Synthesis, biological activity, and crystal structure of potent nonnucleoside inhibitors of HIV-1 reverse transcriptase that retain activity against mutant forms of the enzyme

Article abstract of DOI:10.1021/jm060103d

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6- difluorophenyl)-4-methylbenzimidazole (1) (IC₅₀ = 0.2 μM, EC ₅₀ = 0.44 μM, and TC₅₀ ≥ 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants.

Full text of DOI:10.1021/jm060103d

J. Med. Chem. 2007, 50, 4003-4015
4003
Synthesis, Biological Activity, and Crystal Structure of Potent Nonnucleoside Inhibitors of
HIV-1 Reverse Transcriptase That Retain Activity against Mutant Forms of the Enzyme†
Marshall L. Morningstar,‡ Thomas Roth,‡ David W. Farnsworth,*,‡ Marilyn Kroeger Smith,‡,§ Karen Watson,#
Robert W. Buckheit, Jr.,# Kalyan Das,| Wanyi Zhang,| Eddy Arnold,| John G. Julias, Stephen H. Hughes, and
Christopher J. Michejda*,‡
Molecular Aspects of Drug Design Section and RetroViral Replication Laboratory, National Cancer InstitutesFrederick, P.O. Box B,
Frederick, Maryland 21702, McDaniel College, Westminster, Maryland 21157, Southern Research InstitutesFrederick Research Center,
431 AViation Way, Frederick, Maryland 21701, and Center for AdVanced Biotechnology and Medicine and Department of Chemistry and
Chemical Biology, Rutgers UniVersity, Piscataway, New Jersey 08854
ReceiVed January 31, 2006
In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors
that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted
benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led
to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 ) 0.2
µM, EC50 ) 0.44 µM, and TC50 g 100 against WT). This paper describes how substitution on the
benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced
potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A
7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided
the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives
were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants.
Introduction
propylamino)-2-pyridinyl]piperazine (delaviridine),6 and more
recently 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2, 6-dimethylphenyl]-
amino]-2-pyrimidinyl]amino]benzonitrile (R278474, rilpivirine)
of the DAPY7 class of NNRTIs, inhibit HIV-1 RT by binding
to a site near the polymerase active site. All anti-HIV-1 drugs,
including NNRTIs, select for drug resistance. In the case of the
NNRTIs, resistance involves mutation of critical residues in the
binding pocket.8 However, because the NNRTI binding pocket
is specific for HIV-1 RT, NNRTIs can be designed to target
HIV-1 RT; thus NNRTIs are less likely to exhibit systemic
toxicity than NRTIs.
The rapid global spread of human immunodeficiency virus
type-1 (HIV-1a) and the emergence of drug resistance make
the development of new drugs for the treatment of acquired
immunodeficiency syndrome (AIDS) an important problem. The
replicative cycle of HIV-1 provides several potential targets for
chemotherapeutic intervention. One of these is the viral enzyme
reverse transcriptase (RT), which catalyzes the conversion of
the single-stranded viral genomic RNA into double-stranded
DNA. Nucleoside analogue inhibitors of RT (NRTIs), such as
3′-azido-2′,3′-dideoxythymidine (AZT), 2′,3′-dideoxyinosine
(ddI), and 2′,3′-dideoxycytidine (ddC) are commonly used in
the treatment of HIV-infected patients. However, because
NRTIs are substrates for host polymerases, their utility is
compromise by serious side effects.1 The nonnucleoside RT
inhibitors (NNRTIs), such as N-11-cyclopropyl-4-methyl-5,11-
dihydro-6H-dipyrido[3,2-b:2′3′-e]diazepin-6-one (nevirapine,
BI-RG-587),2 R-anilinophenylacetamide (R-APA),3 and 8-chloro-
4,5,6,7-tetrahydro-5-methylimidazo-[4,5,1-jk][1,4]benzodiazepine-
2(1H)-one (8-Cl TIBO),4 (-)-6-chloro-4-cyclopropylethynyl-4-
trifluoromethyl-1,4-dihydro-2H-3, 1-benzoxazin-one (efavirence),5
1-(5-methanesulphonamido)-1H-indol-2-yl-carbonyl)-4-[3-(iso-
Crystallographic analysis of HIV-1 RT has defined the details
of binding of the NNRTIs and delineated important structural
differences between unliganded RT, RT bound to nucleic acids,
and RT bound to NNRTIs.9 Although all of the NNRTIs bind
in the same pocket, they are quite diverse in part because the
pocket is flexible. This makes it difficult to predict a priori which
inhibitors will bind tightly and how an individual resistance
mutation will affect the binding of a specific NNRTI.
We previously reported the discovery of a new class of
NNRTIs that has activity against wild-type RT and some
important variants, the 1-(2,6-difluorobenzyl)-2-(2,6-difluo-
rophenyl)benzimidazoles10 (BPBIs). This class of inhibitors was
designed to be more flexible than analogues of 1-(2,6-difluo-
† This paper is dedicated to the memory of Dr. Christopher J. Michejda.
* To whom correspondence should be addressed. Fax: (301) 846-6231.
Phone: (301) 846-1224. E-mail: farnswor@ncifcrf.gov.
‡ Molecular Aspects of Drug Design Section, National Cancer Institutes
Frederick.
rophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole (TZB),11-13
a
known inhibitor of wild-type (WT) HIV-1 RT. It is now
generally recognized that NNRTIs that have the structural
flexibility and the ability to adapt to changes in the geometry
of the binding pocket caused by resistance mutations are
important in discovering NNRTIs that will be effective against
resistant viruses.10,14,15 Removal of the thiazolo ring from TZB
and optimization of the lead compound resulted in the develop-
ment of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-meth-
ylbenzimidazole (1) (IC50 ) 0.2 µΜ against HIV-1 RT and EC50
) 0.44 µΜ in a cytoprotection assay16,17 using wild-type virus).
We showed that this novel NNRTI was active in cytoprotection
§ McDaniel College.
# Southern Research InstitutesFrederick Research Center.
| Rutgers University.
Retroviral Replication Laboratory, National Cancer InstitutesFrederick.
a Abbreviations. AIDS, acquired immunodeficiency syndrome; HIV-1,
human immunodeficiency virus type 1; RT, reverse transcriptase; TZB,
1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a]benzimidazole; NNRTI, non-
nucleoside reverse transcriptase inhibitor; BPBI, 1-(2,6-difluorobenzyl)-2-
(2,6-difluorophenyl)benzimidazole; WT, wild type; 8-Cl TIBO, 8-chloro-
4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-
one; SAR, structure-activity relationship.
10.1021/jm060103d CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/31/2007

4004 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Morningstar et al.
Scheme 1. Synthesis of 1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazoles (BPBI)
assays against clinically relevant HIV-1 variants that have amino
acid substitutions in the NNRTI binding pocket of RT.
outlined in Scheme 1. A variety of substituted 6-nitroanilides
are commercially available, and this starting material was used
to prepare a variety of differentially substituted benzimidazole
derivatives. Depending on the type and regiochemistry of the
different substitutents, the desired monoacylated product could
be obtained in good yields using 1.2 equiv of 2,6-difluoroben-
zoyl chloride. In the case of electron-withdrawing substituents,
benzoylation led to a mixture of monoacylated and bis-acylated
products. Variations in time, temperature, equivalents of reac-
tants, and concentration were examined in unsuccessful efforts
to produce only the desired mono-2,6-difluorobenzoyl products.
These intermediate compounds were obtained in high yields by
synthesizing the bis-2,6-difluorobenzoyl derivatives with excess
2,6-difluorobenzoyl chloride followed by selective base-
catalyzed deacylation to the monobenzoyl product. Depending
on the desired regiochemistry of the final benzimidazoles, the
monoacylated 6-nitroanilides were either reductively cyclized
with iron (method B) and alkylated with 2,6-difluorobenzyl
bromide (method A) or alkylated with 2,6-difluorobenzyl
bromide and then reductively cyclized. For instance, 2-substi-
tuted 6-nitroanilides were reductively cyclized to 4-substituted
benzimidazoles. Alkylation of the benzimidazole N1 position
with 2,6-difluorobenzyl bromide proceeded with >92%
regiochemical purity, for a variety of 4-substituted compounds
Crystallographic analysis of compound 1 in the HIV-1 RT
binding pocket was then used as a guide to synthesize analogues
with better antiviral properties. The C4 desmethyl analogue (2)
showed a consistent 3- to 4-fold better inhibitory activity against
wild type and against clinically important RT variants.10 To
understand the significance of this difference in inhibitory
activity, we systematically varied the substituents at the 4, 5,
6, and 7 positions of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophe-
nyl)benzimidazoles. The results of these combined studies led
to the discovery of derivatives with improved activity and
allowed us to formulate relevant structure-activity relationships.
Chemistry
The general synthetic route to the substituted 1-(2,6-difluo-
robenzyl)-2-(2,6-difluorophenyl)benzimidazole derivatives is

Inhibitors of HIV-1 ReVerse Transcriptase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4005
Table 1. Enzyme Inhibition Data for Substituted Benzimidazolesa
(30-36). In the case of 5-, 6-, and 7-substituted benzimidazoles,
regiochemical control was maintained by alkylation of the
monoacylated derivative (method A) to N-(2,6-difluorobenzyl)-
N-(2,6-difluorophenyl)nitroanilides (23-28) followed by reduc-
tive cyclization (method B).
Nonreductive acid-catalyzed cyclization, in contrast, was used
to synthesize 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-
nitrobenzimidazole (48), a key intermediate for many C4
derivatives. Monoacylation of 3-nitro-1,2-phenylenediamine
with 2,6-difluorobenzoyl chloride yielded a single product (11)
that was subsequently cyclized with only acid and heat. Since
cyclization of either N1 or N2 acylated product would lead to
the desired 2-arylbenzimidazole, detailed regiochemical analysis
was not carried out on 11.
compd
X
% inhibitionb (1 µM)
(A) 5, 6, or 7-Methyl-Substituted
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazoles
The 4-nitro group 1-(2,6-difluorobenzyl)-2-(2,6-difluorophe-
nyl)-4-nitrobenzimidazole (48) was reduced subsequently with
tin(II) chloride in acetic acid to yield the 4-amino compound
(53). Treatment of the 4-amino compound (53) with either HBr
or HCl under Sandmeyer conditions yielded the 4-bromo (54)
or 4-chloro (55) product, respectively. Alternatively, reaction
of the 4-amino compound (53) with formaldehyde and sodium
borohydride provided the 4-dimethylamino derivative (59).
Monoacetylation of the 4-amino (53) with acetic anhydride gave
the 4-acetamido product (56). Subsequent alkylation of the
4-acetamido (56) with methyl iodide yielded a mixture of
N-methyl-N-acetamido product (57) and starting material.
Deacylation of 57 under acidic conditions yielded the 4-me-
thylamino compound (58).
2
1
H
50
71
22
42
1
4-CH3
5-CH3
6-CH3
7-CH3
4,5-diCH3
4,6-diCH3
41
42
43
45
46
26
37
(B) 4, 5, or 6-Substituted
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazoles
54
38
55
39
40
48
49
4-Br
5-Br
4-Cl
5-Cl
78
43
75
48
69
51
30
6-Cl
4-NO2
5-NO2
The 4-methyl ester (50) was a key intermediate for a number
of 4-substituted benzimidazole derivatives (60-66). Treatment
of the 4-methyl ester derivative (50) with dimethylaluminum
amine18 resulted in the 4-nitrile derivative (60). The 4-nitrile
(60) was used subsequently to prepare the 4-hydroxyamidino
(61) and 4-amido (62) compounds by hydrolysis with hydroxy-
lamine and hydrogen peroxide, respectively. Bis methylation
of the 4-amido derivative (62) with sodium hydride and methyl
iodide yielded the desired 4-(N,N-dimethyl)amido derivative
(63). Alternatively, reaction of the 4-methyl ester derivative (50)
with barium hydroxide or methyl lithium yielded the 4-car-
boxylate (64) and 4-isopropanol (65) derivatives, respectively.
Hydrolysis of the 4-isopropanol derivative (65) with strong acid
led to the 4-isopropenyl compound (66) in low yields. Reduction
of the 4-methyl ester (50) to the 4-hydroxymethyl derivative
(67) resulted in low yields under a number of reducing
conditions (LAH, NaBH4, borane). Consequently, the reduction
step was carried out earlier in the synthesis with the precursor
methyl N-(2,6-difluorobenzoyl)-6-nitro-2-anilidecarboxylate (18).
Protection of the 2-hydroxymethyl-6-nitroanilide (19) with acetic
anhydride and subsequent modification as done with the
4-methyl ester led to the desired 4-hydroxymethylbenzimidazole
derivative (67). This product was subsequently converted to the
4-formyl (68), 4-chloromethyl (69), 4-azidomethyl (70), 4-ami-
nomethyl (71), 4-acetamidomethyl (72), 4-(N-methyl)acetamido
(73), 4-methoxymethyl (74), and 4-cyanomethyl (75).
a Enzyme assays done with WT RT. b % inhibition is the average of
two experiments done in triplicate.
2,6-difluorobenzyl bromide (22) occurred regiospecifically to
yield the 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-5-ni-
trobenzimidazole (49).
Results and Discussion
An in vitro HIV-1 RT assay using rC-dG template primer,
as described previously, was used as a screen for the activity
of compounds 38-43, 45, 46, 48, 49, 54, 55, and the data are
presented in Table 1. This initial screen for activity was done
at 1 µM (10-fold lower than used in our earlier publication).10
As illustrated in Table 1, the level of inhibitory activity differs
dramatically as the regiochemistry of substituents changes from
the 4 to 7 position. Substitution at the 4 position always
enhanced the inhibitory activity relative to the other sites of
substitution, regardless of the type of substituent [CH3 (1 vs
41-43), Br (54 vs 38), Cl (55 vs 39 and 40), NO2 (48 vs 49)].
A combination of a C4 substituent (CH3) with an additional
substituent at the 5 or 6 position (1 vs 45 and 1 vs 46) reduced
the inhibitory activity, suggesting a preference for monosub-
stitution at C4. Substitution at the 7 position with a CH3 (43),
however, resulted in complete loss of activity. Molecular
modeling of the 7-CH3 (43) vs the 4-CH3 (1) suggested an
alternative and possibly nonproductive conformation for the
7-CH3 (43) as a consequence of steric interactions between the
C7 substituent and the N1-2,6-difluorobenzyl group. This was
confirmed by structure determination of (1) in complex with
HIV-1 RT by X-ray crystallography (Table 2, Figure 1). Most
NNRTIs bind to the hydrophobic binding pocket in a “butterfly-
like” conformation.14,15 Like a butterfly, the two wings of the
inhibitor, wing 1 and wing 2, generally contain π-systems that
interact with aromatic amino acids (Y181, Y188, F227, and
W229) and hydrophobic amino acids (L100 and L234) through
favorable π-stacking interactions. The X-ray data suggest
Other compounds prepared for this series were made via
synthetic modification of intermediates described in our earlier
publication.10 Bromination of 1-(2,6-difluorobenzoyl)-2-nitroa-
nilide yielded 1-(2,6-difluorobenzoyl)-4-bromo-2-nitroanilide
(23). This derivative was then benzylated and reductively
cyclized with standard conditions to yield 1-(2,6-difluorobenzyl)-
2-(2,6-difluorophenyl)-5-bromobenzimidazole (38). Mononitra-
tion of 2-(2,6-difluorophenyl)benzimidazole with nitric acid at
room temperature yielded 5-nitrobenzimidazole (37) as the
only product. We subsequently found that alkylation with

