Syntheses and anti-HIV and human cluster of differentiation 4 (CD4) down-modulating potencies of pyridine-fused cyclotriazadisulfonamide (CADA) compounds

Article abstract of DOI:10.1016/j.bmc.2020.115816

CADA compounds selectively down-modulate human cell-surface CD4 protein and are of interest as HIV entry inhibitors and as drugs for asthma, rheumatoid arthritis, diabetes and some cancers. Postulating that fusing a pyridine ring bearing hydrophobic subst

Full text of DOI:10.1016/j.bmc.2020.115816

Journal Pre-proofs
Syntheses and Anti-HIV and Human Cluster of Differentiation 4 (CD4)
Down-Modulating Potencies of Pyridine-Fused Cyclotriazadisulfonamide
(CADA) Compounds
Liezel A. Lumangtad, Elisa Claeys, Sunil Hamal, Amarawan Intasiri,
Courtney Basrai, Expedite Yen-Pon, Davison Beenfeldt, Kurt Vermeire,
Thomas W. Bell
PII:
S0968-0896(20)30646-5
https://doi.org/10.1016/j.bmc.2020.115816
BMC 115816
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 August 2020
6 October 2020
8 October 2020
Please cite this article as: L.A. Lumangtad, E. Claeys, S. Hamal, A. Intasiri, C. Basrai, E. Yen-Pon, D. Beenfeldt,
K. Vermeire, T.W. Bell, Syntheses and Anti-HIV and Human Cluster of Differentiation 4 (CD4) Down-
Modulating Potencies of Pyridine-Fused Cyclotriazadisulfonamide (CADA) Compounds, Bioorganic &
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115816
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Syntheses and Anti-HIV and Human Cluster of Differentiation 4
CD4) Down-Modulating Potencies of Pyridine-Fused
Cyclotriazadisulfonamide (CADA) Compounds
(
a,b
c
a
a,d
Liezel A. Lumangtad, Elisa Claeys, Sunil Hamal, Amarawan Intasiri, Courtney
a
a
a
c
Basrai, Expedite Yen-Pon, Davison Beenfeldt, Kurt Vermeire, and Thomas W.
,
a
Bell*
aDepartment of Chemistry, University of Nevada, Reno, Nevada, 89557-0216, USA
cKU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute,
Laboratory of Virology and Chemotherapy, 3000 Leuven, Belgium
dDepartment of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330
Thailand
ABSTRACT
CADA compounds selectively down-modulate human cell-surface CD4 protein and are of interest
as HIV entry inhibitors and as drugs for asthma, rheumatoid arthritis, diabetes and some cancers.
Postulating that fusing a pyridine ring bearing hydrophobic substituents into the macrocyclic
scaffold of CADA compounds may lead to potent compounds with improved properties, 17
macrocycles were synthesized, 14 with 12-membered rings having an isobutylene head group, two
arenesulfonyl side arms, and fused pyridine rings bearing a para substituent. The analogs display
a wide range of CD4 down-modulating and anti-HIV potencies, including some with greater
potency than CADA, proving that a highly basic nitrogen atom in the 12-membered ring is not
required for potency and that hydrophobic substituents enhance potency of pyridine-fused CADA
compounds. Cytotoxicities of the new compounds compared favorably with those of CADA,
showing that incorporation of a pyridine ring into the macrocyclic scaffold can produce selective
compounds for potently down-modulating proteins of medicinal interest.
ARTICLE INFO
*
Corresponding author. E-mail address: twb@unr.edu
bPresent address: Nanosyn, 3100 Central Expressway, Santa Clara, California 95051, USA

Keywords: cell-surface protein, CD4, fused pyridines, HIV, signal peptide, translocation inhibitors
. Introduction
Cyclotriazadisulfonamide (CADA) compounds selectively down-modulate expression of
1
the cell-surface cluster of differentiation 4 (CD4) protein on primate cells and inhibit replication
of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV).1–6 CD4 is
expressed on T cells and other immune cells; it is required for entry of HIV into host cells.7,8
Hence, CADA compounds act as HIV entry inhibitors, a class of compounds that are of continuing
interest for treatment of HIV infection.5,9–16 They are unique in that they do not simply inhibit
direct binding of the virus to the cell; they reduce CD4 levels below the concentration required for
2
17
entry of HIV. Monoclonal antibodies that target CD4, such as iMabm36, and small-molecule
inhibitors18–21 of the binding of viral gp120 to CD4 are of current interest as candidates for
prophylactic and therapeutic drugs for HIV and acquired immunodeficiency syndrome (AIDS).
The small-molecule drug fostemsavir (BMS663068),19–21 which inhibits attachment by blocking
the GP120-CD4 interaction, is in a phase III clinical trial for treatment of HIV-1 infected patients.22
GSK3732394, a tri-specific entry inhibitor targeting CD4, viral gp41, and -helical peptide fusion,
has also entered clinical trials.23,24
Because antiretroviral therapy does not fully eradicate the virus, current research is aimed
at a “kick and kill” strategy; the virus is first activated by an anti-latency drug, then eliminated by
1
5
immune activation and/or antiviral or cytotoxic drugs. Recent clinical trials of this approach have
2
5
failed. Combinations of anti-HIV drugs operating by different mechanisms may be required, as
2
6
15
in current therapeutic regimens, so there is a continuing demand for new HIV entry inhibitors.
CADA compounds are radically different from conventional anti-HIV agents that target viral
2
7
proteins and are susceptible to resistance by mutation and selection mechanisms. Instead, CADA
2

compounds target a host biochemical pathway for cellular expression of CD4, so it is difficult to
generate a CADA-resistant virus with high infectivity.28
The availability of many conventional anti-HIV drugs suggests that CADA compounds
may be less significant as anti-HIV agents and may be more significant as drugs for other diseases.
Anti-CD4 antibodies have long been of interest for chemotherapy of various immune-based
diseases involving CD4+ T cells,29–31 including asthma, rheumatoid arthritis,32–35 and diabetes.
They are also being investigated as therapeutic agents for various cancers, such as neuroblastoma36
and T cell lymphomas.37,38 Thus, as CD4 receptor down-modulating drugs, CADA compounds are
of potential interest as therapeutic agents for numerous autoimmune diseases and cancers. While
CD4 down-modulation may at first appear to be a therapeutic strategy that could compromise the
immune system, the African green monkey avoids progression to AIDS when infected by SIVagm
–
through decreased expression of CD4 on its T cells, and the resulting CD4 cells are
immunocompetent.39
It has been found that the lead compound (CADA, 1, Figure 1) down-modulates cell-
4
0
surface CD4 by binding the signal peptide (SP) of the CD4 preprotein. In mammals, more than
one third of all proteins are bound to the cell membrane.41,42 These proteins are translated by
ribosomes that are docked onto the endoplasmic reticulum (ER) membrane. Cell-surface and
secreted proteins consisting of more than 100 amino acids enter the interior (lumen) of the ER by
crossing its membrane during translation, a process known as co-translational translocation.43–50
Most type-1 transmembrane proteins, such as CD4, are targeted to the ER membrane by the SP,
consisting of the first 15-40 amino acids at the N-terminus of the nascent protein (preprotein). For
some membrane-bound proteins, such as G-protein coupled receptors (GPCRs), the first
transmembrane domain serves as the signal sequence and is targeted in a similar manner. CADA
3

recognizes and binds to the SP of the human CD4 preprotein, preventing co-translational
translocation of CD4 into the ER lumen, thus inhibiting cell-surface expression of this protein.40
The significance of CADA compounds may go far beyond CD4 down-modulation in that the
results of structure-activity and mechanistic studies could eventually enable the design of SP-
targeting drugs for inhibiting expression of many cell-surface proteins of medicinal interest.51
Figure 1. Structures of CADA (1), CK147 (2), pyridine-fused analog SH28 (3) and its
retrosynthesis to key intermediate, diol 7 (R = H).
CADA specifically down-modulates CD4 among the numerous antigens expressed on T
cells, but another human protein, sortilin, has been discovered as a secondary target for inhibition
5
2
of ER translocation by CADA. Sortilin has recently been identified as a potential drug target for
5
3
54
55
56
many disorders, including frontotemporal lobar degeneration, autism, Alzheimer’s disease,
atherosclerosis,57,58 and breast cancer. The structure-activity relationships (SARs) described here
focus on CD4 down-modulation potencies of pyridine-fused CADA compounds, but these
compounds also contribute to the library of analogs that will be available for future SAR studies
on sortilin down-modulation.
59
Previous SAR studies of CADA compounds focused on modifications of CADA’s benzyl
tail group,2,6,60 its isobutylene head group, and the two arenesulfonamide side arms.
2
2,6,60–62
6
0
Increasing the hydrophobicity of the tail and the size of the dipole moment of one of the side
4

