Probing Mercaptobenzamides as HIV Inactivators via Nucleocapsid Protein 7

Article abstract of DOI:10.1002/cmdc.201700141

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein 7 (NCp7), a zinc finger protein, plays critical roles in viral replication and maturation and is an attractive target for drug development. However, the development of drug-like molecules that inhibit NCp7 has been a significant challenge. In this study, a series of novel 2-mercaptobenzamide prodrugs were investigated for anti-HIV activity in the context of NCp7 inactivation. The molecules were synthesized from the corresponding thiosalicylic acids, and they are all crystalline solids and stable at room temperature. Derivatives with a range of amide side chains and aromatic substituents were synthesized and screened for anti-HIV activity. Wide ranges of antiviral activity were observed, with IC₅₀ values ranging from 1 to 100 μm depending on subtle changes to the substituents on the aromatic ring and side chain. Results from these structure–activity relationships were fit to a probable mode of intracellular activation and interaction with NCp7 to explain variations in antiviral activity. Our strategy to make a series of mercaptobenzamide prodrugs represents a general new direction to make libraries that can be screened for anti-HIV activity.

Full text of DOI:10.1002/cmdc.201700141

Accepted Article
Title: Probing Mercaptobenzamides as HIV Inactivators via
Nucleocapsid Protein 7
Authors: Mrinmoy Saha, Michael T Scerba, Nathaniel I Shank, Tracy L
Hartman, Caitlin A Buchholz, Robert W Buckheit, Jr., Stewart
R Durell, and Daniel H. Appella
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201700141
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700141
A Journal of
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10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
Probing Mercaptobenzamides as HIV Inactivators via
Nucleocapsid Protein 7
Mrinmoy Saha[a], Michael T. Scerba[a], Nathaniel I. Shank[a], Tracy L. Hartman[b], Caitlin A. Buchholz[b],
Robert W. Buckheit, Jr. [b], Stewart R. Durell[c], Daniel H. Appella*[a]
[a]
Dr. Daniel H. Appella, Dr. Mrinmoy Saha, Dr. Michael T. Scerba, Dr. Nathaniel I. Shank
Synthetic Bioactive Molecules Section
LBC, NIDDK, NIH
8 Center Dr., Room 404, Bethesda, MD 20892
E-mail: appellad@niddk.nih.gov
[b]
Dr. Robert W. Buckheit, Jr., Ms. Tracy L. Hartman, Ms. Caitlin A. Buchholz
ImQuest Biosciences
7340 Executive Way, Suite R, Frederick, MD 21704
Dr. Stewart R. Durell
[c]
Laboratory of Cell Biology, NCI, NIH
9000 Rockville Pike, Bethesda, MD 20892
Supporting information for this article is given via a link at the end of the document.
Abstract: Human immunodeficiency virus type
1
(HIV-1)
replication steps, such as strand transfers during reverse
transcription of viral RNA and genome packaging during virion
assembly.[11] The principle role of NCp7 is to bind viral RNA and
protect it from degradation. The NCp7 protein is part of the larger
HIV polyprotein Gag which contains matrix and capsid proteins.
To successfully mature, HIV protease must cleave peptide bonds
at specific locations within Gag to liberate the matrix, capsid, and
NCp7 proteins.[11]
nucleocapsid protein 7 (NCp7), a zinc finger protein, plays critical
roles in viral replication and maturation and represents an attractive
target for drug development. However, development of drug-like
molecules that inhibit NCp7 has been a significant challenge. In this
report, a series of novel 2-mercaptobenzamide prodrugs have been
investigated for anti-HIV activity in the context of NCp7 inactivation.
The molecules are synthesized from the corresponding thiosalicylic
acids, and they are all crystalline solids and stable at room
temperature. Derivatives with a range of amide side chains and
aromatic substituents were synthesized and screened for anti-HIV
activity. Wide ranges of antiviral activity were observed, with IC50
values ranging from 1 to 100 μM depending on subtle changes to the
substituents on the aromatic ring and the sidechain. Results from
these structure-activity relationships were fit to a probable mode of
intracellular activation and interaction with NCp7 to explain variations
Inhibition of NCp7 yields immature virions that are non-infectious.
A number of compounds in the literature are known to inhibit
,
12 13
]
NCp7,[
and these molecules typically have electrophilic
groups that react with the most nucleophilic cysteine in NCp7
(shown to be Cys49). These inhibitors are also generally toxic due
to non-specific reactions with other cellular targets. In general,
]
NCp7 has proven to be a difficult drug target[14 as there is no
distinct three-dimensional structure for the protein other than the
zinc-coordinated regions. Furthermore, traditional medicinal
chemistry approaches to target the active sites of enzymes are
not applicable to a protein like NCp7. As a result, there are
currently no NCp7 inhibitors that are used clinically to treat HIV
in antiviral activity.
Our strategy to make
a
series of
mercaptobenzamide prodrugs represents a general new direction to
make libraries that can be screened for anti-HIV activity.
]
infection.[15
HIV continues to be
a major public health issue with
approximately 37 million people affected globally.[1,2] According to
the WHO, 1.1 million people died from HIV-related causes in
2015.[3] The therapies being used to manage HIV infection inhibit
the essential viral enzymes reverse transcriptase, protease, or
C-terminal Knuckle
N-terminal Knuckle
20
E
K
G
E
G
K
40
G
G
H
H
45
Q
I
]
integrase.[4 The standard treatment for HIV is HAART (highly
C
25
C
M
A
Zn
N
Zn
active antiretroviral therapy) in which combinations of drugs
simultaneously target these viral enzymes.[5] Infected individuals
who adhere to HAART can expect a normal life span. However,
HAART requires regular and lifelong access to costly medication
that often impedes many HIV-infected people from receiving
proper treatment. Furthermore, HAART is hampered by viral
resistance[6] and may also predispose patients to cardiovascular
K
K
K
F
N
W
D
C
C
C
C
TERQAN
55
MQKGNFRNQRKTVK
5
RAPRKKG
30 35
15
50
1
10
Figure 1. Primary sequence of NCp7 with Zn coordinating residues specified
with spherical shapes. N-terminal knuckle represented with blue color and C-
terminal knuckle represented with brown color. Zn atoms are represented with
green spheres.
,
]
8
and neurological diseases.[
Therefore, it is essential to
7
continue development of affordable and mutation-resistant small
molecule inhibitors of HIV.
Nucleocapsid protein 7 (NCp7, Figure 1), a 55-amino acid protein
Our lab is interested in the potential of mercaptobenzamide (MB)-
based molecules to inhibit HIV-1 through the inactivation of NCp7.
Previously, we have shown that the S-acetyl forms of these
]
containing two highly conserved zinc-knuckle motifs,[ is an
9
attractive target for antiretroviral drugs due to its essential role in
]
viral replication and maturation.[ 10 NCp7 facilitates essential
1
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10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
molecules (MB1, Figure 2) are NCp7 inactivators that promote the
]
unfolding of the protein.[
In contrast to other electrophilic
The general synthetic route for the 2-mercaptobenzamide
prodrugs is shown in Scheme 1. The mercaptobenzamides were
synthesized in two steps from thiol 1. In most cases, the
carboxylic acid 1 was first coupled with amine 2, using suitable
peptide coupling reagents to form the amide bond.[21] Then, the
16
inhibitors of NCp7, MB1 reacts selectively at Cys36 (in the C-
terminal knuckle) of the protein (Figure 2).17 In this mechanism,
the sulfur of Cys36 attacks MB1 as a nucleophile at the acetyl
group of the thioester. This results in an intermolecular acetyl
transfer from MB1 to the cysteine and release of a free thiol (MB2). free thiol intermediate was protected with chloromethyl butyrate
Next, an intramolecular acetyl transfer takes place between
acetylated Cys36 and an epsilon nitrogen on the sidechain of a
proximal lysine residue (typically Lys38). The sulfur to nitrogen
acetyl transfer creates a very stable amide and is likely
irreversible. Once a lysine is acetylated, zinc coordination is
disrupted, allowing further modification of the protein and zinc
ejection. As the binding of zinc is specifically required for the
function and stability of NCp7, loss of zinc causes disruption of
the NCp7 structure, loss of function, and ultimately the formation
of immature non-infectious virions.
(3) in the same pot to provide the desired prodrug 4. The
sequence of reactions had to be reversed when electron
withdrawing groups were attached to the aromatic ring. The
prodrugs are mostly crystalline white solids that are purified by
column chromatography and can be stored at room temperature
without any special precautions.
Scheme 1. Synthesis of Mercaptobenzamidesa
O
R1-NH2
SH
O
O
S
O
2
OH
R1
O
The molecule MB2 also has the same anti-HIV activity as MB1 in
cell-based assays due to a unique intracellular mechanism where
MB2 is acetylated in cells by acetyl coenzyme A to generate MB1.
The MB1 generated by activation inside cells then inactivates
N
H
R
Cl
O
1
R
3
4
aReagents and conditions: 1, HATU, HBTU or HCTU, DIPEA,
DMF, rt, 10-15 min; 2, 16-24 h; 3, rt, 12-24 h (X = Cl or TFA)
]
NCp7 by the same mechanism described previously.[16, Both
MB1 and MB2 are too chemically unstable to develop into drugs
due to hydrolysis of the thioester and oxidation of the free thiol. In
addition, these molecules display EC50 values in the low
micromolar range in cell-based assays that test for anti-HIV
activity. Testing different analogs of MB1 and MB2 could lead to
the discovery of new mercaptobenzamides with better anti-HIV
activity. However, the chemical instabilities of the thioester and
thiol complicate the synthesis of analogs. Recently, we found that
introduction of a prodrug group improves the chemical stability
without compromising anti-viral activity. Attaching an S-methyl
butyrate prodrug to the sulfur of MB2 affords MB3, which has
18
If the thiols were not commercially available, the precursor thiols
for the mercaptobenzamide prodrugs were synthesized in three
]
steps (Scheme 2).[22 Amine 5 was first diazotized and the diazo
compound was treated with freshly prepared Na2S2 (from
Na2S•9H2O and elemental sulfur) to give disulfide 7. The disulfide
bond in 7 was then reduced with either NaBH4 or TCEP to
produce the mercapto-benzoic acid 1.
Scheme 2. Synthesis of Thiolsb
Cl
NH2
O
N2
O
(i)
OH
OH
]
exactly the same anti-HIV activity in cell-based assays.[19 The
prodrug of MB3 is sensitive to esterase enzyme-promoted
hydrolysis that yields butyric acid, formaldehyde, and MB2. The
toxicity of MB3 is remarkably low.[19] Attaching the S-methyl
butyrate group to a series of prodrugs facilitates the chemical
synthesis of stable analogs to screen for anti-HIV activity. Herein,
we report a series of 2-mercaptobenzamide prodrugs and their
corresponding anti-HIV activity in cell-based assays. By making a
series of mercaptobenzamide prodrugs,[20] it was easier to isolate
and purify analogs compared to their corresponding free thiols or
S-acetyl forms. The results from this screen allowed us to probe
the influence of mercaptobenzamide substitution on anti-HIV
activity, and how it may be rationalized by interaction with the
NCp7 target.
R
R
6
5
(ii)
R
SH
O
HO
R
O
(iii) or (iv)
OH
S
S
R
O
OH
1
7
bReagents and conditions: (i) NaNO2, Conc. HCl, H2O, 0 °C, 1h,
(ii) Na2S•9H2O, S8, 70-80 °C, ~30 min; add 6, 0 °C to rt, (iii) TCEP,
DMF/H2O (9:1), rt, 14-18h, (iv) NaBH4, MeOH or THF/MeOH, rt,
2-12 h.
O
O
MB1
CH3
His
NH
Table 1 describes the anti-HIV data of 2-mercaptobenzamide
prodrugs containing different functional groups on the aromatic
ring. These assays employ CEMMSS cells infected with a
laboratory-adapted strain of HIV to provide an initial gauge of anti-
HIV activity relative to cellular toxicity.[19] For comparison, the
activity of MB3 is listed in entry 1. Adding an electron donating
methoxy group (OMe, entry 2) group had little effect on EC50
values of anti-HIV activity and also did not influence the toxicity.
However, a p-hydroxy (OH, entry 3) was not active nor toxic.
Adding electron-withdrawing groups (entries 4 to 8) at the para
position of the aromatic ring improves the anti-HIV activity slightly,
but only at the expense of increasing toxicity to the cells in the
assay. An additional electron-withdrawing group at the 4 position
H2N
Cys
S
O
O
HN
S
N
N
H
NH2
Zn
S
S
Cys
Cys
Acetylated NCp7
Deactivated
C-terminal zinc knuckle of NCp7
O
SH
O
O
O
S
O
O
N
H
NH2
N
H
NH2
MB2
MB3
Figure 2. Illustration of how MB1 acetylates NCp7. The molecules MB1, MB2,
and MB3 all display similar anti-HIV activity in cell-based assays.
2
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10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
Table 2. Anti-HIV activity and toxicity data with variable amide side chains.[d]
(entry 6) does not further improve the activity. An o-methyl group
to sulfur (entry 9) eliminated all anti-HIV activity, but remained
non-toxic. We envisioned that the o-methyl group could obstruct
the acetyl group transfer from the sulfur of the
mercaptobenzamide to Cys36 of NCp7 or perhaps interfere with
acetylation at sulfur by acetyl CoA. Therefore, we added a
relatively smaller o-fluoro group to sulfur (entry 10) expecting a
different result. However, we found the o-fluoro group also has
no anti-HIV activity and displays moderate toxicity that is
consistent with the other derivatives with electron withdrawing
groups.
O
H3C
O
S
O
R2
R1
Entry
1
Compoun
d
R1
H
R2
CEMSS
EC50 (µM)
CEMSS
TC50
(µM)
MB13
MB14
NH2 2.93 ± 1.92
>100
N
H
O
Table 1. Anti-HIV activity (EC50) data for substituents on the aromatic ring.[c]
2
3
4
H
H
H
2.24 ± 2.05
>100
H3C CH3
N
NH2
O
H
O
O
S
O
O
R3
MB15
MB16
2.18 ± 1.43
>100
>100
N
H
NH2
NH2
N
H
R2
O
R1
Entry
Compoun
d
R1
H
R2
R3
CEMSS
EC50 (µM)
CEMSS
TC50 (µM)
3.60 ± 0.95
NH2
N
H
1
2
MB3
MB4
MB5
H
H
H
H
H
H
2.38 ± 2.23
1.50 ± 0.33
>100
>100
>100
>100
O
OMe
OH
323
5
6
MB17
MB18
H
H
8.20 ± 4.20
>100
4
MB6
OCF3
F
H
H
F
H
1.11 ± 0.60
1.16 ± 0.95
1.26 ± 0.53
1.64 ± 0.35
2.60 ± 1.44
>100
32.1 ± 7.9
>5
NH2
N
H
5
MB7
H
O
6
MB8
F
H
39 ± 28
14.8 ± 5.7
14.5 ± 0.2
>100
>100
34.8±
23.7
7
MB9
CN
Cl
H
H
H
H
H
8
MB10
MB11
MB12
H
NH2
N
H
9
H
Me
O
10
H
F
>100
>6
7
8
MB19
MB20
OMe
2.20 ± 0.89
>100
H3C CH3
N
[c]Inhibition of HIVRF in CEMSS cells were performed using published
procedures. In each individual antiviral assay, efficacy and toxicity values are
derived from a minimum of three replicate wells.[19]
NH2
H
O
OCF3
3.7 ± 1.7
56.1±
0.8
H3C CH3
N
Next, we moved our attention to functionalizing the amide side
chain on the 2-mercaptobenzamide prodrugs with different
geminal substitutions on the α position of the amides (Table 2).
The anti-HIV activity of the mercaptobenzamide with aβ-alanine
amide side chain (MB3) is in entry 1, table 1.[19] The glycinamide
side chain also showed similar activity to MB3 (table 2, entry 1).
Geminal bis-substitution was tolerated for anα-methylalanine
amide (entry 2) and up to ring size of 5 (entry 3-5). Within this
series it is interesting that the EC50 values gradually increase as
the ring size increases from a three-, to four-, to five-membered
ring. At the same time, the cellular toxicity remains about the
same within this series. Then there is a dramatic change for the
derivative with a ring size of 6: this molecule has no antiviral
activity and displays a significant increase in cellular toxicity (entry
7). We also briefly evaluated the anti-HIV activity of two prodrugs
with the α-methylalanine amide chain and substitution on the
aromatic ring (entry 7-8). Neither the electron-donating nor the
electron-withdrawing group affected the EC50 values compared to
the non-substituted form (entry 2). Cellular toxicity was increased
with the electron-withdrawing group in a manner consistent with
the results in Table 1. Except for the geminal cyclohexyl amide
and aromatic OCF3 groups, the rest of the compounds in Table 2
are non-toxic.
NH2
H
O
[d]Inhibition of HIVRF in CEMSS cells were performed using published
procedures. In each individual antiviral assay, efficacy and toxicity values are
derived from a minimum of three replicate wells.[19]
The assays based on CEMSS cells were used to provide an initial
gauge of antiviral activity and cellular toxicity. At the same time,
we tested a selection of compounds in an assay that more closely
represents HIV infection in which human peripheral blood
mononuclear cells (PBMCs) are infected with a clinical isolate of
HIV-1.[ 24 The selected compounds (MB4, MB15, MB19 and
]
MB21) were tested in both CEMSS and PBMC assays and a
comparison of data between the two assays is in Table 3. We
also included tests of a mercaptobenzamide with a pyridine ring
(compound MB21). All the compounds showed no toxicity (TC50
>100 µM) in PBMCs. The EC50 values were comparable for all the
prodrugs, with only slightly weaker antiviral activity in the PMBC
assay compared to the CEMSS. Interestingly, MB21, which has a
pyridine as the core aromatic building block, showed significantly
weaker antiviral activity in the PMBC assay.
3
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10.1002/cmdc.201700141
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COMMUNICATION
Table 3. Comparison of cytopathic effect inhibition assay (inhibition of cell lysis in CEM-SS cells) to full active HIV assay (PBMC/HIV-192HT599
[e]
)
O
O
O
O
nPr
O
S
O
nPr
O
S
O
O
Me Me
N
nPr
O
nPr
O
O
O
S
O
S
NH2
N
H
NH2
NH2
H
N
H
N
N
H
NH2
O
O
MB19
OMe
MB4
MB21
MB15
OMe
Compound
CEMSS EC50 (µM)
CEMSS TC50 (µM)
PBMC HIV-1 EC50 (µM)
PBMC HIV-1 TC50 (µM)
MB4
1.50 ± 0.33
>100
4.23 ± 2.89
>100
MB15
MB19
MB21
2.18 ± 1.43
2.20 ± 0.89
2.37 ± 0.34
>100
>100
>100
6.80 ± 2.59
14.1 ± 6.8
67.8 ± 23.5
>100
>100
>100
[e]Inhibition of HIVRF in CEMSS cells and inhibition HIV-192HT599 in human PBMCs were performed using previously published procedures. In each individual antiviral
assay, efficacy and toxicity values are derived from a minimum of three replicate wells.[19]
From the antiviral data, there is clearly a subtle interplay between
substituents on the aromatic ring and the overall antiviral activity
and toxicity. Substituents ortho to the sulfur atom effectively
eliminate activity. For instance, MB11 (which has an ortho methyl
group) did not have any antiviral activity or toxicity. Electron
withdrawing groups on the aromatic ring consistently increase the
toxicity of these molecules. Based on our prior mechanistic work,
the thiol of the mercaptobenzamides must be acetylated by acetyl
coenzyme A for antiviral activity (Figure 3). It is likely that there is
a distinct equilibrium between the free thiol MB2 and the
acetylated derivative MB1 that exists within cells, mediated by the
interaction of MB2 with acetyl CoA. A possible explanation for the
poor activity of MB11 is that the acetylation at sulfur is blocked
due to steric hindrance from the ortho methyl group, and that
without acetylation there is no antiviral activity. The introduction of
electron withdrawing groups on the aromatic ring may also
interfere with acetylation by decreasing the nucleophilicity at the
sulfur atom. This situation would perturb the equilibrium between
free thiol and S-acetyl forms in favor of the free thiol, possibly
increasing the general toxicity.
The correlation of lowered antiviral activity with increases in the
steric size of sidechains as seen with compounds MB13, MB15,
MB16, MB17 and MB18 indicated that there could be steric
interaction with the NCp7 protein. Molecular models were
generated to probe the interaction of this series of compounds
with the C-terminal finger of NCp7 (Figure 4). As described earlier,
the prodrugs are first removed by esterases to afford the free thiol
forms of these molecules, followed by S-acetylation by acetyl
CoA. For this comparison, we assumed that the S-acetyl thioester
compounds 8-12 all form in the cell and we modeled their potential
to inhibit NCp7. In the model, the primary amide of the
glycinamide side chain of thioester 8 may form hydrogen-bonds
with Lys34 and His44 of the C-terminal knuckle. At the same time
the aromatic ring of 8 may engage in a π-stacking interaction with
Trp37. This arrangement positions the carbonyl carbon of 8 for
nucleophilic attack by the sulfur of Cys36. Another hydrogen-
bonding interaction between the oxygen of the thioester carbonyl
of 8 and Met46 may also help to facilitate the nucleophilic attack
by increasing the electrophilicity of the carbonyl. A similar model
could also be constructed for geminal cyclopropyl (9), cyclobutyl
(10) and cyclopentyl (11) glycinamide side chains, but as the
sidechains increase in size they sterically clash with the carbonyl
oxygen of Lys34 and Gln45. These increases in steric hindrance
between mercaptobenzamide and NCp7 may explain why the
antiviral activity decreases as the size of the ring increase from 3,
to 4, to 5. A model with compound 12 shows that the cyclohexyl
group clearly cannot interact with NCp7 in the same manner as
the previous compounds as the ring sterically clashes with Lys34,
Gln45, and His44. This steric interaction probably eliminates the
antiviral activity of compound 12 in the cell-based assays.
O
SH
O
O
S
O
O
Acetyl CoA
Acetyl CoA
Acetyl CoA
N
NH2
N
H
NH2
H
MB1
MB2
SH
O
O
H3C
No Reaction
N
NH2
H
MB11
Ortho Methyl
O
SH
O
O
S
O
O
N
H
NH2
Electron
N
H
NH2
Withdrawing
Group
EWG
EWG
Figure 3. Illustration of how mercaptobenzamide substitution may
affect reaction with acetyl coenzyme.
4
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10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
8
9
W37
M46
H44
C36
K34
10
11
12
O
O
S
S
O
O
Compounds
in Models:
NH2
O
N
H
8
O
S
O
NH2
O
NH2
N
H
N
H
O
X
9
10
X
O
S
O
O
S
O
NH2
NH2
O
N
H
N
H
O
11
12
Figure 4. Models of NCp7 interacting with the series of inactivators with different geminal-disubstituted side chains (glycinamide (8), cyclopropyl (9), cyclobutyl (10),
cyclopentyl (11), cyclohexyl (12) glycinamide side chains). NCp7 is shown as grey surface with oxygens in red, nitrogen in blue, hydrogen in white, and sulfur in
yellow. The skeleton of the compounds (8 – 12) are portrayed as magenta sticks with blue, red, yellow and white representing nitrogen, oxygen, sulfur and hydrogen
atoms respectively. Key amino acid residues in NCp7 are labeled in the model with compound 8. Green X’s represent steric clashes that may inhibit interactions.
In conclusion, a series of novel 2-mercaptobenzamide prodrugs
was synthesized and evaluated for their anti-viral activity. Cell
Experimental Section
1H NMR spectra were recorded in deuterated solvents and are reported in
ppm relative to tetramethylsilane and referenced internally to the residually
protonated solvent. 13C NMR spectra were recorded in deuterated
solvents and are reported in ppm relative to tetramethylsilane and
referenced internally to the residually protonated solvent. Routine
monitoring of reactions was performed using EM Science DC-Alufolien
silica gel TLC plates. Flash chromatography was performed with the
indicated eluents on EM Science Gedurian 230-400 mesh silica gel. Air
and/or moisture sensitive reactions were performed under usual inert
atmosphere conditions. Reactions requiring anhydrous conditions were
performed under a blanket of nitrogen, in glassware dried in an oven at
130 °C or by flame, then cooled under nitrogen. Dry acetonitrile, DMF, THF
and DCM were obtained via a solvent purification system. All other
solvents and commercially available reagents were either purified via
literature procedures or used without further purification.
viability studies in the same cell line indicate that most of these
molecules are non-toxic (TC50>100 μM). These prodrugs are
stable at room temperature, crystalline, and easily synthesized in
two steps from the corresponding aromatic substituted thiols. The
substituted thiols were synthesized from inexpensive starting
materials. We speculate that ortho substitution next to the sulfur
atom may hinder acetylation of the thiol by acetyl CoA. It is
possible that electron withdrawing groups decrease the
nucleophilicity of the sulfur atom and lead to alternative modes of
reactivity that are toxic. We found that the introduction of gem-
disubstitution on the amide side chain may be tolerated to an
extent, but large rings like cyclohexyl likely block interaction with
NCp7. Further improvement and applications of the
mercaptobenzamide prodrugs will continue.
5
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10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
General Procedure for Synthesis of Prodrugs 4: Method A: To a stirred
solution of thiosalicylic acid 1 in DMF (0.2 M) at rt was added HBTU or
HATU or HCTU (1 equiv.) and Pr2NEt (3.5 equiv.) sequentially. After 10
2H), 2.57 (t, J = 5.8 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 1.62 (q, J = 7.4 Hz,
2H), 0.92 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 176.29,
175.22, 169.32, 141.59, 135.94, 135.02, 133.73, 132.92, 130.42, 69.81,
38.35, 38.14, 36.87, 20.50, 15.86 ppm; HRMS (ES+) calcd. for
C15H19ClN2O4SNa (M+Na) 381.0652, found 381.0659.
MB11: Method B, 31 mg, 61% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.33 – 7.27 (m, 3H), 6.56 (s, 1H), 5.94 (s, 1H), 5.45 (s, 1H), 5.25
(s, 2H), 3.71 (q, J = 6.0 Hz, 2H), 2.59 (m, 2H), 2.53 (s, 3H), 2.32 (t, J = 7.4
Hz, 2H), 1.66-1.57 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (100
MHz, CDCl3) δ 174.01, 173.50, 169.80, 144.07, 144.00, 131.83, 129.69,
128.59, 125.71, 69.52, 36.17, 35.86, 34.87, 21.73, 18.26, 13.78 ppm;
HRMS (ES+) calcd. for C16H22N2O4NaS (M+Na) 361.1198, found
361.1200.
i
min, the amine 2 (1.05 equiv.) was added to the previous solution. After
stirring for 16-24 h, chloromethyl butyrate 3 (0.99 equiv.) was added to the
reaction mixture and stirred for 12-18 h. Then, the reaction was
concentrated in vacuo and treated with H2O. The aq. layer was extracted
with EtOAc (3x) and the combined organic layer was washed with 1 M HCl
(2x), sat. NaHCO3 (2x) and brine. The dried (Na2SO4) extract was then
concentrated in vacuo and the crude product was purified by
chromatography over silica gel, eluting with EtOAc / hexanes or MeOH /
EtOAc or MeOH / CH2Cl2, to give 4.
Method B: To a stirred solution of thiosalicylic acid 1 in DMF (0.2 M) at rt
was added iPr2NEt (2.5 equiv.) and chloromethyl butyrate 3 (0.99 equiv.).
After stirring for 18-24 h, HBTU or HATU or HCTU (1 equiv.), amine 2 (1.05
equiv.) and iPr2NEt (1 equiv.) were added to the previous solution
sequentially and stirred for 16-24 h. Then, the reaction was concentrated
in vacuo and treated with H2O. The aq. layer was extracted with EtOAc
(3x) and the combined organic layer was washed with 1 M HCl (2x), sat.
NaHCO3 (2x) and brine. The dried (Na2SO4) extract was then concentrated
in vacuo and the crude product was purified by chromatography over silica
gel, eluting with EtOAc / hexanes or MeOH / EtOAc or MeOH / CH2Cl2, to
give 4.
MB4: Method B; 21 mg, 44% yield; white solid; 1H NMR (400 MHz, DMSO-
d6) δ 8.34 (t, J = 5.7 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.37 (s, 1H), 7.01
(m, 1H), 6.95 (d, J = 2.9 Hz, 1H), 6.85 (s, 1H), 5.33 (s, 2H), 3.78 (s, 3H),
3.39 – 3.35 (m, 2H), 2.35-2.29 (m, 4H), 1.59-1.19 (m, 2H), 0.88 (t, J = 7.4
Hz, 3H) ppm; 13C NMR (100 MHz, DMSO-d6) δ 172.39, 172.23, 167.31,
158.57, 141.19, 133.69, 122.44, 115.69, 113.32, 68.01, 55.47, 35.76,
35.37, 34.83, 17.81, 13.34 ppm; HRMS (ES+) calcd. for C16H22N2O5NaS
(M+Na) 377.1147, found 377.1141.
MB12: Method B, 59 mg, 35% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.34 (m, 1H), 7.26 (d, J =
6.2 Hz, 1H), 7.16 (td, J = 1.3, 8.4 Hz, 1H), 6.96 - 6.89 (m, 1H), 6.07 (s, 1H),
5.69 (s, 1H), 5.27 (s, 2H), 3.67 (dd, J = 5.1, 5.5 Hz, 2H), 2.57 (t, J = 5.7 Hz,
2H), 2.30 (t, J = 7.4 Hz, 2H), 1.66 (sex, J = 7.4 Hz, 2H), 0.90 (t, J = 7.4 Hz,
3H) ppm; 13C NMR (100 MHz, CDCl3) δ 176.20, 175.57, 171.10, 169.83,
166.48, 164.02, 162.06, 146.19, 133.55, 133.46, 133.10, 125.93, 122.16,
119.66, 119.42, 114.44, 70.17, 38.25, 38.12, 36.92, 20.35, 15.85 ppm; 19
F
NMR (376 MHz, CDCl3) δ -104.62 (dd, J = 5.1, 8.7 Hz, 1F) ppm; HRMS
(ES+) calcd. for C15H19FN2O4SNa (M+Na) 365.0947, found 365.0952.
MB13: Method A, 228 mg, 33% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.60 (d, J = 18.4 Hz, 1H), 7.55 (dd, J = 1.2, 7.6 Hz, 1H), 7.42 (td,
J = 1.6, 7.7 Hz, 1H), 7.31 (td, J = 1.2, 7.5 Hz, 1H), 7.09 (t, J = 5.4 Hz, 1H),
6.60 (s, 1H), 5.79 (s, 1H), 5.39 (s, 2H), 4.15 (d, J = 5.2 Hz, 2H), 2.31 (t, J
= 7.4 Hz, 2H), 1.62 (sex, J = 7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm; 13
C
NMR (100 MHz, CDCl3) δ 173.08, 171.21, 168.64, 136.95, 133.37, 131.46,
131.28, 128.44, 127.70, 77.47, 77.35, 77.15, 76.83, 67.74, 43.42, 36.20,
18.34, 13.70. ppm; HRMS (ES+) calcd. for C14H18N2O4NaS (M+Na)
333.0885, found 333.0883.
MB14: Method A, 310 mg, 39% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.60 (dd, J = 0.8, 6.8 Hz, 1H), 7.54 (dd, J = 1.5, 7.2 Hz, 1H), 7.43
(td, J = 1.5, 7.5 Hz, 1H), 7.35 (td, J = 1.1, 7.5 Hz, 1H), 6.71 (s, 1H), 6.57
(s, 1H), 5.41 (s, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.69 (s, 6H), 1.63 (q, J = 7.4
Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 176.84,
173.25, 168.33, 139.02, 132.52, 132.23, 131.27, 128.56, 128.36, 68.24,
58.30, 36.41, 25.72, 18.55, 13.92 ppm; HRMS (ES+) calcd. for
C16H22N2O4SNa (M+Na) 361.1198, found 361.1193.
MB5: Method B; 10 mg, 20% yield from SI-2; white solid; 1H NMR (400
MHz, CD3CN) δ 7.43 (dd, J = 0.8, 8.0 Hz, 1H), 7.01 (s, 1H), 6.99 – 6.84
(m, 2H), 6.25 (s, 1H), 5.70 (s, 1H), 5.27 (s, 2H), 3.54 – 3.49 (m, 2H), 2.44
(t, J = 6.5 Hz, 2H), 2.29 (t, J = 7.3 Hz, 3H), 1.63 – 1.53 (m, 2H), 0.91 (t, J
= 7.4 Hz, 3H) ppm; 13C NMR (100 MHz, CD3CN) δ 174.20, 173.60, 168.81,
158.07, 143.02, 136.71, 121.78, 118.08, 115.67, 69.68, 36.63, 36.58,
35.40, 18.93, 13.75 ppm; HRMS (ES+) calcd. for C15H20N2O5NaS (M+Na)
363.0991, found 363.0989.
MB6: Method A, 61 mg, 32% yield; white solid; 1H NMR (400 MHz, CDCl3)
δ 7.61 (d, J = 8.6 Hz, 1H), 7.36 (dq, J = 0.9, 2.8 Hz, 1H), 7.30 – 7.20 (m,
1H), 7.01 (t, J = 5.6 Hz, 1H), 5.83 (s, 1H), 5.59 (s, 1H), 5.37 (s, 2H), 3.71
(dddd, J = 2.0, 4.5, 6.5, 7.8 Hz, 3H), 2.58 (t, J = 5.8 Hz, 2H), 2.33 (t, J =
7.4 Hz, 2H), 1.65 (dt, J = 7.4, 14.8 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H) ppm;
13C NMR (Some carbons are not resolved due to extensive C-F coupling.
Major peaks are listed.) (100 MHz, CDCl3) δ 173.83, 173.03, 166.88,
148.54, 139.89, 133.46, 131.46, 124.28, 122.97, 121.71, 120.83, 119.14,
67.71, 36.18, 35.91, 34.61, 18.34, 13.69 ppm; 19F NMR (376 MHz, CDCl3)
δ -57.87 ppm; HRMS (ES+) calcd. for C16H19F3N2O5S (M+H) 409.1045,
found 409.1039.
MB15: Method A, 68 mg, 37% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.49 - 7.44 (m, 2H), 7.42 - 7.40 (m, 1H),
7.01 (s, 1H), 6.59 (s, 1H), 5.88 (s, 1H), 5.36 (s, 2H), 2.29 (t, J = 7.4 Hz,
2H), 1.65 – 1.57 (m, 4H), 1.12 (dd, J = 4.5, 7.7 Hz, 2H), 0.90 (t, J = 7.4 Hz,
3H) ppm; 13C NMR (100 MHz, CDCl3) δ 173.63, 169.62, 164.97, 133.13,
132.30, 131.05, 130.88, 127.47, 126.58, 68.62, 43.73, 36.11, 18.26, 17.62,
13.69 ppm; HRMS (ES+) calcd. for C16H20N2O4SNa (M+Na) 359.1041,
found 359.1046.
MB16: Method A, 71 mg, 46% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.61 (dd, J = 1.0, 7.8 Hz, 1H), 7.54 (dd, J = 1.4, 7.6 Hz, 1H), 7.43
(td, J = 1.6, 7.4 Hz, 1H), 7.35 (td, J = 1.3, 7.6 Hz, 1H), 7.07 (s, 1H), 6.82
(s, 1H), 5.41 (s, 2H), 2.84 – 2.77 (m, 2H), 2.40 – 2.25 (m, 4H), 2.12 – 1.99
(m, 2H), 1.63 (sex, J = 7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm; 13C NMR
(100 MHz, CDCl3) δ 175.41, 172.99, 168.31, 137.90, 132.45, 132.32,
131.28, 128.57, 128.12, 68.07, 60.18, 36.17, 31.50, 18.33, 15.86, 13.70
ppm; HRMS (ES+) calcd. for C17H22N2O4SNa (M+Na) 373.1198, found
373.1195.
MB8: Method B; 31 mg, 61% yield; white solid; 1H NMR (400 MHz, CDCl3)
δ 7.45 – 7.38 (m, 2H), 7.01 (m, 1H), 5.69 (s, 1H), 5.46 (s, 1H), 5.35 (s, 2H),
3.71 (q, J = 6.0 Hz, 2H), 2.58 (m, 2H), 2.35 (t, J = 7.4 Hz, 2H), 1.70 – 1.61
(m, 2H), 0.95 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 173.77,
172.96, 166.19, 134.87, 129.67, 121.23, 121.04, 118.05, 117.86, 67.78,
36.18, 35.90, 34.56, 18.36, 13.71 ppm; 19F NMR (376 MHz, CDCl3) δ -
133.1 (d, J = 21.7 Hz, 1F), -137.4 (d, J = 21.7 Hz, 1F) ppm; HRMS (ES+)
calcd. for C15H18N2O4F2NaS (M+Na) 383.0853, found 383.0851.
MB9: Method B, 151 mg, 23% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.72 – 7.60 (m, 3H), 7.07 (t, J = 5.6 Hz, 1H), 5.76 (s, 1H), 5.61
(s, 1H), 5.46 (s, 2H), 3.72 (q, J = 5.9 Hz, 2H), 2.60 (t, J = 5.8 Hz, 2H), 2.34
(t, J = 7.4 Hz, 2H), 1.66 (sex, J = 7.5 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H) ppm;
13C NMR (100 MHz, CDCl3) δ 176.03, 174.93, 168.37, 143.96, 138.85,
135.88, 133.42, 130.93, 120.02, 112.24, 67.69, 38.28, 38.07, 36.57, 20.50,
15.83 ppm; HRMS (ES+) calcd. for C16H19N3O4SNa (M+Na) 372.0994,
found 372.0994.
MB17: Method A; 16 mg, 6% yield; white solid; 1H NMR (400 MHz, CDCl3)
δ 7.61 (dd, J = 1.2, 7.9 Hz, 1H), 7.51 (dd, J = 1.8, 7.5 Hz, 1H), 7.44 (td, J
= 1.8, 7.6 Hz, 1H), 7.37 (td, J = 1.2, 7.6 Hz, 1H), 7.08 (s, 1H), 6.35 (s, 1H),
5.41 (m, 3H), 2.43 – 2.39 (m, 2H), 2.31 (t, J = 7.4 Hz, 2H), 2.14 – 2.09 (m,
2H), 1.86 - 1.81 (m, 4H), 1.67-1.58 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm;
13C NMR (100 MHz, CDCl3) δ 176.16, 173.03, 168.65, 138.93, 132.80,
131.75, 131.20, 128.44, 128.42, 68.25, 68.21, 37.24, 36.19, 24.35, 18.34,
13.72 ppm; HRMS (ES+) calcd. for C18H24N2O4NaS (M+Na) 387.1354,
found 387.1358.
MB10: Method B, 129 mg, 25% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.53 – 7.42 (m, 2H), 7.33 (dd, J = 2.4, 8.4 Hz, 1H), 7.11 (t, J =
6.0 Hz, 1H), 6.10 (s, 1H), 5.83 (s, 1H), 5.34 (s, 2H), 3.68 (q, J = 6.0 Hz,
MB18: Method A, 31 mg, 12% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.61 (dd, J = 1.5, 7.7 Hz, 1H), 7.54 (ddd, J = 1.6, 7.6 Hz, 1H),
7.43 (td, J = 1.6, 7.4 Hz, 1H), 7.36 (td, J = 1.2, 7.5 Hz, 1H), 7.03 (s, 1H),
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
6.18 (s, 1H), 5.41 (s, 2H), 2.30 (t, J = 7.4 Hz, 2H), 2.25 (d, J = 14.3 Hz,
2H), 2.03 – 1.91 (m, 2H), 1.74 – 1.59 (m, 6H), 1.54 – 1.47 (m, 2H), 0.92 (t,
J = 7.4 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 176.54, 173.00, 168.49,
139.00, 132.66, 131.07, 128.30, 68.09, 61.18, 36.17, 32.30, 25.27, 21.68,
18.33, 13.71 ppm; HRMS (ES+) calcd. for C19H26N2O4SNa (M+Na)
401.1511, found 401.1515.
MB19: Method B; 6 mg, 13% yield; white solid; 1H NMR (400 MHz, CDCl3)
δ 7.51 (d, J = 8.6 Hz, 1H), 7.10 (d, J = 2.9 Hz, 1H), 6.95 (dd, J = 2.9, 8.6
Hz, 1H), 6.80 (s, 1H), 6.75 (s, 1H), 5.59 (s, 1H), 5.30 (s, 2H), 3.84 (s, 3H),
2.30 (t, J = 7.4 Hz, 2H), 1.67 (s, 6H), 1.66 – 1.56 (m, 2H), 0.91 (t, J = 7.4
Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 176.93, 173.11, 167.84,
160.42, 141.74, 136.92, 120.62, 117.35, 113.74, 69.41, 58.08, 55.79,
36.19, 25.59, 18.32, 13.74 ppm; HRMS (ES+) calcd. for C17H25N2O5S
(M+H) 369.1484, found 369.1487.
MB20: Method A, 65 mg, 51% yield; white solid; 1H NMR (400 MHz,
CDCl3) δ 7.63 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 2.6 Hz, 1H), 7.28 – 7.22 (m,
1H), 6.86 (s, 2H), 6.48 (s, 1H), 5.50 (s, 1H), 5.39 (s, 2H), 2.32 (t, J = 7.4
Hz, 2H), 1.70 (s, 6H), 1.61 (q, J = 7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm;
13C NMR (100 MHz, CDCl3) δ 176.36, 172.91, 166.32, 148.87, 140.52,
134.18, 131.32, 130.61, 123.08, 121.69, 120.94, 119.12, 115.83, 111.53,
67.89, 58.12, 36.14, 25.28, 18.32, 13.68 ppm; 19F NMR (376 MHz, CDCl3)
δ -57.83 ppm; HRMS (ES+) calcd. For C17H22N2O5F3S (M+H) 423.1202,
found 423.1201.
MB21: Method A; 25 mg, 6% yield; white solid; 1H NMR (400 MHz, DMSO-
d6) δ 8.73 (t, J = 5.9 Hz, 1H), 8.43 (dd, J = 1.3, 4.5 Hz, 1H), 8.04 (dd, J =
1.4, 8.3 Hz, 1H), 7.59 (dd, J = 4.5, 8.3 Hz, 1H), 7.38 (s, 1H), 6.87 (s, 1H),
5.53 (s, 2H), 3.45 – 3.41 (m, 2H), 2.36-2.29 (m, 4H), 1.52 (m, 2H), 0.84 (t,
J = 7.4 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 173.64, 172.83, 165.69,
144.49, 144.23, 136.31, 134.19, 126.20, 64.29, 36.22, 35.48, 35.35, 18.42,
13.71 ppm; HRMS (ES+) calcd. for C14H19N3O4NaS (M+Na) 348.0994,
found 348.0092.
General Procedure for Syntheses of Disulfides 7: An Erlenmeyer flask
was charged with 2-aminobenzoic acid 5 (11.5 mmol) and 25 mL water.
The mixture was cooled to ~ 0° C with an ice bath, and 4.5 mL of
concentrated hydrochloric acid was added. A blast shield was installed and
then a solution of sodium nitrite (1.61 g, 23.3 mmol) in 5 mL water was
carefully added drop wise (CAUTION: Diazonium salts are potentially
explosive; the use of a blast shield and appropriate personal protective
gear is required.) The reaction was stirred for 1 h and carefully maintained
at ~ 0° C. In the meantime, a solution of disodium disulfide was prepared
under a stream of N2 by mixing sodium sulfide nonahydrate (Na2S•9H2O)
(5.93 g, 24.6 mmol) with 12.5 mL water at 72° C. Elemental sulfur was
added (780 mg, 24.3 mmol) and the mixture was stirred at 72° C for 40
min. The resulting dark orange solution was cooled to ~ 0° C and treated
with a solution of sodium hydroxide (900 mg, 22.5 mmol) in 7.5 mL water.
The mix was stirred for an additional 5 min before being used in the next
step. The freshly prepared disodium disulfide (Na2S2) solution was poured
into a large Erlenmeyer flask maintained at ~ 0° C via an external ice water
bath. A handful of ice chips were added, and a blast shield placed in front
of the flask. Using the utmost caution, the cooled (0° C) diazonium solution
was carefully and slowly poured into the cooled (0° C) solution of Na2S2
and the reaction was allowed to stir at 0° to rt overnight. The reaction was
then acidified to ~ pH 1 with concentrated HCl, and the resulting precipitate
was filtered and washed thoroughly with cold water. The solids were then
transferred to an Erlenmeyer flask, combined with saturated aqueous
potassium carbonate and heated gently to produce a heterogeneous
mixture of dark brown liquor (basic to pH paper) and cream-colored solids.