4006 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Morningstar et al.
Table 2. X-ray Crystallographic Data and Refinement Statistics
X-ray Diffraction Data
PDB code
2B6A
CHESS F1
268
10
0.918
C2
X-ray source
data collection temp (K)
no. of crystals
wavelength (Å)
space group
CHESS F1
104
1
0.918
C2
cell constants
a (Å)
227.8
223.9
b (Å)
70.2
68.8
c (Å)
106.7
103.0
â (deg
105.7
107.2
resolution range (Å)
no. of unique reflections
no. of observations
completeness (%)
40.0-2.5
49 714
334 752
88.2
40-2.7
38 261
377 187
92.0
Figure 2. Orientation of 4-methyl BPBI in the binding pocket of HIV-1
RT allows for expansion of substituents in the 4 position of the
benzimidazole ring.
R
merge (in last shell)
0.087
|F| < 1.0σ(|F|)
0.084
|F| < 1.0σ(|F|)
I/σ cutoff
Refinement Statistics
total no. of atoms
total no. of inhibitors
resolution range (Å)
no. of reflections
7933
27
20.0-2.65
44 766
Rfree set
2245
completeness (in last shell, 2.82-2.65 Å) (%)
cutoff criterion
Rcryst
94.6 (82.5)
|F| < 1.00σ(|F|)
0.226
Rfree
0.277
rms deviation
bond length (Å)
bond angle (deg)
0.012
1.8
Figure 3. Orientation of 4-methoxy BPBI (44) in the binding pocket
of HIV-1 RT.
to some chemical or structural property of the substituent. The
compounds were tested for their ability to inhibit HIV-1 (NL4-
3) replication.16,17 The data are presented as concentrations to
reduce the cell killing by the virus by 50% (EC50). We also
carried out enzymatic assays on the inhibition of HIV-1 RT by
selected analogues as described previously.10 These data are
reported as IC50. Our compounds exhibit relatively low toxicity
(see TC50 values as determined in CEM cells) in comparison
to their activities (EC50 and IC50 values). All data are reported
in Table 3. While a variety of C4 substituents [(NH2 (53), Br
(54), Cl (55), CN (60)] showed a modest increase in potency
relative to the unsubstituted (2) or methyl (1) parent compounds,
the methoxyl (44) substituent showed a dramatic 25-fold
increase in the inhibitory activity against WT RT (IC50 ) 7.3
nM) relative to the lead compound (1). Preliminary X-ray crystal
structure data for this analogue (Figure 3) show increased
favorable interactions with binding pocket residues, thus
explaining the increase in potency of this analogue. In contrast,
the isosteric ethyl (47) and monomethylamino (58) compounds
were significantly poorer inhibitors than the lead compound.
Analogues 50, 56-59, and 61-64 were synthesized and
tested to determine whether an oxygen or any other type of
substituent in the 4-position would enhance the potency of the
compound. An N-methylacetamide (57) at the C4 position
significantly enhanced inhibitory potency, while other carbonyl
groups [CO2Me (50), CNHNHOH (61), CONH2 (62), CON-
(CH3)2 (63), and CO2H (64)] were clearly detrimental to
potency. Synthesis of compounds 65-73 and determination of
each analogue’s EC50 value focused attention on whether a
hydrophobic or methylene inserted at the C4 position would
have potencies similar to the those of des-methylene analogues
Figure 1. Electron density of 4-methyl BPBI (1) in the binding pocket
of HIV-1 RT. The piece of electron density covering the inhibitor is
from a |Fo| - |Fc| map calculated prior to inclusion of the inhibitor in
structure refinement.
(Figures 1-3) that a substituent in the 7 position would have
unfavorable steric interactions, especially with residue F227,
which would force a repositioning of aromatic wing 1 and loss
of π-stacking interaction with Y188 and Y181.
The crystal structure of 1 in HIV-1 RT can explain why
substituents in position 4 are particularly favorable. These
substituents point into space surrounded by the backbone and
side chain groups of residues 100-103 and 318. This can be
seen in Figure 2, where the 4-methyl group of 1 is oriented
into that space. Thus, 4-substituents that are capable of making
favorable contacts, especially with the backbone atoms, would
favor tighter binding of the inhibitor and, in general, be more
effective against drug-resistant HIV-1 RTs.
In light of these results, analogues in Table 3 were synthesized
and tested to determine whether the enhancement of potency
caused by a C4 group was general for any substituent or related

Inhibitors of HIV-1 ReVerse Transcriptase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4007
Table 3. Enzyme Inhibition Data for 4-Substituted
viruses, especially the K103N mutant. This mutation is one of
the most commonly observed in patients treated with NNRTIs.
In fact, most of our inhibitors failed against that mutation, with
the dramatic exception of compound 51. This derivative was
more active against the virus carrying the K103N mutation than
against the WT virus. However, compound 51 was inactive
against other NNRTI-resistant viruses, specifically Y181C and
V106A. One derivative, compound 57, showed excellent activity
against the V106A mutation and was only moderately effective
against Y181C. It appears that mutations of tyrosine at the 181
position are particularly deleterious to the binding of this class
of inhibitors. The reason for this is apparent in the crystal-
lographic structure of 1 in complex with HIV-1 RT, where the
difluorobenzyl moiety of the inhibitor is π-stacked with tyrosine
181. Interestingly, compound 51 effectively inhibits the Y188C
mutant, which is a frequently observed variant in viruses treated
with NNRTIs. The other analogues in Table 3, including the
cyanomethylene derivative 75 that showed excellent activity
against WT RT, were much less active against the NNRTI-
resistant viruses.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazolesa
compd
R
IC50 (µM)
EC50 (µM)
TC50 (µM)
1
2
CH3
H
OCH3
NAcCH3
CH2CH3
NO2
CO2Me
CH2OAc
OH
NH2
Br
Cl
NHAc
NHCH3
N(CH3)2
CN
CNH(NHOH)
CONH2
CON(CH3)2
CO2H
isopropyl
isoprenyl
CH2OH
CHO
0.2
0.44
1.7
0.06
0.02
0.82
2.4
>100
18.8
44.3
>100
26.1
30.1
57.7
55.8
>100
26.3
24.5
30.4
39.3
27.2
>100
26.7
23.1
>100
80.1
23.5
27.0
28.3
58.1
0.82
13.4
>100
49.3
20.8
20.6
NDb
NDb
NDb
0.11
0.0073
0.32
17.5
1.88
157.8
NDb
NDb
0.28
1.61
2.63
54.7
4.94
40
44
57
47
48
50
51
52
53
54
55
56
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
NRVCc
0.059
0.65
0.46
0.37
0.44
1.48
1.7
2.37
0.43
0.19
It should be noted that the acetoxymethylene derivative 51
can be hydrolyzed to the hydroxymethylene analogue 67.
Indeed, 51 is a substrate for porcine liver esterase (data not
shown). However, the observed spectrum of activities of 51
against the HIV-1 RT variants is distinctly different from that
of 67. This suggests that the intact acetoxymethylene group is
responsible for the overall activity of 51. Likewise, it could be
argued that the potent inhibitors such as 51, the chloromethylene
derivative 69, and the azidomethylene derivative 70, which are
potential electrophiles, react covalently with nucleophilic resi-
dues in the binding pocket. While a proteomic analysis of
possible covalent modifications of the protein was not carried
out, the markedly different mutation profiles for these derivatives
(Table 4) argue against the importance of this reaction.
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
4.9
NRVCc
NRVCc
6.34
1.44
0.023
0.12
CH2Cl
CH2N3
0.18
0.045
0.037
NRVCc
0.58
0.06
0.06
CH2NH2
CH2NHAc
CH2NCH3Ac
CH2OCH3
CH2CN
The experimental activities of some of the 4-substituted
derivatives were compared with the computationally derived
binding energies (∆Gbinding).19 The data in Table 5 show that in
general the modeled free energies correspond to the experimental
values (less than (1 kcal). This increases our confidence that
the computational method can be used to predict which
compounds will be the most active in advance of their syntheses.
nevirapine
0.035
a IC50 is the quantity of drug required to reduce WT RT enzyme activity
by 50%. EC50 is the quantity of drug required to reduce cell killing of HIV-1
infected CEM cells by 50%. TC50 is the quantity of drug required to reduce
cell viability by 50%. b ND: not determined. c NRVC: no reduction in viral
cytopathic effect.
In summary, the following conclusions can be drawn about
the structure-activity relationship involved in the variation of
the benzimidazole moiety of 1. The structure-activity data
clearly show that substituents on C4 of the benzimidazole
nucleus are generally beneficial for activity against HIV-1 RT.
However, there are limitations. Compounds that have a carbonyl
group or larger group substituted at the C4 position are generally
inactive. The best activity is obtained with small heteroatom-
bearing substituents, which presumably act as hydrogen-bond
acceptors. The structure-activity data are fully supported by
the X-ray crystallographic data for HIV-1 RT bound to 4-methyl
BPBI. The level of activity of the compounds in the present
study against the various variant forms of RT was more difficult
to predict. The most active derivatives, such as compound 51,
are apparently able to make significant interactions with the
RT backbone so that the variations in the binding pocket caused
by NNRTI-resistant mutations can be reasonably well tolerated.
It is possible that the ability to inhibit the different HIV-1 virus
isolates tested reflects the flexible nature of the 1-(2,6-
difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole system to
better compensate for the different structural changes arising
as a consequence of amino acid changes in the RT binding
pocket. Thus, while no single compound in our series is able to
(53-57). These results indicated that bulkier alkyl substituents
at C4 decreased activity. However, compounds 51, 67-71, 74,
and 75 showed excellent activity against the WT virus. This
was especially true for the hydroxymethylene derivative 67, the
acetoxymethylene derivative 51, the azidomethylene derivative
70, the aminomethylene derivative 71, the methoxymethylene
derivative 74, and the cyanomethylene derivative 75. These
compounds were the most active inhibitors in our panel along
with the 4-N-(acetyl)methyl derivative 57 and the 4-methoxy
derivative 44. As determined by crystallography, to be effective,
the substituent can point toward the main chain of amino acids
100-103 or toward the Pro236. However, it is clear that bulky
substituents can cause steric hindrance with the 100-103 loop
unless the binding pocket is significantly enlarged. Small
substitutents bearing heteroatoms that can make hydrogen bond
interactions with backbone residues of the 100-103 loop should
enhance binding, as predicted by molecular modeling.19
In addition to obtaining EC50 values against WT virus, we
tested some of the compounds against MT-4 cells infected with
HIV-1 variants containing NNRTI-resistant RTs (Table 4). The
4-methoxy compound 44, which is an order of magnitude more
active against the WT virus than the prototypic compound 1,
was however ineffective against most of the NNRTI-resistant

