); MS (FAB, t-
glyc) 243 (M+H); meso-3e: 1H NMR (D2O) d 0.89±0.91
(d, 6H), 1.06±1.13 (m, 1H), 1.23±1.30 (m, 1H), 1.33±1.42
(m, 2H), 1.61±1.64 (m, 4H), 3.18±3.25 (m, 4H); MS
(FAB, t-glyc) 243 (M+H).
Hept-2-yn-1,7-dimesylate (7). Method F. A solution of 6
(6.4 g, 50 mmol) and triethylamine (15.2 g, 150 mmol) in
500 mL of methylene chloride was cooled in an ice±salt
bath and mesyl chloride (12.6 g, 110 mmol) was added
over a period of 10 min with vigorous stirring. After an
additional 15 min, the reaction mixture was washed
successively with cold 10% HCl, saturated NaHCO3
and brine, dried over Na2SO4, and evaporated to a€ord
11.5 g (81%) of 7 as a colorless oil, which moved as a
single component on TLC (2/3, ethyl acetate/hexane, Rf
0.26): 1H NMR (CDCl3) d 1.64±1.71 (m, 2H), 1.84±1.91
(m, 2H), 2.31±2.36 (m, 2H), 3.02 (s, 3H), 3.10 (s, 3H),
4.26 (t, 2H), 4.84 (s, 2H).
4-Methyl-1-amino-7-guanidinoheptane dihydrochloride (4c).
Method C. A mixture of diamine 2c (free base, 0.288 g,
2 mmol) and S-methylisothiuronium sulfate (0.278 g,
2 mmol) in 3 mL of water was heated at re¯ux for 1 h.
Puri®cation of 4c was carried out by ion exchange
chromatography on a 1Â12.5 cm column of Dowex 50
X 2 (H+, 200±400 mesh) using a logarithmic gradient
generated from water in a 100 mL constant volume
chamber by displacement with 2.5 N HCl. 4c emerged
from the column between 75 and 140 mL well separated
from unreacted 2c and the fully guanylated compound
3c. The oil that remained after removal of solvent crys-
tallized after long standing at room teꢀmperature to
Hept-2-yn-1,7-diazide (8). Method G. To a solution of
7 (8.5 g, 30 mmol) in 60 mL of DMSO was added
powdered NaN3 (5.85 g, 90 mmol) portionwise with
stirring. Stirring was continued overnight. The reaction
was diluted with 120 mL of water and extracted with
ether (3Â100 mL). The combined extracts were washed
with water and dried over Na2SO4. The residue was
¯ash chromatographed (1/9, ethyl acetate/hexane) to
a€ord 2.8 g (52%) of 8 as a slightly yellow oil.
1
a€ord 0.224 g (43%) of product: mp 140 C; H NMR
(D2O) d 0.89±0.91 (d, 3H), 1.20±1.28 (m, 2H), 1.33±1.44
(m, 2H), 1.47±1.54 (m, 1H), 1.57±1.74 (m, 4H), 2.99 (t,
2H), 3.18 (t, 2H); MS (FAB, t-glyc) 187 (M+H). Simi-
1
larly prepared was 4d: H NMR (D2O) d 0.89 (s, 6H),
1.24±1.31 (m, 4H), 1.54±1.65 (m, 4H), 2.98 (t, 2H), 3.17
(t, 2H); MS (FAB, t-glyc) 201 (M+H).
7-(2-Tetrahydropyranyloxy)-2-heptyn-1-ol (5). Method D.
To a solution of ethyl magnesium bromide (1 M in
THF, 103 mL, 0.103 mol) under argon at room tem-
perature was added dropwise with stirring 1-(2-tetra-
hydropyranyloxy)-5-hexyne (16.5 g, 90.6 mmol). The
mixture was then heated at re¯ux for 15 min and cooled
to room temperature. After addition of DMPU
(103 mL), formaldehyde produced in an outside vessel
by heating of paraformaldehyde (6.2 g, 206 mmol) at
1,7-Diaminohept-2-yne dihydrochloride (9). Method H.
A mixture of 8 (2.7 g, 15 mmol) and triphenylphosphine
(7.8 g, 30 mmol) in 60 mL of THF containing water
(0.81 g, 45 mmol) and a few boiling stones was allowed
to react overnight at room temperature. After removal
of solvent, the solid residue was transfered to a mortar
with the aid of approximately 20 mL of water and pul-
verized. Sucient HCl solution (approximately 6 mL of
5 N) was added with stirring to render the mixture
strongly acidic (congo red test paper). The supernatant
was collected by suction ®ltration and the residue
washed with water. The combined extracts and wash-
ings were reduced in volume to about 10 mL, cooled,
basi®ed (solid NaOH) and extracted with methylene
chloride (5Â20 mL). The combined extracts were dried
over Na2SO4. The oil obtained was ¯ash chromato-
graphed (7/2/1, methylene chloride/methanol/NH4OH,
lower layer used as eluant) to give 1.53 g (81%) of free
amine. A portion of this amine in ethanol was treated
ꢀ
165±170 C was forced into the reaction mixture over a
period of 30±45 min by a very slow stream of argon. The
temperature of the reaction was allowed to rise during
this period. After an additional 30 min, the mixture was
poured into a solution of ammonium chloride (12 g) in
500 mL of water and the crude product was extracted
with ether (3Â500 mL). The combined extracts were
washed with brine, dried over Na2SO4 and evaporated.