arms62 were found to improve activity, leading to potent analog CK147 (2, Figure 1).62
Hydrophobic tail groups decrease water solubility of CADA compounds, limiting biological
studies. Introducing a pyridine ring into the structure was attractive for a number of reasons.
6
3
Pyridine is second in abundance only to benzene as a 6-membered ring in drugs. There are
numerous examples of pyridine-containing drugs,64,65 partly because its moderate pK (5.23)
a
enables facile protonation/deprotonation during cell permeation and its metabolic reaction are
readily adjustable via substitution.66,67 It also has a large dipole moment (2.23 Debye) and its
nitrogen atom is a good hydrogen bond acceptor, enhancing attractive charge-dipole, dipole-dipole
and hydrogen bonding interactions with drug targets. Of potential concern to this study was that
pyridine is very polar and miscible with water in all proportions. Yet, the first pyridine-fused
analog (SH28, 3) had measurable CD4 down-modulation potency. We hypothesized that adding
nonpolar groups to the para position of the pyridine ring would increase potency, without
sacrificing water solubility. The retrosynthesis of 3 shown in Figure 1 was applied to each new
analog, reducing the synthetic challenge for each target to obtaining diol 7 as the key intermediate.
68
2
. Results and discussion
2
.1 Synthesis
The routes used for synthesis of the para substituted diols (7) are shown in Scheme 1. The
shortest approach is reduction of diesters of pyridine-2,6-dicarboxylic acids, but only the
unsubstituted dimethyl ester 8a is commercially available at a price that is attractive for multistep
synthesis. Reduction of 8a and some para-substituted esters, including 8e–h, with sodium
borohydride in methanol or ethanol generally proceeds in high yields.69–78 Dimethyl 4-
ethylpyridine-2,6-dicarboxylate (8b) and diethyl 4-t-butylpyridine-2,6-dicarboxylate (8c) were
prepared by free radical substitution reactions using H O , FeSO and propionaldehyde or
2
2
4
5

pivaldehyde, respectively.79,80 The 4-alkoxypyridinedicarboxylates 8e–h were prepared by
deprotonation and alkylation of 8d (dimethyl 4-hydroxypyridine-2,6-dicarboxylate70,72,78 or
diethyl 4-hydroxypyridine-2,6-dicarboxylate).71,75 Sodium borohydride reduction of all diesters
8
a–c and 8e–h gave the corresponding diols 7a–c and 7e–h in very high yields, as shown in
Scheme 1.
Scheme 1
Five additional para substituted diols with methyl, propyl, butyl, isobutyl, and phenyl
substituents (7i–m, Scheme 1) were prepared by the Hantzsch pyridine synthesis81–86 with the
direct method, condensing the corresponding aldehydes with t-butyl acetoacetate and ammonium
hydroxide.81,83,84,86,87 Because of their ease of hydrolysis under acidic conditions, t-butyl esters
were chosen for the general synthetic approach, modifying reactions reported for the 4-phenyl
series.88,89 Mild oxidizing conditions reported by Bai et al. were chosen for standard conversion
of 10 to 11, rather than employing the traditional oxidant, nitric acid.87 4-Phenyl-1,4-
dihydropyridine diester 10l, obtained in 52% yield from benzaldehyde, was oxidized quantitatively
under these conditions to 11l. The HCl salt produced by acidic hydrolysis of this t-butyl diester
was insoluble in the convenient pyrolysis solvent DowthermTM A, and t-butyl esters of this type
6
90
91

are resistant to alkaline saponification. Fortunately, 11l88,89 underwent quantitative
dealkylcarboxylation to 12l in boiling DowthermTM A. In contrast, saponification of the
corresponding diethyl ester and neat pyrolysis of the intermediate diacid has been reported to give
1
2l in 66% yield, overall.92 This sequence of reactions was applied also to butyraldehyde,
isobutyraldehyde, valeraldehyde, isovaleraldehyde and cyclohexanecarboxaldehyde, giving yields
ranging from 34–72% for the condensation step (9 to 10). Excellent yields were obtained for the
oxidation step (10 to 11), except that dealkylation of secondary substituents (isopropyl and
cyclohexyl) was observed, apparently as a consequence of the single-electron transfer mechanism
of this reaction.90 Diesters 11i–k underwent pyrolytic dealkylcarboxylation to 4-alkyl-2,6-
dimethylpyridines 12i–k in useful yields (51–100%).
Methyl groups in the 2 and 6 positions of pyridines can be selectively converted to aldehyde
substituents by condensation with benzaldehyde, followed by ozonolysis.93,94 As described in the
9
4
literature, this sequence of reactions was used to convert collidine (2,4,6-trimethylpyridine, 12m)
to 4-methyl-2,6-pyridinedicarboxaldehyde (14m) in 40% overall yield. As shown in Scheme 1,
this route was also used to convert 12i–l to dialdehydes 14i–l. The intermediate 4-phenyl-2,6-
9
5
distyrylpyridine (13l) was obtained in 83% yield, while the yields of 4-alkyl analogs 13i–k were
lower due to side-products from condensation in the 4-position. All distyrylpyridines 13i–m
9
6
underwent ozonolysis in high yields and the resulting dialdehydes, including 14l, were reduced
in very high yields to 14i–m, including known diols 14l69,73 and 14m.74
As shown in Scheme 2, twelve diols (7a–c, e–m) were converted to the corresponding
open-chain ditosylamides to prepare for macrocyclization to the target fused pyridine CADA
analogs. Preparation of 2,6-bis(bromomethyl)pyridine (6a) from diol 7a has been reported
frequently, generally with HBr in water97–99 or acetic acid,77,100 but PBr in CHCl
75,78
was chosen
3
3
7

for the standard approach to the entire series, considering the potential lability of 4-
alkoxypyridines under protic conditions. Conversions of 7e–g75,76,78,101 and 7l to 6e–g,l have also
69
been reported. The dibromides (6) were converted to dinitriles (5) via S 2 displacement by
N
cyanide, as described for 6a to 5a.73,97 Dinitrile 5a was reduced to diamine 15a with BH THF,
73,97
3
1
02
but higher yields were obtained in the remaining cases with NaBH and NiCl . Finally, the
4
2
diamines were tosylated in high yield with p-toluenesulfonyl chloride and triethylamine in
dichloromethane, as described for 15a to 4a.73
Scheme 2
2
R = H (a), Et (b), t-Bu (c), OMe (e), OEt (f), OBn (g), OCH Cy (h), Pr (i), Bu (j), i-Bu (k), Ph (l), Me (m).
As shown in Scheme 3, the 12 open-chain disulfonamides 4a-c,e-m underwent palladium-
catalyzed cyclization with 3-methylene-1,3-propanediyl bis(t-butylcarbonate), as previously
reported for 4a to 3a.103 To examine biological effects of a 4-hydroxypyridine (or the 4-pyridone
tautomer), para benzyloxy analog 3g was dealkylated to give compound 3d. Also, Atkins-Richman
cyclization104,105 of para t-butyl open-chain disulfonamide 4c with 1,2-dibromoethane gave 11-
membered macrocycle 16 for further structural diversity. While an isobutylene head group has
been essential for maximal potency in CADA compounds tested so far, 16 tests the combination
of an ethylene head group with a fused pyridine ring. In an attempt to prepare a 10-membered
macrocycle, Pd-catalyzed cyclization of disulfonamide 17 was attempted, but 20-membered 2+2
product 18 was isolated, as previously reported.103 Considering that the electron donating
dimethylamino side-arm substituent in compound 2 imparts high potency for CD4 down-
modulation,62 2,6-bis(2-aminoethyl)-4-ethylpyridine 15b was converted to the bis(4-
8

dimethylaminobenzenesulfonamide) 19, which was then cyclized to analog 20 (Scheme 3, inset).
Finally, replacement of the pyridine ring with acridine was considered desirable because acridine
is a common scaffold in drugs.106 For this purpose, readily available 1,8-bis(bromomethyl)acridine
(
21)107 was converted to disulfonamide 22 by reaction with Boc-protected p-
toluenesulfonamide,108 then 22 was cyclized to acridine analog 23.
Scheme 3
2
.2 Potency
A total of 17 macrocycles were synthesized, 14 with 12-membered rings having an
isobutylene head group, two arenesulfonyl side arms, and fused pyridine rings bearing an alkyl,
aryl, hydroxy or alkoxy substituent in the para position. Each of these 17 compounds, including
9

