An efficient and practical synthesis of 2,4-substituted pyrido[4,3-d]pyrimidin-5(6H)-ones

Article abstract of DOI:10.1016/j.tetlet.2019.151312

We describe an efficient synthesis of 2,4-substituted pyrido[4,3-d]pyrimidin-5(6H)-ones, which involves the acid-promoted cyclization of cyano enamine 15 to afford 2,4-bis(methylthio)pyrido[4,3-d]pyrimidin-5(6H)-one 17 as a key intermediate. Selective displacement of the 4-methylthio group by a wide range of anilines followed by oxidation of the 2-methylthio group and subsequent substitution by amines enabled the synthesis of a variety of 2,4-disubstituted pyrido[4,3-d]pyrimidin-5(6H)-ones.

Full text of DOI:10.1016/j.tetlet.2019.151312

Journal Pre-proofs
An Efficient and Practical Synthesis of 2,4-Substituted Pyrido[4,3-d]pyrimi-
din-5(6H)-ones
Xinrong Tian, Dominic P. Suarez, William Hoi Hong Li, Allison K. McSherry,
Robert M. Sanchez, Michael L. Moore, Jeffrey M. Axten
PII:
S0040-4039(19)31096-2
DOI:
https://doi.org/10.1016/j.tetlet.2019.151312
TETL 151312
Reference:
Tetrahedron Letters
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20 October 2019
21 October 2019
Please cite this article as: Tian, X., Suarez, D.P., Li, W.H.H., McSherry, A.K., Sanchez, R.M., Moore, M.L., Axten,
J.M., An Efficient and Practical Synthesis of 2,4-Substituted Pyrido[4,3-d]pyrimidin-5(6H)-ones, Tetrahedron
Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.151312
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An Efficient and Practical Synthesis of 2,4-Substituted Pyrido[4,3-
d]pyrimidin-5(6H)-ones
Xinrong Tian*, Dominic P. Suarez, William Hoi Hong Li, Allison K. McSherry, Robert
M. Sanchez, Michael L. Moore, Jeffrey M. Axten
GlaxoSmithKline, Research and Development, 1250 South Collegeville Road, Collegeville,
PA 19426
Abstract: We describe an efficient synthesis of 2,4-substituted pyrido[4,3-d]pyrimidin-
5(6H)-ones, which involves the acid-promoted cyclization of cyano enamine 15 to afford
2,4-bis-thiomethyl pyrido[4,3-d]pyrimidin-5(6H)-one 17 as a key intermediate. Selective
displacement of the 4-methylthio group by a wide range of anilines followed by
oxidation of the 2-methylthio group and subsquent substitution by amines enabled the
synthesis of a variety of 2,4-disubstituted pyrido[4,3-d]pyrimidin-5(6H)-ones.
Bicyclic pyrido[4,3-d]pyrimidin-5(6H)-one is an important scaffold that can be found in a
number of biologically active compounds. Recently, a series of publications and patent
applications have disclosed the synthesis of pyrido[4,3-d]pyrimidin-5(6H)-ones,
developed as GAA activators1 and kinase inhibitors2-6. In addition, pyrido[4,3-
d]pyrimidin-5(6H)-ones serve as pivotal intermediates for the synthesis of 2,4-
disubstituted pyrido[4,3-d]pyrimidines as autotaxin inhibitors7 and Syk kinase inhibitors
8. While unsubstituted pyrido[4,3-d]pyrimidin-5(6H)-one could be derived from the
reaction of triazine and ethyl acetoacetate in the presence of sodium ethoxide in a low
yield1, substituted pyrido[4,3-d]pyrimidin-5(6H)-ones have been largely prepared by an
approach involving the cyclization of an enamino ester3-9.
In the course of a drug discovery program optimizing kinase inhibitors, we sought an
efficient synthetic route to quickly prepare analogs of biologically active hit molecule 1.
Optimally, we envisioned a synthesis of 2,4-substituted pyrido[4,3-d]pyrimidin-5(6H)-
ones that was amenable to flexible late stage analog derivatizations at the 2- and 4-
positions. When we embarked on our own synthetic campaign, the synthesis of

pyrido[4,3-d]pyrimidin-5(6H)-ones bearing 2,4-diamino substituents had been scarcely
reported in the literature.
In 1979, Baldwin and co-workers10 reported the synthesis of naphthyridinone via an
enamine cyclization, where the treatment of the enamine of 4-methylnicotinonitrile with
30% HBr-HOAc afforded naphthyridinone in 41% yield. Inspired by their work, we
explored extension of their synthetic approach to a more complex pyrimidine ring
system, such as 4-methyl-2-(methylthio)-6-(m-tolylamino)pyrimidine-5-carbonitrile (8a).
Herein we describe the development of our initial approach to the synthesis of 2,4-
substituted pyrido[4,3-d]pyrimidin-5(6H)-ones, as outlined in Scheme 1.

