Preclinical Optimization of gp120 Entry Antagonists as anti-HIV-1 Agents with Improved Cytotoxicity and ADME Properties through Rational Design, Synthesis, and Antiviral Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.9b02149

We previously reported a milestone in the optimization of NBD-11021, an HIV-1 gp120 antagonist, by developing a new and novel analogue, NBD-14189 (Ref1), which showed antiviral activity against HIV-1_HXB2, with a half maximal inhibitory concentr

Full text of DOI:10.1021/acs.jmedchem.9b02149

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Article
Preclinical optimization of gp120 entry-antagonists as anti-
HIV-1 agents with improved cytotoxicity and ADME properties
through rational design, synthesis, and antiviral evaluation
Francesca Curreli, Shahad Ahmed, Sofia Mary Benedict Victor, Ildar Iusupov,
Pavel Markov, Alexander V. Kurkin, Andrea Altieri, and Asim Kumar Debnath
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02149 • Publication Date (Web): 07 Feb 2020
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Preclinical optimization of gp120 entry-antagonists as anti-HIV-1 agents
with improved cytotoxicity and ADME properties through rational design, synthesis,
and antiviral evaluation
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Francesca Currelia, Shahad Ahmeda, Sofia M. Benedict Victora, Ildar R. Iusupovb, Pavel O.
Markovb, Alexander V. Kurkinb, Andrea Altierib, Asim K. Debnath*a
aLaboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball Research Institute, New
York Blood Center, 310 E 67th Street, New York, NY 10065, USA
bEDASA Scientific, Scientific Park, Moscow State University, Leninskie Gory Bld. 75, 77-101b,
119992 Moscow, Russia
*Corresponding author
Keywords
HIV-1, ENV-pseudovirus, gp120 entry-antagonist, cytotoxicity, broad-spectrum, structure-
activity relationship (SAR), selectivity index (SI), ADME
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ABSTRACT
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We previously reported a milestone in the optimization of NBD-11021, an HIV-1 gp120
antagonist, by developing a new and novel analog, NBD-14189 (Ref1), which showed antiviral
activity against HIV-1HXB2, with an IC50 of 89 nM. However, cytotoxicity remained high, and the
ADME data showed relatively poor aqueous solubility. To optimize these properties, we
replaced the phenyl ring in the compound with a pyridine ring and synthesized a set of 48
novel compounds. One of the new analogs, NBD-14270 (8), showed a marked improvement
in cytotoxicity, with a 3-fold and 58-fold improvements in SI values compared with that of Ref1
and NBD-11021, respectively. Furthermore, the in vitro ADME data clearly showed
improvements in aqueous solubility and other properties compared with those for Ref1. The
data for 8 indicated that the pyridine scaffold is a good bioisostere for phenyl, allowing the
further optimization of this molecule.
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INTRODUCTION
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According to the Joint United Nations Programme on HIVAIDS (UNAIDS) report, published in
2018, 37 million people are living with HIV, and almost 2 million new infections are diagnosed
each year. Despite the availability of more than 30 Food and Drug Administration (FDA)-
approved drugs for acquired immunodeficiency syndrome (AIDS) treatment, AIDS remains a
major global public health concern; although, combination antiretroviral therapies (cART) can
help manage this devastating disease. However, no vaccine has yet been developed to
manage the transmission of the human immunodeficiency virus (HIV), and despite many years
of effort, no cure has been identified for HIV. Therefore, drug-based treatments remain the
only option for those who have been infected with the virus. Furthermore, HIV patients must
engage in life-long therapy regimens. Unfortunately, due to the very high heterogeneity among
HIV strains, drug-associated toxicities and difficulties with adherence often result in treatment
failure. Long-term use of HIV treatment drugs may also lead to the emergence of drug-
resistant HIV. As a result, treatment-experienced patients may exhaust their treatment
options. Therefore, the development of novel drugs, with fewer side effects and high antiviral
potencies against a large array of drug-resistant HIV-1, is urgently needed. To achieve this
goal, the continued discovery of new classes of drugs that target novel HIV components is
critical.
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HIV fusion and attachment (often collectively termed as “entry”) are the most critical
steps necessary for HIV to initiate its life cycle in the host cell1-4. Despite the importance of
these steps, only two FDA-approved drugs target the HIV-1 entry pathway: Fuzeone
(Enfuvirtide), which targets HIV-1 glycoprotein 41 (gp41), and Maraviroc, which targets C-C
chemokine receptor type 5 (CCR5) co-receptors. Thus far, no FDA-approved drugs target the
HIV-1 envelope glycoprotein gp120. However, the potential of gp120 as a drug target was
validated by reports describing several highly potent HIV-1 attachment inhibitors, from Bristol-
Meyer Squibs (BMS)5-13, our group, and others14-18. One of the BMS attachment inhibitors,
known as Fostemsavir (BMS-663068), is currently in an advanced stage of clinical
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development and demonstrated success in a recent Phase III clinical trial (Bright Study). Until
recently, the target site of this class of attachment inhibitors being developed by BMS was
uncertain. In 2017, Pancera et al. reported first the X-ray crystal structures of these inhibitors,
with a trimeric HIV-1 envelope19. Interestingly, these crystal structures showed that the binding
pocket for this class of inhibitors was distinct from the Phe43 cavity induced by the cluster of
differentiation 4 (CD4), the cellular receptor that binds to gp120. In contrast, Madani et al. first
suggested that NBD-556, which our group identified and reported in 200513, binds to the highly
conserved Phe43 cavity20. In 2012, we solved the X-ray crystal structure of NBD-556 bound
to gp120, confirming that NBD-556 binds to the Phe43 cavity11. Unfortunately, NBD-556 was
shown to be a CD4-agonist that could enhance HIV-1 infection in CD4-CCR5+ cells20. This
undesirable trait motivated us to use the insights gained from the crystal structure to modify
the structure of NBD-556, to convert this compound into a gp120 entry-antagonist. We were
successful and reported the first gp120 entry-antagonist, NBD-11021, in 201221.
Subsequently, a series of X-ray structures, showing gp120 entry-antagonists bound to
monomeric gp120, have confirmed that these antagonists also bind to the Phe43 cavity9, 11, 12,
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Recently, we reported the successful design of our most advanced gp120 antagonist,
NBD-14189 (Ref1, Figure 1), which was tested against a large panel of HIV-1 Env-
pseudotyped viruses, representing a diverse set of clinical isolates and multiple HIV
subtypes10. Ref1 showed remarkable antiviral potency (as low as 63 nM), indicating that this
compound has broad-spectrum inhibitory activity against HIV-110. Furthermore, this inhibitor
had ADME properties comparable to BMS-626529, a prodrug of which, BMS-663068, is
currently undergoing Phase III clinical trials10. However, the in vitro ADME and cellular toxicity
data confirmed that further improvements could be made to the cellular toxicity of Ref1, to
improve the selectivity index (SI = CC50/IC50) and to the ADME properties, especially, aqueous
solubility. Towards that goal, we replaced the phenyl group in Region I (Figure 1) with a
pyridine moiety, a unique aromatic ring. The use of pyridine as a bioisostere of aromatic rings
has been reported in the fields of medicinal chemistry and drug discovery to improve aqueous
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solubility, stability, and the ability to form H-bonds23-25. Here, we report the detailed synthesis,
structure-activity relationships (SARs), antiviral activities, and ADME properties of a large set
of pyridine-based analogs, with considerable improvement over the most potent gp120 entry-
antagonists previously reported by our group. This study explored a new scaffold in Region I
that resulted in many improved properties while still displaying potent antiviral activity.
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CHEMISTRY
The syntheses of the novel inhibitors are described in Schemes 1-7. First, we prepared a
series of 5-arylpyrrole-2-carboxylic acids with pyridine pharmacophoric fragment or its
bioisosteres - pyrimidine and pyridazine (Scheme 1). Acids S4 a-h were prepared using the
general scheme involving Suzuki coupling of pyridine/pyrimidine/pyridazine halogenides with
N-Boc-2-pyrrole boronic acid, followed by Boc cleavage, acylation, and semi-haloform
reaction26. Aryl bromides and aryl chlorides were commercially available. Acid S8 was
prepared using a general procedure A involving Suzuki coupling of 4-Bromo-2-fluoropyridine
and with N-Boc-2-pyrrole boronic acid, followed by Boc cleavage (MeOH-HCl) afforded the 2-
methoxy-4-(1H-pyrrol-2-yl)pyridine S6 in good yield (67%), which was then subjected first to
an acylation and then to a semi-haloform reaction8, 21, 27-29
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5-Aryl-3-methyl-1H-pyrrole-2-carboxylic acids were prepared using commercially
available reagents (2-bromo-5-(trifluoromethyl)pyridine or 2-bromo-5-chloropyridine) and
known compound N-Boc-β-iododehydroamino acid methyl esters R4 as per the reported
procedure29 (Scheme 3). This one-pot, two-step procedure occurs by a Sonogashira coupling
followed by a 5-endo-dig-cyclization, which involves the nitrogen atom of the dehydroamino
acid.