previously reported103 compounds 3a (SH28) and 18, was tested for CD4 down-modulation, either
as its hydrochloride salt, hydrobromide salt, or free base form, as indicated in Table 1. As described
in the Experimental Section, compositions and purities (>95%) were determined by combustion
microanalysis. As for previous CADA compounds,60–62 most of the samples tested were
stoichiometric hydrates of the monohydrochloride salts. The CD4 down-modulation potencies are
given in Table 1 as IC values, which are the concentrations producing a 50% decrease in CD4
5
0
expression, measured in Chinese hamster ovary (CHO) cells stably transfected with CD4 fused to
yellow fluorescent protein (CD4-YFP), after 24 h of drug treatment. The structures, compound
numbers/code names, anti-HIV potencies, and cytotoxicities of the 14 analogs are also given in
Table 1, along with those of the previously reported reference compound 1 (CADA). Three of the
new macrocycles, 16 (LAL039), 18 (LAL012), and 23 (LAL032), as well as all the open-chain
disulfonamides, showed no observable CD4 down-modulation or anti-HIV activity and were not
examined further.
The CD4 down-modulation and anti-HIV potencies given in Table 1 correlate well, as
observed in previous studies of CADA compounds.60–62 As anticipated from previous SAR
6
0
studies, increasing hydrophobicity in the tail region enhances CD4 down-modulation potency.
The least potent fused pyridine CADA analog is 3d, bearing a para-hydroxy group. IC values for
5
0
CD4 down-modulation drop from 9.3 to 0.5 M as the para substituent is modified as follows: OH
> H => Me => Et => Pr => Bu in the 3d, 3a, 3m, 3b, 3i, 3j series. Branched or cyclic para
substituents iBu, t-Bu or Ph in 3c, 3k, and 3l give slightly higher IC values in the range of 0.7 –
=
5
0
1
.0 M. Methoxy and ethoxy substituents in 3e and 3f reduce potency relative to methyl (3m) and
ethyl (3b), increasing IC values by 0.2 and 0.6 M, respectively, perhaps due to increased dipole
5
0
moments of pyridine rings with para electron donating substituents. The more hydrophobic
1
0

benzyloxy group in 3g give potency comparable to that of CADA, while cyclohexylmethoxy
analog 3h has the greatest potency in the entire series. Analog 20 with a para-ethylpyridine ring
and para-dimethylamino groups on the benzenesulfonyl side arms is also more potent than CADA
(
by 50%), with an IC value that is smaller by 0.11 M. Finally, 12 of the fused pyridine CADA
5
0
compounds have undetectable cytotoxicities, and the remaining two, 3j and 3k, have therapeutic
indices (CC /IC for CD4) exceeding 100.
5
0
50
Table 1. CD4 Down-Modulating and anti-HIV Potencies and Cytotoxicities of Pyridine-
Fused CADA Compounds
a
b
IC CD4 (µM)
IC HIV (µM)
5
0
50
c
Compound
R
R’
mean ± SD
0.33 ± 0.06
2.81 ± 0.21
0.86 ± 0.10
0.95 ± 0.14
9.30 ± 0.60
1.88 ± 0.10
1.50 ± 0.17
0.42 ± 0.11
0.13 ± 0.02
0.83 ± 0.12
0.54 ± 0.17
0.66 ± 0.03
0.74 ± 0.15
1.67 ± 0.24
0.22 ± 0.01
mean ± SD
1.68 ± 1.00
> 50
CC (µM)
50
1
(CADA•HCl)
> 100
> 100
3
a (SH28)
b (LAL028•HCl)
c (LAL005•HCl)
d (LAL030•HBr)
e (LAL020•HCl)
f (LAL016•HCl)
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
NMe2
3
Et
3.40 ± 1.84
2.70 ± 0.05
> 50
> 100
3
t-Bu
OH
OMe
OEt
OBn
> 100
3
> 100
3
12.59 ± 0.54
10.40 ± 4.02
2.18 ± 0.32
0.93 ± 0.75
2.81 ± 0.28
2.41 ± 0.62
2.30 ± 0.27
2.47 ± 0.94
> 50
> 100
3
> 100
3
3
g (LAL018•HCl)
> 100
h (LAL036•HCl) OCH Cy
> 100
2
3
i (LAL026•HCl)
Pr
Bu
iBu
Ph
> 100
3
j (LAL022•HCl)
65.8 ± 7.2
3
k (LAL024•HCl)
l (LAL014•HCl)
m (LAL001•HCl)
0 (LAL042•HCl)
87.9 ± 17.1
> 100
3
3
Me
Et
> 100
2
1.10 ± 0.15
> 100
aIC50: inhibitory concentration 50%; concentration at which 50% down-modulation of CD4 expression measured in CD4-YFP
transfected CHO cells after 24 h of drug treatment. Values are the mean ± standard deviation with n = 3.
b
IC50: inhibitory concentration 50%; concentration at which 50% reduction of HIV-1 NL4.3 (X4) replication in MT-4 cells was
measured. Values are mean ± standard deviation with n = 2 or 3.
c
CC50: cytotoxic concentration 50%; concentration required to reduce viability of MT-4 cells by 50%.
1
1

2
.3 Solubility
A key rationale for investigating pyridine-fused CADA analogs was to determine
whether the polar pyridine ring can enhance water solubilities in potent compounds. Drug
solubility is perhaps one of the most important pharmacokinetic properties of an orally
administered drug. A number of assays to determine solubility of drug candidates have been
developed,109 two of which have emerged as common and convenient. Kinetic solubility is
measured by adding a concentrated solution of the drug in an organic solvent (e.g. DMSO) to an
aqueous buffer (e.g. PBS) at various concentrations.110 The drug is soluble at very low
concentrations but becomes increasingly insoluble at higher aqueous concentrations.
Nephelometry is commonly used to determine the kinetic solubility of a drug by measuring
turbidity of mixtures of drug stock solutions diluted with buffer in wells of microtiter plates.111
The concentration at which the nephelometer detects significant light scattering is the kinetic
solubility of the drug. Alternatively, thermodynamic solubility is typically determined by
agitating an excess of the drug and an aqueous buffer to achieve equilibrium between the
crystalline and dissolved states. The concentration of the dissolved drug at equilibrium solubility
can be determined conveniently by UV-Visible spectroscopy. Considering that many relatively
hydrophobic drugs have such low aqueous concentrations that their molar absorptivities
(
extinction coefficients) are difficult to determine in water by Beer-Lambert112 plots, the  value
is often determined in methanol and applied to the absorbance of the drug in water.
Listed in Table 2 are the molar absorptivities () and the thermodynamic and kinetic
solubilities of several selected pyridine-fused CADA compounds, as well as the lead compound,
CADA. Only the thermodynamic or kinetic solubility was determined for some of the new
compounds due to limitations of supply, but all of the results show that each of the fused-
1
2

pyridine analogs tested has greater aqueous solubility than CADA. This is not surprising for the
compounds with small alkyl or alkoxy substituents in the para-pyridine position, but even
LAL036 with the most hydrophobic substituent and greatest potency has greater kinetic
solubility than CADA.
Table 2. Molar Absorptivities, Thermodynamic and Kinetic Solubilities of Pyridine-Fused
CADA Compounds
 a
23,141
Therm. sol. (M)b Kinetic Sol. (M)
c
Compound
(CADA•HCl)
R
R’

max (nm)
230
1
11 ± 1
20 ± 1
27 ± 1
28 ± 1
ND
43 ± 1
ND
2
30
3
c (LAL005•HCl)
d (LAL030•HBr)
e (LAL020•HCl)
f (LAL016•HCl)
t-Bu
OH
Me
Me
Me
Me
35,200
27,150
32,950
ND
3
230
ND
64 ± 2
54 ± 2
65 ± 3
ND
2
2
2
2
2
2
30
30
30
30
30
30
3
OMe
OEt
3
3
3
h (LAL036•HCl) OCH Cy Me
ND
ND
2
i (LAL026•HCl)
Pr
iBu
Ph
Me
Me
Me
34,000
29,650
37,099
18 ± 1
29 ± 1
ND
3
k (LAL024•HCl)
l (LAL014•HCl)
ND
3
61 ± 1
a
Molar absorptivity determined from Beer-Lambert Law plots of absorbances measured at the max value listed for the major
absorption.
b
Thermodynamic solubility measured by UV-Visible absorption spectroscopy in aqueous PBS buffer (pH 7.4).
Kinetic solubility measured by nephelometry in aqueous PBS buffer (pH 7.4)/2.5% DMSO.
c
3
. Conclusions
A total of 17 pyridine-fused macrocycles were synthesized with a wide range of structural
variation. Many useful synthetic intermediates are reported, including intermediates in Hantzsch
syntheses of para-propyl, butyl, isobutyl, and phenyl analogs. The new CADA analogs display a
wide range of CD4 down-modulating and anti-HIV potencies, including some with greater potency
than CADA. These results prove that a highly basic nitrogen atom in the 12-membered ring is not
required for high potency and that hydrophobic substituents enhance the potency of pyridine-fused
1
3

