A structure-activity study of spermicidal and anti-HIV properties of hydroxylated cationic surfactants

Article abstract of DOI:10.1016/S0968-0896(02)00245-6

The syntheses of 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethylhexadecan-1-aminium chloride [1(16)Cl] and iodide [1(16)I], 2-hydroxy-N,N,N-trimethylhexadecan-1-aminium chloride (6), N-(2-hydroxyethyl)-N,N-dimethylhexadecan-1-aminium chloride (8), N,N-bis(2-hydroxyethyl)-N-methylhexadecan-1-aminium chloride (11), and 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxahexadecan-1-aminium chloride (14) are reported along with the critical micelle concentrations (cmcs), as measured by conductivity at 25°C, of 1(16)Cl, 1(16)I, 6, 8, 11, and N,N,N-trimethylhexadecan-1-aminium chloride (12). All compounds display spermicidal and virucidal activity. A plot of minimum effective concentration (MEC) in the Sander-Cramer spermicidal assay and cmc shows that 1(16)Cl and 6 have the best spermicidal activity and highest cmcs. Compounds 8, 11, and 1(16)Cl are the most active at 0.05 mg mL^-1 against cell-free and cell-associated virus. In conclusion, 1(16)Cl shows the best combination of dual activity against sperm and HIV; it is a promising candidate for further preclinical studies as a topical, contraceptive microbicide.

Full text of DOI:10.1016/S0968-0896(02)00245-6

Bioorganic & MedicinalChemistry 10 (2002) 3599–3608
A Structure–Activity Study of Spermicidal and Anti-HIV
Properties of Hydroxylated Cationic Surfactants
Yue-Ling Wong,a,y M. Patricia Hubieki,a,{ Christopher L. Curfman,a,x Gustavo
F. Doncel,b Travis C. Dudding,a,k Prashant S. Savlea,{ and Richard D. Gandoura,*
aDepartment of Chemistry, Virginia Tech, Blacksburg, VA 24061-0212, USA
bDepartment of Obstetrics and Gynecology, CONRAD Program, Eastern Virginia Medical School, Norfolk, VA 23507, USA
Received 1 March 2002; accepted 7 May 2002
Abstract—The syntheses of 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethylhexadecan-1-aminium chloride [1(16)Cl] and iodide
[1(16)I], 2-hydroxy-N,N,N-trimethylhexadecan-1-aminium chloride (6), N-(2-hydroxyethyl)-N,N-dimethylhexadecan-1-aminium
chloride (8), N,N-bis(2-hydroxyethyl)-N-methylhexadecan-1-aminium chloride (11), and 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-
4-oxahexadecan-1-aminium chloride (14) are reported along with the critical micelle concentrations (cmcs), as measured by con-
ductivity at 25 ꢀC, of 1(16)Cl, 1(16)I, 6, 8, 11, and N,N,N-trimethylhexadecan-1-aminium chloride (12). All compounds display
spermicidaland virucidalactivity. A pol t of minimum effective concentration (MEC) in the Sander–Cramer spermicidalassay and
cmc shows that 1(16)Cland 6 have the best spermicidalactivity and highest cmcs. Compounds 8, 11, and 1(16)Clare the most
active at 0.05 mg mLÀ1 against cell-free and cell-associated virus. In conclusion, 1(16)Clshows the best combination of dualactivity
against sperm and HIV; it is a promising candidate for further preclinical studies as a topical, contraceptive microbicide.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
N-9, a detergent that disrupts cell walls by solubilizing
membranes, has severaldisadvantages. N-9 increases
The worldwide increase in human-immunodeficiency-
virus (HIV) infections among women1 has made devel-
oping user-controlled, topical vaginal microbicides that
provide protection against sexually transmitted patho-
gens an urgent global priority.2 Most heterosexual
women want to reduce the risk of acquiring a sexually
transmitted disease;3 many want to controltheir ferti-
lity. New dual-active agents (spermicide and micro-
bicide) that do not have the disadvantages of
nonoxynol-9 (N-9), currently the most widely used
spermicide in the United States, occupy the efforts of
the risk of urinary tract infections,13,14 vulvovaginal
16
candidiasis,15 and genitaluclers.
As human trials
show, N-9 may increase the risk of HIV transmission,17
perhaps by initiating interleukin-1-mediated NF-kB
activation, which leads to cytokine-induced recruitment
of HIV-1 host cells and increased HIV-1 replication.18
N-9, a mixture of oligomers,19À23 may violate future
federalregual tions; pure compounds or mixtures whose
individualcomponents have met safety standards will
become the norm. Furthermore, the breakdown pro-
ducts of N-9 pose serious health and environmental
risks.24
4À11
severalresearch groups.
These new agents must be
safe, effective, acceptable, and affordable.12
Quaternary ammonium salts, well-known microbicides,25
can act as spermicides (e.g., European and Canadian
spermicidalproducts contain benzaklonium cholride, a
*Corresponding author. Tel.: +1-540-231-3731; fax: +1-540-231-
3255; e-mail: gandour@vt.edu
yPresent address: Computer Science Department, Wake Forest U.,
Winston-Salem, NC, USA.
popular microbicide). Although comparatively less sper-
26
micidalthan N-9,
tialas an anti-HIV, topicalagent.
benzalkonium chloride has poten-
27
Alkyl quaternary
{Present address: J-Star Research, Inc., South Plainfield, NJ, USA.
xPresent address: Needle & Rosenberg, P.C., Atlanta, GA, USA.
kPresent address: Department of Chemistry, Johns Hopkins U., Balti-
more, MD, USA.
ammonium salts have potent microbicidal activity, but
they have modest solubility in water and can irritate
sensitive tissue. We aim to find or synthesize quaternary
ammonium salts that minimize these shortcomings,
{Present address: Rhodia ChiRex, Inc., Malvern, PA, USA.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00245-6

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Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
11
especially active compounds that (1) have high critical
micelle concentrations (cmcs) and (2) that can be made
relatively inexpensively.
from our initialstudy. Reaction of 2-[(N-methyl)ami-
no]ethanolwith a mixture of bromohydrins ( 3) gave the
desired long-chain alkyl tertiary aminodiol (4),28 which
reacted with methyliodide to give 1(16)I.29 Reaction of
tetradecyloxirane (5) with hydrogen chloride followed
by addition of 2-(dimethylamino)ethanol30 gave 1(16)Cl.
Compound 1(16)Clwas the most active of the three
halides in initial spermicidal assays. (data not shown)
We then made 2(16)Cl31 from the reaction of 5 with
hydrogen chloride followed by addition of 2-{(2-hydroxy-
ethyl)methylamino}ethanol. As 1(16)Br is more potent
spermicide than 2(16)Br,11 we needed to verify that
1(16)Clis more potent than 2(16)Cl.
Our recent report11 shows that the water-soluble qua-
ternary ammonium bromides, 1(p)Br and 2(p)Br, act as
spermicides and anti-HIV agents. The best compounds
— 1(17)Br and 1(18)Br — have spermicidalactivities at
minimum effective concentrations (MECs) slightly
lower than that of N-9. Compounds 1(16)Br and
2(17)Br show the best anti-HIV activity in in-vitro, cell-
free and cell-associated virus inactivation assays. Com-
pound 1(16)Br shows the best combination of dual
activity against sperm and HIV.
To probe the structure–function of hydroxylation, we
synthesized 6, 8, and 11 (Scheme 2), which structurally
resemble 1(16)Cland 2(16)Cl, but lack one or two
hydroxylgroups.