The Suzuki approach could not be used with the methylpyrrole intermediates S15 and
S16 due to chemical feasibility issues related to the synthesis of the methyl pyrrole boc-
protected derivative needed as a substrate. Except for S23, S32, and S37, all the required
intermediary protected secondary amines have been prepared using an enantioselective
pathway reported previously9. Instead, S23 and S32, due to the extended and different side
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chains, have been prepared accordingly to Schemes 4 and 5, respectively, but still exploiting
the enantioselective step as per the General Procedure E. While the synthetic approach for
S37 is depicted on Scheme 6, it was obtained as a racemate. Since the corresponding final
compound 31 showed no improvement in anti-HIV-1 activity, but rather a loss of activity,
therefore no attempts were wasted in the isolation of the corresponding enantiomers.
The synthesis of all final 48 pyridine analogs (1-48, Table 1) was performed as per
Scheme 7. The amines S38-S40 were prepared using a method described in our earlier work.
Similarly, amines S41-S43 and S44 and S45 were synthesized, starting from the appropriate
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thiazole derivative and following the protocols as reported earlier10, 28
r
All the alloc protected intermediates were worked-up as per the general procedure H10,
and the corresponding alloc products (1a-48a) were used directly in the next step without
characterization.
RESULTS AND DISCUSSION
Optimization strategy
In recent years, we have made significant progress converting NBD-556, the first-in-class
CD4-agonist, into gp120 entry-antagonists, such as NBD-11021, NBD-14010, and Ref1, while
simultaneously dramatically improving the anti-HIV potencies of these new molecules (Figure
1)9, 10, 21. Despite this success, we realized that our most potent inhibitor, Ref1, still requires
further improvements in cytotoxicity and SI values. Furthermore, the in vitro ADME study
indicated that Ref1 requires improved solubility10. Poor aqueous solubility can lead to slow
drug absorption, which may result in the poor bioavailability of drugs. Therefore, we reasoned
that improved aqueous solubility and reduced toxicity are urgently necessary for our best
inhibitors. However, the X-ray crystal structures of CD4 bound to gp120 and those of Phe43
cavity-targeted inhibitors bound to gp120 have confirmed that the hydrophobic cavity where
Phe43 binds is narrow9, 22; therefore, the structural manipulation of the phenyl ring in our best
inhibitors required careful consideration. We used a pyridine scaffold as a bioisostere,
replacing the phenyl ring, due to its aromatic characteristics and increased basicity compared
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with the phenyl ring, to improve aqueous solubility, minimize reactive metabolite formation,
and mitigate toxicity liability23-25. While this article was in preparation, Kobayakawa et al.
reported successful improvements in aqueous solubility and cytotoxicity by replacing the
phenyl ring of NBD-556 with a pyridine scaffold30.
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Anti-HIV-1 screening and structure-activity relationships (SARs)
Our previously reported data for Ref1, the best gp120 entry-antagonist that we have identified
to date, showed an anti-HIV-1 activity of 89 nM against HIV-1HXB2; however, Ref1 showed high
cytotoxicity, with a SI value of 24610. Furthermore, although the SI value was considered to be
reasonable, the cytotoxicity and ADME data indicated that further improvements in cytotoxicity
and ADME properties, especially aqueous solubility, were necessary to develop this molecule
into a preclinical candidate for further assessment. Until recently, our group and others have
avoided altering the phenyl group in Region I (Figure 1) because all of the X-ray crystal
structures of this class of compounds bound to HIV-1 gp120 have confirmed that this
hydrophobic ring was inserted deep inside the Phe43 cavity, which is surrounded by
hydrophobic residues. Pyridine was used by BMS as a substitute for the phenyl ring in the
inhibitor that prevents the attachment of HIV gp120 to CD4 host cells, which led to the
development of the potent clinical candidate BMS-48804324. BMS-488043 showed a better
pharmacokinetic profile, including improved solubility, and better metabolic profiles than
previously described inhibitors. To explore other alternative bioisosteres, we opted to utilize a
pyridine scaffold for replacing the phenyl ring (Figure 1).
We made a concerted effort to synthesize pyridine analogs for some of our most active
phenyl-containing inhibitors, produced 48 novel pyridine-based compounds, and determined
comprehensive SARs for these compounds. In general, we observed that cytotoxicity
improved considerably for these compounds compared with Ref1 and a structurally similar
analog, NBD-1413610 (Ref2, Figure 1), irrespective of the position of “N” within the pyridine
ring. The introduction of CH2OH or higher congeners at position R1 (15-34) improved
cytotoxicity, but antiviral activity was dependent on other substituents in Regions I, II, and III.
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For example, the introduction of the CH3 group at positions R4 and R5 in Region III generally
retained antiviral potency, but the toxicities of these compounds were higher (9-12, 23, and
24). An electron-withdrawing substituent at position R7 generally improved or maintained
antiviral potency; however, antiviral activity reduced considerably when an electron-donating
substituent, such as CH3, was introduced at position R7 (19 and 20).
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Although fluorine is an electron-withdrawing substituent and is hydrophobic, its
presence at R7 reduced antiviral activity, most likely due to its small size. When the “N” atom
in pyridine was at positions Y or W (see the structures in Table 1), the antiviral potencies of
these compounds dropped dramatically. Similarly, the presence of a bulkier group at position
R8 also had detrimental effects on antiviral activity, most likely due to steric limitations in the
narrow hydrophobic cavity.
Interestingly, the presence of a CH3 group at position R6 improved antiviral activity,
with no detrimental effects on toxicity. We also introduced a branched alcohol group
(CHOHCH2OH) at position R1, which resulted in the loss of antiviral activity (13, 14, and 29,
30), although the cytotoxicity of these compounds remained low (higher CC50 values).
Similarly, we introduced longer alcohol groups, such as (CH2)2OH (25, 26) and (CH2)3OH (27,
28) at R1, and observed no substantial reductions in antiviral activity; however, cytotoxicity
was somewhat higher for some of these compounds (25, 26 and 28).
The most notable improvements we observed were for NBD-14270 (8), with an IC50 of
0.16 µM and a CC50 of 109.3 µM. The IC50 of 8 reduced by approximately 2-fold compared
with Ref1, while the cytotoxicity (CC50) improved by approximately 5-fold. However, the activity
and cytotoxicity of 8 improved by 2-fold and 2.5-fold, respectively, compared with those for
Ref2. The SI value for Ref1 was 246, whereas the SI value for 8 reached 683, an almost 3-
fold improvement. This remarkable improvement in the cytotoxicity and SI values prompted
more in-depth assessments of the antiviral potency and ADME properties of this inhibitor.
Kobayakawa et al., in their recent report describing the replacement of the phenyl ring
with pyridine, also showed measurable improvements in cytotoxicity for their pyridine analogs
of NBD-55630. However, the closest analog of NBD-556 (5-chloro-2-substituted pyridine)
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showed an antiviral potency (IC50) reduction of approximately 358-fold, whereas the
cytotoxicity (CC50) improved by approximately 3-fold. Besides, some other reported analogs,
such as 2-chloro-4-substituted pyridine and 2-chloro-5-substituted pyridine, showed antiviral
potency reductions of 13-fold and 23-fold, respectively, whereas the cytotoxicity improvements
were also approximately 3-fold for these compounds30.
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The new generation of inhibitors showed entry antagonist traits
Our first-generation NBD-compound, NBD-556 13, was reported to facilitate HIV infection into
CD4-negative cells that expressed the coreceptor CCR5 by mimicking CD4 and inducing a
conformational change in gp120, promoting CCR5 binding and enhancing HIV-1 entry31, 32
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Subsequently, we verified that our new inhibitors did not possess this undesirable trait and
behaved as entry antagonists9, 10. In this study, we initially tested all 48 pyridine-containing
molecules in a single-cycle assay against HIV-1HXB2. We selected 8, which exhibited the best
anti-HIV-1 activity and a higher SI value (SI = 683), and NBD-14235 (33), which has a SI of
243 and close structural similarity to 8 to confirm that they are HIV-1 gp120 entry-antagonists.
CD4-negative and CCR5-positive Cf2TH-CCR5 cells were infected with the recombinant CD4-
dependant HIV-1ADA virus, in the presence of escalating concentrations of these inhibitors.
