5-Phenyl substituted 1-methyl-2-pyridones and 4′-substituted biphenyl-4-carboxylic acids. synthesis and evaluation as inhibitors of steroid-5α-reductase type 1 and 2

Article abstract of DOI:10.1016/S0968-0896(01)00293-0

The synthesis of a series of 5-phenyl substituted 1-methyl-2-pyridones (I) and 4′-substituted biphenyl-4-carboxylic acids (II) as novel A-C ring steroidomimetic inhibitors of 5α-reductase (5αR) is described. Compounds 1-4 (I) were synthesized by palladium catalyzed cross coupling (Ishikura) reaction between diethyl(3-pyridyl)borane and aryl halides (1b-4b) followed by α-oxidation with sodium ferrocyanate of the 1-methyl-pyridinium salt. Inhibitors II (5-18) were obtained either by two successive Friedel-Crafts acylations from biphenyl (5a-10a) followed by saponification to yield the corresponding carboxylic acids (5-10) or by Suzuki cross coupling reaction to give the 4′-substituted biphenyl 1-4-carbaldehydes 11a-18a. The latter compounds were subjected to a Lindgren oxidation to yield compounds 11-18. The compounds were tested for inhibitory activity toward human and rat 5 αR1 and 2. The test compounds inhibited 5αR, showing a broad range of inhibitory potencies. The best compound in series I was the N-(dicyclohexyl)-4-(1,2-dihydro-1-methyl-2-oxopyrid-5-yl) benzamide 4 exhibiting an IC₅₀ value for the human type 2 enzyme of 10 μM. In series II, the most active compound toward human type 2 isozyme was the 4′-(dicyclohexyl)acetyl-4-biphenyl carboxylic acid (10; IC₅₀ = 220 nM). Both series showed only marginal activity toward the human type 1 isozyme. In conclusion, the biphenyl carboxylic acids (II) are more appropiate for 5αR inhibition than the 5-phenyl-1-methyl-2-pyridones (1). Especially the 4′-carbonyl compounds 5-10 represent new lead structures for the development of novel human type 2 inhibitors. Copyright

Full text of DOI:10.1016/S0968-0896(01)00293-0

Bioorganic & Medicinal Chemistry10 (2002) 437–448
5-Phenyl Substituted 1-Methyl-2-pyridones and 40-Substituted
Biphenyl-4-carboxylic Acids. Synthesis and Evaluation as
Inhibitors ofSteroid-5 ꢀ-reductase Type 1 and 2
Franck Picard, Tobias Schulz and Rolf W. Hartmann*
8.5 Pharmaceutical and Medicinal Chemistry, Saarland University, PO Box 15 11 50, D-66041 Saarbrucken, Germany
¨
Received 9 July2001; accepted 15 August 2001
Abstract—The synthesis of a series of 5-phenyl substituted 1-methyl-2-pyridones (I) and 40-substituted biphenyl-4-carboxylic acids
(II) as novel A–C ring steroidomimetic inhibitors of 5a-reductase (5aR) is described. Compounds 1–4 (I) were synthesized by pal-
ladium catalyzed cross coupling (Ishikura) reaction between diethyl(3-pyridyl)borane and aryl halides (1b–4b) followed by a-oxi-
dation with sodium ferrocyanate of the 1-methyl-pyridinium salt. Inhibitors II (5–18) were obtained either bytwo successive
Friedel–Crafts acylations from biphenyl (5a–10a) followed by saponification to yield the corresponding carboxylic acids (5–10) or
0
bySuzuki cross coupling reaction to give the 4 -substituted biphenyl-4-carbaldehydes 11a–18a. The latter compounds were sub-
jected to a Lindgren oxidation to yield compounds 11–18. The compounds were tested for inhibitoryactivitytoward human and rat
5aR1 and 2. The test compounds inhibited 5aR, showing a broad range of inhibitorypotencies. The best compound in series I was
the N-(dicyclohexyl)-4-(1,2-dihydro-1-methyl-2-oxopyrid-5-yl)benzamide 4 exhibiting an IC50 value for the human type 2 enzyme of
10 mM. In series II, the most active compound toward human type 2 isozyme was the 40-(dicyclohexyl)acetyl-4-biphenyl carboxylic
acid (10; IC50=220 nM). Both series showed only marginal activity toward the human type 1 isozyme. In conclusion, the biphenyl
carboxylic acids (II) are more appropriate for 5aR inhibition than the 5-phenyl-1-methyl-2-pyridones (I). Especially the 40-carbonyl
compounds 5–10 represent new lead structures for the development of novel human type 2 inhibitors. # 2001 Elsevier Science Ltd.
All rights reserved.
Introduction
side effects reported so far bythe use of finasteride 9 and
to increase the limited in vivo activityof this compound.
5a-Reductase (5aR) is a NADPH-dependent, mem-
brane bound enzyme which catalyzes the conversion of
testosterone (T) to the more potent androgen dihy-
drotestosterone (DHT). Two isozymes, named type 1
and 2, have been described with a different tissue dis-
tribution pattern and with distinct biochemical and
pharmacological properties.1 Both are able to convert T
into DHT. Despite a decrease in plasma testosterone
level with aging, the DHT concentration remains at a
constant level in the prostate.2 This is believed to playa
crucial role (permissive or/and causative) in benign
prostatic hyperplasia (BPH). Searching for a non inva-
sive treatment for BPH, the 5aR appears to be a pro-
mising therapeutic target. The first and so far only
compound on the market is finasteride, a steroidal type
2 inhibitor.3 Lots of efforts have been undertaken to
develop non steroidal inhibitors4ꢀ8 in order to avoid the
We previously reported that 5-aryl substituted 1-methyl-
2-pyridones (I, Chart 1), non steroidal compounds
mimicking the steroidal A and C ring, are inhibitors of
5aR.7 In the class of N-substituted piperidine-4-(ben-
zylidene-4-carboxylic acids) — new 5aR inhibitors
designed as substrate analogues using molecular
modeling — we recentlyobserved that the introduction
of hydrophobic and bulky substituents strongly
8
increased the inhibitoryactivity. This finding encour-
aged us to introduce some of these substituents into I
(1–4). The biphenyl structure is another steroidal A–C
ring mimetic, which has been successfullyused byour
group for the design of CYP 17 (P450 17) inhibitors.10
Consequently, we now describe the syntheses of a series
of 40-substituted biphenyl-4-carboxylic acids designated
as 5aR inhibitors (II, Chart 1, 5–18). Identical sub-
stituents were introduced in II (13–16) as theyhave been
used in I (1–4) to compare the two non steroidal
skeletons. In addition to the alkylketones 5–10 and the
*Corresponding author. Tel.:+49-681-302-3424; fax:+49-681-302-
4386; e-mail: rwh@mx.uni-saarland.de
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00293-0

438
F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
Chart 1. General structures.
amides 11–16 two halogenated derivatives were synthe-
sized (17, 18), as the corresponding tricyclic analogues
(III, Chart 1) have been reported to be 5aR type 1
inhibitors.11 In the following, we describe the syntheses
of compounds 1–18 as well as their evaluation as inhi-
bitors of human and rat 5aR type 1 and 2.
carbonylbiphenylketones 5a–10a (method D) from
which the corresponding carboxylic acids 5–10 were
obtained bysaponification (method E). For route B, a
Suzuki cross coupling reaction between 4-formyl-boro-
nic acid and the corresponding N-substituted 4-bromo-
benzamides using Pd(Ph3P)4 as catalyst14 was applied
(method F). Compounds 17a and 18a were prepared as
described.15 The 40-substituted biphenyl-4-carbox-
aldehydes 11a–18a were subjected to a Lindgren oxida-
tion using NaClO2 as oxidative agent and 2-methyl-2-
butene (11a–16a)16 or H2O2 (35%; 17a, 18a)17 as a
hypochloric acid scavenger to yield the corresponding
carboxylic acids 11–18 (method G).
Chemistry
We previouslyreported on the synthetic strategyof the
title compounds 1–4 (Scheme 1).7 Briefly, the first step
was the preparation of the diethyl(3-pyridyl)borane 20
which was synthesized from a solution of 3-lithiopyr-
idine and triethylborane as described.12 The 4-bromo-
benzoyl chloride, prepared from 4-bromobenzoic acid
and thionyl chloride, was reacted with the appropriate
amines to give the corresponding N-substituted 4-
bromobenzamides 1b–4b. The 3-arylpyridines 1a–4a
were synthesized by a cross coupling reaction between
20 and 1b–4b using Pd(Ph3P)4 as a catalyst and KOH as
a base (method A).13 Subsequentlycompounds 1a–4a
were subjected to methylation and K3[Fe(CN)6] oxida-
tion (method B). The regioisomers 1–4 and 10–40
obtained were separated bycolumn chromatography.
Biological Results
The inhibitoryactivityof compounds 1–18 and finas-
teride as a reference was determined using rat prostate
homogenates (pH 6.6, type 1; pH 5.5, type 2) and
human prostate homogenate (BPH tissue for type 2)
according to the method of Liang et al.,18 and the DU
145 cell line (for human type 1 enzyme) as described in
the literature.19,20
As seen in Table 1, the N-methylpyridones 1–4 dis-
played no or only a weak inhibitory activity toward
human type 2 isozyme. The most active compound was
the dicyclohexyl compound 4 with an IC50 value of
10 mM. The highest activityfor the human isozymes was
displayed by compound 2 for type 1 5aR. Notably, 2
(IC50 value 2.6 mM) consists of an analogous 3,5-bis(tri-
fluoromethyl)phenyl amide substituent as does the well
Two routes A and B were used for the syntheses of the 40-
substituted biphenyl-4-carboxylic acids 5–18 (Scheme 2).