CADA compounds. Moreover, thermodynamic and kinetic water solubilities were measured for
many of the new compounds and they all compare favorably with those of CADA, validating the
hypothesis that incorporation of a pyridine ring can enhance some drug-like properties of CADA
compounds. In addition, the pyridine-fused analogs were determined to have low cytotoxicities
and high therapeutic indices. These results may lead to improved candidates for studying the
effects of CD4 down-modulation in vivo and may be useful for the design of novel SP-targeting
drugs for down-modulating other proteins of medicinal interest.
4
4
4
. Experimental section
.1 Biological Evaluation
.1.1 CD4 down-modulation
To study the effect of the CADA analogs on CD4 expression, CHO cells, stably expressing
CD4-YFP (human CD4 fused at its COOH-terminus to the yellow fluorescent protein), were
treated for 24 h with serial dilutions (1:5) of the compounds at 37 °C. Cells were then washed,
fixed in 1% formaldehyde and analyzed immediately. Data were acquired with a FACSCanto flow
cytometer (BD Biosciences) using the 488 nm laser line and the BD FACSDiva software
application (BD Biosciences). Data were analyzed with FLOWJO software (Tree Star, San Carlos,
CA). Down-modulation of CD4 was evaluated by the decrease in fluorescence intensity on CADA-
treated cells relative to matched, untreated cells. To calculate the efficiency of CD4 down-
modulation, the mean fluorescence intensity (MFI) for YFP for each sample was expressed as a
percentage of the MFI of control cells (after subtracting the background MFI of the non-transfected
control cells).
4
.1.2 Viral replication
1
4

MT-4 cells (obtained from the American Type Culture Collection) were seeded in 96-well
5
plates at 3.75×10 cells/mL (150 µL/well) filled with culture medium (Roswell Park Memorial
Institute 1640, 10% (v/v) fetal bovine serum, 2 mM L-glutamine) and test compounds. Cells were
pre-incubated with the compounds for 15 min before addition of the laboratory HIV-1 strain NL4.3
(obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH) at 50 µL (30 pg/well) and incubation at 37 °C. Four days after infection, cell viability was
quantified by the MTS/phenazine ethosulfate (PES) method. Absorbance was measured at 490 and
7
00 nm and used to calculate the 50% inhibitory concentration (IC ) of each compound for viral
50
replication.
4
.1.3 Cytotoxicity
MT-4 cells were seeded in transparent 96-well plates at 10 cells per well in Dulbecco’s
4
Modified Eagle Medium (Life Technologies, Waltham, MA, USA) with 10% (v/v) fetal bovine
serum and 10 mM HEPES. Subsequently, compounds were added and the cell/compound mixture
was incubated at 37 °C for 48 h. The 50% cytotoxic concentration (CC ) of each compound was
5
0
determined from the reduction of viability of cells exposed to the compound, as measured by the
MTS/phenazine ethosulfate (PES) method.
4
4
.2 Solubility
.2.1 Thermodynamic solubility
Solutions of five different concentrations of each compound in methanol were prepared
and analyzed by UV-Visible spectroscopy. The absorbance values obtained were plotted as a
function of concentration and the ε value of each compound was determined by linear least
squares curve fitting of the data to obtain the slope (ε). Thermodynamic solubility was analyzed
using the traditional shake flask method.109,110 For each compound tested in this assay, a small
1
5

amount (3-5 mg) of the compound was added to a glass vial containing 5 mL of PBS buffer (pH
7.4) and agitated on a mechanical shaker for 72 h. In order to ensure the solutions remained
=
saturated, the solutions were visually inspected at 24 and 48 h to ensure that some of the
compound remained insoluble. After 72 h, the solutions were filtered using a PTFE syringe filter
(0.45 µL) and the filtrate was analyzed by the standard addition method using UV-Visible
spectroscopy in conjunction with the Beer-Lambert law to calculate the concentration of each
compound.
4
.2.2 Kinetic solubility
The following procedure109,110 was used to determine the kinetic solubility of each
compound tested. Solutions of each compound with various starting concentrations in 2.5%
DMSO with a final volume of 100 μL were prepared in quadruplicate by serial dilution on
sterile, 96-well microtiter plates, with wells arranged in 12 columns (1-12) and 8 rows (A-H).
Using a micropipette, the appropriate amount of a 20 mM DMSO stock solution for each
compound was added into a vial contained 858 μL of PBS buffer (pH = 7.4) and the appropriate
amount of DMSO in order to keep the DMSO concentration constant. A second starting solution
with lower concentration of the CADA compound was prepared in the same manner. Then, 100
μL of a solution of 2.5% DMSO in PBS buffer (pH = 7.4) was added into wells (A-H)2-8 and
(A-H)11,12. Into wells (A-D)1, 200 μL of the first starting solution for each CADA compound
tested was added. Also 200 μL of the second starting solution for each CADA compound tested
was added to wells (E-H)1. Each solution in column 1 was mixed by pipetting 100 μL up and
down five times, then 100 μL was transferred to the adjacent well in column (A-H)2. Each
solution in column 2 was mixed by pipetting 100 μL up and down 5 times then 100 μL was
transferred to well (A-H)3, and so on through column (A-H)8, after which 100 μL was removed
1
6

from each well in column 8 and placed in the waste container. Columns (A-H)11,12 served as
blanks, containing only 100 μL of a solution of 2.5% DMSO in PBS buffer (pH = 7.4).
Therefore, serial dilutions were performed from the starting solution with each well containing a
final volume of 100 μL. Furthermore, four more solutions having CADA compound
concentrations between the concentration of the first starting solution and half of the
concentration of the first starting solution were prepared in the same manner. Aliquots (100 μL)
of these solutions were placed in quadruplicate into wells (A-H)9,10. Plates were shaken for 2
min, incubated for 1 h at 37 ºC, and shaken again for 3 min before being analyzed using a
nephelometer. Prior to use, a Nepheloskan Ascent nephelometer was allowed to warm up for at
least 0.5 h. Each plate was placed in the nephelometer with the lid off, shaken for 2 min,
incubated for 1 h at 37 ºC, and shaken again for 3 min before measuring each well. The raw data
was adjusted by subtracting the average blank value and analyzed using Microsoft Excel.
4
4
.3 Chemistry
.3.1 General Methods
All reactions were performed under an atmosphere of dry nitrogen. Reagents and solvents
purchased from Aldrich Chemical Company, Acros Organics, or Fisher Scientific were of ACS
reagent grade or better and were used without purification, unless indicated otherwise. Anhydrous
acetonitrile (AN) and THF were distilled from CaH and from Na/benzophenone, respectively.
2
Solutions of 2 N HCl in methanol/water was prepared from 42 mL of concentrated aq. HCl (12.1
N) and 210 mL of methanol. Trituration of HCl salts was done by sonication in anhydrous diethyl
ether (5 – 15 mL, unless indicated otherwise) for 5 min and filtration, repeating the process two
more times. “Overnight” periods are ca. 16 h. Organic solutions were dried with anhydrous
Na SO , and sample drying in vacuo was performed at 0.1 mm at room temperature. Column
2
4
1
7

chromatography was performed with Sorbent Technologies neutral alumina (50-200 m) or
standard grade silica (32-63 m), unless noted otherwise. Melting points were measured on a
1
Thomas-Hoover or Mel-Temp apparatus and are uncorrected. H NMR (400 MHz or 500 MHz)
1
3
and C NMR (100 MHz or 125 MHz) spectra were acquired on a Varian 400 or a Varian Unity+
00 spectrometer. All chemical shifts () are reported in ppm units relative to solvent resonances,
5
1
13
as follows: H, CDCl /TMS = 0.00, DMSO-d = 2.50, CD OD = 3.31; C, CDCl = 77.16, DMSO-
3
6
3
3
d = 39.7, CD OD = 49.15 ppm. Infrared spectra (IR) were recorded neat on a Nicolet 6700 FTIR
6
3
spectrometer. Low-resolution mass spectra (MS) were acquired on a Waters Micromass ZQ
electrospray ionization quadrupole mass spectrometer with positive ion detection (capillary
voltage = 3.5 kV). High-resolution mass spectra (HRMS) were determined on an Agilent 6230
TOF mass spectrometer. Samples for combustion analysis were dried at 78 ºC (0.1 mm) for 2 d,
unless stated otherwise, and microanalysis was performed by NuMega Resonance Labs, Inc.
Samples for biological testing were at least 95% pure, as shown by combustion microanalysis.
4
.3.2 Synthesis
-Ethyl-2,6-bis(hydroxymethyl)pyridine (7b). A solution of 1.6 g (7.2 mmol) of
4
7
9
dimethyl 4-ethylpyridine-2,6-dicarboxylate in 100 mL of absolute ethanol was stirred at 0 ºC as
.0 g (0.1 mol) of NaBH was added in portions, then the mixture was stirred at 0 ºC for 1 h, at
4
4
room temperature for 1 h, then boiled under reflux for 24 h. Acetone (150 mL) was added and the
mixture was boiled for 1 h, then concentrated by rotary evaporation. A mixture of the residue and
6
0 mL of saturated aq. Na CO solution (60 mL) was boiled under reflux for 1 h, then cooled to
2 3
room temperature and extracted with DCM (5 x 80 mL). The combined extracts were dried, filtered
and concentrated to dryness by rotary evaporation. The residue was dried in vacuo overnight,
yielding 1.2 g (100%) of 7b as a viscous oil, which was used in the next step without further
1
8