In this paper, we report a limited structure–activity
study, using 1(16)Br as the lead compound. We use
spermicidaland anti-HIV assays to probe structure–
activity as a function of different halide counterions, the
amount of hydroxylation, and oxygen replacement in
the hexadecylchain. For the most active compounds,
we compare the minimum effective concentration
(MEC) in the spermicidalassay to the cmc.
Reaction of 5 with trimethylamine hydrochloride gave
6.32 Reaction of 1-chlorohexadecane (7) with 2-(dime-
thylamino)ethanol gave 8.33,34 The three-step synthesis
of 11 began with the synthesis of N-methyl-1-hexa-
decanamine (9) from 7 and excess methylamine.35
Reaction of 9 with 2-chloroethanol gave 10,36,37 which
we isolated by chromatography. Treatment of 10 in a
sealed tube with excess 2-chloroethanol gave 11.38 We
recrystallized a commercial sample of N,N,N-trimethyl-
1-hexadecanaminium chloride (12), which served as the
reference, non-hydroxylated alkyl quaternary ammo-
nium salt.
Results
Chemistry
To explore the effect of counterions, we synthesized
1(16)Cl, 1(16)I, and 2(16)Cl(Scheme 1). We had 1(16)Br
Scheme 2. (a) Á, 16 h, MeOH; (b) K2CO3, KI, Á, 3 days, EtOH;
(c) K2CO3, Á, 1 day, MeOH; (d) Á, 5 days, MeOH.
Scheme 1. (a) Á, 16 h, MeOH; (b) CH3I, Et2O, 16 h; (c) aq HCl, Á, 3
h; (d) Á, 3 h; (e) Á, 16 h.

Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
3601
cmc by 15 and 8%, respectively. Comparison of two
pairs of compounds 11/12 and 2/6 shows that replacing
two methyls with two 2-hydroxyethyl groups decreases
the cmc by 22 and 12%, respectively. In summary,
hydroxylation at carbon 2 on the hexadecyl group increa-
ses the cmc by a greater amount than replacement of a
methylwith a 2-hydroxyethylgroup decreases the cmc.
Biology
Scheme 3. (a) NaH, THF, 15 ꢀC!rt,
4 h; (b) rt, 16 h; (c)
Table 2 presents MECs for spermicidal activity in a
modified Sander–Cramer assay45 for 1(16)X (X=Cl, Br,
I), 2(16)Cland N-9. MECs represent a measure of
potency — the lower the MEC, the more potent the
compound. The Sander–Cramer assay determines the
minimum concentration required to stop all sperm
motility in 20 s. The MEC decreases; that is, the potency
increases, as the size of the halide counterion decreases
to 1(16)Cl. The MEC of 1(16)Cl, which is the most
potent compound in this series, is nearly half the MEC
of N-9. As with the bromide salts,11 1(16)Clis more
potent than 2(16)Cl. Compounds 1(16)Br and 2(16)Cl
are as effective spermicides as N-9.
.
HOCH2CH2NMe2 HCl, Á, 16 h, MeOH.
To test how hydrophobicity affected activity, we syn-
thesized 14 (Scheme 3), which structurally resembles
1(16)Cl but has one oxygen in place of the methylene at
carbon 4 of the hexadecylgroup. Reaction of (bromo-
methyl)oxirane with the in-situ generated sodium dode-
can-1-oxide39 gave oxirane 13. Reaction of 13 with
2-(dimethylamino)ethanol in the presence of an equivalent
of 2-(dimethylamino)ethanol hydrochloride yielded 14.
The cmcs determined from conductivities of dilute
solutions of 1(16)X (X=Cl, Br, I), 2(16)Cl, 6, 8, 11, and
12 are given in Table 1. Two reported40,41 values for the
cmc (1.36 and 1.41Â10À3 moldm À3) of 12 in water
agree with the value determined by our procedure. As
we anticipated from others,42,43 the cmcs and alpha for
1(16)X (X=Cl, Br, I) decrease as halide counterion
increases in size. As we anticipated from previous data
on bromide44 and chloride38 salts, hydroxylation has
small but significant effects on the cmc.
Table 3 presents the MECs for spermicidal activity in a
modified Sander–Cramer assay for 6, 8, 11, 12, 14, and
Table 2. Spermicidalactivity of 1(16)X (X=Cl, Br, I) and 2(16)Clin
the Sander–Cramer assay
Compd
Highest spermicidal
dilution (HSD) (1/X)
MEC
(mg mLÀ1
)
1(16)Cl362.7
1(16)Br
1(16)Ia
ꢁ44.8
250.7ꢁ46.8
245.3ꢁ58.2
93.3ꢁ16.6
213.3ꢁ40.9
0.072ꢁ0.012
0.120ꢁ0.022
0.221ꢁ0.061
0.140ꢁ0.019
0.124ꢁ0.014
Replacing a hydrogen at carbon 2 of a hexadecyl group
with a hydroxylgroup increases the cmc; repalcing a
methylwith a 2-hydroxyethylgroup decreases the cmc.
Comparison of three pairs of compounds 6/12, 1/8, and
2/11 shows that replacing a hydrogen at carbon 2 of a
hexadecylgroup with a hydroxylgroup increases the
cmc by 41, 51, and 58%, respectively. Comparison of two
pairs of compounds 8/12 and 1/6 shows that replacing
one methylwith one 2-hydroxyethylgroup decreases the
2(16)Clb
N-9
Sperm samples (n=12) were mixed with 2-fold serial dilutions of the com-
pounds in 0.9% NaCl(initialconcentration of compound in distiled water,
20 mg mLÀ1) and observed under the microscope for 20 s. Those dilutions
that completely immobilized all screened spermatozoa were further diluted
in excess buffer and incubated for 1 h to verify lack of motility recovery. The
highest dilution that successfully passed both assessments was considered the
HSD and was used to calculate the MEC.
aRequires warm water (40 ꢀC) to Àre1main in solution at 20 mg mLÀ1
.
Table 1. Critical micelle concentrations (cmcs) and alpha (ratio of
slopes before and after the cmc) for 1(16)X, 2(16)Cl, 6, 8, 11, and 12 at
25 ꢀC as measured by conductance in distilled water
bInitialconcentration, 10 mg mL
.
Compd
103 cmc
(moldm
Alpha
Table 3. Spermicidalactivity of 6, 8, 11, 12, 14, and N-9 in the San-
der–Cramer assay
À3
)
1(16)Cl1.77
ꢁ0.01
0.290ꢁ0.002
0.299ꢁ0.002
0.305ꢁ0.002
0.213ꢁ0.003
0.264ꢁ0.006
0.181ꢁ0.004
0.160ꢁ0.002
0.299ꢁ0.003
0.346ꢁ0.004
0.350ꢁ0.005
0.33ꢁ0.01
Compd
HSD (1/X)
MEC (mg mLÀ1
)
1.791ꢁ0.009
1.75ꢁ0.01
1.36ꢁ0.02
1.34ꢁ0.02
0.88ꢁ0.01
0.833ꢁ0.006
ꢁ0.03
6
8
256.0ꢁ0.0
320.0ꢁ32.0
170.7ꢁ17.4
320.0ꢁ32.0
61.3ꢁ2.6
0.078ꢁ0.000
0.068ꢁ0.005
0.130ꢁ0.011
0.068ꢁ0.005
0.339ꢁ0.025
0.081ꢁ0.016
1(16)Br
1(16)I
2(16)Cl1.70
6
11
12
14
N-9a
181.3ꢁ25.9
1.68ꢁ0.02
1.87ꢁ0.03
1.97ꢁ0.01
1.94ꢁ0.02
1.176ꢁ0.007
1.162ꢁ0.009
1.055ꢁ0.01
1.091ꢁ0.009
1.37ꢁ0.03
Sperm samples (n=12) were mixed with 2-fold serial dilutions of the com-
pounds in 0.9% NaCl(initialconcentration of compound in distiled water,
20 mg mLÀ1) and observed under the microscope for 20 s. Those dilutions
that completely immobilized all screened spermatozoa were further diluted
in excess buffer and incubated for 1 h to verify lack of motility recovery. The
highest dilution that successfully passed both assessments was considered the
HSD and was used to calculate the MEC.