The residue was ¯ash chromatographed (3/7, ethyl
acetate/hexane) to a€ord 16.3 g (85%) of 5 as a colorless

266
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
with 2 N HCl in ethanol. The solid obtained upon eva-
poration was recrystallized from ethanol-ether to give 9:
2H), 3.77±3.79 (d, 2H), 5.48±5.57 (m, 1H), 5.71±5.80 (m,
1H); MS (FAB, t-glyc) 213 (M+H), 311
(M+H+H2SO4).
mp 204±205 ꢀC; H NMR (D2O) d 1.59±1.66 (m, 2H),
1
1.73±1.81 (m, 2H), 2.31±2.35 (m, 2H), 3.03 (t, 2H), 3.80
(s, 2H); MS (FAB, t-glyc) 127 (M+H).
1,7-Diguanidino-cis-hept-2-ene sulfate (14). Prepared
1
from 11 using Method K: H NMR (D2O) d 1.43±1.50
1,7-Diamino-trans-hept-2-ene dihydrochloride (10). Method
I. A solution of 9 (free base, 0.63 g, 5 mmol) in 15 mL
of THF was added slowly to LiAlH4 (1 M, 15 mL,
15 mmol) in THF at room temperature. The mixture was
heated to re¯ux for 18 h and cooled. To this was added
carefully in succession, water (0.6 mL), NaOH (15%,
0.6 mL) and water (1.8 mL). The mixture was ®ltered by
suction and the precipitate washed well with water and
methylene chloride. The ®ltrate and washings were com-
bined, reduced in volume to about 10 mL, cooled, made
strongly basic (solid NaOH) and extracted with methy-
lene chloride (5Â20 mL). The puri®ed amine obtained
upon ¯ash chromatographed (7/2/1, methylene chloride/
methanol/ NH4OH, lower layer used as eluant) was
dissolved in ethanol and treated with 2 N HCl in ethanol
(m, 2H), 1.58±1.63 (m, 2H), 2.12±2.18 (m, 2H), 3.19 (t,
2H), 3.85±3.87 (d, 2H), 5.48±5.57 (m, 1H), 5.69±5.78 (m,
1H); MS (FAB, t-glyc) 213 (M+H), 311
(M+H+H2SO4).
7-(2-Tetrahydropyranyloxy)-3-heptyn-1-ol (15a). Method
L. A solution of 1-(2-tetrahydropyranyloxy)-4-pentyne
(12.6 g, 75 mmol) in 70 mL of THF was cooled in an ice±
salt bath and n-butyllithium (2.5 M in hexane, 33 mL,
82.5 mmol) was added slowly under argon with stirring.
The ice-salt bath was replaced by an ice bath and the
reaction mixture was allowed to stir for 2 h, cooled to 50
ꢀC and 70 mL of DMPU was added. This was followed by
the rapid addition of ethylene oxide (10 g, 225 mmol)
from an inverted cylinder. The mixture was allowed to
come to room temperature over a period of 6 h and
stirring was continued for 18 h. The mixture was poured
into 750 mL of ice water and extracted with ether
(3Â100 mL). The combined extracts were washed with
water, and with brine, dried over MgSO4, and evapo-
rated. Flash chromatography (2/3, ethyl acetate/hexane)
of the residue a€orded 11.9 g (75%) of 15a as a light-pink
oil: 1H NMR (CDCl3) d 1.51±1.83 (m, 8H), 2.23±2.32
(m, 2H), 2.39±2.47 (m, 2H), 3.44±3.55 (m, 2H), 3.66±
3.77 (m, 2H), 3.78±3.90 (m, 3H), 4.59±4.64 (m, 1H).
1
to a€ord 0.302 g (30%) of 10: H NMR (D2O) d 1.45±
1.54 (m, 2H), 1.63±1.73 (m, 2H), 2.12±2.19 (m, 2H),
2.98±3.03 (t, 2H), 3.56±3.58 (d, 2H), 5.57±5.66 (m,1H),
5.90±5.99 (m, 1H); MS (FAB, t-glyc) 129 (M+H).
1,7-Diamino-cis-hept-2-ene dihydrochloride (11). Method
J. A solution of 9 (free base, 0.63 g, 5 mmol) in 100 mL
of ethanol was hydrogenated over activated Lindlar
catalyst (100 mg) at atmospheric pressure and room
temperature. When the hydrogen uptake reached the
level of one molar equivalent (112 mL), the catalyst was
removed by ®ltration and the solvent was evaporated.
Isolation of the product by ¯ash chromatography (7/2/
1, methylene chloride/methanol/NH4OH, lower layer
used as eluant) and treatment with alcoholic HCl in the
usual manner a€orded 0.72 g (72%) of 11: 1H NMR
(D2O) d 1.49±1.54 (m, 2H), 1.67±1.71 (m, 2H), 2.15±2.22
(m, 2H), 2.99±3.04 (t, 2H), 3.67±3.70 (d, 2H), 5.50±5.58
(m, 1H), 5.81±5.97 (m, 1H); MS (FAB, t-glyc) 129
(M+H).
8-(2-Tetrahydropyranyloxy)-4-octyn-2-ol (15b and (S)-
15b). Obtained as colorless oils using Method L with
the exceptions that DMPU was replaced by HMPA
(10.5 mL) and ethylene oxide by propylene oxide: yields;
racemic 15b, 68%; (S)-15b, 65%; for (S)-15b, [a]D+9.9ꢀ
1
(c 1.2, CHCl3); H NMR (CDCl3) d 1.22±1.24 (d, 3H),
1.52±1.82 (m, 8H), 2.27±2.36 (m, 4H), 3.44±3.52 (m,2H),
3.79±3.87 (m, 1H), 3.88±3.92 (m, 1H), 4.59 (t, 1H)
(spectra identical for 15b and (S)-15b).
1,7-Diguanidinohept-2-yne sulfate (12). Method K. To a
solution of 9 (0.2 g, 1 mmol) in 2 mL of methanol was
added a solution of O-methylisourea sulfate (0.52 g,
3 mmol) and 2 mL of triethylamine in 2 mL of water.
After standing overnight, ethanol was added to com-
plete crystallization. Recrystallization from water±etha-
nol a€orded 0.124 g (40%) of 12: 1H NMR (D2O) d
1.58±1.61 (m, 2H), 1.65±1.72 (m, 2H), 2.29±2.30 (m,
2H), 3.21 (t, 2H), 4.00 (s, 2H); MS (FAB, t-glyc) 211
(M+H), 309 (M+H+H2SO4).
1-(2-Tetrahydropyranyloxy)-4-nonyn-7-ol (15c). Prepared
by Method L: in 40% yield, using HMPA and 1,2-
epoxybutane, respectively, in place of DMPU and ethyl-
ene oxide: H NMR (CDCl3) d 0.94 (t, 3H), 1.49±1.85
(m, 10H), 2.09±2.42 (m, 4H), 3.48±3.49 (m, 2H), 3.59±
3.62 (m, 1H), 3.73±3.89 (m, 2H), 4.59 (s, 1H).
1
Hept-3-yn-1,7-diol (16a). Prepared from 15a in 86%
yield using Method E: 1H NMR (CDCl3) d 1.70±1.79
(m, 2H), 2.27±2.33 (m, 2H), 2.39±2.45 (m, 2H), 3.66±
3.70 (t, 2H), 3.74±3.77 (t, 2H).
1,7-Diguanidino-trans-hept-2-ene sulfate (13). Prepared
1
from 10 using Method K: H NMR (D2O) d 1.45±1.50
(m, 2H), 1.57±1.62 (m, 2H), 2.10±2.14(m, 2H), 3.18 (t,
Oct-3-yn-1,7-diol (16b and (S)-16b). Prepared in 85%
and 80% yields, respectively, from 15b and (S)-15b

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
267
using Method E: for (S)-16b, [a]D+15.4ꢀ (c 1.8, CHCl3);
1H NMR (CDCl3) d 1.22±1.24 (d, 3H), 1.72±1.79 (m,
2H), 2.28±2.36 (m, 4H), 3.75 (t, 2H), 3.88±3.94 (m, 1H).
(Spectra identical for 16b and (S)-16b).
1,7-Diaminonon-3-yne dihydrochloride (19c). From 18c
1
by Method H: H NMR (D2O) d 0.99 (t, 3H), 1.71±1.81
(m, 2H), 1.83±1.93 (m, 2H), 2.36 (t, 2H), 2.53±2.70 (m,
2H), 3.11 (t, 2H), 3.32±3.38 (m, 1H); MS (FAB, t-glyc)
155 (M+H).
Non-4-yn-1,7-diol (16c). Prepared from 15c in 77% yield
using Method E and ¯ash chromatographed in 1/1,
acetone/hexane for puri®cation: 1H NMR (CDCl3) d
0.95 (t, 3H), 1.50±1.60 (m, 2H), 1.73±1.80 (m, 2H), 2.28±
2.34 (m, 2H), 2.38±2.44 (m, 2H), 3.61±3.65 (m, 1H), 3.76
(t, 2H).