1
purification. H NMR (400 MHz, CDCl ) δ 7.03 (s, 2 H, Py), 4.70 (d, 8.1 Hz, 4 H, CH O), 4.09
3
2
1
3
(
br, 2 H, OH), 2.64 (q, 7.4 Hz, 2 H, CH Me), 1.24 (t, 7.8 Hz, 3 H, CH ). C NMR (125 MHz,
2
3
CDCl ) 159.1, 154.8, 118.8, 64.0, 28.0, 13.9.
3
Diethyl 4-tert-butylpyridine-2,6-dicarboxylate (8c).79,80 A solution of 20.8 g (90 mmol)
of diethyl pyridine-2,6-dicarboxylate100 in 185 mL of 30% (v/v) aq. H SO was stirred at 0 ºC as
2
4
2
5 g (0.29 mol) of pivaldehyde was added dropwise over 45 min. The reaction mixture was stirred
for 1 h at 0 ºC, initially forming a white solid that re-dissolved. A solution of 10.4 g (70 mmol) of
FeSO •7H O in 75 mL of water was added dropwise over 45 min and the reaction mixture was
4
2
stirred for 1 h at 0 ºC. A solution of 30% aq. hydrogen peroxide (23.3 mL, 25.7 g, 0.19 mol) was
added dropwise over 45 min, the reaction mixture was stirred at 0 ºC for 2 h, then allowed to warm
to room temperature and was stirred for 48 h. The reaction mixture was adjusted to pH 3 by
dropwise addition of a saturated aq. solution of K CO . The mixture (5 L) was divided into 10
2
3
parts, which were separately extracted with ethyl acetate (5 x 100 mL). The combined extracts
were dried, filtered and concentrated by rotary evaporation with a water bath temperature of 60
ºC. The resulting brownish residue was dried in vacuo overnight giving a brownish solid, which
was purified by column chromatography on silica, eluting with 4:6 (v/v) ethyl acetate/cyclohexane,
8
0
1
yielding 6.8 g (26%) of 8c as a white solid, mp 66-68 ºC; lit. 68 ºC. H NMR (400 MHz, CDCl )
3
δ 8.27 (s, 2 H, Py), 4.50 (q, 7.6 Hz, 4 H, OCH ), 1.47 (t, 7.1 Hz, 6 H, CH CH ), 1.39 (s, 9 H,
2
2
3
1
3
C(CH ) ). C NMR (100 MHz, CDCl ) δ 14.0, 26.9, 30.3, 35.2, 62.2, 124.8, 148.5, 162.9, 165.0.
3
3
3
-1
+
IR (cm ) 2972 (m), 2060 (w), 1719 (s), 1371 (m), 1248 (s), 1023 (s). MS m/z 280.54 (MH) . Anal.
calcd for C H NO : C, 63.47; H, 7.64; N, 4.93; Found: C, 63.44; H, 7.95; N, 5.32.
1
5
21
4
4
-tert-Butyl-2,6-bis(hydroxymethyl)pyridine (7c). A solution of (0.20 g, 0.72 mmol) of
1
9

diethyl 4-tert-butylpyridine-2,6-dicarboxylate (8c) in 10 mL of absolute ethanol was stirred at 0
ºC as a suspension of 0.15 g (4.0 mmol) of NaBH in 10 mL of absolute ethanol was added in
4
portions. The reaction mixture was stirred for 3 h at room temperature, boiled under reflux for 5
h, cooled to room temperature and concentrated by rotary evaporation. A mixture of the residue
and 100 mL of saturated aq. K CO solution was stirred at 60 ºC for 2 h, cooled to room
2
3
temperature, and extracted with chloroform (8 x 25 mL). The combined extracts were dried,
filtered, and concentrated to dryness by rotary evaporation. The residue was dried in vacuo
1
overnight to give 0.13 g (93%) of 7c as a brown solid, mp 105-107 ºC. H NMR (400 MHz, DMSO-
d ) δ 7.32 (s, 2 H, Py), 5.33 (t, 6.3 Hz, 2 H, OH), 4.50 (d, 5.9 Hz, 4 H, CH ), 1.28 (s, 9 H, CH ).
6
2
3
1
3
-1
C NMR (100 MHz, DMSO-d ) δ 26.2, 30.5, 64.5, 115.1, 160.4, 160.9. IR (cm ) 3349 (br), 2965
6
+
(
w), 2911 (w), 2748 (w), 2060 (w), 1607 (s). MS m/z 218.24 (M+Na) . Anal. calcd for C H NO :
1
1
17
2
C, 67.66; H, 8.78; N, 7.17; Found: C, 67.29; H, 9.00; N, 7.17.
2
,6-Bis(hydroxymethyl)-4-methoxypyridine (7e).72,73,101,113,114 By the method described
for 7b, 4.0 g (18 mmol) of dimethyl 4-methoxypyridine-2,6-dicarboxylate72,73,101,113 gave 3.0 g
100%) of 7e as a white solid, mp 121 ºC; lit.114 121-122 ºC; lit.113 125-127 ºC. H NMR (500
1
(
MHz, DMSO-d ) δ 6.86 (s, 2 H, Py), 5.35 (t, 5.4 Hz, 2 H, OH), 4.46 (d, 5.2 Hz, 4 H, CH ), 3.82
6
2
1
3
(
s, 3 H, OMe). C NMR (125 MHz, DMSO-d ) δ 166.6, 162.9, 103.9, 64.0, 55.1.
6
7
5
4
-Ethoxy-2,6-bis(hydroxymethyl)pyridine (7f). By the method described for 7b, 5.3 g
7
5
(
19 mmol) of diethyl 4-ethoxypyridine-2,6-dicarboxylate gave 3.30 g (97%) of 7f as a white
1
solid, mp 117-118 ºC. H NMR (400 MHz, DMSO-d ) δ 6.83 (s, 2 H, Py), 5.32 (t, 5.9 Hz, 2 H,
6
OH), 4.44 (d, 6.8 Hz, 4 H, CH OH), 4.10 (q, 6.7 Hz, 4 H, OCH CH ), 1.34 (t, 7.1 Hz, 3 H,
2
2
3
OCH CH ).
2
3
4
-Benzyloxy-2,6-bis(hydroxymethyl)pyridine (7g).70–72,76,78,115 A mixture of 0.18 g (4.8
2
0

7
0–72,76,78
mmol) of NaBH , 0.28 g (0.92 mmol) of dimethyl 4-benzyloxypyridine-2,6-dicarboxylate,
4
and 11 mL of anhydrous THF was stirred and heated to boiling, then 2.5 mL of MeOH was added
over 10 min. The reaction mixture was boiled under reflux for 3 h then cooled to room temperature
and 5 mL of water was added dropwise. Most of the MeOH was removed by rotary evaporation,
resulting in a cloudy solution, which was diluted with 100 mL of water and extracted with ethyl
acetate (3 x 50 mL). The combined organic extracts were dried, filtered, and concentrated by rotary
evaporation. The residue was dried in vacuo overnight, yielding 0.23 g (100%) of 7g as a white
7
6
115
1
solid, mp 295-298 ºC (dec); lit. 298 ºC (dec); lit. 268 ºC. H NMR (400 MHz, CD OD) δ 7.43-
3
7
.38 (m, 5 H, Ph), 7.06 (s, 2 H, Py), 5.20 (s, 2 H, OCH Ph), 4.63 (s, 4 H, CH OH).
2 2
Dimethyl 4-cyclohexylmethoxy-2,6-dicarboxylate (8h). A mixture of 0.30 g (1.42 mmol)
7
0
of dimethyl chelidamate, 0.80 g (5.8 mmol) of K CO , and 15 mL of anhydrous DMF was stirred
2
3
at 90 ºC for 30 min. After cooling to room temperature, 1.0 g (5.7 mmol) of cyclohexylmethyl
bromide was added in one portion. The tube was sealed and the reaction mixture was stirred at 90
ºC for 48 h. Water (200 mL) was added, then the mixture was extracted with DCM (5 x 50 mL).
The combined extracts were dried, filtered, and concentrated by rotary evaporation. A solution of
the resulting oil in 200 mL of diethyl ether was washed with water (3 x 100 mL). The organic layer
was dried, filtered, and concentrated by rotary evaporation, then the resulting yellow oil was dried
in vacuo overnight. Recrystallization from diethyl ether gave 0.10 g (23%) of pure product as a
white solid. Concentration of the mother liquor by rotary evaporation gave a white residue, which
was dried in vacuo for 3 h, then purified by column chromatography on silica gel, eluting with
DCM, followed by 9:1 (v/v) DCM/EtOAc, giving an additional 0.22 g of the product as a white
1
solid. Total yield: 0.32 g (73%), mp 105 ºC. H NMR (400 MHz, CDCl ) δ 7.80 (s, 2 H, Py), 4.01
3
1
3
(
s, 6 H, CH ), 3.93 (d, 6.0 Hz, 2 H, OCH Cy), 1.88-1.04 (m, 11 H, Cy). C NMR (100 MHz,
3
2
2
1