Route A is based on two successive Friedel–Crafts acy-
lations. In the first one, biphenyl was reacted with the
corresponding acid chlorides to yield compounds 5b–10b
(method C). Subsequent reaction with oxalylchloride
and methanol gave the corresponding 40-methoxy-
Scheme 1. Synthesis of 5-phenyl and 3-phenyl substituted 1-methyl-2-pyridones 1–4 and 10–40.

F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
439
known steroidal inhibitor dutasteride.21 None of these
compounds was able to inhibit the rat type 2 isozyme to
a strong extent. Contraryto this, most of them showed
a good inhibitory activity toward the rat type 1 isozyme.
Except for compound 2, the inhibitors displayed IC50
values in the range of 10 to 1.3 mM for compounds 3
and 4, respectively. None of the isomers 10-40 (Scheme 1)
showed inhibitoryactivity(data not given) which also
had been observed with the analogous compounds.7
11% inhibition at a concentration of 10 mM (16) to an
IC50 value of 220 nM for the most active compound 10
(Table 2). Compounds 9, 13, and 15 also showed good
inhibition values (IC50 values of 0.64, 2.33, and 1.9 mM,
respectively). Contrary to the results observed for type 2
isozyme, most of the biphenyl compounds exhibited a
poor inhibitory activity versus human type 1 isozyme.
The most active compound toward this enzyme was the
dicyclohexyl amide 14 exhibiting 44% inhibition. Sur-
prisinglythe halogenated compounds 17 and 18 did not
show significant inhibition toward human type 1 enzyme
(DU 145 cells). Although their tricyclic analogues had
The biphenyl compounds showed a large variety of
potencies toward human type 2 isozyme ranging from
Scheme 2. Synthesis of 40-substituted biphenyl-4-carboxylic acids 5–16.
Table 1. Inhibition of rat and human 5aR type 1 and 2 in vitro by 5-phenyl-1-methyl-2-pyridones 1–4 (I) in comparison to corresponding biphenyl
compounds 13–16 (II)
R
Class No.
RVP:a (IC50, mM) or % inhibition (10 mM)
Human: (IC50, mM) or % inhibition (10 mM)
Type 1c
Type 2c
BPHdc
DU 145bce
N(Phenyl)2
I
II
I
II
I
II
I
II
1
13
2
16
3
15
4
14
(3.5)
(2.44)
19
(0.46)
(10)
(5.6)
(1.3)
(1.4)
24
23
11
(3)
23
(10)
34
n.i.
11
7
(2.33)
36
(1.9)
(10)
(4.7)
37
40
(2.6)
28
21
16
43
44
NH(3,5-bis(trifluoromethyl)phenyl)
NH(Adamantyl)
N(Cyclohexyl)2
30
aEnzyme of rat ventral prostate, 200–250 mg protein, substrate [1b, 2b-3H] testosterone 0.21 mM.
bSubstrate:[3H] androstenedione 5 nM.
cMean value; tests have been run in duplicate. The standard deviation for IC50 is 20%, for percent inhibtion isꢁ10%.
dEnzyme from BPH tissue (type 2), 200–300 mg protein, substrate[1b,2b-3H] testosterone, 0.21 mM.
eProstatic tumor cell line expressing type 1 enzyme.

440
F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
shown strong activityin CHO cells expressing human
isozyme 1.11 The rat type 2 isozyme was not inhibited
stronglybythe biphenyl compounds either. The highest
activityshowed compound 13 exhibiting an IC50 value
of 3 mM. Interestingly, compounds 13–16 inhibited the
rat type 1 enzyme stronger than the rat type 2 isozyme.
For instance, compound 13 displayed IC50 values of
0.46 and 3 mM in rat type 1 and type 2 isozyme,
respectively.
compound 2 being a much stronger inhibitor than
compound 16.
It is striking that the biphenyl compounds are weak
inhibitors of the human type 1 enzyme. This might be
due to the fact that the benzoic acid derivatives, which
under physiological conditions are deprotonated, can-
not easilypermeate the intact DU 145 cells. To clarify
this issue, a parallel artificial membrane permeation
23
assay(PAMPA) was performed. PAMPA mimics the
Comparing the inhibition values of series I and II com-
pounds with identical substituents, it becomes apparent
that the prostatic therapeutic target, the human type 2
enzyme, is inhibited by series II compounds stronger
than by 1–4 (Table 1).
passive diffusion through a cell membrane. Compound
9 showed a high flux rate (41%), even higher than
finasteride (32%), indicating that the poor activityis not
caused bya low permeation rate. From this, it has to be
concluded that the biphenyl-4-carboxylic acids are
indeed poor 5aR type 1 inhibitors.
Compounds inhibiting both types of 5aR, so-called dual
inhibitors, might be advantageous for the therapyof
BPH, since it has been shown that the dual inhibitor
dutasteride is more powerful in reducing the DHT
plasma concentration than selective type 1 or type 2
inhibitors.21
Discussion and Conclusion
The present paper demonstrates that there is a sig-
nificant increase in the inhibitoryactivityprofiles of
compounds 1–4 in comparison with those reported in
our previous paper.7 The most active compound A in
that series (Chart 2) had shown 61% inhibition at a
concentration of 100 mM, whereas the most active com-
pound 4 of this paper displayed an IC50 value of 10 mM
toward human type 2 isozyme. This result obviously is
due to the bulkycarboxamide substituents. It supports
the hypothesis of the existence of a hydrophobic pocket
The carboxylic acid groups are essential for activity, as
the aldehyde intermediates 13a and 14a (Scheme 2) of
compounds 13 and 14 did not show anyactivityat a
concentration of 10 mM toward both human isozymes
(less than 10% inhibition at a concentration of 10 mM,
data not given). Obviously, the carboxylate is required
for mimicking the transition state (enolate) formed
during the conversion of T to DHT.
22
in the active site of 5aR proposed byothers.
Comparing the steroidal A ring mimetics, it becomes
apparent that in general the benzoic acid moiety(type
II) is superior to the 1-methyl-2-pyridone structure (type
I). This is especially true for the human type 2 enzyme.
An exception observed for the human type 1 enzyme is
In the ketone compounds 5–10 of series II, the role of
the alkyl substituents was investigated. This study was
performed because MK-386, an azasteroid bearing a
Table 2. Inhibition of rat and human 5aR type 1 and 2 in vitro by compounds 5–18 and finasteride
R
Route No.
RVP:a (IC50, mM) or % inhibition (10 mM)
Human: (IC50, mM) or % inhibition (10 mM)
Type 1c
Type 2c
BPHcd
DU 145bce
(CH2)2CH3
(CH2)3CH3
CH(CH3)2
CH2CH(CH3)2
Cyclohexyl
CH(Cyclohexyl)2
N(iPr)2
N(iBu)2
A
A
A
A
A
A
B
B
B
B
B
B
B
B
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
24
15
22
31
31
4
24
(0.46)
(1.4)
(5.6)
(2.44)
28
33
28
34
36
34
30
10
37
(3)
30
(10)
23
29
27
(0.011)
(10)
60
(10)
25
35
34
11
19
27
9
16
28
44
16
40
7
(3.3)
(0.64)
(0.22)
15
(4.0)
(2.33)
(4.7)
(1.9)
11
N(Phenyl)2
N(Cyclohexyl)2
NH(Adamantyl)
NH(3,5-bis(trifluoromethyl)phenyl)
Br
Cl
Finasteride
65
65
24
(0.01)
8
(0.039)
(2–3 nM)
aꢀeSee Table 1.

