Modifications of C-2 on the pyrroloquinoline template aimed at the development of potent herpesvirus antivirals with improved aqueous solubility

Article abstract of DOI:10.1016/j.bmcl.2010.03.115

A series of C-2 pyrroloquinoline analogs designed to improve aqueous solubility were examined for herpesvirus polymerase and antiviral activity. Several analogs were identified that maintained the antiviral activity of the previous development candidate against HCMV, HSV-1 and VZV, but with significantly improved aqueous solubility.

Full text of DOI:10.1016/j.bmcl.2010.03.115

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3039–3042
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modifications of C-2 on the pyrroloquinoline template aimed at the
development of potent herpesvirus antivirals with improved aqueous solubility
b
a
a
a
James A. Nieman a, , Sajiv K. Nair , Steven E. Heasley , Brenda L. Schultz , Herbert M. Zerth ,
Richard A. Nugent c, Ke Chen a, Kevin J. Stephanski a, Todd A. Hopkins b, Mary L. Knechtel a, Nancee L. Oien a,
Janet L. Wieber a, Michael W. Wathen a
*
a Global Research and Development, Pfizer, Inc., 7000 Portage Road, Kalamazoo, MI 49007, USA
b Global Research and Development, Pfizer, Inc., 10770 Science Center Drive, San Diego, CA 92121, USA
c Global Research and Development, Pfizer, Inc., 620 Memorial Drive, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of C-2 pyrroloquinoline analogs designed to improve aqueous solubility were examined for her-
pesvirus polymerase and antiviral activity. Several analogs were identified that maintained the antiviral
activity of the previous development candidate against HCMV, HSV-1 and VZV, but with significantly
improved aqueous solubility.
Received 13 February 2010
Revised 27 March 2010
Accepted 31 March 2010
Available online 3 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pyrroloquinoline
Cytomegalovirus
HCMV
Varicella-zoster
Herpesvirus
The Herpesvirus family contains eight known viruses that infect
humans.1 These members of Herpesviridae are the herpes simplex
viruses (HSV-1 and HSV-2), varicella zoster virus (VZV), Epstein-
Barr Virus (EBV), human cytomegalovirus (HCMV), and human her-
pes viruses (HHV-6, HHV-7 and HHV-8). Infections by members of
this family are responsible for a wide range of diseases. Although
troublesome, infections in immunocompetent patients are typi-
cally minor and may involve periodic reactivation of a latent state.
However, for patients with suppressed immune systems as a result
of chemotherapy, HIV infection or old-age, serious morbidity and
mortality can occur.2
Safe and effective treatments have been developed for infec-
tions resulting from some members of the Herpesviridae family,
for example treatment of HSV-2 with acyclovir.3 The treatment
of other Herpesvirus infections has not proven as successful. For
HCMV, agents such as ganciclovir are associated with a range of
serious adverse effects and display only modest efficacy.4 Even
with these complications, ganciclovir is considered the gold stan-
dard compared to other less ideal agents such as forscarnet, fomi-
virsen and cidofovir.1 The emergence of drug-resistant strains also
poses an increasing problem for disease management.4a Therefore,
the discovery of a new class of treatments for immunocompro-
mised patients infected with HCMV represents a serious unmet
medical need. Previously disclosed DNA polymerase inhibitors 1,5
26 and 37 (Fig. 1) are broad-spectrum anti-herpetic compounds
that show activity against ganciclovir resistant isolates.5c One con-
sistent issue observed for these and related templates was poor
aqueous solubility.3,8 This Letter describes our C-2 analog effort
O
O
N
N
H
O
O
N
O
Cl
1
O
O
N
H
N
N
N
O
S
Cl
Cl
2
N
H
O
N
2
3
OH
* Corresponding author. Tel.: +1 780 462 4044; fax +1 780 461 0196.
E-mail addresses: jnieman@naeja.com, jnieman@shaw.ca (J.A. Nieman).
Figure 1. Previously reported polymerase inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.115

3040
J. A. Nieman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3039–3042
OH
O
OH O
Reference 7
a
N
N
O
N
N
H
O
O
O
NH2
N
N
Cl
4
I
I
5
6
b
b, c
O
O
O
O
N
O
a, c
N
N
O
O
H
N
N
Cl
R
R
7
8-24
Scheme 1. Synthesis of C-2 pyrroloquinoline analogs 8–24. Reagents and conditions: (a) 4-chlorobenzylamine, 135 °C, 4 h; (b) CuI, PdCl2(PPh3)2, triethylamine, alkyne, EtOH,
reflux, (c) if deprotection was required, HCl in dioxanes or methanol for Boc removal or in THF/water for ketal hydrolysis.
to alleviate the poor aqueous solubility shown by development
candidate 3, while attempting to improve, or at least retain, potent
antiviral activity.
Compounds 8 and 9 are analogs that included an additional hy-
droxyl group in the C-2 side-chain. Although compound 8 and 9 had
improved aqueous solubility compared to 3, they both had dimin-
ished antiviral activity, likely associated with a decrease in perme-
ability. Entry 10, with an increase chain length and ethereal oxygen
demonstrated a significant improvement, almost 10-fold, in aque-
The synthesis of key intermediate 5 from 4 (Scheme 1) has pre-
viously been reported and is amenable to pilot plant synthesis.7a
Conversion of 5 to pyrroloquinoline analogs 8–24 (Table 1) was
accomplished by two routes. Reaction of 5 with 4-chlorobenzyl-
amine generated compound 6.7,9 Sonogashira coupling of 6 with
the appropriate alkyne and concomitment annulation generated
the C-2 pyrroloquinoline analogs.10 Although this approach al-
lowed for late stage diversity, removing Pd from the target com-
pound proved difficult in some cases.7a An alternate approach
from 5, that allowed an extra step for Pd removal, involved initial
Sonogashira coupling/annulation generating 7, followed by 4-chlo-
robenzylamide formation and, if required, deprotection.
The alkynes used in the Sonogashira couplings in Scheme 1
were commercially available or prepared in the following manners.
For compound 8, the protected alkyne (Fig. 2) was generated by
periodate cleavage of the diketal of mannitol (25).11 Attempted
conversion of the resulting aldehyde to an alkyne by a Corey–Fuchs
reaction resulted in a low yield of 26, but the alternate dia-
zophosphonate protocol resulted in a reasonable yield of 26.12
The ether-containing alkynes used in Scheme 1 were generated
according to literature.13 The alkynes with amine linkers (entries
11–24) were prepared simply by reacting propargyl bromide or
3-butynyl p-toluenesulfonate and the corresponding amine in the
presence of diisopropylethylamine or potassium carbonate in
dichloromethane or ethanol.14
ous solubility (P5 lg/mL was the observation-based criteria for
the team) and comparable HCMV polymerase and antiviral activity.
Acyclic amine derivatives 11, 12, 14 and 15 all displayed an
improvement in aqueous solubility compared to 3, but interest-
ingly compound 13, despite its basic amine, displayed no solubility
improvement. Unfortunately, compound 11 showed a slight loss in
HCMV polymerase inhibition, and 12 and 14 demonstrated mar-
ginally lower antiviral activities. Compound 15 was similar in
HCMV polymerase and plaque reduction activity when compared
to 3, but with almost an order of magnitude improvement in aque-
ous solubility.
The cyclic amine containing C-2 analogs are entries 16–24 in
Table 1. Entries 17, 20, 21, 23 and 24 showed significantly better
aqueous solubility than 3, but only 17 maintained antiviral activity.
In contrast, poor solubility was observed for entries 16, 18, 19 and
22. It is interesting to note the dramatic difference (31-fold) in sol-
ubility between 16 and its homolog 17. Two aqueous solubility
prediction programs employed (data not shown) calculated that
16 and 17 would have similar solubility, less than threefold differ-
ence, and both programs overestimated the solubility of these
compounds. This exemplifies the difficulty in accurately predicting
thermodynamic aqueous solubilities.
The HCMV DNA Polymerase and Plaque Reduction assays re-
sults are shown in Table 1 and the details of these assays and their
statistical error have previously been disclosed.3,5,6,7b The C-2 ana-
logs in Table 1 were designed to improve the thermodynamic
aqueous solubility15 while also attempting to pick up additional
interactions with the protein. The results for compound 3 are
shown for comparison purposes to its C-2 analogs.
In many examples in Table 1, as one would expect, incorporat-
ing polar functionality to improve solubility appears to have nega-
tively impacted permeability. This is supported by the higher
HCMV plaque reduction IC50 values despite similar DNA polymer-
ase inhibition. It is also noteworthy that pyrroloquinoline com-
pounds 3, 8–24 showed micromolar or better HCMV DNA
polymerase inhibition. This implies that the C-2 substituent is
not in close contact with the polymerase protein when bound. Un-
like the tight structure activity relationship of the 4-chlorobenzyla-
mide moiety, the structural tolerance displayed by the C-2 region
supports the choice of this region to refine physical properties.
An alternate method to improve aqueous solubility, besides
introducing polarity or removing ‘grease’ is to decrease the crystal
packing capabilities of the analog. One means to accomplish this is
to reduce the planarity of the template. Towards that goal, entry 3
and 10 were reduced generating racemic 27 and 28 ( Fig. 3),16
H
O
O
1) NaIO4, CH2Cl2
rt, 50%
OH
O
O
P
O
HO
2)
O
O
O
O
O
N2
MeOH, K2CO3, 66%
which showed acceptable solubilities of 7.1 and 4.9
tively. However, a slight loss of HCMV DNA polymerase activity
was observed (IC50 = 1.5
M for both).17
lg/mL, respec-
26
25
Figure 2. Preparation of alkyne 26 for generation of compound 8.
l

J. A. Nieman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3039–3042
3041
Table 1
HCMV polymerase and plaque reduction and aqueous solubility results for compounds 3, 8–24
O
O
N
N
H
O
N
Cl
2
R
a
a,b
Entry
R
HCMV Pol. IC50
(lM)
HCMV Plaq. IC50
(l
M)
Aq Sol. pH 7.0c
0.6
(lg/mL)
3
0.35
0.04
OH
OH
8
9
0.29
0.20
0.20
1.4
5.8
OH
OH
0.44 ( 0.02)
O
OH
O
10
11
0.51 ( 0.03)
0.80
0.03
nd
5.8
55
OH
N
H
N
12
13
0.35
0.46
0.20
0.10
16.1
0.37
OH
OH
N
N
OH
OH
OH)2
14
0.52
0.10
2.4
(
15
16
0.55 ( 0.03)
0.65
0.06
0.03
5.2
0.2
N
N
O
N
17
18
0.45 ( 0.06)
0.90
0.04
nd
6.2
0.1
O
OH
N
OH
N
19
0.80
nd
0.1
NH
20
21
1.1 ( 0.0)
nd
80.3
23
N
N
N
0.39 ( 0.02)
0.35
NH
O
22
23
24
0.72
0.39
0.43
nd
0.4
NH
N
N
0.20
0.14
168
34.5
NH
NH
a
b
C
Ref. 6a (nd = not determined).
Determined by plaque reduction assay (Davis strain).
Thermodynamic method used from Ref. 15.
The compounds that showed antiviral activity on par with 3 and
vir against VZV. For HSV-1, acyclovir and the pyrroloquinoline
compounds had comparable antiviral activity. However, com-
pound 17 showed human a-DNA polymerase inhibition and com-
pound 15 displayed cytotoxicity. Compound 10 had good
selectivity, no cytotoxicity and also demonstrated almost identical
pharmacokinetics to 3 in rats (F = 66%, T1/2 = 0.8 h, CL = 2.5 L/h/kg,
and Vss = 1.7 L/kg). Overall, compound 10 had a very similar profile
to the develop candidate 3, but with dramatically improved
solubility.
had improved aqueous solubility (Table 1, entries 10, 15, and 17)
were then examined for broad-spectrum activity against HSV-1
and VZV in the DNA Polymerase Inhibition and Plaque Reduction
assays as well as selectivity over human
a-DNA polymerase and
cytotoxicity (Table 2). As observed for other templates3,5,6,8 all
show good broad-spectrum anti-herpetic activity. In the Plaque
Reduction assays the pyrroloquinoline compounds in Table 2 had
better antiviral activity than ganciclovir against HCMV and acyclo-

3042
J. A. Nieman et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3039–3042
In addition, we would also like to recognize the yet unpublished
pioneering pyrroloquinoline work by Joseph Strohbach, Audris
Huang, Sandra Staley and Valerie Vaillancourt.
O
N
O
N
N
H
O
Cl
3 (R = CH2OH)
10 (R = O(CH2)2OH)
Supplementary data
R
Pd/BaSO4,
H2 (balloon),
DMF
Supplementary data (additional cytotoxicity data, synthetic
procedures, and characterization data for 8–21, 26 and 28) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.03.115.
O
O
N
N
H
O
References and notes
N
Cl
27 (R = CH2OH)
28 (R = O(CH2)2OH)
1. Wathen, M. W. Rev. Med. Virol. 2002, 12, 167.
2. Villarreal, E. C. Prog. Drug Res. 2003, 60, 263.
R
3. Oien, N. L.; Brideau, R. J.; Hopkins, T. A.; Wieber, J. L.; Knetchel, M. L.; Shelly, J.
A.; Anstadt, R. A.; Wells, P. A.; Poorman, R. A.; Huang, A.; Vaillancourt, V. A.;
Clayton, T. L.; Tucker, J. A.; Wathen, M. W. Antimicrob. Agents Chemother. 2002,
46, 724.
Figure 3. Reduction of 3 and 10 generating 27 and 28, respectively.
4. (a) Mercorelli, B.; Sinigalia, E.; Loregian, A.; Palu, G. Rev. Med. Virol. 2008, 18,
177; (b) Buhles, W. C. In Ganciclovir Therapy for Cytomegalovirus Infections;
Spector, S. A., Ed.; Marcel Dekker: New York, 1991; pp 31–40.
5. (a) Brideau, R. J.; Knetchel, M. L.; Huang, A.; Vaillancourt, V. A.; Vera, E. E.; Oien,
N. L.; Hopkins, T. A.; Wieber, J. L.; Wilkinson, K. F.; Rush, B. D.; Schwende, F. J.;
Wathen, W. M. Antiviral Res. 2002, 54, 19; (b) Knechtel, M. L.; Huang, A.;
Vaillancourt, V. A.; Brideau, R. J. J. Med. Virol. 2002, 28, 234; (c) Hartline, C. B.;
Harden, E. A.; Williams-Aziz, S. L.; Kushner, N. L.; Brideau, R. J.; Kern, E. R.
Antiviral Res. 2005, 65, 97.
6. (a) Schnute, M. E.; Cudahy, M. M.; Brideau, R. J.; Homa, F. L.; Hopkins, T. A.;
Knechtel, M. L.; Oien, N. L.; Pitts, T. W.; Poorman, R. A.; Wathen, M. W.; Wieber,
J. L. J. Med. Chem. 2005, 48, 5794; (b) Schnute, M. E.; Anderson, D. J.; Brideau, R.
J.; Ciske, F. L.; Collier, S. A.; Cudahy, M. M.; Eggen, M.; Genin, M. J.; Hopkins, T.
A.; Judge, T. M.; Kim, E. J.; Knechtel, M. L.; Nair, S. K.; Nieman, J. A.; Oien, N. L.;
Scott, A.; Tanis, S. P.; Vaillancourt, V. A.; Wathen, M. W.; Wieber, J. L. Bioorg.
Med. Chem. Lett. 2007, 17, 3349. and references within.
7. (a) Dorow, R. L.; Herrinton, P. M.; Hohler, R. A.; Maloney, M. T.; Mauragis, M. A.;
McGhee, W. E.; Moeslein, J. A.; Strohbach, J. W.; Veley, M. F. Org. Process Res.
Dev. 2006, 10, 493; (b) Vaillancourt, V. A.; Staley, S.; Huan, A.; Nugent, R. A.;
Chen, K.; Nair, S. K.; Nieman, J. A.; Strohbach, W. A. U.S. Patent 6,525,049, 2003
8. (a) Tanis, S. P.; Strohbach, J. W.; Parker, T. T.; Moon, M. W.; Thaisrivongs, S.;
Perrault, W. R.; Hopkins, T. A.; Knechtel, M. L.; Oien, N. L.; Wieber, J. L.;
Stephanski, K. J.; Wathen, M. W. Bioorg. Med. Chem. Lett. 2010, 20, 1994; (b)
Schnute, M. E.; Brideau, R. J.; Collier, S. A.; Cudahy, M. M.; Hopkins, T. A.;
Knechtel, M. L.; Oien, N. L.; Sackett, R. S.; Scott, A.; Sephan, M. L.; Wathen, M.
