US warplanes have bombed pro-government forces in Syria to repel “an unprovoked attack” on allied forces in the country’s east in one of the most serious clashes between the Washington-led coalition and those loyal to the country’s President Bashar al-Assad.

It is a sign of the escalating risk of confrontations between the various players in Syria’s complicated civil war, particularly as the territory once held by Isis – the one common enemy – shrinks, leaving competing factions butting up against each other on the battlefield.

US officials said that more than 100 pro-government troops had been killed in the wake of more 500 troops launching an attack on territory held by the US-backed Syrian Democratic Forces (SDF) alliance of Kurdish and Arab militias around the Euphrates River. Syria state media accused Washington of a “war crime”.

In a sign of the dangers of escalating conflict across a nation where a number of groups and nations are now facing off against each other as Isis, which was the common enemy, continues to lose territory.

The SDF control areas east of the Euphrates River in Deir Ezzor province, including most of the oil and gas fields, having forced out Isis from the area last year. Meanwhile Syrian government forces are based in the west. The oil-rich province had been a major source of income for Isis and the resources there would be a boon for the government of Syrian President Bashar al-Assad. Mr Assad has repeatedly said he wants to claim back every inch of his country.

Mr Assad is backed by Russia, as well as Shia militia groups themselves backed by Iran, while the US is supporting the Kurdish SDF forces who have been a crucial ally in battling Isis – the major aim in Syria for the administration of President Donald Trump. Turkey has also started an offensive to push the US-backed Kurdish forces out of the northern town of Afrin, near the Turkish border, which has also increased tensions between Washington and Ankara.

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“Pro-regime forces initiated hostilities with artillery pieces (howitzers). Additionally, Syrian pro-regime forces manoeuvred T-55 and T-72 main battle tanks with supporting mortar fire in what appears to be a co-ordinated attack on Syrian Democratic Forces approximately five miles east of the Euphrates River de-confliction line in Khusham, Syria,” Colonel Thomas F Veale, of the US-led coalition forces said of the latest clash.

The US Defense Secretary Jim Mattis called the attack “perplexing” during a press conference on Thursday.

By crossing the river, the pro-government forces would have violated the existing de-confliction agreement that had been agreed between Washington and Moscow as they had sought to reduce the threat of confrontation in a country that has become a complicated patchwork of potential clashes.

In a letter to the United Nations, Syria’s Foreign Ministry described the strike as a “war crime” and called for the coalition to be dismantled, Syrian state news agency SANA said. “We demand [that the international community] condemn this massacre and hold the coalition responsible for it.”

Russia’s Interfax news agency cited the country’s defence ministry as saying the incident showed the US goal in Syria was not to battle Isis but “the capture and withholding of the economic assets”, an apparent reference to the Khusham oil field. Russia itself had entered the war in Syria saying that it was also looking to defeat “terrorists”, although not just Isis, but has become a crucial ally for Mr Assad.

Video shows the moment a bomb hits a building in Syria as new onslaught new onslaught causes mass civilian casualties

Russia’s UN envoy, Vassily Nebenzia, described the deadly US-led coalition strike as “regrettable”.

In a later statement, Pentagon spokeswoman Dana White stressed the coalition strikes were purely defensive. Washington was not seeking to fight Syrian forces, she said.

“We are not looking for a conflict with the regime [of President Assad],” Ms White said.

Adding to the tension, the latest battlefield clashes come amid a backdrop of escalating tension over allegations of the use of chemical weapons by President Assad’s forces, and increased violence around Damascus. Syria forces are pounding besieged pockets of rebel-held towns in Eastern Ghouta, with the Syrian Observatory for Human Rights, a Britain-based monitoring group, alleging that dozens had been killed in the aerial bombardment.

The US State Department has backed a UN call for a month-long pause in hostilities to allow for humanitarian aid and an evacuation of citizens. It also reiterated allegations of recent use of chemical weapons such as chlorine against his own people by Assad. While Syria denies such claims, they are being investigated by the Organisation for the Prohibition of Chemical Weapons (OPCW).

On Thursday State Department Spokesperson Heather Nauert said: “We are yet again appalled by the recent reports of the Assad regime’s use of chemical weapons and the escalation of bombings that has resulted in dozens of civilian deaths in the last 48 hours.”