Scheme 1. Reagents and Conditions: a) acetic acid, 90 oC, 66%; b) S-methyl-
isothiouronium sulfate, NaOMe, CH3OH, 74%; c) CuCl2, tert-butylnitrite, MgSO4,
acetonitrile, 80 oC, 68%; d) ArNH2, DIEA, DMF, 75 oC; e) dimethylformamide
dimethylacetal, DMF, 100-110 oC, for 7a-d; or 2.5 eq. tert-butoxy
bis(dimethylamino)methane (BBDM), DMF, 110 oC, for 7e; f) 33% HBr/HOAc; g) 6N
HCl (aq.)/HOAc, 80 oC; h) mCPBA, DMF; i) tert-butyl (3-aminopropyl)carbamate; j)
TFA, CH2Cl2.
The synthesis started with condensation of malononitrile and triethyl orthoacetate to give
(1-ethoxylethylidene)malononitrile 4. Reaction of 4 with s-methylisothiourea and sodium
methoxide in methanol afforded 4-amino-6-methyl-2-(methylthio)-5-
pyrimidinecarbonitrile 5. Diazotization of 5 with tert-butyl nitrite in the presence of
cuprous chloride generated the 2-chloro intermediate 6, which was then reacted with m-

toluidine in the presence of N,N-diisopropylethylamine (DIEA) in DMF at 65 oC to yield
7a. With 7a in hand, we set out to explore pyrido[4,3-d]pyrimidin-5(6H)-one formation
using the synthetic protocols reported by Baldwin and co-workers10. To this end,
treatment of 7a with dimethylformamide dimethylacetal (DMF-DMA) in DMF at 75 oC
afforded enamine 8a. Interestingly, when 8a was reacted with a solution of 33% HBr in
acetic acid, we obtained exclusively the bromopyrido[4,3-d]pyrimidine 9a and did not
observe the expected pyrido[4,3-d]pyrimidin-5(6H)-one 10a. However, 9a could be
readily hydrolyzed using 6N HCl in acetic acid at 80 oC to afford the pyrido[4,3-
d]pyrimidin-5(6H)-one 10a in 80% yield over two steps. Oxidation of 10a with mCPBA,
displacement of the resulting sulfone with amines, followed by Boc-deprotection
afforded desired pyrido[4,3-d]pyrimidin-5(6H)-one analog 1. We further expanded the
scope and demonstrated the utility of the four step transformation of chloropyrimidine 6
to disubstituted pyrido[4,3-d]pyrimidin-5(6H)-ones 10b-e using a variety of substituted
anilines.
While the use of DMF-DMA to generate the enamines was generally functional-group
tolerant, in certain cases we did encounter undesired alkylation of functional groups as a
significant byproduct. For example, treatment of 7e with DMF-DMA in DMF at 100 oC
produced a 1:6 mixture of 8e and the N-methylated product 13 (Scheme 2). We thereafter
discovered that the use of tert-butoxy bis(dimethylamino) methane (BBDM)11 to replace
DMF-DMA overcame this issue, affording exclusively the desired enamine 8e.
Scheme 2. Enamine formation using DMF-DMA.

