A one-pot preparation of N-2-mercaptobenzoyl-amino amides

Article abstract of DOI:10.1016/j.tetlet.2011.05.115

The HIV-1 nucleocapsid (NCp7), structurally defined by zinc-binding domains, participates in crucial stages of the HIV-1 lifecycle and is mutationally nonpermissive, making it an attractive anti-HIV target. Mode of action studies have shown that the secondary structure and activity of NCp7 can be disrupted by acyl transfer from N-2-mercaptobenzoyl-amino amides. We have developed an improved one-pot reaction that affords N-2-mercaptobenzoyl-amino acids on multi-gram scales. This synthetic route allows for rapid modular construction and has greatly expanded the scope of easily accessible potential NCp7 inhibitors.

Full text of DOI:10.1016/j.tetlet.2011.05.115

Tetrahedron Letters 52 (2011) 4103–4105
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Tetrahedron Letters
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A one-pot preparation of N-2-mercaptobenzoyl-amino amides
⇑
Robert J. Bahde, Daniel H. Appella, William C. Trenkle
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The HIV-1 nucleocapsid (NCp7), structurally defined by zinc-binding domains, participates in crucial
stages of the HIV-1 lifecycle and is mutationally nonpermissive, making it an attractive anti-HIV target.
Mode of action studies have shown that the secondary structure and activity of NCp7 can be disrupted by
acyl transfer from N-2-mercaptobenzoyl-amino amides. We have developed an improved one-pot reac-
tion that affords N-2-mercaptobenzoyl-amino acids on multi-gram scales. This synthetic route allows for
rapid modular construction and has greatly expanded the scope of easily accessible potential NCp7
inhibitors.
Received 13 April 2011
Revised 20 May 2011
Accepted 24 May 2011
Available online 12 June 2011
Keywords:
HBTU
Amide formation
Thioester
Published by Elsevier Ltd.
Topical microbicide
Ettore Appella and co-workers have previously reported a class
of antiviral agents that attack the highly conserved retroviral
nucleocapsid protein, NCp7, as an alternative to combinational
therapies for combating the hyper-mutable HIV.1–4 Nucleocapsid
NCp7 is a short protein (72 amino acid) which is structurally de-
fined by two zinc-binding domains and participates in crucial
stages of the HIV-1 lifecycle. Since its activity is mutationally non-
permissive, disruption of NCp7 is a target for discovery of topical
anti-HIV microbicides, which should not suffer from the develop-
ment of resistance to treatment.5,6 Mode of action studies have
demonstrated that acyl transfer from N-2-mercaptobenzoyl-amino
amides (SAMT, general structure 1, Fig. 1) can affect one of the zinc
finger motifs, disrupt the secondary structure, and result in loss of
activity of NCp7.4 Shattock and co-workers demonstrated that sus-
pensions of SAMT 2 provided protection in a study using rhesus
macaque vaginal challenge models with mixed R5 and X4 sim-
ian-HIV infection as well as preventing transmission of HIV-1 in
cell-based antiviral assays.7 Development of these anti-HIV com-
pounds was hindered by the laborious original synthesis of SAMT
analogues, which required seven overall steps.3,8 In order to pre-
pare larger quantities to facilitate formulation, cell-to-cell trans-
mission assays, and additional biological studies, an improved
shorter route was desired. Herein, we wish to report a one-pot
preparation of N-2-mercaptobenzoyl-amino amides, which per-
mits the facile generation of molecular diversity and provides high
purity material in multi-gram quantities. The new synthetic strat-
egy will allow us to advance SAMT molecules as anti-HIV-1 topical
microbicides and further elucidate their biological activity.
Analysis of the SAMT structure offers two convergent synthetic
routes (Scheme 1), which lead to the common, commercially avail-
able, starting material of thiosalicylic acid (3). Both synthetic strat-
egies would eliminate unnecessary protecting group manipulation,
which would save resources and facilitate large-scale preparation.