F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
441
Chart 2. General structures.
long alkyl substituent in 17b position (Chart 2), has
been described to be a strong inhibitor of the human
isozyme 1.24 Most of these compounds, however, turned
out to be weak inhibitors of human type 1 isozyme in
spite of a rather good fit obtained byoverlaying MK-
386 with compound 6 (minimum energyconforma-
tion—Hyperchem program). But they are good inhibi-
tors of the human type 2 isozyme. This is in accordance
with the finding of Holt et al. who described compound
B (Chart 2) to be a strong inhibitor of the human type 2
enzyme.25 The most active compounds of this series
were the cyclohexyl compound 9 and the dicyclohex-
ylmethyl compound 10 showing IC50 values of 0.64 and
0.22 mM, respectively. Comparing the ketone com-
pounds 5–10 with the amides 11–16, it seems as if the
ketone compounds were more active. This originates
from the observation that the dicyclohexylmethyl com-
pound 10 is bya factor of 21 more active than the
dicyclohexylamide 14 (IC50 values of 0.22 and 4.7 mM,
respectively). As the most active compounds toward the
human type 2 enzyme did not show sufficient activity
toward the rat enzyme an in vivo test could not be
performed using our rat model.8
Diethyl(3-pyridyl)borane (20).12 A 1 M solution of trie-
thylborane in hexane (21 mL) was added dropwise to a
drydiethlyether solution (30 mL) of 3-lithiopryidine
[prepared from 3-bromopyridine (20a, 21 mmol) and n-
BuLi (21 mmol, 13 mL of 1.6 M solution in hexane)]
under a nitrogen atmosphere at ꢀ78 ꢂC. The mixture
was stirred at the same temperature for 2 h, and at room
temperature overnight. Then a solution of iodine
(21 mmol) in THF (20 mL) was added slowlyat room
temperature. After stirring for 2 h, the mixture was
diluted with ethyl acetate and washed with 10%
Na2S2O3 aq. solution and twice with brine. The organic
phase was dried over MgSO4. After evaporation the
crude compound was purified bychromatographywith
benzene as eluent to yield 20 (65%) as white crystals,
mp 160–161 ꢂC. H NMR (400 MHz, CDCl3): d 0.47 (t,
1
6H, 3J=7.5 Hz, 2-CH3), 0.65 (m, 4H, 2-CH2–), 7.24 (dd,
1H, 3J=7 Hz, 3J=6 Hz, C5–H), 7.60 (s, 1H, C2–H), 7.71
(ddd, 1H, 3J=7 Hz, 4J=1.5 Hz, 4J=1.5 Hz, C4–H), 7.98
(d, 1H, 3J=6 Hz, C6-H). IR (KBr): n=3015, 2930, 2900,
2860, 2810, 1600, 1475, 1405, 1260, 1195, 1085, 1045,
1025, 900, 875, 800, 710. Anal. C9H14BN14 H2O.
General procedure for the synthesis of 1b–4b, 11b, 12b
As shown in the present paper, we have obtained new
non steroidal inhibitors of 5aR. In the class of A-C ring
steroidomimetics the biphenyl carboxylic acid (series II)
is more appropriate for 5aR inhibition than the 5-
phenyl-1-methyl-2-pyridone structure (series I). Espe-
4-Bromo-N,N-diphenylbenzamide (1b). A mixture of 4-
bromobenzoic acid (23 g, 0.1 mol), one drop of N,N-
dimethylformamide and thionyl chloride (50 mL) was
refluxed for 2 h. After cooling to ambient temperature,
SOCl2 was removed bydistillation. The crude acid
0
ciallythe 4 -carbonyl compounds represent promising
new leads for the development of novel human type 2
chloride was dissolved in 20 mL of dryCH Cl2 and was
2
5aR inhibitors.
added dropwise to a suspension of diphenylamine (18 g,
0.1 mol) and drytriethylamine (40 mL, 0.3 mol) in dry
CH2Cl2 (170 mL). The solution was stirred for 2 h. The
organic phase was washed with water (2ꢃ20 mL) and
dried over MgSO4. After filtration, the solvent was eva-
porated in vacuo and the crude product was purified by
recrystallization from hexane/ethyl acetate to yield 1b
Experimental
1H NMR spectra were recorded on a Bruker AM-400
(400 MHz) in DMSO-d6 or CDCl3. Chemical shifts are
reported as d values (ppm) relative to internal tetra-
methylsilane (d 0 ppm). Elemental analyses were per-
formed in the Department of Inorganic Chemistry,
Universityof the Saarland. IR spectra were performed
with KBr disks or films as indicated on a Perkin-Elmer
398 infrared spectrometer. Melting points were deter-
mined on a Kofler melting point apparatus Thermopan
(Reichert) and are uncorrected. Column chromato-
graphywas performed on Merck silicagel 60 (40–63 mm)
or (50–200 mm). All reactions were followed bythin
layer chromatography using Alugram1 Silica gel 60.
Chemicals and solvents used were commerciallyavailable
(Lancaster, Fluka, Acros) and were used without
further purification.
(18.3 g, 50%) as white crystals, mp 150 ꢂC. H NMR
1
(400 MHz, CDCl3): d 7.12 and 7.14 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 7.19 (t, 2H, 3J=6 Hz, aromat. H),
7.25–7.36 (m, 8H, aromat. H). IR (KBr): n=3450 (br),
3040, 1670, 1590, 1490, 1450, 1395, 1345, 1310, 1180,
1110, 1010, 860, 830, 770, 750, 700. C19H14NOBr
(352.23).
4-Bromo-N-3,5-bis(trifluoromethyl)phenylbenzamide (2b).
Synthesized from 4-bromobenzoic acid and 3,5-bis(tri-
fluoromethyl)aniline. Purified by recrystallization from
hexane/ethyl acetate. Yield 55%, white crystals, mp
169 ꢂC. 1H NMR (400 MHz, CDCl3): d 7.64 and 7.74 (d,
AA0BB0, 4H, 3J=8 Hz, aromat. H, and 1H, phenyl-H4),

442
F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
8.15 (s, 2H, phenyl-H2,-H6), 8.19 (s, 1H, NH). IR (KBr):
n=3280 (NH), 3100, 1660, 1600, 1560, 1470, 1450,
1380, 1310, 1280, 1230, 1180, 1150, 1110, 1020, 990,
890, 850, 750, 700. C15H8NOF6Br (317.13).
pyr. C6–H), 8.82 (s, 1H, pyr. C2–H). IR : n=3450 (br),
3040, 1670, 1590, 1490, 1450, 1395, 1345, 1310, 1180,
1110, 1010, 860, 830, 770, 750, 700. C24H18N2O
(350.41).
4-Bromo-N-adamantylbenzamide (3b). Synthesized from
4-bromobenzoic acid and adamantylamine. Purified by
recrystallization from hexane/ethyl acetate. Yield 44%,
white crystals, mp 160 ꢂC. 1H NMR (400 MHz, CDCl3):
d 1.72 (s, 6H, ada. H), 2.11 (s, 9H, ada. H), 5.71 (s,
broad, 1H,–NH–), 7.52 and 7.57 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H). IR (KBr): n=3340 (NH), 2920,
2860, 1650, 1600, 1540, 1490, 1460, 1360, 1310, 1260,
1160, 1070, 1020, 860, 850, 760. C17H20NOBr (334.25).
N-(3,5-Bis(trifluoromethyl)phenyl)-4-(3-pyridyl)benzamide
(2a). Synthesized from 4-bromo-N-3,5-bis(trifluoro-
methyl)benzamide (2b) and diethyl-3-(pyridyl)borane
(20) and purified byFCC in petrol ether/acetone 9:3.
Yield 30%, white solid, mp 226–227 ꢂC. 1H NMR
3
3
(400 MHz, CDCl3): d 7.44 (dd, 1H, J=8 Hz, J=5 Hz,
pyr. C5–H), 7.67 (s, 1H, phenyl-H4), 7.73 and 8.01 (d,
AA0BB0, 4H, 3J=8 Hz, aromat. H), 7.93 (d, 1H,
3J=8 Hz, pyr. C4–H), 8.22 (s, 2H, phenyl–H2,–H6), 8.27
3
(s, 1H,–NH–), 8.66 (d, 1H, J=5 Hz, pyr. C6–H ) 8.86
4-Bromo-N,N-dicyclohexylbenzamide (4b). Synthesized
from 4-bromobenzoic acid and dicylohexylamine and
recrystallized from hexane/ethyl acetate. Yield 52%,
white crystals, mp 138 ꢂC. 1H NMR (400 MHz, CDCl3):
d 1.15–1.78 (m, 20H, cyclohexyl–H), 2.58 and 3.18 (s,
broad, 2H,–N(CH)2–), 7.16 and 7.49 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H). IR (KBr): n=3400 (br), 2960,
2940, 2860, 1640, 1590, 1470, 1440, 1390, 1370, 1320,
1250, 1190, 1130, 1105, 1070, 1020, 1000, 940, 900, 850,
830, 760, 750, 700. C19H26NOBr (364.32).
(s, 1H, pyr. C2–H). IR (KBr): n=3320 (NH), 3100,
1660, 1610, 1560, 1470, 1450, 1390, 1280, 1230, 1210,
1180, 1130, 1000, 940, 890, 860, 820, 760, 700.
C20H12N2OF6 (410.32).
N-Adamantyl-4-(3-pyridyl)benzamide (3a). Synthesized
from 4-bromo-N-adamantylbenzamide (3b) and diethyl-
3-(pyridyl)borane (20) and purified byFCC in petrol
ether/acetone 6:4 and recrystallized from hexane/ethyl
acetate. Yield 89%, slightlyyellow solid, mp 149 ꢂC. H
1
NMR (400 MHz, CDCl3): d 1.74 (s, 6H, ada. H), 2.15
(s, 9H, ada. H), 5.85 (s, 1H,–NH–), 7.53 (s, 1H, pyr. C5-
H), 7.63 and 7.83 (d, AA0BB0, 4H, 3J=8 Hz, aromat. H)
4-Bromo-N,N-diisopropylbenzamide (11b).26 Synthesized
from 4-bromobenzoic acid and diisopropylamine. Pur-
ified byrecrystallization from petrol ether. Yield 64%,
3
7.90 (d, 1H, J=8 Hz, pyr. C4–H), 8.64 (s, 1H, broad,
mp 72–73 ꢂC. H NMR (400 MHz, CDCl3): d 1.43 (s,
pyr. C6–H), 8.87 (s, 1H, broad, pyr. C2–H). IR (KBr):
n=3340 (NH), 3040, 2920, 2850, 1660, 1590, 1540,
1510, 1470, 1450, 1360, 1340, 1310, 1260, 1190, 1130,
1030, 1000, 860, 820, 760, 730, 700. C22H24N2O
(332.44).