W.; Wieber, J. L. Bioorg. Med. Chem. Lett. 2008, 18, 3856; (c) Larsen, S. D.; Zhang,
Z.; DiPaolo, B. A.; Manninen, P. R.; Rohrer, D. C.; Hageman, M. J.; Hopkins, T. A.;
Knechtel, M. L.; Oien, N. L.; Rush, B. D.; Schende, F. J.; Stefanski, K. J.; Wieber, J.
L.; Wilkinson, K. F.; Zamora, K. M.; Wathen, M. W.; Brideau, R. J. Bioorg. Med.
Chem. Lett. 2007, 17, 3840.
Table 2
Comparison of select compounds, including 3, and established therapies for broad-
spectrum herpesvirus activity, selectivity and cytotoxicity
a
c
Compound
DNA polymerase IC50
(l
M)
Plaque reduction
CC50
M)
a,b
IC50
(lM)
(
l
HCMV HSV-1 VZV
Human
a HCMV HSV-1 VZV
3
10
15
17
0.35
0.51
0.55
0.45
0.12
0.37
0.75
0.36
0.07
0.38
0.23
0.19
>50
>50
>20
26
0.04
0.03
0.06
0.04
1.3
0.8
1.2
1.7
2.1
nd
0.05 >75
0.10 >75
nd
0.03 nd
nd
23
Ganciclovir
Acyclovir
Forscarnet 2.5
Aphidicolin 0.487 0.438 0.473 2.6
AZT-TPd
22.1 3.3 5.8
>100
8.1 >100
>20
2.1
a
Ref. 6a (nd = not determined).
Determined by plaque reduction assay with Davis strain (HMCV), KOS strain
b
(HSV-1) and Webster strain (VZV).
c
Ref. 8b.
d
AZT-TP = Azidovudine triphosphate.
In conclusion, this examination of analogs at C-2 of the pyrrol-
oquinoline template successfully demonstrated that solubilizing
groups could be incorporated and the HCMV antiviral activity
maintained. Analogs 10, 15 and 17 demonstrated broad-spectrum
anti-herpetic activity that was equal or better than current thera-
pies with improved aqueous solubility over previous development
9. General procedure for amide bond formation: To the desired ester was added
4-chlorobenzylamine (3–10 equiv) and the mixture was heated to 135 °C for
4 h. Excess 4-chlorobenzylamine was removed by Kugelrohr distillation at
120 °C (0.1 Torr). The product was purified by silica gel chromatography.
10. General procedure for coupling/cyclization reaction: To
a solution of the
candidate 3. Compound 10 also had good selectivity over human a-
desired aryl iodide, copper(I) iodide (0.1 equiv), and PdCl2(PPh3)2 (0.05 equiv)
in EtOH (5 mL) was added sequentially triethylamine (2 equiv) and the
appropriate alkyne (1.5 equiv) at room temperature. The reaction mixture is
heated to reflux (0.5–2 h) and cooled to room temperature. The solvent is
removed in vacuo. The product was purified by silica gel chromatography.
11. Schmid, C. R.; Bradley, D. A. Synthesis 1992, 2540–2541.
DNA polymerase, no cytotoxicity and acceptable rat pharmacoki-
netics. The focus of our next publication will be the exploration
of the C-8 position of the pyrroloquinoline system, in combination
with select C-2 substituents presented here.
12. Pietruszka, J.; Witt, A. J. Chem. Soc., Perkin Trans. 1 2000, 4293.
13. Hernandez, E.; Galan, A.; Rovira, C.; Veciana, J. Synthesis 1992, 1164.
Acknowledgements
14. Entries 20, 21 and 24 had
a Boc protected nitrogen and thus required
deprotection to form the final compound.
15. For the protocol used to measure thermodynamic solubility see: Guo, J.;
Elzinga, P. A.; Hageman, M. J.; Herron, J. N. J. Pharm Sci. 2008, 97, 1427.
With much appreciation, we acknowledge Roberta Dorow and
Mark Lyster for providing additional quantities of 5 and 6 and Ste-
ven Tanis for supplying 25. We thank the Analytical Chemistry
group in Kalamazoo and Eric Seest for their work. We also thank
Steven Tanis and Michael Ennis for helpful discussions and advice.
16. Alternatively, intermediate
chlorobenzylamide formation.
7
could be reduced followed by 4-
17. The enantiomers of 28 were separated and displayed HCMV DNA polymerase
IC50 values of 3.0 and 0.7 M.
l