0.303ꢁ0.008
0.387ꢁ0.004
0.374ꢁ0.001
0.3626ꢁ0.0007
0.375ꢁ0.003
0.371ꢁ0.002
8
11
12
aInitialconcentration, 10 mg mL
.
À1

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Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
N-9. The MECs for 6, 8, and 12 are better than the
MEC for N-9. In this particular assay, N-9 has a lower
MEC than is typically observed (i.e., 0.12 mg mLÀ1).
This batch of sperm is apparently more sensitive to
spermicidal agents. Regardless, the trends are clear.
Compound 14 is much less effective than the others.
Compound 11 is less active than 6, 8, 12, and N-9.
Figure 1 presents the data for anti-HIV activity of
1(16)X (X=Cl, Br, I), 2(16)Cland N-9 in two in-vitro
assays:46 cell-free and cell-associated virus inactivation.
All five compounds are effective, that is, ꢂ3 log units of
infectious titer reduction at 1.0 and 0.5 mg mLÀ1 in
both assays. (data not shown) At 0.05 mg mLÀ1
,
1(16)Br and 1(16)I are more potent than 1(16)Clagainst
both cell-free and cell-associated HIV. Compound
Hydroxylation can either not change or decrease the
MEC compared to the reference compound, 12. In
comparing 6 and 12, we find that replacing a hydrogen
at carbon 2 of a hexadecylgroup with a hydroxylgroup
does not change the potency. In comparing 8 and 12, we
find that replacing one methyl with one 2-hydroxyethyl
group does not change the potency. In comparing 11
and 8, we find that replacing a second methyl with a 2-
hydroxyethylgroups decreases potency (i.e., increases
the MEC). A similar result is seen (Table 2) when com-
paring 1(16)Cland 2(16)Cl; a second 2-hydroxyethyl
group decreases potency. We conclude that 1(16)Cl, 6,
8, and 12 are similarly potent spermicides and that
hydroxylation at carbon 2 of a hexadecyl group and
replacing one methyl with one 2-hydroxyethyl group do
not affect the potency of this series of alkyl quaternary
ammonium chlorides.
1(16)Br is more potent than N-9 against cell-free HIV at
0.05 mg mLÀ1
.
Figure 2 presents the data for anti-HIV activity of 6, 8,
11, 12, 14, 1(16)Cl, 1(16)Br, and N-9 in two in-vitro
assays: cell-free and cell-associated virus inactivation.
All compounds are effective, that is ꢂ4 log units of
infectious titer reduction at 1.0 and 0.5 mg mL-1 in both
assays. At 0.05 mg mLÀ1, 6 and 1(16)Clare more potent
than N-9 against cell-free HIV; 11, 1(16)Cl, 8, and
1(16)Br are more potent than N-9 against cell-asso-
ciated HIV. In these two assays, 1(16)Clis the onyl
compound that is consistently more potent than N-9.
Hydroxylation increases or decreases the antiviral
activity. In comparing 6 and 12, we find that replacing a
hydrogen at carbon 2 of a hexadecylgroup with a
Figure 1. In vitro cell-free (a) and cell-associated (b) inactivation of
HIV assays for 1(16)X (X=Cl, Br, I), 2(16)Cl, and N-9. Cell-free or
cell-associated HIV-1 was incubated with multiple concentrations of
the test agents for 2 min at 37 ꢀC. Compound effect was then termi-
nated by 10-fold serial dilutions, and the treated virus was further
incubated with infection-susceptible MT-2 cells for 6 days. Virus-
induced cytopathic effects (formation of cell syncytium) were recorded
under microscopic observation. The values presented in the body of
the table represent the reduction in the infectious titer of the untreated
virus (in logs) effected by each compound concentration.
Figure 2. In vitro cell-free and cell-associated inactivation of HIV
assays for 6, 8, 11, 12, 14, 1(16)Cl, 1(16)Br, and N-9. Cell-free or cell-
associated HIV-1 was incubated with multiple concentrations of the
test agents for 2 min at 37 ꢀC. Compound effect was then terminated
by 10-fold serial dilutions, and the treated virus was further incubated
with infection-susceptible MT-2 cells for 6 days. Virus-induced cyto-
pathic effects (formation of cell syncytium) were recorded under
microscopic observation. The values presented in the body of the table
represent the reduction in the infectious titer of the untreated virus (in
logs) effected by each compound concentration.

Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
3603
hydroxylgroups; 11 and 6 have one hydroxylgroup
each; 1(16)Clhas two hydroxylgroups. From these
data, we conclude that (1) hydroxylation affects the cmc
and MEC independently and (2) the position and
amount of hydroxylation either increase or decrease
spermicidalpotency and cmc.
Compounds 14, which is quite water-soluble, is not a
potent spermicidal agent (Table 3) Although we did not
measure the cmc of 14, we can estimate it from the cmc
of 2-hydroxy-N,N,N-trimethyl-4-oxahexadecan-1-ami-
nium chloride, which is 5.6Â10À3 M.50 Given the slight
decrease in cmc expected for replacing a methyl with a
2-hydroxyethylgroup, we estimate that the cmc of 14 is
ꢃ5.1Â10À3 M, which is 3-fold greater than the cmc for
Figure 3. Comparison of MECs and cmcs of 1(16)Cl, 2(16)Cl, 6, 8, 11,
and 12.
hydroxylgroup improves potency against ce-lfree HIV
but not against cell-associated HIV. In comparing 8 and
12, we find that replacing one methyl with one 2-hydroxy-
ethylgroup improves potency against both ce-lfree and
cell-associated HIV. In comparing 11 and 8, we find that
replacing a second methyl with a 2-hydroxyethyl groups
increases potency against cell-associated HIV. In con-
trast, when comparing 1(16)Cland 2(16)Cl(Fig. 1), a
second 2-hydroxyethylgroup decreases potency. We
conclude that 1(16)Cl, 8, and 11 are similarly potent
antiviralagents. Furthermore, hydroxyaltion at carbon
2 of a hexadecylgroup increases potency against ce-ll
free HIV, but decreases potency against cell-associated
HIV. Replacing a methyl with a 2-hydroxyethyl group
increases potency against both cell-free and cell-asso-
ciated HIV. Hydroxylation at carbon 2 of a hexadecyl
group in combination with replacing methyl with a
2-hydroxyethylgroup produces the best resutl.
1(16)Cl(Tabel 1). As a spermicide,
1(16)Clis nearyl
5-fold more potent than 14. We conclude that replacing
a methylene with oxygen at carbon 4 of a hexadecyl
chain decreases the spermicidalpotency more than it
increases the cmc.