1
12H, –CH(CH3)2), 3.71 (s, 2H, broad,–CH(CH3)2), 7.20
and 7.51 (d, AA0BB0, 4H, 3J=8 Hz, aromat. H).
C13H18NOBr (284.19).
4-Bromo-N,N-diisobutylbenzamide (12b). Synthesized
from 4-bromobenzoic acid and diisobutylamine. Pur-
ified byrecrystallization first from petrol ether, then from
hexane. Yield 61%, mp 59 ꢂC. 1H NMR (400 MHz,
CDCl3): d 0.74 and 0.98 (2s, 12H,–CH(CH3)2), 1.84 and
2.11 (2s, 2H,–CH(CH3)2), 3.07 and 3.34 (2d, 4H,
3J=6 Hz,–NCH2–), 7.23 and 7.52 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H). C15H22NOBr (312.25).
N,N-Dicyclohexyl-4-(3-pyridyl)benzamide (4a). Synthe-
sized from 4-bromo-N,N-dicyclohexylbenzamide (4b)
and diethyl(3-pyridyl)borane (20) and purified bycol-
umn chromatography(CC) in petrol ether/acetone 1:1
and recrystallized from hexane/ethyl acetate. Yield
35%, white crystals, mp 167 ꢂC. H NMR (400 MHz,
1
CDCl3): d 1.05–1.79 (m, 20H, cyclohexyl-H), 2.61 and
3.16 (s, broad, 2H,–N(CH)2–), 7.39 (s, 1H, pyr. C5–H)
7.41 and 7.59 (d, AA0BB0, 4H, J=8 Hz, aromat. H),
3
Method A
7.92 (d, 1H, 3J=8 Hz, pyr. C4–H), 8.61 (d, 1H,
3J=4 Hz, pyr. C6–H), 8.87 (s, 1H, pyr. C2–H). IR
(KBr): n=3420 (br), 3020, 2940, 2860, 1640, 1450, 1440,
1390, 1370, 1320, 1250, 1190, 1130, 1000, 940, 900, 860,
805, 760, 720, 710. C24H30N2O (362.51).
N,N-Diphenyl-4-(3-pyridyl)benzamide (1a). To a stirred
mixture of 4-bromo-N,N-diphenylbenzamide (1b)
(10 mmol, 1.2 mol equiv, 3.52 g), and Pd(Ph3P)4
(500 mg) in THF under nitrogen atmosphere at room
temperature, were added tetra-n-butylammonium bro-
mide (0.82 mmol, 0.1 mol equiv, 264 mg), powdered
KOH (25 mmol, 3 mol equiv, 1.4 g) and diethyl-3-(pyr-
idyl)borane (20) (8.2 mmol, 1.2 g). The mixture was
heated under reflux for 2 h. After cooling to room tem-
perature the suspension was diluted with ethyl acetate.
The combined organic phases were washed with brine
and dried over MgSO4. After removal of the solvent, the
residue was purified byflash column chromatography
(FCC) (hexane/ethyl acetate, 1:1) to give 800 mg (28%)
Method B
The H NMR spectrum of compound 30 is given as an
1
example. In case of the isomers 10, 20, 40 the data corre-
sponding to the (1,2-dihydro-1-methyl-2-oxo-5-pyr-
idyl)benzamide moiety are very similar to that described
in 30. All the isomers 10–40 appeared on TLC as a
fluorescent UV visible spot.
1
of 1a as a brown oil. H NMR (400 MHz, CDCl3): d
N,N-Diphenyl-4-(1,2-dihydro-1-methyl-2-oxo-5-pyridyl)-
benzamide (1). Dimethyl sulfate (2.28 mmol, 288 mg)
was slowlyadded dropwise to 2.28 mmol of N,N-diphe-
nyl-4-(3-pyridyl)benzamide (1a) and heated for 1 h on a
7.17 and 7.23 (d, AA0BB0, 4H, J=8 Hz, aromat. H),
3
7.44–7.58 (m, 10H, aromat. H, and 1H, pyr. C5–H), 7.99
3
3
(d, 1H, J=8 Hz, pyr. C4–H), 8.61 (d, 1H, J=5 Hz,

F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
443
steam bath. The product was oxidized byadding an
aqueous solution of K3[Fe(CN)6] (5.70 mmol, 1.78 g) in
H2O (5.70 mL) under stirring and cooling. Powdered
KOH (18.2 mmol, 1.02 g) was added slowlykeeping the
temperature at 5–10 ꢂC. After adding CH2Cl2 (6 mL),
the mixture was stirred for 30 min before additional
portions of K3[Fe(CN)6] (2.85 mmol, 935 mg) in H2O
(2.85 mL) and powdered KOH (9.12 mmol, 500 mg)
were added. The mixture was left overnight. The bipha-
sic mixture was filtered and the layers were separated.
The aqueous layer was extracted with CH2Cl2. The
combined organic layers were dried over MgSO4, fil-
tered and evaporated to dryness. The brown oily residue
was purified byCC in petrol ether/ethyl acetate 1:1 to
yield 1 (41%) and 10 (26%). 1: slightlyyellow powder,
N,N-Dicyclohexyl-4-(1,2-dihydro-1-methyl-2-oxo-5-pyri-
dyl)benzamide (4). Synthesized from N,N-dicyclohexyl-
4-(3-pyridyl)benzamide (4a). Purified byFCC in hex-
ane/ethyl acetate 1:9 and recrystallized form hexane/
ethyl acetate. Yield 51%, white crystals, mp 232 ꢂC. H
1
NMR (400 MHz, CDCl3): d 1.10–1.78 (m, 20H, cyclo-
hexyl–H), 2.61 and 3.20 (s, broad, 2H,–N(CH)2–), 3.64
3
(s, 3H,–NCH3), 6.69 (d, 1H, J=9 Hz, pyr. C3–H), 7.34
and 7.41 (d, AA0BB0, 4H, 3J=8 Hz, aromat. H), 7.54 (d,
1H, 4J=2.5 Hz, pyr. C6–H), 7.65 (dd, 1H, 3J=9 Hz,
4J=2.5 Hz, pyr. C4–H). IR (KBr): n=3420 (br), 2940,
2860, 1670, 1620, 1540, 1470, 1440, 1370, 1320, 1300,
1260, 1190, 1170, 1130, 1000, 940, 900, 830, 770, 710.
Anal. C25H32N2O2 (392.54).
ꢂ
1
mp 200–201 C. H NMR (400 MHz, CDCl3): d 3.60 (s,
3H,–NCH3), 6.67 (d, 1H, J=9 Hz, pyr. C3–H), 7.15
Route A: method C
3
and 7.49 (d, AA0BB0, 4H, J=8 Hz, aromat. H), 7.17–
4-Biphenylyldicyclohexylmethylketone (10b). Under
anhydrous conditions, a stirred mixture of biphenyl
(6.50 g, 0.028 mol), carbon disulfide (150 mL), and finely
powdered anhydrous aluminum chloride (4.13 g,
0.031 mol) was treated with dicyclohexylacetylchloride
(7.00 g, 0.028 mol). The reaction mixture was refluxed
for 4 h. After cooling to ambient temperature the car-
bon disulfide was removed bydistillation under reduced
pressure and the residue was decomposed bycarefully
pouring onto a mixture of ice and dilute hydrochloric
acid. The solid was filtered off, dried and purified by
FCC in hexane/CH2Cl2 6:1 to yield 3.40 g (33%) of 10b
as a white solid, mp 140 ꢂC. 1H NMR (400 MHz,
CDCl3): d 0.86–1.91 (m, 22H, cyclohexyl–H), 3.34 (m,
3
7.31 (m, 10H, aromat. H), 7.51 (s, 1H, pyr. C6-H), 7.57
(dd, 1H, 3J=9 Hz, 4J=2.5 Hz, pyr. C4–H). IR : n=3450
(br), 3060, 1660, 1600, 1540, 1490, 1450, 1400, 1350,
1310, 1190, 1160, 1080, 1010, 860, 830, 770, 700. Anal.
C25H20N2O2 (380.44).
N-(3,5-Bis(trifluoromethyl)phenyl)-4-(1,2-dihydro-1-methyl-
2-oxo-5-pyridyl)benzamide (2). Synthesized from N-3,5-
bis(trifluoromethyl)phenyl-4-(3-pyridyl)benzamide (2a).
Purified byCC in n-hexane/ethyl acetate 1:1. Yield
ꢂ
1
30%, white crystals, mp 284 C. H NMR (400 MHz,
3
CDCl3): d 3.64 (s, 3H, ꢀNCH3), 6.73 (d, 1H, J=9 Hz,
pyr. C3–H), 7.46 (s, 1H, phenyl–H4), 7.52 and 7.66 (d,
AA0BB0, 4H, 3J=8 Hz, aromat. H), 7.56 (s, 1H,
4J=2.5 Hz, pyr. C6–H), 7.64 (dd, 1H, 3J=9 Hz,
4J=2.5 Hz, pyr. C4–H) 8.25 (s, 2H, phenyl–H2,–H6),
8.49 (s, 1H, NH). IR (KBr): n=3320 (NH), 3100, 1660,
1610, 1560, 1470, 1450, 1390, 1280, 1230, 1210, 1180,
1130, 1000, 940, 890, 860, 820, 760, 700. Anal.