4008 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Morningstar et al.
Table 4. Cross-Resistance Profile of NNRTI with Resistant HIV-1 Isolates As Determined by Cytopathic Cell Killing Assaya
R group
CH3 OCH3
Cl
55
Br
54
NH2
53
Et
47
NHAc NAcCH3 CH2N3 CH2OAc CH2OH CH2NH2 CH2Cl CH2CN
isolate
1
44
56
57
70
51
67
71
69
75
NVPb
NL4-3 (WT) 0.5
0.06
0.16
0.5
0.3
0.5
0.5
0.4
1.2
0.9
1.97
0.04
0.045
0.08
0.6
1.48
0.026
0.28
0.22
0.61
0.062
0.068
0.025
0.027
0.013
2.32
0.048
0.02
5
13.6
0.023
0.72
0.17
5.9
1.36
0.16
0.08
0.46
3.34
0.21
2.38
0.57
4.7
0.06
0.1
0.72
1.1
1.09
0.2
4.9
L100I
0.3
2.48 3.25
1.66
0.64
>10
>10
0.4
NDc
1.49
>50
2.51
12.8
11.6
9.23
NDc
NDc
4.2
K101E
K103N
V108I
Y181C
Y188C
V106A
16.7 2.82
>20 >20 >20 >50 NDc
8.1
2.8
6
>50
2.82
0.26
NDc
10.2 >20 3.1
>50 >100
>50 NDc
7.18 >100
NDc NDc
3.2
8.4
3.4
4.2
10.7 9.2
3.7
3.3
NDc
2.2
2.3
2
0.54
0.05
0.033
4.5
NDc
NDc
0.1
2.36
NDc NDc
NDc NDc NDc NDc NDc
a Antiviral data are reported as the quantity of drug required to reduce cell killing or virus production by 50% (EC50). All values reported in µM. b NVP:
nevirapine. c ND: not determined.
Table 5. Comparison of Experimental and Calculated Binding Energies for Several BPBI Analoguesa
b
c
b
c
b
c
EC50
CH2OAc
∆Gexpt
∆Gcalc
EC50
CH2CN
∆Gexpt
∆Gcalc
EC50
CH2N3
∆Gexpt
∆Gcalc
isolate
CH2OAc
CH2OAc
CH2CN
CH2CN
CH2N3
CH2N3
NL4-3 (WT)
L100I
K101E
K103N
V108I
Y181C
Y188C
V106A
0.062
0.068
0.025
0.027
0.013
2.32
-10.24
-10.28
-10.29
-10.43
0.06
NDd
-10.24
-9.48
-9.99
0.045
0.08
0.6
1.48
0.026
0.28
0.22
0.61
-10.43
-10.6
NDd
4.2
2.2
-7.67
-8.09
NDd
-7.48
-7.77
-6.97
-8.27
-7.85
-8
-8.15
-6.63
0.033
4.5
-7.59
NDd
a All values reported in µM. b ∆Gexpt ) -RT ln EC50. c See ref 19. d ND: not determined.
the mixture was concentrated, redissolved in ethyl acetate, washed
with water, NaHSO4 (10% solution), and NaCl (saturated aqueous),
dried (Na2SO4), filtered, and concentrated. The product was purified
by flash chromatography, eluting with 1:4 ethyl acetate/hexane (1.79
g, 10.65 mmol, 82% yield of white crystals): 1H NMR (300 MHz,
CD2C12) δ 7.69 (dd, J ) 1.4, 8.9 Hz, 1H, H5), 6.92 (dd, J ) 1.4,
7.8 Hz, 1H, H3), 6.62 (dd, J ) 8.9, 7.8 Hz, 1H, H4), 6.42 (br, 2H,
NH2), 3.91 (s, 3H, OCH3).
effectively block the replication of viruses carrying all of the
common NNRTI-resistant mutations in RT, each of the muta-
tions can be inhibited efficiently by at least one of the active
compounds. The reported systematic studies on the BPBI series
of compounds can be used in designing improved NNRTIs and
may help in understanding the role of NNRTI-resistant
mutations.
N,N-Bis-(2,6-difluorobenzoyl)-4-chloro-6-nitroanilide (5). To
4-chloro-2-nitroaniline (1.05 g, 6.08 mmol) dissolved in THF/
pyridine (1:1) (20 mL) were added 2,6-difluorobenzoyl chloride
(4) (1.9 mL, 15.1 mmol, 250 M %) and a second addition (0.6
mL, 4.77 mmol, 78 M %) after 6 h. After being stirred overnight
at room temperature, the mixture was concentrated to dryness. The
residue was redissolved in CH2Cl2, washed with NaHCO3 (saturated
aqueous) and NaCl (saturated aqueous), dried (Na2SO4), filtered,
and concentrated. The product was recrystallized from ethyl acetate
(2.67 g, 5.90 mmol, 97% yield of white crystals): 1H NMR (300
MHz, CD2C12) δ 8.15 (d, J ) 2.4 Hz, 1H, H3), 7.67 (dd, J ) 2.4,
8.5 Hz, 1H, H5), 7.51 (d, J ) 8.5 Hz, 1H, H6), 7.36 (m, 2H, H4′,4′),
6.89 (m, 4H, H3′,5′,3′,5′).
N-(2,6-Difluorobenzoyl)-4-chloro-6-nitroanilide (6). To 5 (1.00
g, 4.42 mmol) dissolved in methanol/dioxane (1:1) (40 mL) was
added NaOH (solid) (0.27 g, 6.75 mmol, 150 M %). After the
mixture was stirred for 30 min at room temperature, the reaction
was quenched with NaHSO4, diluted with CH2Cl2, washed with
NaHCO3 (saturated aqueous) and NaCl (saturated aqueous), dried
Experimental Section
General. Where analyses are indicated by symbols of the
elements, results were within 0.4% of the theoretical values.
Elemental analyses were determined by Atlantic Microlab, Inc.,
Norcross, GA. Melting points were determined on an Electrothermal
apparatus using the supplied, stem-corrected thermometer and are
1
as read. H NMR spectra were recorded on a Varian 200 or 300
MHz spectrometer with Me4Si as the internal standard. Yields were
not optimized. Merck silica gel, 230-400 mesh, was used for flash
chromatography. Priming nomenclature in the identification of the
protons refers in the case of a double prime to the aromatic protons
on the N1 benzyl substitutents and a single prime to the protons
on the aromatic substitutents on the C2 aromatic in the final
benzimidazoles or the N1 benzoates in the N-(2,6-difluorobenzoyl)-
N-(2,6-difluorobenzyl)nitroanilide derivatives.
2-Methoxyl-6-nitroaniline (3). To 2-amino-3-nitrophenol (2.00
g, 12.98 mmol) dissolved in acetone (20 mL) were added K2CO3
(2.15 g) and methyl iodide (1.00 mL). After being stirred overnight,

Inhibitors of HIV-1 ReVerse Transcriptase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4009
(Na2SO4), filtered, and concentrated. The product (1.28 g, 4.09
mmol, 93% yield) was recrystallized from diethyl ether/hexane (3:
1): 1H NMR (300 MHz, CD2Cl2) δ 10.72 (s, 1H, NH), 8.91 (d, J
) 9.1 Hz, 1H, H6), 8.27 (d, J ) 2.5 Hz, 1H, H3), 7.71 (dd, J )
9.1, 2.5 Hz, 1H, H5), 7.52 (m, 1H, H4′), 7.08 (m, 2H, H3′,5′).
N,N-Bis-(2,6-difluorobenzoyl)-5-chloro-3-nitroanilide (7). To
5-chloro-2-nitroaniline (1.02 g, 5.91 mmol) dissolved in pyridine/
THF (1:1) (20 mL) was added 4 (1.50 mL, 11.9 mmol, 200 M %).
After being stirred overnight at room temperature, the mixture was
concentrated to dryness. The residue was redissolved in CH2Cl2,
washed with NaHCO3 (saturated aqueous) and NaCl (saturated
aqueous), dried (Na2SO4), filtered, and concentrated. The product
was recrystallized from ethyl acetate (2.50 g, 5.52 mmol, 93% yield
of white crystals): 1H NMR (300 MHz, CD2Cl2) δ 8.12 (dd, J )
8.5, 1.1 Hz, 1H, H3), 7.58 (AB, J ) 2.4, 1.1 Hz, 1H, H6), 7.57
(AB, J ) 2.4, 8.4 Hz, 1H, H4), 7.36 (m, 2H, H4′,4′), 6.89 (m, 4H,
acetate/hexane (1:1), 7.07 g (23.1 mmol, 90% yield) of white
crystals: 1H NMR (200 MHz, CD2Cl2) δ 8.41 (br, 1H, NH), 7.68
(br d, J ) 2.6 Hz, 1H, H5), 7.48 (m, 1H, H4′), 7.42 (d, J ) 2.6 Hz,
1H, H3), 7.04 (m, 2H, H3′,5′), 2.42 (s, 3H, CH3), 2.39 (s, 3H, CH3).
N-(2,6-Difluorobenzoyl)-2-ethyl-6-nitroanilide (14). 2-Ethyl-
6-nitroaniline (3.10 g, 18.65 mmol) and 4 (3.50 mL, 27.83 mmol,
150 M %) gave, after being stirred overnight at room temperature
and flash chromatography with 2% methanol/CH2Cl2 and recrys-
tallization from ethyl acetate/hexane (1:4), 2.34 g (7.64 mmol, 41%
yield) of a white solid: 1H NMR (200 MHz, CD2Cl2) δ 8.25 (br,
1H, NH), 7.85 (dd, J ) 1.7, 8.1 Hz, 1H, H5), 7.66 (dd, J ) 1.7, 7.9
Hz, 1H, H3), 7.49 (m, 1H, H4′), 7.45 (dd, J ) 7.9, 8.1 Hz, 1H, H4),
7.06 (m, 2H, H3′,5′), 2.81 (q, J ) 7.6 Hz, 2H, CH2), 1.29 (t, J ) 7.6
Hz, 3H, CH3).
N-(2,6-Difluorobenzoyl)-4-methyl-2-nitroanilide (15). 4-Meth-
yl-2-nitroaniline (4.95 g, 32.5 mmol) and 4 (4.50 mL, 41.8 mmol,
130 M %) gave, after recrystallization from ethyl acetate, 6.69 g
(22.9 mmol, 70% yield) of yellow crystals: 1H NMR (300 MHz,
CD2Cl2) δ 10.64 (s, 1H, NH), 8.75 (d, J ) 8.6, 1H, H6), 8.06 (s,
1H, H3), 7.52 (m, 1H, H4′), 7.48 (d, J ) 8.6, 1H, H5), 7.06 (m, 2H,
H3′,5′,3′,5′).
N-(2,6-Difluorobenzoyl)-5-chloro-2-nitroanilide (8). To 7 (1.00
g, 2.20 mmol) dissolved in methanol/dioxane (1:1) (20 mL) was
added NaOH (solid) (92 mg, 2.30 mmol, 105 M %). After the
mixture was stirred for 30 min at room temperature, additional
NaOH (92 mg, 2.30 mmol, 105 M %) was added. After an
additional 15 min, the reaction was quenched with NaHSO4, diluted
with CH2Cl2, washed with NaHCO3 (saturated aqueous) and NaCl
(saturated aqueous), dried (Na2SO4), filtered, and concentrated. The
product was recrystallized from diethyl ether/hexane (3:1) (0.55 g,
1.85 mmol, 84% yield of white crystals): 1H NMR (200 MHz,
CD2Cl2) δ 10.91 (s, 1H, NH), 9.04 (d, J ) 2.3 Hz, 1H, H6), 8.23
(d, J ) 9.0 Hz, 1H, H3), 7.52 (cm, 1H, H4′), 7.26 (dd, J ) 2.3, 9.0
Hz, 1H, H4), 7.08 (m, 1H, H3′,5′).
N,N-Bis-(2,6-difluorobenzoyl)-2-methoxyl-6-nitroanilide (9).
To 3 (13.75 g, 81.8 mmol) dissolved in THF/pyridine (1:1) (300
mL) was added 4 (22.0 mL, 174.4 mmol, 215 M %). After being
stirred overnight at room temperature, the mixture was concentrated
to dryness. The residue was suspended in water and filtered, and
the filtered solid was resuspended in hot methanol and filtered (33.7
g, 75.2 mmol, 92% yield of white crystals): 1H NMR (300 MHz,
CD2C12) δ 7.70 (dd, J ) 1.4, 8.4 Hz, 1H, H5), 7.48 (dd, J ) 8.4,
8.4 Hz, 1H, H4), 7.35 (2H, m, H4′,4′), 7.15 (dd, J ) 1.4, 8.4 Hz,
1H, H3), 6.87 (m, 4H, H3′,5′,3′,5′), 3.85 (s, 3H, OCH3).
H3′,5′), 2.42 (s, 3H, CH3).
N-(2,6-Difluorobenzoyl)-5-methyl-2-nitroanilide (16). 5-Meth-
yl-2-nitroaniline (4.95 g, 32.5 mmol) and 4 (4.50 mL, 41.8 mmol,
130 M %) gave, after recrystallization from ethyl acetate, 8.71 g
(29.8 mmol, 92% yield) of yellow crystals: 1H NMR (300 MHz,
CD2Cl2) δ 10.85 (s, 1H, NH), 8.75 (s, 1H, H6), 8.16 (d, J ) 8.6,
1H, H3), 7.50 (m, 1H, H4′), 7.48 (d, J ) 8.6, 1H, H4), 7.06 (m, 2H,
H3′,5′), 2.50 (s, 3H, CH3).
Methyl N,N-Bis(2,6-difluorobenzoyl)-6-nitro-2-anilidecarbox-
ylate (17). To methyl 2-amino-6-nitrobenzoate (12.75 g, 65.0 mmol)
dissolved in THF/pyridine (1:1) (300 mL) was added 4 (18.0 mL,
142.7 mmol, 220 M %). After a second addition at 7 h of 4 (8.0
mL, 64 mmol, 100 M %) and after being stirred overnight at room
temperature, the mixture was concentrated. The residue was
suspened in water and filtered. The filtered solid was then suspended
in boiling methanol and filtered yielding 28.26 g (59.3 mmol, 91%
yield) of a white solid: 1H NMR (200 MHz, DMSO-d6) δ 8.41
(dd, J ) 1.5, 8.2 Hz, 1H, H5), 8.29 (dd, J ) 1.5, 8.0 Hz, 1H, H3),
7.86 (dd, J ) 8.0, 8.2 Hz, 1H, H4), 7.55 (m, 2H, H4′,4′), 7.13 (m,
4H, H3′,5′,3′,5′), 3.86 (s, 3H, CO2Me).
N-(2,6-Difluorobenzoyl)-2-methoxyl-6-nitroanilide (10). To 9
(33.7 g, 75.2 mmol) dissolved in pyridine (500 mL) was added
hydrazine (4.50 mL, 92.6 mmol, 120 M %). After being stirred
overnight at room temperature, the mixture was concentrated, and
the residue was redissolved in CH2Cl2, washed with NaHCO3
(saturated aqueous), and NaCl (saturated aqueous), dried (Na2SO4),
filtered, evaporated, and recrystallized from methanol, yielding
22.39 g (72.6 mmol, 96% yield) of a white solid: 1H NMR (200
MHz, CD2C12) δ 8.82 (br, 1H, NH), 7.51 (dd, J ) 1.6, 8.2 Hz,
1H, H5), 7.45 (m, 2H, H4′), 7.35 (dd, J ) 8.2, 8.3 Hz, 1H, H4),
7.12 (dd, J ) 8.3, 1.6 Hz, 1H, H3), 7.01 (m, 2H, H3′,5′), 3.92 (s,
3H, OMe).
N-(2,6-Difluorobenzoyl)-2-amino-3-nitroanilide (11). 3-Nitro-
1,2-phenylenediamine (13.3 g, 86.85 mmol) and 4 (15.33 g, 10.95
mL, 86.85 mmol) gave, after being stirred overnight and recrys-
tallization from ethyl acetate/hexane (1:1), 12 g (41 mmol, 48%
yield) of yellow crystals: 1H NMR (300 MHz, DMSO-d6) δ 10.23
(s, 1H, NH), 7.99 (dd, J ) 8.7, 1.4 Hz, 1H, H4), 7.71 (dd, J ) 7.6
Hz, 1.4 Hz, 1H, H6), 7.62 (m, 1H, H4′), 7.28 (m, 2H, H3′,5′), 6.91
(s, 2H, NH2), 6.76 (dd, J ) 8.7, 7.6 Hz, 1H, H5).
N-(2,6-Difluorobenzoyl)-2,3-dimethyl-6-nitroanilide (12). 2,3-
Dimethyl-6-nitroaniline (2.00 g, 12.04 mmol) and 4 (1.50 mL, 13.93
mmol, 115 M %) and a second addition of 4 (0.50 mL, 4.64 mmol,
40 M %) at 3 h gave, after 5 h and recrystallization from ethyl
acetate/hexane (1:4), 2.71 g (8.85 mmol, 74% yield) of yellow
crystals: 1H NMR (300 MHz, CD2Cl2) δ 8.67 (s, 1H, NH), 7.81-
(d, J ) 8.4 Hz, 1H, H6), 7.48 (m, 1H, H4′), 7.28 (d, J ) 8.4 Hz,
1H, H5), 7.05 (m, 2H, H3′,5′), 2.44 (s, 3H, CH3), 2.31 (s, 3H, CH3).
N-(2,6-Difluorobenzoyl)-2,4-dimethyl-6-nitroanilide (13). 4,6-
Dimethyl-2-nitroaniline (4.25 g, 25.6 mmol) and 4 (4.25 mL, 33.80
mmol, 130 M %) gave, after 5 h and recrystallization from ethyl
Methyl N-(2,6-Difluorobenzoyl)-6-nitro-2-anilidecarboxylate
(18). To 17 (28.20 g, 59.2 mmol) suspended in pyridine (300 mL)
was added hydrazine (3.0 mL, 61.7 mmol, 105 M %). After 6 h at
room temperature, the mixture was concentrated and the residue
was redissolved in CH2Cl2, washed with NaHCO3 (saturated
aqueous) and NaCl (saturated aqueous), dried (Na2SO4), filtered,
evaporated, and recrystallized from methanol, yielding 18.31 g (54.5
mmol, 92% yield) of a white solid: 1H NMR (200 MHz, CD2C12)
δ 10.81 (br, 1H, NH), 8.22 (dd, J ) 1.7, 8.1 Hz, 1H, H5), 8.15 (dd,
J ) 1.7, 8.1 Hz, 1H, H3), 7.50 (m, 1H, H4′), 7.42 (dd, J ) 8.1, 8.1
Hz, 1H, H4), 7.05 (m, 2H, H3′,5′), 3.94 (s, 3H, CO2Me).
2,6-Difluorobenzoic Acid (2-Hydroxymethyl)-6-nitroanilide
(19). To 18 (4.50 g, 13.4 mmol) dissolved in THF (50 mL) at 0 °C
was added lithium aluminum hydride (0.50 g, 13.2 mmol, 100 M
%). After 30 min, the mixture was allowed to warm to room
temperature followed by a second addition of lithium aluminum
hydride (0.20 g, 5.26mmol, 40 M %) at 2 h. After 4 h, the mixture
was concentrated and the residue was redissolved in EtOAc, washed
with NaHSO4 (10% solution), NaHCO3 (saturated aqueous), and
NaCl (saturated aqueous), dried (Na2SO4), filtered, and evaporated.
The product was purified by flash chromatograph, eluting with 4%
MeOH/CH2Cl2 and recrystallizing from CH2Cl2, yielding 2.47 g
(8.01 mmol, 60% yield) of a white solid: 1H NMR (200 MHz,
CD2C12) δ 7.92 (dd, J ) 1.4, 8.2 Hz, 1H, H5), 7.79 (dd, J ) 1.4,
7.8 Hz, 1H, H3), 7.54-7.43 (m, 2H, H4′,H4), 7.06 (m, 2H, H3′,5′),
4.74 (s, 2H, CH2O).
2,6-Difluorobenzoic Acid (2-Acetoxymethyl)-6-nitroanilide
(20). To 19 (6.00 g, 19.5 mmol) dissolved in THF (85 mL) and
triethylamine (4.00 mL) was added acetic anhydide (4.00 mL, 42.4
mmol, 220 M %). After being stirred overnight at room temperature,
the mixture was concentrated and the residue was redissolved in