Initially, we examined each route in a stepwise fashion to deter-
mine if we could perform a two-step preparation of the desired
SAMT structures, which would be a dramatic improvement over
the previously reported route. It was unclear at the outset whether
it would be more advantageous to perform the amide coupling of
salicylic acid followed by thiol acylation or the reverse. We exam-
ined both routes during the course of our studies.
Initially, studies examined amide formation using thiosalicylic
acid (3) with an unprotected thiol to prepare SAMTs without resort-
ing to protecting group manipulation (route a, Scheme 1). Thiosal-
icyclic acid and the hydrochloride salt of amino amides were easily
coupled to form amides using HBTU and DIEA in DMF (Scheme 2).9
Unfortunately, there was substantial side reaction during aqueous
work up which afforded significant amounts of disulfide 8. The for-
mation of disulfides proved problematic during all attempts to iso-
late and purify the free thiol 7 with varying amounts of disulfide
developing over time. Disulfides with similar functionality were
originally reported by researchers at Warner–Lambert to have
activity as inhibitors of HIV nucleocapsid protein zinc fingers, but
were an undesired byproduct in this case.10–14 Liebeskind and co-
workers have coupled alkyl- and aryl-amines to make disulfides
analogous to 8 then cleaved the disulfide with sodium borohydride
to access the reactive thiols (general structure 4, R = -Me, -Et, -t-Bu,
-aryl, -morpholino).15,16 Unfortunately, the free thiols of our amino-
amides (general structure 7) were exceptionally prone to disulfide
formation even under anaerobic conditions and this method was
unsuccessful in our system for the preparation of pure thiols and
⇑
Corresponding author.
E-mail address: wtrenkle@niddk.nih.gov (W.C. Trenkle).
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2011.05.115

4104
R. J. Bahde et al. / Tetrahedron Letters 52 (2011) 4103–4105
Figure 1. Structures of N-2-mercaptobenzoyl-amino amides.
Scheme 3. Amide coupling with acylated substrates.
aryl-boronic acids.15,16 Future studies will clarify the ability of
these thioesters to act as activated esters for amide coupling.
The successful solution to our synthetic challenge was the rec-
ognition that the amide formation with free thiosalicylic acid pro-
ceeds cleanly, but that the free thiol needed to be capped prior to
deleterious disulfide formation. This synthetic plan was realized
using a two-step one-pot reaction of amide coupling followed by
thiol acylation to minimize disulfide by-products and avoid thioes-
ter acyl transfer (Scheme 4). The amide coupling was carried out at
room temperature using thiosalicyclic acid (3), b-alanine amide
hydrochloride salt, HBTU, and i-Pr2EtN in DMF. After completion
of the amide formation, an activated acid was added directly to
the reaction mixture to acylate the intermediate thiol 7, which pro-
vided the desired thioester 5 in modest to excellent yield after
aqueous workup and recrystallization (Scheme 4).21 Careful opti-
mization of reaction work-up for thioester 5 revealed a facile isola-
tion by precipitation and recrystallization. These conditions were
utilized to produce 5 on multi-gram scale (17 g) in high yield
(70%) without the need for column purification.
The one-pot preparation allows the facile variation of the amine
and the acid chloride. To demonstrate the flexibility of our route, we
prepared two brief series of SAMT analogues by varying the amine
or the thiol acylating agents. The first series maintained the amine
coupling partner with a variety of activated esters (Scheme 5). Var-
ious aroyl acid chlorides were effective in the preparation of SAMTs.
Alkyne 18 was prepared by trapping of the thiol with a mixture of 4-
pentynoic acid and additional amide coupling agent (Scheme 5,
(iii)).22 Combination of the free aliphatic carboxylic acid and HBTU
forms the activated ester in situ to trap the thiol intermediate albeit
under longer reaction times (Scheme 5). Introduction of other
commercially available carboxylic acids would allow preparation
of a diversity of analogues.