C21H14N2O2F6 (440.34).
3
3
1H, J=7 Hz,–COCH–), 7.38 (t, 1H, J=7 Hz, aromat.
H), 7.46 (t, 2H, 3J=7 Hz, aromat. H), 7.62 (d, 2H,
3J=8 Hz, aromat. H), 7.66 and 8.03 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H). IR (KBr): n=1680 (CO stretch).
C26H32O (360.54).
4-Biphenylyl-n-propylketone (5b). Synthesized from
biphenyl and butanoyl chloride. Yield 58%, white solid,
mp 95 ꢂC, (lit. 94 ꢂC).27 1H NMR (400 MHz, CDCl3): d
N-Adamantyl-4-(1,2-dihydro-1-methyl-2-oxo-5-pyridyl)-
benzamide (3). Synthesized from N-adamantyl-4-(3-pyr-
idyl)benzamide (3a). Purified byCC in petrol ether/ethyl
acetate 1:1 and recrystallized from petrol ether/ethyl
acetate. Yield 62%, white crystals, mp 260 ꢂC. 1H NMR
(400 MHz, CDCl3): d 1.73 (s, 6H, ada. H), 2.14 (s, 9H,
ada. H), 3.64 (s, 3H,–NCH3), 5.82 (s, 1H,–NH–), 6.69
(d, 1H, 3J=9 Hz, pyr. C3–H), 7.44 and 7.76 (d, AA0BB0,
3
1.02 (t, 3H, J=7 Hz,–CH2CH2CH3), 1.80 (sextett, 2H,
3J=7 Hz,–CH2CH2CH3), 2.97 (t, 2H, 3J=7 Hz,–CH2
3
CH2CH3), 7.39 (t, 1H, J=7 Hz, aromat. H), 7.46 (t,
2H, 3J=7 Hz, aromat. H), 7.62 (d, 2H, 3J=7 Hz, aromat.
H), 7.67 and 8.03 (d, AA0BB0, 4H, 3J=8 Hz, aromat. H).
IR (KBr): n=1690 (CO stretch). C16H16O (224.30).
3
4
4H, J=8 Hz, aromat. H) 7.56 (d, 1H, J=2.5 Hz, pyr.
4-Biphenylyl-n-butylketone (6b). Synthesized from
biphenyl and pentanoyl chloride. Yield 61%, white
solid, mp 73 ꢂC, (lit. 76–78 ꢂC).27 1H NMR (400 MHz,
CDCl3): d 0.97 (t, 3H, 3J=7 Hz,–CH2CH2CH2CH3),
1.43 (sextett, 2H, 3J=7 Hz,–CH2CH2CH2CH3), 1.74
(quintett, 2H, 3J=7 Hz,–CH2CH2CH2CH3), 2.99 (t,
2H, 3J=7 Hz,–CH2CH2CH2CH3), 7.39 (t, 1H,
3
4
C6–H), 7.64 (dd, 1H, J=9 Hz, J=2.5 Hz, pyr. C4–H).
IR (KBr): n=3350 (NH), 3040, 2905, 2860, 1660, 1600,
1560, 1505, 1510, 1450, 1410, 1360, 1330, 1310, 1250,
1200, 1160, 1090, 1010, 860, 840, 770, 730, 710. Anal.
C23H26N2O2 (362.47).
3
N-Adamantyl-4-(1,2-dihydro-1-methyl-2-oxo-3-pyridyl)-
benzamide (30). Synthesized from N-adamantyl-4-(3-
3J=7 Hz, aromat. H), 7.47 (t, 2H, J=7 Hz, aromat.
H), 7.62 (d, 2H, 3J=7 Hz, aromat. H), 7.68 and 8.03 (d,
pyridyl)benzamide (3a). Yield 26%, colourless oil. H
AA0BB0, 4H, J=8 Hz, aromat. H). IR (KBr): n=1670
(CO stretch). C17H18O (238.33).
1
3
NMR (400 MHz, CDCl3): d 1.73 (s, 6H, ada. H), 2.13
(s, 9H, ada. H), 3.62 (s, 3H, –NCH3), 5.86 (s, 1H, –NH–
), 6.27 (t, 1H, 3J=7 Hz, pyr. C5–H), 7.34 (dd, 1H,
3J=7 Hz, 4J=2 Hz, pyr. C4–H), 7.52 (dd, 1H, 3J=7 Hz,
4J=2 Hz, pyr. C6–H), 7.72 and 7.75 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H).
4-Biphenylylisopropylketone (7b). Synthesized from
biphenyl and isobutyryl chloride. Yield 54%, white pow-
der, mp 49–50 ꢂC. (lit. 62 ꢂC).27 1H NMR (400MHz,
CDCl3): d 1.24 (d, 6H, 3J=7Hz, –CH(CH3)2), 3.58

444
F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
(septett, 1H, 3J=7 Hz, –CH(CH3)2), 7.39 (t, 1H,
40-Isobutanoylbiphenyl-4-carboxylic acid methyl ester
(7a). Synthesized from 4-biphenylylisopropylketone
(7b) and oxalylchloride. Purified by FCC in hexane/
ethyl acetate 9:1. Yield 53%, white solid, mp 101 ꢂC.
3
3J=7 Hz, aromat. H), 7.46 (t, 2H, J=7 Hz, aromat.
H), 7.62 (d, 2H, 3J=7 Hz, aromat. H), 7.68 and 8.03 (d,
AA0BB0, 4H, J=8 Hz, aromat. H). IR (KBr): n=1640
3
3
(CO stretch). C16H16O (224.30).
1H NMR (400 MHz, CDCl3): d 1.25 (d, 6H, J=7 Hz,
3
–CH(CH3)2), 3.59 (septett, 1H, J=7 Hz,–CH(CH3)2),
4-Biphenylylisobutylketone (8b). Synthesized from
biphenyl and 3-methylbutyryl chloride. Yield 65%,
white powder, mp 73 ꢂC, (lit. 74–76.5 ꢂC).27 1H NMR
3.95 (s, 3H, COOCH3), 7.70 (2ꢃoverlapp., d, 4H,
3J=8 Hz, aromat. H), 8.05 and 8.13 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H). C18H18O3 (282.34).
(400 MHz, CDCl3):
d
1.01 (d, 6H, 3J=7 Hz,–
CH(CH3)2), 2.32 (septett, 1H, 3J=7 Hz,–CH(CH3)2),
2.86 (d, 2H, 3J=7 Hz,–COCH2–), 7.39 (t, 1H, 3J=7 Hz,
40-(3-Methylbutanoyl)biphenyl-4-carboxylic acid methyl
ester (8a). Synthesized from 4-biphenylyl-(3-methylpro-
pyl)ketone (8b) and oxalylchloride. Purified by FCC in
hexane/ethyl acetate 9:1. Yield 12%, slightly yellow
3
aromat. H), 7.47 (t, 2H, J=7 Hz, aromat. H), 7.62 (d,
2H, J=7 Hz, aromat. H), 7.67 and 8.02 (d, AA0BB0,
3
4H, 3J=8 Hz, aromat. H). IR (KBr): n=1670 (CO
stretch). C17H18O (238.33).
solid, mp 121 ꢂC. H NMR (400 MHz, CDCl3): d 1.02
1
(d, 6H, 3J=7 Hz,–CH(CH3)2), 2.32 (septett, 1H,
3J=7 Hz,–CH(CH3)2), 2.86 (d, 2H, 3J=7 Hz,–COCH2–),
3.95 (s, 3H, COOCH3), 7.69 (2ꢃoverlapp., d, 4H,
3J=8 Hz, aromat. H), 8.04 and 8.13 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H). C19H20O3 (296.36).
4-Biphenylylcyclohexylketone (9b). Synthesized from
biphenyl and cyclohexyl carboxylic acid chloride. Yield
52%, white crystals, mp 86 ꢂC. 1H NMR (400 MHz,
CDCl3): d 1.27–1.93 (m, 10H, cylohexyl–H), 3.29 (m,
1H,–COCH–), 7.40 (t, 1H, J=8 Hz, aromat. H), 7.47
40-Cyclohexanoylbiphenyl-4-carboxylic acid methyl ester
(9a). Synthesized from 4-biphenylylcyclohexylketone
(9b) and oxalylchloride. Purified by FCC in hexane/
ethyl acetate 9:1. Yield 54%, white crystals, mp 119 ꢂC.
1H NMR (400 MHz, CDCl3): d 1.27–1.93 (m, 10H,
cylohexyl–H), 3.29 (m, 1H,–COCH–), 3.95 (s, 3H,–
COOCH3), 7.70 (2ꢃoverlapp., d, 4H, 3J=8 Hz, aromat.
3
(t, 2H, 3J=8 Hz, aromat. H), 7.62 (d, 2H, 3J=8 Hz, aro-
mat. H), 7.68 and 8.02 (d, AA0BB0, 4H, 3J=8 Hz, aromat.
H). IR (KBr): n=1680 (CO stretch). C19H20O (264.36).
Method D
40-Butanoylbiphenyl-4-carboxylic acid methyl ester (5a).
In an ice bath, anhydrous aluminum chloride (4.00 g,
30.0 mmol) was added in one portion to a mixture of 4-
biphenylyl-n-propylketone 5b (2.20 g, 9.90 mmol), and
oxalylchloride (1.70 mL, 2 equiv, 20.0 mmol) in 40 mL
of anhydrous dichloromethane. The mixture was stirred
for 45 min at 4–10 ꢂC, then poured carefullyonto 50 g of
ice. The solution was extracted several times with ethyl
acetate. The organic layer was washed with brine and
dried over MgSO4. After removal of the solvent the
residue was dissolved in drydichloromethane and added
dropwise to a solution of drymethanol (10 mL) in dry
dichloromethane. An exothermic reaction occurs. The
black mixture was stirred for 1 h, diluted with CH2Cl2
and washed with water. The combined organic phases
were dried over MgSO4 and evaporated. The residue was
purified by FCC in hexane/ethylꢂacetate 9:1 to yield the
title compound 5a (18%), mp 158 C. 1H NMR (400MHz,
H), 8.03 and 8.13 (d, AA BB , 4H, J=8 Hz, aromat.
0
0
3
H). C21H22O3 (322.40).
40-Dicyclohexylacetylbiphenyl-4-carboxylic acid methyl
ester (10a). Synthesized from 4-biphenylyldicyclohex-
ylmethylketone (10b) and oxalylchloride. Purified by
FCC in hexane/diethyl ether 8.5:1. Yield 50%, white
crystals, mp 149 ꢂC. 1H NMR (400 MHz, CDCl3): d
0.88–1.80 (m, 22H, cyclohexyl–H), 3.33 (t, 1H,
3J=7 Hz,–COCH–), 3.95 (s, 3H,–COOCH3), 7.69 and
0
0
3
8.05 (d, AA BB , 4H, J=8 Hz, aromat. H), overlapped
with 7.69 and 8.13 (d, AA0BB0, 4H, J=8 Hz, aromat.
3
H). C28H34O3 (418.57).
Method E
40-Butanoylbiphenyl-4-carboxylic acid (5). A mixture of
40-butanoylbiphenyl-4-carboxylic acid methyl ester 5a
(500 mg, 1.77 mmol) and potassium carbonate
(3.45 mmol, 485 mg) in methanol/water 9:1 (33 mL) was
refluxed for 3 h, then stirred at room temperature over-
night. The reaction was acidified with 1 N hydrochloric
acid and was extracted with CH2Cl2 (3ꢃ30 mL). The
combined organic phases were washed with water and
dried over MgSO4. The solvent was removed in vacuo
and the residue recrystallized from hexane/ethyl acetate
to yield 350 mg (73%) of 5 as white crystals.
3
CDCl3): d 0.95 (t, 3H, J=7 Hz,–(CH2)2CH3), 1.63 (sex-
tett, 2H, 3J=7 Hz,–CH2CH2CH3), 3.04 (t, 2H,
3J=7 Hz,–CH2CH2CH3), 3.96 (s, 3H, COOCH3), 7.86
(2ꢃoverlapp. d, 4H, 3J=8 Hz, aromat. H), 7.90 and 8.03
(d, AA0BB0, 4H, 3J=8 Hz, aromat. H). C18H18O3
(282.34).
40-Pentanoylbiphenyl-4-carboxylic acid methyl ester (6a).
Synthesized from 4-biphenylyl-n-butylketone (6b) and oxa-
lylchloride. Purified by FCC in hexane/ethyl acetate 9:1.
mp 221 ꢂC. H NMR (400 MHz, DMSO-d6): d 0.95 (t,
1
Yield 20%, white solid, mp 150ꢂC. H NMR (400 MHz,
3H, 3J=7 Hz,–CH2CH2CH3), 1.66 (sextett, 2H,
3J=7 Hz,–CH2CH2CH3), 3.04 (t, 20H, 3J=7 Hz,–
CH2CH2CH3), 7.87 and 7.88 (d, AA0BB , 4H, 3J=8 Hz,
1
3
CDCl3): d 0.97 (t, 3H, J=7 Hz, –(CH2)3CH3), 1.44 (sex-
tett, 2H, 3J=7 Hz, –CH2CH2CH2CH3), 1.75 (quintett, 2H,
3J=7 Hz, –CH2CH2CH2CH3), 3.00 (t, 2H, 3J=7 Hz, –CH2
CH2CH2CH3), 3.95 (s, 3H, COOCH3), 7.70 (2ꢃoverlapp.,
3
aromat. H), 8.06 (2ꢃoverlapp., d, 4H, J=8 Hz, aro-
mat. H), 13.03 (s, 1H, COOH). IR (KBr): n=2960,
2840, 1680, 1610, 1430, 1400, 1300, 1220, 1130, 1000,
900, 830, 780, 750. Anal. C17H16O3 (268.31).
0
0
3
d, 4H, J=8 Hz, aromat. H), 8.05 and 8.13 (d, AA BB ,
3
4H, J=8 Hz, aromat. H). C19H20O3 (296.36).

F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
445
40-Pentanoylbiphenyl-4-carboxylic acid (6). Synthesized
Route B: method F
from 40-pentanoylbiphenyl-4-carboxylic acid methyl
ester (6a). Purified bytwo recrystallizations from hex-
ane/ethyl acetate. Yield 53%, white solid, mp 202–
Compounds 17a and 18a were prepared according to
the literature.15
203 ꢂC. H NMR (400 MHz, DMSO-d6): d 0.91 (t, 3H,
1
3J=7 Hz,–CH2CH2CH2CH3), 1.36 (sextett, 2H,
3J=7 Hz,–CH2CH2CH2CH3), 1.62 (quintett, 2H,
3J=7 Hz,–CH2CH2CH2CH3), 3.05 (t, 2H, 3J=7 Hz,-
CH2CH2CH2CH3), 7.88 and 7.87 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 8.06 (2ꢃoverlapp., d, 4H,
3J=8 Hz, aromat. H), 13.03 (s, 1H, COOH). IR (KBr):
n=2980, 2860, 1680, 1610, 1440, 1420, 1300, 1240, 1130,
1000, 900, 830, 780, 750. Anal. C18H18O3 (282.34).
N,N-Diisopropyl-4-(4-formylphenyl)benzamide
(11a).
Under nitrogen aqueous potassium carbonate (646 mL
of a 2 M solution) was added to a stirred solution of 4-
bromo-N,N-diisopropylbenzamide (11b) (7.07 mmol,
2.00 g)
and
tetrakis(triphenylphosphine)palladium
(200 mg) in 20 mL anhydrous toluene. After this a solu-
tion of 4-formylphenyl boronic acid (1.00 g, 6.67 mmol)
in methanol (10 mL) was dropped into the reaction
mixture, which was stirred at 85 ꢂC for 4 h and then left
at room temperature overnight. The phases were sepa-
rated and the organic phase was washed with 5% aqu-
eous HCl and saturated aqueous sodium hydrogen
carbonate and dried over MgSO4. The solvent was
evaporated under reduced pressure to give a brown
solid which was recrystallized from hexane/chloroform
to yield the title compound 11a (1.00 g, 48%), mp
40-Isobutanoylbiphenyl-4-carboxylic acid (7). Synthesized
from 40-isobutanoylbiphenyl-4-carboxylic acid methyl
ester (7a). Purified bytwo recrystallizations from hex-
ane/ethyl acetate. Yield 62%, white solid, mp 234–
ꢂ
1
235 C. H NMR (400 MHz, DMSO-d6): d 1.24 (d, 6H,
3J=7 Hz,–CH(CH3)2), 3.71 (septett, 1H, 3J=7 Hz,–
CH(CH3)2), 7.81 (2ꢃoverlapp., d, 4H, 3J=8 Hz, aromat.
H), 8.05 and 8.10 (d, AA0BB0, 4H, J=8 Hz, aromat.
184 ꢂC. H NMR (400 MHz, CDCl3) : d 1.25 (s, broad,
3
1
H), 13.04 (s, 1H, COOH). IR (KBr): n=2980, 2880,
1680, 1610, 1430, 1300, 1230, 1160, 1000, 980, 850, 750.
Anal. C17H16O3 (268.31).
12H,–CH(CH3)2), 3.70 (s, broad, 2H,–CH(CH3)2), 7.42
and 7.65 (d, AA0BB0, 4H, J=8 Hz, aromat. H), 7.75
3
and 7.96 (d, AA0BB0, 4H, J=8 Hz, aromat. H), 10.06
3
(s, 1H, CHO). IR (KBr): n=2960, 2940, 1700, 1640,
1600, 1440, 1370, 1350, 1210, 1170, 1040, 1000, 840,
820, 770. C20H23NO2 (309.41).
40-(3-Methylbutanoyl)biphenyl-4-carboxylic acid (8).