4010 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Morningstar et al.
EtOAc, washed with NaHCO3 (saturated aqueous) and NaCl
(saturated aqueous), dried (Na2SO4), filtered, and evaporated. The
product was purified by flash chromatography eluting with 4%
MeOH/CH2Cl2 and titration from Et2O, yielding 5.09 g (14.5 mmol,
75% yield) of a white solid: 1H NMR (200 MHz, CD2C12) δ 9.16
(br, 1H, NH), 7.99 (dd, J ) 1.5, 8.3 Hz, 1H, H5), 7.79 (dd, J )
1.5, 7.7 Hz, 1H, H3), 7.50 (m, 1H, H4′), 7.49 (dd, J ) 7.7, 8.3 Hz,
1H, H4), 7.06 (m, 2H, H3′,5′), 5.20 (s, 2H, CH2O), 2.09 (s, 3H, OAc).
N-(2,6-Difluorobenzoyl)-4-bromo-2-nitroanilide (21). To N-(2,6-
difluorobenzoyl)-2-nitroanilide1 (1.20 g, 8.69 mmol) suspended in
10 mL of pyridine/THF (1:1) was added bromine (0.5 mL) dissolved
in acetic acid (0.5 mL). After the mixture was stirred for 1 h at
room temperature, the reaction was quenched with NaHCO3
(saturated aqueous). The solution was extracted with CH2Cl2. and
the organic extract was washed with NaCl (saturated aqueous), dried
(Na2SO4), filtered, and concentrated. The product was recrystallized
from ethyl acetate (1.55 g, 4.34 mmol, 50% yield of yellow
crystals): 1H NMR (200 MHz, CD2Cl2) δ 10.72 (br s, 1H, NH),
8.85 (d, J ) 9.1 Hz, 1H, H5), 8.42 (d, J ) 2.4 Hz, 1H, H3), 7.84
(ddd, J ) 0.5, 2.4, 9.1 Hz, 1H, H6), 7.52 (m, 1H, H4′), 7.07
(m, 2H, H3′,5′).
Method A: N-(2,6-Difluorobenzoyl)-N-(2,6-difluorobenzyl)-
4-bromo-2-nitroanilide (23). To 21 (0.26 g, 0.73 mmol) and 2,6-
difluorobenzyl bromide (22) (0.27 g, 1.30 mmol, 180 M %)
dissolved in THF (2 mL) was added NaH (0.15 g, 500 M %). After
6 h, the reaction was quenched with methanol and concentrated.
The residue was redissolved in CH2Cl2, washed with NaHCO3
(saturated aqueous) and NaCl (saturated aqueous), dried (Na2SO4),
filtered, and concentrated. The product was purified by flash
chromatography eluting with ethyl acetate/hexane (1:4) and recrys-
tallization from diethyl ether/hexane (3:1) (0.26 g, 0.54 mmol, 74%
yield of white crystals): 1H NMR (200 MHz, DMSO-d6) (rotamers)
δ 8.30 (d, J ) 2.3 Hz, 1H, H3, rotamer 1), 8.25 (d, J ) 2.3 Hz,
1H, H3, rotamer 2), 8.02 (dd, J ) 2.3, 8.5 Hz, 1H, H5, rotamer 1),
7.85 (dd, J ) 2.3, 8.5 Hz, 1H, H5, rotamer 2), 7.68 (m, 1H, H4′,
rotamer 1), 7.54-7.27 (m, 5H, H6,4′ rotamer 1 and H6,4′,4′ rotamer
2), 7.16-6.94 (m, H3′,5′,3′,5′ rotamers 1 and 2), 5.63 (d, J ) 14.3
Hz, 1H, CH2PhF2, rotamer 2), 4.99 (br, 1H, CH2PhF2, rotamer 1),
4.87 (br, 1H, CH2PhF2, rotamer 1), 4.86 (d, J ) 14.3 Hz, 1H, CH2-
Ph, rotamer 2).
5H, H5 rotamer 2, H3′,5′ rotamer 1, H4′ rotamers 1 and 2) 7.21 (d,
J ) 8.2 Hz, 1H, H6 rotamer 1), 7.07 (m, 2H, H3′,5′ rotamer 2),
7.01-6.90 (m, 4H, H3′,5′ rotamers 1 and 2), 6.83 (br d, J ) 7.7 Hz,
1H, H6 rotamer 2), 5.71 (d, J ) 14.4 Hz, 1H,CH2PhF2 rotamer 2),
4.97 (d, J ) 14.4 Hz, 1H, CH2PhF2 rotamer 1), 4.83 (d, J ) 14.4
Hz, 1H,CH2PhF2 rotamer 1), 4.78 (d, J ) 14.4 Hz, 1H, CH2PhF2
rotamer 2), 2.41 (s, 3H, CH3 rotamer 1), 2.27 (s, 3H, CH3
rotamer 2).
N-(2,6-Difluorobenzoyl)-N-(2,6-difluorobenzyl)-5-methyl-2-ni-
troanilide (27). 16 (2.00 g, 6.84 mmol) and 22 (2.12 g, 10.2 mmol,
150 M %) gave, after 3 h and recrystallization from diethyl ether/
hexane (3:1), 1.92 g (4.59 mmol, 67% yield) of white crystals: 1H
NMR (200 MHz, CD3OD) (rotamers) δ 7.92 (d, J ) 8.5 Hz, 1H,
H6, rotamer 1), 7.89 (d, J ) 8.5, 1H, H6, rotamer 2), 7.60 (m, 1H,
H4′, rotamer 1), 7.44-6.72 (m, 15H, H3,4,3′,5′,3′,4′,5′ rotamers 1 and
H3,4,3′,4′,5′,3′,4′,5′ rotamer 2), 5.86 (d, J ) 14.3 Hz, 1H, CH2PhF2,
rotamer 2), 4.98 (br, 2H, CH2PhF2, rotamer 1), 4.88 (d, J ) 14.3
Hz, 1H, CH2PhF2, rotamer 2), 2.35 (s, 3H, CH3, rotamer 1), 2.35
(s, 3H, CH3, rotamer 2).
N-(2,6-Difluorobenzoyl)-N-(2,6-difluorobenzyl)-2-methyl-6-ni-
troanilide (28). 2,6-Difluorobenzoyl-2-methyl-6-nitroanilide1 (450
mg, 1.54 mmol) and 22 (351 mg, 1.69 mmol) gave, after
recrystallization from diethyl ether/methanol, 490 mg (1.17 mmol,
76% yield) of colorless crystals: 1H NMR (300 MHz, CD2C12) δ
7.82 (dd, J ) 8.0, 1.5 Hz, 1H, H5), 7.52 (m, 1H, H4′), 7.51 (dd, J
) 7.9, 1.6 Hz, 1H, H3), 7.42 (t, J ) 7.9 Hz, 1H, H4), 7.25 (m, 1H,
H4′), 7.12 (m, 2H, H3′,5′), 6.74 (m, 2H, H3′,5′), 4.80 (s, 2H, CH2),
2.17 (s, 3H, CH3).
Method B: 2-(2,6-Difluorophenyl)-4-methoxylbenzimidazole
(30). To 10 (22.04 g, 71.5 mmol) suspended in acetic acid (250
mL) was added iron powder (29) (22.0 g). After 1 h at reflux, the
mixture was concentrated and the residue was redissolved in CH2-
Cl2, washed with NaHCO3 (saturated aqueous) and NaCl (saturated
aqueous), dried (Na2SO4), filtered, evaporated, purified by flash
chromatography with 2% methanol/CH2Cl2, and recrystallized from
methanol to yield 16.08 g (61.8 mmol, 86% yield) of white
crystals: 1H NMR (300 MHz, CD2Cl2) δ 10.07 (br d, J ) 36 Hz,
1H, NH), 7.40 (cm, 1H), 7.29-7.00 (cm, 4H), 6.75 (dd, J ) 8.0,
13.8 Hz, 1H), 4.00 (d, J ) 4.2 Hz, 3H, OCH3).
The following compounds were prepared by method B.
2-(2,6-Difluorophenyl)-4,5-dimethylbenzimidazole (31). 12
(1.40 g, 4.57 mmol) and 29 (1.05 g) gave, after 1 h at reflux and
recrystallization from ethyl acetate, 1.07 g (4.14 mmol, 91% yield)
of white crystals: 1H NMR (300 MHz, CD2Cl2) δ 7.49-7.37 (m,
2H, H4′,7), 7.16-7.07 (m, 3H, H3′,5′,6), 2.54 (br s, 3H, CH3), 2.42
(s, 3H, CH3).
2-(2,6-Difluorophenyl)-4,6-dimethylbenzimidazole (32). 13
(4.55 g, 14.9 mmol) and 29 (3.45 g) gave, after 0.5 h at reflux and
recrystallization from diethyl ether/hexane (3:1), 3.35 g (12.97
mmol, 87% yield) of white crystals: 1H NMR (200 MHz, CD2-
Cl2) δ 9.69 (br, 1H, NH), 7.43 (m, 1H, H4′), 7.26 (br s, H5), 7.09
(m, 2H, H3′,5′), 6.95 (br s, 1H, H7), 2.59 (s, 3H, CH3), 2.45 (s, 3H,
CH3).
2-(2,6-Difluorophenyl)-4-ethylbenzimidazole (33). 14 (2.00 g,
6.53 mmol) and 29 (2.00 g) gave, after 0.5 h at reflux, flash
chromatography with 2% methanol/CH2Cl2, and recrystallization
from CH2Cl2, 1.65 g (6.39 mmol, 98% yield) of white crystals:
1H NMR (200 MHz, CD2Cl2) δ 9.78 (br, 1H, NH), 7.46 (br, 1H,
H5), 7.45 (m, 1H, H4′), 7.23 (dd, J ) 7.4, 7.9 Hz, H6), 7.17-7.04
(m, 3H, H3′,5′,7), 3.03 (br, 2H, CH2), 1.39 (t, J ) 7.7 Hz, 3H, CH3).
Methyl 2-(2,6-Difluorophenyl)benzimidazole-4-carboxylate
(34). 18 (30.38 g, 90.35 mmol) and 29 (30.80 g) gave, after 0.5 h
at reflux and recrystallization from EtOAc, 18.47 g (64.07 mmol,
71% yield) of white crystals: 1H NMR (200 MHz, CD2Cl2) δ 10.92
(br, 1H, NH), 8.06 (d, J ) 8.1 Hz, 1H, H5), 7.97 (d, J ) 7.7 Hz,
1H, H7), 7.49 (m, 1H, H4′), 7.37 (dd, J ) 7.7,8.1 Hz, H6), 7.13 (m,
2H, H3′,5′), 4.01 (s, 3H, CO2CH3).
The following compounds were prepared by method A.
N-(2,6-Difluorobenzoyl)-N-(2,6-difluorobenzyl)-4-chloro-2-ni-
troanilide (24). 6 (600 mg, 1.92 mmol) and 22 (478 mg, 2.3 mmol)
gave, after recrystallization from diethyl ether/hexane (3:1), 620
mg (1.41 mmol, 74%) of white crystals: 1H NMR (300 MHz, CD2-
Cl2) (rotamers) δ 8.02 (d, J ) 2.4 Hz, 1H, H3, rotamer 1), 7.95 (d,
J ) 2.4 Hz, 1H, H3, rotamer 2), 7.57 (dd, J ) 8.6, 2.4 Hz, 1H, H5,
rotamer 1), 7.50 (m, 1H, H4′, rotamer 1), 7.30 (dd, J ) 8.4, 2.4 Hz,
1H, H5, rotamer 2), 7.35-7.03 (m, 5H, H4′ rotamer 2 and H6,4′
rotamers 1 and 2), 6.94-6.68 (m, 8H, H3′,5′,3′,5′, rotamers 1 and 2),
5.84 (d, J ) 14.4, 1H, CH2PhF2, rotamer 2), 4.94 (br s, 2H, CH2-
PhF2, rotamer 1), 4.82 (d, J ) 14.4 Hz, 1H, CH2PhF2, rotamer 2).
N-(2,6-Difluorobenzoyl)-N-(2,6-difluorobenzyl)-5-chloro-2-ni-
troanilide (25). 8 (1.95 g, 6.24 mmol), 22 (1.40 g, 6.76 mmol,
110 M %), and additional 22 (1.30 g, 100 M % and 0.75 g, 60 M
%, respectively at 2 and 5 h) gave, after 8 h and purification by
flash chromatography eluting with ethyl acetate/hexane (1:4), 2.06
g (4.69 mmol, 75% yield) of white crystals: 1H NMR (200 MHz,
DMSO-d6) (rotamers) δ 8.08 (d, J ) 9.4 Hz, 1H, H6, rotamer 1),
8.03 (d, J ) 8.8 Hz, 1H, H6, rotamer 2), 7.79-6.91 (m, 16H,
H
rotamers 1 and 2), 5.55 (d, J ) 14.7, 1H, CH2PhF2,
3,4,3′,4′,5′,3′, 4′,5′
rotamer 2), 5.12 (br, 1H, CH2PhF2, rotamer 1), 4.99 (d, J ) 14.7
Hz, 1H, CH2PhF2, rotamer 2), 4.87 (br, 1H, CH2PhF2, rotamer 1).
N-(2,6-Difluorobenzoyl)-N-(2,6-difluorobenzyl)-4-methyl-2-ni-
troanilide (26). 15 (2.00 g, 6.84 mmol) and 22 (2.12 g, 10.2 mmol,
150 M %) gave, after 3 h and recrystallization from diethyl ether/
hexane (3:1), 2.80 g (6.69 mmol, 98% yield) of white crystals: 1H
NMR (300 MHz, DMSO-d6) (rotamers) δ 7.89 (dd, J ) 2.0, 0.9
Hz, 1H, H3 rotamer 1), 7.87 (dd, J ) 2.0, 0.8 Hz, 1H, H3 rotamer
2), 7.66 (m, 1H, H4′ rotamer 1), 7.56 (ddd, J ) 8.2, 2.0, 0.9 Hz,
1H, H5 rotamer 1), 7.43 (m, 1H, H4′ rotamer 2), 7.37-7.27 (m,
2-(2,6-Difluorophenyl)-4-acetoxymethylbenzimidazole (35). 20
(2.82 g, 8.05 mmol) and 29 (2.50 g) gave, after 0.5 h at relux,
flash chromatography with 2% methanol/CH2Cl2, and titration from
3:1 Et2O/hexane, 1.83 g (6.05 mmol, 75% yield) of white crystals:

Inhibitors of HIV-1 ReVerse Transcriptase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4011
1H NMR (200 MHz, CD2Cl2) δ 10.64 and 9.97(rotamers) (br, 1H, NH),
7.83 (m, 1H), 7.45 (m, 1H, H4′), 7.29 (m, 2H), 7.13 (m, 2H, H3′,5′),
5.61 and 5.46(rotamers) (s, 2H, CH2O), 2.11 (s, 3H, Ac).
2H, H7, 4′), 7.14-7.03 (m, 3H, H5, 3′,5′), 6.81 (m, 2H, H3′,5′), 5.33
(s, 2H, CH2PhF2), 2.48 (s, 3H, CH3). Anal. (C21H14F4N2) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-7-methylbenz-
imidazole (43). 28 (300 mg, 0.72 mmol) and 29 (50 mg) gave,
after recrystallization from ethyl acetate, 149 mg (0.15 mmol, 56%
yield) of colorless crystals: mp 177-178 °C; 1H NMR (300 MHz,
CD2Cl2) δ 7.62 (d, J ) 8.2 Hz, 1H, H4), 7.45 (m, 1H, H4′), 7.18
(m, 1H, H4′), 7.17 (dd, J ) 7.3, 8.2 Hz, 1H, H5), 7.08 (d, J ) 7.3
Hz, 1H, H6), 6.95 (m, 2H, H 3′,5′), 6.70 (m, 2H, H3′,5′), 5.64 (s, 2H,
CH2PHF2), 2.74 (s, 3H, CH3). Anal. (C21H14F4N2) C, H, N.
The following compounds were prepared by method A.
2-(2,6-Difluorophenyl)-4-nitrobenzimidazole (36). 11 (11.73
g, 40.0 mmol) was dissolved in 150 mL of acetic acid and heated
to reflux. After being heated overnight, the reaction mixture was
cooled to room temperature, concentrated, redissolved in CH2Cl2,
washed with NaHCO3 (saturated aqueous) and NaCl (saturated
aqueous), dried (Na2SO4), filtered, and evaporated. The residue was
purified by flash chromatography eluting with ethyl acetate/hexane
(1:1) and recrystallization from ethyl acetate/hexane (1:4), yielding
7.90 g (28.7 mmol, 72%): 1H NMR (300 MHz, CD2C12) δ 11.00
(s, 1H, NH), 8.23 (dd, J ) 8.2, 0.8 Hz, 1H, H5), 8.21 (dd, J ) 8.0,
0.8 Hz, 1H, H7), 7.54 (m, 1H, H4′), 7.45 (dd, J ) 8.0, 8.2 Hz, 1H,
H6), 7.13 (m, 2H, H3′,5′).
2-(2,6-Difluorophenyl)-5-nitrobenzimidazole (37). To 2-(2,6-
difluorophenyl)benzimidazole1 (2.00 g, 8.70 mmol) dissolved in
H2SO4 (5.0 mL) was added HNO3 (5.0 mL). After 2 h at room
temperature the reaction was quenched with ice (50 mL), filtered,
and washed with water, yielding a white solid (1.92 g, 80% yield):
1H NMR (300 MHz, CD2Cl2) δ 8.60 (d, J ) 2.2 Hz, 1H, H4), 8.25
(dd, J ) 2.2, 8.9 Hz, 1H, H7), 7.78 (d, J ) 8.9 Hz, 1H, H6), 7.65
(m, 1H, H4′), 7.24 (m, 2H, H3′,5′).
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-methoxylben-
zimidazole (44). 30 (1.25 g, 4.80 mmol) and 22 (1.30 g, 6.28 mmol,
130 M %) gave, after flash chromatography eluting with 2%
methanol/CH2Cl2 and recrystallization from diethyl ether/hexane
(3:1), 1.68 g (4.35 mmol, 91% yield) of white crystals: mp 154-
1
155 °C; H NMR (200 MHz, CD2C12) δ 7.52 (m, 1H, H4′), 7.25
(m, 1H, H4′), 7.19 (dd, J ) 7.8, 1.0 Hz, 1H, H5), 7.13-7.00 (m,
3H, H3′,5′,6), 6.81 (m, 2H, H3′,5′), 6.72 (dd, J ) 7.8, 1.0 Hz, H7),
5.33 (s, 2H, CH2PhF2), 4.00 (s, 3H, OCH3). Anal. (C21H14F4N2O)
C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4,5-dimethyl-
benzimidazole (45). 31 (0.25 g, 0.97 mmol) and 22 (0.44 g, 2.12
mmol, 220 M %) gave, after flash chromatography eluting with
4% methanol/CH2Cl2 and recrystallization from ethyl acetate/hexane
(1:1), 0.38 g (0.81 mmol, 83% yield) of white crystals: mp 176-
The following compounds were prepared by method B.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-5-bromobenz-
imidazole (38). To 23 (0.26 g, 0.54 mmol) dissolved in glacial
acetic acid (5 mL) was added 29 (0.55 g). After 30 min, the mixture
was concentrated to dryness, diluted with ethyl acetate, and adjusted
to pH 7 with NaHCO3 (saturated aqueous). The organic solution
was collected and washed with NaHCO3 (saturated aqueous) and
NaCl (saturated aqueous), dried (Na2SO4), filtered, and concentrated.
The product was purified by flash chromatography eluting with
2% methanol/CH2Cl2 and then recrystallization from 3:1 diethyl
ether/hexane (0.14 g, 0.33 mmol, 62% yield of white crystals): mp
1
177 °C; H NMR (300 MHz, CD3OD) δ 7.63 (cm, 1H, H4′), 7.34
(cm, 1H, H6), 7.30 (cm, 1H, H4′), 7.16 (cm, 1H, H7), 7.13 (cm,
2H, H3′,5′), 6.85 (cm, 2H, H3′,5′), 5.40 (s, 2H, CH2PhF2), 2.54 (s,
3H, CH3), 2.40 (s, 3H, CH3). Anal. (C22H16F4N2) C, H, N
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4,6-dimethyl-
benzimidazole (46). 32 (0.50 g, 1.94 mmol) and 22 (0.52 g, 2.51
mmol, 130 M %) gave, after flash chromatography eluting with
2% methanol/CH2Cl2 and recrystallization from hexane, 0.68 g (1.77
1
mmol, 91% yield) of white crystals: mp 153-154 °C; H NMR
1
155-156 °C; H NMR (300 MHz, CD2Cl2) δ 8.11 (dd, J ) 0.6,
(200 MHz, CD2Cl2) δ 7.51 (cm, 1H, H4′), 7.23 (m, 1H, H4′) 7.11
1.9 Hz, 1H, H4), 7.56 (cm, 1H, H4′), 7.41 (AB, J ) 1.9, 8.7 Hz,
1H, H7), 7.40 (AB, J ) 0.6, 8.7 Hz, 1H, H6), 7.26 (cm, 1H, H4′),
7.10 (cm, 2H, H3′,5′), 6.83 (cm, 2H, H3′,5′), 5.35 (s, 2H, CH2PhF2).
Anal. (C20H11BrF4N2‚3/4H2O) C, H, N.
(m, 1H, H5), 7.05 (cm, 2H, H3′,5′), 6.93 (m, 1H, H7), 6.80 (cm, 2H,
H3′,5′), 5.29 (s, 2H, CH2PhF2), 2.58 (s, 3H, CH3), 2.44 (s, 3H, CH3).
Anal. (C22H16F4N2) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-ethylbenzimi-
dazole (47). 33 (0.70 g, 2.71 mmol) and 22 (0.72 g, 3.48 mmol,
130 M %) gave, after flash chromatography eluting with 2%
methanol/CH2Cl2 and recrystallization from diethyl ether/hexane
(3:1), 0.79 g (2.06 mmol, 76% yield) of white crystals: mp 165-
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-5-chlorobenz-
imidazole (39). 24 (620 mg, 1.41 mmol) and 29 (200 mg) after 3
h gave 250 mg (0.64 mmol, 45%) of colorless crystals: mp 136-
1
137 °C; H NMR (300 MHz, CD2C12) δ 7.77 (d, J ) 1.95 Hz,
H4), 7.55 (m, 1H, H4′), 7.42 (d, J ) 8.7 Hz, 1H, H7), 7.27 (dd, J )
8.7, 1.95 Hz, 1H, H6), 7.26 (m, 1H, H4′), 7.09 (m, 2H, H3′,5′), 6.82
(m, 2H, H3′,5′), 5.34 (s, 2H, CH2PHF2). Anal. (C20H11ClF4N2) C,
H, N.
1
166 °C; H NMR (200 MHz, CD2C12) δ 7.53 (m, 1H, H4′), 7.32
(d, J ) 8.2 Hz, 1H, H5), 7.29-7.16 (m, 2H, H6,4′), 7.14-7.01 (m,
1H, H7), 7.07 (m, 2H, H3′,5′), 6.81 (m, 2H, H3′,5′), 5.33 (s, 2H, CH2-
PHF2), 3.07 (q, J ) 7.6 Hz, 2H, CH2), 1.35 (t, J ) 7.6 Hz, 3H,
CH3). Anal. (C22H16F4N2‚1/5H2O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-6-chlorobenz-
imidazole (40). 25 (0.57 g, 1.30 mmol) and 29 (0.43 g) gave, after
1 h and purification by flash chromatography eluting with 2%
methanol/CH2Cl2 and recrystallization from 3:1 diethyl ether/
hexane, 0.43 g (1.10 mmol, 85% yield) of white crystals: mp 152-
153 °C; 1H NMR (300 MHz, CD2Cl2) δ 7.73 (dd, J ) 0.9, 8.6 Hz,
1H, H4), 7.56 (m, 1H, H4′), 7.51 (d, J ) 1.9 Hz, 1H, H7), 7.29 (dd,
J ) 1.9, 8.6 Hz, 1H, H5), 7.27 (m, 1H, H4′), 7.09 (m, 2H, H3′,5′),
6.84 (m, 2H, H3′,5′), 5.33 (s, 2H, CH2PhF2). Anal. (C20H11ClF4N2)
C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-5-methylbenz-
imidazole (41). 26 (1.55 g, 3.71 mmol) and 29 (0.79 g) gave, after
recrystallization from 3:1 diethyl ether/hexane, 0.85 g (2.30 mmol,
62% yield) of white crystals: mp 147-149 °C; 1H NMR (300 MHz,
CD2Cl2) δ 7.60-7.49 (cm, 2H, H4,4′), 7.37 (d, J ) 8.4 Hz, 1H,
H7), 7.24 (m, 2H, H 4′), 7.14 (m, 1H, H6), 7.09 (m, 2H, H3′,5′), 6.81
(m, 2H, H3′,5′), 5.34 (s, 2H, CH2PhF2), 2.47 (s, 3H, CH3). Anal.
(C21H14F4N2) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-nitrobenzimi-
dazole (48). 36 (7.7 g, 28 mmol) and 22 (6.95 g, 33.6 mmol) gave,
after recrystallization from diethyl ether/hexane (3:1), 9.8 g (24.4
1
mmol, 87%) of white crystals: mp 168-170 °C; H NMR (300
MHz, CD2C12) δ 8.13 (dd, J ) 8.1, 0.9 Hz, 1H, H5), 7.86 (dd, J
) 8.1, 0.9 Hz, 1H, H7), 7.59 (m, 1H, H4′), 7.43 (dd, J ) 8.1 Hz,
H6), 7.28 (m, 1H, H4′), 7.12 (m, 2H, H3′,5′), 6.84 (m, 2H, H3′,5′),
5.44 (s, 2H, CH2). Anal. (C20H11F4N3) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-5-nitrobenzimi-
dazole (49). 37 (0.91 g, 3.31 mmol), 22 (1.08 g, 5.22 mmol, 160
M %), and a second addition of 22 (0.47 g, 2.27 mmol, 70 M %)
after 1 h gave, after flash chromatography eluting with ethyl acetate/
hexane (1:4), 1.09 g (2.71 mmol, 82% yield) of white crystals: mp
1
168-169 °C; H NMR (300 MHz, CD2Cl2) δ 8.69 (dd, J ) 0.5,
2.2 Hz, 1H, H4), 8.23 (dd, J ) 2.2, 9.0 Hz, 1H, H6), 7.59 (dd, J )
0.5, 9.0 Hz, 1H, H7), 7.59 (m, 1H, H4′), 7.28 (m, 1H, H4′), 7.12 (m,
2H, H3′,5′), 6.84 (m, 2H, H3′,5′), 5.44 (s, 2H, CH2PhF2). Anal.
(C20H11F4N3O2) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-6-methylbenz-
imidazole (42). 27 (1.56 g, 3.73 mmol) and 29 (0.79 g) gave, after
recrystallization from 3:1 diethyl ether/hexane, 0.89 g (2.41 mmol,
64% yield) of white crystals: mp 172-173 °C; 1H NMR (300 MHz,
CD2Cl2) δ 7.64 (cm, 1H, H4), 7.52 (m, 1H, H4′), 7.30-7.19 (m,
Methyl 1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimi-
dazole-4-carboxylate (50). 34 (16.15 g, 56.0 mmol) and 22 (14.67
g, 70.86 mmol, 125 M %) gave, after flash chromatography eluting
with 2% methanol/CH2Cl2 and recrystallization from diethyl ether,