Anti-HIV properties
Those compounds — 8, 11, and 1(16)Cl— that are
active (ꢂ3 log units of infectious titer reduction) at 0.05
mg mLÀ1 against cell-free and cell-associated virus have
at least one 2-hydroxyethyl group (Fig. 2). Compound
14 has one 2-hydroxyethylgroup. Athl ough it was
dropped from consideration as a dualagent because of
relatively low potency in the spermicidal assay, it shows
excellent activity against cell-free virus. Compound 6
differs the most in activity between the cell-free and cell-
associated virus. At 0.05 mg mLÀ1, 6 is the most potent
against the cell-free virus and the least potent against
the cell-associated virus. Compound 11, which has two
2-hydroxyethylgroups, is the most active against the
cell-associated virus. Of the three best compounds in
this assay, 1(16)Clhas the highest cmc (Fig. 1).
Discussion
Spermicidal activity and micellar concentration
Figure 1 presents the MECs for spermicidalactivity and
cmcs for 1(16)Cl, 2(16)Cl, 6, 8, 11, and 12. We show the
cmcs in mg mLÀ1 to facilitate comparison with the
bioassays. The cmcs, which were determined in distilled
water, represent an upper limit of the cmc under the
conditions of the Sander–Cramer assay. That is, ꢃ1%
bovine serum albumin (BSA), 0.9% saline, and Baker’s
(phosphate and glucose) solution might decrease the
cmc. Studies47,48 on the binding of 12 to BSA and
human serum albumin reveal ꢃ100 binding sites on
each protein. This surfactant–protein binding should
increase the totalamount of surfactant needed to form a
micelle. Salt significantly decreases the cmc of hydroxy-
functionalized alkyl quaternary ammonium chlorides.49
In the presence of salt, the cmcs would probably be lower.
Comparison with previous work
The antibacterialactivities and appilcations in shampoo
formulations of 1(16)Cl, 2(16)Cl, and 6 are known from
the patent literature.30À32 Cognis markets 1(16)Clas the
active ingredient in Dehyquart1 E-CA as wetting agent
for hair-care products. The antimicrobialactivities of
(2-hydroxyethyl)-N,N-dimethyl-1-alkanaminium halides
have been well known since the 1950s.51À53 Compound 8
has excellent bacteriostatic53,54 and fungiostatic53 activ-
ities. However, 8 is quite cytotoxic to Chinese hamster
lung fibroblasts and a strong irritant in the Draize eye
test.55 Our study extends these previous results to spermi-
cidaland antiviralactivities. Furthermore, it compares
these compounds in a manner previously unreported.
We group the compounds in Figure 3 by cmc to show
the structuralsimailrities that produce higher cmcs.
Each compound in the left triad — 1(16)Cl, 6, and
2(16)Cl-has a hydroxyl at carbon 2 of a hexadecyl
group; compounds in the right triad — 11, 8, and 12-do
not. The two compounds at the ends, 11 and 2(16)Cl,
are 2-fold less potent as spermicides than the four com-
pounds in the middle. Both of these compounds have
two 2-hydroxyethylgroups. The four compounds in the
middle have similar potency despite different amounts
and positions of hydroxylation. Compound 12 has no
Conclusion
Hydroxylation and oxygen replacement affect the cmc,
spermicidalpotency, and antiviralactivity of akl ylqua-
ternary ammonium salts. Replacing a hydrogen with a
hydroxylgroup at carbon 2 of a hexadecylgroup
increases the cmc, improves spermicidalpotency, and

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Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
has little effect on antiviral activity. Replacing a methyl
with a 2-hydroxyethylgroup decreases the cmc, has ilt-
tle effect on spermicidal potency, and improves antiviral
activity. Replacing two methyls with two 2-hydroxy-
ethylgroups decreases the cmc, decreases spermicidal
potency, and improves activity against cell-associated
virus. Replacing one oxygen for a methylene group at
carbon 4 in the hexadecylchain increases the cmc,
decreases spermicidalactivity, and has ilttel effect on
activity against cell-free virus. For this limited set of
compounds, hydroxylation and oxygen replacement
affect the cmc, spermicidalpotency, and antiviralactiv-
ity independently.
mL) was stirred at room temperature under N2 over-
night. The reaction mixture was diluted with ether (10
mL) and filtered to collect the white solid. The white
solid was recrystallized twice in EtOAc/CH2Cl2 (10:1,
1
11 mL) to give colorless crystals (1.20 g, yield 83%): H
NMR (400 MHz, CDCl3) d 4.36 (br m, 1H), 4.16 (br m,
3H), 3.90 (br m, 2H), 3.84 (d, 1H, Japp=6.7 Hz), 3.64
(dd, 1H, Japp=13.5, 10.0 Hz), 3.45 (comp, 7H), 1.58–
1.24 (comp, 26H), 0.87 (t, 3H, Japp=6.8 Hz); 13C NMR
(100 MHz, CDCl3) d 70.1, 66.7, 65.6, 56.0, 53.9, 53.7,
35.9, 31.9, 29.7, 29.64, 29.59, 29.5, 29.3, 25.1, 22.6, 14.1.
Anal. calcd for C20H44NO2I: C, 52.51; H, 9.69; N, 3.06.
Found: C, 52.45; H, 9.62; N, 3.09.
The best candidate for further evaluation as a dual
agent is 1(16)Cl. It has the best combination of spermi-
cidaland antiviralactivities, which are beol w the cmc.
Compound 1(16)Clis a racemic mixture; it has spermi-
cidaland antiviralpotencies that are indistinguishabel
from the individualenantiomers, which we synthesized
and assayed. (data not shown) Further preclinical tests12
are warranted to see if 1(16)Clcan be formual ted into a
safe, effective topicalmicrobicide that wilprotect against
mucosalinfections by sexualy transmitted pathogens.
2-Hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-1-hexa-
decanaminium chloride, 1(16)Cl. A mixture of 5 (10.55 g
of 85% tech, 37.30 mmol) and aq HCl (8 mL, 6 M
solution) was heated to reflux for 3 h. 2-(Dimethylamino)
ethanol(8.97 g, 100.63 mmo)l was added. The resutling
mixture was heated to reflux for 3 h. The warm reaction
mixture was poured into CH3CN (350 mL), which was
then heated to dissolve the reaction mixture. EtOAc
(150 mL) was added while the CH3CN was still hot. The
solution was cooled at room temperature for 1 h; crys-
tals formed slowly. The crystals were collected by
vacuum filtration, and washed with EtOAc/CH3CN
(2:1, 300 mL). The filtrate stood at room temperature
overnight; a second crop of crystals formed and were
collected as before. The combined crystals were dried in
vacuo for 3 days to give a white crystalline powder
Experimental
General
1
Reagents and HPLC-grade solvents were used as
received. Bromohydrins 3 were prepared as described.11
Tetradecyloxirane (5) and 1-chlorohexadecane (7)
were used as received. Solutions were concentrated by
rotary evaporation. Flash chromatography was per-
formed with silica gel, 60 X, 230–400 mesh. TLC was
performed with PE SIL G/UV 250-mm plates. Apparent
coupling constants, Japp, were the measured peak
widths. Atlantic MicroLab Inc. performed the elemental
analyses.
(10.22 g, yield 75%): H NMR (400 MHz, CDCl3) d
5.49 (br t, 1H, Japp=5.3 Hz), 5.32 (d, 1H, Japp=7.3 Hz),
4.23 (br m, 1H), 4.09 (br m, 2H), 3.83 (m, 2H), 3.61 (dd,
1H, Japp=13.6, 10.1 Hz), 3.41 (s, 6H), 3.32 (d, 1H,
Japp=13.0 Hz), 1.58–1.24 (comp, 26H), 0.87 (t, 3H,
Japp=6.9 Hz); 13C NMR (100 MHz, CDCl3) d 70.4,
66.8, 65.6, 56.0, 53.5, 53.1, 36.0, 31.9, 29.7, 29.65, 29.59,
29.3, 25.3, 22.6, 14.1. Anal. calcd for C20H44NO2Cl: C,
65.63; H, 12.12; N, 3.83. Found: C, 65.56, H, 12.09, N,
3.90.