The newly developed synthetic route described above allowed us to readily prepare
analogs for exploring structure-activity relationship (SAR) at the 2 and 4-positions of the
initial hit 1. However, the major drawback of this approach lies in the fact that the
anilines had to be introduced early in the synthesis prior to the ring closure to form
pyrido[4,3-d]pyrimidin-5(6H)-one core, which increased the likelihood of side reactions
associated with the functional group incompatibility.
To develop a more efficient synthetic approach to analog preparation, our subsequent
efforts focused on constructing an advanced pyrido[4,3-d]pyrimidin-5(6H)-one
intermediate which would allow for late stage independent functionalization of the 2- and
4-positions. Considering leaving groups at these positions needed to be reactive to aniline
displacement as well as inert to the conditions for enamine formation and subsequent
cyclization, we reasoned that the methylthio group could satisfy these requirements. The
methylthio functionality is not only relatively stable toward acids but also could be
oxidized if necessary to a more reactive sulfone or sulfoxide leaving group. Thus, the
reaction of chloropyrimidine 6 with sodium thiomethoxide efficiently produced 4-
methyl-2,6-bis(methylthio)pyrimidine-5-carbonitrile 14, which was converted to the
enamine 15 by heating with DMF-DMA in DMF. The enamino nitrile 15 then underwent
cyclization upon treatment with 33% HBr in acetic acid to yield 5-bromo pyrido-
pyrimidine 16, which was subsequently hydrolyzed to pyrido[4,3-d]pyrimidin-5(6H)-one
17 in 72% yield over two steps. To further simplify the synthesis, we attempted the
direct cyclization of 15 of 17 under acidic conditions and eventually found that heating
enamine 15 in acetic acid with 10% aqueous HBr or 6N HCl effected cyclization to give
the desired pyrido[4,3-d]pyrimidin-5(6H)-one 17 in good yield.12 With 17 in hand, we
then explored conversion of methylthioether to the methylsulfoxide or methylsulfone.
However, oxidation with mCPBA initially led to the formation of sulfoxide and sulfone
as indicated by LC-MS, but the products decomposed as reaction progressed, making it
impossible to separate and isolate the pure sulfoxide and sulfone products. This result
prompted us to explore direct displacement of the 2- or 4-methylthio group. After some
experimentation, we were pleased to find that the selective replacement of 4-methylthio
group could be achieved by heating 17 with anilines in the presence of HCl in

isopropanol. This reaction proceeded smoothly over a broad range of anilines with high
functional group tolerance (10a, 18b-k) including heterocycles such as aminopyrrazole,
exemplified by 18k.
Scheme 3. Reagents and Conditions: a) Sodium thiomethoxide, DMF, 81%; b) DMF-
DMA, DMF, 110 oC, 91%; c) 33% HBr/HOAc; d) 6N HCl (aq.)/HOAc, 80 oC, 72% over
two steps; f) 10% HBr, acetic acid, 83 oC, 70%; g) ArNH2, conc. HCl, isopropanol,
reflux; h) mCPBA, DMF; i) tert-butyl (3-aminopropyl)carbamate then amine for 10a,
18c; (R)-tert-butyl 3-aminopyrrolidine-1-carboxylate for 18h-k; j) TFA, CH2Cl2.
In addition to the propylamine moiety contained in 1, a variety of amines had been
readily incorporated to the 2-position of pyrido[4,3-d]pyrimidin-5(6H)-one core by
means of the reaction sequence shown in Scheme 4. The synthesis of target analogs

Scheme 4. Reagents and Conditions: Method A: i) mCPBA, DMF; ii) RNH2; Method B:
i) mCPBA, DMF; ii) RNH2,; iii) 4N HCl in dioxane, DCM.
using this novel synthetic route and their biological activity will be reported elsewhere in
due course.
In conclusion, we have demonstrated the development of an efficient synthesis of 2,4-
disubstituted pyrido[4,3-d]pyrimidin-5(6H)-ones. The approach features a versatile 2,4-
bis(methylthio)pyrido[4,3-d]pyrimidin-5(6H)-one intermediate which was initially
prepared from enamino nitrile 15 using a high yielding two-step process. The synthetic
efficiency was further improved by achieving the direct acid-promoted cyclization of 15.
Selective displacement of the 4-methylthio group in 17 by a wide range of anilines was
successfully accomplished under acidic conditions. Subsequent oxidation of the 2-
methylthio ether followed by reaction with amines and deprotection (if necessary), led to
the synthesis of a variety of 2,4-disubstituted pyrido[4,3-d]pyrimidin-5(6H)-ones.

Acknowledgments
The authors would like to thank Victoria Magaard for performing HRMS analyses, and
Minghui Wang and Grazyna Graczyk-Milbrandt for carrying out NMR analyses.
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An Efficient and Practical Synthesis of 2,4-Substituted Pyrido[4,3-
d]pyrimidin-5(6H)-ones
Xinrong Tian, Dominic P. Suarez, William Hoi Hong Li, Allison K. McSherry, Robert
M. Sanchez, Michael L. Moore, Jeffrey M. Axten
• Efficient synthesis of a versatile pyrido[4,3-d]pyrimidin-5(6H)-one intermediate
• Selective displacement of the 4-methylthio group by various anilines under the acid
conditons
• Facile replacement of the 2-methylthio group with diverse amines to biologically
significant analogous.
• BBDM is more functional-group tolerant than DMF-DMA for enamine formation

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Substituted Pyrido[4,3-d]pyrimidin-5(6H)-ones
Xinrong Tian, Dominic P. Suarez, William Hoi Hong Li, Allison K. McSherry, Robert M. Sanchez, Michael L. Moore,
Jeffrey M. Axten

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests: