Synthesis of non-nucleoside anti-viral cyclopropylcarboxacyl hydrazones and initial anti-HSV-1 structure-activity relationship studies

Article abstract of DOI:10.1016/j.bmcl.2020.127559

The synthesis of a lead anti-viral cyclopropyl carboxy acyl hydrazone 4F17 (5) and three sequential arrays of structural analogues along with the initial assessment and optimization of the antiviral pharmacophore against the herpes simplex virus type 1 (HSV-1) are reported.

Full text of DOI:10.1016/j.bmcl.2020.127559

Bioorganic&MedicinalChemistryLetters30(2020)127559
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of non-nucleoside anti-viral cyclopropylcarboxacyl hydrazones
and initial anti-HSV-1 structure-activity relationship studies
⁎
James McNultya, , Chanti Babu Dokuburraa, Leonardo D'Aiutob, Matthew Demersb,
⁎
Lora McClainb, Paolo Piazzab, Kelly Williamsonb, Wenxiao Zhengb, Vishwajit L. Nimgaonkarb,
a Department of Chemistry & Chemical Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada
b Department of Psychiatry, WPIC, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cyclopropanes
Hydrazones
Anti-viral
The synthesis of a lead anti-viral cyclopropyl carboxy acyl hydrazone 4F17 (5) and three sequential arrays of
structural analogues along with the initial assessment and optimization of the antiviral pharmacophore against
the herpes simplex virus type 1 (HSV-1) are reported.
HSV-1
Herpesvirus
The discovery of novel anti-viral agents targeting herpesviruses such
as herpes simplex virus (HSV-1), varicella zoster virus (VZV) and
human cytomegalovirus (HCMV) is of increasing concern given their
prevalence (global infection rates 60–95%) and the severity of effects
on the central nervous system.1 Current treatment options are limited to
nucleoside analogues acyclovir (ACV) 1 and the pro-drug valacyclovir
2.2 Both molecules cause serious side effects, including nephrotoxicity,3
and drug-resistant viral strains continue to be reported.4 These factors
have provided a strong impetus for the discovery of molecules that
exhibit alternative modes of action. We recently investigated the anti-
viral activity of a collection non-nucleoside library-array compounds
resulting in the discovery of several lead molecules that demonstrated
broad-spectrum inhibition of human herpesviruses.5a Screening this
collection of compounds5b revealed that the three derivatives 30 N12
(3), 16F19 (4) and 4F17 (5) (Fig. 1) demonstrated broad-spectrum
activity against human herpesviruses.6 While all three compounds de-
monstrated potent anti-viral activity to HSV-1 and varicella zoster virus
(VZV), 4F17 (5) was distinguished by demonstrating low cytotoxicity in
the respective neuronal or ARPE-19 host cells.
modulating local pH.7 Structurally similar acylhydrazones 6 have been
shown to inhibit Dengue viral entry through similar pH modulation of
endosomes,7 while another set of analogues 7, developed as alternatives
to the lysomotropic agent chloroquine, have been shown to possess
antimalarial activity.8 Lastly, NMR-investigation of the library com-
pound 4F17 was complicated by the presence of two sets of overlapping
signals indicative of either decomposition or the existence of rotamers.
In view of the valuable biological activity and chemical issue described,
compound 4F17 became a high priority target in our anti-viral drug
discovery program.9 In this communication, we report the synthesis,
and confirmation of the structure and stability of 4F17 (5) as the ro-
tamers 5 and 8. The synthesis and optimization of anti-viral activity
through three consecutive rounds of structure–activity (HSV-1) in-
vestigations is described.
The synthesis of 4F17 (5) is outlined in Scheme 1. Cyclopropanation
of commercially available 1,1,-diphenylethylene 9 with ethyl diazoa-
cetate 10 was initially attempted under heterogeneous conditions with
copper (II) salts10 with limited success. In order to develop an in-
expensive initially racemic version of the synthesis, we prepared a
homogeneous copper (II) complex from racemic N-tosyltryptophan 18,
reacting with copper sulfate monohydrate 19.
Most importantly, compound 5 was shown to significantly inhibit
reactivation of quiescent HSV-1 infection in induced pluripotent stem
cell (iPSC)-derived neurons, conferring a privileged status to this mo-
lecule toward the discovery of selective anti-viral therapeutics that
target viral reactivation. While acyclovir 1 and valacyclovir 2 target
viral replication, the mechanism of action of the non-nucleoside com-
pound 4F17 is uncertain at present. The compound may act as a lyso-
motropic agent inhibiting viral packaging and maturation through
This method is based upon earlier unpublished observations from
our group and results in the formation of a complex that is highly so-
luble in organic media, tentatively described as 20. This homogeneous
complex proved extremely efficient and the cyclopropane derivative 11
was isolated in good yield. Reaction with hydrazine hydrate 12 in re-
fluxing ethanol provided the acylhydrazide 13 that was condensed with
⁎ Corresponding authors.
E-mail addresses: jmcnult@mcmaster.ca (J. McNulty), vishwajitNL@upmc.edu (V.L. Nimgaonkar).
https://doi.org/10.1016/j.bmcl.2020.127559
Received 14 July 2020; Received in revised form 12 September 2020; Accepted 13 September 2020
Availableonline19September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

J. McNulty, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127559
Fig. 1. Structure of current approved anti-herpesvirus drugs acyclovir 1, vala-
cyclovir 2 and novel non-nucleoside hits 3–5, anti-Dengue 6 and antimalarial 7
acylhydrazones.
Fig. 2. Anti-viral assessment of the initial set of analogues 21–31 in HSV-1
infected iPSC neurons.
compounds, such as the indole derivative 25 and the highly substituted
trimethoxy-derivative 27 proved more active. Nonetheless, the con-
clusion derived from the SAR on the initial array was that no derivative
exhibited potency close to 4F17, indicating the requirement of a small,
electron-rich heterocycle on the hydrazone portion of the molecule.
These results with the array of aryl derivatives in comparison to the
furano-substituted parent compound 5, focussed our attention on the
preparation of a second generation of analogues containing small,
electron-rich heterocyclic rings. This second-generation compound
array was readily accessed from the penultimate hydrazide 13 con-
densing with a range of commercially available heterocyclic aldehydes,
as shown in Fig. 3.
Scheme 1. Synthesis of library target 4F17 (5) proceeding via homogeneous
copper (II) catalysed cyclopropanation with 20, hydrazinolysis and hydrazone
condensation with furfural.
furfural 14 under acid catalysis to give synthetic 4F17, compound 5.
Analysis of synthetic 5 by NMR showed the pure molecule to exist as a
3:1 mixture of rotamers 5:8, identical to that observed for the com-
mercial library product 4F17, thus confirming the structure and stabi-
lity and validating the initial anti-viral activity described for this
compound. Synthetic 5 also exhibited anti-HSV1 activity indis-
tinguishable from the library compound 4F17.
This second-generation array compounds 32–38, was also screened
in the same assay as described above and the overall results presented
in Fig. 3. The 2-thiofurano analogue 32 showed substantial activity,
similar to the positive control acyclovir. To our delight, the corre-
sponding 2-pyrrolo analogue 33 proved highly potent. Interestingly, the
two constitutionally isomeric imidazoles 34 and 35 proved significantly
less active, indicating that a basic nitrogen atom is detrimental, perhaps
due to solubility and/or basicity of the side chain. In order to probe
basicity further, we prepared the corresponding 2-pyridyl analogue 36
which proved highly potent, and the isomeric 3-pyridyl derivative 37
which was less active. The bromofurano analogue of 5, compound 38,
proved less active than 5. Overall, the second-generation structure-an-
tiviral assessment allowed identification of the 2-pyrrolo- and 2-pyr-
idinyl- substituted compounds 33 and 36 as being significantly and
slightly more potent that acyclovir and the parent 2-furano derivative 5
itself.
The synthetic route developed toward 5 outlined in Scheme 1 was
designed to allow the preparation of multiple analogues for structure-
antiviral activity investigations following our late-stage (last step) di-
versity-oriented approach that has proven successful elsewhere.11 The
first array of hydrazone derivatives was readily accessed from the pe-
nultimate intermediate 13 through condensation with a range of alde-
hydes and ketones to generate the acyl hydrazones 21–31 shown in
Fig. 2. The anti-HSV-1 activity of these analogues was investigated in
iPSC-neurons following the previously developed protocol.5 The assay
read-out is based on a recombinant HSV-1 strain derived from the KOS
virus expressing the enhanced green fluorescent protein (EGFP) from
the early ICP0 promoter and a monomeric red fluorescent protein (RFP)
from the glycoprotein C promoter.6 Uninfected cells as well as those
treated with the recombinant HSV-1 in the presence of the positive
control acyclovir (ACV) or synthetic 4F17 exhibited a low proportion of
fluorescent cells, while those infected with HSV-1 alone or any of the
initial compound screening set 21–31, demonstrated relatively low
inhibition of viral infection, with compounds 25, 27 and 29–31
showing slight inhibition.6
These results set the stage for the preparation of the third generation
of compounds that would allow probing the contribution of the 1,1-
diarylmethano fragment to the anti-viral activity. Strategically, the
synthetic approach followed the original synthetic protocol, allowing
access to arrays through late-stage incorporation of 2-furanyl, 2-pyridyl
and 2-pyrrolyl substituents. The synthesis of the required substituted
1,1-diarylethylenes required is shown in Scheme 2. The reaction of a 4-
substituted benzaldehyde 39 or 40 with the corresponding 4-
Overall, the structure–activity correlation of the variously sub-
stituted benzaldehyde derived hydrazones disclosed no linear electronic
effect, however, on the whole, electron poor analogues were seen to be
somewhat less active (e.g. 23, 28). Conversely, electron rich
2

J. McNulty, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127559
Scheme 3. Synthesis of the substituted hydrazides 54-56 and the three furano
hydrazides 57–59. The reaction of 54–56 with 2-pyrrolocarboxaldehyde and
separately 2-pyridylcarbox-aldehyde similarly yielded array compounds 60–65.
Fig. 3. Second generation heterocyclic hydrazones 32–38 containing modified
heterocycles and their anti-viral assessment.
Fig. 4. Third generation array compounds 57–65 containing modified 1,1-
Scheme 2. Reparation of 1,1 diaryl substituted ethylenes.
diarylmethano-substituents and their anti-viral activity.
substituted Grignard reagent 41 and 42 yielded the diarylmethanols 43
and 44. These were oxidised to the corresponding benzophenone de-
rivatives 45 and 46, along with the commercially available 1,1-bis-
pyridyl-benzophenone analogue 49, were converted to the 1,1-diaryl
ethylenes 47, 48 and 50 respectively, through Wittig olefination with
the ylide derived from triphenylmethyl phosphonium bromide.
carboxaldehyde independently yielded the hydrazones 60, 62 and 64,
and reaction with pyridine-2-carboxaldehyde yielded analogues 61, 63
and 65 completing the third generation of array summarized in Fig. 4.
The third-generation array compounds 57–65, was screened in the
same assay as described above and the overall results presented in
Fig. 4. Once again, the anti-viral activity of the 2-pyrrolyl compounds
stood out with compounds 62 and 63 proving highly active, and
especially 62 being significantly more potent than acyclovir.
Conversion of each of these three substituted ethylene derivatives to
their corresponding hydrazides was accomplished following the meth-
odology developed earlier. Thus, the reaction of the 1,1-diarylethylene
with ethyl diazoacetate in the presence of catalyst 20, yielded the
corresponding cyclopropanes 51–53 in good yield, as shown in Scheme
3. Interestingly, we observed that the cyclopropanation of the 1,1-bis-
pyridyl ethylene 50 with ethyl diazoacetate did not require any cata-
lyst. The reaction of this substrate ,alone of all derivatives investigated -
was serendipitously found to proceed rapidly without the addition of
any catalyst, indicating that the pyridyl substituent may be playing a
role in an autocatalytic or organocatalytic cyclopropanation process.12
Hydrazinolysis converted these to the three penultimate hydrazides 54,
55 and 56 were then independently reacted with furfural to produce
the array of hydrazones 57, 58 and 59 as shown (Sch. 3). Similarly, the
reaction of the three hydrazides 54, 55 and 56 with pyrrole-2-
In order to interpret the structure–activity relationships within the
third set of compounds further we employed a Radar Plot analysis of the
major pharmacological parameters as depicted in Fig. 5.13 Normalised
values for molecular weight (MW), calculated cLogP, tPSA and number
of H-bond donors (HBD) and acceptors (HBA) were plotted with acy-
clovir and the initial hit compound 5. The overall structural parameters
of the cyclopropylcarboxacylhydrazone derivatives differ considerably
from acyclovir as expected. The most revealing insights were gained
from the analysis of the central horizontal and vertical analogues as
shown in Fig. 4. Anti HSV-1 activity of the pyrrole derivatives 60, 62
and 64 correlated strongly with cLogP, the major factor being con-
tributed by the left-hand lipophilic trifluoromethoxy-containing dia-
rylmethano-fragment. Lipophilicity alone is not sufficient for potent
3

J. McNulty, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127559
Fig. 6. Antiviral IC50 determination of compound 62, 14.1 µM, R2 = 0.9675, in
comparison to acyclovir (ACV), 27.4 µM, R2 = 0.9648 in HSV-1 infected
neuronal progenitor cells.
cycloaddition reactions are underway. The synthetic route allowed for
the rapid preparation of series of analogues and three successive rounds
of structure-antiviral activity assessment identified compound 62 as an
optimised anti HSV-1 derivative, about twice as potent as the standard
therapeutic agent ACV. The SAR analysis demonstrates that anti-viral
activity correlates with a small H-bond donor, such as pyrrole, as the
right-hand fragment and liphophilic substituent on the diarylmethano-
fragment of the cyclopropyl core. Further investigations to determine
the target and mechanism of action of these potent HSV-1 derivatives
and investigation of their anti-viral activity to emerging viruses is un-
derway in our laboratories.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Correspondence concerning chemistry should be addressed to
jmcnult@mcmaster.ca and anti-viral activity to nimga@pitt.edu. All
compounds were synthesized by CBD. LD developed the iPSC neuronal
HSV-1 assay and LM, MD, PP, KW and WZ conducted the anti-viral
assessments. The viral construct was donated by Paul Kinchington,
Ph.D., University of Pittsburgh. This work was supported by funding
from the Stanley Foundation grant 13R-002 (JMcN), NIH
R01 MH063480 and Stanley Foundation grant 07R-1712 (VLN)
and R21 NS096405 (LD).
Fig. 5. Radar Plot analyses of the main pharmacological parameters along the
vertical (60-62-64) and horizontal (58-62-63) sets of analogues as defined in
Fig. 4.
HSV-1 inhibition, however, as the series 58, 62 and 63 demonstrates. A
strong synergistic effect is seen with the small H-bond donor pyrrole 62,
as the right-hand fragment, and to a lesser extent with the pyridine 63.
This data is also consistent with the activity of the pyrrole 33, in
comparison to imidazole substituted derivatives 34 and 35 (Fig. 3).
Overall, this analysis indicates that potent anti HSV-1 activity correlates
with an enhanced lipophilic substituent on the left–hand diaryl frag-
ment and small electron-rich H-bond donor on the right-hand hetero-
aryl fragment of the molecule.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127559.
Lastly, the antiviral potency of compound 62 was determined in
HSV-1-infected neuronal progenitor cells (NPCs) in comparison to ACV
(Fig. 6). Compound 62 exhibited an IC50 of 14.1 µM, whilst the IC50 of
ACV was estimated to be 27.4 µM. In terms of host cell viability,
compound 62 demonstrated no toxicity to NPCs at a concentration of
25 µM.
Reference
1. (a) Schmutzhard E. J Neurol. 2001;248:469
(b) Steiner I, Kennedy PG, Pachner AR. Lancet Neurol. 2007;6:1015
(b) Treml J, Gazdová M, Šmejkal K, Šudomová M, Kubatka P, Hassan STS. Viruses.
2020;12:154.
2. (a) Kanai R, Wakimoto H, Cheema T, Rabkin SD. Future Oncol. 2010;6:619
(b) Flint SJ, Enquist LW, Racaniello VR, Skalka AM. Principles of Virology. Third Ed.
Washington DC: ASM Press; 2009:278–308.
In conclusion, we report the total synthesis of the cyclopropyl
-carboxyacylhydrazone 5, confirmation of its structure (rotamers) and
anti HSV-1 activity.5a A new homogeneous copper (II) catalyst has been
developed that is highly efficient in promoting the [2+1]-cycloaddition
leading to the cyclopropyl core. Attempts to confirm the structure of
catalyst 20 and investigate its potential in copper-catalyzed [2+1]-
3. Izedine H, Launay-Vacher V, Deray G. Am J Kidney Dis. 2005;45:804.
4. (a) Strasfield L, Chou S. Infect Dis Clin North Am. 2010;24:809–833
(b) Burrel S, Aime C, Hermet L, Ait-Arkoub Z, Agut H, Boutolleau D. Antiviral Res.
2013;100:365
(c) Stranska R, Schuurman R, Nienhuis E, et al. M. J Clin Virol. 2005;32:7
4

J. McNulty, et al.
(d) Malvy D, Treilhaud M, Bouee S, et al. Clin Infect Dis. 2005;41:320.
Bioorganic&MedicinalChemistryLetters30(2020)127559
(c) Brown CE, Kong T, McNulty J, et al. Bioorg Med Chem Lett. 2017;27:4601–4605
(d) Brown CE, Kong T, Britten JF, et al. ACS Omega. 2018;3:11469
(e) D’Aiuto L, McNulty J, Hartline C, et al. Sci Rep. 2018;8:16662.
10. (a) Tayama E, Saito S. Synlett. 1880;2015:26
5. (a) McClain, L.; Zhi, Y.; Cheng, H.; Gosh, A.; Piazza P.; Yee, M. B.; Kumar, S.;
Milosevic, J.; Bloom, D. C.; Arav-Boger, R.; Kinchington, P. R.; Yolken, R.;
Nimgaonkar, V.; Aiuto, L. D’. Anti-viral Res. 2015, 121, 16. (b) Library compounds
were purchased from ChemBridge Corporation, San Diego, CA 92121, USA.
6. D’Aiuto L, Williamson K, Dimitrion P, et al. Antiviral Res. 2017;142:136.
7. Schmidt AG, Lee K, Yang PL, Harrison SC. PLoS Pathog. 2012;8:1002627.
8. Filho, J. M. D. S.; Silva, D. M. A. D. Q.; Macedo, T. S.; P. Teixeira, H. M.; Moreira, D.
R. M.; Challal, S.; Wolfender, J.-L.; Queiroz, E. F.; Soares, M. B. P. Bioorg. & Med.
Chem. 2016, 24, 5693.
(b) Nozaki H, Takaya H, Moriuti S, Noyori R. Tetrahedron. 1968;24:3655
(c) Yates P. J Am Chem Soc. 1952;74:5376.
11. McNulty, J., Keskar, K.; Bordon, C.; Yolken, R.; Jones-Brando, L. Chem. Comm. 2014,
50, 8904. (b) McNulty, J. Keskar, K.; Jenkins, H. A.; Werstiuk, N. H.; Bordon, C.;
Yolken, R.; Jones-Brando, L. Org. & Biomolec. Chem. 2015, 13, 10015.
12. (a) For auto catalysis and organocatalysis of related cyclopropanations see: A.P.
Gray H. Kraus J. Org. Chem. 1966, 31, 399.
9. (a) McNulty J, D’Aiuto L, Zhi Y, et al. ACS Med Chem Lett. 2016;7:46
(b) Revu O, Zepeda-Velazquez C, Nielsen AJ, McNulty J, Yolken RH, Jones-Brando
L. Chem Select. 2016;1:5895
(b) Shim SY, Kim JY, Nam M, Hwang G-S, Ryu DH. Org Lett. 2016;18:160
13. Ritchie TR, Ertl P, Lewis R. Drug Disc Today. 2011;16:65.
5