Scheme 1. Potential synthetic routes.
subsequent thioesters. Although we were unable to avoid the for-
mation of disulfide, we could drive disulfide formation to comple-
tion by modification of the workup conditions.17
A facile
precipitation and filtration provided pure disulfide 8, which is spar-
sely soluble in most organic and aqueous solutions.
Alternatively, reversal of reaction order, with selective acylation
of the thiol followed by amide coupling, could provide the desired
SAMTs (route b, Scheme 1). Acylation of the free thiol was readily
accomplished in high yield to afford thioesters 9 and 11.18–20 How-
ever, the subsequent amide coupling attempts with acylated 9
were unsuccessful (Scheme 3). Curiously, the unexpected thioke-
tene acetal 10 was isolated as a byproduct under standard condi-
tions for amide coupling of 9. This byproduct is the result of
enolization and intramolecular trapping of acyl thioester furnish-
ing thioketene acetal 10, which is stable to silica gel chromatogra-
phy (Scheme 3). Since benzoyl 2-mercaptobenzoic acid (11) lacks
enolizable protons, it was envisioned that the benzoyl would not
interfere with amide coupling. However, application of the stan-
dard coupling conditions to thioester 11 and b-alanine methyl es-
ter induced benzoyl transfer to produce N-benzoyl amide 12. The
scope of this reaction was briefly probed with thioester 11 and
benzylamine, which afforded amide 13 in good yield (Scheme 3).
The benzoyl transfer proceeds in both the presence and absence
of coupling reagent (EDC), albeit in slightly reduced yield when
EDC is omitted (Scheme 3). Thioesters 9 and 11 offered interesting
yet undesirable reactivity in the context of our studies, although
the facile benzoyl transfer hints at the potential of these reagents
in amide formation. The reactivity of a similar system has been
exploited by Liebeskind and co-workers for copper catalyzed
carbon–carbon bond formation between the thioester and
The second series of analogues was prepared with variation of
the amine component while maintaining the acid chloride used
Scheme 2. Initial amide coupling results. Reagents and conditions: (a) HBTU, i-
Pr2EtN, b-ALA-NH2ÁHCl, DMF, rt, 12 h, 70%.
Scheme 4. Optimized one-pot coupling. Reagents and conditions: (i) HBTU, i-
Pr2EtN, b-ALA-NH2ÁHCl, DMF, rt; (ii) (MeO)3Bz-Cl, 70%.

R. J. Bahde et al. / Tetrahedron Letters 52 (2011) 4103–4105
4105
References and notes
1. Goel, A.; Mazur, S. J.; Fattah, R. J.; Hartman, T. L.; Turpin, J. A.; Huang, M.; Rice,
W. G.; Appella, E.; Inman, J. K. Bioorg. Med. Chem. Lett. 2002, 12, 767–770.
2. Song, Y.; Goel, A.; Basrur, V.; Roberts, P. E. A.; Mikovits, J. A.; Inman, J. K.;
Turpin, J. A.; Rice, W. G.; Appella, E. Bioorg. Med. Chem. 2002, 10, 1263–1273.
3. Srivastava, P.; Schito, M.; Fattah, R. J.; Hara, T.; Hartman, T.; Buckheit, R. W.;
Turpin, J. A.; Inman, J. K.; Appella, E. Bioorg. Med. Chem. 2004, 12, 6437–6450.
4. Miller Jenkins, L.; Hara, T.; Durell, S. R.; Hayashi, R.; Inman, J. K.; Piquemal, J. P.;
Gresh, N.; Appella, E. J. Am. Chem. Soc. 2007, 129, 11067–11078.
5. Dorfman, T.; Luban, J.; Goff, S. P.; Haseltine, W. A.; Gottlinger, H. G. J. Virol. 1993,
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6. (a) Housset, V.; De Rocquigny, H.; Roques, B. P.; Darlix, J. L. J. Virol. 1993, 67,
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Hayashi, R.; Neurath, A. R.; Appella, E.; Shattock, R. J. J. Virol. 2009, 83, 9175–
9182.