Synthesized from 40-(3-methylbutanoyl)biphenyl-4-car-
boxylic acid methyl ester (8a). Purified bytwo recrys-
tallizations from hexane/ethyl acetate. Yield 60%, white
N,N-Diisobutyl-4-(4-formylphenyl)benzamide (12a). Syn-
thesized from 4-bromo-N,N-diisobutylbenzamide (12b)
and 4-formylphenylboronic acid. Yield 53%, white
crystals, mp 151 ꢂC. 1H NMR (400 MHz, CDCl3): d
0.77 and 1.01 (2s, 12H,–CH(CH3)2), 1.89 and 2.14 (2s,
broad, 2H,–CH(CH3)2), 3.15 and 3.38 (2s, 4H,–NCH2–
solid, mp 230–231 ꢂC. H NMR (400 MHz, DMSO-d6):
1
3
d 0.96 (d, 6H, J=7 Hz,–CH(CH3)2), 2.18 (septett, 1H,
3J=7Hz,–CH(CH3)2), 2.93 (d, 2H, 3J=7 Hz,–COCH2–),
7.88 and 7.89 (d, AA0BB0, 4H, J=8 Hz, aromat. H),
3
8.05 and 8.07 (d, AA0BB0, 4H, J=8 Hz, aromat. H),
3
0
0
3
13.03 (s, 1H, COOH). IR (KBr): n=2960 (br), 1680,
1610, 1430, 1400, 1370, 1300, 1240, 1180, 1130, 1000,
950, 850, 820, 780. Anal. C18H18O3 (282.34).
), 7.46 and 7.65 (d, AA BB , 4H, J=8 Hz, aromat. H),
7.77 and 7.96 (d, AA0BB0, 4H, J=8 Hz, aromat. H),
3
10.07 (s, 1H, CHO). IR (KBr): n=2960, 2880, 1700,
1640, 1600, 1470, 1380, 1320, 1220, 1170, 1100, 1000,
840, 820, 760. C22H27NO2 (337.46).
40-Cyclohexanoylbiphenyl-4-carboxylic acid (9). Synthe-
sized from 40-cyclohexanoylbiphenyl-4-carboxylic acid
methyl ester (9a). Purified bytwo recrystallizations from
hexane/ethyl acetate. Yield 54%, white crystals,
mp 250 ꢂC. 1H NMR (400 MHz, DMSO-d6): d 1.16–
1.84 (m, 10H, cylohexyl-H), 3.45 (m, 1H,–COCH–),
N,N-Diphenyl-4-(4-formylphenyl)benzamide (13a). Syn-
thesized from 4-bromo-N,N-diphenylbenzamide (1b)
and 4-formylphenylboronic acid. Yield 14%, white
crystals, mp 180–181 ꢂC. H NMR (400 MHz, CDCl3):
1
0
0
3
7.88 and 7.89 (d, AA BB , 4H, J=8 Hz, aromat. H),
d 7.17–7.33 (m, 10H, aromat. H), 7.49 and 7.57 (d,
8.06 and 8.07 (d, AA0BB0, 4H, J=8 Hz, aromat. H),
AA0BB0, 4H, J=8 Hz, aromat. H), 7.69 and 7.92 (d,
3
3
13.02 (s, 1H, COOH). IR (KBr): n=2940, 2860, 1680,
1610, 1430, 1400, 1300, 1250, 1220, 1170, 1130, 1000,
980, 950, 850, 770, 750. Anal. C20H20O3 (308.37).
AA0BB0, 4H, 3J=8 Hz, aromat. H), 10.04 (s, 1H, CHO).
IR (KBr): n=3050, 2820, 1700, 1650, 1600, 1490, 1320,
1300, 1210, 1170, 1110, 1000, 850, 770, 700. C26H19NO2
(377.44).
40-Dicyclohexylacetylbiphenyl-4-carboxylic acid (10).
Synthesized from 40-dicyclohexylacetylbiphenyl-4-car-
boxylic acid methyl ester (10a). Purified bytwo recrys-
tallisations from hexane/ethyl acetate. Yield 56%, white
N,N-Dicyclohexyl-4-(4-formylphenyl)benzamide (14a).
Synthesized from 4-bromo-N,N-dicyclohexylbenzamide
(4b) and 4-formylphenylboronic acid. Yield 38%, white
crystals, mp 231–232 ꢂC. H NMR (400 MHz, DMSO-
crystals, mp 235–236 ꢂC. H NMR (400 MHz, CDCl3):
1
1
d6): d 0.82–1.80 (m, 22H, cylohexyl–H), 3.48 (t, 1H,
3J=7 Hz,–COCH–), 7.87 and 7.87 (d, AA00BB00, 4H,
3J=8 Hz, aromat. H), 8.06 and 8.09 (d, AA BB , 4H,
3J=8 Hz, aromat. H), 13.03 (s, 1H, COOH). IR (KBr):
n=2920, 2840, 1680, 1610, 1450, 1430, 1280, 1250,
1200, 1130, 1000, 940, 840, 770, 760. Anal. C27H32O3
(404.55).
d 1.09 and 1.79 (m, 20H, cyclohexyl-H), 2.62 and 3.18
(2s, broad, 2H,–NCH2–), 7.41 and 7.65 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 7.78 and 7.97 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 10.07 (s, 1H, CHO). IR (KBr):
n=2940, 2840, 1700, 1630, 1450, 1370, 1320, 1220,
1130, 1000, 940, 900, 840, 830, 770. C26H31NO2
(389.53).

446
F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
N-Adamantyl-4-(4-formylphenyl)benzamide (15a). Syn-
thesized from 4-bromo-N-adamantylbenzamide (3b)
and 4-formylphenylboronic acid. Yield 37%, white
N,N-Dicyclohexyl-4-(4-carboxyphenyl)benzamide (14).
Synthesized from N,N-dicyclohexyl-4-(4-formylphenyl)-
benzamide (14a). Yield 42%, white crystals, mp 266 ꢂC.
1H NMR (400 MHz, DMSO-d6): d 1.09 and 1.98 (m,
22H, cyclohexyl–H), 7.38 and 7.79 (d, AA00BB00, 4H,
3J=8 Hz, aromat. H), 7.84 and 8.03 (d, AA BB , 4H,
3J=8 Hz, aromat. H), 12.98 (s, 1H, COOH). IR (KBr):
n=2920, 2860, 1680, 1640, 1610, 1430, 1360, 1310, 1180,
1140, 1000, 900, 840, 750. Anal. C26H31NO3 (405.53).
crystals, mp 182–183 ꢂC. H NMR (400 MHz, CDCl3):
1
d 1.74 (s, 6H, ada. H), 2.15 (0s, 9H, ada. H), 5.83 (s, 1H,–
NH–), 7.67 and 7.76 (d, AA BB0, 4H, 3J=8 Hz, aromat.
H), 7.82 and 7.97 (d, AA0BB0, 4H, J=8 Hz, aromat.
3
H), 10.07 (s, 1H, CHO). IR (KBr): n=3400 (NH), 2940,
2840, 1700, 1620, 1450, 1350, 1320, 1200, 1130, 1000,
940, 840, 830, 700. C24H25NO2 (359.47).
N-Adamantyl-4-(4-carboxyphenyl)benzamide (15). Syn-
N-(3,5-Bis(trifluoromethyl)phenyl)-4-(4-formylphenyl)-
benzamide (16a). Synthesized from 4-bromo-N-3,5-bis(tri-
fluoromethyl)benzamide (2b) and 4-formylphenylboronic
thesized
benzamide (15a). Yield 42%, white crystals, mp
from
N-adamantyl-4-(4-formylphenyl)-
>300 ꢂC. H NMR (400 MHz, DMSO-d6): d 1.66 (s,
1
acid. Yield 44%, white crystals, mp 209 ꢂC. H NMR
6H, ada. H), 2.06 (s, 9H,0 ada. H), 7.65 (s, 1H,–NH–),
1
0
3
(400 MHz, CDCl3): d 7.67 (s, 1H, phenyl–H4), 7.79 and
7.79 and 7.84 (d, AA BB , 4H, J=8 Hz, aromat. H),
7.80 (d, AA0BB0, 4H, 3J=8 Hz, aromat. H), 8.0 and 8.01
7.89 and 8.03 (d, AA0BB0, 4H, J=8 Hz, aromat. H),
3
(d, AA0BB0, 4H, J=8 Hz, aromat. H), 8.17 (s, 1H,–
12.97 (s, 1H, COOH). IR (KBr): n=3400 (NH), 2910,
2860, 1690, 1660, 1610, 1510, 1430, 1300, 1100, 1000,
950, 850, 750. Anal. C24H25NO3 (375.47).
3
NH–), 8.22 (s, 2H, phenyl–H2,–H6), 10.09 (s, 1H,
CHO). IR (KBr): n=3380 (NH), 2920, 2860, 1680,
1610, 1550, 1470, 1440, 1390, 1280, 1170, 1140, 1000,
900, 830, 770, 700. C22H13NO2F6 (437.34).
N-(3,5-Bis(trifluoromethyl)phenyl)-4-(4-carboxyphenyl)-
benzamide (16). Synthesized from N-(3,5-bis(tri-
fluoromethyl)phenyl)-4-(4-formylphenyl)benzamide (16a).