1,7-Diamino-trans-hept-3-ene dihydrochloride (20a). From
19a (free base) by Method I: 1H NMR (D2O) d 1.71±1.81
(m, 2H), 2.12±2.19 (m, 2H), 2.36±2.43 (m, 2H), 2.98±3.08
(m, 4H), 5.49±5.56 (m, 1H), 5.64±5.71 (m, 1H); MS
(FAB, t-glyc) 129 (M+H).
Hept-3-yn-1,7-dimesylate (17a). Obtained as an oil
1
1,7-Diamino-trans-oct-3-ene dihydrochloride (20b). Method
M. To a solution of racemic 19b (free base, 0.28 g,
2 mmol) in 8 mL of diglyme was added slowly with stir-
ring under argon LiAlH4 (0.5 M in diglyme, 13 mL,
6.5 mmol). The mixture was heated at re¯ux for 18 h,
cooled and hydrolyzed carefully with water using exter-
nal cooling. After ®ltration with the use of celite, the
solvent was removed under high vacuum. The residue
was dissolved in 10 mL of water, basi®ed (solid NaOH)
and extracted with methylene chloride (5Â20 mL).
Satisfactorily puri®ed product could not be obtained by
¯ash chromatography. Therefore the material was
chromatographed by the ion exchange procedure
described in Method C. Compound 20b emerged from
the column between 50 and 75 mL and was obtained as a
(97%) from 16a using Method F: H NMR (CDCl3) d
1.90±1.95 (m, 2H), 2.32±2.36 (m, 2H), 2.61±2.67 (m,
2H), 3.04 (s, 3H), 3.06 (s, 3H), 4.27 (t, 2H), 4.35 (t, 2H).
Oct-3-yn-1,7-dimesylate (17b and (S)-17b). By Method F
from 16b and (S)-16b (98% and 100%, respectively): 1H
NMR (CDCl3) d 1.46±1.49 (d, 3H), 1.88±1.96 (m, 2H),
2.34 (t, 2H), 2.54±2.56 (m, 2H), 3.03 (s, 3H), 3.04 (s,
3H), 4.34 (t, 2H), 4.81±4.86 (m, 1H). (Spectra identical
for 17b and (S)-17b).
Non-3-yn-1,7-dimesylate (17c). By Method F from 16c
1
(99%): H NMR (CDCl3) d 1.01 (t, 3H), 1.77±1.85 (m,
2H), 1.89±1.97 (m, 2H), 2.32±2.34 (m, 2H), 2.59±2.62 (m,
2H), 3.03 (s, 3H), 3.06 (s, 3H), 4.35 (t, 2H), 4.65±4.69
(m, 1H).
1
colorless oil (0.085 g, 24%) upon removal of solvent: H
NMR (D2O) d 1.28±1.31(d, 3H), 1.71±1.81 (m, 2H),
2.13±2.20 (m, 2H), 2.33±2.38 (m, 2H), 3.00 (t, 2H), 3.37±
3.44 (m, 1H), 5.48±5.57 (m, 1H), 5.64±5.74 (m, 1H); MS
(FAB, t-glyc) 143 (M+H).
Hept-3-yn-1,7-diazide (18a). From 17a using Method G
(70%): 1H NMR (CDCl3) d 1.71±1.81 (m, 2H), 2.26±
2.33 (m, 2H), 2.43±2.50 (m, 2H), 3.34±3.44 (t, 4H).
1,7-Diamino-cis-hept-3-ene dihydrochloride (21a). From
19a free base by Method J: 1H NMR (D2O) d 1.71±1.81
(m, 2H), 2.16±2.23 (m, 2H), 2.43±2.50 (m, 2H), 2.99±
3.08 (m, 4H), 5.46±5.51 (m, 1H), 5.63±5.71 (m, 1H); MS
(FAB, t-glyc) 129 (M+H).
Oct-3-yn-1,7-diazide (18b and (S)-18b). By Method G
1
from 17b and (S)-17b (65% and 70%, respectively): H
NMR (CDCl3) d 1.31±1.33 (d, 3H), 1.72±1.81 (m, 2H),
2.31±2.33 (m, 2H), 2.36±2.39 (m, 2H), 3.41 (t, 2H), 3.57-
3.63 (m, 1H). (Spectra identical for 18b and (S)-18b).
1,7-Diamino-cis-oct-3-ene dihydrochloride (21b). From
1
Non-3-yn-1,7-diazide (18c). By Method G from 17c
1
racemic 19b free base by Method J: H NMR (D2O) d
(52%): H NMR (CDCl3) d 1.00 (t, 3H), 1.56±1.71 (m,
2H), 1.73±1.81 (m, 2H), 2.28±2.33 (m, 2H), 2.40±2.43
(m, 2H), 3.34±3.38 (m, 1H), 3.42 (t, 2H).