4012 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Morningstar et al.
17.63 g (42.55 mmol, 76% yield) of white crystals: mp 184-
186 °C; 1H NMR (200 MHz, CD2C12) δ 7.94 (dd, J ) 1.1, 7.6 Hz,
1H, H5), 7.72 (dd, J ) 1.1, 8.2 Hz, 1H, H7), 7.56 (m, 1H, H4′),
7.37 (dd, J ) 7.6, 8.2 Hz, 1H, H6), 7.26 (m, 1H, H4′), 7.09 (m, 2H,
(dd, J ) 8.0, 1.1 Hz, 1H, H7), 7.31 (dd, J ) 7.8, 1.1 Hz, 1H, H5),
7.26 (m, 1H, H4′), 7.24 (dd, J ) 8.0, 7.8 Hz, 1H, H6), 7.09 (m, 2H,
H
3′,5′), 6.83 (m, 2H, H3′,5′), 5.36 (s, 2H, CH2). Anal. (C20H11ClF4N2)
C, H, N.
H3′,5′), 6.82 (m, 2H, H3′,5′), 5.39 (s, 2H, CH2PhF2), 3.95 (s, 3H,
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-acetamido-
CO2CH3). Anal. (C22H14F4N2O2) C, H, N.
benzimidazole (56). To 53 (0.30 g, 0.81 mmol) dissolved in THF
(3 mL) was added acetic anhydride (100 mL, 1.06 mmol). After 3
h, additional acetic anhydride (20 mL, 0.21 mmol) was added. After
5 h, the mixture was concentrated to dryness, diluted with ethyl
acetate, washed with NaHCO3 (saturated aqueous) and NaCl
(saturated aqueous), dried (Na2SO4), filtered, and concentrated. The
product was recrystallized from diethyl ether/hexane (3:1) (0.32 g,
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-acetoxymeth-
ylbenzimidazole (51). 35 (3.55 g, 11.74 mmol) and 22 (3.40 g,
16.4 mmol, 140 M %) gave, after flash chromatography eluting
with 2% methanol/CH2Cl2 and recrystallization from diethyl ether,
5.94 g (13.9 mmol, 76% yield) of white crystals: mp 126-
1
127 °C; H NMR (300 MHz, CD2C12) δ 7.54 (m, 1H, H4′), 7.48
1
(m, 1H, H6), 7.33-7.27 (m, 2H, H5,7), 7.25 (m, 1H, H4′), 7.08 (m,
2H, H3′,5′), 6.82 (m, 2H, H3′,5′), 5.54 (s, 2H, CH2PhF2), 5.36 (s, 2H,
CH2O), 2.08 (s, 3H, OAc). Anal. (C23H16F4N2O2) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-hydroxylben-
zimidazole (52). To 44 (0.30 g, 0.78 mmol) and hexadecyltrim-
ethylammonium bromide (0.30 g) dissolved in acetic acid (9.0 mL)
was added HBr (1.0 mL). After 7 h at reflux, the mixture was
concentrated and the residue was diluted with EtOAc, washed with
NaHCO3 (saturated aqueous) and NaCl (saturated aqueous), dried
(Na2SO4), filtered, and concentrated. The product was purified by
flash chromatography eluting with 2% methanol/CH2Cl2 and
recrystallization from diethyl ether, yielding 0.25 g (0.67 mmol,
0.77 mmol, 95% yield of white crystals): mp 194-195 °C; H
NMR (300 MHz, CD2Cl2) δ 8.49 (1H, br, NH), 8.20 (d, J ) 7.8
Hz, 1H, H5), 7.56 (m, 1H, H4′), 7.26 (t, J ) 7.9 Hz, 1H, H6), 7.26
(m, 1H, H4′), 7.19 (d, J ) 7.9 Hz, 1H, H7), 7.10 (m, 2H, H3′,5′),
6.82 (m, 2H, H3′,5′), 5.34 (s, 2H, CH2PhF2), 2.21 (s, 3H, Ac). Anal.
(C22H15F4N3O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(N-methylac-
etamido)benzimidazole (57). To 56 (2.44 g, 5.90 mmol) and
methyl iodide (0.60 mL, 9.69 mmol) dissolved in THF (30 mL)
was added excess NaH. After being stirred overnight, the solution
was concentrated to dryness, diluted with CH2Cl2, washed with
water, NaHSO4 (10% solution), and NaCl (saturated aqueous), dried
(Na2SO4), filtered, concentrated, and purified by flash chromatog-
raphy eluting with 2% methanol in CH2Cl2 (0.89 g, 2.08 mmol,
and 1.07 g recovered starting material, 63% yield): mp 155-
1
86% yield): mp 257-259 °C; H NMR (200 MHz, CD2C12) δ
7.57 (m, 1H, H4′), 7.25 (m, 1H, H4′), 7.17 (dd, J ) 7.9, 8.3 Hz,
1H, H6), 7.12 (m, 2H, H3′,5′), 6.99 (d, J ) 8.3 Hz, 1H, H7), 6.82
(m, 2H, H3′,5′), 6.73 (dd, J ) 7.9, 1.0 Hz, H5), 5.35 (s, 2H, CH2-
PhF2). Anal. (C20H12F4N2O) C, H, N.
1
156 °C; H NMR (200 MHz, CD2Cl2) δ 7.55 (m, 1H, H4′), 7.48
(br d, J ) 7.4 Hz, 1H, H5), 7.31 (dd, J ) 7.4, 7.7 Hz, 1H, H6),
7.27 (m, 1H, H4′), 7.14 (dd, J ) 1.1, 7.7 Hz, 1H, H7), 7.09 (m, 2H,
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-aminobenz-
imidazole (53). To 48 (2.50 g, 6.63 mmol) dissolved in acetic acid
(25 mL) was added SnC12‚2H2O (9.80 g) dissolved in HC1
(concentrated) (10 mL). After being stirred for 30 min at room
temperature, the mixture was concentrated. The residue was diluted
with CH2Cl2, washed with water, NaHCO3 (saturated aqueous), and
NaCl (saturated aqueous), dried (Na2SO4), filtered, concentrated,
and recrystallized from methanol (2.23 g, 5.99 mmol, 90% yield):
H3′,5′), 6.84 (m, 2H, H3′,5′), 5.38 (s, 2H, CH2PhF2), 3.34 (s, 3H,
NCH3), 1.83 (s, 3H, NAc). Anal. (C23H17F4N3O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(N-methy-
lamino)benzimidazole (58). To 57 (0.50 g, 1.17 mmol) suspended
in water (9.0 mL) was added HCl (1.0 mL). After 3 h at reflux, the
solution was concentrated to dryness, diluted with ethyl acetate,
washed with NaHCO3 (saturated aqueous) and NaCl (saturated
aqueous), dried (Na2SO4), filtered, concentrated, and purified by
flash chromatography eluting with 2% methanol in CH2Cl2 (0.35
1
mp 178-179 °C; H NMR (300 MHz, CD2C12) δ 7.52 (m, 1H,
H4′), 7.23 (m, 1H, H4′), 7.07 (m, 2H, H3′,5′), 7.06 (dd, J ) 8.1, 7.7
Hz, 1H, H6), 6.82 (d, J ) 8.1 Hz, H7), 6.81 (m, 2H, H3′,5′), 6.52
(dd, J ) 7.7, 0.9 Hz, 1H, H5), 5.29 (s, 2H, CH2), 4.42 (s, 2H, NH2).
Anal. (C20H13F4N3) C, H, N.
1
g, 78% yield): mp 194-195 °C; H NMR (200 MHz, CD2Cl2) δ
7.52 (m, 1H, H4′), 7.23 (m, 1H, H4′), 7.13 (dd, J ) 8.3, 8.0 Hz,
1H, H6), 7.07 (m, 2H, H3′,5′), 6.80 (m, 2H, H3′,5′), 6.76 (d, J ) 8.3
Hz, 1H, H5), 6.36 (d, J ) 8.0 Hz, 1H, H7), 5.29 (s, 2H, CH2PhF2),
2.96 (s, 3H, NCH3). Anal. (C21H15F4N3) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-bromobenz-
imidazole (54). To 53 (300 mg, 0.81 mmol) suspended in 48%
HBr (3 mL) at 0 °C was slowly added NaNO2 (88 mg, 1.26 mmol,
160 M %) in water (1.0 mL). After being stirred for 30 min at
0-5 °C, the mixture was added to CuBr (140 mg, 0.98 mmol, 120
M %) dissolved in 48% HBr (1 mL). After 30 min at room
temperature, water (60 mL) was added and the pH adjusted to 7
with NaOH (solid). The mixture was extracted with ethyl acetate,
washed with NaHCO3 (saturated aqueous) and NaCl (saturated
aqueous), dried (Na2SO4), filtered, evaporated, purified by flash
chromatography eluting with 2% methanol in CH2Cl2, and recrys-
tallized with diethyl ether/hexane (3:1) (267 mg, 0.61 mmol, 75%
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(N,N-dimethy-
lamino)benzimidazole (59). To a mixture of 3 M H2SO4 (0.80
mL) and 37% H2CO (0.50 mL) was added a slurry of 53 (0.37 g,
1.0 mmol) and sodium borohydride (0.27 g). After the addition
was complete, the mixture was concentrated to dryness, diluted with
ethyl acetate, washed with Na2CO3 (saturated aqueous) and NaCl
(saturated aqueous), dried (Na2SO4), filtered, concentrated, and
recrystallized from diethyl ether/hexane (3:1) (0.34 g, 0.85 mmol,
85% yield of white crystals): mp 155-156 °C; 1H NMR (300 MHz,
CD2Cl2) δ 7.51 (m, 1H, H4′), 7.23 (m, 1H, H4′), 7.13 (dd, J ) 8.0
Hz, 1H, H6), 7.06 (m, 2H, H3′,5′), 6.90 (d, J ) 8.0 Hz, 1H, H5),
6.80 (m, 2H, H3′,5′), 6.48 (d, J ) 8.0 Hz, 1H, H7), 5.30 (s, 2H,
CH2PhF2), 3.18 (s, 6H, N(CH3)2). Anal. (C22H17F4N3) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole-
4-carbonitrile (60). To 50 (1.02 g, 2.46 mmol) suspended in xylene
(60 mL) was added freshly prepared 1.0 M AlMe2NH2 (20 mL).
After 3 h at reflux, the mixture was concentrated. The residue was
redissolved in CH2Cl2, washed with NaHSO4 (10% solution),
NaHCO3 (saturated aqueous), and NaCl (saturated aqueous), dried
(Na2SO4), filtered, and concentrated. The product was purified by
flash chromatography eluting with 2% methanol/CH2Cl2 and
recrystallization from diethyl ether/hexane (3:1) to give 1.72 g (4.51
1
of white powder): mp 147-148 °C; H NMR (300 MHz, CD2-
C12) δ 7.56 (m, 1H, H4′), 7.49 (dd, J ) 7.7, 0.87 Hz, 1H, H5), 7.47
(d, J ) 7.9 Hz, H7), 7.26 (m, 1H, H4′), 7.18 (dd, J ) 8.2, 7.7 Hz,
1H, H6), 7.09 (m, 2H, H3′,5′), 6.82 (m, 2H, H3′,5′), 5.35 (s, 2H, CH2).
Anal. (C20H11BrF4N2‚1/4H2O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-chlorobenz-
imidazole (55). To 53 (800 mg, 2.15 mmol) dissolved in 37% HC1
(7 mL) and water (5 mL) at 0 °C was slowly added NaNO2 (193
mg, 2.8 mmol) in water (1.5 mL). After 30 min at 0-5 °C, the
mixture was added to CuC1 (256 mg, 2.6 mmol) in HCl
(concentrated) (2 mL) at 0 °C. After rising to room temperature
over 40 min, the pH was adjusted to pH 5, diluted with water (80
mL), extracted with ethyl acetate, dried (Na2SO4), filtered, and
evaporated. The residue was purified by gravity chromatography
eluting with hexane/acetone (2:1) and recrystallized from acetone/
hexane (350 mg of yellow crystals, 0.90 mmol, 42%): mp 163-
1
mmol, 61% yield) of white crystals: mp 156-157 °C; H NMR
(200 MHz, CD2C12) δ 7.76 (dd, J ) 1.0, 8.6 Hz, 1H, H5), 7.63
(dd, J ) 1.0, 7.6 Hz, 1H, H7), 7.58 (m, 1H, H4′), 7.37 (dd, J ) 7.6,
8.6 Hz, 1H, H6), 7.28 (m, 1H, H4′), 7.11 (m, 2H, H3′,5′), 6.83 (m,
2H, H3′,5′), 5.40 (s, 2H, CH2PhF2). Anal. (C21H11F4N3) C, H, N.
1
164 °C; H NMR (300 MHz, CD2C12) δ 7.56 (m, 1H, H4′), 7.43