-1
CDCl ) δ 167.4, 165.4, 149.8, 114.7, 74.5, 53.4, 37.5, 29.8, 26.4, 25.8. IR (cm ) 2930 (s), 2853
3
+
(
s), 1744 (s), 1718 (s), 1592 (s), 1436 (s). MS m/z 308.3 (MH) . Anal. calcd for C H NO ·2H O:
1
6
21
5
2
C, 55.97; H, 7.34; N, 4.08; Found: C, 56.27; H, 7.20; N, 4.00.
4
-Cyclohexylmethoxy-2,6-bis(hydroxymethyl)pyridine (7h). By the method described
for 7b, 1.8 g (5.86 mmol) of dimethyl 4-cyclohexylmethoxy-2,6-dicarboxylate (8h) gave 1.4 g
1
(
92%) of 7h as a white solid, mp 114-115 ºC. H NMR (400 MHz, CDCl ) δ 6.67 (s, 2 H, Py),
3
4
.67 (s, 4 H, CH O), 3.79 (d, 6.1 Hz, 2 H, OCH Cy), 3.31 (s, 2 H, OH), 1.84-0.94 (m, 11 H, Cy).
2 2
1
3
-1
C NMR (100 MHz, CDCl ) δ 166.9, 160.2, 105.7, 73.7, 64.6, 37.6, 29.9, 26.5, 25.8. IR (cm )
3
+
3
340 (m), 3070 (br), 2927 (m), 2850 (m), 1600 (s), 1566 (m). HRMS calcd for C H NO (MH) :
1
4
21
3
m/z 252.1600; Found: 252.1593. Anal. calcd for C H NO ·0.25H O: C, 65.73; H, 8.47; N, 5.48;
1
4
21
3
2
Found: C, 65.32; H, 7.86; N, 5.38.
,5-Di(tert-butoxycarbonyl)-2,6-dimethyl-4-propyl-1,4-dihydropyridine (10i).
3
A
solution of 18 g (0.11 mol) of t-butyl acetoacetate, 3.6 g (50 mmol) of butyraldehyde and 15 mL
of ethanol (15 mL) was stirred at room temperature as 3.8 mL (56 mmol) of concentrated aq.
ammonium hydroxide solution was added slowly. The mixture was boiled under reflux for 6 h,
cooled to room temperature and concentrated by rotary evaporation. Aq. 2 N HCl solution (100
mL) and 200 mL of DCM were added to the residue and the layers were separated. The aqueous
phase was extracted with DCM (3 x 75 mL). The combined organic solutions were washed with
saturated aq. NaHCO solution (2 x 100 mL), saturated aq. NaCl solution (100 mL), then dried,
3
filtered, and concentrated by rotary evaporation. The residue was dried in vacuo overnight then
recrystallized from aq. isopropanol to give 6.04 g (34%) of 10i as a pale yellow, crystalline solid,
1
mp 133-134 ºC. H NMR (400 MHz, CDCl ) δ 5.40 (s, 1 H, NH), 3.85 (t, 5.7 Hz, 1 H, CH), 2.23
3
2
2

(
s, 6 H, C=CCH ), 1.49 (s, 18 H, t-Bu), 1.24 (m, 4 H, CH CH ), 0.85 (t, 7.0 Hz, 3 H, CH CH CH ).
3 2 2 2 2 3
1
3
C NMR (100 MHz, CDCl ) δ 167.8, 143.5, 105.1, 79.4, 39.5, 33.4, 28.5, 19.5, 18.5, 14.6. IR
3
-1
+
(
cm ) 3322 (s), 2968 (s), 2931 (m), 1691 (s), 1645 (s). MS m/z 374.69 (M+Na) . Anal. calcd for
C H NO ·0.25H O: C, 67.48; H, 9.49; N, 3.93; Found: C, 67.18; H, 9.16; N, 4.07.
2
0
33
4
2
3
,5-Di(tert-butoxycarbonyl)-2,6-dimethyl-4-propylpyridine (11i). A mixture of 5.1 g
(15 mmol) of 3,5-di(tert-butoxycarbonyl)-2,6-dimethyl-4-propyl-1,4-dihydropyridine (10i), 5.3 g
(
30 mmol) of Na S O and 380 mL of EtOAc was stirred at room temperature as 8.7 mL (8.2 g,
2
2
4
6
0 mmol) of 70% t-butyl hydroperoxide was added in one portion. The reaction mixture was stirred
at room temperature for 4 d, then 150 mL of saturated aq. NaHCO solution and 60 mL of H O
3
2
were added, and the mixture was stirred for 30 min at room temperature. The layers were separated
and the aqueous layer was extracted with EtOAc (3 x 75 mL). The combined organic solutions
were washed with saturated aq. NaHCO solution (2 x 100 mL) and saturated aq. NaCl solution
3
(100 mL), then dried, filtered, and concentrated by rotary evaporation. The residue was dried in
1
vacuo for 2 d, yielding 5.1 g (100%) of 11i as a pale orange oil. H NMR (400 MHz, CDCl ) δ
3
2
.57 (m, 2 H, CH Py), 2.51 (s, 6 H, CH Py), 1.60 (s, 18 H, t-Bu), 1.25 (m, 2 H, CH CH CH ), 0.98
2 3 2 2 3
1
3
(
t, 7.53 Hz, 3 H, CH CH CH ). C NMR (100 MHz, CDCl ) δ 167.8, 154.0, 145.1, 128.4, 82.6,
2 2 3 3
-1
3
3.3, 28.1, 24.3, 22.6, 14.6. IR (cm ) 2970 (m), 2933 (w), 2875 (w), 1719 (s), 1567 (s). HRMS
+
calcd for C H NO (MH) : m/z 350.2331; Found: 350.2349.
2
0
31
4
4
-Propyl-2,6-distyrylpyridine (13i).116,117 A mixture of 10.3 g (30 mmol) of 3,5-di-t-
butoxycarbonyl-2,6-dimethyl-4-propylpyridine (11i) and 150 mL of Dowtherm A was stirred and
boiled under reflux for 30 min (in an open apparatus to allow emission of white fumes) and then
boiled at reflux under nitrogen for 24 h. After cooling to room temperature, 200 mL of 6 N aq.
HCl solution and 100 mL of DCM were added and the mixture was stirred for 18 h at room
2
3

temperature. The mixture was filtered, washing the residue with water (2 x 100 mL). The layers
of the filtrate were separated and the aqueous layer was washed with DCM (5 x 100 mL). The
aqueous layer was basified (pH 14) with NaOH pellets added in portions with shaking, then
saturated with NaCl. The resulting solution was stirred with 300 mL of DCM at room temperature
for 18 h. The layers were separated and the aqueous layer was extracted with DCM (3 x 75 mL).
The combined organic solutions were dried, filtered, and concentrated by rotary evaporation. The
residue was dried in vacuo overnight, yielding 2.88 g (65%) of 2,6-dimethyl-4-propylpyridine
12i)116 as a brown oil, which was not purified further. H NMR (400 MHz, CDCl ) δ 6.78 (s, 2 H,
1
(
3
Py), 2.48 (m, 7.3 Hz, 8 H, CH Py, CH Py), 1.62 (sext, 7.6 Hz, 2 H, CH CH CH ), 0.93 (t, 7.4 Hz,
3
2
2
2
3
1
3
3
H, CH CH ). C NMR (100 MHz, CDCl ) δ 157.4, 152.0, 120.5, 37.2, 24.4, 23.5, 13.8. A
2 3 3
solution of 2.9 g (20 mmol) of 2,6-dimethyl-4-propylpyridine (12i), 11 g (0.10 mol) of
benzaldehyde, and 40 mL of acetic anhydride was stirred and boiled under reflux for 87 h. After
cooling to room temperature, 100 mL of 5% (w/v) aq. sodium hydroxide solution was added and
the mixture was stirred at room temperature for 3 h. The layers were separated and the aqueous
layer was extracted with DCM (5 x 50 mL). The combined organic solutions were dried, filtered,
and concentrated by rotary evaporation. A mixture of the residue, 11 g (0.11 mol) of NaHSO , 80
3
mL of ethyl acetate, 50 mL of EtOH, and 17 mL of water (17 mL) was stirred at 40 ºC for 24 h,
then filtered, washing with DCM (3 x 50 mL). The filtrate was concentrated by rotary evaporation
and extracted with DCM (3 x 100 mL). The combined extracts were washed with 100 mL of
saturated aq. NaCl solution, dried, filtered, and concentrated by rotary evaporation. A mixture of
the residue and 300 mL of 2 N HCl in aq. MeOH was stirred at room temperature for 6 h then
stored in a refrigerator overnight. The precipitate was collected by filtration, washed with cold
MeOH (2 x 30 mL) and dried in vacuo overnight, yielding 2.0 g (27%) of 4-propyl-2,6-
2
4