1.30±1.32 (d, 3H), 1.71±1.81 (m, 2H), 2.16±2.23 (m, 2H),
2.39±2.45 (m, 2H), 3.01 (t, 2H), 3.39±3.46 (m, 1H), 5.45±
5.54 (m, 1H), 5.65±5.73 (m, 1H); MS (FAB, t-glyc) 143
(M+H).
1,7-Diaminohept-3-yne dihydrochloride (19a). From 18a
1
using Method H: H NMR (D2O) d 1.85±1.90 (m, 2H),
2.35 (t, 2H), 2.61 (t, 2H), 3.09±3.17 (m, 4H); MS (FAB,
t-glyc) 127 (M+H).
1,7-Diguanidinohept-3-yne sulfate (22a). From 19a by
Method K: 1H NMR (D2O) d 1.75±1.82 (m, 2H), 2.29 (t,
2H), 2.48 (t, 2H), 3.27±3.36 (m, 4H); MS (FAB, t-glyc)
211 (M+H), 309 (M+H+H2SO4).
1,7-Diaminooct-3-yne dihydrochloride (19b and (S)-19b).
1
From 18b and (S)-18b by Method H: H NMR (D2O) d
1,7-Diguanidinooct-3-yne dihydrochloride (22b and (S)-
22b). Method N. To solutions of 19b and (S)-19b (free
bases, 0.14 g, 1 mmol) in 1 mL of DMF were added 1H-
pyrazole-1-carboxamidine.HCl (0.31 g, 2.1 mmol) and
diisopropylethylamine (0.383 mL, 2.2 mmol) under
1.36±1.38 (d, 3H), 1.84±1.93 (m, 2H), 2.35±2.40 (m, 2H),
2.50±2.67 (m, 2H), 3.12 (t, 2H), 3.50±3.57 (m, 1H); MS
(FAB, t-glyc) 141 (M+H). (Spectra identical for 19b
and (S)-19b).

268
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
argon. After standing overnight, the mixtures were
diluted with 10 mL portions of ether. The oils that
formed were washed well with ether, dissolved in water
and chromatographed by the ion exchange procedure
described in Method C. Products, which eluted between
150±250 mL, were obtained as colorless somewhat
hygroscopic foams (0.248 g, 83% for 22b and 0.196 g,
66% for (S)-22b) upon removal of solvent and drying
under high vacuum: 1H NMR (D2O) d 1.27±1.29 (d,
3H), 1.74±1.82 (m, 2H), 2.28±2.34 (m, 2H), 2.41±2.58
(m, 2H), 3.30 (t, 2H), 3.73±3.79 (m, 1H); MS (FAB, t-
glyc) 225 (M+H), 261 (M+H+HCl). (Spectra iden-
tical for 22b and (S)-22b).
8-(2-Tetrahydropyranyloxy)-4-octyn-2-mesylate (27). Pre-
pared from racemic 15b (4.2 g, 18.6 mmol) by Method F
using 1.5 equiv of triethylamine and 1.1 equiv of
mesylchloride. Cold 5% citric acid replaced cold 10%
HCl in washing the reaction mixture. The product
(5.2 g, 92%) was obtained as a colorless oil, which
moved as a single component on TLC (2/3, ethyl acet-
1
ate/hexane, Rf 0.57): H NMR (CDCl3) d 1.47±1.49 (d,
3H), 1.53±1.81 (m, 8H), 2.24±2.29 (m, 2H), 2.52±2.59
(m, 2H), 3.04 (s, 3H), 3.44±3.52 (m, 2H), 3.79±3.89 (m,
2H), 4.58 (t, 1H), 4.79±4.85 (m, 1H).
8-(2-Tetrahydropyranyloxy)-4-octyn-2-azide (28). The app-
lication of Method G to 27 (5.1 g, 16.8 mmol) a€orded
2.81 g (67%) of 28 as a slightly yellow oil: 1H NMR
(CDCl3) d 1.31±1.33 (d, 3H), 1.53±1.84 (m, 8H),
2.26±2.32 (m, 2H), 2.35±2.39 (m, 2H), 3.44±3.53
(m, 2H), 3.56±3.60 (m, 1H), 3.76±3.88 (m, 2H), 4.60 (t,
1H).
1,7-Diguanidino-trans-hept-3-ene sulfate (23). From 20a
by Method K: 1H NMR (D2O) d 1.62±1.72 (m, 2H),
2.07±2.14 (m, 2H), 2.26±2.32 (m, 2H), 3.15±3.25 (m,
4H), 5.47±5.54 (m, 1H), 5.56±5.64 (m, 1H); MS (FAB, t-
glyc) 213 (M+H), 311 (M+H+H2SO4).
1,7-Diguanidino-cis-hept-3-ene sulfate (24). From 21a by
1
1-(2-Tetrahydroxypyranyloxy)-7-aminooctane
(29).
Method K: H NMR (D2O) d 1.62±1.71 (m, 2H), 2.11±
Method P. A solution of 28 (2.76 g, 11 mmol) in 25 mL
of ethanol was hydrogenated over Adams catalyst
(25 mg) at 40 lbs/in2 for 6 h. Removal of catalyst by ®l-
tration and evaporation of solvent a€orded 2.2 g (96%)
of the saturated amine 29 as a colorless oil that chro-
matographed as a single material on TLC (17/2/1,
methylene chloride/methanol/NH4OH, lower layer
used for solvent, Rf 0.43); 1H NMR (CDCl3) d 1.07±1.09
(d, 3H), 1.22±1.58 (m, 14H), 1.67±1.82 (m, 2H), 2.49 (s,
2H), 2.90±2.92 (m, 1H), 3.33±3.52 (m, 2H), 3.67±3.88
(m, 2H), 4.56 (t, 1H).
2.18 (m, 2H), 2.31±2.38 (m, 2H), 3.17±3.27 (m, 4H),
5.44±5.52 (m, 1H), 5.57±5.65 (m, 1H); MS (FAB, t-glyc)
213 (M+H) 311 (M+H+H2SO4).
1,7-Diaminooctane dihydrochloride (25b and (S)-25b).
Method O. Solutions of 19b and (S)-19b (free bases,
0.28 g, 2 mmol) in 100 mL portions of ethanol were
hydrogenated over activated Adams catalyst (25 mg) at
atmospheric pressure and room temperature. When the
hydrogen uptake reached the theoretical level (2 mol
equiv, 90 mL), catalyst was removed by ®ltration and
the solvent evaporated. Conversion of the free amines to
the dihydrochlorides was conducted in the usual manner
1 - (2 - Tetrahydropyranyloxy) - 7 - tert - butyloxycarbonyl-
aminooctane (30). To a solution of amine 29 (2.1 g,
9.16 mmol) in 2.5 mL of DMF was added in portions
with stirring a solution of di-tert-butyl dicarbonate (2 g,
9.2 mmol) in 2.5 mL of DMF. After stirring for an
additional 30 min the solvent was removed under high
vacuum. The oil that remained was dissolved in ether,
washed with cold 5% citric acid, saturated NaHCO3,
water and brine, and dried over MgSO4. Removal of
ether a€orded 2.85 g (94%) of 30 as a colorless oil: TLC
(2/3, ethyl acetate/hexane, Rf 0.74); 1H NMR (CDCl3) d
1.09±1.11 (d, 2H), 1.33±1.58 (m, 14H), 1.44 (s, 9H),
1.71±1.84 (m, 2H), 3.34±3.54 (m, 2H), 3.61±3.66
(m, 1H), 3.69±3.90 (m, 2H), 4.29±4.33 (m, 1H), 4.58 (t,
1H).
1
to a€ord 25b and (S)-25b: H NMR (D2O) d 1.28±1.30
(d, 3H), 1.33±1.61 (m, 6H), 1.59±1.75 (m, 4H), 3.00 (t,
2H), 3.33±3.40 (m, 1H); MS (FAB, t-glyc) 145 (M+H).
(Spectra identical for 25b and (S)-25b).
1,7-Diaminononane dihydrochloride (25c). From 19c free
1
base by Method O: H NMR (D2O) d 0.97 (t, 3H), 1.40
(m, 6H), 1.59±1.76 (m, 6H), 3.00 (t, 2H), 3.18±3.26 (m,
1H); MS (FAB, t-glyc) 159 (M+H).
1,7-Diguanidinooctane sulfate (26b and (S)-26b). From
1
25b and (S)-25b, respectively, by Method K: H NMR
(D2O) d 1.17±1.20 (d, 3H), 1.30±1.40 (m, 6H), 1.55±1.65
(m, 6H), 3.18 (t, 2H), 3.50±3.57 (m, 1H); MS (FAB, t-
glyc) 229 (M+H), 327 (M+H+H2SO4). (Spectra iden-
tical for 26b and (S)-26b).
7-tert-Butyloxycarbonylaminooctane-1-ol (31). Obtained
from 30 (2.8 g, 8.5 mmol) with the use of Method E.
Puri®cation by ¯ash chromatography (2/3, ethyl acet-
ate/hexane) provided 31 (1.8 g, 86%) as a colorless oil:
1H NMR (CDCl3) d 1.09±1.12 (d, 3H), 1.34 (m, 8H),
1.44 (s, 9H), 1.54±1.58 (m, 2H), 3.61 (m, 1H), 3.64 (t,
1H).