2-Hydroxy-N,N-bis(2-hydroxyethyl)-N-methyl-1-hexade-
canaminium chloride, 2(16)Cl. A mixture of 5 (2.06 g,
8.57 mmol) in aq HCl (16 mL, ꢃ0.6 M solution) was
heated to reflux for 3 h. 2-[(2-Hydroxyethyl)methylamino]
ethanol(2.47 g, 20.73 mmo)l was added. The hetero-
geneous reaction mixture was heated to reflux over-
night. The reaction mixture turned into a clear,
homogeneous, light-yellow solution; turned opaque
when it was cooled to room temperature. The reaction
mixture was washed with EtOAc (2Â25 mL). The aqu-
eous layer was collected, concentrated, and dried in
vacuo to give a thick oil. To purify the product, several
crystallizations were attempted. The oil was dissolved in
boiling EtOAc/CH3CN (2:1, 105 mL) and cooled at
room temperature. A layer of semi-solid, sticky oil was
formed on the glass surface. Scraping the beaker with a
spatula induced solidification. The solid was collected
by vacuum filtration and washed with CH3CN (2Â10
mL) to give a white powder (1.47 g, yield 43%).
Syntheses
2-Hydroxy-N-(2-hydroxyethyl)-N-methyl-1-hexadecana-
mine, 4. A solution of bromohydrins 3 (2.00 g, 6.22
mmol) and 2-[(N-methyl)amino]ethanol (1.12 g, 14.9
mmol) in CH3OH (40 mL) was heated to reflux over-
night. The solvent was removed under reduced pressure
to give a colorless oil, which was purified twice with
flash column chromatography (1.75-inch diameterÂ5.5-
inch height) by eluting with CHCl3/CH3OH (10:1) to
give a thick, colorless oil that solidified in vacuo over-
night to give a white solid (1.51 g, yield 77%): 1H NMR
(400 MHz, CDCl3) d 3.70–3.61 (comp, 3H), 2.67 (ddd,
1H, Japp=13.0, 6.8, 4.7 Hz), 2.52 (ddd, 1H, Japp=13.0,
5.6, 4.4 Hz), 2.35 (m, 2H), 2.31 (s, 3H), 1.46–1.20
(comp, 26H), 0.87 (t, 3H, Japp=6.9 Hz; 13C NMR
(100 MHz, CDCl3) d 67.6, 64.1, 59.6, 59.3, 42.4, 34.9,
31.9, 29.7, 29.63, 29.61, 29.55, 29.3, 25.6, 22.6, 14.1.
2-Hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-1-hexa-
decanaminium iodide, 1(16)I. A solution of 4 (1.00 g,
3.17 mmol) and CH3I (2.14 g, 15.1 mmol) in ether (10
1H NMR (400 MHz, CDCl3) d 5.21 (two overlapping
triplets, 2H), 5.06 (d, 1H, Japp=7.0 Hz), 4.20 (m, 1H),

Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
3605
4.07 (m, 4H), 3.91–3.81 (comp, 4H), 3.63 (m, 1H), 3.38
(overlapping d and s, 4H and NCH3), 1.53–1.24 (comp,
26H), 0.87 (t, 3H, Japp=6.8 Hz); 13C NMR (100 MHz,
CDCl3) d 68.4, 65.6, 65.3, 65.1, 55.9, 55.8, 51.0, 36.1,
31.9, 29.83, 29.80, 29.78, 29.76, 29.69, 29.4, 25.4, 22.7,
14.1. Anal. calcd for C21H46NO3Cl: C, 63.69; H, 11.71;
N, 3.54. Found: C, 63.74; H, 11.71; N, 3.52.
0.88 (t, 3H, Japp=6.9 Hz); 13C NMR (100 MHz, CDCl3)
d 52.2, 36.6, 31.9, 30.0 29.7, 29.60, 29.58, 29.3, 27.3,
22.7, 14.1.
N-(2-Hydroxyethyl)-N-methyl-1-hexadecanamine, 10. A
mixture of 9 (1.00 g, 3.91 mmol), 2-chloroethanol (711
mg, 8.83 mmol) and K2CO3 (677 mg, 4.90 mmol) in
CH3OH (10 mL) was heated to reflux for 24 h. The
solution was concentrated, and ether (200 mL), EtOAc
(100 mL), and water (100 mL) were added. The organic
layer was separated and dried over Na2SO4, filtered,
and concentrated to give a mixture of oiland crystasl.
This crude product was purified with flash column
chromatography (1.75-inch diameterÂ4.5-inch height)
by eluting with CHCl3/CH3OH (10:1, 1000 mL) to give
2-Hydroxy-N,N,N-trimethyl-1-hexadecanaminium chloride,
6. A mixture of 5 (3.69 g, 15.3 mmol) (not very soluble
.
in CH3OH at room temperature) and (CH3)3N HCl
(1.23 g, 12.9 mmol) in CH3OH (50 mL) was heated to
reflux overnight. The reaction mixture was concentrated
to give a sticky solid, which was recrystallized in
CH3CN (250 mL). A solid started forming after 1 h at
room temperature. The solid was collected by vacuum
filtration, and washed with CH3CN, to give a white
solid (2.54 g, yield 59%): 1H NMR (400 MHz, CDCl3) d
5.69 (d, 1H, Japp=7.0 Hz), 4.22 (br m, 1H), 3.53 (dd,
1H, Japp=13.2, 10.3 Hz), 3.45 (s, 9H), 3.32 (m, 1H),
1.56–1.21 (comp, 26H), 0.84 (t, 3H, Japp=6.8 Hz); 13C
NMR (100 MHz, CDCl3) d 71.5, 65.5, 54.8, 35.8, 31.9,
29.7, 29.62, 29.59, 29.56, 29.3, 25.3, 22.6, 14.1. Anal.
calcd for C19H42NOCl: C, 67.92; H, 12.60; N, 4.17.
Found: C, 67.87; H, 12.60; N, 4.12.
1
an oily crystalline solid (630 mg, yield 54%): H NMR
(400 MHz, CDCl3) d 3.59 (t, 2H, Japp=5.0 Hz), 2.54 (t,
2H, Japp=5.0 Hz), 2.42 (t, 2H, Japp=7.2 Hz), 2.26 (s,
3H), 1.47 (m, 2H), 1.28–1.25 (comp, 26H), 0.88 (t, 3H,
1
Japp=6.5 Hz); (lit.36,37 60 MHz H NMR) 13C NMR
(100 MHz, CDCl3) d 58.7, 58.2, 57.7, 41.5, 31.9, 29.7,
29.64, 29.60, 29.5, 29.3, 27.3, 27.1, 22.7, 14.1.
N,N-Bis(2-hydroxyethyl)-N-methyl-1-hexadecanaminium
chloride, 11. A solution of 10 (630 mg, 2.10 mmol) and
2-chloroethanol (687 mg, 8.53 mmol) in MeOH (12 mL)
was heated to boiling in a heavy-walled sealed tube for 5
days. The solvent was removed under reduced pressure
to give a colorless oil, which was triturated in EtOAc.