The mixture was passed through a pad of celite, and the filtrate was
carefully acidified to ~ pH 1 with drops of concentrated HCl. The resulting
precipitate was isolated by filtration, washed with cold water and dried in
vacuo to give 7 of a crude tan solid that was carried forward to the next
step without further purification.
pH = ~ 1 with drops of concentrated HCl to give a pale yellow precipitate.
The precipitate was filtered, washed with ice cold water (5X) and dried on
a Büchner funnel overnight under nitrogen atmosphere to give 1 as a crude
yellowish white solid that was used immediately in the next step.
General Procedure for Syntheses of Thiols: Method B: To a solution
of disulfide 2 in 9:1 DMF/H2O (0.1 M) was added TCEP hydrochloride (1.5
equiv.) and Et3N (3 equiv.) at rt. After stirring for 18 h, the reaction was
concentrated in vacuo at 45 °C and treated with cold water. The mixture
was stirred vigorously for 2h and acidified (pH = ~ 1) with conc. HCl to give
yellow precipitate. The precipitate was filtered, washed with ice cold water
(5X) and dried on a Büchner funnel overnight under nitrogen atmosphere
to give 1 as a crude yellowish white solid that was used immediately in the
next step.
MB7: To a stirred solution of thiophenol SI-7 (54 mg, 0.206 mmol) in DMF
(0.25 mL) at rt was added triethylamine (57 µL, 0.309 mmol). After 3 min,
chloromethyl butyrate 3 (40 uL, 0.309 mmol) was added and the mixture
was stirred for 18 h. The reaction was quenched with 5 mL of water and
extracted with EtOAc (3 x 10 mL). The combined organic layers were
washed with brine, dried (Na2SO4) and concentrated in vacuo. The
resulting residue was purified by silica gel chromatography
(EtOAc/hexanes) to give MB7 (24 mg, 34% yield) as a light tan solid. 1H
NMR (400 MHz, DMSO-d6) δ 8.46 (t, J = 5.7 Hz, 1H), 7.61 (dd, J = 5.3, 8.7
Hz, 1H), 7.37 – 7.26 (m, 3H), 6.85 (s, 1H), 5.41 (s, 2H), 3.41-3.36 (m, 2H),
2.32 (td, J = 4.7, 7.4 Hz, 4H), 1.59-1.49 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H)
ppm; 13C NMR (100 MHz, DMSO-d6) δ 172.32, 172.23, 166.20, 161.96,
159.51, 140.42, 140.36, 132.69, 132.61, 128.56, 128.53, 117.24, 117.02,
114.98, 114.75, 66.99, 35.82, 35.31, 34.77, 17.80, 13.31 ppm; 19F NMR
(376 MHz, DMSO-d6) δ -114.71 ppm; HRMS calcd. for C15H19N2O4FNaS
(M+Na) 365.0947, found 365.0940.
Anti-HIV Cytoprotection Evaluation - Assay Methodology: Inhibition of
virus-induced cytopathic effects (CPE) and cell viability following HIV
replication in CEM-SS cells was measured by XTT tetrazolium dye. CEM-
SS cells (2.5 x 103 cells per well) were seeded in 96-well U-bottomed tissue
culture plates in RPMI medium supplemented with 10% FBS, 2 mM L-
glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin. Serially
diluted compounds (6 concentrations) and HIV-1RF diluted to a pre-
determined titer to yield 85 to 95% cell killing at 6 days post-infection were
be added to the plate. AZT was evaluated in parallel as a positive control.
Following incubation at 37°C, 5% CO2 for six days, cell viability was
measured by XTT staining. The optical density of the cell culture plate will
be determined spectrophotometrically at 450 and 650 nm using Softmax
Pro 4.6 software. Percent CPE reduction of the virus-infected wells and
the percent cell viability of uninfected drug control wells were calculated to
define the EC50, TC50 and therapeutic index (TI50) using Microsoft Excel
Xlfit4. Anti-HIV Evaluation in Human PBMCs – Assay Methodology:
PHA-P stimulated PBMCs from three donors were pooled together and
resuspended in fresh tissue culture medium 1x106 cells/mL and plated in
the interior wells of a 96 well round bottom microplate at 50 µL/well. A 100
µL volume of
9 concentrations of compound serially diluted were
transferred to the round-bottom 96 well plate containing the cells in
triplicate. 50 µL of HIV-1 at a pre-determined dilution was added. Each
plate contained cell control wells and virus control wells in parallel with
experimental wells. After 7 days in culture, efficacy was evaluated by
measuring the reverse transcriptase in the culture supernatants and the
cells were stained with the tetrazolium dye XTT to evaluate cytotoxicity.
General Method for Docking: Models of the different inhibitor compounds
were developed with the CHARMM software package.[25] The structure of
the C-terminal finger of NCp7 was obtained from the NMR-derived
ensemble determined by Lee et al. (PDB: 1MFS).[ 26 ] Images of the
“manually”-docked complexes were developed with the UCSF Chimera
software program.[27]
General Procedure for Syntheses of Thiols: Method A: Disulfide 2 was
suspended in MeOH (0.1 M) under N2 atmosphere at rt and sodium
borohydride (4 equiv.) was added in small portions over the course of 30
min. Then, the reaction mixture was stirred for 2-12 h and the yellow slurry
became a clear solution during the reaction. The reaction was carefully
quenched with drops of water and the solvents were removed under
reduced pressure. The residue was combined with water and acidified to
Acknowledgements
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
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Keywords: HIV • Nucleocapsid Protein 7 • Mercaptobenzamide
compositions
as
anti-HIV
and
anti-retroviral
agents.
• Prodrug • Antiviral
WO 2015/035003 A1, March 12, 2015.
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This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700141
ChemMedChem
COMMUNICATION
Entry for the Table of Contents
Insert graphic for Table of Contents here.
Prodrug
O
Steric Effect
O
S
O
R
R
NH2
N
H
O
R
Electronic Effect
Insert text for Table of Contents here.
Mercapotobenzamide derivatives could become the next generation of drugs to treat HIV infection by targeting and inactivating
nucleocapsid protein 7 (NCp7). This protein contains two zinc-coordinated knuckles that bind to HIV RNA and are essential for viral
replication. Certain mercaptobenzamide derivatives promote the acetylation of NCp7, loss of zinc coordination, and eliminate the
ability of NCp7 to bind HIV RNA. This manuscript describes how to quickly make mercaptobenzamide analogs to screen for antiviral
activity.
9
This article is protected by copyright. All rights reserved.