Scheme 5. Variation of the ester coupling partner. Reagents and conditions: (i)
HBTU, i-Pr2EtN, b-ALA-NH2ÁHCl, DMF, rt; (ii) acid chloride (2 equiv), 2 h; (iii)
pentynoic acid, HBTU, 12 h.
8. The reported preparation is seven total steps with a longest linear sequence of
four steps.3 This route initiated from the disulfide dimer of 3; preparation and
isolation of the bis-N-hydroxysuccimide ester; coupling and isolation of the
diamide (such as 8, Scheme 2); disulfide cleavage; and final thioester
preparation plus a three step preparation of the free base of the aminoamide.
9. HBTU: O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate.
10. Rice, W. G.; Schaeffer, C. A.; Harten, B.; Villinger, F.; South, T. L.; Summers, M.
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Mendeleyev, J.; Kun, E. Nature 1993, 361, 473–475.
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Graham, L.; Schaeffer, C. A.; Turpin, J. A.; Domagala, J.; Gogliotti, R.; Bader, J. P.;
Halliday, S. M.; Lori, C.; Sowder, R. C.; Arthur, L. O.; Henderson, L. E. Science
1995, 270, 1194–1197.
12. Domagala, J. M.; Bader, J. P.; Gogliotti, R. D.; Sanchez, J. P.; Stier, M. A.; Song, Y.;
Vara Prasad, J. V. N.; Tummino, P. J.; Scholten, J.; Harvey, P.; Holler, T.;
Gracheck, S.; Hupe, D.; Rice, W. G.; Schultz, R. Bioorg. Med. Chem. 1997, 5, 569–
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13. Loo, J. A.; Holler, T. P.; Sanchez, J.; Goliotti, R.; Maloney, L.; Reily, M. D. J. Med.
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14. Miller Jenkins, L. M.; Ott, D. E.; Hayashi, R.; Coren, L. V.; Wang, D.; Xu, Q.;
Schito, M. L.; Inman, J. K.; Appella, D. H.; Appella, E. Nat. Chem. Biol. 2010, 6,
887–889.
15. Villalobos, J. M.; Srogl, J.; Liebeskind, L. S. J. Am. Chem. Soc. 2007, 129, 15734–
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Scheme 6. Variation of the amine coupling partner. Reagents and conditions: (i)
HBTU, i-Pr2EtN, AA-NH2ÁHCl, DMF, rt; (ii) (MeO)3Bz-Cl.
16. Liebeskind, L. S.; Yang, H.; Li, H. Angew. Chem., Int. Ed. 2009, 48, 1417–1421.
17. After dilution with EtOAc, CH2Cl2, and water, the reaction mixture was
submitted to vigorous stirring under air for 1 h. This exposure led to the
complete conversion of free thiol intermediate 7 into crystalline disulfide 8,
which precipitated from the mixture.
18. Taiyoung, S. J.; Russell, M. P.; Howard, J. Eur. Pat. Appl. EP 54936, 1982.
19. Jung, J.-C.; Kim, J.-C.; Park, O.-S. Synth. Commun. 2000, 30, 1193–1203.
20. Wurster, J. A.; Yee, R. C. Indol Kinase Inhibitors. U.S. Patent 7,439,371 B2,
October 21, 2008.
to cap the thiol (Scheme 6). These analogues were successfully pre-
pared using the standard condition and demonstrated that the
route tolerates steric congestion and
a-branching of the amine
component. The reactions proceeded uneventfully and provided
the desired analogues after purification.
The flexibility and efficiency of the synthetic route allows the
rapid preparation of new and unexplored SAMT analogues
(Schemes 5 and 6).23 Key aspects of this route, which allow facile
access to molecular diversity, include modularity, simple building
blocks, and lack of protecting group manipulation in a one-pot
reaction. Our new route and expanded library of SAMT structures
will be used to probe the pharmacology and structure–activity
relationships of the SAMTs as anti-HIV compounds. We will report
on the biological activity of these analogues in due course. This bio-
logical data will further our understanding of the acyl transfer and
aid in the design of a new generation of therapeutic molecules,
which are resistant to viral mutation.