NBD-556 was used as a control. As shown in Figure 2, NBD-556 significantly enabled the
infection of the Cf2Th-CCR5 cells, whereas 8 and 33 did not, indicating that these compounds
maintained the HIV-1 entry antagonist property. Additionally, these compounds did not show
toxicity at the doses used in this assay. We used these two molecules for further antiviral
evaluation.
Antiviral activities of 8 and 33 against a large and diverse panel of HIV-1 Env-
pseudotyped clinical isolates.
We previously reported systematic improvements in the anti-HIV-1 activities of our gp120
entry-antagonists against a large panel of diverse HIV-1 Env-pseudotyped clinical isolates10.
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Some of our best inhibitors against HIV-1HXB2 showed broad-spectrum antiviral activities
against these clinical isolates9, 10, 21, 27. We selected 8 and 33 to evaluate the anti-HIV-1 activity
against a set of 50 HIV-1 clinical isolates belonging to diverse subtypes, including primary,
transmitted, and early founder HIV-1 isolates and a selection of 12 recombinant HIV-1 clones.
Except for two dual-tropic (CCR5/CXCR4) clinical isolates, all of the clones tested use the
CCR5 co-receptor for entry. We compared the antiviral activity of the new generation of
inhibitors with that of the previously described inhibitor Ref210, which has structural similarity
to 8. 33 exhibited anti-HIV-1 activity similar to that of Ref2, as demonstrated by their overall
mean IC50 values and their antiviral activities against the different HIV subtypes (Table 2). The
overall mean IC50 value for Ref2 was 0.39 ± 0.02 µM (IC50 values ranged from 0.19-0.79 µM),
whereas the overall mean IC50 value determined for 33 was 0.43 ± 0.02 µM (IC50 values ranged
from 0.13-0.99 µM). Ref2 showed better antiviral activities against subtype D viruses (mean
IC50 value of 0.26 ± 0.03 µM), whereas 33 showed better antiviral activities against subtype C
and D viruses (mean IC50 values of 0.37 ± 0.03 µM and 0.35 ± 0.07 µM, respectively). In
contrast, the respective SI values (CC50/IC50) determined for the panel of pseudoviruses were
higher for 33 than for Ref2, with 1.5–2.1-fold improvements, depending on the viral subtype.
Furthermore, this new generation compounds exhibited a better cytotoxicity profile (8: CC50 of
109.3 ± 2 µM and 33: CC50 of 85 ± 3 µM) than that for Ref2 (CC50 of 42.4 ± 1.0 µM). 8 showed
better SI values than both Ref2 and 33, as well as better anti-HIV-1 activities and a better
cytotoxicity profile. The overall mean IC50 value for 8 was 0.18 ± 0.008 µM (IC50 values ranged
from 0.11-0.33 µM). Except for subtype A (mean IC50 value of 0.22 ± 0.003 µM), 8 worked
equally well against all of the subtypes tested (mean IC50 value of 0.17-0.18 µM). The SI value
calculated using the overall mean IC50 value was 607.2, and depending on the viral subtype,
the SI values varied from 497 to 643, representing 4.9–5.6-fold improvements compared with
Ref2. Overall, 8 and 33 were active against all of the tested clinical isolates, regardless of the
viral subtypes (A-D), indicating that these compounds have broad-spectrum inhibitory activity,
unlike BMS-378806, an early stage attachment inhibitor, which despite its low nM potency
showed weaker activity against A, C and D subtypes33. Furthermore, 8 and 33, similar to earlier
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reports for Ref2, were poorly active against the pseudovirus VSV-G, which was used here as
a control, suggesting that the inhibitory activities of these compounds are specific to HIV-1.
Additionally, 8 and 33 did not induce toxicity in the U87-CD4-CXCR4 cell line at the doses
used for this assay.
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Moreover, we tested the anti-HIV-1 activity of 8 and 33 against an HIV-1 panel,
comprised of paired infant and maternal clones belonging to subtypes A and D/A. These HIV
clones were isolated from chronically infected mothers and their respective infected infants34.
Previous studies have shown that vertically transmitted HIV infant variants were more difficult
to neutralize using combinations of broadly neutralizing antibodies (bNAbs) 2G12, biz, 2F5,
and 4E1034. In this study, we found that Ref2, 8, and 33 equally neutralized both the infant
and maternal HIV-1 variants (Table 3), as shown by their overall mean IC50 values and the
means for infant and maternal viruses. 8, the most effective compound with an overall mean
IC50 value of 0.37 ± 0.07 µM, was slightly more efficient against the maternal clones (mean
IC50 value of 0.31 ± 0.04 µM) than against the infant clones (mean IC50 value of 0.43 ± 0.07
µM). These findings suggest that the new gp120 entry-antagonists can neutralize both infant
and maternal HIV-1 variants.
Inhibitory activity of 8 and 33 against a large panel of FDA-approved-drug-resistant
viruses
To further evaluate the neutralizing activities of 8 and 33, we assessed these compounds
against a large set of drug-resistant viruses, including 5 Enfuvirtide (T-20)-resistant viruses, 7
multi-drug, non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant viruses, which
carry mutations that confer resistance to both NNRTIs and nucleoside inhibitors (NRTIs), 3
Raltegravir-resistant viruses, and 9 protease inhibitor(PI)-resistant viruses (Table 4). As a
reference, the activities of the gp120 entry-antagonists were assessed against the
reconstructed wild type (WT) HIV-1NL4-3 clone, from which the drug-resistant clones were
obtained. For this assay, the WT HIV-1NL4-3 control virus or the drug-resistant viruses were
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pretreated with the gp120 entry-antagonists and then used to infect TZMb-l cells. We found
that 8 and 33 both inhibited WT HIV-1NL4-3, with IC50 values of 2.1 and 1.1 µM, respectively.
Moreover, both compounds neutralized the T-20-resistant viruses, with IC50 values similar to
the IC50 values determined for the control virus, WT HIV-1NL4-3. Similar results were observed
for the NNRTI-resistant viruses, and in some cases, the drug-resistant viruses appeared to be
more sensitive to the gp120 entry-antagonists than the WT virus. The Raltegravir-resistant
viruses and the PI-resistant viruses were also highly sensitive to 8 and 33, as shown by their
low IC50 values. For 33, the IC50 values detected against these clones were 1.8–10.5-fold lower
than the IC50 value detected for WT HIV-1NL4-3, and for 8, the IC50 values were 1.8–11.7-fold
lower than the IC50 value detected for WT HIV-1NL4-3. In conclusion, we found that 8 and 33
were active against all of the tested drug-resistant viruses, indicating that these compounds
could potentially be successfully used in combination with other antiviral agents.
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However, we are currently conducting experiments to identify the possible generation
of drug-resistant mutants in response to the use of the most potent gp120 entry-antagonists
examined in this study.
gp120 entry-antagonists inhibited cell-to-cell HIV-1 transmission
Another important feature described for the earlier generation of gp120 entry-antagonists was
their ability to inhibit cell-to-cell HIV-1 transmission10, which has been reported to be more
efficient than HIV-1 cell-free infection35, 36. Multiple viral particles can be transmitted to non-
infected cells simultaneously. Additionally, cell-to-cell HIV-1 transmission in vitro has been
shown to be resistant to some potent, bNAbs, including CD4 binding site (CD4bs) antibodies36-
38 and NRTIs, but not to other antiretrovirals, including entry inhibitors, NNRTIs, and protease
inhibitors39.
Here, we evaluated the activities of the new generation of gp120 entry-antagonists, 8
and 33, against cell-to-cell HIV-1 transmission and compared their activities to that of Ref2,
which has previously been reported to inhibit cell-to-cell HIV-1 transmission10 H9 cells,
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chronically infected with HIV-1IIIB (CXCR4-tropic), and MOLT-4 cells, chronically infected with
HIV-1ADA (CCR5-tropic), were used as donor cells, and TZM-bl cells were used as acceptor
cells. BMS-626529 was used as a control treatment drug. Our results (Table 5) indicated that
both 8 and 33 have similar activities to that for the previous generation compound, Ref2,
against cell-to-cell HIV transmission. All of the tested compounds, including BMS-626529,
showed better activities against the CXCR4-tropic virus HIV-1IIIB than against the CCR5-tropic
virus HIV-1ADA. In particular, the IC50 values for the gp120 entry-antagonists calculated against
HIV-1IIIB in the CXCR4-tropic assay were 1-fold lower than the IC50 values calculated against
HIV-1ADA in the CCR5-tropic assay.
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In vitro ADME assessment
The in vitro assessment of ADME properties has played an important role, especially for the
pharmaceutical industry, in reducing the drug attrition rate. In 1997, the major causes of failure
for drugs that advanced to clinical trials were poor ADME properties40. However, the judicious
use of ADME assessments during the early stages of drug development has dramatically
improved the failure rate of drugs during recent years41. Drug failures during the later stages
of drug development can be very costly. Therefore, the major goal of the pharmaceutical
industry with regards to drug development programs is to “fail early, fail cheaply41.” Therefore,
we also adopted in vitro ADME assessments in our early optimization phase of developing
these entry-antagonists as future clinical candidates. We previously reported the ADME
properties of our most active inhibitor, Ref1, which targets the Phe43 cavity of gp120, and
compared our results with the data obtained for BMS-626529, a prodrug of which is showing
promising results in Phase III clinical trials10. The ADME properties of Ref1 indicated room for
further improvements.
Since this is the first time we decide to use pyridine scaffold as aromatic ring in Region
I and this bioisostere was used before in medicinal chemistry to improve physicochemical and
ADME properties, it was also imperative for us to evaluate these properties by selecting one
of our most active inhibitors, 8, which has a better cytotoxicity profile and SI values than the
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other compounds examined in this study, and compared with Ref1 previously reported10. The
data presented in Table 6 shows that 8 (a pyridine analog) has approximately 3-fold improved
solubility compared with Ref1 (a phenyl analog) and BMS-626529, which was expected and
was the basis of the design strategy used in this study. Since these inhibitors are expected to
be used orally, we performed the Caco-2 bidirectional permeability experiment [apical to
basolateral (A-B) and basolateral to apical (B-A) across the Caco-2 cell monolayer], which can
be used to measure the efflux ratio and predict the human intestinal permeability of orally
administered drugs. The data shown in Table 6 indicates that the apparent permeabilities of
8 and the clinical candidate, BMS-626529, are similar. However, the permeability of Ref1 was
poor compared with both of these inhibitors. We used digoxin, a P-gp substrate, as a positive
control to identify whether active efflux was mediated by P-gp. It is apparent that the efflux of
8, Ref1 and digoxin was mediated by P-gp. However, we were unable to directly compare the
effects of P-gp inhibitors on permeability parameters because different P-gp inhibitors were
used in the current study (1 M valsopodar) and the previous study (100 M verapamil)10.
However, the data confirmed that all three compounds had efflux ratios greater than 2,
suggesting the potential involvement of an efflux transporter, which can mediate the transport
of these inhibitors from the basolateral side to the apical side. Interestingly, the efflux ratios
for all three cases were reduced in the presence of a P-gp inhibitor. For 8, BMS-626529, and
digoxin (positive control) the efflux ratios were reduced to less than 2, indicating that these
compounds could be P-gp substrates. However, for Ref1, the efflux ratio was not reduced to
less than 2, indicating the possible involvement of other transporters, such as breast cancer-
resistant protein (BCRP) or multi-drug resistance-associated protein 2 (MRP2).
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We also examined the metabolic stability of 8 in the human liver microsome because
the liver is the primary site of drug metabolism. The data in Table 6 shows that 8 has the
highest stability (93.5%) among the examined compounds, followed by Ref1, whereas BMS-
626529 was metabolized to some degree (71.5%) in our earlier study. The clearance data
(Clint) indicated that all three compounds are low-clearance compounds, and 8 had a half-life
longer than 120 minutes. We did not calculate half-life data in our earlier reported study. It is
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worthwhile to mention that compounds with high clearance values may not be considered
favorable because they may be cleared rapidly from the body, and the drugs may have a short
duration of action and may need multiple dosing. We also examined the protein-binding
potential of these inhibitors in human plasma. The data indicated that both 8 and Ref1 were
highly bound (>99%), whereas BMS-626529 had a binding potential of 86.9%.
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The oxidative biotransformation of many lipophilic drugs into hydrophilic counterparts
is critical, facilitating the elimination of drugs from the body. The cytochrome P450 enzyme
system plays a critical role in these oxidative biotransformation reactions associated with drug
metabolism. More than 50 CYP450 enzymes have been identified, but CYP1A2, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 metabolize almost 80
percent of all drugs. Therefore, we decided to use this set of eight CYP450 enzymes to
determine whether any of our potent inhibitors have inhibitory effects that may help predict
potential drug-drug interactions when co-administered with other treatment agents. Earlier, we
reported the inhibitory effects of Ref1 and BMS-626529, which showed no inhibitory activities
for doses as high as 25 µM. In this study, we escalated the tested doses to as high as 100
µM, and our most potent inhibitor, 8, showed no inhibition at > 50 µM dose levels against
CYP1A2, CYP2B6, and CYP3A. No inhibitory activities were observed against the other
CYP450 enzymes at the 100 µM dose level.
CONCLUSIONS
Here, we reported a significant shift in the design of gp120 antagonists, based on our
previously reported and optimized inhibitor, Ref1. The phenyl ring in this class of inhibitors has
been accepted to represent the critical moiety for antiviral potency because this ring is located
deep inside the narrow hydrophobic cavity, according to X-ray crystallography data. In this
study, we deployed pyridine, a bioisostere of phenyl, as a scaffold to replace the phenyl ring.
We synthesized 48 novel compounds and determined a comprehensive SAR to identify the
best possible gp120 entry-antagonists that display improved cytotoxicity and ADME
properties. We identified the two best inhibitors, 8 and 33, as determined by antiviral activity
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against HIV-1HXB2. These inhibitors were confirmed to be gp120 entry-antagonists and showed
broad-spectrum activity against a large panel of HIV-1 Env-pseudotyped viruses, representing
diverse subtypes. Although the antiviral potency of these two new inhibitors remained similar
to that for Ref1, the cytotoxicity of 8 was substantially improved, with an overall SI value of
607 against 50 diverse clinical isolates. This improvement was quite substantial compared to
the SI value for 33, which was 198. The ADME data for 8 also showed a considerable
improvement in aqueous solubility. All other ADME properties for 8 were comparable to those
for the clinical candidate BMS-626529, a prodrug of which is currently being tested in Phase
III clinical trials. Overall, the pyridine substitution of the phenyl ring has been the most effective
alteration in our quest to identify a clinically relevant gp120 antagonist. This finding is expected
to improve the antiviral potency, cytotoxicity, and ADME properties of this class of inhibitors
for subsequent preclinical studies.
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EXPERIMENTAL SECTION
Cells and viruses
TZM-bl cells 42, U87CD4+CXCR4+ cells 43, HIV-1 IIIB, infected H9 Cells 44, and MOLT-
4 CCR5+ Cells 45 were obtained through the NIH ARP. HEK 293T cells were purchased from
ATCC. CD4-negative Cf2Th-CCR5+ cells and Env expression vector pSVIIIenv-ADA were
kindly provided by Dr. J. G. Sodroski 46. HIV-1 Env molecular clone expression vector pHXB2-
env (X4) DNA was also obtained through the NIH ARP 47. HIV-1 Env molecular clones of
gp160 genes for HIV-1 Env pseudovirus production were obtained as follows: clones
representing the standard panels A, A/D, A2/D, D, and C (QB099.391M.Env.B1) were
obtained through the NIH ARP from Dr. J. Overbaugh 48, 49. The HIV-1 Env molecular clones
panel of subtype A/G, A/E, and G Env clones were obtained through the NIH ARP from Drs.
D. Ellenberger, B. Li, M. Callahan, and S. Butera 50. The HIV-1 Env panel of standard reference
subtype B Env clones was obtained through the NIH ARP from Drs. D. Montefiori, F. Gao and
M. Li (PVO, clone 4 (SVPB11) TRO, Clone 11 (SVPB12), QH0692, clone 42 (SVPB6),
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SC422661, clone B (SVPB8)); from Drs. B. H. Hahn and J. F. Salazar-Gonzalez (
5
6
pRHPA4259, clone 7 (SVPB14)); from Drs. B. H. Hahn and D. L. Kothe (pTHRO4156 clone
7
8
9
51,
18 (SVPB15), pCAAN5342 clone A2 (SVPB19))
pWEAUd15.410.5017, p1058_11.B11.1550,
52. The subtype B clones
p1006_11.C3.1601,
p1054.TC4.1499,
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p1056.TA11.1826 and p9021_14.B2.4571 were obtained through the NIH ARP from Drs. B.
H. Hahn, B. F. Keele, and G. M. Shaw 53. The subtype C HIV-1 reference panel of Env clones
was also obtained through the NIH ARP from Drs. D. Montefiori, F. Gao, S. A. Karim, and G.