Inhibitors of HIV-1 ReVerse Transcriptase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4013
N-Hydroxy 1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)ben-
zimidazole-4-carboxamidine (61). To 60 (0.60 g, 1.57 mmol)
dissolved in ethanol (15 mL) and triethylamine (0.5 mL) was added
hydroxylamine (0.14 g, 130 M %). After 24 h at reflux, the mixture
was concentrated. The residue was redissolved in CH2Cl2, washed
with NaHSO4 (10% solution), NaHCO3 (saturated aqueous), and
NaCl (saturated aqueous), dried (Na2SO4), filtered, and concentrated.
The product was purified by flash chromatography eluting with
4% methanol/CH2Cl2 and recrystallization from methanol to give
0.57 g (1.37 mmol, 87% yield) of white crystals: mp 214-
216 °C; 1H NMR (200 MHz, CD2C12) δ 7.84 (dd, J ) 1.1, 7.7 Hz,
1H, H5), 7.57 (m, 1H, H4′), 7.54 (dd, J ) 1.1, 8.2 Hz, 1H, H7),
7.30 (dd, J ) 7.7, 8.2 Hz, 1H, H6), 7.28 (m, 1H, H4′), 7.11 (m, 2H,
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-isopropenyl-
benzimidazole (66). 65 (0.45 g, 1.09 mmol) was added to H2SO4
(1.00 mL). After 15 min at room temperature, the mixture was
diluted with EtOAc, washed with NaHCO3 (saturated aqueous) and
NaCl (saturated aqueous), dried (Na2SO4), filtered, and concentrated.
The product was purified by flash chromatography eluting with
1% MeOH/CH2Cl2 and recrystallization from diethyl ether/hexane
(3:1), yielding 0.11 g (0.28 mmol, 30% yield) of white crystals:
1
mp 156-158 °C; H NMR (200 MHz, CD2C12) δ 7.53 (m, 1H,
H4′), 7.44-7.16 (cm, 4H, H4′,5,6,7), 7.07 (m, 2H, H3′,5′), 6.81 (m,
2H, H3′,5′), 6.03 (dq, J ) 0.8, 2.4 Hz, 1H, vinyl), 5.37 (s, 2H, CH2-
PhF2), 5.36 (dq, J ) 1.5, 2.4 Hz, 1H, vinyl), 2.33 (dd, J ) 0.8, 1.5
Hz, 3H, CH3). Anal. (C23H16F4N2) C, H, N.
H3′,5′), 6.83 (m, 2H, H3′,5′), 6.61 (br, 2H, NH2), 5.39 (s, 2H, CH2-
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-hydroxymeth-
ylbenzimidazole (67). To 51 (1.45 g, 3.38 mmol) dissolved in
methanol (19 mL) and water (1.0 mL) was added K2CO3 (0.40 g).
After 30 min, the mixture was concentrated, redissolved in EtOAc,
washed with NaHSO4 (10% solution) and NaCl (saturated aqueous),
dried (Na2SO4), filtered, concentrated, and recrystallized from
diethyl ether/hexane (3:1), yielding 1.25 g (3.24 mmol, 96% yield)
of white crystals: mp 157-158 °C; 1H NMR (300 MHz, CD2C12)
δ 7.54 (m, 1H, H4′), 7.43 (d, J ) 7.9 Hz, 1H, H7), 7.30-7.22 (m,
2H, H6,4′), 7.18 (d, J ) 7.5 Hz, 1H, H5), 7.09 (m, 2H, H3′,5′), 6.82
(m, 2H, H3′,5′), 5.37 (s, 2H, CH2PhF2), 5.06 (d, J ) 5.4 Hz, 2H,
CH2O), 3.51 (t, J ) 5.4 Hz, 1H, OH). Anal. (C21H14F4N2O) C, H,
N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole-
4-carbaldehyde (68). To CrO3 (1.26 g) dissolved in pyridine (1.0
mL) and CH2Cl2 (9.0 mL) was added 67 (0.49 g, 1.27 mmol)
dissolved in pyridine/CH2Cl2 (1:1) (2 mL). After being stirred for
6 h, the mixture was filtered. The filtrate was dilitued with CH2-
Cl2, washed with NaHCO3 (saturated solution) and NaCl (saturated
aqueous), dried (Na2SO4), filtered, concentrated. The product was
purified by flash chromatography eluting with 1% methanol/CH2-
Cl2 and recrystallized from diethyl ether/hexane (3:1), yielding 0.38
g (0.99 mmol, 78% yield) of white crystals: mp 151-153 °C; 1H
NMR (300 MHz, CD2C12) δ 10.82 (d, J ) 0.6 Hz, 1H, CHO),
7.83 (dd, J ) 7.5, 1.1 Hz, 1H, H7), 7.78 (dd, J ) 8.1, 1.1 Hz, H5),
7.58 (m, 1H, H4′), 7.44 (ddd, J ) 8.1, 7.5, 0.6 Hz, 1H, H6), 7.27
(m, 1H, H4′), 7.11 (m, 2H, H3′,5′), 6.84 (m, 2H, H3′,5′), 5.42 (s, 2H,
CH2PhF2). Anal. (C21H12F4N2O) C, H, N.
PhF2). Anal. (C21H14F4N4O‚1/4H2O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole-
4-carboxylic Acid Amide (62). To 60 (1.10 g, 2.88 mmol)
dissolved in ethanol (100 mL) and 3 M Na2CO3 (10 mL) was added
30% hydrogen peroxide (10 mL). After being stirred overnight at
room temperature, the mixture was concentrated and redissolved
in EtOAc, washed with NaHSO4 (10% solution), NaHCO3 (satu-
rated aqueous), and NaCl (saturated aqueous), dried (Na2SO4),
filtered, and concentrated. The product was purified by flash
chromatography eluting with 2% methanol/CH2Cl2 and recrystal-
lization from methanol, yielding 0.83 g (2.08 mmol, 72% yield) of
white crystals: mp 260-261 °C; 1H NMR (200 MHz, CD2C12) δ
8.10 (dd, J ) 1.1, 7.7 Hz, 1H, H5), 7.71 (br d, J ) 8.1 Hz, 1H,
H7), 7.59 (m, 1H, H4′), 7.43 (dd, J ) 7.7, 8.1 Hz, 1H, H6), 7.28
(m, 1H, H4′), 7.13 (m, 2H, H3′,5′), 6.84 (m, 2H, H3′,5′), 5.43
(s, 2H, CH2PhF2), 1.92 (s, 2H, NH2). Anal. (C21H13F4N3O) C, H,
N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole-
4-carboxylic Acid Dimethylamide (63). To 62 (1.10 g, 2.88 mmol)
and methyl iodide (0.12 mL) suspended in THF (5 mL) was added
sodium hydride (0.15 g). After being stirred overnight at room
temperature, the mixture was diluted with EtOAc, washed with
NaHSO4 (10% solution), NaHCO3 (saturated aqueous), and NaCl
(saturated aqueous), dried (Na2SO4), filtered, and concentrated. The
product was purified by flash chromatography eluting with 5%
methanol/CH2Cl2, yielding 0.20 g (0.47 mmol, 90% yield) of white
crystals: mp 213-214 °C; 1H NMR (300 MHz, CD2C12) δ 7.59-
7.43 (m, 2H, H4′,5), 7.36-7.20 (m, 3H, H6.7,4′), 7.08 (m, 2H, H3′,5′),
6.83 (m, 2H, H3′,5′), 5.37 (s, 2H, CH2PhF2), 3.12 (s, 3H, CH3), 2.88
(s, 3H, CH3). Anal. (C23H17F4N3O‚1/4H2O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole
4-Carboxylate (64). To 50 (1.02 g, 2.46 mmol) dissolved in
methanol (20 mL) was added barrium hydroxide (1.20 g). After 2
h, acetic acid was added and the mixture concentrated. The residue
was redissolved in CH2Cl2, washed with NaHCO3 (saturated
aqueous) and NaCl (saturated aqueous), dried (Na2SO4), filtered,
and concentrated. The product was purified by flash chromatog-
raphy eluting with 8% methanol/CH2Cl2 and recrystallization from
diethyl ether/hexane (3:1), yielding 0.71 g (1.77 mmol, 72% yield)
of white crystals: mp 178-180 °C; 1H NMR (200 MHz, CD2C12)
δ 8.06 (d, J ) 7.8 Hz, 1H, H5), 7.76 (d, J ) 8.1 Hz, 1H, H7), 7.62
(m, 1H, H4′), 7.47 (dd, J ) 7.8, 8.1 Hz, 1H, H6), 7.29 (m, 1H, H4′),
7.14 (m, 2H, H3′,5′), 6.85 (m, 2H, H3′,5′), 5.45 (s, 2H, CH2PhF2).
Anal. (C21H12F4N2O2) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-chlorometh-
ylbenzimidazole (69). To 67 (0.54 g, 1.40 mmol) dissolved in
CHCl3 (3.0 mL) was added SOCl2 (0.15 mL, 2.06 mmol, 150 M
%). After 3 h at room temperature, the mixture was diluted with
CHCl3, washed with NaHCO3 (saturated solution) and NaCl
(saturated aqueous), dried (Na2SO4), filtered, and concentrated. The
product was purified by flash chromatography eluting with 1%
MeOH/CH2Cl2 and recrystallized from diethyl ether/hexane (3:1),
yielding 0.52 g (1.28 mmol, 92% yield) of white crystals: mp 143-
1
144 °C; H NMR (300 MHz, CD2C12) δ 7.55 (m, 1H, H4′), 7.48
(dd, J ) 7.7, 1.1 Hz, 1H, H7), 7.36 (dd, J ) 7.6 Hz, 1.1 Hz, 1H,
H5), 7.30 (dd, J ) 7.7, 7.6 Hz, 1H, H6), 7.25 (m, 1H, H4′), 7.09
(m, 2H, H3′,5′), 6.82 (m, 2H, H3′,5′), 5.36 (s, 2H, CH2PhF2), 5.07 (s,
2H, CH2Cl). Anal. (C21H13ClF4N2) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(azidometh-
ylene)benzimidazole (70). To 69 (1.31 g, 3.23 mmol) dissolved
in DMF (10 mL) was added sodium azide (0.68 g, 10.5 mmol, 325
M %). After 1 h at room temperature, the mixture was diluted with
CH2Cl2, washed with NaHCO3 (saturated solution) and NaCl
(saturated aqueous), dried (Na2SO4), filtered, concentrated, and
recrystallized from diethyl ether/hexane (3:1), yielding 1.23 g (2.99
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-benzimidazoyl-
2-propanol (65). To 50 (1.02 g, 2.46 mmol) suspended in THF (5
mL) was added 1.4 M methyllithium (0.95 mL). After 0.5 h, the
mixture was diluted with EtOAc, washed with NaHSO4 (10%
solution) and NaCl (saturated aqueous), dried (Na2SO4), filtered,
and concentrated. The product was purified by flash chromatog-
raphy eluting with 1:1 ethyl acetate/hexane and recrystallization
from hexane, yielding 0.11 g (0.27 mmol, 55% yield) of white
1
mmol, 93% yield) of white crystals: mp 150-152 °C; H NMR
(300 MHz, CD2C12) δ 7.55 (m, 1H, H4′), 7.50 (dd, J ) 7.7, 1.6
Hz, 1H, H7), 7.32 (m, 1H, H6), 7.30 (m, 2H, H5,4′), 7.09 (m, 2H,
H3′,5′), 6.82 (m, 2H, H3′,5′), 5.37 (s, 2H, CH2PhF2), 4.81 (s, 2H,
1
CH2N3). Anal. (C21H13F4N5) C, H, N.
crystals: mp 149-150 °C; H NMR (200 MHz, CD2C12) δ 7.55
(m, 1H, H4′), 7.48 (d, J ) 8.1 Hz, 1H, H5), 7.25 (m, 2H, H6,4′),
7.16 (dd, J ) 1.1, 7.6 Hz, 1H, H7), 7.10 (m, 2H, H3′,5′), 6.82 (m,
2H, H3′,5′), 5.82 (s, 1H, OH), 5.37 (s, 2H, CH2PhF2), 1.67 (s, 6H,
CH3). Anal. (C23H18F4N2O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(aminometh-
ylene)benzimidazole (71). To 70 (0.55 g, 1.34 mmol) dissolved
in acetic acid (10 mL) was added zinc (0.55 g, 6.27 mmol, 630 M
%). After 1 h at room temperature, the mixture was concentrated,