21. Some reactions were purified by passing through a plug of silica gel prior to
recrystallization to remove HOBT esters. Complete experimental details are
reported in the Supplementary data.
22. One can envision a sequence of initial addition of multiple equivalents of HBTU
to the amine and 3, followed by subsequent addition of carboxylic acid to react
with the excess HBTU. In practice, we found that excess tetramethyluronium
coupling agent will react with the primary amine to provide the corresponding
tetramethylguanidine which was difficult to remove without silica gel
chromatography. This deleterious reactivity necessitates the sequential
addition of HBTU with the free acid after the initial amide formation is
complete.
23. Representative procedure for one-pot coupling. S-(2-((3-amino-3-
oxopropyl)carbamoyl)phenyl)3,4,5-trimethoxybenzothioate (5). Diisopropylethy-
lamine (36.0 mL, 204 mmol) was added to 2-mercaptobenzoic acid (9.00 g,
58.4 mmol), HBTU (23.2 g, 61.3 mmol), and b-alanine amide hydrochloride
(7.63 g, 61.3 mmol) in DMF (42.0 mL). The mixture slowly became
homogenous orange solution with stirring and maintained at rt for 12 h.
Addition of 3,4,5-trimethoxybenzoyl chloride (27.6 g, 120 mmol) gave
a
Acknowledgments
a
This research was supported in part by the Intramural Research
Program of the NIH, National Institute of Diabetes and Digestive
and Kidney Diseases. We thank Ettore Appella, and Lisa M. Miller
Jenkins (NIH, NCI) and Hans F. Luecke and Dongwook Kang (NIH,
NIDDK) for helpful discussion. We thank Noel Whittaker for able
assistance with Mass Spectral Analyses.
suspension. Rapid dissolution of the suspended material provided an orange
solution, which was followed by precipitation of an off white solid over the
course of 2 h. The mixture was diluted with CH2Cl2 (100 mL) and H2O (100 mL)
resulting in a biphasic mixture of two clear layers. The aqueous layer was
extracted with CH2Cl2 (4 Â 200 mL). The organic layers were combined, dried
(MgSO4), and concentrated under reduced pressure to furnish an orange oil. The
residue was redissolved in EtOAc (300 mL) and the solution was washed with
aqueous NaHCO3 (0.5 M, 100 mL) to remove trace DMF. The EtOAc layer was
chilled to 4 °C and the product crystallized to afford a white solid, S-2-(3-amino-
3-oxopropylcarbamoyl)phenyl 3,4,5-trimethoxybenzothioate (5) (17.0 g,
Supplementary data
40.6 mmol, 70% yield): mp 183 °C; 1H NMR (400 MHz, DMSO)
d 8.36 (t,
J = 5.4 Hz, 1H), 7.67–7.45 (m, 4H), 7.39–7.28 (m, 1H), 7.21 (s, 2H), 6.81 (s, 1H),
3.87 (s, 6H), 3.77 (s, 3H), 3.40–3.33 (m, 2H), 2.30 (t, J = 7.3 Hz, 2H); 13C NMR
(100 MHz, DMSO) d 187.8, 172.3, 167.0, 153.0, 142.4, 141.7, 136.6, 131.3, 129.8,
129.5, 128.1, 124.7, 104.4, 60.2, 56.1, 35.8, 34.8; IR (neat) 3421, 3338, 3244, 1677,
1662, 1633, 1584, 1414 cmÀ1; HRMS (ESI) m/z calcd for C20H23N2O6S [M+H]
419.1277, found 419.1267.
Supplementary data (general experimental methods and proce-
dures for the preparation of 5, 8, 11, 13, 15–18, 20–22, detailed char-
acterization data, and copies of the spectral data) associated with
this article can be found, in the online version, at doi:10.1016/
j.tetlet.2011.05.115.