Ramjee ( Du172.17); from Drs. D. Montefiori, F. Gao, C. Williamson, and S. A. Karim
(Du422.1), from Drs. B. H. Hahn, Y. Li and J. F. Salazar-Gonzalez (ZM197M.PB7;
ZM214M.PL15, ZM249M.PL1); from Drs. E. Hunter and C. Derdeyn (ZM53M.PB12;
ZM109F.PB4); from Drs. L. Morris, K. Mlisana, and D. Montefiori, (CAP210.2.00.E8) 54-56. The
HIV-1 Subtype C Panel of Indian gp160 Env Clones HIV-16055-2 clone 3, HIV-16936-2 clone
21, HIV-25711-2 clone 4 and HIV-225925-2 clone 22 were obtained through the NIH ARP
from Drs. R. Paranjape, S. Kulkarni and D. Montefiori 50. The panel of paired Infant and
maternal HIV-1 Env Molecular Clones were obtained through the NIH ARP from Dr. J.
Overbaugh 57. The Env pseudotyped genes of BG505.T332N, KNH1144, and B41 were kindly
provided by Dr. J. P. Moore of the Weil Cornell Medical College, NY.
The Env-deleted proviral backbone plasmids pNL4-3.Luc.R-.E-DNA (from Dr. N.
42, 52
Landau) 58, 59, the pSG3Δenv DNA (from Drs. J. C. Kappes and X. Wu)
and the pNL4-3
60
(from Dr. Malcolm Martin) were obtained through the NIH ARP Division of AIDS, NIAID,
NIH. The following molecular clones were also obtained through the NIH ARP, Division of
AIDS, NIAID, NIH: the panel of Enfuvirtide (T-20) resistant viruses from Trimeris, Inc. 60, 61; the
panel Multi-Drug Resistant NNRTI Infectious Clones from Dr. R. Shafer 62; the Raltegravir-
resistant infectious molecular clones from Dr. R. Shafer and E. Reuman, M.S 63 and the multi-
drug Protease inhibitor-resistant infectious clones from Dr. R. Shafer 64.
MLV gag-pol-expressing vector pVPack-GP, Env-expressing vector pVPack-VSV-G,
and a pFB-Luc vector were obtained from Stratagene (La Jolla, CA).
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Pseudovirus preparation
Pseudoviruses capable of single-cycle infection were prepared as previously described 27, 31
.
7
Briefly, 5 x 106 HEK293T cells were transfected with an HIV-1 Env-deleted pro-viral backbone
plasmid pSG3Δenv or pNL4-3.Luc.R-.E- DNA, and an HIV-1 Env-expression plasmid by using
FuGENE6 (Promega). The control VSV-G pseudovirus was prepared by transfecting the
HEK293T cells with a combination of the Env-expressing plasmid pVPack-VSV-G, the MLV
gag-pol-expressing plasmid pVPack-GP, the pFB-Luc plasmid, and FuGENE6. Pseudovirus-
containing supernatants were collected two days after transfection, filtered, tittered, and stored
in aliquots at −80 °C.
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Measurement of antiviral activity
Single-cycle infection assay in TZM-bl cells. The antiviral activity of the gp120 entry-
antagonists was evaluated in single-cycle infection assay by infecting TZM-bl cells with HIV-1
pseudotyped with the Env from the lab-adapted HIV-1HXB-2 (CXCR4-tropic). Additionally, Ref2,
33, and 8 were tested against a large group of HIV-1 pseudotyped with the Env from the panel
of clinical isolates as previously described 27, 31. Briefly, TZM-bl cells were plated at 1 x 104 /
well in a 96-well tissue culture plate and cultured overnight. On the following day, HIV-1
pseudovirus was pre-treated with graded concentrations of the small molecules for 30 min and
added to the cells. Following three days of incubation, the cells were washed and lysed with
50 µl of lysis buffer (Promega). 20 µl of the lysates were transferred to a white plate and mixed
with the luciferase assay reagent (Promega). The luciferase activity was measured
immediately with a Tecan Spark reader, and the percent inhibition by the compounds and the
IC50 (the half-maximal inhibitory concentration) values were calculated using the GraphPad
Prism software.
Single-cycle infection assay in U87-CD4-CXCR4 cells. The antiviral activity of Ref2, 33,
and 8 was tested against the control pseudovirus VSV-G in U87-CD4-CXCR4 cells. Briefly,
U87-CD4-CXCR4 cells were plated in a 96-well tissue culture plate at 1 x 104/well and cultured
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at 37 ºC. The following day, aliquots of pseudovirus pre-treated with graded concentrations of
the small molecules for 30 min, were added to the cells and incubated for three days. Cells
were washed and lysed with 40 µl of lysis buffer. The lysates were then transferred to a white
plate and mixed with the luciferase assay reagent. The luciferase activity was immediately
measured to calculate the percent of inhibition and IC50 values by using the GraphPad Prism
software.
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Assay in Cf2Th-CCR5 cells
CD4-negative Cf2Th-CCR5 cells were plated at 6 × 103 cells/well in a 96-well tissue culture
plate and incubated at 37 C. The cells were infected with the recombinant CD4-dependent
pseudovirus HIV-1ADA as previously described 18. Briefly, aliquots of HIV-1ADA pseudovirus pre-
treated with graded concentrations of compounds for 30 min were added to the cells and
cultured for 48 h. Cells were washed with PBS and lysed with 40 µl of cell lysis reagent.
Lysates were transferred to a white 96-well plate and mixed with the luciferase assay reagent.
The luciferase activity was immediately measured to obtain the relative infection compared to
the untreated control. The Relative virus infectivity indicates the ratio of the amount of infection
detected in the presence of the compounds and the amount of infection detected in the
absence of the compounds.
Measurement of antiviral activity against drug-resistant viruses in TZM-bl cells
The antiviral activity of the gp120 entry-antagonists against a panel of drug-resistant viruses
was evaluated by infecting TZM-bl cells. Briefly, TZM-bl cells were plated at 104/well in a 96
well plate and cultured overnight. On the following day, HIV-1 drug-resistant viruses were pre-
treated with graded concentrations of the small molecules for 30 min and added to the cells.
Following 48 h incubation, the cells were washed and lysed. The cellular lysates were
transferred to a white plate, and the luciferase activity was immediately measured with a Tecan
Spark reader. The percent inhibition by the compounds and the IC50 values were calculated
using the GraphPad Prism software, as reported above.
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The cell-to-cell HIV-1 transmission inhibition assay was performed as previously described 65,
66, with few modifications. Briefly, TZM-bl cells (used as acceptor cells) were plated at 104/well
in a 96 well plate 24 h before the assay. As transmitting cells, we used H9 cells chronically
infected with HIV-1IIIB at 2 × 103 cells/well for the CXCR4-tropic assay and MOLT-4/CCR5 cells
chronically infected with HIV-1ADA at 2 × 103 cells/well for the CCR5-tropic assay. The
transmitting cells were pre-treated with 200 μg/mL mitomycin C (Sigma) for 1 h at 37 °C,
washed with PBS, and incubated with the acceptor cells in the presence of escalating
concentrations of compounds for 24 h. Therefore, the cells were washed and lysed. The
lysates were mixed with the luciferase assay reagent. The luciferase activity was immediately
measured to calculate the percent of inhibition and IC50 values by using the GraphPad Prism
software.
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Evaluation of cytotoxicity
TZM-bl cells and U87-CD4-CXCR4 cells. The cytotoxicity of the gp120 entry-antagonists in
TZM-bl and U87-CD4-CXCR4 cells was measured by using the colorimetric CellTiter 96®
AQueous One Solution Cell Proliferation Assay (MTS) (Promega) following the manufacturer’s
instructions. Briefly, the cells were plated at 1 x 104 / well and cultured at 37 °C. Following
overnight incubation, the cells were incubated with 100 µl of the compounds at graded
concentrations and cultured for three days. The MTS reagent was added to the cells and
incubated for four h at 37 ºC. The absorbance was recorded at 490 nm. The percent of
cytotoxicity and the CC50 (the concentration for 50 % cytotoxicity) values were calculated as
above.
Cf2Th-CCR5 cells. The cytotoxicity of the small molecules in Cf2Th-CCR5 cells was also
measured with the colorimetric CellTiter 96® AQueous One Solution Cell Proliferation Assay.
Briefly, Cf2Th-CCR5 cells were plated in a 96-well plate and cultured at 37 °C overnight. Next,
the cells were incubated with 100 µl of the compounds at graded concentrations and cultured
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for 48 h. The MTS reagent was added to the cells, and 4 h later, the absorbance was recorded
at 490 nm. The percent of cytotoxicity and the CC50 values were calculated as above.
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In vitro ADME study
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Details of the In vitro ADME study and data analyses can be found in the “Supplemental
Information.”