1,7-Diguanidinononane sulfate (26c). From 25c by
Method K: 1H NMR (D2O) d 0.97 (t, 3H), 1.38 (m, 6H),
1.55±1.73 (m, 6H), 3.16±3.23 (m, 3H); MS (FAB, t-glyc)
243 (M+H), 341 (M+H+H2SO4).

Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
269
7-tert-Butyloxycarbonylaminooctane-1-mesylate
(32).
turation with ether and ¯ash chromatographed (7/2/1,
methylene chloride/methanol/NH4OH, lower layer used
for elution) to a€ord 37 (0.63 g, 91%) as a colorless oil:
1H NMR (CDCl3) d 1.14±1.16 (d, 3H), 1.32±1.50 (m,
10H), 2.97±3.02 (m,1H), 3.15±3.22 (m, 2H), 5.10 (s, 2H),
7.36 (s, 5H).
Application of Method F (as used in preparation of 27)
to 31 (1.7 g, 7 mmol) a€orded 32 (1.96 g, 87%) as a
white solid: mp 54 ꢀC after recrystallization from hex-
1
ane: TLC (2/3, ethyl acetate/hexane, Rf 0.47); H NMR
(CDCl3) d 1.10±1.12 (d, 3H), 1.36±1.39 (m, 8H), 1.45 (s,
9H), 1.73±1.78 (m, 2H), 3.01 (s, 3H), 3.61 (m, 1H), 4.23
(t, 2H); Anal. (C14H29NO5S) C,H,N,S.
7-Guanidino-1-benzyloxycarbonylaminooctane (38). Guan-
ylation of amine 37 (0.42 g, 1.5 mmol) was conducted
according to Method N using 1 equiv each of reagent
and diisopropylethylamine. Flash chromatography (7/2/
1, methylene chloride/methanol/NH4OH, lower layer
used for elution) a€orded 38 (0.3 g, 62%) as a strongly
basic oil: 1H NMR (CDCl3) d 1.05±1.46 (m, 13H), 2.99±
3.14 (m, 2H), 3.52±3.61 (m, 1H), 5.06 (s, 2H), 7.32 (s,
5H).
7-tert-Butyloxycarbonylaminooctane-1-azide (33). From
32 (1.93 g, 6 mmol) by Method G as a colorless oil
(1.45 g, 89%): 1H NMR (CDCl3) d 1.09±1.11 (d, 3H),
1.34 (m, 8H), 1.44 (s, 9H), 1.54±1.62 (m, 2H), 3.25 (t,
2H), 3.62 (m, 1H), 4.28 (br, 1H).
1-Amino-7-tert-butyloxycarbonylaminooctane (34). The
application of Method P to azide 33 (1.36 g, 5 mmol)
a€orded the t-Boc diamine 34 (1.2 g, 98%) as an oil:
TLC (17/2/1, methylene chloride/methanol/NH4OH,
lower layer used for solvent, Rf 0.40); 1H NMR (CDCl3)
d 1.07±1.10 (d, 3H), 1.31 (m, 8H), 1.42 (s, 9H), 1.47±1.49
(m, 2H), 2.72 (t, 2H), 2.90 (s, 2H), 3.60 (m, 1H), 4.34±
4.37 (d, 1H).
7-Amino-1-guanidinooctane sulfate (39). Removal of the
t-Boc group from 35 (0.2 g, 0.6 mmol) was carried out
by Method Q. The residual oil obtained upon removal
of acid was crystallized under ether and recrystallized
1
from water±ethanol to give 39 (0.07 g, 46%): H NMR
(D2O) d 1.28±1.30 (d, 3H), 1.38 (m, 6H), 1.61 (m, 4H),
3.18 (t, 2H), 3.32±3.39 (m, 1H).
1-Guanidino-7-tert-butyloxycarbonylaminooctanehemi-
sulfate(35). Guanylation of amine 34 (0.37 g, 1.5 mmol)
using Method K followed by ¯ash chromatography (2/2/
1, methylene chloride/methanol/NH4OH, Rf 0.31)
a€orded 35 (0.24 g, 48%) as a white powder after drying
under high vacuum : mp 184 ꢀC dec.; 1H NMR (CDCl3)
d 1.09±1.11 (d, 3H), 1.32 (m, 8H), 1.43 (s, 9H), 1.59 (m,
1-Amino-7-guanidinooctane dihydrochloride (40). A solu-
tion of 38 (0.25 g, 7.8 mmol) in a mixture of 5 mL of
methanol and 2 mL of N HCl was hydrogenated over
Pd black (50 mg) at 40 lbs/in2 for 2 h. The catalyst was
removed by ®ltration and the residue that remained
upon evaporation of solvents was dissolved in water and
chromatographed on an ion exchange column using the
conditions outlined under Method C. The target com-
pound 40 emerged from the column between 80 and
150 mL and was obtained as a hygroscopic glass (0.16 g,
76%) upon removal of solvent and drying under high
.