4014 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Morningstar et al.
redissolved with EtOAc, washed with NaHCO3 (saturated solution)
and NaCl (saturated aqueous), dried (Na2SO4), filtered, and
concentrated. The product was purified by flash chromatography
eluting with 4% MeOH/CH2Cl2 increasing to 100% MeOH, yielding
0.16 g (0.42 mmol, 31% yield) of white crystals: mp 257-
Cornell High Energy Synchrotron Source (CHESS) F1 beam line.
One data set was collected from a cryocooled crystal, and the other
was from 10 crystals at 268 K. The data were processed and scaled
using Denzo and Scalepack.22 The previously reported HIV-1 RT/
TIBO structure (PDB code 1HNV) was used as a template in
obtaining molecular replacement solutions. Multiple crystal form
averaging21 was carried out to improve the electron density to which
the initial model was fitted using the graphics program O.23 Cycles
of model building and structure refinement were carried out using
O and XPLOR 3.1, respectively. The data set collected from crystals
at 268 K was used to refine the final model. The final cycles of
structure refinement was carried out using CNS 1.124 The structure
was refined at 2.65 Å resolution to an R and Rfree of 0.226 and
0.277, respectively (Table 2). The coordinates and structure factor
are deposited in Protein Data Bank (PDB code 2B6A).
Molecular Modeling. Computer modeling of RT complexed
with 4-methyl BPBI was based on crystallographic data. A
representative model of the binding site was constructed as
described previously.19 Calculations using this model were carried
out using the MCPRO (Monte Carlo calculations of proteins)
software program.20 Visualization of final structures was carried
out using the Insight software program.
1
260 °C; H NMR (300 MHz, CD2C12) δ 7.59 (m, 1H, H4′), 7.55
(m, 1H, H7), 7.35 (m, 2H, H5,6), 7.27 (m, 1H, H4′), 7.12 (m, 2H,
H3′,5′), 6.83 (m, 2H, H3′,5′), 5.40 (s, 2H, CH2PhF2), 4.49 (s, 2H,
CH2N). Anal. (C21H15F4N3) C, H, N.
N-(1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(N-acety-
laminomethylene)benzimidazole (72). To 71 (0.28 g, 0.73 mmol)
dissolved in THF (10 mL) and triethylamine (0.2 mL) was added
acetic anhydride (0.20 mL, 2.12 mmol, 300 M %). After 1.5 h at
room temperature, the mixture was concentrated, redissolved with
EtOAc, washed with NaHCO3 (saturated solution) and NaCl
(saturated aqueous), dried (Na2SO4), filtered, and concentrated. The
product was purified by flash chromatography eluting with 4%
MeOH/CH2Cl2 increasing to 8% MeOH/CH2Cl2, yielding 0.20 g
1
(0.47 mmol, 65% yield) of white crystals: mp 178-179 °C; H
NMR (300 MHz, CD2C12) δ 7.56 (m, 1H, H4′), 7.42 (m, 1H, H7),
7.30-7.18 (m, 3H, H5,6,4′), 7.10 (m, 2H, H3′,5′), 6.90 (br, 1H, NH),
6.82 (m, 2H, H3′,5′), 5.37 (s, 2H, CH2PhF2), 4.79 (d, J ) 5.9 Hz,
2H, CH2N), 1.92 (s, 3H, NAc). Anal. (C23H17F4N3O‚1/2H2O) C, H,
N.
Biological Assays. The reverse transcriptase inhibition experi-
ments were carried out as described previously.10
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(N-acetyl-N-
methylaminomethylene)benzimidazole (73). To 72 (0.25 g, 0.58
mmol) and methyl iodide (0.065 mL, 1.04 mmol, 180 M %)
dissolved in THF (10 mL) was added sodium hydride (50 mg, 1.25
mmol, 215 M %). After being stirred overnight at room temperature,
the mixture was diluted with EtOAc, washed with NaHSO4 (10%
solution) and NaCl (saturated aqueous), dried (Na2SO4), filtered,
and concentrated. The product was purified by flash chromatog-
raphy eluting with 2% MeOH/CH2Cl2 increasing to 4% MeOH/
CH2Cl2, yielding 0.24 g (0.54 mmol, 94% yield) of white crystals:
Acknowledgment. This research was supported [in part] by
the Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research. E.A. is grateful to NIH
MERIT Award AI 27690 and Grant P01 GM 56671 for support
of RT structural studies. The content of this publication does
not necessarily reflect the views or policies of the Department
of Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsements by
the U.S. Government. We thank the personnel at the Cornell
High Energy Synchrotron Source for their help in providing
X-ray diffraction data collection resources. We also thank the
Biophysics Resource in the Structural Biophysics Laboratory,
NCIsFrederick, for assistance with LC-MS studies.
1
mp 171-172 °C; H NMR (300 MHz, CD2C12) δ 7.55 (m, 1H,
H4′), 7.44 and 7.40 (rotamers) (d, J ) 8.3 Hz, 1H, H7), 7.32-7.20
(m, 2H), 7.15-7.04 (m, 3H), 6.82 (m, 2H, H3′,5′), 5.37 and 5.35
(rotamers) (s, 2H, CH2PhF2), 5.00 and 4.98 (rotamers) (s, 2H,
CH2N), 3.04 and 2.96 (rotamers) (s, 3H, NCH3), 2.15 and 2.11
(rotamers) (s, 3H, NAc). Anal.(C24H19F4N3O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(methoxym-
ethylene)benzimidazole (74). To 67 (0.20 g, 0.52 mmol) dissolved
in THF (2 mL) was added methyl iodide (40 µL, 0.64 mmol, 120
M %) and sodium hydride (20 mg). After 2 h at room temperature,
additional methyl iodide (20 µL, 0.32 mmol, 60 M %) was added.
After 3 h, the mixture was diluted with EtOAc, washed with
NaHCO3 (saturated solution) and NaCl (saturated aqueous), dried
(Na2SO4), filtered, and concentrated. The product was purified by
flash chromatography eluting with 2% MeOH/CH2Cl2, yielding 0.10
g (0.25 mmol, 48% yield) of white crystals: mp 122-124 °C; 1H
NMR (300 MHz, CD2C12) δ 7.53 (m, 1H, H4′), 7.43 (dd, J ) 7.1,
2.2 Hz, 1H, H7), 7.34-7.28 (m, 2H, H5,6), 7.24 (m, 1H, H4), 7.08
(m, 2H, H3′,5′), 6.81 (m, 2H, H3′,5′), 5.35 (s, 2H, CH2PhF2), 4.90 (s,
2H, CH2O), 3.45 (s, 3H, OCH3). Anal. (C22H15F4N2O) C, H, N.
1-(2,6-Difluorobenzyl)-2-(2,6-difluorophenyl)-4-(cyanometh-
ylene)benzimidazole (75). To 69 (0.125 g, 0.309 mmol) partially
dissolved in acetonitrile (0.7 mL) were added 18-crown-6 (0.006
g, 0.022 mmol, 7 M %) and potassium cyanide (0.203 g, 0.312
mmol, 101 M %). This was vigorously stirred at room temperature
for 24 h, and the mixture was diluted with CH2Cl2, washed with
H2O, dried over Na2SO4, filtered, and concentrated. The product
was purified by chromatography eluting with (1:1) hexane/ethyl
acetate, yielding 0.109 g (0.275 mmol, 89% yield) of white
Supporting Information Available: Elemental analysis data
of all new compounds described here. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) De Clercq, E. Highlights in the development of new antiviral agents.
Mini-ReV. Med. Chem. 2002, 2, 163-175.
(2) Merluzzi, V. J.; Hargrave, K. D.; Labadia, M.; Grozinger, K.; Skoog,
M.; Wu, J. C.; Shih Ch.-K.; Eckner, K.; Hattox, S.; Adams, J.;
Rosenthal, A. S.; Franes, R.; Eckner, R.; Koup, R. A.; Sullivan, J.
L. Inhibition of HIV-1 replication by a nonnucleoside reverse
transcriptase inhibitor. Science 1990, 250, 1411-1413.
(3) Pauwels, R.; Andries, K.; Debyser, Z.; Van Daele, P.; Schols, D.;
Stoffels, P.; De Vreese, K.; Woestenborghs, R.; Vandamme, A.;
Janssen, C. G. M.; Anne, J.; Cauwenbergh, G.; Desmyter, J.; Hekants,
J.; Janssen, M. A. C.; De Clercq, E.; Janssen, P. A. J. Potent and
highly selective human immunodeficiency virus type 1 (HIV-1)
inhibition by a series of R-anilinophenylacetamide derivatives targeted
at HIV-1 reverse transcriptase. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 1711-1715.
(4) Pauwels, R.; Andries, K.; Desmyter, J.; Schols, D.; Kukla, M. J.;
Breslin, H. J.; Raeymaeckers, A.; Van Gelder, J.; Woestenbogs, R.;
Heykants, J.; Schellekens, K.; Janssen, M. A. C.; De Clercq, E.;
Janssen, P. A. J. Potent and selective inhibition of HIV-1 replication
in vitro by a novel series of TIBO derivatives. Nature 1990, 343,
470-474.
1
crystals: mp 124-127 °C; H NMR (300 MHz, CD2C12) δ 7.55
(m, 1H, H4), 7.50 (dd, J ) 7.7, 1.6 Hz, 1H, H7), 7.32 (m, 1H, H6),
7.28 (m, 2H, H5,4), 7.11 (m, 2H, H3′,5′), 6.83 (m, 2H, H3′,5′), 5.38
(s, 2H, CH2PhF2), 4.2 (s, 2H, CH2CN). Anal. (C22H15F4N3) C, H,
N.
(5) Young, S. D.; Britcher, S. F.; Tran, L. O.; Payne, L. S.; Lumma, W.
C.; Lyle, T. A.; Huff, J. R.; Anderson, P. S.; Olsen, D. B.; Carroll,
S. S.; Pettibone, D. J.; O’Brien, J. A.; Ball, R. G.; Balani, S. K.;
Lin, J. H.; Chen, I.-W.; Schleif, W. A.; Sardane, V. V.; Long, W. J.;
Byrnes, V. W.; Emini, E. A. L-743,726 (DMP-266): a novel, highly
potent nonnucleoside inhibitor of the human immunodeficiency virus
type 1 reverse transcriptase. Antimicrob. Agnents Chemother. 1995,
39, 2602-2605.
Structure Determination. Compound 1 was crystallized as
described previously.21 The diffraction data sets (Table 2) for the
complex were collected using synchrotron radiation sources at the

Inhibitors of HIV-1 ReVerse Transcriptase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4015
(6) Romero, D. L.; Morge, R. A.; Biles, C.; Berrios-Pena, N.; May, P.
D.; Palmer, J. R.; Johnson, P. D.; Smith, H. W.; Busso, M.; Tan,
C.-K.; Voorman, R. L.; Reusser, F.; Althaus, I. W.; Downey, K. M.;
So, A. G.; Resnick, L.; Tarpley, W. G.; Aristoff, P. A. Discovery,
synthesis, and bioactivity of bis(heteroaryl)piperazines. 1. A novel
class of non-nucleoside HIV-1 reverse transcriptase inhibitors. J. Med.
Chem. 1994, 37, 999-1014.
(7) Janssen, P. A. J.; Lewi, P. J.; Arnold, E.; Daeyaert, F.; de Jonge,
M.; Heeres, J.; Koymans, L.; Vinkers, M.; Guillemont, J.; Pasquier,
E.; Kukla, M.; Ludovici, D.; Andries, K.; de Bethune, M.-P.; Pauwels,
R.; Das, K.; Clark, A. D., Jr.; Frenkel, Y. V.; Hughes, S. H.; Medaer,
B.; De Knaep, F.; Bohets, H.; De Clerck, F.; Lampo, A.; Williams,
P.; Stoffels, P. In search of a novel anti-HIV drug: multidisciplinary
coordination in the discovery of 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2,6-
dimethylphenyl]amino]-2-pyrimidinyl]amino]benzonitrile (R278474,
rilpivirine). J. Med. Chem. 2005, 48, 1901-1909.
(8) Campiani, G.; Ramunno, A.; Maga, G.; Nacci, V.; Fattorusso, C.;
Catalanotti, B.; Morelli, E.; Novellino, E. Non-nucleoside HIV-1
reverse transcriptase (RT) inhibitors: past, present, and future
perspectives. Curr. Pharm. Des. 2002, 8, 615-657.
(9) Das, K.; Clark, A. D., Jr.; Lewi, P. J.; Heeres, J.; de Jonge, M. R.;
Koymans, L. M. H.; Vinkers, H. M.; Daeyaert, F.; Ludovici, D. W.;
Kukla, M. J.; De Corte, B.; Kavash, R. W.; Ho, C. Y.; Ye, H.;
Lichtenstein, M. A.; Andries, K.; Pauwels, R.; de Bethune, M.-P.;
Boyer, P. L.; Clark, P.; Hughes, S. H.; Janssen, P. A. J.; Arnold E.
Roles of conformational and positional adaptability in structure-based
design of TMC125-R165335 (etravirine) and related non-nucleoside
reverse transcriptase inhibitors that are highly potent and effective
against wild-type and drug-resistant HIV-1 variants. J. Med. Chem.
2004, 47, 2550-2560.
(10) Roth, T.; Morningstar, M. L.; Boyer, P. L.; Hughes, S. H.; Buckheit,
R. W.; Michejda, C. J. Synthesis and biological activity of novel
nonnucleoside inhibitors of HIV-1 reverse transcriptase. 2-Aryl-
substituted benzimidazoles. J. Med. Chem. 1997, 40, 4199-
4207.
(11) Chimirri, A.; Grasso, S.; Monforte, A.-M.; Monforte, P.; Zappala,
M. Anti-HIV agents. I. Synthesis and in vitro anti-HIV evaluation
of novel 1H,3H-thiazolo[3,4-a]benzimidazoles. Farmaco 1991, 46,
817-823.
(12) Buckheit, R. W.; Hollingshead, M. G.; Germany-Decker, J.; White,
E. L.; McMahon, J. B.; Allen, L. B.; Ross, L. J.; Decker, W. D.;
Westbrook, L.; Shannon, W. M.; Weislow, O.; Bader, J. P.; Boyd,
M. R. Thiazolobenzimidazole: biological and biochemical anti-
retroviral activity of a new nonnucleoside reverse transcriptase
inhibitor. AntiViral Res. 1993, 21, 247-265.
(13) El Dareer, S. M.; Tillery, K. F.; Rose, L. M.; Posey, C. F.; Struck,
R. F.; Stiller, S. W.; Hill, D. L. Metabolism and disposition of a
thiazolobenzimidazole active against human immunodeficiency virus-
1. Drug Metab. Dispos. 1993, 21, 231-235.
(14) Das, K.; Lewi, P. J.; Hughes, S. H.; Arnold, E. Crystallography and
the design of anti-AIDS drugs: conformational flexibility and
positional adaptability are important in the design of non-nucleoside
HIV-1 reverse transcriptase inhibitors. Prog. Biophys. Mol. Biol.
2005, 88, 209-231.
(15) Ding, J.; Das, K.; Moereels, H.; Koymans, L.; Andries, K.; Janssen,
P. A.; Hughes, S. H.; Anold, E. Structure of HIV-1 RT/TIBO R86183
complex reveals similarity in the binding of diverse nonnucleoside
inhibitors. Nat. Struct. Biol. 1995, 2, 407-415.
(16) Weislow, O. S.; Kiser, R.; Fine, D. L.; Bader, J.; Shoemaker, R. H.;
Boyd, M. R. New soluble-formazan assay for HIV-1 cytopathic
effects: Application to high-flux screening of synthetic and natural
products for AIDS-antiviral activity. J. Natl. Cancer Inst. 1989, 81,
557-586.
(17) Yang, S.; Fliakas-Boltz, V.; Bader, J. P.; Buckheit, R. W. Charac-
teristics of a group of nonnucleoside reverse transcriptase inhibitors
with structural diversity and potent anti-human immunodeficiency
virus activity. Leukemia 1995, Suppl. 1, S75-S85.
(18) Wood, J. L.; Khartri, N. A.; Weinreb, S. M. A direct conversion of
esters to nitriles. Tetrahedron Lett. 1979, 51, 4907-4910.
(19) Kroeger Smith, M. B.; Hose, B. M.; Hawkins, A.; Lipchock, J.;
Farnsworth, D. W.; Rizzo, R. C.; Tirado-Rives, J.; Arnold, E.; Zhang,
W.; Hughes, S. H.; Jorgensen, W. L.; Michejda, C. J.; Smith, R. H.,
Jr. Molecular modeling calculations of HIV-1 reverse transcriptase
nonnucleoside inhibitors: Correlation of binding energy with biologi-
cal activity for novel 2-aryl substituted benzimidazole analogues. J.
Med. Chem. 2003, 46, 1940-1947.
(20) Jorgensen, W. L. MCPRO, version 2.0; Yale University: New Haven,
CT.
(21) Das, K.; Ding, J.; Hsiou, Y.; Clark, A. D., Jr.; Moereels, H.; Koymans,
L.; Andrias, K.; Pauwels, R.; Janssen, P. A.; Boyer, P. L.; Clark, P.;
Smith, R. H., Jr.; Kroeger Smith, M. B.; Michejda, C. J.; Hughes, S.
H.; Arnold, E. Crystal structure of 8-Cl and 9-Cl TIBO complexed
with wild-type HIV-1 RT and 8-Cl TIBO complexed with the
Tyr181Cys HIV-1 RT drug-resistant mutant. J. Mol. Biol. 1996, 264,
1085-1100.
(22) Otwinowski, Z.; Minor, W. DENZO and SCALEPACK. In The
International Union of Crystallography: Crystallography of Biologi-
cal Macromolecules; Rossmann, M. G., Arnold, E., Eds.; Kluwer
Academic: Boston, MA, 2001; Vol. F, pp 226-235.
(23) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Improved
methods for building protein models in electron density maps and
the location of errors in these models. Acta Crystallogr. 1991, A47,
110-119.
(24) Brunger, A. T.; Adams, P. D.; Rice, L. M. Recent developments for
the efficient crystallographic refinement of macromolecular structures.
Curr. Opin. Struct. Biol. 1998, 8, 606-611.
JM060103D