Chemistry
General. We used commercial reagents and solvents without further purification. We also
performed all reactions in the air atmosphere unless otherwise stated. Reactions were
monitored by thin-layer chromatography (TLC) carried out on Merck TLC Silica gel plates (60
F254), using a UV light for visualization and basic aqueous potassium permanganate or iodine
fumes as a developing agent. 1H and 13C NMR spectra were recorded on Bruker Avance 400
instrument with operating frequency of 400 and 100 MHz, respectively, and calibrated using
residual undeuterated chloroform (δH = 7.28 ppm) and CDCl3 (δC = 77.16 ppm) or
undeuterated DMSO (δH = 2.50 ppm) and DMSO-d6 (δC = 39.51 ppm) as internal references.
The following abbreviations are used to set multiplicities: s = singlet, d = doublet, t = triplet, q
= quartet, m = multiplet, br = broad. The purity of the final compounds was checked by LCMS
in a Shimadzu LCMS-2010A using three types of detection systems such as EDAD, ELSD,
and UV and was found to be ≥95%.
General procedure A: for Suzuki coupling
To a solution containing appropriate bromide (or chloride) (50 mmol, 1 equiv), (1-(tert-
butoxycarbonyl)-1H-pyrrol-2-yl)boronic acid (50 mmol, 1 equiv) in THF-H2O (1:1, 100 mL),
Na2CO3 (100 mmol, 2 equiv) and Pd(Ph3P)Cl2 (1 mol. %) were added under a nitrogen
atmosphere. The mixture was stirred at reflux for 8-15 h (TLC-control). After cooling to the
room temperature, water (50 mL) and CH2Cl2 (50 mL) were added. The organic layer was
separated; the aqueous layer was extracted with CH2Cl2 (3x50 mL). The combined organic
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layers were dried over Na2SO4, filtered, and concentrated. Purification by flash
chromatography using the hexane-EtOAc mixture as eluent afforded the desired compound.
Compounds S1a-h were obtained following the general procedure A. Compounds S1g and
S1h were obtained from the corresponding chlorides.
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Tert-butyl 2-(5-methylpyridin-2-yl)-1H-pyrrole-1-carboxylate (S1a)
Eluent: Hex-EtOAc (from 10:1 to 5:1), Rf=0.2 (5:1, Hex-EtOAc). Yield = 63%.
1H NMR (CDCl3, 400 MHz): δ= 1.37 (s, 9 H), 2.34 (s, 3 H), 6.22 (t, J=3.3 Hz, 1 H), 6.36 (dd,
J=3.2, 1.7 Hz, 1 H), 7.28 (d, J=8.1 Hz, 1 H), 7.33 (dd, J=3.2, 1.8 Hz, 1 H), 7.48 (dd, J=7.9, 1.8
Hz, 1 H), 8.42 - 8.45 (m, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 18.3, 27.6 (3C), 83.5, 110.5, 115.3, 123.2, 123.2, 131.3,
134.2, 136.3, 149.2, 149.3, 150.2.
Tert-butyl 2-(5-chloropyridin-2-yl)-1H-pyrrole-1-carboxylate (S1b)
Eluent: Hex-EtOAc (from 20:1 to 10:1), Rf=0.3 (10:1, Hex-EtOAc). Yield = 84%.
1H NMR (CDCl3, 400 MHz): δ= 1.41 (s, 9 H), 6.25 (t, J=3.3 Hz, 1 H), 6.43 (dd, J=3.3, 1.7 Hz,
1 H), 7.34 - 7.38 (m, 2 H), 7.66 (dd, J=8.4, 2.5 Hz, 1 H), 8.57 (d, J=2.4 Hz, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 27.8 (3C), 84.1, 110.8, 116.3, 124.0, 124.3, 130.2, 133.1,
135.6, 147.8, 149.2, 151.0.
Tert-butyl 2-(5-fluoropyridin-2-yl)-1H-pyrrole-1-carboxylate (S1c)
Eluent: Hex-EtOAc (from 20:1 to 10:1), Rf=0.3 (10:1, Hex-EtOAc). Yield = 68%.
1H NMR (CDCl3, 400 MHz): δ= 1.39 (s, 9 H), 6.23 (t, J=3.3 Hz, 1 H), 6.39 (dd, J=3.3, 1.7 Hz,
1 H), 7.35 (dd, J=3.2, 1.7 Hz, 1 H), 7.39 (d, J=1.8 Hz, 1 H), 7.40 - 7.42 (m, 1 H), 8.47 (t, J=1.7
Hz, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 27.7 (3С), 83.9, 110.6, 115.8, 122.7 (d, J=18.6 Hz), 123.6,
124.6 (d, J=4.2 Hz), 133.1, 137.0 (d, J=23.8 Hz), 149.2 (d, J=1.5 Hz), 149.3, 158.4 (d, J=256.0
Hz).
Tert-butyl 2-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-1-carboxylate (S1d)
Eluent: Hex-EtOAc (from 30:1 to 20:1), Rf=0.3 (30:1, Hex-EtOAc). Yield = 65%.
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1H NMR (CDCl3, 400 MHz): 1.42 (s, 9 H), 6.28 (t, J=3.3 Hz, 1 H), 6.54 (dd, J=3.4, 1.7 Hz, 1
H), 7.40 (dd, J=3.2, 1.7 Hz, 1 H), 7.54 (d, J=8.2 Hz, 1 H), 7.92 (dd, J=8.3, 2.2 Hz, 1 H), 8.87
(s, 1 H).
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13C NMR (CDCl3, 100 MHz): δ= 27.6 (3C), 84.3, 110.9, 117.3, 122.8, 123.8 (q, J=272.0 Hz),
124.4 (q, J=33.0 Hz), 124.8, 132.9 (q, J=3.5 Hz), 133.0, 145.8 (q, J=4.1 Hz), 149.1, 156.0 (q,
J=1.5 Hz).
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Tert-butyl 2-(6-chloropyridin-3-yl)-1H-pyrrole-1-carboxylate (S1e)
Eluent: Hex-EtOAc (from 20:1 to 10:1), Rf=0.4 (10:1, Hex-EtOAc). Yield = 76%.
1H NMR (CDCl3, 400 MHz): δ= 1.44 (s, 9 H), 6.25 - 6.29 (m, 2 H), 7.33 (d, J=8.2 Hz, 1 H),
7.41 (t, J=2.5 Hz, 1 H), 7.66 (dd, J=8.2, 2.5 Hz, 1 H), 8.38 (d, J=2.3 Hz, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 27.8 (3C), 84.6, 111.1, 116.1, 123.1, 123.8, 129.3, 130.0,
139.4, 149.0, 149.5, 150.0.
Tert-butyl 2-(6-(trifluoromethyl)pyridin-3-yl)-1H-pyrrole-1-carboxylate (S1f)
Eluent: Hex-EtOAc (from 30:1 to 20:1), Rf=0.5 (30:1, Hex-EtOAc). Yield = 84%.
1H NMR (CDCl3, 400 MHz): δ= 1.43 (s, 9 H), 6.30 (t, J=3.3 Hz, 1 H), 6.32 - 6.35 (m, 1 H), 7.45
(dd, J=3.2, 1.8 Hz, 1 H), 7.68 (d, J=8.1 Hz, 1 H), 7.86 (dd, J=8.1, 1.8 Hz, 1 H), 8.72 (d, J=1.7
Hz, 1 H).
13C NMR (CDCl3, 400 MHz): δ= 27.7 (3C), 84.8, 111.3, 116.8, 119.5 (q, J=2.8 Hz), 121.8 (q,
J=273.9 Hz), 124.3, 130.0, 133.2, 137.3, 146.3 (q, J=34.8 Hz), 148.9, 150.0.
Tert-butyl 2-(6-(trifluoromethyl)pyridazin-3-yl)-1H-pyrrole-1-carboxylate (S1g)
Eluent: Hex-EtOAc (from 20:1 to 5:1), Rf=0.4 (5:1, Hex-EtOAc). Yield = 61%.
1H NMR (CDCl3, 400 MHz): δ= 1.45 (s, 9 H), 6.35 (t, J=3.4 Hz, 1 H), 6.73 (dd, J=3.4, 1.6 Hz,
1 H), 7.48 (dd, J=3.2, 1.7 Hz, 1 H), 7.75 - 7.79 (m, 2 H).
13C NMR (CDCl3, 400 MHz): δ= 27.8 (3C), 85.1, 111.6, 119.2, 121.7 (q, J=274.2 Hz), 122.8
(q, J=2.4 Hz), 125.8, 127.7, 129.9, 148.9, 149.5 (q, J=35.0 Hz), 157.2.
Tert-butyl 2-(5-chloropyrimidin-2-yl)-1H-pyrrole-1-carboxylate (S1h)
Eluent: Hex-EtOAc (from 30:1 to 20:1), Rf=0.5 (30:1, Hex-EtOAc). Yield = 58%..