Fine crystals were collected by vacuum filtration to
N-(2-Hydroxyethyl)-N,N-dimethyl-1-hexadecanaminium
chloride, 8. A mixture of 7 (3.01 g, 11.5 mmol) (inso-
luble in CH3OH) and 2-(dimethylamino)ethanol (1.64 g,
18.4 mmol) in CH3OH (35 mL) was heated to reflux
overnight. The solvent was removed under reduced
pressure to give a white gummy solid. EtOAc (400 mL)
was added to triturate the product. The EtOAc solution
was heated to the bp for 30 min, but some solid
remained. The EtOAc solution was cooled at room
temperature overnight. Crystals were collected by
vacuum filtration, and washed with EtOAc (100 mL)
and then hexanes (50 mL) to give colorless crystals (2.12
1
give a powder. H NMR showed that the powder was
a mixture of the desired product and the hydrochloride
salt of amine 10. The powder was dissolved in CHCl3
(10 mL) and treated with deactivated basic alumina
(160 mg). The mixture was allowed to stand at room
temperature for 4 days with occasionalswirilng. The
basic alumina particles were removed by filtration
through a small cotton wool in a 6-inch pipet. The fil-
trate was concentrated to give a white, oily solid, which
was recrystallized in EtOAc (50 mL) to give white
crystals (399 mg, yield 50%). 1H NMR (400 MHz,
CDCl3) d 5.47 (t, 1H, Japp=5.4 Hz), 4.07 (br m, 4H),
3.69 (m, 4H), 3.51 (m, 2H), 3.30 (s, 3H), 1.72 (br m,
2H), 1.32–1.24 (comp, 26H), 0.86 (t, 3H, Japp=6.9
Hz); 13C NMR (100 MHz, CDCl3) d 64.2, 64.0, 55.8,
50.3, 31.9, 29.7, 29.6, 29.53, 29.48, 29.34, 29.25, 26.4,
22.7, 22.6, 14.1. Anal. calcd for C21H46NO2Cl: C,
66.37; H, 12.20; N, 3.69. Found: C, 66.43; H, 12.18; N,
3.73.
1
g, yield 52%): H NMR (400 MHz, CDCl3) d 5.81 (t,
1H, Japp=5.6 Hz), 4.08 (br m, 2H), 3.70 (m, 2H), 3.51
(m, 2H), 3.34 (s, 6H), 1.72 (m, 2H), 1.32–1.23 (comp,
26H), 0.85 (t, 3H, Japp=6.9 Hz); 13C NMR (100 MHz,
CDCl3) d 66.0, 65.7, 55.9, 51.9, 31.9, 29.7, 29.62, 29.57,
29.5, 29.4, 29.3, 29.2, 26.3, 22.8, 22.6, 14.1. Anal. calcd
for C20H44NOCl: C, 68.63; H, 12.67; N, 4.00. Found: C,
68.54; H, 12.65; N, 3.93.
N-Methyl-1-hexadecanamine, 9. A mixture of 7 (2.29 g,
8.78 mmol), CH3NH2 (30 mL of 8.03 M solution in abs
EtOH, 241 mmol), K2CO3 (1.21 g, 8.75 mmol) and KI
(1.70 g, 10.21 mmol) was heated to reflux for 3 days.
The reaction mixture was diluted with ether (150 mL),
and washed with brine (100 mL). The etherealsoul tion
was dried over Na2SO4, filtered, and concentrated to
give a pale-yellow liquid. The crude product was puri-
fied with flash column chromatography (1.75-inch dia-
meterÂ5.5-inch height) by eluting with CHCl3/CH3OH
(10:1, 5:1, and 3:1, 1000 mL each) to give dihexa-
decylmethylamine (0.81 g) as a side product, which was
Recrystallization of N,N,N-Trimethyl-1-hexadecanami-
nium chloride, 12. Compound 12 (1.02 g, Eastman
Kodak) was added to acetone/EtOAc/EtOH (80:80:1,
80.5 mL), which was heated to boiling. Not all the solid
dissolved. The hot solution was decanted and filtered
through a filter paper; some crystals formed on the filter
paper and some crystals formed on cooling the filtrate.
These crystals were combined and recrystallized with
acetone/EtOAc (1:1, 80 mL) then washed with EtOAc
(20 mL) and hexanes (20 mL) to give colorless crystals
1
identified by H NMR, and the desired product as an
off-white solid (1.00 g, yield 45%): 1H NMR
(400 MHz, CDCl3) d 2.55 (t, 2H, Japp=7.2 Hz), 2.42 (s,
3H), 1.49–1.43 (comp, 2H), 1.28–1.25 (comp, 26H),
(210 mg): H NMR (400 MHz, CDCl3) d 3.52 (m, 2H),
3.45 (s, 9H), 1.73 (m, 2H), 1.34–1.19 (comp, 26H), 0.87
1
(t, 3H, Japp=6.8 Hz); 13C NMR (100 MHz, CDCl3) d

3606
Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
67.0, 53.2, 31.9, 29.65, 29.63, 29.60, 29.5, 29.4, 29.3,
29.2, 26.2, 23.2, 22.6, 14.1. Anal. calcd for C19H42NCl:
C, 71.31; H, 13.23; N, 4.38. Found: C, 71.32; H, 13.18;
N, 4.41.
Determination of critical micelle concentrations by
conductivity
As described previously in detail,11 conductivity mea-
surements were made with a conductance meter con-
nected to a conductance cell, containing platinum-
iridium electrodes coated with platinum black and gold
soldered to platinum lead wires, a dip-type cell made
2-Oxatetradecyloxirane, 13. To a suspension of NaH
(500 mg, 20.8 mmol) in THF (10 mL) at 15 ꢀC was
added a solution of 1-dodecanol (1.972 g, 10.58 mmol)
in THF (20 mL) dropwise over 10 min. After stirring at
room temperature for 4 h, (bromomethyl)oxirane (2.479
g, 18.10 mmol) was added. The reaction mixture was
stirred at room temperature overnight. The reaction
mixture was added to brine (50 mL) in a 500-mL
separatory funnel. The aqueous solution was extracted
with ether (2Â100 mL). The combined ether extracts
were washed with water (50 mL), dried over Na2SO4,
filtered and concentrated to give an oil. The crude pro-
duct was purified with flash column chromatography
(1.75-inch diameterÂ5.5-inch height) by eluting with
hexanes/EtOAc (5:1) to give a clear colorless oil (2.18 g,
from ABS plastic with a cgs cell constant of 1.0 cmÀ1
.
The meter gave readings in siemens/cm (S/cm). A tem-
perature probe connected directly to the meter mea-
sured the temperature of the sample solution before and
after each set of data was collected. A temperature of
25 ꢀC was maintained with a water bath. Typically, 20
data points were collected for each set; data points up to
but not exceeding 5 times the cmc were collected.
Semen samples
Semen samples were collected by healthy volunteers by
masturbation and allowed to liquefy for 30 min at room
temperature. If completely liquefied, they were eval-
uated for sperm concentration and motions parameters
with a computer-assisted semen analyzer (Hamilton
Thorne Research, Beverly, MA, USA). Only specimens
with >60Â106 motile sperm/mL and 50% motility were
used.
1
yield 85%). H NMR (400 MHz, CDCl3) d 3.70 (dd,
1H, Japp=11.5, 3.1 Hz), 3.48 (m, 2H), 3.38 (dd, 1H,
Japp=11.5, 5.8 Hz), 3.14 (m, 1H), 2.79 (dd, 1H,
Japp=5.2, 4.3 Hz), 2.60 (dd, 1H, Japp=5.2, 2.7 Hz), 1.57
(m, 2H), 1.35–1.25 (comp, 18H), 0.87 (t, 3H, Japp=6.9
Hz); 13C NMR (100 MHz, CDCl3) d 71.7, 71.4, 50.9,
44.3, 31.9, 29.7, 29.63, 29.60, 29.57, 29.56, 29.4, 29.3, 26.0,
22.7, 14.1. (lit.56 1H and 13C NMR, MHz unspecified).
In vitro spermicidal activity
2-Hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-4-oxahexa-
decan-1-aminium chloride, 14. To a solution of 13 (600
mg, 2.47 mmol) and 2-(dimethylamino)ethanol (206 mg,
2.31 mmol) in CH3OH (8 mL) was added a solution of
2-(dimethylamino)ethanol hydrochloride in CH3OH
(2.5 mL of 0.92 M). The pH of the resulting solution
was about 8. This reaction mixture was heated to reflux
overnight and then concentrated to give a colorless
thick oil, which was dissolved in boiling CHCl3 (10 mL).
The solution was boiled for 10–15 min to concentrate to
about 5 mL. After the solution had been cooled to room
temperature and EtOAc (90 mL) added, the solution
turned white. Scratching the beaker with spatula
induced the formation of a solid. The white solid was
collected by vacuum filtration, washed with EtOAc/
CHCl3 (9:1, 50 mL), and then ether (20 mL). The white
solid was dissolved in CHCl3 (1 mL) and filtered
through a small amount of flash silica gel (1-cm
heightÂ0.7-cm diameter) in a 6-inch pipet, and rinsed
with CHCl3 (2Â1 mL). EtOAc (170 mL) was added; the
solution was allowed to stand at room temperature
overnight. The fine crystals were collected by vacuum
filtration, washed with EtOAc (100 mL), and then hex-
anes (50 mL), to give white crystals (0.453 g, Yield
53%): 1H NMR (400 MHz, CDCl3) d 5.46 (d, 1H,
Japp=6.4 Hz), 5.40 (t, 1H, Japp=5.3 Hz), 4.39 (m, 1H),
4.09 (br m, 2H), 3.79 (m, 2H), 3.61 (dd, 1H, Japp=13.8,
9.4 Hz), 3.45–3.48 (comp, 2H), 3.41 (t, 2H, Japp=6.9
Hz), 3.38 (comp, 7H), 1.56–1.48 (comp, 2H), 1.30–1.24
(comp, 18H), 0.86 (t, 3H, Japp=6.9 Hz); 13C NMR
(100 MHz, CDCl3) d 72.4, 71.8, 68.7, 66.8, 64.5, 56.0,
53.6, 53.2, 31.9, 29.7, 29.6, 29.54, 29.50, 29.3, 26.0, 22.7,
14.1. Anal. calcd for C19H42NO3Cl: C, 62.01; H, 11.50;
N, 3.81. Found: C, 61.92; H, 11.48; N, 3.79.
Spermicidalactivities of the compounds were evaul ated
at once using a modified version to the protocolorigin-
ally described by Sander and Cramer.45 Semen samples
were adjusted with a buffered glucose solution (Baker’s
buffer) supplemented with 0.5–1.0% BSA in order to
contain 60Â106 motile sperm/mL; 2-fold serial dilutions
of the compounds were prepared in 0.9% NaCl(sailne).
Fifty microliters of semen were pipetted into 250 mL of
compound/saline dilutions and gently vortexed for 3 s.
A drop of this mixture was placed on a pre-warmed
glass slide, coverslipped, and analyzed under a dark-
field microscope with a 10Â objective during 30 s. If any
motile spermatozoon was seen, the dilution was con-
sidered as negative. Positive dilutions (i.e., all observed
sperm immotile) were further incubated at 37 ꢀC for 1 h
with two volumes of Baker’s buffer, and then re-exam-
ined for sperm motility. If no motile sperm were seen,
the positive score was maintained. The MEC of a com-
pound was calculated using the highest sperm-immobi-
lizing dilutions and the initial concentration of the
compound. Severaldifferent donor semen sampels were
assayed to ensure consistency of results. N-9 was used
as positive controlin alassays.
Statistical analysis
Comparisons between pairs of spermicidalresutls were
done with the non-parametric Mann–Whitney test.
In vitro anti-HIV activity
Following the method of Resnick et al.,46 cell-free (RF
strain) and cell-associated (RF-infected H9 cells) HIV-1

Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
3607
stocks, displaying titers around 6 Àlog10 TCID50, were
incubated with half-log serial dilutions of the test agents
(starting at 0.1%) for 2 min. Compound effect was ter-
minated by 10-fold serial dilutions, which were then
incubated with plated MT-2 cells for 4–6 days (in
quadruplicates). Endpoint: syncytium formation and
compound cytotoxicity (agents+MT-2 cells without
virus). Results were calculated by the Reed–Muench
method.57 N-9 was used as positive control.
15. Geiger, A. M.; Foxman, B. Epidemiology 1996, 7, 182.
16. Feldblum, P. J. Genitourin. Med. 1996, 72, 451.
17. Stephenson, J. JAMA 2000, 284, 949.
18. Fichorova, R. N.; Tucker, L. D.; Anderson, D. J. J. Infect.
Dis. 2001, 184, 418.
19. Black, D. B.; Dawson, B. A.; Ethier, J. C.; Neville, G. A.
J. Pharm. Biomed. Anal. 1990, 8, 527.
20. Walter, B. A.; Digenis, G. A. Pharm. Res. 1991, 8, 409.
21. Walter, B. A.; Hawi, A. A.; Zavos, P. M.; Digenis, G. A.
Pharm. Res. 1991, 8, 403.
22. Zavos, P. M.; Correa, J. R.; Nosek, D.; Mohammadi, F.;
Digenis, G. A. Contraception 1996, 54, 39.
23. Zavos, P. M.; Correa, J. R.; Nosek, D.; Mohammadi, F.;
Digenis, G. A. Fertil. Steril. 1996, 66, 729.
Acknowledgements
24. Scott, M. J.; Jones, M. N. Biochim. Biophys. Acta 2000,
1508, 235.
25. Merianos, J. J. In Disinfection, Sterilization, and Pre-
servation, 4th ed.; Block, S. S., Ed.; Lea & Febiger: Malvern,
1991; p 225.
26. Furuse, K.; Ishizeki, C.; Iwahara, S. J. Pharmacobiodyn.
1983, 6, 359.
27. Tevi-Benissan, C.; Makuva, M.; Morelli, A.; Georges-
Courbot, M. C.; Matta, M.; Georges, A.; Belec, L. J. AIDS
2000, 24, 147.
28. Tsubone, K.; Uchida, N.; Mimura, K. J. Am. Oil Chem.
Soc. 1990, 67, 451.
29. Tsubone, K.; Uchida, N. J. Am. Oil Chem. Soc. 1990, 67,
394.
30. Rutzen, H. Process for the Preparation of Quaternary
Ammonium Compounds; (HenkelK.-G.a.A., Fed. Rep. Ger.).
US Patent 4,480,126, Oct 30, 1984.
This work was supported by contracts from the Contra-
ceptive Research and Development (CONRAD) pro-
gram, Eastern Virginia MedicalScho,ol under a
Cooperative Agreement with the US Agency for Inter-
nationalDeveolpment (USAID). The views expressed
by the authors do not necessarily reflect those of CON-
RAD or USAID. We thank Atochem of North America
for their generous gift of tetradecyloxirane. We thank
PersonalProducts Company, a division of Ortho-
McNeilPharmaceutica,l Inc. (Raritan, NJ, USA) for
generously providing samples of N-9. We thank Pro-
fessor Richey M. Davis, for the use of the conductivity
meter and Professor Thomas C. Ward for the thermo-
stat bath.
31. Hensen, H.; Rutzen, H.; Busch, P.; Stuhrmann, D.; Thiele,
K. Quaternary Ammonium Compound Hair Conditioners;
(HenkelK.-G.a.A., Fed. Rep. Ger.). US Patent 4,744,977,
May 17, 1988.
References and Notes
1. UNAIDS/WHO AIDS Epidemic Update: December 2001;
http://www.unaids.org/worldaidsday/2001/Epiupdate2001/
Epiupdate2001_en.pdf (accessed, Feb 3, 2002).
2. McCormack, S.; Hayes, R.; Lacey, C. J. N.; Johnson, A. M.
Br. Med. J. 2001, 322, 410.
3. Elias, C.; Coggins, C. J. Womens Health Gend. Based Med.
2001, 10, 163.
4. Savle, P. S.; Doncel, G. F.; Bryant, S. D.; Hubieki, M. P.;
Robinette, R. G.; Gandour, R. D. Bioorg. Med. Chem. Lett.
1999, 9, 2545.
5. Gagne, N.; Cormier, H.; Omar, R. F.; Desormeaux, A.;
Gourde, P.; Tremblay, M. J.; Juhasz, J.; Beauchamp, D.;
Rioux, J. E.; Bergeron, M. G. Sex. Transm. Dis. 1999, 26, 177.
6. D’Cruz, O. J.; Venkatachalam, T. K.; Uckun, F. M. Biol.
Reprod. 2000, 62, 37.
7. Belec, L.; Tevi-Benissan, C.; Bianchi, A.; Cotigny, S.; Beu-
mont-Mauviel, M.; Si-Mohamed, A.; Malkin, J. E. J. Anti-
microb. Chemother. 2000, 46, 685.
8. D’Cruz, O. J.; Venkatachalam, T. K.; Uckun, F. M. Biol.
Reprod. 2000, 63, 196.
9. Weber, J.; Nunn, A.; O’Connor, T.; Jeffries, D.; Kitchen,
V.; McCormack, S.; Stott, J.; Almond, N.; Stone, A.; Darby-
shire, J. AIDS 2001, 15, 1563.
32. Rutzen, H.; Nikolaus, P.; Bischoff, M.; Lehmann, R.
Process for Manufacture of Quaternary Ammonium Com-
pounds; (HenkelK.-G.a.A., Fed. Rep. Ger.; Degussa A.-G.).
US Patent 4,492,802, Jan 8, 1985.
33. Le Bihan, H.; Rayet, P.; Urbain, M. Arch. Intern. Phar-
macodynamie 1949, 79, 481.
34. Tishkova, E. P.; Fedorov, S. B.; Kudryavtseva, L. A.;
Bel’skii, V. E.; Ivanov, B. E. J. Gen. Chem. USSR (Engl.
Transl.) 1990, 60, 2016.
35. Westphal, O.; Jerchel, D. Ber. 1940, 73B, 1002; Chem.
Abstr. 1941, 35, 22748.
36. Tonellato, U. J. Chem. Soc., Perkin Trans. 2 1977, 821.
37. Moss, R. A.; Ihara, Y. J. Org. Chem. 1983, 48, 588.
38. Ralston, A. W.; Eggenberger, D. N.; Harwood, H. J.; Du
Brow, P. L. J. Am. Chem. Soc. 1947, 69, 2095.
39. Najem, L.; Borredon, M. E. Synth. Commun. 1994, 24,
3021.
40. Fouda, A. A. S.; Madkour, L. H.; Evans, D. F. Indian J.
Chem., Sect. A 1986, 25A, 1102.
41. Bacaloglu, R.; Bunton, C. A.; Ortega, F. J. Phys. Chem.
1989, 93, 1497.
42. Mukerjee, P.; Ray, A. J. Phys. Chem. 1966, 70, 2150.
43. Perche, T.; Auvray, X.; Petipas, C.; Anthore, R.; Perez,
E.; Rico-Lattes, I.; Lattes, A. Langmuir 1996, 12, 863.
44. Wilk, K. A.; Burczyk, B.; Kubica, H. Colloids Surf. 1990,
50, 363.
45. Sander, F. V.; Cramer, S. D. Hum. Fertil. 1941, 6, 134.
46. Resnick, L.; Busso, M. E.; Duncan, R. C. Anti-HIV
Screening Technology. In Heterosexual Transmission of AIDS:
Proceedings of the Second Contraceptive Research and
Development (CONRAD) Program International Workshop,
held in Norfolk, Virginia, Feb 1–3, 1989; Alexander, N. J.,
Gabelnick, H. L., Spieler, J. M., Eds.; Wiley-Liss: New York,
1990; p 311.
10. Raghuvanshi, P.; Bagga, R.; Malhotra, D.; Gopalan, S.;
Talwar, G. P. Indian J. Med. Res. 2001, 113, 135.
11. Wong, Y.-L.; Curfman, C. L.; Doncel, G. F.; Patricia
Hubieki, M.; Dudding, T. C.; Savle, P. S.; Gandour, R. D.
Tetrahedron 2002, 58, 45.
12. Mauck, C.; Doncel, G. Curr. Infect. Dis. Rep. 2001, 3, 561.
13. Fihn, S. D.; Boyko, E. J.; Normand, E. H.; Chen, C. L.;
Grafton, J. R.; Hunt, M.; Yarbro, P.; Scholes, D.; Stergachis,
A. Am. J. Epidemiol. 1996, 144, 512.
14. Fihn, S. D.; Boyko, E. J.; Chen, C. L.; Normand, E. H.;
Yarbro, P.; Scholes, D. Arch. Intern. Med. 1998, 158, 281.

3608
Y.-L. Wong et al. / Bioorg. Med. Chem. 10 (2002) 3599–3608
47. Gelamo, E. L.; Tabak, M. Spectrochim. Acta, Part A
2000, 56, 2255.
48. Gelamo, E. L.; Silva, C. H. T. P.; Imasato, H.; Tabak, M.
Biochim. Biophys. Acta 2002, 84, 84.
Molodykh, Z. V.; Kucherova, N. L.; Yakubov, S. M.; Gor-
bunov, S. M.; Zotova, A. M.; Teplyakova, L. V.; Abram-
zon, A. A. Pharm. Chem. J. USSR (Engl. Transl.) 1989, 23,
418.
49. Wilk, K. A.; Burczyk, B.; Wilk, T. Pol. J. Chem. 1993, 67,
1675.
54. Ma, J.; Teng, F. Nanjing Huagong Xueyuan Xuebao 1994,
16, 115; Chem. Abstr. 1995, 122, 284523.
50. Zhao, S.; Zhang, G.; Zheng, G.; Niu, C. Riyong Huaxue.
Gongye 1997, 7.
55. Tachon, P.; Cotovio, J.; Dossou, K. G.; Prunieras, M. Int.
J. Cosmet. Sci. 1989, 11, 233.
51. Zissmann, E. Compt. Rend. 1954, 238, 1843.
52. Zissmann, E. Compt. Rend. 1957, 245, 237.
53. Tishkova, E. P.; Fedorov, S. B.; Kudryavtseva, L. A.;
56. Van Elburg, P. A.; Ormskerk, O. A.; De Kloe, K. P.;
Boogaard, P. J. J. Label. Compd. Radiopharm. 2000, 43, 147.
57. Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493.