2H), 3.21 (m, 2H), 3.55 (m, 1H); Anal. (C14H30N4O2 0.5
H2SO4) C, H, N, S.
1 - Benzyloxycarbonylamino - 7 - tert - butyloxycarbonyl-
aminooctane (36). To a solution of amine 34 (0.73 g,
3.25 mmol) in 5 mL of CHCl3 at ice bath temperature
under argon was added dropwise with stirring a solu-
tion of N-(benzyloxycarbonyloxy)succinimide (0.81 g,
3.25 mmol) in 5 mL of CHCl3. The mixture was allowed
to warm to room temperature and kept overnight. Upon
extraction successively with cold 5% citric acid, satu-
rated NaHCO3 and brine and removal of solvent, a
solid residue was obtained and recrystallized from ethyl
acetate-pentane to a€ord the di€erentially blocked di-
amine 36 (1.05 g, 85%): mp 52±53 ꢀC; TLC (2/3, ethyl
1
vacuum: H NMR (D2O) d 1.19±1.21 (d, 3H), 1.36 (m,
6H), 1.54 (m, 2H), 1.67 (m, 2H), 3.00 (t, 2H), 3.51±3.57
(m, 1H).
Enzyme assay and determination of cellular hypusine
biosynthesis
Human recombinant deoxyhypusine synthase was pre-
pared as outlined29. The enzyme was assayed by a pub-
lished procedure30. The assay mixtures included, in a
total of 0.02 mL, 0.2 M glycine±NaOH bu€er, pH 9.5,
10 mM eIF-5A precursor protein (prepared by over-
expression of a human eIF-5A cDNA in Escherichia
coli), 1 mM dithiothreitol, 0.5 mM NAD,+ 5.4 mM
[1,8-3H]spermidine, 25 mg of bovine serum albumin and
3±15 units of enzyme. Incubations were conducted at
pH 9.5 and 37 ꢀC for 1 h. Labeled deoxyhypusine was
measured after ion exchange chromatographic separa-
tion from acid hydrolysates of the precipitated protein
fractions. Test compounds were dissolved in water and
1
acetate/hexane, Rf 0.66); H NMR (CDCl3) d 1.09±1.11
(d, 3H), 1.31±1.49 (m, 10H), 1.44 (s, 9H), 3.18 (m, 2H),
3.61 (m, 1H), 4.29 (s, 1H), 4.69 (s, 1H), 5.10 (s, 2H), 7.36
(s, 5H); Anal. (C21H34N2O4) C, H, N.
7-Amino-1-benzyloxycarbonylaminooctane (37). Method
Q. To 4 mL of tri¯uoroacetic acid at 0 ꢀC was added 36
(0.95 g, 2.5 mmol). After 1 h at this temperature, the acid
was removed by rotary evaporation without external
heating. The residue was washed several times by tri-

270
Y. B. Lee, J. E. Folk/Bioorg. Med. Chem. 6 (1998) 253±270
their solutions, adjusted to pHꢁ9, were added to assay
mixtures prior to addition of enzyme. Compounds were
tested for inhibition at concentrations from 0.1 to
1000 mM. IC50 values (50% inhibitory concentrations)
were calculated by ®tting the data to the equation,
IC50=I[(100-x)/x], where I=inhibitor concentration
and x=the percentage inhibition6, using the Kaleida-
Graph program. Methods for cultures of Chinese ham-
ster ovary cells and for the determination of hypusine in
these cells are given elsewhere8. Values for hypusine
11. Katahira, J.; Ishizaki, T.; Sakai, H.; Adachi, A.; Yama-
moto, K.; Shita, H. J. Virol. 1995, 69, 3125.
12. Liao, D.-I.; Wol€, E. C.; Park, M. H.; Davies, D. R.
Structure 1998, 6, 23.
13. Franke, A.; Kroupa, A.; Schweitzer, F.; Winischhoper, H.;
Kelin-Lohr, M.; Just, M.; Hackl, M.; Reyher, I.V.; Bader, R.
Monatsh. 1936, 69, 167.
14. Hanschke, E. Ber. 1955, 88, 1048.
15. Blomquist, A. T.; Wheder, E. S.; Chu, Y. J. Am. Chem.
Soc. 1955, 77, 6307.
3
formed at 18 h after addition of H-labeled spermidine
are given as percentage reduction in the corresponding
value in untreated cells.
16. Allinger, N. L. J. Am. Chem. Soc. 1959, 81, 232.
17. Whitehead, E. V.; Dean, R. A.; Fidler, F. A. J. Am. Chem.
Soc. 1951, 73, 3632.
18. Smiley, R. A.; Arnold, C. J. Org. Chem. 1960, 25, 257.
19. Tufariello, J. J.; Trybalski, E. J. J. Org. Chem. 1974, 39,
3378.
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