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1H NMR (CDCl3, 400 MHz): δ= 1.44 (s, 9 H), 6.26 (t, J=3.3 Hz, 1 H), 6.78 (dd, J=3.4, 1.7 Hz,
1 H), 7.35 (dd, J=3.1, 1.7 Hz, 1 H), 8.67 (s, 2 H).
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13C NMR (CDCl3, 400 MHz): δ= 27.8 (3C), 84.3, 110.9, 118.8, 125.7, 128.4, 131.8, 149.2,
155.2 (2C), 158.8.
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General procedure B: for Boc-deprotection
To a solution containing Boc-protected compound (30 mmol) in MeOH (15 mL, 2M solution),
1M HCl solution in MeOH (45 mL) was added in a one portion. The mixture was stirred at
reflux for 7-8 h. After cooling to the room temperature, solvent was evaporated. Then 10%
aqueous K2CO3 (50 mL) was added carefully (CO2 evolution) and mixture was extracted with
CH2Cl2 (3x50 mL). The combined organic layers were dried over Na2SO4, filtered, and
concentrated. Crude product was used in the next step without purification. Compound S2a-
h were obtained following the general procedure B.
5-Methyl-2-(1H-pyrrol-2-yl)pyridine (S2a)
Yield= 94 %.
1H NMR (CDCl3, 400 MHz): δ= 2.33 (s, 3 H), 6.31 - 6.35 (m, 1 H), 6.72 - 6.75 (m, 1 H), 6.89 -
6.91 (m, 1 H), 7.48 (dd, J=8.2, 1.9 Hz, 1 H), 7.53 (d, J=8.1 Hz, 1 H), 8.27 - 8.38 (m, 1 H), 10.73
(br. s., 1 H).
13C NMR (CDCl3, 100 MHz): δ= 18.2, 106.8, 109.9, 118.1, 119.9, 129.9, 131.8, 137.4, 148.5,
148.9.
5-Chloro-2-(1H-pyrrol-2-yl)pyridine (S2b)
Yield= 92 %.
1H NMR (CDCl3, 400 MHz): δ= 6.31 - 6.34 (m, 1 H), 6.71 - 6.74 (m, 1 H), 6.92 (td, J=2.6, 1.4
Hz, 1 H), 7.50 (d, J=8.6 Hz, 1 H), 7.60 (dd, J=8.6, 2.4 Hz, 1 H), 8.42 (d, J=2.2 Hz, 1 H), 9.90
(br. s., 1 H).
13C NMR (CDCl3, 100 MHz): δ= 107.9, 110.6, 118.9, 120.5, 128.3, 130.7, 136.4, 147.7, 148.9.
5-Fluoro-2-(1H-pyrrol-2-yl)pyridine (S2c)
Yield= 93 %.
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1H NMR (CDCl3, 400 MHz): δ= 6.33 (dd, J=6.2, 2.7 Hz, 1 H), 6.66 - 6.71 (m, 1 H), 6.88 - 6.94
(m, 1 H), 7.38 (td, J=8.5, 2.8 Hz, 1 H), 7.56 (dd, J=8.8, 4.3 Hz, 1 H), 8.35 (d, J=2.8 Hz, 1 H),
9.96 (br. s., 1 H).
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13C NMR (CDCl3, 100 MHz): δ= 107.1, 110.3, 119.1 (d, J=4.1 Hz), 120.2, 123.9 (d, J=19.3
Hz), 130.9, 136.7 (d, J=23.9 Hz), 147.4 (d, J=3.2 Hz), 157.8 (d, J=253.2 Hz).
2-(1H-Pyrrol-2-yl)-5-(trifluoromethyl)pyridine (S2d)
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Eluent: Hex-EtOAc (from 20:1 to 10:1), Rf=0.5 (10:1, Hex-EtOAc). Yield = 88%.
1H NMR (CDCl3, 400 MHz): δ= 6.32 - 6.37 (m, 1 H), 6.80 - 6.86 (m, 1 H), 6.94 - 7.03 (m, 1 H),
7.61 (d, J=8.4 Hz, 1 H), 7.83 (dd, J=8.4, 2.1 Hz, 1 H), 8.71 (s, 1 H), 9.74 (br. s., 1 H).
13C NMR (CDCl3, 100 MHz): δ= 109.6, 111.0, 117.6, 121.6 122.9 (q, J=33.0 Hz), 124.0 (q,
J=271.6 Hz), 130.5, 133.7 (q, J=3.5 Hz), 146.1 (q, J=4.4 Hz), 153.5 (q, J=1.5 Hz).
2-Chloro-5-(1H-pyrrol-2-yl)pyridine (S2e)
Yield = 93%.
1H NMR (CDCl3, 400 MHz): δ= 6.33 - 6.37 (m, 1 H), 6.57 - 6.60 (m, 1 H), 6.94 - 6.98 (m, 1 H),
7.32 (d, J=8.4 Hz, 1 H), 7.74 (dd, J=8.3, 2.6 Hz, 1 H), 8.52 (d, J=2.4 Hz, 1 H), 8.84 (br. s., 1
H).
13C NMR (CDCl3, 100 MHz): δ= 107.9, 110.6, 120.8, 124.5, 127.4, 128.2, 134.2, 144.6.
5-(1H-Pyrrol-2-yl)-2-(trifluoromethyl)pyridine (S2f)
Yield = 92%
1H NMR (CDCl3, 400 MHz): δ= 6.29 - 6.41 (m, 1 H), 6.64 - 6.74 (m, 1 H), 6.92 - 7.04 (m, 1 H),
7.63 (d, J=8.2 Hz, 1 H), 7.89 (dd, J=8.2, 1.8 Hz, 1 H), 8.82 (d, J=1.8 Hz, 1 H), 9.32 (br. s., 1
H).
13C NMR (CDCl3, 400 MHz): δ= 109.2, 111.0, 120.9 (q, J=2.9 Hz), 121.8, 121.8 (q, J=273.7
Hz), 127.2, 131.5, 131.7, 144.7 (q, J=35.1 Hz), 144.9.
3-(1H-Pyrrol-2-yl)-6-(trifluoromethyl)pyridazine (S2g)
Yield = 95%
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1H NMR (DMSO-d6, 400 MHz): δ= 6.25 - 6.29 (m, 1 H), 7.07 - 7.14 (m, 2 H), 8.10 (d, J=9.0
Hz, 1 H), 8.20 (d, J=9.0 Hz, 1 H), 12.15 (br. s., 1 H).
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13C NMR (DMSO-d6, 100 MHz): δ= 110.5, 112.7, 122.0 (q, J=273.5 Hz), 122.9, 124.2, 124.8
(q, J=2.2 Hz), 126.9, 147.3 (q, J=33.7 Hz), 154.8.
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5-Chloro-2-(1H-pyrrol-2-yl)pyrimidine (S2h)
Yield = 90%.
1H NMR (DMSO-d6, 400 MHz): δ= 6.20 (s, 1 H), 6.93 (d, J=1.8 Hz, 1 H), 6.97 (s, 1 H), 8.78
(s, 2 H), 11.77 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 110.1, 112.3, 123.2, 125.7, 129.4, 155.6 (2C), 157.1.
General procedure C: for acylation
Crude pyrrole from the previous step (1 equiv) was dissolved in CH2Cl2 (0.5 M solution), and
pyridine (1.2 equiv) was added, followed by dropwise addition of TFAA (1.2 equiv). After
completion of the addition, the mixture was stirred for 1 h, and the solvent was evaporated.
The product was triturated in aqueous, and the precipitate was filtered, washed with aqueous
twice, and dried on a filter. Compound S3a-h were obtained following the general procedure
C.
2,2,2-Trifluoro-1-(5-(5-methylpyridin-2-yl)-1H-pyrrol-2-yl)ethanone (S3a)
Yield = 89%.
1H NMR (DMSO-d6, 400 MHz): δ= 2.34 (s, 3 H), 7.05 (d, J=4.2 Hz, 1 H), 7.28 (dd, J=3.9, 1.9
Hz, 1 H), 7.75 (dd, J=8.0, 1.4 Hz, 1 H), 8.09 (d, J=8.1 Hz, 1 H), 8.49 - 8.52 (m, 1 H), 12.87 (br.
s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 17.8, 111.8, 117.0 (q, J=290.3 Hz), 120.6, 122.9 (q, J=3.5
Hz), 126.1, 133.4, 137.8, 142.6, 145.3, 149.8, 168.1 (q, J=35.0 Hz).
1-(5-(5-Chloropyridin-2-yl)-1H-pyrrol-2-yl)-2,2,2-trifluoroethanone (S3b)
Yield = 94%.