Yield 30%, white crystals, mp>300 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d 7.83 (s, 1H, phenyl–H4), 7.91
Method G
Compounds were prepared following the procedure
described in the literature.16,17 For substances 17 and 18
THF was used as a solvent instead of acetonitrile. Com-
pounds were recrystallized twice from hexane/ethyl acetate.
and 7.96 (d, AA0BB0, 4H, J=8 Hz, aromat. H), 8.07
3
and 8.14 (d, AA0BB0, 4H, 3J=8 Hz, aromat. H), 8.56 (s,
2H, phenyl–H2,–H6), 10.91 (s, 1H,–NH–), 13.02 (s, 1H,
COOH). IR (KBr): n=3450 (NH), 2940 (br), 1680,
1610, 1550, 1450, 1380, 1270, 1250, 1180, 1140, 1000,
940, 890, 850, 750, 700. Anal. C22H13NO3F6 (453.34).
N,N-Diisopropyl-4-(4-carboxyphenyl)benzamide (11).
Synthesized from N,N-diisopropyl-4-(4-formyl-
phenyl)benzamide (11a). Yield 48%, white crystals, mp
270–271 ꢂC. H NMR (400 MHz, DMSO-d6): d 1.29 (s,
40-Bromobiphenyl-4-carboxylic acid (17). Synthesized
from 40-bromobiphenyl-4-carbaldehyde (18a). Yield
37%, white crystals, mp 292–293 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d 7.56 and 7.76 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 7.80 and 8.02 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 12.99 (s, 1H, COOH). IR (KBr):
n=2950 (br), 1670, 1610, 1480, 1430, 1300, 1200, 1100,
1080, 1000, 930, 870, 820, 770. Anal. C13H9O2Br
(277.11).
1
broad, 12H,–CH(CH3)2), 3.66 (s, 2H,–CH(CH3)2), 7.38
0
0
3
and 7.77 (d, AA BB , 4H, J=8 Hz, aromat. H), 7.82
and 8.02 (d, AA0BB0, 4H, J=8 Hz, aromat. H), 12.93
3
(s, 1H, COOH). IR (KBr): n=2920 (br), 2860, 1680,
1640, 1610, 1430, 1360, 1310, 1180, 1140, 1000, 900,
840, 750. Anal. C20H23NO3 (325.41).
N,N-Diisobutyl-4-(4-carboxyphenyl)benzamide
(12).
Synthesized from N,N-diisobutyl-4-(4-formylphenyl)-
benzamide (12a). Yield 45%, white crystals, mp 179 ꢂC.
1H NMR (400 MHz, DMSO-d6): d 0.69 and 0.94 (2s,
12H,–CH(CH3)2), 1.83 and 2.07 (2s, 2H,–CH(CH3)2),
3.01 and 3.12 (2s, 4H,–NCH2-), 7.43 and 7.80 (d,
40-Chlorobiphenyl-4-carboxylic acid (18). Synthesized
from 40-chlorobiphenyl-4-carbaldehyde (19a). Yield
42%, white crystals, mp 293–294 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d 7.55 and 7.76 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 7.80 and 8.03 (d, AA0BB0, 4H,
3J=8 Hz, aromat. H), 13.01 (s, 1H, COOH). IR (KBr):
n=2990 (br), 1670, 1610, 1480, 1430, 1330, 1300, 1100,
1000, 830, 770. Anal. C13H9O2Cl (232.66).
AA0BB0, 4H, J=8 Hz, aromat. H), 7.84 and 8.03 (d,
3
AA0BB0, 4H, 3J=8 Hz, aromat. H), 12.97 (s, 1H,
COOH). IR (KBr): n=2980 (br), 1700, 1640, 1610,
1470, 1430, 1280, 1100, 1010, 930, 840, 760. Anal.
C22H27NO3 (353.46).
Enzyme inhibition test
N,N-Diphenyl-4-(4-carboxyphenyl)benzamide (13). Syn-
thesized
from
N,N-diphenyl-4-(4-formylphenyl)-
Reagents. [1,2-3H]Androstenedione (4-androstene-3,17-
dione, AD), and [1,2-3H]testosterone (17b-hydroxy-4-
androstene-3-one, T) were purchased from DuPont,
Bad Homburg, Germany.
benzamide (13a). Yield 31%, white crystals, mp 266–
267 ꢂC. 1H NMR (400 MHz, DMSO-d6): d 7.20–7.37
(m, 10H, aromat. H), 7.53 and 7.63 (d, AA00BB00, 4H,
3J=8 Hz, aromat. H), 7.77 and 7.99 (d, AA BB , 4H,
3J=8 Hz, aromat. H), 12.97 (s, 1H, COOH). IR (KBr):
n=3000 (br), 1680, 1650, 1600, 1490, 1410, 1340, 1280,
1180, 1100, 1000, 850, 760, 700. Anal. C26H19NO3
(393.44).
Preparation oftissue. Rat prostatic enzyme was pre-
pared according to the method of Liang et al.18 with
slight modifications.8 Male rats were sacrificed and
prostates were taken within 5 min and put in ice cold

F. Picard et al. / Bioorg. Med. Chem. 10 (2002) 437–448
447
0.9% NaCl solution. All the following operations were
performed at 0–4 ꢂC. The prostates were dissected free
from fat and connective tissue, cut into pieces and
weighed. Per 1 g of tissue, 3 mL of 20 mM phosphate
buffer pH 6.5 containing 0.32 mM sucrose and 1 mM
DTT were added. The tissue was homogenized byten
10-s strokes at 20,500 rpm of an ultraturax (IKA) in 60-
s intervals, filtered through cheesecloth and centrifuged
for 60 min at 105,000g. The pellet obtained was resus-
pended in phosphate buffer. The centrifugation was
repeated, the final pellet resuspended in a minimum
volume of phosphate buffer and stored in 300 mL por-
tions at ꢀ70 ꢂC. The 105,000-g pellet contains nuclei,
mitochondria and microsomes and is referred to as the
enzyme preparation. The protein content was deter-
mined and was in the range of 15–25 mg/mL. Human
prostatic tissue from BPH patients was processed in the
same wayusing citrate buffer pH 5.5.
HPLC procedure. The procedure was carried out8 simi-
lar to the method of Cook et al.28 The steroids were
dissolved in 50 mL methanol and 25 mL injected into the
computer-controlled HPLC system, which was checked
before using labelled reference controls. Radioactivity
was measured using a Berthold LB 506C monitor.
Using methanol/water (55:45, w/w) for T and DHT,
with a flow of 0.4 ml/min and an additive flow of 1.0 mL
for scintillator, base-line-separation of T and DHT was
achieved within 20 min. For the steroids androstene-
dione and dihydroandrostenedione methanol/water
(50:50, w/w) was used.
Calculation procedure. The amount of DHT formed was
calculated (% DHT). The zero value was subtracted
from the control (cv) and inhibition (iv) value (cvcorr
and ivcorr).
Inhibition (I) was calculated using the following equa-
tion:%
Incubation procedure. The assaywas performed as
described18 with modifications.8 All values were run in
duplicate. The incubation was carried out for 30 min at
37 ꢂC in a total volume of 250 mL. In the case of rat
enzyme preparation phosphate buffer (40 mM, pH 6.6)
and in the case of human enzyme preparation citrate
buffer (40 mM, pH 5.5) was used. The incubation mix-
ture contained approximately250 mg rat protein (125 mg
human protein), 200 mM NADPH (human enzyme:
100 mM NADPH), 0.21 mM T including 45 nCi [1,2-3H]-
T, and 2% DMSO with or without test compound
(100 mM). The reaction was started byadding the pro-
static enzyme preparation and stopped by addition of
50 mL NaOH (10 M). The steroids were extracted using
500 mL of diethylether. The mixture was shaken for
10 min and centrifuged 10 min at 4000 rpm. The water
layer was frozen and the ether layer was decanted in
fresh tubes and evaporated to dryness.
%I ¼ ð1 À ivcorr=cvcorrÞ100
Acknowledgements
Thanks are due to the Fonds der Chemischen Industrie,
who supported this work bygrants. Franck Picard is
grateful to the Deutsche Forschungsgemeinschaft
(DFG) for financial support (Graduiertenstipendium).
We thank Mrs. Anja Palusczak for performing the bio-
logical tests and Pharmacelsus, CRO, Saarbrucken,
Germany, for performing the PAMPA assay.
Human type 1 inhibition: DU 145-assay20. Intact
human prostatic carcinoma DU 145 cells were used as
the source of type 1 5a-reductase.19 The inhibitory
potencies of the compounds were determined bymon-
itoring the conversion of the tritiated substrate andros-
tenedione (5 nM) to androstanedione during an
incubation period of 6 h. A daybefore the experiment,
DU 145 cells were seeded in a 24-multiwell-plate at a
densityof 180,000 cells/well and allowed to become
adherent overnight. Compounds to be tested were dis-
solved in DMSO and 5 mL of each were added to the
cells in a final volume of 0.5 mL complete medium.
Inhibitors are first screened at concentrations of 10 mM
in an initial test and in case of exceeding 80% inhibi-
tion, three concentrations were choosen for measure-
ment of IC50 values. As control of conversion (typically
about 35% under these conditions) served a triplicate of
wells without inhibitors and as positive control for
inhibition finasteride (80, 60, 40, 20 nM) was used. After
the 6 h incubation period in 5% CO2 at 37 ꢂC, the
medium samples were extracted twice with 1 mL die-
thylether and the steroids were separated by HPLC.
Results are expressed as amount of formed androstane-
dione as percentage of control values.
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