distyrylpyridine HCl salt (13i•HCl) as a greenish yellow solid, mp 222-223 ºC; lit.117 225 ºC
CHCl ). The HCl salt was converted to the free base by stirring with 200 mL of DCM, 200 mL
(
3
of saturated aq. NaCl solution, and 200 mL of 2 N aq. NaOH solution for 5 h. The layers were
separated and the aqueous layer was extracted with DCM (3 x 50 mL). The combined organic
solutions were dried, filtered, and concentrated by rotary evaporation. The residue was dried in
vacuo overnight giving 1.80 of a yellow solid. The filtrate from collection of the HCl salt was
concentrated by rotary evaporation to give a black oil, which was dried in vacuo for 3 d, then
mixed with diethyl ether and acetone, but no precipitate formed. The solution was again
concentrated by rotary evaporation. The residue was dried in vacuo overnight then stirred with 200
mL of DCM, 200 mL of 2 N aq. NaOH solution, and 200 mL of saturated aq. NaCl solution for 7
h. The layers were separated and the aqueous layer was extracted with DCM (3 x 100 mL). The
combined organic solutions were dried, filtered, and concentrated by rotary evaporation. The
residue was dried in vacuo overnight then subjected to column chromatography on silica, eluting
with DCM/hexane (1:1, 7:3, then 9:1 v/v), yielding 0.76 g of 4-propyl-2,6-distyrylpyridine (13i)
1
as a glassy yellow solid. Total yield: 2.6 g (41%). H NMR (400 MHz, CDCl ) δ 7.71 (d, 16.1 Hz,
3
2
H, C=CHPy), 7.56 (d, 8.4 Hz, 4 H, o-Ph), 7.31 (m, 7.4 Hz, 6 H, m,p-Ph), 7.14 (d, 16.1 Hz, 2 H,
C=CHPh), 6.96 (s, 2 H, Py), 2.46 (t, 7.6 Hz, 2 H, CH Py), 1.59 (sext, 8.0 Hz, 2 H, CH CH CH ),
2
2
2
3
1
3
0
1
1
.90 (t, 7.6 Hz, 3 H, CH CH CH ). C NMR (100 MHz, CDCl ) δ 155.1, 152.0, 136.8, 132.4,
2 2 3 3
-1
28.6, 128.4, 128.0, 127.0, 120.8, 37.1, 23.3, 13.7. IR (neat, cm ) 2962 (w), 2871 (w), 2234 (s),
952 (s), 1616 (s), 1494 (m), 1451 (s).
2
,6-Bis(hydroxymethyl)-4-propylpyridine (7i). A solution of 1.77 g (5.44 mmol) of 4-
propyl-2,6-distyrylpyridine (13i), 40 mL of DCM, and 10 mL of MeOH was cooled to -78 ºC by
means of a dry ice/acetone bath, and a stream of O /O was bubbled through the solution until it
3
2
2
5

turned a pale greenish color. Nitrogen was bubbled through the solution for 15 min, and then 4.2
g (70 mmol) of Me S was added in one portion. The solution was stirred at room temperature
2
overnight then concentrated by rotary evaporation. The resulting oil was dried in vacuo overnight
and subjected to column chromatography on silica, eluting with 95:5 then 9:1 (v/v) hexane/ethyl
acetate, yielding 0.79 g (82%) of 4-propylpyridine-2,6-dicarboxaldehyde (14i) as a pale yellow
1
oil, which was used in the next step without further purification. H NMR (400 MHz, CDCl ) δ
3
1
0.16 (s, 2 H, CHO), 8.01 (s, 2 H, Py), 2.80 (t, 7.6 Hz, 2 H, CH Py), 1.76 (sext, 8.1 Hz, 2 H,
2
1
3
CH CH CH ), 1.00 (t, 7.8 Hz, 3 H, CH CH CH ). C NMR (100 MHz, CDCl ) δ 192.7, 154.8,
2
2
3
2
2
3
3
-1
1
53.1, 125.5, 37.2, 23.3, 13.6. IR (cm ) 2962 (m), 2933 (m), 2872 (m), 1708 (s), 1600 (s), 1452
(m). A solution of 0.63 g (3.6 mmol) of 4-propylpyridine-2,6-dicarboxaldehyde (14i) in 40 mL of
absolute ethanol was stirred at 0 ºC as 1.2 g of NaBH (30 mmol) was added in one portion. The
4
mixture was stirred at 0 ºC for 1 h, at room temperature for 1 h, then boiled and stirred under reflux
for 22 h. After cooling, 40 mL of acetone was added and the mixture was boiled for 1 h, then
concentrated by rotary evaporation. A mixture of the residue and 25 mL of saturated aq. Na CO
2
3
solution (25 mL) was boiled under reflux for 1 h. After cooling to room temperature, the mixture
was diluted with 10 mL of water and extracted with chloroform (8 x 30 mL). The combined
extracts were dried, filtered, and concentrated by rotary evaporation. The residual oil was dried in
vacuo overnight, yielding 0.60 g (94%) of 2,6-bis(hydroxymethyl)-4-propylpyridine (7i) as a pale
1
yellow oil, which was used in the next step without further purification. H NMR (400 MHz,
CDCl ) δ 7.01 (s, 2 H, Py), 4.70 (s, 2 H, CH O), 4.16 (br, 2 H, OH), 2.58 (t, 7.5 Hz, 2 H,
3
2
1
3
CH CH CH ), 1.64 (sext, 7.5 Hz, 2 H, CH CH CH ), 0.93 (t, 7.4 Hz, 3 H, CH CH CH ). C NMR
2
2
3
2
2
3
2
2
3
-1
(
100 MHz, CDCl ) δ 158.7, 153.6, 119.8, 64.5, 37.4, 23.5, 13.8. IR (cm ) 3246 (br), 2959 (m),
3
+
2
930 (m), 2869 (m), 1610 (s), 1564 (s), 1427 (s). HRMS calcd for C H NO (MH) : m/z
1
0
15
2
2
6

1
82.1181; Found: 182.1204.
,5-Di(tert-butoxycarbonyl)-4-butyl-2,6-dimethyl-1,4-dihydropyridine (10j). By the
3
method described for 10i, 18 g (0.11 mol) of tert-butyl acetoacetate and 4.3 g (50 mmol) of
1
valeraldehyde gave 12.5 g (68%) of 10j as a yellow solid, mp 143-144 ºC. H NMR (400 MHz,
CDCl ) δ 5.39 (s, 1 H, NH), 3.85 (t, 5.0 Hz, 1 H, CH), 2.24 (s, 6 H, C=CCH ), 1.49 (s, 18 H, tBu),
3
3
1
3
1
.24 (m, 6 H, CH CH CH CH ), 0.86 (t, 7.0 Hz, 3 H, CH CH CH CH ). C NMR (100 MHz,
2
2
2
3
2
2
2
3
-1
CDCl ) δ 167.6, 143.4, 104.9, 79.2, 36.7, 33.5, 28.4, 27.6, 23.0, 19.4, 14.2. IR (neat, cm ) 3323
3
(
s), 3237 (m), 2968 (s), 2925 (s), 1692 (s), 1644 (s), 1486 (s). Anal. calcd for C H NO : C, 69.01;
21 35 4
H, 9.65; N, 3.83; Found: C, 68.98; H, 10.00; N, 4.03.
3
,5-Di(tert-butoxycarbonyl)-4-butyl-2,6-dimethylpyridine (11j). By the method
described for 11i, 12 g (30 mmol) of 3,5-di(tert-butoxycarbonyl)-4-butyl-2,6-dimethyl-1,4-
dihydropyridine (10j) gave 12 g (100%) of 11j as an orange oil, which was used in the next step
1
without further purification. H NMR (400 MHz, CDCl ) δ 2.59 (m, 2 H, CH Py), 2.51(s, 6 H,
3
2
PyCH ), 1.60 (s, 18 H, tBu), 1.36 (m, 4 H, CH CH CH CH ), 0.93 (t, 7.5 Hz, 3 H,
3
2
2
2
3
1
3
CH CH CH CH ). C NMR (100 MHz, CDCl ) δ 167.9, 154.0, 145.2, 128.4, 82.6, 33.0, 31.1,
2
2
2
3
3
-1
2
8.1, 23.4, 22.7, 13.8. IR (neat, cm ) 2962 (s), 2931 (m), 2874 (m), 1720 (s), 1565 (s), 1457 (s).
+
HRMS calcd for C H NO (MH) : m/z 364.2489; Found: 364.2469.
2
1
33
4
4
-Butyl-2,6-bis(hydroxymethyl)pyridine (7j). By the method described for conversion
of 11i to 12i, 12 g (0.03 mol) of 3,5-di(tert-butoxycarbonyl)-4-butyl-2,6-dimethylpyridine (11j)
gave 2.65 g (51%) of 4-butyl-2,5-dimethylpyridine (12j)118 as a brown oil, which was used in the
1
next step without further purification. H NMR (400 MHz, CDCl ): δ 6.78 (s, 2 H, Py), 2.51 (m,
3
7
0
.8 Hz, 8 H, CH Py, CH Py), 1.57 (m, 2 H, CH CH CH CH ), 1.34 (m, 2 H, CH CH CH CH ),
2 3 2 2 2 3 2 2 2 3
1
3
.93 (t, 7.5 Hz, 3 H, CH CH CH CH ). C NMR (100 MHz, CDCl ) δ 157.4, 152.2, 120.4, 34.9,
2
2
2
3
3
2
7