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1H NMR (DMSO-d6, 400 MHz): δ= 7.09 (dd, 1 H), 7.29 (dt, J=4.1, 2.0 Hz, 1 H), 8.07 (dd, J=8.6,
2.5 Hz, 1 H), 8.23 (d, J=8.6 Hz, 1 H), 8.69 (d, J=2.4 Hz, 1 H), 13.04 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 112.5, 117.0 (q, J=290.25 Hz), 122.0, 122.7 (q, J=3.5 Hz),
126.6, 130.7, 137.0, 141.5, 146.7, 148.3, 168.5 (q, J=35.0 Hz).
2,2,2-Trifluoro-1-(5-(5-fluoropyridin-2-yl)-1H-pyrrol-2-yl)ethanone (S3c)
Yield = 91%.
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1H NMR (DMSO-d6, 400 MHz): δ= 7.04 (dd, J=4.1, 2.3 Hz, 1 H), 7.28 (dt, J=4.1, 2.0 Hz, 1 H),
7.87 (td, J=8.8, 2.9 Hz, 1 H), 8.27 (dd, J=8.9, 4.4 Hz, 1 H), 8.65 (d, J=2.8 Hz, 1 H), 12.96 (br.
s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 112.0, 117.0 (q, J=289.9 Hz), 122.4 (d, J=4.8 Hz), 122.7
(q, J=3.5 Hz), 124.1 (d, J=19.0 Hz), 126.4, 138.0 (d, J=24.5 Hz), 141.8, 144.9 (d, J=3.9 Hz),
158.7 (d, J=256.2 Hz), 168.3 (q, J=35.0 Hz).
2,2,2-Trifluoro-1-(5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrol-2-yl)ethanone (S3d)
Yield = 92%.
1H NMR (DMSO-d6, 400 MHz): δ= 7.19 (dd, J=4.0, 2.2 Hz, 1 H), 7.27 - 7.32 (m, 1 H), 8.33
(dd, J=8.4, 1.7 Hz, 1 H), 8.40 (d, J=8.3 Hz, 1 H), 8.99 (s, 1 H), 13.19 (br. s., 1 H)
13C NMR (DMSO-d6, 100 MHz): δ= 113.7, 117.2 (q, J=289.8 Hz), 120.9, 122.7 (q, J=1.5 Hz),
124.0 (q, J=271.5 Hz), 124.5 (q, J=32.2 Hz), 127.5, 135.1, 141.2, 146.7, 152.0, 169.1 (q,
J=34.4 Hz).
1-(5-(6-Chloropyridin-3-yl)-1H-pyrrol-2-yl)-2,2,2-trifluoroethanone (S3e)
Yield = 87%.
1H NMR (DMSO-d6, 400 MHz): δ= 7.08 (dd, J=4.2, 2.4 Hz, 1 H), 7.33 (dt, J=4.1, 2.0 Hz, 1 H),
7.64 (d, J=8.4 Hz, 1 H), 8.43 (dd, J=8.4, 2.6 Hz, 1 H), 9.02 (d, J=2.4 Hz, 1 H), 13.14 (br. s., 1
H).
13C NMR (DMSO-d6, 100 MHz): δ= 111.9, 117.1 (q, J=289.9 Hz), 123.2 (q, J=3.3 Hz), 124.5,
125.5, 126.8, 137.0, 139.1, 147.6, 150.2, 168.3 (q, J=35.0 Hz).
2,2,2-Trifluoro-1-(5-(6-(trifluoromethyl)pyridin-3-yl)-1H-pyrrol-2-yl)ethanone (S3f)
Yield = 93%.
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1H NMR (DMSO-d6, 400 MHz): δ= 7.14 (d, J=4.3 Hz, 1 H), 7.26 - 7.33 (m, 1 H), 7.96 (d, J=8.3
Hz, 1 H), 8.61 (dd, J=8.2, 1.8 Hz, 1 H), 9.32 (d, J=1.7 Hz, 1 H), 13.24 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 112.6, 116.9 (q, J=289.8 Hz), 120.8 (q, J=2.2 Hz), 121.6
(q, J=273.7 Hz), 122.9 (q, J=3.7 Hz), 127.2, 129.1, 135.1, 138.4,145.7 (q, J=34.4 Hz), 147.7,
168.5 (q, J=35.1 Hz).
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2,2,2-Trifluoro-1-(5-(6-(trifluoromethyl)pyridazin-3-yl)-1H-pyrrol-2-yl)ethanone (S3g)
Yield = 85%.
1H NMR (DMSO-d6, 400 MHz): δ= 7.34 - 7.39 (m, 2 H), 8.39 (d, J=9.0 Hz, 1 H), 8.70 (d, J=9.0
Hz, 1 H), 13.50 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 114.1, 116.7 (q, J=289.8 Hz), 121.6 (q, J=274.0 Hz), 122.3
(q, J=3.1 Hz), 125.4 (q, J=2.0 Hz), 125.5, 128.0, 137.8, 149.2 (q, J=34.1 Hz), 153.8, 169.1 (q,
J=35.4 Hz).
1-(5-(5-Chloropyrimidin-2-yl)-1H-pyrrol-2-yl)-2,2,2-trifluoroethanone (S3h)
Yield = 86%.
1H NMR (DMSO-d6, 400 MHz): δ= 7.10 (d, J=3.9 Hz, 1 H), 7.24 (s, 1 H), 8.97 (s, 2 H), 13.07
(br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 114.2, 116.7 (q, J=289.8 Hz), 122.2 (q, J=2.9 Hz), 127.2,
129.2, 139.6, 155.3, 156.2 (2C), 168.7 (q, J=35.1 Hz).
General procedure D: for haloform reaction
Appropriate trifluoroethanone (1 equiv) was added to a solution of NaOH (5 equiv) in dioxane-
H2O mixture (1:1, 0.5M solution). The resulting reaction mixture was refluxed for 20 h and
cooled to the room temperature. A concentrated aqueous HCl solution (~12 M, 5 equiv) was
added dropwise. The resulting precipitate was filtered off, washed with H2O and dried on filter.
Compound S4a-h were obtained following the general procedure D.
5-(5-Methylpyridin-2-yl)-1H-pyrrole-2-carboxylic acid (S4a)
Yield = 82%.
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Journal of Medicinal Chemistry
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1H NMR (DMSO-d6, 400 MHz): δ= 2.28 (s, 3 H), 6.77 - 6.84 (m, 2 H), 7.61 (dd, J=8.1, 2.1 Hz,
1 H), 7.88 (d, J=8.1 Hz, 1 H), 8.38 (d, J=1.8 Hz, 1 H), 11.69 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 17.9, 109.2, 116.5, 119.1, 124.8, 131.6, 136.4, 137.6,
147.1, 149.7, 162.0.
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5-(5-Chloropyridin-2-yl)-1H-pyrrole-2-carboxylic acid (S4b)
Yield = 73%.
1H NMR (DMSO-d6, 400 MHz): δ= 6.78 - 6.93 (m, 2 H), 7.94 (dd, J=8.6, 2.4 Hz, 1 H), 8.06 (d,
J=8.6 Hz, 1 H), 8.58 (d, J=2.2 Hz, 1 H), 11.96 (br. s., 1 H), 12.57 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 110.3, 116.2, 120.3, 125.8, 128.8, 135.0, 136.8, 147.9,
148.2, 161.8.
5-(5-Fluoropyridin-2-yl)-1H-pyrrole-2-carboxylic acid (S4c)
Yield = 85%.
1H NMR (DMSO-d6, 400 MHz): δ= 6.80 - 6.85 (m, 2 H), 7.74 (td, J=8.8, 2.9 Hz, 1 H), 8.08 (dd,
J=8.8, 4.3 Hz, 1 H), 8.53 (d, J=2.8 Hz, 1 H), 11.86 (br. s., 1 H), 12.52 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 109.7, 116.3, 120.6 (d, J=4.4 Hz), 124.1 (d, J=18.8 Hz),
125.1, 135.4, 137.4 (d, J=24.0 Hz), 146.4 (d, J=3.7 Hz), 158.0 (d, J=253.4 Hz), 161.8.
5-(5-(Trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxylic acid S4d)
Yield = 80%.
1H NMR (DMSO-d6, 400 MHz): δ= 6.76 - 6.95 (m, 1 H), 6.95 - 7.08 (m, 1 H), 8.12 - 8.31 (m, 2
H), 8.89 (s, 1 H), 12.18 (br. s., 1 H), 12.71 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 111.7, 116.3, 119.0, 122.7 (q, J=32.2 Hz), 124.0 (q,
J=272.2 Hz), 126.7, 134.4 (q, J=3.7 Hz), 134.8, 146.2 (q, J=4.4 Hz), 153.1, 161.8.
5-(6-Chloropyridin-3-yl)-1H-pyrrole-2-carboxylic acid (S4e)
Yield = 91%.
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