3
2.5, 24.4, 22.4, 13.9. The method described for conversion of 12i to 13i was applied to 2.64 g (20
mmol) of 12j, except that after aq. workup and treatment with NaHSO , the residue from the
3
combined DCM extracts were subjected to column chromatography on alumina, eluting with
DCM/hexane (1:4, 1:1, then 4:1, v/v), yielding 3.37 g (61%) of 4-butyl-2,6-distyrylpyridine (13j),
1
a yellow solid, mp 65-67 ºC, which was used in the next step without further purification. H NMR
(
400 MHz, CDCl ): δ 7.70 (dd, 2.3 Hz, 16.4 Hz, 2 H, C=CHPy), 7.60 (d, 7.6 Hz, 4 H, o-Ph), 7.38
3
(td, 2.4 Hz, 7.5 Hz, 4 H, m-Ph), 7.29 (m, 2 H, p-Ph), 7.20 (dd, 2.3 Hz, 16.1 Hz, 2 H, C=CHPh),
7
.10 (s, 2 H, Py), 2.62 (t, 7.6 Hz, 2 H, CH Py), 1.66 (m, 2 H, CH CH CH CH ), 1.39 (m, 2 H,
2 2 2 2 3
1
3
CH CH CH CH ), 0.96 (t, 7.5 Hz, 3 H, CH CH CH CH ). C NMR (100 MHz, CDCl ) δ 155.5,
2
2
2
3
2
2
2
3
3
1
52.4, 137.1, 132.7, 128.8, 128.7, 128.3, 127.2, 121.0, 35.2, 32.7, 22.5, 14.1. By the method
described for conversion of 13i to 14i, 3.2 g (9.5 mmol) of 4-butyl-2,6-distyrylpyridine (13j) gave
1
.4 g (77%) of 4-butylpyridine-2,6-dicarboxaldehyde (14j) as a pale yellow oil, which was used
1
without further purification. H NMR (400 MHz, CDCl ) δ 10.16 (s, 2 H, CHO), 8.00 (s, 2 H, Py),
3
2
.81 (t, 7.5 Hz, 2 H, CH Py), 1.69 (m, 7.6 Hz, 2 H, CH CH CH CH ), 1.38 (m, 7.4 Hz, 2 H,
2 2 2 2 3
CH CH CH CH ), 0.96 (t, 7.4 Hz, 3 H, CH CH CH CH ). By the method described for 7i, 0.95 g
2
2
2
3
2
2
2
3
(5.0 mmol) of 14j gave 0.98 g (100%) of 4-butyl-2,6-bis(hydroxymethyl)pyridine (7j) as a
1
colorless oil, which was used in the next step without further purification. H NMR (500 MHz,
CDCl ) δ 7.06 (s, 2 H, Py), 5.32 (br, 2 H, OH), 4.63 (s, 4 H, CH O), 2.55 (t, 7.5 Hz, 2 H, CH Py),
3
2
2
1
3
.77 (m, 7.7 Hz, 2 H, CH CH CH CH ), 1.30 (m, 7.5 Hz, 2 H, CH CH CH CH ), 0.89 (t, 7.4 Hz,
2 2 2 3 2 2 2 3
+
H, CH CH CH CH ). HRMS calcd for C H NO (MH) : m/z 196.1337; Found: 196.1335.
2
2
2
3
11 17
2
3
,5-Di-(tert-butoxycarbonyl)-4-isobutyl-2,6-dimethyl-1,4-dihydropyridine (10k). By
the method described for 10i, 18 g (0.11 mol) of tert-butyl acetoacetate, 4.3 g (50 mmol) of
1
isovaleraldehyde gave 13.2 g (72%) of a (10k) as a yellow solid, mp 157-158 ºC. H NMR (400
2
8

MHz, CDCl
3
): δ 5.53 (s, 1 H, NH), 3.90 (t, 7.0 Hz, 1 H, C=CCH), 2.25 (s, 6 H, C=CCH ), 1.49
3
(
m, 20 H, tBu, CH CH(CH ) ), 1.08 (t, 7.0 Hz, 1 H, CH CH(CH) ), 0.90 (d, 6.5 Hz, 6 H,
2
3 2
2
3
1
3
CH CH(CH ) ). C NMR (100 MHz, CDCl
3
) δ 167.8, 143.5, 105.7, 79.4, 46.9, 31.5, 28.5, 23.7,
3.5, 19.5. IR (neat, cm ) 3318 (s), 2969 (s), 1690 (s), 1643 (s), 1491 (s). Anal. calcd for
: C, 69.01; H, 9.65; N, 3.83; Found: C, 68.90; H, 10.05; N, 4.08.
,5-Di-(tert-butoxycarbonyl)-4-isobutyl-2,6-dimethylpyridine (11k). By the method
2
3 2
-
1
2
C
21
H35NO
4
3
described for 11i, 20 g (55 mmol) of 3,5-di-(tert-butoxycarbonyl)-4-isobutyl-2,6-dimethyl-1,4-
dihydropyridine (10k) gave 19.5 g (98%) of 11k as pale orange oil, which was used in the next
1
step without further purification. H NMR (500 MHz, CDCl
3
): δ 2.62 (d, 7.4 Hz, 2 H, CH Py),
2
2
.52(s, 6 H, PyCH ), 1.93 (m, 1 H, CH CH(CH ) ), 1.60 (s, 18 H, tBu), 0.89 (d, 7.2 Hz, 6 H,
2 3 2
3
1
3
CH CH(CH ) ). C NMR (125 MHz, CDCl
3
) δ 168.0, 154.1, 144.6, 128.9, 82.7, 39.1, 29.5, 28.1,
2
3 2
-1
2
2.9, 22.7. IR (neat, cm ) 2968 (s), 1718 (s), 1565 (s), 1457 (s). HRMS calcd for C21
H33NO
4
+
(
MH) : m/z 364.2488; Found: 364.2458.
4
-Isobutyl-2,6-distyrylpyridine (13k). By the method described for conversion of 11i to
1
2i, 11 g (30 mmol) of 3,5-di-(tert-butoxycarbonyl)-4-isobutyl-2,6-dimethylpyridine (11k) gave
2
.5 g (51%) of 4-isobutyl-2,6-dimethylpyridine (12k),119 which was used in the next step without
1
further purification. H NMR (500 MHz, CDCl ): δ 6.75 (s, 2 H, Py), 2.49 (s, 6 H, CH Py), 2.38
3
3
1
3
(
d, 7.3 Hz, 2 H, CH Py), 1.88 (m, 1 H, CH CH(CH ) ), 0.90 (d, 7.0 Hz, 6 H, CH CH(CH ) ). C
2 2 3 2 2 3 2
NMR (125 MHz, CDCl3) δ 157.3, 151.2, 121.2, 44.7, 29.6, 24.4, 22.5. By the method described
for conversion of 12j to 13j, 2.5 g (20 mmol) of 4-isobutyl-2,6-dimethylpyridine (12k) gave 2.3 g
1
(
43%) of 4-isobutyl-2,6-distyrylpyridine (13k) as a yellow solid, mp 79-80 ºC. H NMR (400 MHz,
CDCl ): δ 7.71 (dd, 2.3 Hz, 16.1 Hz, 4 H, C=CHPy), 7.61 (d, 7.6 Hz, 4 H, o-Ph), 7.38 (t, 7.1 Hz,
3
4
H, m-Ph), 7.29 (m, 2 H, p-Ph), 7.18 (dd, 2.3 Hz, 16.1 Hz, 2 H, C=CHPh), 7.07 (s, 2 H, Py), 2.50
2
9